Citation
Linkage between Biogeochemical Properties and Microbial Activities in Lake Sediments

Material Information

Title:
Linkage between Biogeochemical Properties and Microbial Activities in Lake Sediments Biotic Control of Organic Phosphorus Dynamics
Creator:
Torres, Isabela Claret
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (279 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Soil and Water Science
Committee Chair:
Reddy, Konda R.
Committee Co-Chair:
Ogram, Andrew V.
Committee Members:
Phlips, Edward J.
Havens, Karl E.
Brenner, Mark
Graduation Date:
12/14/2007

Subjects

Subjects / Keywords:
Carbon ( jstor )
Carbon dioxide ( jstor )
Lakes ( jstor )
Methane production ( jstor )
Microbial biomass ( jstor )
Nutrients ( jstor )
Phosphates ( jstor )
Phosphorus ( jstor )
RNA ( jstor )
Sediments ( jstor )
Soil and Water Science -- Dissertations, Academic -- UF
biogeochemistry, carbon, eutrophication, isotopes, lake, methanogenesis, microorganisms, nitrogen, phosphodiesterase, phosphomonoesterase, phosphorus, respiration, sediment
Lake Okeechobee ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Soil and Water Science thesis, Ph.D.

Notes

Abstract:
In lakes, deposition of allochthonous and autochthonous particulate matter to sediments can alter the physico-chemical properties and associated biogeochemical processes. Coupling and feedback between sediment biogeochemistry and water column primary productivity often depends on biogeochemical processes within sediments and associated microbial communities. The current investigation was conducted to link biogeochemical properties of benthic sediments and microbial communities and their activities in sub-tropical lakes of different trophic state (Lake Annie: oligo-mesotrophic, Lake Okeechobee: eutrophic, and Lake Apopka: hypereutrophic). The central hypothesis of this study was that lakes with contrasting trophic states have sediments with different biogeochemical properties that have a selection pressure (i.e., C, N and P availability or limitation) on the microbial community that is reflected in their activities. Sediments sampled from sixteen different sites revealed that trophic state was not related to nutrient content of sediments. The relative abundance of phosphorus (P) forms in sediments was more important than total P concentration in characterizing the processes occurring in sediments. Laboratory batch incubation studies were conducted to determine the relationships between major sediment P forms, enzyme activity, heterotrophic microbial activity, and nutrient limitation. Results showed that the concentrations of various P compounds changed with sediment depth, indicating that different processes were controlling P reactivity and mobility in these lakes. Also, P-associated enzyme activities were related to sediment microbial biomass and activity, as well as to the different P forms and availability in sediments. Microbial community biomass and activity, as well as incubation experiments, revealed that the Lake Annie sediment microbial community was carbon (C)-limited, while Lake Apopka was P-limited. Lake Okeechobee mud and sandy sediments were C and nitrogen (N) limited, whereas in the peat sediment a co-limitation of C and P was observed. Stable isotope analyses showed that, in each lake, different mechanisms control delta 13C and delta 15N signatures in these sediments, and were closely linked to lake physico-chemical properties, as well as the primary productivity in the water column. Isotopic signatures in the lake sediments showed a trend of enrichment in delta 13C and delta 15N with increasing trophic state. Oligo-mesotrophic Lake Annie sediment had the lowest values of delta 13C and delta 15N. Eutrophic Lake Okeechobee mud sediments displayed intermediate values for both isotopes. And hypereutrophic Lake Apopka had the highest values for both delta 13C and delta 15N. Catabolic response profiles of a wide variety of C-substrates added to sediments indicated that different microbial communities are present in these sediments. The microbial community of hypereutrophic lake sediments had higher efficiency use of energy and higher catabolic diversity. This study highlighted the relationships between sediment biogeochemical properties and the microbial community, how they differ among lakes with different trophic states, and how the physico-chemical conditions of lakes affect sediment properties and microbe-mediated processes. Results suggest that although the microbial community is C/energy limited, C, coupled with N and P availability had a strong influence on microbial communities in these lakes sediments. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2007.
Local:
Adviser: Reddy, Konda R.
Local:
Co-adviser: Ogram, Andrew V.
Statement of Responsibility:
by Isabela Claret Torres.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Torres, Isabela Claret. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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CHAPTER 1
INTTRODUCTION

In freshwater ecosystems an increase in external nutrient input resulting from

anthropogenic activities is frequently the maj or cause of eutrophication (Krug 1993; Straskraba

et al. 1995; Noges et al. 1998). Although some freshwater ecosystems can become eutrophic

naturally, accelerated rate of eutrophication of many lakes is a direct consequence of high

nutrient load from anthropogenic activities, such as agricultural practices and urban activities.

The main paths of anthropogenic eutrophication (also called cultural eutrophication) in

lakes are: increase in input of nutrients (mainly nitrogen and phosphorus), increase of the

phytoplankton biomass, loss of biological diversity, dominance by cyanobacteria, diatom, and

unicellular green algae, occurrence of algae 'blooms' (high biomass production of certain species

of algae at the water surface), reduction in light and oxygen availability, change in heterotroph

community composition, death of fish. All these alterations will lead to an ecosystem change,

loss of species diversity and decrease in water quality. Hence, lakes with different trophic states

(oligotrophic: low productivity, mesotrophic: medium productivity, eutrophic: high productivity

and hypereutrophic: very high productivity) will have distinctive physical, chemical and

biological characteristics (i.e., pH, redox potential, and microbial community).

Sediment Organic Matter

Particulate matter that enters a lake (allochthonous) or is produced within a lake

autochthonouss) is deposited and becomes an integral part of sediments. Consequently, lakes

function as natural traps for particulate matter and associated nutrients. Accumulation and

retention of particulate matter and nutrients in sediments depends on lake morphometry, water

renewal, nutrient loading, edaphic characteristics of the drainage basin, among others (Bostroim

et al. 1988) and can alter the physico-chemical properties of sediments and associated









CHAPTER 3
SEDIMENT PHOSPHORUS FORMS INT SUBTROPICAL LAKES

Introduction

Phosphorus (P) is often the limiting nutrient for primary productivity in freshwater

ecosystems. Sources of P to lakes can be external (allochthonous) or internal autochthonouss).

Allochthonous P input originates in the drainage basin, while autochthonous P originates from

primary and secondary productivity within lakes. A maj or portion of P from these sources added

to the water column accumulates in sediments. Sediment P is present in both inorganic and

organic forms. Organic P and cellular constituents of the biota represent 90% of total P (TP) in

freshwater ecosystems (Wetzel 1999), and in sediments 30-80% of TP is typically in organic

form (Williams and Mayer 1972; Bostroim et al. 1982).

Although organic P is an important component of sediment P, it has been relatively under-

studied as compared with the fate of inorganic P (Turner et al. 2005). The reason for this is that

there is no direct way to measure organic P. It is usually estimated by difference (before and after

ignition at high temperature) (Saunders and Williams 1955), or by sequential extraction or

chemical fractionation (Condron et al. 2005; McKelvie 2005). These chemical fractionations are

based on different solubilities of P forms in alkaline and acid extractions with different pH.

Turner et al. (2006) compared two methodologies, chemical fractionation and phosphorus-31

nuclear magnetic resonance (31P NMR) spectroscopy, to measure organic P, and showed that for

wetland soils, alkaline extraction with molybdate colorimetry overestimated organic P (between

30-54%). They concluded that alkaline extraction with 31P NMR spectroscopy is a more accurate

method to quantify organic P. In recent years there have been many studies using this

methodology to distinguish different organic P forms in lake sediments (Hupfer et al. 1995,

2004; Carman et al. 2002; Ahlgren et al. 2005; Ahlgren et al. 2006a, b; Reitzel et. al 2006a, b,









enrichment of autochthonous OM and stratigraphic variation in the 6 5N signature of sediments

in Lake Apopka may indicate periods of N limitation.

Other mechanisms may also influence 6 5N of Lake Apopka sediments. The N isotopic

signature of sediment integrates multiple fractionation processes that occur in the sediment and

water column (Lehmann et al. 2004). Ammonium production through organic matter

mineralization is high in these sediments (Chapter 5). Such mineralization processes can lead to

isotopic enrichment of the remaining OM. Inglett et al. (2007) related 6 "N enrichment in the

Everglades soil that is highly impacted with P, to an increase in microbial processes (i.e.,

respiration, mineralization rates). Moreover, denitrification discriminates against heavy 1N and

increases in denitrification rates have been related to enriched 6 "N signatures in sediments

(Terranes and Bernasconi 2000; Savage et al. 2004). The N isotope signature in Lake Apopka

sediments is generated by multiple factors including the isotopic signature of autochthonous N

sources, the primary producer community, and N related processes in the water column and

sediments (Figure 6-9E).

Samples from each core were plotted in isotope space, i.e., 613C VS. 615N (Figure 6-8).

Each core occupies a distinct region in isotope space. Lake Apopka is relatively enriched in both

813C and 6 5N. Mud zone site M9 in Lake Okeechobee displays intermediate values for both

isotopes. Lake Annie is most depleted in 613C, but similar in 61 N to sites M17 and KR.

Excluding the highly different sediment types, peat (M17) and sand (KR) from the figure, it

seems that the remaining sediments show a gradient in relation to both 813C and 6 "N. Oligo-

mesotrophic Lake Annie is at the bottom of the graph with low values, followed by eutrophic

Lake Okeechobee (mud sediments M9) with intermediate values, and then hypereutrophic Lake











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1800

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o 600

0y 400


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2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0


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2000

1800

1600

1400

1200

1000


13utvrate (
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JNt Basal









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Me Basal















0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20


+ Formate E
11Glucose
Sa Propionate















0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20


Time (days)

Figure 7-3. Microbial activity response to the different carbon source addition in the peat sediments (site M17) of Lake Okeechobee:
A, B and C) Anaerobic respiration (mg CO2-C kg l) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg- ) vs.
time (days). *Different scales.









The M17 sediment from Lake Okeechobee also had an increase in NH4-N concentration

during incubation and highest C:nutrient ratios. These sediments also seem to be P limited. The

primary productivity in the southern region (peat zone) of Lake Okeechobee was reported to be

limited by N and a high frequency of co-limitation by N + P occurs (Aldridge et al. 1995). A

dual limitation by P and C, however, seems to be occurring in M17 sediments. M17 sediments

are characterized by peat deposits formed by incomplete decomposition of higher plants (Reddy

et al. 1991). Extractable C is probably rich in humic substances known to be refractory.

Consequently, although the C:nutrient ratios are high for this site, available C is probably low. If

DOC is not easily available, C can limit heterotrophic bacteria (Vadstein et al. 2003). Vrede

(2005) showed that lakes with high concentrations of humic substances are usually limited by

both C and P. The incubation experiment strongly indicates the refractory nature of C in these

sediments. There was a decrease in DOC concentration during incubation, probably reflecting

high demand for labile C by the microbial community. This site was the only one that showed a

significant accumulation of DRP, and NH4-N following incubation. The decrease in C and

simultaneous increase in inorganic nutrients, N and P, indicates a high demand of these

sediments for labile C.

High availability of P in Lake Okeechobee sites M9 and KR surface sediments is causing

C limitation in the system. Moreover, in M9 sediments, low values of Ext-N:Ext-P indicate that

N can be also limiting in this system. Crisman et al. (1995) reported that temperature and trophic

state variables Secchi disk depth, total P, and total N, had a weak correlation with

bacterioplankton abundance (number of cells mL- ) in a seasonal study of Lake Okeechobee.

They concluded bacterioplankton communities were probably controlled by grazing and/or C

and nutrient availability. Work et al. (2005) reported high bacterioplankton production (mg L-1 h-
















Smears
POR











Figure 8-9. Agarose (2%) gel electrophoresis of RT-PCR of the E. coli added to Lake Apopka
samples (core # 93). (A) old primers; (B) new primers.


Figure 8-10. Agarose gel (1%) electrophoresis of PCR of E coli RNA samples treated with
DNase and not treated with DNase.









Lake Apopka is also a shallow lake with 125 km2 Of surface area, located in central

Florida. Once a clear-water macrophyte-dominated lake, Lake Apopka has changed to a turbid,

algal-dominated lake since 1947 (Clugston 1963). This shift may have been caused by nutrient

input from several sources, including agricultural drainage from adj acent vegetable farms (Baird

and Bateman 1987, Schelske et al. 2000), although some suggest that the proximal 'trigger' for

the switch was a hurricane or tornado (Bachmann et al. 1999). Even though these inputs were

controlled and regulated to some degree, the eutrophication process continued and Lake Apopka

is considered hypereutrophic. Benthic sediments are characterized by unconsolidated material,

which mainly consists of algal deposits (Reddy and Graetz 1991) (Figure 1-4).

Obj ectives

The specific obj ectives of this study were to:

* Determine the biogeochemical properties of sediments and examine relationships among
sediment biogeochemical properties (nutrient concentrations and availability) and
microbial biomass and activity (Chapter 2).

* Determine relative distributions of P compounds in sediment profies using two different
techniques, 31P NMR spectroscopy and a P chemical fractionation scheme. (Chapter 3).

* Characterize P-related enzyme activities in sediment profies and determine relationships
between different P compounds and enzyme activities (Chapter 4).

* Determine stratigraphic biogeochemical properties in sediment cores and evaluate how
they are related to microbial biomass and activity; and establish whether there is nutrient
limitation of the microbial community (Chapter 5).

* Determine the source and long-term accumulation of OM to sediments using 813C and 6 "N
signatures. (Chapter 6).

* Evaluate the catabolic diversity of microbial communities in sediments (Chapter 7).

* Identify microbial communities that utilize acetate through RNA-stable isotope probing
(Chapter 8).

A series of Hield sampling and laboratory studies were conducted to accomplish these

obj ectives. Results of these studies provided insights into the relationships between sediment










Table 2-3. pH, bulk density (BD), organic matter content (LOI loss on ignition), total nitrogen, and total carbon concentration in
sediments from three subtropical lakes. (mean a standard deviation). Sediment depth 0-10 cm.

ED ~OTTotal Nitrogen Total Carbon

Lake Ste pH(g of dry cm' of wet) (%) (g kg-l dw)


South

Annie Central

North

M17

011

M9

K8

Okeechobee FC

J5

TC

KR

J7

South

Central
Apopka
West

North


5.7 & 0.1

5.8 & 0.01

6.0 + 0.1

7.4 & 0.2

7.5 & 0.03

7.6 & 0.03

7.5 & 0.02

7.1 & 0.2

7.6 & 0.1

7.2 & 0.4

7.5 & 0.1

8.1 & 0.2

7.5 & 0.2

7.4 & 0.2

7.7 & 0.1


0.024 & 0.003

0.026 & 0.005

1.64 & 0. 11

0. 19 & 0.02

0. 16 & 0.04

0.261 0.02

0. 16 & 0.04

1.50 + 0.07

1.43 & 0.16

1.35 & 0.12

0.47 & 0.06

1.60 + 0.14

0.022 & 0.005

0.016 & 0.003

0.016 & 0.001


53.8 & 0.8

54.9 & 0.5

0.45 & 0.3

72.2 & 5.3

40.2 & 2.6

28.5 & 2.0

36.5 A 1.7

2.6 & 0.6

1.6 & 0.3

2.4 & 0.0

23.5 & 3.5

2.2 & 0.8

64.2 & 2.9

67.8 & 1.9

69.4 & 2.7


19.1 +1.6

20.2 & 0.7

0.26 & 0.0

21.5 & 2.8

11.9 & 0.6

8.0 + 0.6

11.4 & 0.6

0.2 & 0.1

0.3 & 0.1

0.4 & 0.0

6.4 & 1.2

0.3 & 0.0

29.7 & 1.6

31.5 A1.1

30.5 & 0.4


263 A 11

265 A 10

1.6 & 0.1

403 & 36

186 & 6.5

146 & 8.3

175 & 3.2

1.3 & 0.7

3.7 &1.7

5.1 & 0.2

97 & 15

4.4 & 1.3

335 A 12

349 & 1.7

356 & 5.1


7.6 & 0.03 0.015 & 0.003 69.2 & 0.2


32.9 & 0.04 356 & 6.1










Casper, P., O. C. Chan, A. L. S. Furtado, and D. D. Adams. 2003. Methane in an acidic bog lake:
the influence of peat in the catchment on the biogeochemistry of methane. Aquat. Sci. 65:
36-46.

Castro, H., S. Newman, K. R. Reddy, and A. Ogram. 2005. Distribution and stability of sulfate-
reducing prokaryotic and hydrogenotrophic methanogenic assemblages in nutrient-
impacted regions of the Florida Everglades. Appl. Environm. Microbiol. 71: 2695-2704.

Celi, L., S. Lamacchia, F. A. Marsan, and E. Barberis. 1999. Interaction of inositol
hexaphosphate on clays: Adsorption and charging phenomena. Soil Sci. 164: 574-585.

Chan, O. C., P. Claus, P. Casper, A. Ulrich, T. Lueders, and R. Conrad. 2005. Vertical
distribution of structure and function of the methanogenic archaeal community in Lake
Dagow sediment. Envrion. Microbiol. 7: 1139-1149.

Charlton, M. N. 1980. Hypolimnion Oz COnSumption in lakes: discussion of productivity and
morphometry effects. Can. J. Fish. Aquat. Sci. 37:1531-1539.

Chauhan, A., A. Ogram, and K. R. Reddy. 2004. Syntrophic-methanogenic associations along a
nutrient gradient in the Florida Everglades. Appl. Environm. Microbiol. 70: 3475-3484.

Chauhan, A., and A. Ogram. 2006a. Fatty acid-oxidizing consortia along a nutrient gradient in
the Florida Everglades. Appl. Environ. Microbiol. 72: 2400-2406.

Chauhan, A., and A. Ogram. 2006b. Phylogeny of acetate-utilizing microorganisms in soils
along a nutrient gradient in the Florida Everglades. Appl. Environ. Microbiol. 72: 6837-
6840.

Chimmey, M. J. 2005. Surface seiche and wind set-up on Lake Okeechobee (Florida, USA)
during hurricanes Frances and Jeanne. J. Lake and Reserv. Manag. 21: 465-473.

Chin, K-J., and R. Conrad 1995. Intermediary metabolism in methanogenic paddy soil and the
influence of temperature. FEMS Microbiol. Ecol. 18: 85-102.

Chrost, R. J. 1991. Environmental control of the synthesis and activity of aquatic microbial
ectoenzymes, pp. 29-59. In R. J. Chrost [ed.], Microbial enzymes in aquatic environments.
Spring-Verlag.

Chrost, R. J., and W. Siuda. 2002. Ecology of microbial enzymes in lake ecosystems, p. 35-72.
In R. G. Burns and R. P. Dick [ed.], Enzymes in the environment: activity, ecology and
applications. Marcel Dekker.

Cichra, M. F., S. Badylak, N. Henderson, B. H. Rueter, and E. J. Phlips. 1995. Phytoplankton
structure in the open water zone of a shallow subtropical lake (Lake Okeechobee, Florida,
USA). Arch. Hydrobiol. Spec. Issues Advanc. Limnol. 45: 157-175.

Cimbleris, A. C. P., and J. Kalff. 1988. Planktonic bacterial respiration as a function of C:N:P
ratios across temperate lakes. Hydrobiol. 384: 89-100.









lighter[12C]DNA fractions, demonstrating that E. coli DNA was a successful control for the

separation of the 'heavy' and 'light' DNA. In this study, although similar density gradients were

obtained (Figure 8-13, 8-15) as described by (Manefield et al. (2002b); Whiteley et al. 2007)

(Figure 8-8A, B), E. coli RNA was found throughout the density gradient, demonstrating that

even after extended ultracentrifugation periods, 'heavy' RNA fractions can still contain 'light'

RNA (Figure 8-14). Furthermore, the presence of E coli RNA mixed with the 'heavy' RNA may

be a further problem in the experiment. The creation of a 'heavy' RNA library is the goal of this

method, thus the cross-contamination of 'heavy' and 'light' RNA may produce a large number of

false-positive clones.

Small differences in buoyant densities are usually observed in RNA-SIP experiments.

Typically, unlabeled RNA has a buoyant density of 1.755 g ml-l while labeled RNA has a

buoyant density of between 1.795 and 1.80 g ml l. However, several studies have shown

overlapping of these two fractions (Manefield et al. 2002a; Lueders et al. 2004a). The detection

of 'heavy' and 'light' RNA in the CsTFA density gradient fractions can also be caused by

interactions of RNA molecules forming secondary structures (Lueders et al. 2004a). Recently,

Lueders et al. (2004a) demonstrated that DNA- and RNA-SIP methodologies are distinguished

in the fractioning of 12C and 13C-COntaining targets (Figures 8-17, 8-18). The authors compared

the sensitivity of the two SIP methods with labeled (13C) and unlabeled (12C) pure cultures of

M~ethylobacterium extorquens and 3\ /I'thaml,(l mi inae barkeri. DNA-SIP CsCl density gradient was

effective to separate 'heavy' and 'light' DNA, either when samples were centrifuged separately

(Figure 8-17A) or simultaneously in the same tube (Figure 8-17B).

However, separation of 'heavy' and 'light' RNA using RNA-SIP CsTFA density gradient

could only be achieved when they were centrifuged separately (Figure 8-18A). An incomplete




















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*.
*
-
*
e
-
*
- e
*

- *
*
- e
*
- a

e

*


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Fraction Number


R...8 I--- --


C













..


1.88

1.86

1.84

1.82


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.



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-


1.86

1.84

1.82




S1.78

1.76

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1.84

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1.78

1.76

1.74

1.72


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

FractionNumber FractionNumber

Figure 8-15. Graph illustrating the buoyant density of gradient fractions. (A) Blank (no RNA),

(B) E. coli 10 ng and (C) E. coli 100 ng.



































231











LIST OF FIGURES


Figure page

1-1 Schematic of maj or processes occurring in sediment and water column of lakes. ............34

1-2 Schematic showing draw of chemical and biological P processes in lake sediments. ......34

1-3 Schematic showing mineralization of organic matter through heterotrophic microbial
activities in sediments. .............. ...............35....

1-4 Map of Lake Annie, Lake Okeechobee, and Lake Apopka with their location in
Florida State. .............. ...............35....

2-1 Map of the three subtropical lakes with sampled sites and their location in Florida
State............... ...............64.

2-2 Linear regressions between microbial biomass carbon and microbial biomass
nitrogen and phosphorus of sediments ................. ...............66......___ ...

2-3 Relationship between anaerobic respiration and microbial biomass carbon of
sediments ...._ ................. ........___.........67

2-4 Results of the Principal Component Analysis 1. ............. ...............68.....

2-5 Results of the Principal Component Analysis 2. ................ ..........._. .... 69....__..

2-6 Graphic representation of sediment characteristics of three lakes in relation to their
trophic state. .............. ...............70....

3-1 Map of the three subtropical lakes with sampled sites and their location in Florida
State............... ...............92.

3-2 Fractionating scheme for the characterization of P organic forms. ............. ..................94

3-3 31PNMR spectra of the NAOH/EDTA extracts of sediment depth profile. .....................95

3-4 Results of the Principal Component Analysis. ...........__.....___ .......___........9

4-1 Enzyme activity of sediment depth profile. ....__ ......_____ ......___ ..........15

4-2 Relationship between phosphate monoester concentration and phosphomonoesterase
activity in sediments. ................ ...............116................

4-3 Relationship between phosphate diester concentration and phosphodiesterase activity
in sedim ents. ................ ...............116......... ......












Sediment Properties

Sediment pH ranged from 5.9 to 7.8 (Table 7-1). Lake Annie sediment pH was lower than

other lakes. Both Lake Okeechobee and Lake Apopka sediment pH were circum-neutral to

alkaline (Table 7-1). Surface sediment bulk densities were lowest in Lake Apopka, followed by

Lake Annie, and then Lake Okeechobee sites M9, M17, and KR, respectively (Table 7-1).

Sediment organic matter content was highest at Lake Okeechobee site M17, reflecting high peat

content. Next in order were Lake Apopka, Lake Annie, followed by site M9, and sandy KR in

Lake Okeechobee (Table 7-1). Total C was highest in peat zone sediments of Lake Okeechobee,

followed by sediments from Lake Apopka and Lake Annie. Lake Apopka and peat zone

sediments of Lake Okeechobee exhibited similar values of TN (Table 7-1). Lake Annie

sediments exhibited higher TP concentrations than Lake Okeechobee and Lake Apopka

sediments (Table 7-1).

Extractable organic C and MBC were highest in Lake Apopka sediments (Table 7-1). Lake

Apopka sediments also had the highest concentrations of Ext-ON and Ext-NH4-N (Table 7-1).

Lake Annie sediments, however, had the highest concentrations of labile inorganic P and labile

organic P (Table 7-1). Sediment Ext-C:Ext-N ratio was similar in all lake sediments (Table 7-1).

Lake Annie and sites M9 and KR in Lake Okeechobee exhibited low Ext-C:Ext-P and Ext-

N:Ext-P ratios.

Electron Donors

Dry suspended material content of three lakes (Lake-SS) was characterized as: 34.2 % C

and 2.9% N from Lake Annie; 15.6 % C and 1.5% N from Lake Okeechobee; and 33.6 % C and

3.7% N from Lake Apopka. Addition of electron donors to sediment microcosms stimulated

heterotrophic microbial activity (Figures 7-1, 7-2, 7-3, 7-4, 7-5, Table 7-3). All sediments


Results










response of microbial communities to addition of a wide variety of C-substrates (Degens and

Harris 1997; Degens 1998a). This has been widely applied in soil studies to address differences

in microbial communities in different soil types, disturbance, and land use (Degens and Harris

1997; Lu et al 2000; Degens et al. 2000, 2001; Stevenson et al. 2004). Substrate induced

respiration is often dependent on the size of the microbial biomass pool, however, response of

microbial communities is also related to the catabolic diversity of soil microorganisms (Degens

1998). A greater relative catabolic response to a substrate in one system as compared with

another indicates that the microbial community is more functionally adapted to use that resource

as well as the presence of enzymes capable of their utilization, and previous exposure to different

C-sources (Degens and Harris 1997; Degens 1998; Baldock et al. 2004; Stevenson et al. 2004).

Metabolic response of a microbial community in lake sediment may vary due to several

factors that influence either the microbial community or due to physico-chemical characteristics

of lakes, which include source and chemical composition of particulate matter and

biogeochemical processes in the sediment and water column. Eutrophic and hypereutrophic lakes

usually receive high external loads of nutrients and display high primary productivity and

nutrient concentrations in the water column and these nutrients eventually reach the sediment,

therefore sediments from eutrophic and hypereutrophic lakes are expected to have high

concentrations of OM.

Binford and Brenner (1986) and Deevey et al. (1986) showed that net accumulation rates

of OM and nutrients increase with trophic state for Florida lakes. In contrast small, oligotrophic

lakes are expected to have relatively high proportions of allochthonous C input to their sediments

(Gu et al. 1996). Sediments with different C-sources quality and quantity as well as nutrient

concentration, will have different microbial communities. These communities can display









CHAPTER 8
RNA-STABLE ISOTOPE PROBING OF ACETATE-UTRLIZING MICROORGANISMS IN
SEDIMENTS OF SUBTROPICAL LAKES

Introduction

In anoxic environments, different groups of microorganisms participate in anaerobic

decomposition of organic matter as no single anaerobic microorganism can completely degrade

organic polymers (Zinder 1993, Megonigal et al. 2004). Bacteria hydrolyze organic polymers

through extracellular enzyme production, and under methanogenic conditions, ferment

monomers to alcohols, fatty acids, and hydrogen (H2). Alcohols and fatty acids are converted by

syntrophic bacteria into acetate, H2, and carbon dioxide (CO2), which is used as substrate by

methanogens (Zinder 1993, Conrad 1999, Megonigal et al. 2004). The structures and functions

of anaerobic microbial communities are strongly affected by competition for fermentation

products such as H2 and acetate, and competition favors the following order of reduction

processes, based on highest thermodynamic yield: NO3- > Mn(IV) > Fe(III) > SO4-2 > HCO3

(i.e., methanogenesis) (e.g., Megonigal et al. 2004).

Several microorganisms use acetate as a carbon (C) source, making this compound the

most important intermediate for microbial communities under anaerobic conditions. Acetate is

assimilated into microorganism biomass and converted to methane (CH4) and/or CO2. In

previous study the addition of acetate enhanced both anaerobic CO2 and CH4 prOduction rates in

sediment microcosms of subtropical lakes with different trophic states (Chapter 7). However, the

acetate-utilizing microorganisms in these lake sediments are not known. In a recent study,

Schwarz et al. (2007) used RNA-based stable isotope probing to identify acetate-utilizing

Bacteria and Archaea in sediments of Lake Kinneret (Israel). The authors concluded that acetate

was predominantly consumed by acetoclastic methanogens and was also utilized by a small and

heterogeneous community of anaerobic bacteria. In a previous study in Lake Kinneret sediments









piston corer (Fisher et al. 1992) or by SCUBA divers. One central site (80-cm core) was sampled

in Lake Annie on June 25, 2005 (Figure 3-1A, Table 3-1). Cores were collected at three sites in

Lake Okeechobee on July 16, 2005: M17 = peat (40-cm core), M9 = mud (70-cm core) and KR

= sand (40-cm core) (Figs. 3-1B, Table 3-1). A western site (98-cm core) was sampled in Lake

Apopka on May 28, 2005 (Figure 3-1C, Table 3-1). Cores were sectioned in the field at the

following intervals: 0-5, 5-10, 10-15, 15-20, 20-30, 30-45, 45-60, 60-80, 80-100 cm. Samples

were placed in plastic bags, sealed, and kept on ice. Nine cores were collected from each site.

Three cores were used to make a composite core to obtain sufficient material for all

measurements. The nine cores yielded three replicates of composite sediments from each site.

All measured sediment variables are reported on a dry weight basis (dw). Water quality variables

were described in Chapter 4, and in the present study values were used to characterize the lakes.

Sediment Properties

Samples were transported on ice and stored in the dark at 4 oC. Before each analysis,

samples were homogenized and sub-samples taken. Sediment bulk density (g dry cm-3 wet) was

determined on a dry weight basis at 70 oC for 72 hours, and pH was determined on wet sediments

(1:2 sediment-to-water ratio). Sediment samples were ground in a ball mill and passed through a

# 40 mesh sieve. Organic matter content (LOI-loss on ignition) was determined by weight loss at

550oC. Total P was measured by inition method, followed by acid di estion (6 M HCI) and

measured colorimetrically with a Bran+Luebbe TechniconThl Autoanalyzer II (Anderson 1976;

Method -365.1, EPA 1993).

Sediment Phosphorus Fractionation

Due to high water content of Lake Annie and Lake Apopka sediments, pore water was

extracted (centrifuged at 10,000 x g for 10 min) prior to P fractionation. Pore water TP was

measured after digestion with 11N_ H2SO4 and potassium persulfate (Method 365.1, EPA 1993).





















FAP s'aan
BD Acetate *
L bPo
Acetate + H2 HAP

Bas 1 (CH )
Ext-N




LO TN
**
Ext-C:N Ext-C
Ext-N:P *TC
Ext C:P


-0.2

-0.4

-0.6

-0.8


-I.
-1.2


-1.0 -0.8 -0.6


-0.4 -0.2 0.0 0.2


0.4 0.6 0.8 1.0 1.2


Axis 1 (60.6)


-1.5

-2.0 ti /

-2.5M1


-2.0 -1.5 -1.0 -0.5 0.0 8; ~ 1.0

Axis 1

Figure 2-5. Results of the Principal Component Analysis (PCA-2), A)
B) the plot of the scores of the sites of Lake Okeechobee.


loadings of (n =27), and


BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon, TN: total nitrogen, Ext-
N: extractable labile nitrogen, TP: total phosphorus, LabPi: labile inorganic phosphorus, LabPo: labile organic
phosphorus, IP: HCl-Pi inorganic phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resistant
organic phosphorus, ResP: residual phosphorus, Ext-P: extractable labile phosphorus, MBC: microbial biomass
carbon, CO2: basal anaerobic respiration, Basal (CH4): basal methane production rates, Acetate, H2, and Acetate +
H2, methane production rates from electron donor addition.









C was calculated by the difference between treated and non-treated samples. Extracts from the

untreated samples represent extractable organic carbon (Ext-C).

Electron Donors

Basic catabolic response was characterized by increasing CO2 and CH4 prOduction rates

from sediment samples by addition of different electron donors. Eight different simple organic

compounds (electron donors) were added to each sediment sample. They consisted of two amino

acids (alanine and arginine), four carboxylic acids (acetate, format, butyrate, and propionate),

one polysaccharide (glucose), and lake suspended solids (Lake-SS). Wet sediment (based on 0.5

g of dry weight) was added to incubation bottles, sealed with rubber stoppers and aluminum

crimp seals, and purged with N2 gaS. Alanine, arginine, acetate, format, butyrate, propionate,

and glucose were added from anaerobic sterile stock solutions to sediments on a C-equivalent

basis, reaching a final concentration of 42 mM C (504 Clg of C gl on a dry weight basis) (Degens

1998a). All stock solutions were adjusted to pH around 7.0 using either HCI or NaOH at the time

of preparation to avoid any substrate-pH effects on microbial communities.

Lake-SS was obtained by centrifugation (10,000 x g for 30 min) of water samples collected

at approximately 50 cm depth in the water column of each lake. Lake-SS was characterized for

LOI, TC, TN, and TP as described previously, and was added on the same C-equivalent basis as

the other electron donors. A sample from each Lake-SS was incubated to account for CO2 and

CH4 prOduction of the material. Values obtained were subtracted from CO2 and CH4 prOduction

rates of the Lake-SS treatment for each lake. Sediments from each site were also incubated with

no substrate addition (control) to obtain values of basal anaerobic CO2 and CH4 prOduction rates.

Samples were incubated anaerobically at 30 oC in the dark. Gas samples were taken at 1, 2, 4, 7,

10, and 14 days and quantified for CO2 and CH4. Gas samples from Lake Annie and Lake

Okeechobee sediments were also taken at 20 days of incubation due to low CH4 prOduction









with 3 M1 HCI and CO2 TeleaSed was measured by gas chromatography using a Shimadzu 8A

GC-TCD equipped with Poropak N column (Supelco Inc., Bellefonte, PA), using He as a carrier

gas. For the methanogenesis experiment, samples were placed in a glass vial, closed with rubber

stoppers and aluminum crimp seals, and incubated anaerobically at 30 oC. Gas samples were

obtained at 2, 4, 7, 10 days and analyzed on a Shimadzu gas chromatograph-8A fitted with flame

ionization detector (110 oC), N2 aS the carrier gas and a 0.3 cm by 2 m Carboxen 1000 column

(Supelco Inc., Bellefonte, PA) at 160 oC. Prior to measuring both CO2 and methane (CH4),

head space pressure was determined with a digital pressure indicator (DPI 705, Druck, New

Fairfield, CT). Concentrations of CO2 and CH4 were determined by comparison with standard

concentrations and production rates were calculated by linear regression (T2 > 0.95).

Methane was not detected during the incubation period in Lake Okeechobee samples.

Suspecting substrate limitation for methane production, additional experiments were conducted

to evaluate the effect of naturally present electron donors acetate and hydrogen (H2) On methane

production in sediments. Wet sediment (based on 0.5 g of dry weight) was added to incubation

bottles, sealed, and purged with N2 gaS. One control (no substrate addition) and three treatments

were applied to each sediment type: 1) Acetate, 2) H2, and 3) Acetate + H2. Acetate (20 mM or

480 mg C kgl on a dry weight basis) was added from anaerobic sterile stock solution and H2

addition was done by purging the headspace with 80:20 (vol/vol) H2-CO2 gaS at 150 Kpa.

Samples were incubated anaerobically in the dark at 30 oC. Gas samples were obtained at 2, 4, 6,

8, 10 and 14 days after incubation and analyzed on a Shimadzu gas chromatograph-8A as

described above.

Statistical Analysis

A regression analysis was conducted to compare microbial biomass C and anaerobic

respiration. A Principal Component Analysis (PCA) was performed to address relationships










Neufeld, J. D., M. G. Dumont, J. Vohra, and J. C. Murrell. 2007. Methodological considerations
for the use of stable isotope probing in microbial ecology. Microb. Ecol. 53: 435-442.

Newman, R. H., and K. R. Tate. 1980. Soil phosphorus characterization by 31P nuclear magnetic
resonance. Commun. Soil Sci. Plant Anal. 11: 835-842.

Newman, S., and K. R. Reddy. 1993. Alkaline phosphatase activity in the sediment-water
column of a hypereutrophic lake. J. Environm. Qual. 22: 832-838.

Newman, S., F. J. Aldridge, E. J. Phlips, and K. R. Reddy. 1994. Assessment of phosphorus
availability for natural phytoplankton populations from a hypereutrophic lake. Arch.
Hydrobiol. 130: 409-427.

Noges, P., A. Jarvert, L. Tuvikene, and T. Noges. 1998. The budgets of nitrogen and phosphorus
in a shallow eutrophic Lake Vortsj ary (Estonia). Hydrobiol. 363: 219-227.

Nuisslein, B., and R. Conrad. 2000. Methane production in eutrophic Lake Plunsee: seasonal
change, temperature effect and metabolic processes in the profundal sediment. Arch
Hydrobiol. 149:597-623.

Niisslein, B., K-J. Chin, W. Eckert, and R. Conrad. 2001. Evidence for anaerobic syntrophic
acetate oxidation during methane production in the profundal sediment of subtropical Lake
Kinneret (Israel). Environ. Microbiol. 3: 460-470.

Odum, E. P. 1969. The strategy of ecosystem development. Science. 164: 262-270.

Olila, O. G., and K. R. Reddy. 1997. Influence of redox potential on phosphorus-uptake in
oxidized sediments from two subtropical eutrophic lakes. Hydrobiol. 345:45-57.

Olila, O. G., K. R. Reddy, and W. G. Harris. 1995. Forms and distribution of inorganic
phosphorus in sediments of two shallow eutrophic lakes in Florida. Hydrobiol. 302:147-
1995.

Olsen, L. M., H. Reinertsen, and O. Vadstein. 2002. Can phosphorus limitation inhibit dissolved
organic carbon consumption in aquatic microbial food webs? A study of three food web
structures in microcosms. Microb. Ecol. 43: 353-366.

Oreland, R. S. 1988. Biogeochemistry of methanogenic bacteria, p. 641-705. In A. J. B. Zehnder
[ed.], Biology of anaerobic microorganisms. Wiley.

Ostrom, P. H., N. E. Ostrom, J. Henry, B. J. Eadie, P. A. Meyers, and J. A. Robbins. 1998.
Changes in the trophic state of Lake Erie: discordance between molecular 613C and bulk
613C Sedimentary records. Chem. Geol. 152: 163-179.

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dominant phenol-degrading organisms from an industrial wastewater treatment plant. Schwarz et

al (2007) used RNA-SIP to identify acetate-utilizing Bacteria and Archaea in sediments of Lake

Kinneret (Israel). RNA-SIP was also used to identify acetate-assimilating organisms in an anoxic

rice field (Hori et al. 2007) and cellulolytic bacteria in soils (Haichar et al. 2007). Organisms

responsible for syntrophic oxidation in sludge (Hatamoto et al. 2007) and flooded soil (Lueders

et al. 2004b) were also identified through RNA-SIP. The use of RNA-SIP has proven to be an

effective method, because RNA is produced independently of cellular replication, and the

activity of slow- and non-replicating cells can be detected (Manefield et al. 2007).

Fully labeling the target RNA with 13C iS essential to achieve an optimal separation of

"heavy" and "light" RNA (Whiteley et al. 2005). A substantial amount of stable isotope atoms in

the target RNA facilitates the density gradient separation by ultracentrifugation of labeled and

unlabeled nucleic acids (Manefield et al. 2002a, 2007). High levels of labeled substrate beyond

the naturally occurring concentrations and extended periods of incubation, however, can increase

the chance of labeling non-target organisms through trophic interactions and cross feeding

(Manefield et al. 2007). Thus, the [13C]-acetate concentration and short incubation periods were

chosen to avoid the above mentioned problems. However, RNA from the lake sediments were

probably not fully labeled with 13C. One week pre-incubation was chosen to exhaust naturally

occurring C sources. Thus, when the [13C]-acetate was added to the sediment microcosms, the

microorganisms would assimilate it faster. To verify if the target RNA is fully labeled,

measuring isotope ratio by mass spectrometry has been suggested (Manefield et al. 2002a).

Different [13C]-acetate concentrations must be run along with different periods with incubation to

determine the optimal concentration and incubation period.









pollution (Aumen 1995; Whalen et al. 2002). The northern area of Lake Okeechobee is

characterized by high intra-annual variability in chlorophyll-a concentration (Havens 1994;

Phlips et al. 1995). Nitrogen limits primary productivity (Aldridge et al. 1995), and the

phytoplankton community is dominated by large N2-fixing cyanobacteria (Phlips et al. 1997),

however, N2-fixation is low (Gu et al. 1997; Phlips et al. 1997). Organic matter content in these

sediments is low, and the contribution of autochthonous OM to isotopic signatures does not seem

to be great at this site.

Wastewater and agricultural runoff are usually enriched in 1N (Bedard-Haughn et al.

2003; Anderson and Cabana 2005), and sewage effluents are depleted in 13C (Gearing et al.

1991). Other studies related the enrichment of 8 "N in Florida lakes sediments to agricultural

runoff (Riedinger-Whitmore et al. 2005; Whitmore et al. 2006).

Depletion of 13C and enrichment of 15N, in sediments of Lake Peten Itza (Guatemala) were

related to sewage input (Rosenmeier et al. 2004). In a study of 613C distribution in sediments and

food webs of estuaries, Gearing et al. (1991) reported that sewage C accumulated in sediments,

and 813C Value Of impacted sites (-24.2%o) was significantly lower than the values from non-

impacted sites (-21.6%o). In a study of a sewage dumpsite in the New York Bight, Burnett and

Schaffer (1980) showed that OM from wastewater (-26.2%o) and from marine origin (-22.0%o)

had distinct 813C Signatures. Seasonality also can affect particulate organic carbon 813C ValUeS in

rivers. During periods of high discharge in Sanaga River (Cameroon) 613C ValUeS are high,

caused by an increase in the proportion of contribution of OM derived from C4 plants from the

further savanna region transported overland by wet season rains (Bird et al. 1994; Bird et al.

1998). In the dry season, when discharge is low, 613C ValUeS are low, reflecting OM derived

primarily from C3 plants growing close to the river bank (Bird et al. 1994; Bird et al. 1998).










(20-25 oC) lead to an increase in contribution of CH4 prOduction from H2/CO2 and probably from

methanol. They hypothesized that at low temperatures hydrogenotrophs were unable to compete

with H2-utilizing homoacetogenic bacteria. Moreover, methanogenic degradation of organic

matter should be dominated by homoacetogenesis plus acetoclastic methanogenesis at low

temperatures versus fermentation, syntrophy, H2 prOduction and hydrogenotrophic

methanogenesis at high temperatures. The same results were reported for sediments from

eutrophic Lake Dagow (Germany), however, the change of dominance from acetoclastic to

hydrogenotrophic methanogenesis with an increase in temperature was not followed by a change

in community structure of the maj or phylogentic groups of methanogens (Glissmann et al. 2004).

Lake Annie sediments are acidic and probably maintain fairly constant low temperatures.

Thermal stratification of the water column was detected during sampling in this lake with a

temperature of 17.3 oC below 14 m water column depth (Chapter 4). Sediment temperature is

probably much lower in this deep (20 m) lake. Sediment acidic pH and low temperatures as well

as the high CH4 prOduction rate with addition of acetate and the placement of Lake Annie with

acetate cluster in the PCA-2 suggests that acetoclastic methanogenesis may be an important

pathway for CH4 prOduction in these sediments (Table 7-3, Figure 7-8A, B). Lake Okeechobee

and Lake Apopka had high temperatures at the sediment-water surface (26.3-30.7 oC), and both

lakes had circum-neutral to alkaline sediment pH (Table 7-1), good conditions for

hydrogenotrophic methanogenesis. In Lake Okeechobee sediments it has been determined that

hydrogenotrophic methanogenesis is the main pathway of CH4 prOduction (Chapter 2). The

pathway for methane production in Lake Apopka cannot be determined with the current data,

however, hydrogenotrophic methanogenesis might be an important pathway in this

hypereutrophic lake. Algae deposition is an important source of C to methanogenic activity in









al. 1988). Eutrophic and hypereutrophic lakes typically receive high external loads of nutrients

and display high primary productivity and nutrient concentrations in the water column and these

nutrients eventually reach the sediment, therefore sediments from eutrophic and hypereutrophic

lakes are expected to have high concentrations of organic matter. Binford and Brenner (1986)

and Deevey et al. (1986) showed that net accumulation rates of organic matter and nutrients

increase with trophic state for Florida lakes. In contrast, small oligotrophic lakes are expected to

exhibit a relatively high proportion of allochthonous carbon input to their sediments (Gu et al.

1996).

Lake depth can also affect the quality of organic material reaching the sediment. In deep

lakes, sedimenting organic matter undergoes intense decomposition in the water column due to

the prolonged period of settling. Consequently, low amounts of labile organic carbon reach the

sediment (Suess 1980; Meyers 1997). In shallow lakes, however, the supply of labile carbon and

nutrients can be higher in sediments than in deep lakes, and the latter often can have more

refractory organic matter. Sediments with different C-sources and with different quality and

quantity as well as nutrient concentration will have different microbial communities. These

communities can display distinct catabolic responses as the mineralization rates of a microbial

community are dependent upon the metabolic capacity for a given substrate (Torien and Cavari

1982). The obj ective of this study was to evaluate the short term catabolic response of the

microbial communities in sediments of three subtropical lakes characterized by different trophic

states. The central hypothesis is that sediments with higher C availability will have higher

catabolic diversity.









methanogens for H2/CO2 and aceteate, due to higher substrate affinities, and higher energy and

growth yield (Lovley and Klug 1983; Lovley and Phillips 1986; Conrad et al. 1987; Bond and

Lovley 2002), however, both processes can coexist (Mountfort and Asher 1981; Holmer and

Kristensen 1994; Roy et al. 1997; Holmer et al. 2003; Roden and Wetzel 2003; Wand et al.

2006). Coexistence occurs because of spatial variation in the abundance of terminal electron

acceptors or because the supply of electron donors is non-limiting (Roy et al. 1997; Megonigal et

al. 2004). During the incubation experiment, DOC accumulated in Lake Annie surface sediments

(Table 5-5). In the upper 10 cm of Lake Annie sediment, the concentration of electron donors

must be sufficient for both methanogenesis and other anaerobic metabolic pathways to occur.

Lake Apopka displays high C availability and can thus support a more diverse community

as reflected by anaerobic respiration and methanogenesis in the sediments. Algal deposition has

been shown to increase acetate concentration, with a consequent increase in CH4 prOduction in

sediments (Schulz and Conrad 1995). Several studies have shown that methane production rates

are higher in eutrophic than oligotrophic lakes. (Casper 1992; Rothfuss et al. 1997; Falz et al.

1999; Niiusslein and Conrad 2000; Dan et al. 2004).

The negligible CH4 prOduction in Lake Okeechobee is clearly a consequence of electron

donor limitation (Chapter 2). However, Fisher et al. (2005) reported CH4 in Sediment porewater

of sites M9 and M17 in Lake Okeechobee. They also reported SO4-2 in these sediment

porewaters, and its decline with sediment depth was related to the use of SO4-2 as a terminal

electron acceptor in the oxidation of sediment organic matter. Iron is important in controlling P

solubility in Lake Okeechobee sediments (Moore and Reddy 1994) and Fe-reducers might also

be present. As discussed before, Fe- and SO4-2-reducers outcompete methanogens for substrates,










glucosidase) and N (i.e. protease) related enzymes, would increase the knowledge of nutrient

dynamics and microbial communities in these sediments. One important point that was not

covered by the current study is the seasonal variation of nutrient limitation. Seasonal variation of

nutrient availability occurs in the water column of lakes and can occur in sediments of shallow

lakes like Lake Okeechobee and Lake Apopka. A study encompassing sampling of surficial

sediments in different seasons (i.e. winter and summer) should also be conducted.










LIST OF TABLES


Table page

2-1 Morphometric and limnological variables of the three subtropical lakes. ................... ......59

2-2 Location and sediment type of the sites sampled in the three different lakes. .................. .59

2-3 pH, bulk density, organic matter content, total nitrogen, and total carbon
concentration in sediments from three subtropical lakes. .........._.... ....._.._.............60

2-4 Phosphorus fractionation in sediments from the three lakes. ............. .....................6

2-5 Extractable and microbial biomass C, N, and P concentrations in sediments from
three subtropical lakes............... ...............62.

2-6 Anaerobic respiration and methane production rates in sediments from subtropical
lakes. ............. ...............63.....

3-1 Characteristics of sampled sites in the three different lakes with sampling date,
location, sediment type and water quality parameters. ................... ...............8

3-2 pH, bulk density, organic matter content in sediment profiles of the three lakes. ............88

3-3 Phosphorus fraction concentrations in sediment profiles. ............. .....................8

3-4 Phosphorus composition of the sediment depth profile determined by 31P NMR
spectroscopy ........... ..... .._ ...............91...

4-1 Measured parameters in the water column of the three lakes............. ... .........___...1 12

4-2 Concentration of total phosphorus, soluble reactive phosphorus, total nitrogen,
ammonium-N and dissolved organic carbon in the water column of the three lakes......1 13

4-3 Water extracdissolved organic carbon, and dissolved reactive phosphorus. ................... 114

5-1 Total carbon, total nitrogen, and C:N:P ratios in sediment profiles of the three lakes....13 8

5-2 Pore water dissolved organic carbon, ammonium-N, and dissolved reactive
phosphorus, total nitrogen, and total phosphorus. ............. ...............139....

5-3 Extracorganic carbon, ammonium, labile organic nitrogen, labile inorganic
phosphorus and labile organic phosphorus concentrations in sediment profiles. ............140

5-4 Microbial biomass carbon, nitrogen and phosphorus concentrations in sediment
profiles of the three lakes ................. ...............141........... ...

5-5 Water extracdissolved organic carbon, dissolved reactive P, and ammonium-N
concentrations at time 0 and time 10. ............. ...............142....









also expected to be depleted in 13C. The heterotrophic microbial community can also contribute

to depleted values of 613C

Heterotrophic uptake of DOC preserves the C isotopic signature of the source, and biomass

associated with chemoautrotrophic and methanotrophic microorganisms is generally depleted in

13C (COnway et al. 1994; Kelley et al. 1998). The isotopic study on sediments from Lake

Mendota (Wisconsin) illustrated that mineralization of C by the heterotrophic microbial

community associated with expansion of anoxic conditions in the water column resulted in low

813C ValUeS in sediments (Hollander and Smith 2001). Moreover, seasonal and long term

increases in contribution of depleted microbial biomass to sediments results in depleted values in

the 613C (Hollander and Smith 2001). Lehmann et al. (2002) reported that 13C depleted OM of

sinking particles and sediments resulted from anaerobic decomposition in Lake Lugano (Swiss-

Italian border). Terranes and Bernasconi (2005) associated the 613C Of sedimentary OM in Lake

Baldeggersee (Switzerland) to variation of relative inputs of eukaryotic biomass, which is

enriched in 13C and the contribution of microbial biomass, depleted in 13C, which is produced in

the expanding anoxic bottom waters. In Lake Annie the thermocline has been detected to be

moving to shallower depths (i.e., higher) in the water column during thermal stratification, with

anoxia below 5 m depth (Swain and Gaiser 2005). High sulfate reduction has been reported to

occur in the anoxic layers of the water column (Swain and Gaiser 2005). Increased anoxia in the

water column can be leading to increased anaerobic decomposition of already depleted

suspended OM, and both depleted microbial biomass and OM will eventually reach the

sediment.

The same processes are affecting N isotopic signatures. Plants tend to fractionate against

15N during inorganic N uptake (Handley and Raven 1992). Allochthonous organic matter derived












6 NUTRIENT ACCUMULATION AND STABLE ISOTOPE SIGNATURES IN
SEDIMENTS OF SUBTROPICAL LAKES ................. ...............145...............


Introducti on ................. ...............145................
M material and M ethod s ................. ................. 147........ ....

Study Sites ................. ...............147................
Sediment Properties ................. ...............147................
Isotopic Analyses............... ...............14
210Pb Dating ................. ...............148................
Results and Discussion ................ ...............149................
Core Chronology ................. ...............149......... ......
Lake Annie ................. ................. 149........ ....
Lake Okeechobee .............. ...............150....

Lake Apopka ................... ...............152......... ......
813C and 6 "N Isotope Signatures ................ ...............153........... ...
Lake Annie ................. ................. 153........ ....
Lake Okeechobee .............. ...............156....

Lake Apopka ................. ...............161......... ......
Conclusions............... ..............16


7 HETEROTROPHIC MICROBIAL ACTIVITY IN SEDIMENTS: EFFECTS OF
ORGANIC ELECTRON DONORS ................. ...............177................


Introducti on ................. ...............177................
Materials and Methods .............. ...............180....

Study Sites ................. ...............180................
Field Sampling............... ...............18
Sediment Properties ................. ...............180................
Extractable C, N and P .............. ...............181....
Microbial Biomass Carbon............... ...............18 1
Electron Donors ................. ...............182................

Statistical Analysis .............. ...............183....
Re sults ................ ........... ...............184......

Sediment Properties ................. ...............184................
Electron Donors ................. ...............184................
Discussion ................. ...............187................
Conclusions............... ..............19


8 RNA-STABLE ISOTOPE PROBING OF ACETATE-UTRIZING
MICROORGANISMS IN SEDIMENTS OF SUBTROPICAL LAKES.............................208


Introducti on ................. ...............208................
Materials and Methods ................ ...............210...

Study Sites and Field Sampling............... ...............21
RNA Extraction ................. ...............211................

Pre-Experiment ................. ...............211................
RNA-SIP Experiment ................. ...............212................




10









activities have been reported to be regulated by availability of orthophosphate, acid PMEase is

usually regarded as a constitutive enzyme (Siuda 1984; Jasson et al. 1988).

The production of constitutive enzymes is neither repressed nor stimulated by high or low

orthophosphate availability in the environment. Its production is related to P concentration and

demand inside the cell (Siuda 1984, Jasson et al. 1988). Jasson et al. (1981), however, suggested

that in acidified lakes, acid PMEase may have a similar role to that of alkaline PMEase in neutral

systems, as its production is also inhibited by orthophosphate. In aquatic systems, alkaline

PMEase is by far the most studied enzyme, probably due to the high number of systems with

neutral pH, that are inappropriate for preservation of extracellular acid PMEase (Siuda 1984).

Another important phosphatase is phosphodiesterase (PDEase) that hydrolyzes phosphate diester

and is known to degrade phospholipids and nucleic acids (Hino 1989; Tabatabai 1994; Pant and

Warman 2000). It is the least studied enzyme in freshwater ecosystems. Few studies have

reported on the occurrence and distribution of phosphatases or other organic P hydrolyzing

enzymes in sediments or their association with sediment bacteria (Wetzel 1991; Chrost and

Siuda 2002). As sediment P is important in P cycling in lakes, and it is well known that

microorganisms can influence the organic P dynamics in sediments, the study of different P

compounds and associated enzymes is important for understanding P cycling in sediments.

The primary hypothesis of this study is that enzyme activities will be higher in recently

accreted sediments (surface) as compared to older sediments (sub-surface) and will be related to

P forms and availability, as well as to microbial community activity. The specific objectives of

this study were: (i) measure vertical distribution of PMEase and PDEase activities and relate

them to microbial activity in sediments; and (ii) to explore relationships between different

phosphorus compounds and enzyme activity.









Most methanogenic species use H2/CO2 and a fewer number of species can use acetate

(Garcia et al. 2000). The present data do not allow any conclusions about the maj or pathways of

CH4 prOduction in these sediments. Formate can be used as a substitute for H2/CO2, however,

only about half of the H2-USers are able to use format for CH4 prOduction (Vogels et al. 1988).

Methane production rates from acetate can also result from syntrophic acetate oxidation to CO2

and H2 COupled with methanogenesis from H2/CO2 (Zinder 1994).

In lake sediments the dominance of acetoclastic versus hydrogenotrophic methanogenesis

has been reported to be related to sediment properties (i.e., pH and temperature). In acidic Lake

Grosse Fuchskuhle (Germany), with high humic content, acetate users (. kiberitsr~ll,(lilcl itr'e til)

were the only detected methanogens (Casper et al. 2003). Phelps and Zeikus (1984) reported that

acetoclastic methanogenesis was the maj or pathway for CH4 prOduction in a mildly acidic (pH

6.2) lake (Knaack Lake, Wisconsin). The increase in pH to neutral values enhanced total CH4

production from H2/CO2, but did not affect the CH4 prOduced from acetate (Zeikus 1984).

Acetoclastic methanogenesis is dominant at low temperatures. In mesotrophic Lake Rotsee

(Switerland) sediments, it was reported thatr.\ Oterlenatentllr~ (acetoclastic methanogen) was the

maj or methanogenic population (91%), indicating that in cold sediments acetate is the main CH4

precursor, and hydrogenotrophs were only found in the organic-rich, upper 2 cm of sediment

(Falz et al. 1999). Niisslein and Conrad (2000) reported that CH4 WAS produced from acetate at

low temperatures (4 oC) but it was produced from both acetate and H2/CO2 at higher

temperatures (25 oC) in sediments of eutrophic Lake Plupsee (Germany). Schulz and Conrad

(1996, 1997) reported a change in the methanogenic degradation pathway of organic matter in

sediments of mesotrophic Lake Constance (Germany). The authors showed that CH4 prOduction

in these cold (4 oC) sediments was exclusively from acetate, however, an increase in temperature










Falz, K. Z., C. Holliger, R. Grobkof, W. Liesack, A. N. Nozhevnikova, B. Muller, B. Wehrli, and
D. Hahn. 1999. Vertical distribution of methanogens in the anoxic sediment of Totsee
(Switzerland). Appl. Environm. Microbiol. 65: 2402-2408.

Fenchel, T., L. D. Kristensen, and L. Rasmussen. 1990. Water column anoxia: vertical zonation
of planktonic protozoa. Mar. Ecol. Prog. Ser. 62: 1-10.

Filley, T. R., K. H. Freeman, T. S. Bianchi, M. Baskaran, L. A. Colarusso, and P. G. Hatcher.
2001. An isotopic biogeochemical assessment of shifts in organic matter input to Holocene
sediments from Mud Lake, Florida. Org. Geochem. 32: 1153-1167.

Findlay, S. E. G., R. L. Sinsabaugh, W. V. Sobczack, and M. Hoostal. 2003. Metabolic and
structural response of hyporheic microbial communities to variation in supply of dissolved
organic matter. Limnol. Oceanogr. 48: 1608-1617.

Fisher, M. M, M. Brenner, and K .R. Reddy. 1992. A simple inexpensive, piston corer for
collecting undisturbed sediment/water interface profiles. J. Paleolimnol. 7: 157-161.

Fisher, M. M, K.R. Reddy, and R. T. James. 2001. Long-term changes in the sediment chemistry
of a large shallow subtropical lake. J. Lake and Reserv. Managem. 17: 217-232.

Fisher, M. M, K.R. Reddy, and R. T. James. 2005. Internal nutrient loads from sediments in a
shallow subtropical lake. J. Lake and Reserv. Managem. 21: 338-349.

Flanery, M. S., R. D. Snodgrass, and T. J. Whitmore. 1982. Deepwater sediments and trophic
conditions in Florida lakes. Hydrobiol. 92: 597-602.

Florida Lake Watch. 2001. http://lakewatch. ifas.ufl.edu/

Fogel, M. L., and L. A. Cifuentes. 1993. Isotope fractionation during primary production, pp. 73-
98. In Organic Geochemistry, eds. M H. Engel and S. A. Macko. Plenum Press.

Fogel, M. L., L. A. Cifuentes, D. J. Velinsky, and J. H. Sharp. 1992. Relationship of carbon
availability in estuarine phytoplankton to isotopic composition. Mar. Ecol. Prog. Ser. 82:
291-300.

Francaviglia, R., L. Gataleta, M. Marchionni, A. Trinchera, R. Aromolo, A. Benedetti, L. Nisini,
L. Morselli, B. Brusori, P. Olivieri, and E. Bernardi. 2004. Soil quality and vulnerability in
a Mediterranean natural ecosystem of Central Italy. Chemosphere 55: 455-466..

Frederico, A. C., K. G. Dikson, C. R. Kratzer, and F. E. Davis. 1981. Lake Okeechobee water
quality studies and eutrophication assessment. South Florida Water Management District,
West Palm Beach, FL, USA.

Friedrich, M. W. 2006. Stable-isotope probing of DNA: insights into the function of uncultivated
microorganisms from isotopically labeled metagenomes. Curr. Opin. Biotechnol. 17: 59-
66.










phytoplankton productivity (Wetzel 2001). Deviations in this ratio can indicate nutrient

limitation as well as affect P uptake by microorganisms.

Microbial Communities

Coupling and feedback between sediment biogeochemistry and water column primary

productivity often depends on biogeochemical processes within sediments and associated

microbial communities. Heterotrophic bacteria play an important role in C and nutrient cycling

in lakes. Phytoplankton and/or heterotrophic bacteria are the maj or drivers of C and nutrient

cycling in the water column, while the heterotrophic bacteria dominate in sediments.

Allochthonous and autochthonous particulate OM in the water column is deposited in the

sediment, leading to high concentrations of nutrients and high microbial biomass. Lake depth

affects the quality of organic material reaching the sediment. In deep lakes, sedimenting OM

undergoes intense decomposition in the water column, due to the prolonged period of settling.

Consequently low amounts of labile organic C reach the sediment (Suess 1980; Meyers 1997). In

shallow lakes, the supply of labile C and nutrients can be higher than in deep lakes, and the latter

often can have more refractory OM.

Organic matter deposition is an important source of C to sediments. Organic compounds

and associated nutrients supplied to the sediment surface are mineralized through heterotrophic

decomposition (Gachter and Meyer 1993; Capone and Kiene 1988; Megonigal et al. 2004)

(Figure 1-3). The composition and activities of the microbial community are regulated by the

quality and availability of C. In high depositional environments, such as eutrophic, or deep

thermally stratified lakes, organic content in sediments is often high, oxygen (Oz) COnSumption

occurs rapidly, and 02 is depleted several millimeters below the sediment water interface

(Jarrgensen 1983; Jorrgensen and Revsbrech 1983). In these systems, facultative and strict

anaerobic communities dominate. Complete oxidation of a broad range of organic compounds in











B) Lake Okeechobee M9













10-15 cm




45-60 cm









20 10 0 -10 -20

Chemical shift (ppm)

Figure 3-3B









from the other two lakes, and also displayed a separation of its sediment types. Mud sediment

types (M9, 01 1, K8 sites) of Lake Okeechobee were placed closer to both forms of inorganic P

(labile-Pi and HCl-Pi) with a gradient in relation to the three mud sites that were related to the

KR site (sand sediment). The peat zone (M17) was placed in a different position with extractable

C:N ratio, and was unrelated to any other site sampled. Sandy sediments from both Lake

Okeechobee and Lake Annie were placed with bulk density (Figure 2-4B).

The PCA-2, using only Lake Okeechobee, corroborates the results from Pearson's

correlation (Figure 2-5A, Appendix A-3). Methane production rates were placed with microbial

biomass, showing that the stimulation of methane production was dependent on the original

microbial biomass (MBC). Again, highest methane production rates were placed with P forms.

Axis 1 explained 60.6% of the variability of the data and the variables selected were BD and in

an opposite position all P forms, anaerobic respiration, methane production with electron donor

addition, LOI and MBC. Axis 2 with 20. 1% of the data variability explained selected extractable

C:N, C:P and N:P ratios. The same distribution of Lake Okeechobee sites seen in PCA-1 was

repeated in PCA-2. Peat zone position showed that this site had the highest concentration of the

variables selected by Axis 2. Sandy sediments were placed with the bulk density and opposite to

the other sites and parameters. Again the same distribution of the mud sediments with the KR

site is seen and they were placed with P forms and microbial biomass and activity (Figure 2-5B).

Discussion

In this study commonly applied methods in soil science were used to measure microbial

biomass in lake sediments. The chloroform fumigation-extraction method is a quick and simple

procedure that has been used widely to measure microbial biomass in soils (e.g. Jenkinson et al.

2004). Soil microbial C, N, and P extraction by this method is largely dependent on soil

characteristics and microbial community composition (Jenkinson et al. 2004). Therefore,









production and further break down monomers to alcohols, fatty acids, and hydrogen (H2).

Alcohols and fatty acids are degraded by syntrophic bacteria into acetate, H2, and carbon dioxide

(CO2), which is used as substrate by methanogens (Zinder 1993, Conrad 1999). Consequently,

CO2 and methane (CH4) are important end products of anaerobic organic matter decomposition

and such gas production can be used as a measure of microbial activity in sediments.

Several factors limit bacterial metabolism in sediments, i.e., temperature, biodegradable

organic C, nutrients, and electron acceptors. Most studies of microbial activity in sediments

focus on C limitation and the effect of electron donors or acceptors in production of CO2 and/or

CH4 (CapOne and Kiene 1988; Schulz and Conrad 1995; Maassen et al. 2003; Thomsen et al.

2004). Little work has been done relating production of CO2 and CH4 with biogeochemical

properties of sediments such as nutrient availability. Studies in the water column of lakes have

shown that several factors can limit bacterial metabolism (Gurung and Urabe 1999; Jasson et al.

2006). Although it has been generally accepted that the heterotrophic community is C/energy

limited, recent studies have shown that inorganic nutrients, especially phosphorus (P) can be the

most limiting nutrient for the bacterial community (Gurung and Urabe 1999; Vadstein 2000;

Olsen et al. 2002; Vadstein et. al. 2003; Smith and Prairie 2004; Jasson et al. 2006). Reviewing

data from freshwater ecosystems, Vadstein (2000) showed that P limitation is a common

phenomenon. Phosphorus limitation occurred in 86% of the cases, while nitrogen or C limitation

was identified in 15% and 20%, respectively (percentages add up to more than 100% due to

methodological aspects, Vadstein 2000). Heterotrophic microbial metabolism can be limited by a

single factor or multiple variables. Limitation varies among lakes and depends on lake

characteristics and biogeochemical properties of sediments.










Table 2-6. Anaerobic respiration and methane production rates in sediments from subtropical lakes. Control are values for basal
methane production without substrate addition, and acetate*, hydrogen* and acetate + hydrogen* results from electron
donor addition experiment only for Lake Okeechobee sediments. (mean a standard deviation).

Anaerobic Respiration Methane Production (CH4-C mg kg- d' dw)
La~ Ste(CO2-Cmg kg- ddw) Control Acetate* Hydrogen* Acetate + Hydrogen*


South

Central

North

M17

011

M9

K8


362 & 48

283 & 32

3.8 &1.2

76 & 17

117 & 26

54 & 14

98 & 11

5.6 & 0.5

15 & 2.8

13 A 1.7

78 & 19

11 & 3.3

563 & 28

654 & 87

455 & 54

1170 & 47


48 & 10

118 &17

0.15 & 0.02


Annie


N.D.

N.D.

N.D.

N.D.

N.D.

0.26

N.D.

N.D.

N.D.


5.0 & 4.5

27.3 & 5.6

11.6 &5.2

24.8 & 6.9

1.6 &1.5

12.1 +10

0.6 & 0.4

3.4 & 1.0

0.5 & 0.2


3.3 & 2.6

217 & 64

130 & 56

204 & 60

41.6 & 25

21.0 & 17

35.4 & 10

38.6 & 20

48.4 & 7.8


98 & 42

127 & 45

122 & 7.1

230 & 30

23.7 & 14

33.2 & 13

43.5 & 3.5

76.2 & 22

55.2 & 6.5


Okeechobee











Apopka


J5

TC

KR

J7

South


31 & 4.5

40 & 13

34 & 24

52 & 19


Central

West


North
N.D. = Not Detected.










Degens, B. P. 1998b. Decreases in microbial functional diversity do not result in corresponding
changes in the decomposition under different moisture conditions. Soil. Biol. Biochem. 30:
1989-2000.

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Degens, B. P., L. A. Shipper, G. P. Sparling and M. Vojvodic-Vukovic. 2000. Decreases in
organic C reserves in soils can reduced catabolic diversity of soil microbial communities.
Soil. Biol. Biochem. 32: 189-196.

Degens, B. P., L. A. Shipper, G. P. Sparling, and L. C. Duncan. 2001. Is the microbial
community in a soil with reduced catabolic diversity less resistant to stress or disturbance?
Soil. Biol. Biochem. 33: 1143-1153.

Del Giorgio, P., and R. H. Peters. 1994. Patterns in planktonic P:R ratios in lakes: influence of
lake trophy and dissolved organic carbon. Limnol. Oceanogr. 39: 772-787.

Deppenmeier, U, V. Muller, and G. Gottschalk. 1996. Pathways of energy conservation in
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Drabkova, V. G. 1990. Bacterial production and respiration in lakes of different types. Arch.
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Edzwald, J. K., D. C. Toensing, and M. C. Leung. 1976. Phosphate adsorption reactions with
clay minerals. Environm. Sci. Technol. 10:485-490.

El-Daoushy, F. 1988. A summary on lead-210 cycle in nature and related applications in
Scandinavia. Environ. Internat. 14: 305-319.

Elser, J. J., and B. L. Kimmel. 1986. Alteration of phytoplankton phosphorus status during
enrichment experiments: implications for interpreting nutrient enrichment bioassay results.
Hydrobiol. 133: 217-222.

Elser, J. J., W. F. Fagan, R. F. Denno, D. R. Dobberfuhl, A. Folarin, A. Huberty, S. Interlandi, S.
S. Kilham, E. Mccauley, K. L. Schulz, E. H. Siemann, and R. W. Sterner. 2000. Nutritional
constraints in terrestrial and freshwater food webs. Nature. 408: 578-580.

Engstrom, D. R., S. P. Schottler, P. R. Leavitt, and K. E. Havens. 2006. A reevaluation of the
cultural eutrophication of Lake Okeechobee using mulitproxy sediment records. Ecol.
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EPA. 1993. Methods for the determination of inorganic substances in environmental samples.
Environmental Monitoring Systems Lab, Cincinnati, OH.

Espie, G. S., A. G. Miller, R. A. Kandasamy, and D. T. Canvin. 1991. Active HCO3 transport in
cyanobacteria. Can. J. Bot. 69: 936-944









Table 3-4. Phosphorus composition of the sediment depth profile determined by 31P NMR spectroscopy. (Percentage in relation to
total extracted phosphorus with NaOH/EDTA in parenthesis)
TP
Lale Ste Depth (aHET Phosphate Mlonoester Lipid DNA Pyro-P Poly-P
(cm> mg P kg' dw (%)


ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
93 (11)
61 (9)
64 (8)
ND
ND
ND
ND
ND


ND: not detected, TR: trace (i.e., not quantifiable). TP NaOH/EDTA: total phosphorus in NaOH/EDTA extracts,
orthophosphate, Monoester: phosphate monoester, Lipid: phospholipids, Pyro-P: pyrophosphate, and Poly-P: pol:


552 (35)
551 (36)
483 (33)
505 (34)
367 (23)
438 (34)
321 (31)
248 (29)
210 (24)
147 (27)
ND
ND
ND
320 (28)
181 (22)
136 (20)
191 (22)
87 (25)
31 (14)
18 (12)
ND
ND


TR
TR
TR
TR
TR
ND
ND
ND
ND
ND
ND
ND
ND
37.2 (3)
32.6 (4)
20.1 (3)
30.4 (4)
TR
TR
TR
ND
ND


5
10
15

Central 20
30
45
60
80
5
10
M9 15
30
60
5
10
15
20
West 30
45
60
80
98


1559
1525
1468
1498
1584
1296
1041
854
896
547
405
174
228
1139
824
670
858
349
224
150
135
102


788 (51)
828 (54)
852 (58)
866 (58)
1127 (71)
777 (60)
636 (61)
518 (61)
604 (68)
363 (67)
405 (100)
174 (100)
228 (100)
313 (28)
263 (32)
231 (35)
290 (33)
165 (47)
141 (63)
95 (64)
107 (80)
86 (85)


219 (10)
146 (9)
132 (9)
127 (6)
94 (6)
81 (8)
82 (10)
88 (8)
82 (9)
36 (7)
ND
ND
ND
355 (31)
237 (29)
204 (31)
261 (30)
97 (28)
52 (23)
37 (25)
28 (21)
16(15)


ND
TR
ND
ND
TR
ND
ND
ND
TR
TR
ND
ND
ND
113 (10)
17 (2)
18 (3)
21 (3)
ND
ND
ND
ND
ND
Phosphate :


Annie







Okeechobee







Apopka


yphosphate.





1.90 1.88
1.88 *A .B
~1.86
1.86 e
1.84 e 1.84*

1.822
~91.80 C 1.8 pe
S1.78 1.80
S1.76
1.74 1.78
S1.72 1.7


S1.68 1.74
1.66 1.72
1.64
1.70
1.62
1.60 1.68
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Fraction Number Fraction Number



Figure 8-11. Graph illustrating the buoyant density of gradient fractions. (A) Lake Apopka (core

# 95); (B) Lake Okeechobee-M9 (core # 148).


Figure 8-12. Agarose (2%) gel electrophoresis of RT-PCR of RNA extracted from Lake Apopka
fractions (core # 95). E. coli specific primers were used.









extraction efficiency is corrected by the kec factor. Reported kec values can vary from 0.2 to 0.45

for C, N and P (Bailey et al 2002). In this study, kec factor was not used, to avoid overestimation

of the microbial biomass. For example, Lake Apopka sediments had MBP concentrations

varying from 561 to 103 1 mg kg- If the reported kec factor of 0.37 for MBP (Hedley and

Stewart 1982) was applied, the final MBP concentration in Lake Apopka sediments would be

higher than the TP (1650-2786 mg kg- ). These kec factors were determined for soils with lower

microbial biomass than sediments like Lake Apopka. The efficiency ofP extraction from

samples with high microbial biomass is probably higher, thus resulting in low kec factors.

Therefore the kec factors reported for typical soil samples are probably not suitable for use in

samples containing high labile P in the microbial biomass.

Several studies, however, reported that MBC measured through the chloroform

fumigation-extraction method (not corrected with the kec factor) yields similar results when

compared with other alternative methods to measure microbial biomass in soils. Leckie et al.

(2004), using humic soils, reported a strong positive linear relationship (r2 = 0.96, p = 0.007)

between microbial biomass C measured with chloroform fumigation-extraction (with no

correction factor) and total phospholipids fatty acid analysis, a more accurate methodology to

measure microbial biomass. Bailey et al. (2002), using mineral soils, also reported a strong linear

relationship between these two measurements. Microbial biomass C concentration in eight

different soils, including sewage sludge, were also strongly correlated (r2 = 0.96) with DNA

measurements (Marstorp et al. 2000). The use of microbial biomass concentrations not corrected

by the kec factor is, therefore, a good measure of microbial biomass present in the samples.

Although the chloroform fumigation-extraction has not been used widely in sediment studies









density gradient centrifugation, the 'heavier' 13C migrates faster than the 'light' unlabeled 12C

nucleic acids. The DNA and RNA sequences present in the 'heavy' gradient fractions must be

derived from organisms that have consumed the added 13C labeled substrate. DNA-SIP is the

least sensitive approach because it requires cell division to obtain sufficient label into DNA, thus

requiring longer incubation times where cross-feeding and false results can occur (Wellington et

al 2003; Neufeld et al. 2007). RNA-SIP has proven a more sensitive approach, since in active

cells RNA synthesis occurs at higher rates, and labeling can occur without replication of the

organism (Manefield et al. 2002a).

In the present study, RNA-SIP was used to identify microorganisms that utilize acetate in

sediments of subtropical lakes with different trophic states. This approach, however, did not

work. This chapter was written with the intent to document the methods used at every step and

explore the source of error that contributed to the failure of the proposed study.

Materials and Methods

Study Sites and Field Sampling

Three Florida (USA) lakes ranging in trophic state were selected: Lake Annie (oligo-

mesotrophic), Lake Okeechobee (eutrophic) and Lake Apopka (hypereutrophic). A map of the

lakes with sampling locations as well as descriptions of the three lakes were reported previously

(Chapter 2 and 3) Triplicate sediment cores were collected using a piston corer (Fisher et al.

1992) or by SCUBA divers. The topmost 10 cm of sediment were collected from one central site

in Lake Annie on June 25, 2005 and a western site in Lake Apopka on May 28, 2005. Cores were

collected at three sites in Lake Okeechobee on July 16, 2005: M17 = peat, M9 = mud and KR =

sand.

Samples were transported on ice and stored in the dark at 4 oC. Sub-samples were taken

and frozen and kept at -80 oC. These samples were also used in a previous study in which eight









separation was observed when samples were centrifuged in the same tube (Figure 8-18B).

Several studies have shown overlap between 'heavy' and 'light' RNA in CsTFA density

gradients (Manefield et al. 2002a; Lueders et al. 2004a, b; Haichar et al. 2007; Hatamoto et al.

2007; Schwarz et al. 2007). Although using E. coli RNA as control is a valid approach, it should

not be added to the tube with the 'heavy' RNA sample, but centrifuged in a separate tube. Then,

the density gradient of the E. coli RNA can be compared with the density gradient of the

experimental sample.

Considering the problems that occurred during the RNA-SIP experiment several

suggestions can be made. The first recommendation is to conduct several experiments with

different concentrations and maybe pulses of [13C]-acetate, with different incubation periods.

Second, samples should be checked for amount of labeling by mass spectrometry, since it is

necessary to assure that a sufficient amount of labeled RNA is present. Once this is determined,

samples should be incubated in two different sets: one with [13C]-acetate and another with [12C]

acetate. RT-PCR of [12C]-acetate density gradients can be used to compare with [13C-cte

density gradients. E. coli RNA can be used as a control as long as it is not added in the same tube

of labeled target RNA. Blank density gradients should be used along with other samples during

centrifugation, so the linear distribution of blank density gradients should be verified before

fractionation of the samples. Finally, proper equipment to fractionate the samples after

ultracentrifugation, such as a Beckman Fraction Recovery System, should be used for a more

precise fractioning of the samples.









allochthonous (high ratio) versus autochthonous (low ratio) organic matter (Hutchinson 1957;

Mackereth 1966). Terrestrial autotrophs have higher C:P and C:N ratios than does lacustrine

particulate organic matter (Elser et al. 2000). Autochthonous organic matter has a C:N ratio

around 12:1 (Wetzel 2001). Among all sediment types in this study, peat zone deposits from

Lake Okeechobee had the highest total weight C:N and C:P ratios reflecting its higher plant

origin. Deposits from other sediment types, especially Lake Apopka, with lower C:N, reflect

algal origin.

Extractable nutrient ratios were low for Lake Annie, reflecting high concentrations of

extractable labile nutrients relative to C. High availability of N and P may indicate C limitation

in Lake Annie sediments. Carbon limitation may reflect the recalcitrant nature of C entering the

lake and physical characteristics of this lake. Lake Annie has experienced an increase in color

during the past decades, probably from high dissolved organic carbon (DOC) input to the lake

from adjacent land (Swain and Gaiser 2005). Battoe (1985) reported high input of surface waters

enriched in humic content to Lake Annie during high rainfall periods. This allochthonous DOC,

of humic origin, will be utilized in the water column. Because Lake Annie is deep, the DOC will

be mineralized during its descent to the sediment (Suess 1980). Consequently lower

concentrations of DOC will reach the sediment (also being highly refractory) leading to low

C:nutrient ratios.

Carbon and N limitation was observed in most Lake Okeechobee sediments, especially in

the mud zone. Hence, there is low microbial biomass and activity. Crisman et al. (1995) reported

that temperature and trophic state variables Secchi, total P, and total N, showed a weak

correlation with bacterioplankton abundance (number of cells mL 1) in a seasonal study in Lake

Okeechobee. They concluded that the factors controlling bacterioplankton communities could be










Table A-3. Pearson correlation coefficients of sediment biogeochemical properties significant at p< 0.05.
Carbon Nitrogen Phosphorus


BD LOI


TC ExtC


TN ExtN TP Pi TIP Po FAP HAP Res


LOI
TC
Ext-C
TN
Ext-N
TP
Lab.Pi
IP
Lab.Po
FAP
HAP


-0.91
-0.88
-0.71
-0.80
-0.65
-0.92
-0.47
-0.67
-0.77
-0.67
-0.67


1.00
0.86
0.91
0.78
0.80
0.17*
0.34
0.77
0.65
0.68


0.85
0.91
0.77
0.76
0.13*
0.29
0.74
0.61
0.64


0.92
0.96
0.74
-0.11*
0.08*
0.77
0.64
0.68


0.87
0.75
0.00*
0.21*
0.76
0.63
0.67


0.75
-0.14*
-0.04*
0.78
0.70
0.71


0.47
0.64
0.85
0.81
0.75


0.80
0.29*
0.34
0.19*


0.36
0.35
0.29*


0.85
0.84


0.90


-0.64 0.52 0.50 0.52 0.52 0.47 0.68 0.28* 0.55 0.41 0.14* 0.12*


Res.P
Ratios
Ext-C:Ext-N
Ext-C :Ex-tP
Ext-N:Ext-P


0.30 -0.22* -0.25* -0.25* -0.25* -0.31


-0.38 -0.10* -0.22* -0.34


-0.34 -0.32 -0.19*


-0.39
-0.43


0.64
0.62


0.67
0.63


0.86
0.87


0.78
0.78


0.82
0.91


0.39
0.52


-0.47
-0.40


-0.27*
-0.19*


0.41
0.50


0.29*
0.41


0.35
0.42


0.40
0.47


BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon, TN: total nitrogen, Ext-N: extractable
labile nitrogen, TP: total phosphorus, Lab.Pi: labile inorganic phosphorus, Lab.Po: labile organic phosphorus, IP: inorganic
phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resistant organic phosphorus, Res.P: residual phosphorus, Ext-P:
extractable labile phosphorus. *Not significant at p< 0.05.




























































0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
FractionNumber FractionNumber

Figure 8-13. Graph illustrating the buoyant density of gradient fractions. (A) Blank (no RNA),
(B) Lake Apopka (core #94) and (C) Lake Apopka (core # 95).


A






SPlateau

***
*

*
**


0 5 10


0


C




Plateaus
* .1


***

*


15 2(
Fraction Number
1.88


1.84

1.82





1.2


S1.70
16 "


25 30


B


e
*
*




.











































































Figure 6-4. Lake Okeechobee mud zone (site M9) sediment depth profile of: A) Total phosphorus, B) TC:TN ratio, C) TN:TP ratio,

D) 613Corg Of sediment organic carbon, E) sediment 8 "N, and F) organic matter content (LOI %).


UIo(/o)
16 18 20 22 24 26 28 30 32 34 36 38 40 42





E


811


Total Phosphorus (mg kg )
0 200 400 600 800 1000


TC:TN
16 18 20 22 24 26 28 30


TN:TP
4 6 8 10 12 14 16 18 20 22 24 26 28 30


14


8 13Corg (%o)
-26.4 -26.2 -26.0 -25.8 -25.6 -25.4 -25.2 -25.0 -24.8-


VI


a 15N (%o)
3.0 3.2 3.4


2.4 2.6 2.8


V


3.6 3.8 4.0


24.6


D


to C


50


60












Incubation and RNA extraction............... ..............21
Escherichia coli RNA .............. ...............212....

Isopycnic centrifugation ................. ...............212................
RT- PCR ................. ...............215......_.. .....
Re sults.................. ...............215........ ......
RNA Extraction ................. ...............215........ ......
Pre-Experiment .......__................. ........_.. .........21
RNA-SIP Experiment .......__................. ..........__..........21
Escherichia coli RNA .............. ...............216....

Isopycnic centrifugation .................. ...............216......_ ......
Discussion, Conclusions and Recommendations .............. ...............218....

9 SUMMARY AND CONCLUSIONS ................. ....._.. ...............234 ....


Biogeochemical properties and microbial activity of sediments (Obj ective 1) ................... .23 5
Sediment phosphorus forms (Obj ective 2) ......... ......._.__._ ......... ............3
Enzyme activities in sediments (Objective 3) .............. ... ...............237..
Microbial biomass and activity in sediments (Obj ective 4) ................. .. ....... ........ ..........23 8
Long-term OM accumulation and stable isotope signatures in sediments (Obj ective 5).....239
Microbial activity in sediments: effects of organic electron donors (Obj ective 6) ........._....240
RNA-stable isotope probing of acetate-utilizing microorganisms (Obj ective 7) .................24 1
Synthesis............... ...............24
Lake Annie .............. ...............242....
Lake Okeechobee .............. ...............243....
Lake Apopka .............. ...............243....


APPENDIX


A Supplemental Tables............... ...............250

LIST OF REFERENCE S ................. ...............254................


BIOGRAPHICAL SKETCH .............. ...............279....









these lakes might be expected to have higher concentrations of organic matter and nutrients than

oligo-mesotrophic lakes. The results from this study, however, showed that trophic state

conditions were not related to the nutrient content of sediments. Organic matter, N and P

concentrations were higher in sediments with lower bulk density, independent of the trophic state

of the lake. Sediment composition therefore reflects an integrative effect of trophic state

conditions and diagenesis over a long period of time, relative to water column physico-chemical

variables.

The relative importance of P forms present in the sediments seemed to be more important

than total P concentration in characterizing the sediment of each of the studied lakes. The oligo-

mesotrophic Lake Annie organic sediments contained maj or P forms in moderate to highly

resistant organic P (NaOH soluble) and HCl-Pi, suggesting P in this lake is old and stable. The

Lake Okeechobee sediment maj or P form was HCl-Pi, which constituted approximately 60-91%

of the total P, while hypereutrophic Lake Apopka sediment had > 50% of the total P in the

microbial biomass.

Extractable nutrient ratios seemed to have stronger influence on sediment microbial

communities than total concentrations. Extractable nutrient ratios were low for Lake Annie,

reflecting high concentrations of extractable labile nutrients relative to C, indicating C limitation

in these sediments. High labile inorganic P availability resulted in low extractable C:P and N:P

ratios, and C and N limitation in most Lake Okeechobee sediments, especially in the mud zone,

followed by low microbial biomass and activity. Moreover, low C availability with concomitant

presence of Fe- SO4-2-reducers appears to be inhibiting the methanogenic community in Lake

Okeechobee sediments. Limitation of the methanogenic community in these sediments is

supported by the positive effect of the addition of electron donors on methane production. The









concentration and contribution to TP, of organic P extracted with NaOH (FAP and HAP), but

poly-P was not detected with 31P NMR (Tables 3-3 and 3-4). Moreover, pyro-P that can include

degradation products of poly-P (Hupfer et al. 1995), was absent in Lake Annie. Several studies

showed that microbial biomass correlates with enzyme activity (Davis and Goulder 1993; Massik

and Cotello 1995; Barik et al. 2001). Since a high correlation between both FAP and HAP and

PMEase activity (Chapter 4) exists, it seems that the correlation between NaOH-P and microbial

biomass reflects the fact that these fractions are used as a P source by microorganisms through

enzyme activity.

Lake Apopka was the only lake where poly-P was detected. Gachter and Meyer (1993)

postulate that if sufficient organic carbon and PO4-3 are available under aerobic conditions,

bacteria can store poly-p. The occurrence of poly-P and the identification of phosphate-

accumulating organisms come from studies in wastewater treatment plants with enhanced

biological P removal (Seviour 2003). In lakes, the mechanism of poly-P formation is poorly

understood, although poly-P has been detected in several recent studies (Hupfer et al. 1995,

2004; Carman et al. 2002; Reitzel et al. 2006a, b, 2007). Alternation of aerobic/anaerobic

conditions, combined with available carbon and phosphate, leads to dominance of bacteria that

can store poly-P (Mino et al. 1998; Seviour 2003). Khoshmanesh et al. (2001) used 31P NMR and

transmission electron microscopy, on a sediment spiked with acetate, and concluded that under

aerobic conditions when acetate was available, microorganisms accumulated phosphate as poly-



Some sediments have ideal conditions for poly-P formation, such as oscillating

aerobic/anaerobic conditions, labile dissolved organic carbon and labile phosphorus. These

conditions exist in Lake Apopka (Chapter 4 and 5). Lake Annie, has available DOC, but low









concentrations of soluble reactive P (SRP) in pore water increased under reduced conditions, and

were low near neutral (pH 6.5 and 7.5), but higher under slightly acidic (pH 5.5) or basic (pH

8.5) conditions. Furthermore, Olila and Reddy (1997) reported that SRP increases exponentially

with a decrease in redox potential in sediments from the mud zone of Lake Okeechobee.

Consequently, P solubility in Lake Okeechobee mud sediments is controlled by abiotic

processes, either pH, redox potential, or both (Moore and Reddy 1994; Olila and Reddy 1997).

The control of P solubility in other sediment types of Lake Okeechobee has not been

studied. Nevertheless, the dominance of inorganic P in all Lake Okeechobee sites and lack of

organic P found in 31P NMR, suggests that pH and redox potential also regulate P solubility in

M17 and KR sediments. Labile Pi follows the HCl-Pi distribution in sites of Lake Okeechobee

(especially M9 and KR). Considering that the maj or P forms in Lake Okeechobee are HCl-Pi, as

well as the fact that these sediments had low enzyme and microbial activities (Chapter 4), it is

reasonable to speculate that abiotic processes control P solubility in these sediments.

In contrast to Lake Annie and Lake Okeechobee, in which either biotic or abiotic processes

alone control P solubility respectively, in Lake Apopka sediments, P solubility is controlled by a

combination of biotic and abiotic processes. Dominant P forms were MBP and HCl-Pi (chemical

fractionation), and orthophosphate, phosphate monoester and DNA-P (31P NMR). The high

contribution of organic P forms in relation to total P in Lake Apopka results from deposition of

algal primary producers to the sediment. Gale and Reddy (1994) reported gross primary

productivity in Lake Apopka of 1400 g C m-2 -1l Of which approximately 1034 g C m-2 -1l is

deposited in sediments. The Lake Apopka phytoplankton community is dominated by

cyanobacteria, (Synechococcus sp., Synechocystis sp., and M~icrocystis incerta), with little

variation throughout the year (Carrick 1993; Carrick and Schelske 1997). Brunberg (1995)









Water extracts were centrifuged at 10,000 x g for 10 min, filtered through a 0.45 Clm

membrane filter, and analyzed for dissolved reactive P (DRP) and DOC with the same method

used for water samples.

Enzyme Activity

Enzyme activities including PMEase and PDEase were determined colorimetrically using

as substrate p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate, respectively (Tabatabai

1994; Alef et al. 1995), both from Sigma Chemical Co (St Louis, MO). Assays were conducted

using three replicates and a control for each sample to account for non-enzymatic color

development. As PMEase activity depends on pH range (Tabatabai 1994; Alef et al. 1995),

alkaline phosphatase activity was measured in Lake Okeechobee and Lake Apopka; while the

acid phosphatase activity was measured in Lake Annie sediments (see Table 3-2). A known

amount of wet sample, 0.5 g for high organic sediment, and 1 g for mineral sediment, was added

to polypropylene centrifuge bottles with the artificial substrate (1 ml of 0.05 M p-nitrophenyl

phosphate for PMEase, and bis-p-nitrophenyl phosphate for PDEase), toluene (to inhibit

microbial growth during measurement), a pH buffer (pH = 11 for alkaline, pH = 6.5 for acid

phosphatase, and pH = 8 for PDEase) and incubated at 37 oC for 1 hour. Enzymatic activity was

sto ped after incubation by addition of 1 mL of 0.5 M CaCl2 and 4 mL 0.5 M NaOH (for

PMEase) and 0.1 M/0.5 M THAM/NaOH (THAM: tris hdrox meth aminomethane)

extractant solution (for PDEase). Samples were centrifuged and filtered through a Whatman # 1

paper filter and analyzed at 420 rim using a UV-VIS spectrophotometer (Shimadzu Model UV -

160) (Tabatabai 1994; Alef et al. 1995). Absorbance was compared with standards. Control

values were subtracted from sample values to account for non-enzymatic substrate hydrolysis.









sediment layers. P solubility in these sediments is controlled by a combination of abiotic (pH)

and biotic processes (Figure 9-4). High concentration of diester P resulted in high PDEase

activity. The activity of PMEase was also high and its production repressed by inorganic P

availability, however, it seems that in these sediments PMEase is also related to C acquisition by

the heterotrophic microbial community. Sediments were characterized by high extractable C and

labile-N and low labile-Pi concentration, which resulted in high C:P and N:P ratios, and

indicated P limitation in these sediments. Microbial biomass and activity were high in these

sediments. High C availability in these sediments probably accounts for efficient use of energy

that it is used for biomass (growth) as well as respiration. The heterotrophic microbial

community in these sediments has high catabolic diversity.

Results from these studies demonstrated the mutual dependency of C, N and P

transformations in regulating the sediment microbial community and nutrient bioavailability,

especially P. Activity of the heterotrophic microbial community can be limited by a range of

properties and will depend on limnological characteristics of lakes and sediment biogeochemical

properties. The results also highlighted the significance of the relationships between sediment

biogeochemical properties and microbial community activities in lakes with different trophic

states, and showed how the physico-chemical conditions of lakes affect sediment properties and

microbial mediated processes. Moreover, it illustrated the importance of measuring several

variables, such as C, N and P, to address questions related to microbial communities.

Future studies should focus on identifying communities that regulate the OM turnover and

nutrient mobilization. Controlled experiments addressing the effect of C, N and P addition to

sediment microbial biomass and activity can strengthen the conclusions about nutrient limitation

in each of these lake sediments. The study of other enzyme activities, such as C (i.e.,
































Mineralization p
Biomass


Mi neral izati on


Annie N2 Fixation ?


,lumn
Phytoplankton
\613C ~15N


~t[N]

B acteri a
\13C L 15



/t[NH4+] \15N Biomass
/t[CO2] \13C


Mi neral izati on


OM


A) Lake

Water Co














Anoxia


OM
\613C ~15N



Mineralization


Sediment \L13 15~lN


\13C ~15N




Groundwater


Allochthonous ?
B) Lake Okeechobee M9 Agricultural runoff
\13C /\15N
Water Column P "~'h to ,lankton


y pI ~l~lCI
Non N2-Fixer
?613C /\15N



C and N
limitation


813C /\15N


Sediment


Figure 6-9. Maj or mechanisms affecting the sediment 813C and 61 N signatures in: A) Lake
Annie, Lake Okeechobee B) site M9, C) site M17 and D) site KR, and E) Lake
Apopka.









The magnitude of CO2 and CH4 prOduction following addition of different electron donors

was strongly related to microbial biomass at each site. There was a strong significant positive

correlation between MBC and CO2, and MBC and CH4 prOduction rates (Figure 7-6A, B).

Two Principal Component Analyses (PCA) were conducted, PCA-1 was performed using

the effect of electron donor additions on CO2 prOduction rates, and PCA-2 on CH4 prOduction

rates. The PCA-1 indicated that 40.7% of the data variability was explained by Axis 1 while Axis

2 explained 20.1% (Figure 7-7A). Anaerobic respiration with the additions of acetate, butyrate,

format, and Lake-SS were the variables selected by Axis 1. Basal anaerobic CO2 prOduction

was selected by Axis 2. The position of sites in relation to variable loadings in PCA-1 showed

that sediments from each lake and site are separated into different groups (Figure 7-7B). Lake

Annie sediments were plotted in the position of basal CO2 prOduction (Figure 7-7B). Lake

Apopka sediments with Lake-SS cluster (Lake-SS, butyrate, acetate, format, and propionate)

opposite from Lake Annie. Lake Okeechobee site M17 was plotted close to Lake Apopka

sediments, while the KR site was in the position with glucose and alanine additions. Lake

Okeechobee mud zone (site M9) was not placed with any specific carbon addition (Figure 7-7B).

The PCA-2 had 33.6% of the data variability explained by Axis 1 while Axis 2 explained

27.4% (Figure 7-8A). Methane production rates with additions of alanine, butyrate, and glucose

were the variables selected by Axis 1. Methane production rates from arginine, and basal

production rate were selected by Axis 2. The position of the sites in relation to the variable

loadings in PCA-2 showed a separation of sediments from each lake and site (Figure 7-8B). Lake

Annie sediment was placed with the basal production, arginine, and acetate cluster. Lake

Okeechobee M9 site was plotted in the position of propionate and format and close to the KR









Table 2-1. Morphometric and limnological variables of the three subtropical lakes.
Lake

Annica,b Okeechobeec Apopkac
Surface Area (km2) 0.366 1800 125

Mean depth (m) 9.1 2.7 1.6

Maximum depth (m) 20.7

Electrical Conductivity (CIS cm l) 43.7 447.7 384

Chlorophyll-a (Clg L^1) 3.6 26 90

Total Nitrogen (Clg L 1) 373 1510 4890

Total Phosphorus (Clg L^1) 5.0 100 190

Secchi Transparency (m) 3.4 0.5 0.23

Trophic Classification Olg-. Eutrophic Hypereutrophic
mesotrophic
aFlorida Lake Watch (2001), bArchbold Station (2005), aHavens et al. (1999)


Table 2-2. Location and sediment type of the sites sampled in the three different lakes.

Lake Date Sediment Type Site Latitude Longitude

Mud/Clay South 27ol2'l18" 81o21'40"
Annie July/04 Mud/Clay Central 27ol2'27" 81o21'44"
Sand North 27ol2'32" 81o20'57"
Peat M17 26o45'24.4" 80o46'36.8"
Mud 011 26o5 5'l14.8" 80o41 '53 .8"
Mud M9 2605 8' 17.6" 80o45'38.4"
Mud K8 27000' 16.6" 80o49'38.1"
Okeechobee May/03 Littoral/Sand FC 26o5 8'll 1.5" 80000'5 1.8"
Littoral/Sand J5 27005'28.1" 80051'28.8"
Sand TC 27ol l'55" 80o47'40"
Sand KR 27o5 8'll 1.5" 80000'5 1.8"
Sand J7 27o02'll1" 80o51'l9.8"
Organic South 28o3 5'00" 81o36'22"
Organic Central 28o37'31" 81o37'24"
Apopka Jan/04
Organic West 28o3 8'01" 81o39'36"
Organic North 28o39'43" 81o37'25"










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archaeal methanogen unrelated to all other known methanogens. Syst. Appl. Microbiol. 14:
346-351.

Burnett, W. C., and O. A. Schaffer. 1980. Effect of ocean dumping on 13 /12C ratios in marine
sediments for the New York Bight. Est. Coast. Mar. Sci. 2: 605-611.

Capone, D. G., and R. P. Kiene. 1988. Comparison of microbial dynamics in marine and
freshwater sediments: contrasts in anaerobic carbon catabolism. Limnol. Oceanogr. 33:
725-749.

Carman, R., G. Edlund, and C. Damberg. 2002. Distribution of organic and inorganic
phosphorus compounds in a marine and lacustrine sediments: a 31P NMR study. Chem.
Geol. 163: 101-114.

Carrick, H. J., and C. L. Schelske. 1997. Have we overlooked the importance of small
phytoplankton in productive waters? Limnol. Oceanogr. 42: 1613-1621.

Carrick, H. J., F. J. Aldridge, and C. L. Schelske. 1993. Wind influences phytoplankton biomass
and composition in a shallow, productive lake. Limnol. Oceanogr. 38: 1179-1192.

Casper, P. 1992. Methane production in lakes of different trophic state. Arch. Hydrobiol. Beith.
Ergebn. Limnol. 37: 149-154.












O-CM. barkedi DNA
f'C IM. extorquens DNA








Total DNA
-eCM. barkedi rRNA genes
a -1- i M~ extorquens rRNA genes







1,68 1.701 1.72 1.74 1.76 17
CsCI buoyant annsrly [g rnI ']

Figure 8-17. CsCl density gradient centrifugation of isotopically distinct DNA species and
qantitative evaluation of nucleic acid distribution within gradient fractions. 12C- and
1C-DNA was centrifuged individually (A) or simultaneously (B) and detected
fluorometrically (full symbols) or via domain-specific real-time PCR (empty
symbols). Figures and captions are from Lueders et al. (2004a).

t'C M. barkedi RNA
tCM. extorquens RNA


102 ,r r j




E Total RNA
E 1'20M. barked rRNA
4 aC M. extorquenarRNA




0 8


1.75 1.Tf 1.70 1.81 1 83
OsTFA buoyant density [g rnl-]
Figure 8-18. CsTFA density gradient centrifugation of isotopically distinct rRNA species and
quantitative evaluation of nucleic acid distribution within gradient fractions. 12C- and
13C-rRNA was centrifuged individually (A) or simultaneously (B) and detected
fluorometrically (full symbols) or via domain-specific real-time RT-PCR (empty
symbols). Figures and captions are from Lueders et al. (2004a).








































Sediment


External


ssolved a Nutrient
and OM


Water Column


Living Particles


Living Particles


Sediment
Labile
a Dissolved
Slowly Available

I i Microbial Community


Refractory OM

Figure 1-1. Schematic of maj or processes occurring in sediment and water column of lakes.


Chemical I
Inorganic P DRP
P Mineralization
Organic P Microbial Activity~ ers *y
Labile Regulators: activity
wly Available Enzyme activity Accumulation of
Recalcitrant Eh Poly-P


Sediment
P release


Slo


Figure 1-2. Schematic showing draw of chemical and biological P processes in lake sediments.










Whiteley, A. S., M. Manefield, and T. Lueders. 2006. Unlocking the 'microbial black box' using
RNA-based isotope probing technologies. Curr. Opin. Biotechnol. 17: 67-71.

Whiticar, M. J. 1999. Carbon and hydrogen isotope systematics of bacterial formation and
oxidation of methane. Chem. Geol. 161: 291-3 14.

Whitmore, T. J., M. Brenner, and C. L. Schelske. 1996. Highly variable sediment distribution: a
case for sediment mapping surveys in paleolimnological studies. J. Paleolimnol. 15: 207-
221.

Whitmore, T. J., M. Brenner, K. V. Kolasa, W. Kenney, M. A. Riedinger-Whitmore, J. H. Curtis,
and J. M. Smoak. 2006. Inadvertent alkalization of a Florida lake caused by increase ionic
and nutrient loading to its watershed. J. Paleolimnol. 36: 353-370.

Widell, F. 1988. Microbiology and ecology of sulfate-and sulfur-reducing bacteria, p. 469-585.
Dr A. J. B. Zehnder [ed.], Biology of anaerobic microorganisms. Wiley.

Williams, J. D. H., and T. Mayer. 1972. Effects of sediment diagenesis and regeneration of
phosphorus with special reference to lakes Erie and Ontario, p. 281-315. Dr E. Allen and J.
R. Kramer, [eds.], Nutrients in natural water, Willey.

Wisniewski, R. J., and M. Planter 1985. Exchange of phosphorus across sediment water interface
(with special attention to the influence of biotic factors) in several lakes of different trophic
status. Verh. Int. Ver. Limnol 22: 3345-3349.

Wobus, A., C. Bleul, S. Maassen, C. Scheerer, M. Schuppler, E. Jacobs, and I. Roiske. 2003.
Microbial diversity and functional characterization of sediments from reservoirs of
different trophic state. FEMS Microb. Ecol. 46: 331-347.

Work, K. A., and K. E. Havens. 2003. Zooplankton grazing on bacteria and cyanobacteria in a
eutrophic lake. J. Plank. Res. 25: 1301-1307.

Work, K. A., K. E. Havens, B. Sharfstein, and T. East. 2005. How important is bacterial carbon
to planktonic grazers in a turbid subtropical lake? J. Plank. Res. 27: 357-372.

Wright, A. L., and K. R. Reddy. 2001. Heterotrophic microbial activity in northern Everglades's
wetland soils. Soil. Sci. Soc. Am. J. 65: 1856-1864.

Yiyong, Z., L. Jianqi, and Z. Min. 2001. Vertical variations in kinetics of alkaline phosphatase
and P species in sediments of a shallow Chinese eutrophic lake (Lake Donghu). Hydrobiol.
450: 91-98.

Zinder, S. H. 1993. Physiological ecology of methanogens, p. 128-206. Dr J.G. Ferry [ed.],
Methanogenesis: Ecology, Physiology, Biochemistry and Genetics. Chapman and Hall.

Zinder, S. H. 1994. Syntrophic acetate oxidation and 'reversible acetogenesis', p. 386-415. Dr H.
L. Drake [ed.], Acetogenesis. Chapman and Hall.










Table 4-2. Concentration of TP: total phosphorus, DRP: soluble reactive phosphorus, TN: total
nitrogen, NH4-N: ammonium-N and DOC: dissolved organic carbon in the water
column of Lake Annie, Lake Okeechobee, and Lake Apopka.

Depth TP DRP TN NH4-N DOC
Lake Site
(m) (pg L ) (mg L )


14.3

15.2

15.3

12.3

12.4

13.1

14.1

16.1

13.5

13.8

20.2

16.0

17.6

20.2

18.8

20.4

14.5

25.1


22.8

22.0

16.1

10.6

7.8

8.8

144.2

211.3

258.3

298.0

224.6

247.4

317.7

113.9

118.4

206.9

60.0

72.6


9.5

7.8

5.5

6.5

5.2

5.5

11.8

90.5

93.4

87.4

121.5

107.3

110.3

64.3

59.9

63.5

15.3

10.2


1484

1374

1264

1154

1099

1319

4955

3192

3192

3934

2938

2883

4266

2717

2717

3436

5505

6056

21884


51.8

102.5

66.7

48.2

111.3

183.4

707.6

92.3

130.9

85.8

53.5

69.4

58.3

80.7

79.5

90.8

233.9

74.9

50.0


Annie


Central


M9


Okeechobee


M17


Apopka


West


2 76.3


53.9









indispensable help with field sampling and the good times we spent in those lakes. Thanks to my

colleagues and dear friends that voluntarily helped me during field sampling, Dr. Noel Cawley,

Kathleen McKee, Andrea Albertin and Jason Smith. I am deeply thankful to Jason Smith who

taught and helped me with most of the molecular biology procedures, and for our discussions

about science and life. Also, I want to expand my thanks to members of Microbial Ecology

Laboratory (Abid, Hiral, Moshik, and Yun) for welcoming me to the lab. Especially to previous

members Dr. Hector Castro and Dr. Ashvini Chahaun for their guidance with the electron donor

experiment, and for sharing their knowledge of soil microbiology. My thanks to Dr. Syed

Noorwez and Dr. Mark P. Krebs (Department of Ophthalmology/UF) for helping with the

ultracentrifuge, special thanks to Dr. Krebs for discussing the methodology for the SIP

experiment and for his help in solving practical problems. My deepest thanks to Dr. Andrew S.

Whiteley (Molecular Microbial Ecology CEH Oxford/UK), for a number of emails

exchanged to help me solve problems with the SIP experiment, and for sharing his knowledge

and his kindness. I also want to thank Bill Reve for providing and setting up the HPLC pump for

the SIP experiment.

My sincere thanks to Dr. Benj amin Turner (Smithsonian Tropical Research

Institute/Panama) for his teachings on 31P NMR analysis, and interpretation and discussion of the

data. Also to Dr. Michael Hupfer (Leibniz-Institute of Freshwater Ecology and Inland Fisheries,

Berlin/Germany) for his time and advice in improving the extraction for 31P NMR. My thanks to

Dr. Kanika S. Inglett (Dr. Sharma!) for her constant support, her help with discussing and setting

up experiments, and her friendship. I really appreciated all those endless conversations we had

and the guidance she provided during the difficult times. My thanks to Dr. Patrick Inglett for his

guidance, and help with isotope analysis.









and deeper sediment layers had a significant decrease in DOC concentration with time. This

suggests that other factors are influencing accumulation of DOC and NH4-N in these sediments.

Several studies have indicated that DOC accumulation is a reflection of P limitation in

freshwater ecosystems. Gurung and Urabe (1999) concluded from controlled experiments on the

bacterial planktonic community from eutrophic Lake Biwa (Japan) that DOC accumulation in

surface water during summer is induced by the high bacterial growth rate and P limitation. Also,

Olsen et al. (2002) studied nutrient limitation of aquatic food webs and showed that DOC

accumulated in experiments where P was limiting, i.e., with high C:P ratios. Other studies also

reported that in lakes where there is P limitation of heterotrophic bacteria, labile DOC

accumulates (Vadstein et al. 2003). Jasson et al. (2006) did controlled experiments with

bacterioplankton in subarctic Lake Diktar Erik, Sweden, and showed that growth of the

heterotrophic community was controlled by DOC and inorganic nutrients. In their experiments,

bacterial production was stimulated by the DOC supply, but the use of DOC for growth was

dependent on the DOC:Pi ratio. Furthermore, DOC was used for growth under C-limited

conditions, but used for respiration under Pi limitation, when bacterioplankton communities tend

to respire large portions of assimilated C.

The increase in NH4-N at the end of incubation is another indication of P limitation.

Bacteria preferably utilize N in the form of amino acids over NH4' (Kirchman 1990), and this

preference has been reported to be stronger when bacterial growth is P-limited (Schweitzer and

Simon 1995, Gurung and Urabe 1999). This can lead to an accumulation of NH4' and other

inorganic N forms in P limited systems (Gurung and Urabe 1999). In Lake Apopka sediments

there was a general increase in DOC and NH4-N with time, strongly indicating that there is P

limitation during summer.









CHAPTER 5
MICROBIAL BIOMASS AND ACTIVITY INT SEDIMENTS OF SUBTROPICAL LAKES

Introduction

Phytoplankton and/or heterotrophic bacteria are the maj or drivers of carbon (C) and

nutrient cycling in the water column of lakes, while the heterotrophic bacteria dominate in

sediments. Allochthonous and autochthonous particulate organic matter in the water column is

deposited in the sediment. Water column depth affects the quality of organic material reaching

the sediment. In deep lakes, detrital organic matter undergoes intense decomposition in the water

column, due to the prolonged period of settling. Consequently low amounts of labile organic C

reach the sediment (Suess 1980; Meyers 1997). In shallow lakes, the supply of labile C and

nutrients can be higher than in deep lakes, and the latter often can have more refractory organic

matter (Suess 1980; Meyers 1997).

As bacteria are the dominant group in sediments, organic compounds and associated

nutrients supplied to the sediment surface are mineralized through heterotrophic decomposition

(Gachter and Meyer 1993; Capone and Kiene 1988). Complete oxidation of a broad range of

organic compounds occurs through the sequential activity of a variety of anaerobic bacteria

(Capone and Kiene 1988). In high depositional environments, such as eutrophic, or deep

thermally stratified lakes, organic content in sediments is often high, oxygen (Oz) COnSumption

occurs rapidly, and 02 is depleted several millimeters below the sediment water interface

(Jarrgensen 1983; Jorrgensen and Revsbrech 1983). In these systems, facultative and obligate

anaerobic communities dominate. In methanogenic habitats, i.e., in the absence of inorganic

electron acceptors, different groups of microorganisms participate in decomposition of organic

matter as no single anaerobic microorganism can completely degrade organic polymers (Zinder

1993, Megonigal et al. 2004). Fermenting bacteria hydrolyze organic polymers through enzyme










Rau, G. H., J.-L. Teyssie, F. Rassoulzadegan, and S. W. Fowler. 1990. 13 /12C and 15N/14N
variations among size-fractionated marine particles: implications for their origin and
trophic relationships. Mar. Ecol. Prog. Ser. 59: 33-38.

Reddy, K. R., O. A. Diaz, L. J. Scinto, and M. Agami. 1995. Phosphorus dynamics in selected
wetlands and streams. Ecol. Engin. 5: 183-207.

Reddy, K. R., and D. A Graetz. 1991. Internal nutrient budget for Lake Apopka. Special Publ.
SJ91-SP6. St Johns River Water Mgt District. Palatka, Florida.

Reddy, K. R., M. Brenner, M. M. Fisher, and D. B. Ivanoff. 1991. Lake Okeechobee phosphorus
dynamics study. Biogeochemical processes in the sediment. Vol. III. Final report to the
South FL Water Mgt District. West Palm Beach, FL. Contract No. 531-m88-0445-A4.

Redfield, A. C., B. H. Ketchum, and F. A. Richards. 1963. The influence of organisms on the
composition of sweater, p. 26-77. In M. N. Hill [ed.] The Sea, Vol. 2. Wiley Interscience.

Reichardt, W. 1971. Catalytic mobilization of phosphate in lake water as by Cyanophyta.
Hydrobiol. 38: 377-394.

Reitzel, K., J. Ahlgren, A. Gogoll, and E. Rydin. 2006a. Effects of aluminum treatment on
phosphorus, carbon and nitrogen distribution in lake sediment: A 31P NMR study. Water.
Res. 40: 647-654.

Reitzel, K., J. Ahlgren, A. Gogoll, H. S. Jensen, and E. Rydin. 2006b. Characterization of
phosphorus in sequential extracts from lake sediments using P-31 nuclear magnetic
resonance spectroscopy. Can. J. Fish. Aquat. Sci. 63: 1686-1699.

Reitzel, K., J. Ahlgren, H. Debrabandere, M. Waldebak, A. Gogoll, and L. Tranvik. 2007.
Degradation rates of organic phosphorus in lake sediments. Biogeochem. 82: 15-28.

Rejmankova, E., and D. Sirova. 2007. Wetland macrophyte decomposition under different
nutrient conditions: Relationships between decomposition rate, enzyme activities and
microbial biomass. Soil Biol. Biochem. 39: 526-538.

Riedinger-Whitmore, M. A., T. J. Whitmore, J. M. Smoak, M. Brenner, A. Moore, J. Curtis, and
C. L. Schelske. 2005. Cyanobacteria proliferation in recent response to eutrophication in
many Florida lakes: a paleolimnological assessment. J. Lake and Reserv. Manag. 21: 423-
435.

Roden, E. E., and R. G. Wetzel. 2003. Competition between Fe(III)-reducing and methanogenic
bacteria for acetate in iron rich freshwater sediments. Microb. Ecol. 45: 252-258.

Rodusky, A. J., A. D. Steinman, T. L. East, B. Sharfstein, and R. H. Meeker. 2001. Periphyton
nutrient limitation and other potential growth-controlling factors in Lake Okeechobee,
U.S.A. Hydrobiol. 448: 27-39.









forms found in the sediment, strongly indicated that biotic processes play an important role in P

solubility in these mud sediments. Lake Okeechobee sediments were dominated by inorganic P

(HCl-Pi) (chemical fractionation) and orthophosphate (31P NR\), indicating abiotic processes

control P solubility in these sediments. Dominant P forms in Lake Apopka were MBP and HCl-

Pi (chemical fractionation), and orthophosphate, phosphate monoester and DNA-P (31P NMR).

Almost 50% of the total P was in microbial biomass in surface sediments. The presence of poly-

P and pyro-P in these sediments also indicated high activity of microorganisms involved in

biological P cycling. Low concentrations of labile-Pi, and its low percent contribution to total P

in surface sediments in hypereutrophic Lake Apopka, probably reflects a high P demand by the

microbial community. This study also showed that the results of 31P NMR spectroscopy were in

agreement with the results of chemical P fractionation, and that the determination of the relative

abundance of different P forms in sediments is important to understand sediment P processes.

























































24(


0
002800 3200 3600 4000


Microbial Biomass Carbon (mg kg l)
Figure 2-2. Linear regressions between 1) microbial biomass carbon and microbial biomass
nitrogen, and 2) microbial biomass carbon and microbial biomass phosphorus of
sediments from A) all lakes and B) data from Lake Annie and Lake Okeechobee only.


_____________ ______


_


400 800 1200 1600 2000


1500

4000

3500


1200


1000


800


600


400


200
o
B
0
00 "a

70 L





30





5 0


3000 E


,'
r
`+.


/ \ M


2500

2000

1500

1000

500


MIBN = 8.28+ 0.17 MBC
1=0.996, p < 0.00001
BP = -4.26+ 0.04 MBC
'=0.973,p <0.00001


280


1


4000 8000 12000 16000


20000 24000


B /


-+ ./





//


300

250

200

150

100


tr. MBN = -1.44 + 0.18 MBC
4=0.95, p < 0.00001

SMBP =-0.78 +0.03 MBC
-0.89, p < 0.00001






































































Axis 1

Figure 4-6. Results of the Principal Component Analysis, A) loadings of different phosphorus
compounds measured by 31P NMR and P fractionation, enzymes and microbial
activities (n = 25), and B) the plot of the scores of the sites and sediment depth
(numbers cm) from Lake Annie (circles), Lake Okeechobee: M9 (squares), M17
(diamonds), KR (crosses), and Lake Apopka (triangles).


*** *


Residual P
Lipids-P
+ Water Ext-DOC:P
MBP *
- PDEase *Polyphosphate

CO2 Pymphosphate

*DNA-P


~Water Ext-P
Water Ext-DOC*
S PMEase Labile Inorganic P

*Labile Organic P Inog9anic P
FAP en
IIAP *P-monoester
Orthophosphate


0.6

0.4

0.2


-0.2

-0.4

-0.6

-0.8


0


-1.0 L
-1.(


-0.8 -06 -0.4


-0.2 0.0 0.2 0.4 0.6 0.8 1.0


Axis 1 (38.6%)


S5 10 20
15
10
-o

30 55
~45 60
a CO6,0






80
j600
iO
10 1520 45!
- O O 030 O


1.5


1.0


0.5


0.0


-0.5


-1.0


-1.5


-2.0 L
-2.5


-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5










To my best and dearest friend Jeremy Bright, without whom it would have been impossible

to do many of the measurements. There are no words to describe how I appreciate his friendship

and all the indispensable and high quality help he provided. My beloved friends Lynette M.

Brown (for sharing the hurdles of a Ph.D. program) and Cecilia C. Kennedy, thank you for

sharing the good and bad moments, for your support, and for making our office the best and

happiest office in the department. My dear friend Adrienne Frisbee I truly thank you for your

friendship and support. I also want to acknowledge friends that left the department but have not

been forgotten, Dr. Hari Pant, Dr. John Leader, and Sue Simon. My sincere thanks to Dr.

Natasha Maynard-Pemba (Counseling Center/UF) for taking me in when I needed it the most, for

her time and guidance, and helping me get back on my feet.

Thanks to my husband, Dr. Paulo Henrique Rodrigues (Department of Oral Biology/UF),

who shared all the hurdles and accomplishments during this time, for his love and support. Also,

for sharing his knowledge of molecular biology and helping me with some molecular procedures

and questions.

Last but not least to all my family and friends that endured all this time without my

presence in my beloved Belo Horizonte (Brazil). My special thanks to my grandmother, Higia

Barros Costa, for her constant support, and for being proud of my accomplishments. My deepest

thanks to my sister, Beatriz Claret T~rres, for her friendship and support when I needed it the

most. To my parents, S8nia Barros Costa and Ant8nio Maria Claret T~rres, from whom I derive

my strength and determination, the people that I am most indebted in life. My thanks for guiding

me through life with their ethics, love, teachings and encouragement, for always supporting my

choices, and cheering my accomplishments.









CHAPTER 4
ENZYME ACTIVITIES INT SEDIMENTS OF SUBTROPICAL LAKES

Introduction

Sediment phosphorus (P) is present in both inorganic and organic forms. Organic P and

cellular constituents of the biota represent 90% of total phosphorus (TP) in freshwater

ecosystems (Wetzel 1999). These organic P compounds present in sediments must be hydrolyzed

before their uptake by microorganisms (Chrost 1991; Sinsabaugh et al. 1991). Organic P is

hydrolyzed by enzymes produced by microbial communities (Gachter et al. 1988; Davelaar

1993; Gachter and Meyer 1993), and the product of this enzymatic hydrolysis is orthophosphate

which is readily used by microorganisms (Barik et al. 2001). Consequently the breakdown of

organic P compounds through enzyme activity and release of labile inorganic P is an important

component of P processing in sediments. Enzyme production can be induced by the presence of

organic P and low levels of bioavailable inorganic P (Kuenzler 1965; Aaronson and Patni 1976).

On the other hand, high levels of inorganic P inhibit the synthesis of enzymes (Torriani 1960;

Lien and Knutsen 1973; Elser and Kimmel 1986; Jasson et. al. 1988; Barik et al. 2001).

Three main groups of hydrolytic enzymes are responsible for phosphate release: non

specific and/or partially specific phosphoesterases (mono and diesterase), nucleotidases (mainly

5'-nucleotidase), and nucleases (exo and endonucleases) (Chrost and Siuda 2002).

Phosphomonoesterases (PMEase) are nonspecific enzymes that hydrolyze phosphate monoester,

and are reported to be produced by several microorganisms (e.g., bacteria, algae, fungi, and

protozoan) that are found in the water column and sediment of lakes. Nonspecific PMEases are

divided into two groups, depending on the pH at which they exhibit maximum activity, alkaline

(pH 7.6-10) and acid (pH 2.6-6.8) (Siuda 1984). Both can be found inside or outside the cell, and

the same cell can produce both alkaline and acid PMEase (Siuda 1984). Although both PMEase

















0
4
8
12
16
20
24
~28

S32
C136
40
44
48
52
56
6o.

2000 1980 1960 1940 1920 1900 1880 1860

210 ge(r


. .


* *





Activity (dpm g )
0 5 10 15 20 25 30


0


10


20


S30


CI40


50


60


70


----Cs -137
- Ra -226
-0 Pb 210


Accumulation Rates (mg cml yrl)
20 40 60 80 100


Figure 6-1. Results of 210Pb dating of Lake Annie sediments: A) Radioisotope activities (total
210Pb, 226Ral, and 137CS) Versus depth, B) sediment depth vs. age/date, and C) sediment
and organic matter accumulation rates vs. age/year.


SDry Mass
SOrganic Matter






































Hil


4*








--C -3
-A- a-6
**4P 1

`tB


Activity (dpm g )
0 5 10 15 20 25 30


s-137
-m-Ra-226
m O DPb -210






A


S40


50


C


Activity (dpm g )
2 4 68


10 12 14


Figure 6-2. Radioisotope activities (total 210Pb, 226Ral, and 137CS) Versus depth, in A) Lake
Okeechobee, site M9 and, B) Lake Apopka.





















168









Pore water DOC and DRP concentrations in Lake Annie and Apopka were measured and

described in another study (Chapter 5, Table 5-2), and the data were used to verify if there was a

relation between enzyme production and DOC and DRP pore water concentration. In Lake

Annie, there was no relationship between either acid PMEase or PDEase activities and pore

water DOC and DRP. For Lake Apopka, however, there was an inverse relationship between

DRP pore water concentration and both PMEase and PDEase (Figure 4-4A). Enzyme activities

in Lake Apopka showed a strong linear relationship with pore water DOC (Figure 4-4B).

Anaerobic respiration (CO2 prOduction rates) data was described elsewhere (Chapter 5),

and the values were used to address relations between anaerobic respiration and enzyme activity.

Microbial activity had a positive relationship with both PMEase and PDEase activities. In Lake

Annie, Lake Apopka, and Lake Okeechobee site-M9 sediments, microbial activity had a

significant linear relationship with both enzyme activities (Figure 4-5A, B, E). The same

relationship was observed between microbial activity and PDEase activity in site KR sediments,

although no relationship was observed for PMEase activity (Figure 4-5D). In peat sediments

(site-M17) no relationship between enzyme and microbial activities was observed (Figure 4-5C).

Two Principal Component Analyses (PCA) were conducted. One analysis used data from

31P NMR, chemical fractionation (Chapter 3), and microbial (Chapter 5) and enzyme activities (n

= 25) (Figure 4-6). The other used data from chemical P fractionation (Chapter 3), and microbial

(Chapter 5) and enzyme activities (n = 107) (Figure 4-7). The first PCA had 3 8.6% of the data

variability explained by Axis 1. Axis 2 explained 30.3% of the data variability (Figure 4-6A).

Lipid-P, DNA-P, anaerobic respiration, microbial biomass P (MBP), fulvic acid P (FAP),

PMEase and PDEase activity were the variables selected by Axis 1, while orthophosphate,

phosphate monoester and residual P (Res. P) were selected by Axis 2. Phosphomonoesterase










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Wang, D., Q. Huang, C. Wang, M. Ma, and Z. Wang. 2007. The effects of different electron
donors on anaerobic nitrogen transformations and denitrification processes in Lake Taihu
sediments. Hydrobiol. 581: 71-77.

Waters, M. N., C. L. Schelske, W. F. Kenney, and A. D. Chapman. 2005. The use of sedimentary
pigments to infer historic algal communities in Lake Apopka, Florida. J. Paleolimnol. 33:
53-71.

Wellington, E. M. H, A. Berry, and M. Krsek 2003. resolving functional diversity in relation to
microbial community structure in soil: exploiting genomics and stable isotope probing.
Curr. Opin. Microbiol. 6: 295-301.

Wetzel, R. G. 1991. Extracellular enzymatic interactions; storage, redistribution, and
interespecific communication, p. 6-28. Dr R. J. Chrost [ed.], Microbial enzymes in aquatic
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Wetzel, R. G. 1999. Organic phosphorus mineralization in soils and sediments, p. 225-241. Dr K.
R. Reddy, G. A. O'Connor and C. L. Schelske [eds.], Phosphorus biogeochemistry of
subtropical ecosystems. Lewis.

Wetzel, R. G. 2001. Limnology: Lake and River Ecosystems, 3rd. ed. Saunders.

Whalen, P. J., L. A. Toth, J. W. Koebel, and P. K. Strayer. 2002. Kissimmee River restoration: a
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Whiteley, A. S., B. Thomson, T. Lueders, and M. Manefield. 2007. RNA stable-isotope probing.
Nature Prot. 2: 838-844.









yield OM depleted in 613C and enriched in 61 N (Gearing et al. 1991; Bumnett and Schaffer 1980;

Savage et al. 2004). Stable isotope 6 5N has also been used to study the nitrogen (N)

biogeochemical cycle. Measurement of 8 "N in suspended and sedimented OM was used to

address the source of N, as well as N limitation of, and utilization by the phytoplankton

community in Lake Lugano (Terranes and Bernasconi 2000).

Sediment Phosphorus

Phosphorus (P) is often the limiting nutrient for primary productivity in freshwater

ecosystems. Sources of P to lakes can be external (allochthonous) or internal autochthonouss).

Allochthonous P input originates in the drainage basin, while autochthonous P originates from

primary and secondary productivity within lakes. A maj or portion of P from these sources added

to the water column accumulates in sediments. Sediment P is present in both inorganic and

organic forms. Organic P and cellular constituents of the biota represent 90% of total P (TP) in

freshwater ecosystems (Wetzel 1999), and in sediments 30-80% of TP is typically in organic

form (Williams and Mayer 1972; Bostroim et al. 1982).

Although organic P is an important component of sediment P, it has been relatively under-

studied as compared with the fate of inorganic P (Turner et al. 2005). The reason for this is that

there is no direct way to measure organic P. It is usually estimated by difference (before and after

ignition at high temperature) (Saunders and Williams 1955), or by sequential extraction or

chemical fractionation (Condron et al. 2005; McKelvie 2005). These chemical fractionations are

based on different solubilities of P forms in alkaline and acid extractions with different pH.

Turner et al. (2006) compared two methodologies, chemical fractionation and phosphorus-31

nuclear magnetic resonance (31P NMR) spectroscopy, to measure organic P, and showed that for

wetland soils, alkaline extraction with molybdate colorimetry overestimated organic P by 30-

54%. They concluded that alkaline extraction with 31P NMR spectroscopy is a more accurate









results of electron donor addition also indicated that H2/CO2 is the maj or substrate for methane

production in Lake Okeechobee sediments.

Hypereutrophic Lake Apopka sediments had higher ratios for extractable C:P and N:P, and

the high C concentration in sediments is supporting high microbial biomass and activity. Lake

Apopka sediments are highly influenced by the deposition of the primary production in the water

column. The results from this study suggest that although the microbial community is C/energy

limited, C, coupled with N and P availability has a strong influence in microbial communities in

these lakes sediments. Therefore, studies of sediment heterotrophic microbial communities

should take into account C as well as N and P availability.









availability in the sediments, rather it resulted from a combination of factors such as high Al and

Fe concentrations, high P demand inside microorganism cells, and/or presence of more stable

phosphate monoester in the sediment. High labile-P concentration in these sediments resulted in

low extractable C:P and N:P ratios and a C limitation of the microbial heterotrophic community.

Carbon limitation probably causes inefficient use of energy by the heterotrophic microbial

community, where there is high respiration per microbial biomass. Heterotrophic microbial

communities in these sediments probably have high respiratory demands, with greater C flow to

CO2 rather than to biomass.

Lake Okeechobee

Eutrophic Lake Okeechobee mud sediment had its TP pool dominated by inorganic P

(HCl-Pi) (Figure 9-3). Sediments were characterized by having high labile-Pi concentration and

low enzyme activity. High P availability in these sediments is repressing the production of P

related enzyme activities. P solubility in these sediments is controlled by abiotic processes

(Figure 9-3). High labile-Pi concentration in these sediments resulted in low extractable C:P and

N:P ratios, and a C and N limitation of the microbial heterotrophic community. Carbon and N

limitation is causing low microbial activities in these sediments. Methanogenesis was inhibited

due to low electron donor availability with concomitant presence of iron and sulfate reducers.

Moreover, it was established for these sediments that H2/CO2 is the maj or substrate for methane

production.

Lake Apopka

Hypereutrophic Lake Apopka, with high autochthonous OM input and highly organic

sediments, had the sediment TP pool dominated by diester P (i.e., MBP, DNA-P, Lipid-P)

followed by inorganic P (HCl-Pi), orthophosphate, FAP/HAP and phosphate monoester. An

intrinsic characteristic of these sediments was the presence of polyphosphate in some of the









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LINKAGE BETWEEN BIOGEOCHEMICAL PROPERTIES AND MICROBIAL ACTIVITIES
IN LAKE SEDIMENTS: BIOTIC CONTROL OF ORGANIC PHOSPHORUS DYNAMICS

By

Isabela Claret Torres

December 2007

Chair: K. Ramesh Reddy
Co-chair: Andrew Ogram
Major: Soil and Water Science

In lakes, deposition of allochthonous and autochthonous particulate matter to sediments

can alter the physico-chemical properties and associated biogeochemical processes. Coupling

and feedback between sediment biogeochemistry and water column primary productivity often

depends on biogeochemical processes within sediments and associated microbial communities.

The current investigation was conducted to link biogeochemical properties of benthic sediments

and microbial communities and their activities in sub-tropical lakes of different trophic state

(Lake Annie oligo-mesotrophic, Lake Okeechobee eutrophic, and Lake Apopka -

hypereutrophic). The central hypothesis of this study was that lakes with contrasting trophic

states have sediments with different biogeochemical properties that have a selection pressure

(i.e., C, N and P availability or limitation) on the microbial community that is reflected in their

activities. Sediments sampled from sixteen different sites revealed that trophic state was not

related to nutrient content of sediments. The relative abundance of phosphorus (P) forms in

sediments was more important than total P concentration in characterizing the processes

occurring in sediments. Laboratory batch incubation studies were conducted to determine the

relationships between maj or sediment P forms, enzyme activity, heterotrophic microbial activity,









RNA-SIP Experiment

Incubation and RNA extraction

Triplicate samples from Lake Apopka (core # 93, 94, 95), Lake Annie (core # 120, 121,

122), and Lake Okeechobee sites M9 (core # 147, 148, 149) and M17 (core # 168, 169, 170)

were incubated for 1 week, as described previously. 13C-labeled acetate was added from

anaerobic sterile stock solutions to a Einal concentration of 1 mM. After 24 hours, total RNA was

extracted as described previously.

Escherichia coli RNA

Escherichia coli (E. coli) RNA was extracted to be used as a RNA control (unlabeled 12C

RNA) to evaluate possible mixing of [12C] RNA with 13C labeled bands (Chauhan and Ogram

2006a). E. coli (strain TOP10F') was grown in 10 ml Luria-Bertani (LB) medium at 37oC for

24h. The culture was then transferred to 260 ml LB medium and incubated for 2 hours in a

shaker at 100 rpm at 37 oC. Total RNA was extracted from E. coli culture with TRI Reagent

(Ambion, Austin, TX) according to the manufacturer' s instructions. E. coli RNA was

resuspended in 1.0 ml of nuclease-free water, and the concentration was determined by

spectrophotometry (GeneQuant, Biochem Ltd., Cambridge, UK).

Isopycnic centrifugation

Density gradient centrifugation was performed as described by Manefield et al. (2002a, b)

and Lueders et al. (2004a). A total of four different centrifugations were performed and some

modifications were made in each one to improve the density gradient.

First centrifugation. The gradient medium consisted of 2.56 ml of a 2.0 g mlF cesium

trifluroacetate (CsTFA) (Amersham Pharmacia Biotech, Buckinghamshire, UK), 410 Cll of

nuclease-free water and 120 Cll of formamide. Ten microliters of E. coli RNA (100 ng) and 100

Cll of sample RNA were then added to the gradient medium. Gradient solutions were loaded in









Extractable nutrient ratios also seemed to have stronger influence on sediment microbial

communities than total concentrations. Extractable C:P ratio was low for Lake Annie, reflecting

high concentrations of extractable labile nutrients relative to C, indicating a C limitation in these

sediments. High labile inorganic P availability resulted in low extractable C:P and N:P ratios,

and C and N limitation in most Lake Okeechobee sediments, especially in the mud zone, along

with low microbial biomass and activity. Moreover, low C availability appears to be inhibiting

the methanogenic community in Lake Okeechobee sediments. Limitation of the methanogenic

community in these sediments is supported by the positive effect of the addition of electron

donors on methane (CH4) prOduction, which indicated that H2/CO2 is the maj or substrate for

methane production in Lake Okeechobee sediments.

Hypereutrophic Lake Apopka sediments had higher ratios for extractable C:P and N:P, and

the high C concentration in sediments is supporting high microbial biomass and activity. Lake

Apopka sediments are highly influenced by the deposition of primary producers from the water

column. The results from this study suggest that although the microbial community is C/energy

limited, C, coupled with N and P availability has a strong influence in microbial communities in

these lake sediments.

Sediment phosphorus forms (Objective 2)

Organic P compounds were characterized in sediment profiles using two different

techniques, 31P NMR spectroscopy and a chemical P fractionation scheme. In all study lakes TP

concentration decreased with sediment depth, and although an oligo-mesotrophic lake, Lake

Annie contained more TP in sediments than both eutrophic Lake Okeechobee and

hypereutrophic Lake Apopka. This study showed that the concentrations of various P compounds

changed with sediment depth, indicating that different processes were controlling P reactivity

and mobility in these lakes.









Cyanobacteria are capable of active CO2 transport (Miller et al. 1991) or utilizing HCO3- (Epsie

et al. 1991), and both can result in enriched 813C in phytoplankton biomass. Jones et al. (2001)

reported enrichment of phytoplankton 613C TOSulted from 613C DIC enrichment in Loch Ness

(Scotland). Similar results were found in urban Lake Jyvaskyla (Finland), where heavy 613C DIC

resulted in enriched 813C Of phytoplankton and zooplankton biomass (Syvaranta et al. 2006).

Heavy 613C DIC in the water column, with high demand for inorganic C due to high primary

productivity, will produce autochthonous OM with enriched 813C, which is then deposited in the

sediments .

Recent 15N enrichment in Lake Apopka sediments was surprising as it is generally

expected that eutrophic and hypereutrophic lakes will have depleted 6 "N, as a consequence of

high rates of N2 fixation (Fogel and Cifuentes 1993). Gu et al. (1996) also reported enriched 6 "N

in Lake Apopka sediments. In this lake, N for phytoplankton assimilation is primarily supplied

by transformation of organic N to NH4+ and then to NO3- by nitrification (D'Angelo and Reddy

1993). Although the phytoplankton community is dominated (> 90%) by cyanobacteria (Carrick

et al. 1993), N2 fixation is relatively unimportant in N dynamics (Schelske et al. 1992). High

NO3- availability can lead to autochthonous OM with depleted 6 5N (Meyers 1997). However, if

N incorporation uses a significant amount of the lake' s NO3~ pOol, the residual NO3- will become

enriched, ultimately leading to an increase in the 6 5N of newly produced OM (Terranes and

Bernasconi 2000; Syvaranta et al. 2006). Jones et al. (2004) reported heavier sediment 8 "N

when inorganic N was low in the water column, reflecting reduced isotopic fractionation under N

limitation. Nitrogen is the primary limiting nutrient in Lake Apopka although co-limitation with

P can occur (Aldridge et al. 1993). Periods of N limitation in the water column can lead to









highest respiration per microbial biomass with most of the electron donor additions propionatee,

Lake-SS, butyrate, acetate, and, formate, indicating that these sediments respired most of the C

added (Figure 7-7A, B). This suggests that the catabolic diversity and activity in these sediments

is higher than other sediments. Increased biogeochemical diversity can be present in

environments with high organic matter content, with diversity in organic compounds as well as

increased by-products diversity (Odum 1969). As an example, Castro et al. (2005) studied the

distribution of sulfate (SO4-2)-reducing prokaryotic assemblages in soils of the nutrient impacted

regions of the Florida Everglades. The authors reported that complete oxidizing species, which

are able to use a broader array of electron donors were dominant in eutrophic and transitional

sites while incomplete oxidizers, which are more efficient at taking up low concentrations of a

few substrates, were present in oligotrophic regions. The authors concluded that eutrophic

regions with greater amount of carbon may select for generalists capable of taking advantage of a

greater diversity of carbon substrates. Lake Apopka exhibits high primary production and high

labile C sedimentation (Gale et al. 1992, Gale and Reddy 1994), supporting higher catabolic

diversity .

Others studies, however, have reported that under P limitation heterotrophic bacteria tend

to respire added C. In controlled experiments with bacterioplankton in subarctic Lake Diktar

Erik, Sweden, Jasson et al. (2006) showed that addition of C was used for growth under C-

limited conditions, but used for respiration under Pi limitation. They concluded that

bacterioplankton communities tend to respire large portions of added C under P limitation, and

high respiration rates of "excess C" was partly used to support growth and not only for

maintenance. Lake Apopka exhibited the highest extractable C:P and N:P ratios (Table 7-1). In

another study, I found that microorganisms in Lake Apopka surface sediments are P limited,









PDEase. Lake Apopka had high concentrations of MBP and phosphate diester (lipids and DNA),

as well as PDEase activity.

The mechanisms controlling PMEase activity, however, seemed to vary among studied

lakes. In Lake Annie, high PMEase activity was unrelated to dissolved reactive P (DRP) and

dissolved organic C (DOC) concentration, and probably was controlled by factors such as high

Al and Fe concentrations, high P demand inside microorganism cells, and/or presence of more

stable phosphate monoester (i.e., inositol phosphate) in the sediment. Lake Apopka's PMEase

production seemed to be controlled by both DOC and DRP availability. There was an inverse

relation between pore water DRP and PMEase activity, and a positive relation between pore

water DOC and PMEase activity. In Lake Apopka sediments production of PMEase by the

microbial community was related to organic P hydrolysis, and uptake of associated organic C

moieties.

Microbial biomass and activity in sediments (Objective 4)

The results from this study showed that hypereutrophic Lake Apopka had the highest

microbial biomass and activity (both CO2 and CH4) followed by oligo-mesotrophic Lake Annie.

Microbial activity decreased with sediment depth and was related to decrease in easily

degradable OM. Carbon, N and P concentrations, and especially nutrient ratios, had a strong

influence on microbial communities in these sediments.

The sediment microbial community in each lake, or site, was limited by different variables.

The Lake Apopka' s surface sediment heterotrophic community appears to be P-limited. High

primary production and high labile C sedimentation resulted in high demand for labile P in

surface sediment, as reflected in high C:P ratio. Peat sediments of Lake Okeechobee were limited

by both C and P. Nitrogen and C limitation was observed in mud and sand sediments of Lake

Okeechobee. High availability of P in Lake Okeechobee mud and sand surface sediments









The central hypothesis of this study was that lakes with contrasting trophic states will have

sediments with different biogeochemical properties that will have a selection pressure (i.e., C, N

and P availability or limitation) on the microbial community; that will be reflected in their

activities. The specific obj ectives of this study were to: (i) determine the biogeochemical

properties of benthic sediments in three subtropical Florida lakes with different trophic states

(Lake Annie oligo-mesotrophic, Lake Okeechobee eutrophic, and Lake Apopka -

hypereutrophic), and (ii) examine relationships among sediment biogeochemical properties

(nutrient concentrations and availability) and microbial biomass and activity.

Materials and Methods

Study Sites

Three Florida lakes (USA) were selected for this study based on water quality variables

and trophic status (Table 2-1, Figure 2-1). Lake Annie (Figure 2-1A), a small (0.37 km2) Oligo-

mesotrophic lake, is located in south-central Florida (Highlands County) at the northern end of

the Archbold Biological Station. Lake Annie is characterized by pristine water quality with little

surface water input (most is ground water), and low anthropogenic impact due to the absence of

development around the lake (Layne 1979). This lake has no natural surface streams but two

shallow man made ditches allow surface water to flow into the lake and contribute to water and

nutrient inputs during high rainfall periods (Battoe 1985). Benthic sediments vary from organic

to sand in the littoral zone (Layne 1979). Lake Okeechobee (Figure 2-1B) is a large (1800 km2)

shallow lake located in south Florida. It is considered to be a eutrophic lake that has experienced

cultural eutrophication over the last 50 years (Engstrom et al. 2006). Benthic sediments are

characterized as: mud (representing 44% of the total lake surface area), sand and rock (28%),

littoral (19%), dominated by macrophyte growth, and peat (9%) that refers to partially

decomposed plant tissues (Fisher et al. 2001). Lake Apopka (Figure 2-1C) is also a shallow lake









Lake Okeechobee

Mud zone sediments (site M9) in Lake Okeechobee showed variable TC:TN ratios over the

length of the core. From 70 cm depth TC:TN ratios rose until 50 cm, suggesting increased input

of allochthonous material, or N loss to mineralization (Figure 6-4B). At 50 and 30 cm the ratio

was similar, and from 30 cm to the surface there was a general decrease in TC:TN. Organic

matter content was highly variable over the length of the core, however, there was an increase in

OM content in surface sediments (Figure 6-4F). TN:TP showed a general decrease from the

bottom of the core to the surface, reflecting more rapid increase of TP than TN concentration

(Figure 6-4C). The same trend was reported by Engstrom et al. (2006) for the mud zone and was

attributed to increase in TP content of these uppermost sediments, as a result of the

eutrophication process. The 613C Sediment profile showed a similar pattern of TC:TN ratios

(Figure 6-4B, D). Delta 13C ValUeS Varied from -26.0%o to -29.9%o with surface sediments only

slightly depleted (0.15%o) relative to bottom deposits (Figure 6-4D). Delta 1N varied from 2.6%o

to 3.9%o and showed ~ 1.3%o enrichment in surface deposits relative to bottom deposits (Figure

6-4E). A similar pattern, i.e., depletion of 613C and enrichment of 8 "N, was reported by

Rosenmeier et al. (2004) in a study of recent eutrophication of Lake Peten Itza, Guatemala, in

which changes were related to sewage input (depleted in 613C and enriched in 61 N) and

increased presence of cyanobacteria. Engstrom et al. (2006) also found 6 "N enrichment (1%o) in

the mud zone, but did not discuss the mechanisms responsible. Stratigraphic changes in 613C and

6 "N in the mud zone are probably controlled by autochthonous OM, availability and demand for

C and N and varying intensities of mineralization. Lake Okeechobee mud zone sediments are

probably C and N limited and N demand is high in these sediments (Chapter 5). In the mud zone

of Lake Okeechobee, light is the most limiting factor of the phytoplankton community during

most of the year (Aldridge et al. 1995). During summer months, light limitation is relaxed and N









CHAPTER 6
NUTRIENT ACCUMULATION AND STABLE ISOTOPE SIGNATURES INT SEDIMENTS
OF SUBTROPICAL LAKES

Introduction

Organic matter (OM) that enters a lake from the watershed (allochthonous) or is produced

within the lake autochthonouss) itself will be deposited on the lake bottom and incorporated into

the sediments. Lakes function as natural traps for OM and associated nutrients. Lake sediments

contain an archive of past environmental conditions in and around the water body (Smol 1992)

and can be used to document anthropogenic impacts through time (Smeltzer and Swain 1985).

Sediment OM provides information about past impacts and biogeochemical processes within

lakes, and has been studied extensively using paleolimnological methods (Meyers 1997). The

timing of past events in a basin is based on reliable dating of sediment cores. Sediment dating

provides an age/depth relation from which bulk sediment accumulation rates can be calculated

(Smeltzer and Swain 1985). The lead-210 (210Pb) technique is used routinely to provide

age/depth relations for the last 100-150 years (Appleby et al. 1986), and has been used widely in

studies of Florida lake sediment cores (e.g., Binford and Brenner 1986; Brezonik and Engstrom

1998; Whitmore et al. 1996; Brenner et al. 2006; Schottler and Engstrom 2006). Bulk sediment

accumulation rates in combination with analyses of sediment composition, can be used to

calculate accumulation rates of sediment constituents such as OM and nutrients. Such measures

provide insights into past changes in productivity and human impacts on the aquatic ecosystem.

Nutrient and OM accumulation rates in sediment have been studied in conjunction with

stable isotope analyses (613C and 6 "N) to infer past environmental impacts in marine (e.g.,

Gearing et al. 1991; Savage et al. 2004), lacustrine (e.g., Schelske and Hodell 1991; Gu et al.

1996; Bernasconi et al. 1997; Hodell and Schelske 1998; Ostrom et al. 1998; Brenner et al.

1999), and riverine ecosystems (e.g., McCallister et al. 2004; Anderson and Cabana 2004; Brunet































0 2 4 6 8 10 12 14 16 18 20


Butyrate F
O Lae
4 Basal















0 2 4 6 8 10 12 14 16 18 20


2000

S1800

S1600

DO 1400
1200



. 800

S600

.y400



S0


S1000
so

S800


S600


S400



El200


2200
2000

1800
1600
1400
1200
1000
800

600
400
200
0


1000



800


600



400


200


2000

1800

1600

1400

1200

1000


Butyrate C
'O Lake
. & Basal









---


if- *-


800 I


0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20
Time (days)


0 2 46


8 10 12 14 16 18 20


Figure 7-2. Microbial activity response to the different carbon source addition in the mud sediments (site M9) of Lake Okeechobee: A,
B and C) Anaerobic respiration (mg CO2-C kg- ) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg- ) vs.
time (days). *Different scales.









CHAPTER 2
BIOGEOCHEMICAL PROPERTIES AND MICROBIAL ACTIVITY OF BENTHIC
SEDIMENTS OF SUBTROPICAL LAKES

Introduction

Particulate matter that enters a lake (allochthonous) or is produced within a lake

autochthonouss) is deposited and becomes an integral part of sediments. Consequently, lakes

function as natural traps for particulate matter and associated nutrients. Accumulation of

particulate matter can alter the physico-chemical properties of sediments and associated

biogeochemical processes in the sediment and water column (Rybak 1969). Accumulation and

retention of particulate matter and nutrients in sediments depends on lake morphometry, water

renewal, nutrient loading, edaphic characteristics of the drainage basin, among others (Bostroim

et al. 1988). Lake sediment characteristics can provide evidence of anthropogenic impacts

through time (Smeltzer and Swain 1985) as lake histories are archived in sediments (Smol 1992).

Organically bound nutrients in particulate matter supplied to the sediment are mineralized by

heterotrophic decomposition, resulting in release of nutrients into water column and potential for

stimulation of biological productivity (Capone and Kiene 1988; Gachter and Meyer 1993;

Brooks and Edgington 1994). Consequently benthic sediments may play a critical role in nutrient

cycling by acting as both sources and sinks of nutrients.

Coupling and feedback between sediment biogeochemistry and water column primary

productivity often depends on biogeochemical processes within sediments and associated

microbial communities. Oxygen (Oz) availability in lake sediments typically is restricted to the

uppermost few millimeters below the sediment-water interface due to limited 02 diffusion and

rapid 02 COnSumption by the heterotrophic community (Charlton 1980; Bostroim et al. 1982).

Facultative and strict anaerobic communities typically dominate the sediments. Anoxic

sediments can be a good habitat for bacterial growth as they usually have high concentrations of









method to quantify organic P. In recent years there have been many studies using this

methodology to distinguish different organic P forms in lake sediments (Hupfer et al. 1995,

2004; Carman et al. 2002; Ahlgren et al. 2005; Ahlgren et al. 2006a, b; Reitzel et. al 2006a, b,

2007). Phosphorus-31 NMR spectroscopy can identify different P compounds, based on their

binding properties, as orthophosphate, pyrophosphate (pyro-P), polyphosphate (poly-P),

phosphate monoester, phosphate diester (e.g., DNA, lipids), and phosphonates (Newman and

Tate 1980; Turner et al. 2003).

These different P compounds present in the sediment will be released to the water column

(internal load) due to chemical, physical and biological processes (Figure 1-2). Therefore benthic

sediments may play a critical role in P cycling by acting as sources or as sinks for P. With

reduction and control of the external nutrient load, the internal load can become a maj or issue in

regulating the trophic state and the time lag for recovery of lakes (Petterson 1998).

Determination of the relative abundance of different P forms in sediments is important to

understand sediment P processes and internal loading.

Organic P compounds present in sediments must be hydrolyzed before their uptake by

microorganisms (Chrost 1991; Sinsabaugh et al. 1991). Organic P is hydrolyzed by enzymes

produced by microbial communities (Gachter et al. 1988; Davelaar 1993; Gachter and Meyer

1993), and the product of enzymatic hydrolysis is orthophosphate that can be readily used by

microorganisms (Barik et al. 2001) (Figure 1-2). Enzyme production can be induced by the

presence of organic P and low levels of bioavailable inorganic P (Kuenzler 1965; Aaronson and

Patni 1976). On the other hand, high levels of inorganic P inhibit the synthesis of enzymes

(Torriani 1960; Lien and Knutsen 1973; Elser and Kimmel 1986; Jasson et. al. 1988; Barik et al.

2001).

















































Trophic State
Figure 9-1. Graphic representation of main sediment characteristics of three lakes in relation to
their trophic state. Ext-C: extractable organic carbon, Ext-N: extractable labile nitrogen, TP: total
phosphorus, Inorganic-P: HCl-Pi, FAP: moderate labile organic phosphorus, HAP: highly resistant
organic phosphorus, Res-P: residual phosphorus, Ext-P: extractable labile phosphorus, MBC:
microbial biomass carbon, MBP: microbial biomass phosphorus, MBN: microbial biomass nitrogen,
and microbial activity: CO2 and CH4 prOduction rates.


TP
Poly-P
DNA-P
Ext-C, Ext-N
Ext-C:P
Ext-N:P
MicTObial Biomass
Microbial Activity
PDEase
61 C 61sN


TP
HAP/FAP
Inorganic-P
Ortho-P
P-Monoester
PMEase
qCO2


TP
Labile-Pi
Inorganic-P
Ortho-P


High


Medium














Low


Labile-Pi
Microbial Biomass
Microbial Activity
PDEase






Ext-C:P
Ext-N:P
Res-P
6 3C 61sN


qCO2
6 3C 61sN






Ext-C :P
Ext-N:P
Microbial Biomass
Microbial Activity
PMEase/PDEase

Lake Okeechobee
Mud Zone


PMEase










qCO2

Labile-Pi

Lake Apopka


Lake Annie
Central










Table 2-4. Phosphorus fractionation in sediments from Lake Annie, Lake Okeechobee, and Lake Apopka.
Percentage (%) Total Phosphorus

Lak Ste Total P Mirba Lbl norganic Moderately Highly Residual P
Lak Ste (mg k~g ) .irba Lbl P Available Resi stant
Biomass P. .. .
Organic Inorganic Fulvic Acid-P Humic Acid-P


2.9

2.4

3.5

2.0

1.7

1.1

1.3

0.8

3.3

1.2

1.0

2.1

2.3

2.5

3.2

1.6


4.5

5.5

13.5

4.9

7.7

8.4

8.1

3.9

7.6

5.5

2.5

8.9

0.4

0.2

0.7

0.1


28.1

26.5

16.2

4.9

9.5

2.2

7.8

2.0

11.0

4.5

7.7

3.4

12.4

15.0

15.5

15.4


15.2

16.6

9.4

4.8

3.0

0.6

2.9

1.4

1.3

1.8

2.7

0.0

9.9

9.1

14.6

6.9


South

Annie Central

North

M17

011

M9

K8

Okeechobee FC

J5

TC

KR

J7

South

Central
Apopka
West

North


1428

1435

7.4

374

1166

922

1200

67

30

110

814

60

1221

1417

1215

1635


2.8

3.7

11.7

1.3

1.8

0.4

1.4

1.2

4.6

1.6

0.1

1.6

45.9

52.6

51.9

61.9


36.1

41.4

10.6

59.9

66.0

79.6

71.8

83.1

38.5

86.6

91.1

62.2

16.9

20.3

35.2

12.9


0.3

0.6

13.2

8.8

17.8

15.0

18.2

4.5

14.3

9.2

13.2

13.2

13.5

13.4

15.3

11.3









wastewater from anthropogenic activities, and variable contributions of river-borne

allochthonous input, related to inter-annual rainfall variations.

In Lake Apopka, heavy 613C DIC in the water column, with high demand for inorganic C

due to high primary productivity, produced autochthonous OM with enriched 813C. The enriched

6 "N signature in Lake Apopka sediments was generated by multiple factors including the

isotopic signature of autochthonous N sources, the primary producer community, and N related

processes in the water column and sediments. A more detailed study of 613C and 6 "N isotopes in

several compartments, i.e., dissolved carbon, different N compounds, phytoplankton biomass,

bacteria biomass, particulate OM in the water column and sediment, can confirm the maj or

processes affecting the isotopic signatures of these sediments.

Microbial activity in sediments: effects of organic electron donors (Objective 6)

Microbial functional diversity of surface sediments of the subtropical lakes was

investigated by measuring catabolic response to a wide variety of C-substrates. Addition of

organic electron donors to sediment microcosms from all lakes stimulated heterotrophic activity,

however the extent of the response was strongly related to microbial biomass and catabolic

diversity. Although the magnitude of the response to electron donor addition was related to

microbial biomass, the different response in each sediment was related to the catabolic diversity

of sediment microorganisms. The addition of some electron donors did not stimulate

heterotrophic microbial respiration, and probably resulted in the addition of C into microbial

biomass rather than release via respiratory pathways.

Lake Apopka had the highest respiration per microbial biomass, indicating that these

sediments respired most of the C added, as a consequence of a P limitation. Lake Annie showed

the highest metabolic quotient (qCO2, prOportion of basal respiration per microbial biomass)

indicating inefficient use of energy. The low qCO2 found in Lake Apopka's sediment indicates









of 'heavy' and 'light' RNA. Density gradients were centrifuged as described previously with an

extended period of centrifugation of 46 h.

RT- PCR

RT- PCRs were conducted using an Access RT-PCR System (Promega, Madison, WI)

following manufacturer instructions. E. coli-specific primers ECA75F (5'-

GGAAGAAGCTTGCTTCTTTGCTGAC-3 ') and ECR619R (5'-

AGCCCGGGGATTTCACATCTGACTTA-3 ') were used (Sabat et al. 2000). Bacterial genes

were amplified with universal bacterial primers 16S rRNA gene sequences 27F (5'-

AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3 ')

(Lane 1991). Archaeal 16S rRNA genes were amplified with the universal primer 1492R and

Archaea-specific primer 23F (5' -TGCAGAYCTGGTYGATYCTGCC-3 ') (Burggraf et al.

1991). RT- PCRs were performed in an iCycler PCR system (Bio-Rad, Hercules, CA). RT-PCR

products were analyzed by agarose electrophoresis.

Results

RNA Extraction

RNA extraction was successful for Lake Annie, Lake Apopka and Lake Okeechobee site

M9 sediments but not for sediments from sites M17 and KR since RNA bands could not be

visualized in agarose gels (Figure 8-3). All following experiments were conducted using 1.0 g of

sediment from lakes Annie, Apopka and Okeechobee site M9. For sites KR and M17, the amount

of sediment used was increased to 2.0 g to improve RNA extraction yield.

Pre-Experiment

Neither time nor concentration affected the quality of the RNA extracted from site M9

(Figure 8-4A). No visible RNA was extracted from sediments from site KR (Figure 8-4B).





























To those who fought, and still fight, so that women of my generation and the ones to come can

have choices, opportunities, respect, and equal rights. There is still a long road towards respect

and equality, hopefully it is soon to come.









uptake (Gu et al. 1997). The depletion in 61 N from 36 up to 22 cm may indicate high deposition

of N2-fixer biomass with low 8 "N. With selective degradation of labile autochthonous OM,

isotopically light 8 "N is removed and remaining material is enriched in 1N. Ammonium-N

concentration increases during anaerobic decomposition of OM (Chapter 5). Ammonium derived

from OM decomposition is usually relatively depleted in 1N (Terranes and Bernasconi 2000),

while residual material is left relatively enriched. Isotopic signatures (613C and 6 "N) of sediment

OM are related to several factors, including sediment origin (i.e., plant tissue), intensities of

primary productivity and diagenesis (Figure 6-9C).

Sand zone (site KR) OM content and TC:TN ratios were low (< 16) (Figure 6-6B, F).

Similar to the peat zone, TN:TP ratio increase with greater depth reflecting the decline in TP

with depth in the core (Figure 6-6A, C). Sediment 813C ValUeS Varied throughout the profile (-

25.63%o to -26.36%o) and were most depleted in 13C near the sediment surface (Figure 6-6D).

Nitrogen isotope values (6 5N) varied from ~ 0.63%o to 4.22 %o and show general enrichment

towards the sediment surface (Figure 6-6E). There are two periods where 6 5N declined (from

16-12 cm, and 4-0 cm), and where 6 5N increased (from 10-4 cm). Organic N mineralization is

an important source of inorganic N in these sediments (Fisher et al. 2005).

The KR site differs from other sites in receiving greater influence from allochthonous

material. The KR site is located near the location where the Kissimmee River flows into the lake.

It is the largest inflow to Lake Okeechobee (31% of inflow), and carries a substantial nutrient

load (Frederico et al. 1981; Aumen 1995). Agricultural activities, mainly dairies, are a principal

non-point source of nutrients to the Kissimmee River, and are responsible for the nutrient

enrichment of this lake (Aumen 1995; Reddy et al. 1995; Havens and Gawlik 2005). Other

nutrient sources are sewage from treatment plants, septic tanks, urban runoff, and industrial









Lake Annie had more stable P compounds with greater sediment depth. Dominant P forms

were HCl-Pi, FAP, and HAP, as determined by chemical fractionation, and orthophosphate and

phosphate monoester as determined by 31PNMR. Inorganic P represents P bound to Ca, Mg, Fe,

and Al, and its solubility is controlled by pH and/or redox potential. Lake Annie sediments

(central site) were characterized as having high Fe (3640 mg kg- ) and Al (34640 mg kg- )

concentration, and its mineral particle size composition was clay (48%), silt (49%), and sand

(2%) (Thompson 1981). Lake Annie sediment pH is low, and decreased with depth. Although

redox potential was not measured in this study, these sediments are apparently highly reduced as

they are under persistent anaerobic conditions. Consequently the influence of redox potential and

pH in P solubility in this lake must be minimal, as physical and chemical conditions in Lake

Annie already favor solubilization of inorganic P. There is no increase in labile-Pi with greater

sediment depth, but there is an increase in HCl-Pi contribution to TP. Thus, it seems that total

inorganic P is present in stable forms in deeper sediments. Also, inorganic P can be bound to

clay minerals, in a stable form, as protonation of surface Fe and Al functional groups in clays

increase the P binding capacity of non calcareous sediments (Edzwald et al. 1976). High labile-Pi

in surface sediment of Lake Annie is probably caused by mineralization of organic P through

enzyme activity (Chapter 4). High enzyme and microbial activities in Lake Annie, along with

lake physico-chemical characteristics, and the maj or P forms found in the sediments, strongly

indicate that biotic processes play an important role in P solubility in these mud sediments.

Lake Okeechobee sediments were dominated by HCl-Pi (chemical fractionation) and

orthophosphate (31P NMR). In Lake Okeechobee mud sediments, Fe-P precipitation controls the

behavior of P under oxidizing conditions while Ca-P mineral precipitation governs P solubility

under reducing conditions (Moore and Reddy 1994). Moore and Reddy (1994) reported that the




Full Text

PAGE 1

1 LINKAGE BETWEEN BIOGEOCHEMICAL PROPER TIES AND MICROBI AL ACTIVITIES IN LAKE SEDIMENTS: BIOTIC CONTRO L OF ORGANIC PHOSPHORUS DYNAMICS By ISABELA CLARET TORRES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Isabela Claret Torres

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3 To those who fought, and still fight, so that wo men of my generation and the ones to come can have choices, opportunities, respect, and equal rights. There is st ill a long road towards respect and equality, hopefully it is soon to come.

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4 ACKNOWLEDGMENTS During my journey down this road towards completing my graduate studies a number of people participated in this proce ss in different ways. Some particip ated directly he lping me with field and laboratory procedures, others indirec tly with their friendship and support. Both were essential for the completion of this work. This will not be a short list, as I want here to offer my sincere and deepest thanks to all. First to my advisor, Dr. K. Ramesh Reddy, for providing this unique opportunity to study at the University of Florida and for his financ ial support, teachings, and guidance. Also, thanks to members of my committee Dr. Andrew Ogram, Dr. Mark Brenner, Dr. Edward Phlips and Dr. Karl Havens for their teachings and contribution to this work. My special thanks to Dr. A. Ogram for allowing me to have a great experience of one year of work at the Soil Microbial Ecology Laboratory, and for his guidance. Also, I want to acknowledge Dr. Brenners support to my project, our talks about scien ce, politics and life, data disc ussion, and his help with dating sediments. To William Kenney, (Geology Departme nt/UF) I thank him for his time and help with freeze drying, and for dating the sediments. I am thankful to all employees of Archbold Biological Station for their help and access to Lake Annie, espe cially Hilary Swain. Dr. Evelyn Gaiser (Florida International University), Dr Larry Battoe (SJWMD), and Dr. Robert E. Ulanowicz (University of Maryland) my thanks for providing information related to Lake Annie. My thanks to all members and friends of the Wetland Biogeochemistry Laboratory, especially to Ms. Yu Wang, for her guidance a nd laboratory assistance. Also, Gavin Wilson was always prompt to help solve di fficulties and taught me about e quipment and analysis, and Xiao Wei Gu, Xian Ying Tian, and Hui X Lu for thei r help. My deepest tha nks to Ron Elliot ( in memoriam ), for teaching me to use the Autoanalyzer and for his friendship, he is truly missed. To my colleague Matt Fisher, w ithout whom I would not be able to get my samples, for his

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5 indispensable help with field sampling and the good times we spent in those lakes. Thanks to my colleagues and dear friends that voluntarily helped me during field sampli ng, Dr. Noel Cawley, Kathleen McKee, Andrea Albertin and Jason Smit h. I am deeply thankful to Jason Smith who taught and helped me with most of the molecu lar biology procedures, a nd for our discussions about science and life. Also, I want to expa nd my thanks to members of Microbial Ecology Laboratory (Abid, Hiral, Moshik, an d Yun) for welcoming me to th e lab. Especially to previous members Dr. Hector Castro and Dr. Ashvini Chahaun for their guidance with the electron donor experiment, and for sharing their knowledge of soil microbiology. My thanks to Dr. Syed Noorwez and Dr. Mark P. Krebs (Department of Ophthalmology/UF) for helping with the ultracentrifuge, special thanks to Dr. Kr ebs for discussing the methodology for the SIP experiment and for his help in solving practical problems. My deepest thanks to Dr. Andrew S. Whiteley (Molecular Microbial Ecology CEH Oxford/UK), for a number of emails exchanged to help me solve problems with th e SIP experiment, and for sharing his knowledge and his kindness. I also want to thank Bill Reve for providing and setting up the HPLC pump for the SIP experiment. My sincere thanks to Dr. Benjamin Tu rner (Smithsonian Tropical Research Institute/Panama) for his teachings on 31P NMR analysis, and interpreta tion and discussion of the data. Also to Dr. Michael Hupfer (Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin/Germany) for his time and a dvice in improving the extraction for 31P NMR. My thanks to Dr. Kanika S. Inglett (Dr. Sharma!) for her consta nt support, her help with discussing and setting up experiments, and her friendship. I really appr eciated all those endles s conversations we had and the guidance she provided during the difficult times. My thanks to Dr. Patrick Inglett for his guidance, and help with isotope analysis.

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6 To my best and dearest friend Jeremy Bright without whom it would have been impossible to do many of the measurements. There are no word s to describe how I appreciate his friendship and all the indispensable and high quality help he provided. My belove d friends Lynette M. Brown (for sharing the hurdles of a Ph.D. program) and Cecilia C. Kennedy, thank you for sharing the good and bad moments, for your suppor t, and for making our office the best and happiest office in the department. My dear fr iend Adrienne Frisbee I truly thank you for your friendship and support. I also want to acknowledge friends that left the department but have not been forgotten, Dr. Hari Pant Dr. John Leader, and Sue Simo n. My sincere thanks to Dr. Natasha Maynard-Pemba (Counseling Center/UF) for taking me in when I needed it the most, for her time and guidance, and helping me get back on my feet. Thanks to my husband, Dr. Paulo Henrique Ro drigues (Department of Oral Biology/UF), who shared all the hurdles and accomplishments duri ng this time, for his love and support. Also, for sharing his knowledge of mol ecular biology and helping me with some molecular procedures and questions. Last but not least to all my family and friends that e ndured all this time without my presence in my beloved Belo Ho rizonte (Brazil). My special tha nks to my grandmother, Hgia Barros Costa, for her constant support, and for being proud of my accomplishments. My deepest thanks to my sister, Beatriz Claret Trres, fo r her friendship and suppor t when I needed it the most. To my parents, Snia Barros Costa and Ant nio Maria Claret Trres, from whom I derive my strength and determination, the people that I am most indebted in life. My thanks for guiding me through life with their ethics, love, teachings and encouragement, for always supporting my choices, and cheering my accomplishments.

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7 We wrest secrets from nature by most unlikely routes. Societies will, of course wish to exercise prudence in deciding which applicat ions of science are to be pur sued and which not. But without funding of basic research, wit hout supporting acquisition of know ledge for its own sake, our options become dangerously limited Wit hout vigorous, farsight ed and continuing encouragement of fundamental scie ntific research, we are in the position of eating our seed corn: we may fend off starvation for one more winter, but we have removed the last hope of surviving the following winter. CARL SAGAN (The Dragons of Eden, p. 236, 1977)

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........12 LIST OF FIGURES................................................................................................................ .......14 ABSTRACT....................................................................................................................... ............18 CHAPTER 1 INTRODUCTION..................................................................................................................20 Sediment Organic Matter........................................................................................................20 Sediment Phosphorus............................................................................................................ ..23 Microbial Communities..........................................................................................................27 Site Descriptions.............................................................................................................. .......31 Objectives..................................................................................................................... ..........32 Dissertation Format............................................................................................................ ....33 2 BIOGEOCHEMICAL PROPERTIES AND MI CROBIAL ACTIVITY OF BENTHIC SEDIMENTS OF SUBTROPICAL LAKES..........................................................................36 Introduction................................................................................................................... ..........36 Materials and Methods.......................................................................................................... .38 Study Sites.................................................................................................................... ...38 Field Sampling.................................................................................................................39 Sediment Properties.........................................................................................................39 Sediment Phosphorus Fractionation................................................................................40 Microbial Biomass Carbon, Nitrogen, and Phosphorus..................................................41 Microbial Activity...........................................................................................................41 Statistical Analysis..........................................................................................................42 Results........................................................................................................................ .............43 Sediment Properties.........................................................................................................43 Sediment Phosphorus Forms...........................................................................................44 Microbial Biomass...........................................................................................................44 Microbial Activity...........................................................................................................45 Discussion..................................................................................................................... ..........47 Conclusions.................................................................................................................... .........56 3 SEDIMENT PHOSPHORUS FORM S IN SUBTROPICAL LAKES...................................71 Introduction................................................................................................................... ..........71 Materials and Methods.......................................................................................................... .72 Study Sites.................................................................................................................... ...72

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9 Field Sampling.................................................................................................................72 Sediment Properties.........................................................................................................73 Sediment Phosphorus Fractionation................................................................................73 31P Nuclear Magnetic Resonance....................................................................................75 Statistical Analysis..........................................................................................................75 Results........................................................................................................................ .............76 Sediment Properties.........................................................................................................76 Sediment Phosphorus Forms...........................................................................................76 31P Nuclear Magnetic Resonance....................................................................................77 Discussion..................................................................................................................... ..........79 Conclusions.................................................................................................................... .........85 4 ENZYME ACTIVITIES IN SEDIME NTS OF SUBTROPICAL LAKES............................99 Introduction................................................................................................................... ..........99 Materials and Methods.........................................................................................................101 Study Sites.................................................................................................................... .101 Water Characteristics.....................................................................................................101 Sediment Properties.......................................................................................................101 Enzyme Activity............................................................................................................102 Statistical Analysis........................................................................................................103 Results........................................................................................................................ ...........103 Water Characteristics.....................................................................................................103 Sediment Properties.......................................................................................................104 Enzyme Activity............................................................................................................104 Discussion..................................................................................................................... ........106 Conclusions.................................................................................................................... .......110 5 MICROBIAL BIOMASS AND ACTIVITY IN SEDIMENTS OF SUBTROPICAL LAKES.......................................................................................................................... .......121 Introduction................................................................................................................... ........121 Materials and Methods.........................................................................................................123 Study Sites.................................................................................................................... .123 Sediment Properties.......................................................................................................123 Extractable C, N and P..................................................................................................123 Microbial Biomass C, N and P......................................................................................124 Microbial Activity.........................................................................................................125 Statistical Analysis........................................................................................................126 Results........................................................................................................................ ...........126 Sediment Properties.......................................................................................................126 Extractable and Microbial Biomass C, N and P............................................................126 Microbial Activity.........................................................................................................128 Discussion..................................................................................................................... ........129 Conclusions.................................................................................................................... .......137

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10 6 NUTRIENT ACCUMULATION AND STAB LE ISOTOPE SIGNATURES IN SEDIMENTS OF SUBTROPICAL LAKES........................................................................145 Introduction................................................................................................................... ........145 Material and Methods...........................................................................................................147 Study Sites.................................................................................................................... .147 Sediment Properties.......................................................................................................147 Isotopic Analyses...........................................................................................................148 210Pb Dating...................................................................................................................148 Results and Discussion.........................................................................................................149 Core Chronology...........................................................................................................149 Lake Annie.............................................................................................................149 Lake Okeechobee...................................................................................................150 Lake Apopka..........................................................................................................152 13C and 15N Isotope Signatures..................................................................................153 Lake Annie.............................................................................................................153 Lake Okeechobee...................................................................................................156 Lake Apopka..........................................................................................................161 Conclusions.................................................................................................................... .......165 7 HETEROTROPHIC MICROBIAL ACTIVITY IN SEDIMENTS: EFFECTS OF ORGANIC ELECTRON DONORS.....................................................................................177 Introduction................................................................................................................... ........177 Materials and Methods.........................................................................................................180 Study Sites.................................................................................................................... .180 Field Sampling...............................................................................................................180 Sediment Properties.......................................................................................................180 Extractable C, N and P..................................................................................................181 Microbial Biomass Carbon............................................................................................181 Electron Donors.............................................................................................................182 Statistical Analysis........................................................................................................183 Results........................................................................................................................ ...........184 Sediment Properties.......................................................................................................184 Electron Donors.............................................................................................................184 Discussion..................................................................................................................... ........187 Conclusions.................................................................................................................... .......195 8 RNA-STABLE ISOTOPE PROBING OF ACETATE-UTILIZING MICROORGANISMS IN SEDIMENTS OF SUBTROPICAL LAKES.............................208 Introduction................................................................................................................... ........208 Materials and Methods.........................................................................................................210 Study Sites and Field Sampling.....................................................................................210 RNA Extraction.............................................................................................................211 Pre-Experiment..............................................................................................................211 RNA-SIP Experiment....................................................................................................212

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11 Incubation and RNA extraction..............................................................................212 Escherichia coli RNA............................................................................................212 Isopycnic centrifugation.........................................................................................212 RTPCR.................................................................................................................215 Results........................................................................................................................ ...........215 RNA Extraction.............................................................................................................215 Pre-Experiment..............................................................................................................215 RNA-SIP Experiment....................................................................................................216 Escherichia coli RNA............................................................................................216 Isopycnic centrifugation.........................................................................................216 Discussion, Conclusions and Recommendations.................................................................218 9 SUMMARY AND CONCLUSIONS...................................................................................234 Biogeochemical properties and microbial activity of sediments (Objective 1)....................235 Sediment phosphorus forms (Objective 2)...........................................................................236 Enzyme activities in sediments (Objective 3)......................................................................237 Microbial biomass and activity in sediments (Objective 4).................................................238 Long-term OM accumulation and stable isotope signatures in sediments (Objective 5).....239 Microbial activity in sediments: effects of organic electron donors (Objective 6)..............240 RNA-stable isotope probing of acetateutilizing microorganisms (Objective 7).................241 Synthesis...................................................................................................................... .........241 Lake Annie....................................................................................................................242 Lake Okeechobee..........................................................................................................243 Lake Apopka.................................................................................................................243 APPENDIX A Supplemental Tables............................................................................................................ .250 LIST OF REFERENCES.............................................................................................................254 BIOGRAPHICAL SKETCH.......................................................................................................279

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12 LIST OF TABLES Table page 2-1 Morphometric and limnological variab les of the three subtropical lakes..........................59 2-2 Location and sediment type of the site s sampled in the three different lakes....................59 2-3 pH, bulk density, organic matter cont ent, total nitrogen, and total carbon concentration in sediments fr om three subtropical lakes...................................................60 2-4 Phosphorus fractionation in se diments from the three lakes.............................................61 2-5 Extractable and microbial biomass C, N, and P concentrations in sediments from three subtropical lakes........................................................................................................62 2-6 Anaerobic respiration and methane producti on rates in sediments from subtropical lakes.......................................................................................................................... .........63 3-1 Characteristics of sampled sites in th e three different lakes with sampling date, location, sediment type and water quality parameters.......................................................87 3-2 pH, bulk density, organic matter content in sediment profiles of the three lakes.............88 3-3 Phosphorus fraction concentra tions in sediment profiles..................................................89 3-4 Phosphorus composition of the sedi ment depth profile determined by 31P NMR spectroscopy................................................................................................................... ....91 4-1 Measured parameters in the water column of the three lakes..........................................112 4-2 Concentration of total phosphorus, so luble reactive phosphor us, total nitrogen, ammonium-N and dissolved organic carbon in the water column of the three lakes......113 4-3 Water extracdissolved organic car bon, and dissolved reactive phosphorus....................114 5-1 Total carbon, total nitroge n, and C:N:P ratios in sediment profiles of the three lakes....138 5-2 Pore water dissolved organic car bon, ammonium-N, and dissolved reactive phosphorus, total nitrogen, and total phosphorus............................................................139 5-3 Extracorganic carbon, a mmonium, labile organic ni trogen, labile inorganic phosphorus and labile organic phosphorus c oncentrations in sediment profiles.............140 5-4 Microbial biomass carbon, nitrogen and phosphorus con centrations in sediment profiles of the three lakes.................................................................................................141 5-5 Water extracdissolved organic carbon, dissolved reactive P, and ammonium-N concentrations at time 0 and time 10...............................................................................142

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13 7-1 Sediment biogeochemical pr operties of the three lakes...................................................197 7-2 One-way ANOVA statistics of the effect of the different carbon sources addition to sediment CO2 and CH4 production rates and turnover rates............................................198 7-3 Sediment CO2 and CH4 production, and turnover rates, wi th the addition of different carbon sources................................................................................................................. .199 A-1 Water variables from Lake Annie, Lake Okeechobee and Lake Apopka........................250 A-2 Total, extracand microbial biomass carbon, nitrogen and phosphorus ratio measured in sediments from Lake Annie, Lake Okeechobee, and Lake Apopka............................251 A-3 Pearson correlation coefficients of sediment biogeochemical properties........................252 A-4 Pearsons correlation coefficients of biogeochemical properties and microbial biomass and activity.........................................................................................................253

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14 LIST OF FIGURES Figure page 1-1 Schematic of major proce sses occurring in sediment a nd water column of lakes.............34 1-2 Schematic showing draw of chemical and biological P processes in lake sediments.......34 1-3 Schematic showing mineralization of or ganic matter through heterotrophic microbial activities in sediments........................................................................................................35 1-4 Map of Lake Annie, Lake Okeechobee, and Lake Apopka with their location in Florida State.................................................................................................................. .....35 2-1 Map of the three subtropical lakes with sampled sites and their location in Florida State.......................................................................................................................... ..........64 2-2 Linear regressions between microbi al biomass carbon and microbial biomass nitrogen and phosphorus of sediments...............................................................................66 2-3 Relationship between anaerobic respir ation and microbial biomass carbon of sediments...................................................................................................................... ......67 2-4 Results of the Principa l Component Analysis 1................................................................68 2-5 Results of the Principa l Component Analysis 2................................................................69 2-6 Graphic representation of sediment characte ristics of three lakes in relation to their trophic state.................................................................................................................. ......70 3-1 Map of the three subtropical lakes with sampled sites and their location in Florida State.......................................................................................................................... ..........92 3-2 Fractionating scheme for the ch aracterization of P organic forms....................................94 3-3 31PNMR spectra of the NAOH/EDTA extr acts of sediment depth profile........................95 3-4 Results of the Princi pal Component Analysis...................................................................98 4-1 Enzyme activity of sediment depth profile......................................................................115 4-2 Relationship between phosphate monoest er concentration and phosphomonoesterase activity in sediments........................................................................................................116 4-3 Relationship between phosphate diester concentration and phosphodiesterase activity in sediments................................................................................................................... ..116

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15 4-4 Relationship between enzyme activit y, phosphomonoesterase and phosphodiesterase and pore water dissolved reactive phosphorus and di ssolved organic carbon concentration in sediments from Lake Apopka...............................................................117 4-5 Relationship of different microbial activities..................................................................118 4-6 Results of the Principa l Component Analysis 1..............................................................119 4-7 Results of the Principa l Component Analysis 2..............................................................120 5-1 Microbial activity in sediments from Lake Annie, Lake Okeechobee and Lake Apopka......................................................................................................................... ....143 5-2 Results of the Princi pal Component Analysis.................................................................144 6-1 Results of 210Pb dating of Lake Annie sediments............................................................167 6-2 Radioisotope activities versus dept h, in Lake Okeechobee and Lake Apopka................168 6-3 Lake Annie sedime nt depth profile..................................................................................169 6-4 Lake Okeechobee mud zone (site M9) sediment depth profile.......................................170 6-5 Lake Okeechobee peat zone (sit e M17) sediment depth profile......................................171 6-6 Lake Okeechobee sand zone (sit e KR) sediment depth profile.......................................172 6-7 Lake Apopka sediment depth profile...............................................................................173 6-8 Carbon vs nitrogen isotopic values of sediments ............................................................174 6-9 Major mechanisms affecting the sediment 13C and 15N signatures..............................175 7-1 Microbial activity response to the different carbon s ource addition in Lake Annie sediments...................................................................................................................... ....200 7-2 Microbial activity respon se to the different carbon source addition in the mud sediments (site M9) of Lake Okeechobee........................................................................201 7-3 Microbial activity respon se to the different carbon source addition in the peat sediments (site M17) of Lake Okeechobee......................................................................202 7-4 Microbial activity respon se to the different carbon source addition in the sand sediments (site KR) of Lake Okeechobee........................................................................203 7-5 Microbial activity response to the different carbon s ource addition in Lake Apopka sediments...................................................................................................................... ....204 7-6 Relationship between microbi al biomass carbon and activity.........................................205

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16 7-7 Results of the Principa l Component Analysis 1..............................................................206 7-8 Results of the Principa l Component Analysis 2..............................................................207 8-1 Picture of the apparatus for fractionating the gradients...................................................223 8-2 Photograph of grad ient fractionation...............................................................................223 8-3 Agarose gel electrophoresis of RNA extr acted from the three lakes sediments..............224 8-4 Agarose gel electrophoresis of RNA extr acted from sediments of Lake Okeechobee sites M9 (A) and KR (B)..................................................................................................224 8-5 Agarose gel electrophoresis of RNA extracte d of samples from A) Lake Annie, Lake Apopka, and B) Lake Okeechobee sites M9 and M17....................................................225 8-6 Agarose gel electrophoresis of RNA extracted from E. coli culture...............................225 8-7 Graph illustrating the buoyant density of gradient fractions...........................................226 8-8 Buoyant density of gradient fractions: (A) Manefield et al. (2002 b ); (B) Whiteley et al. (2007)..................................................................................................................... .....226 8-9 Agarose gel electrophoresis of RT-PCR of the E.coli added to Lake Apopka samples (A) old primers; (B) new primers....................................................................................227 8-10 Agarose gel electrophoresis of PCR of E. coli RNA samples treated and not treated with DNase..................................................................................................................... ..227 8-11 Buoyant density of gradient fractions..............................................................................228 8-12 Agarose gel electrophoresis of RT-PCR of RNA extracted fr om Lake Apopka fractions...................................................................................................................... ......228 8-13 Buoyant density of gradient fractions..............................................................................229 8-14 Agarose gel electrophoresis of RT-PCR of RNA extracted fr om Lake Apopka fractions...................................................................................................................... ......230 8-15 Graph illustrating the buoyant density of gradient fractions...........................................231 8-16 Agarose gel electrophoresis of RT-PCR of E. coli RNA extracted from gradient fractions...................................................................................................................... ......232 8-17 CsCl density gradient centrifugation of isotopically distinct DNA species and quantitative evaluation of nucleic acid di stribution within gr adient fractions.................233 8-18 CsTFA density gradient centrifugation of isotopically distinct rRNA species and quantitative evaluation of nucleic acid distribution within gradient fractions.................233

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17 9-1 Graphic representation of main sediment ch aracteristics of three lakes in relation to their trophic state............................................................................................................ ..246 9-2 Summary of the main biogeochemical pr operties and processes occurring in Lake Annie water column and sediments.................................................................................247 9-3 Summary of the main biogeochemical prope rties and processes occurring in the Lake Okeechobee site M9 water column and sediments..........................................................248 9-4 Summary of the main biogeochemical prope rties and processes occurring in the Lake Apopka water column and sediments..............................................................................249

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18 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LINKAGE BETWEEN BIOGEOCHEMICAL PROPER TIES AND MICROBI AL ACTIVITIES IN LAKE SEDIMENTS: BIOTIC CONTRO L OF ORGANIC PHOSPHORUS DYNAMICS By Isabela Claret Torres December 2007 Chair: K. Ramesh Reddy Co-chair: Andrew Ogram Major: Soil and Water Science In lakes, deposition of allochthonous and au tochthonous particulate matter to sediments can alter the physico-chemical properties and a ssociated biogeochemical processes. Coupling and feedback between sediment biogeochemistry and water column primary productivity often depends on biogeochemical processes within sedi ments and associated microbial communities. The current investigation was conducted to link biogeochemical properties of benthic sediments and microbial communities and their activities in sub-tropical lakes of different trophic state (Lake Annie oligo-mesotrophic, Lake Okeechobee eutrophic, and Lake Apopka hypereutrophic). The central hypothesis of this study was that lakes w ith contrasting trophic states have sediments with diffe rent biogeochemical properties th at have a selection pressure (i.e., C, N and P availability or limitation) on the microbial community that is reflected in their activities. Sediments sampled from sixteen differe nt sites revealed that trophic state was not related to nutrient content of sediments. The relative abundance of phosphorus (P) forms in sediments was more important than total P concentration in characterizing the processes occurring in sediments. Laboratory batch incu bation studies were conducted to determine the relationships between major sediment P forms, enzyme activity, heterotrophic microbial activity,

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19 and nutrient limitation. Results showed that the concentrations of vari ous P compounds changed with sediment depth, indicating that different processes were controlling P reactivity and mobility in these lakes. Also, P-associated enzyme activities were related to sediment microbial biomass and activity, as well as to the different P forms and availa bility in sediments. Microbial community biomass and activity, as well as incu bation experiments, revealed that the Lake Annie sediment microbial community was carbon (C)-limited, while Lake Apopka was Plimited. Lake Okeechobee mud and sandy sediments we re C and nitrogen (N) limited, whereas in the peat sediment a co-limitation of C and P was observed. Stable isotope analyses showed that, in each lake, different mechanisms control 13C and 15N signatures in these sediments, and were closely linked to lake physico-chemical properti es, as well as the primary productivity in the water column. Isotopic signatures in the lake sediments showed a trend of enrichment in 13C and 15N with increasing trophic state. Oligo-mesotrophic Lake Annie sediment had the lowest values of 13C and 15N. Eutrophic Lake Okeechobee mud sediments displayed intermediate values for both isotopes. And hypereutrophic Lake Apopka had the highest values for both 13C and 15N. Catabolic response profiles of a wide va riety of C-substrates added to sediments indicated that different microbial communities ar e present in these sediments. The microbial community of hypereutrophic lake sediments ha d higher efficiency use of energy and higher catabolic diversity. This study hi ghlighted the relationships be tween sediment biogeochemical properties and the microbial community, how th ey differ among lakes with different trophic states, and how the physico-chemical conditions of lakes affect sediment properties and microbemediated processes. Results suggest that alt hough the microbial community is C/energy limited, C, coupled with N and P availability had a st rong influence on microbial communities in these lakes sediments.

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20 CHAPTER 1 INTRODUCTION In freshwater ecosystems an increase in external nutrient input resulting from anthropogenic activities is frequently the major cause of eutrophication (Krug 1993; Straskraba et al. 1995; Noges et al. 1998). Although some freshwater ecos ystems can become eutrophic naturally, accelerated rate of eutrophication of many lakes is a direct consequence of high nutrient load from anthropogenic ac tivities, such as agricultural practices and urban activities. The main paths of anthropoge nic eutrophication (also called cultural eutrophication) in lakes are: increase in input of nutrients (mainly nitrogen and phosphorus), increase of the phytoplankton biomass, loss of bi ological diversity, dominance by cyanobacteria, diatom, and unicellular green algae, occurren ce of algae blooms (high biom ass production of certain species of algae at the water surface), reduction in light and oxygen availability, change in heterotroph community composition, death of fish. All these a lterations will lead to an ecosystem change, loss of species diversity and decrease in water qu ality. Hence, lakes with different trophic states (oligotrophic: low productivity, mesotrophic: me dium productivity, eutrophic: high productivity and hypereutrophic: very high productivity) wi ll have distinctive physical, chemical and biological characteristics (i.e., pH, redox potential, and microbial community). Sediment Organic Matter Particulate matter that enters a lake (all ochthonous) or is produced within a lake (autochthonous) is deposited and becomes an integral part of sediments. Consequently, lakes function as natural traps for particulate matte r and associated nutrients. Accumulation and retention of particulate matter and nutrients in sediments depe nds on lake morphometry, water renewal, nutrient loading, edaphic characterist ics of the drainage basin, among others (Bostrm et al. 1988) and can alter the physico-chemical properties of sediments and associated

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21 biogeochemical processes (Rybak 1969). Organi cally bound nutrients in particulate matter supplied to the sediment are mineralized by he terotrophic decomposition, resulting in release of nutrients into the water column and stim ulation of biological productivity (Capone and Kiene 1988; Gchter and Meyer1993; Brooks and Edgington 1994). Consequently benthic sediments play a critical role in nutrient cycling by acting as both sources and sinks of nutrients (Figure 1-1). Lake sediments contain an archive of past environmental conditions in and around the water body (Smol 1992) and can be used to document anthropogenic impacts through time (Smeltzer and Swain 1985). Sediment organic ma tter (OM) provides information about past impacts and biogeochemical processes within la kes, and has been stud ied extensively using paleolimnological methods (Meyers 1997). The timi ng of past events in a basin is based on reliable dating of sediment cores. Sediment dating provides an age/de pth relation from which bulk sediment accumulation rates can be calcu lated (Smeltzer and Swain 1985). The lead-210 (210Pb) technique is used routinely to provide age/depth relations fo r the last 100-150 years (Appleby et al. 1986), and has been used widely in studies of Fl orida lake sediment cores (e.g., Binford and Brenner 1986; Brezonik and Engstr om 1998; Whitmore et al 1996; Brenner et al. 2006; Schottler and Engstrom 2006). Bulk sedime nt accumulation rates in combination with analyses of sediment composition, can be used to calculate accumulation rates of sediment constituents such as OM and nutrients. Such me asures provide insights into past changes in productivity and human impacts on the aquatic ecosystem. Nutrient and OM accumulation rates in sedime nt have been studied in conjunction with stable isotope analyses ( 13C and 15N) to infer past environmental impacts in marine (e.g., Gearing et al. 1991; Savage et al. 2004), lacustrine (e.g., Schels ke and Hodell 1991; Gu et al.

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22 1996; Bernasconi et al. 1997; Hodell and Schels ke 1998; Ostrom et al. 1998; Brenner et al. 1999), and riverine ecosystems (e.g., McCallister et al. 2004; Anderson and Cabana 2004; Brunet et al. 2005). Measurements of 13C and 15N in several lake compartm ents, (i.e., dissolved and particulate matter in the water column and sediment s) have been used to identify the origin of lacustrine OM (Filley et al. 2001; Griffths et al. 2002), infer pa st primary productivity (Schelske and Hodell 1991; Hodell and Schelske 1998; Be rnasconi et al. 1997), document historical eutrophication (Gu et al. 1996; Ostrom et al. 1998; Brenner et al. 1999), elucidate biogeochemical cycles (Terranes and Bernasconi 2000; Jonsson et al. 2001; Lehmann et al. 2004), and shed light on microbial activity (Hol lander and Smith 2001; Lehmann et al. 2002; Gu et al. 2004; Terranes and Bernasco ni 2005; Kankaala et al. 2006). Allochthonous OM usually has more negative 13C values than does autochthonous OM. Values of 13C can also be used to distinguish periods of high versus low primary productivity. Algae fractionate against the heavier isotope, 13C. Consequently, under conditions of low to moderate primary productivity autoch thonous OM displays high negative 13C. During periods of very high primary productivity the preferred 12C in the water column is exhausted and fractionation is diminishe d, yielding OM with higher 13C (Mizutani and Wada 1982; Raul et al. 1990). Hypereutrophic lakes with high rates of primary productivit y have low concentrations of carbon dioxide (CO2) in the water column. Moreover, in alkaline (high-pH) waters bicarbonate (HCO3 -) dominates the dissolved inorganic carbon, and has a 13C that is 8 heavier than dissolved CO2 (Fogel et al. 1992). High demand for inorganic carbon and low free CO2 leads to utilization of HCO3 as a carbon source resulting in heavier 13C of OM (Goericke et al. 1994). Stable isotope signatures of sediment OM can be used to identify im pacts of anthropogenic activities. Sources of OM from wastewater and ag ricultural runoff can be identified because they

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23 yield OM depleted in 13C and enriched in 15N (Gearing et al. 1991; Bu rnett and Schaffer 1980; Savage et al. 2004). Stable isotope 15N has also been used to study the nitrogen (N) biogeochemical cycle. Measurement of 15N in suspended and sedimented OM was used to address the source of N, as well as N limita tion of, and utilization by the phytoplankton community in Lake Lugano (Terranes and Bernasconi 2000). Sediment Phosphorus Phosphorus (P) is often the limiting nutrient for primary productivity in freshwater ecosystems. Sources of P to lakes can be extern al (allochthonous) or in ternal (autochthonous). Allochthonous P input originates in the drainage basin, while autochthonous P originates from primary and secondary productivity within lakes. A major portion of P from these sources added to the water column accumulates in sediments. Sediment P is present in both inorganic and organic forms. Organic P and cellular constituents of the biota represent 90% of total P (TP) in freshwater ecosystems (Wetzel 1999), and in se diments 30-80% of TP is typically in organic form (Williams and Mayer 1972; Bostrm et al. 1982). Although organic P is an important component of sediment P, it has been relatively understudied as compared with the fate of inorganic P (Turner et al. 2005). The reason for this is that there is no direct way to measure organic P. It is usually estimat ed by difference (before and after ignition at high temperature) (Saunders and Williams 1955), or by se quential extraction or chemical fractionation (Condron et al. 2005; McKelvie 2005). Thes e chemical fractionations are based on different solubilities of P forms in al kaline and acid extractio ns with different pH. Turner et al. (2006) compared two methodolog ies, chemical fractionation and phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy, to measure organic P, and showed that for wetland soils, alkaline extracti on with molybdate colorimetry overestimated organic P by 3054%. They concluded that alkaline extraction with 31P NMR spectroscopy is a more accurate

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24 method to quantify organic P. In recent year s there have been many studies using this methodology to distinguish differe nt organic P forms in lake sediments (Hupfer et al. 1995, 2004; Carman et al. 2002; Ahlgren et al. 2005; Ah lgren et al. 2006a, b; Re itzel et. al 2006a, b, 2007). Phosphorus-31 NMR spectroscopy can identif y different P compounds, based on their binding properties, as orthophosphate, pyrophos phate (pyro-P), polyphosphate (poly-P), phosphate monoester, phosphate diester (e .g., DNA, lipids), and phosphonates (Newman and Tate 1980; Turner et al. 2003). These different P compounds present in the sedi ment will be released to the water column (internal load) due to chemical, physical and biol ogical processes (Figure 1-2). Therefore benthic sediments may play a critical role in P cycling by acting as sources or as sinks for P. With reduction and control of the extern al nutrient load, the internal lo ad can become a major issue in regulating the trophic state and the time lag for rec overy of lakes (Petterson 1998). Determination of the relative abundance of diffe rent P forms in sediments is important to understand sediment P proce sses and internal loading. Organic P compounds present in sediments must be hydrolyzed before their uptake by microorganisms (Chrost 1991; Sinsabaugh et al 1991). Organic P is hydrolyzed by enzymes produced by microbial communities (Gchter et al. 1988; Davelaar 1993; Gchter and Meyer 1993), and the product of enzymatic hydrolysis is orthophosphate that can be readily used by microorganisms (Barik et al. 2001) (Figure 1-2) Enzyme production can be induced by the presence of organic P and low levels of bioavailable inorgani c P (Kuenzler 1965; Aaronson and Patni 1976). On the other hand, high levels of i norganic P inhibit the synthesis of enzymes (Torriani 1960; Lien and Knutsen 1973; Elser an d Kimmel 1986; Jasson et. al. 1988; Barik et al. 2001).

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25 Three main groups of hydrolytic enzymes are responsible for phosphate release: non specific and/or partially specific phosphoesterases (mono and diesterase), nucleotidases (mainly 5-nucleotidase), and nucl eases (exo and endonucleases ) (Chrost and Siuda 2002). Phosphomonoesterases (PMEase) are nonspecifi c enzymes that hydrolyze phosphate monoester, and are reported to be produced by several microorganisms (e.g., bacteria, algae, fungi, and protozoa) that are found in the water column and sediment of lakes. Nonspecific PMEases are divided into two groups, depending on the pH at which they exhi bit maximum activity, alkaline (pH 7.6-10) and acid (pH 2.6-6.8) (Si uda 1984). Both can be found inside or outside the cell, and the same cell can produce both alkaline and acid PMEase (Siuda 1984). Although both PMEase activities have been repo rted to be regulated by availability of orthophosphate, acid PMEase is usually regarded as a constitutive enzyme (Siuda 1984; Jasson et al. 1988). The production of constitutive enzymes is not repressed nor stimulated by high or low orthophosphate availability in th e environment. Its production is related to P concentration and demand inside the cell (Siuda 1984, Jasson et al. 1988). Jasson et al. (198 1), however, suggested that in acidified lakes, acid PMEa se may have a similar role to that of alkaline PMEase in neutral systems, as its production is also inhibited by orthophosphate. In aquatic systems, alkaline PMEase is by far the most studied enzyme, pr obably due to the high num ber of systems with neutral pH, that are inappropriate for preservati on of extracellular acid PMEase (Siuda 1984). Another important phosphatase is phosphodieste rase (PDEase) that hydrolyzes phosphate diester and is known to degrade phospholipids a nd nucleic acids (Hino 1989; Tabatai 1994; Pant and Warman 2000). It is the least studied enzyme in freshwater ecosystems. Few studies have reported on the occurrence and distribution of phosphatases or other organic P hydrolyzing

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26 enzymes in sediments or their association with sediment bacteria (Wetzel 1991; Chrost and Siuda 2002). The association of carbon (C), nitrogen (N) and P influences the structure, energetics and function of all life forms. The degradation of organic P is closely related to organic C degradation, as both are consti tuents of the OM. As an example, Siuda and Chrost (2001) demonstrated from controlled experiments that PMEa se activity of bacteria is used for organic P hydrolysis and uptake of associated organic C moieties, concluding that bacterial PMEase contributes substantially to dissolved organic carbon (DOC) decomposition in lake water. Dissolved organic carbon is an important consti tuent of the C pool in an aquatic ecosystem, and due to the bacteria activity it can be converted to particulate organic C (POC) and thus become available to the upper levels of the aquatic food web (Sndergaard 1984, Azam 1998). As C is the major driver and basic const ituent in all living forms, its cy cle is strongly linked to the P cycle. As a result C:P ratios of the sediment-wat er column can influence P uptake by the bacteria community. Nitrogen is also one of the major nutrients required for cell metabolism. Nitrogen is considered, together with P, to be responsible for the eutrophication process. In lakes where P is present in high concentrations, N can become the limiting nutrient for productivity (Wetzel 2001). The main difference between the P and N cy cles is that the N cycle has an important gaseous phase that does not occur in the P cycle. The Redfield ratio, reported by Redfield et al. (1963) with respect to marine pla nkton, stated that there is a cons tancy in the molar C:N:P ratio = 106:16:1 (by weight 41:7.2:1). This ratio can be applied to differe nt ecosystems and to processes such as decomposition of OM. The C:N:P ratio of materials is reflected in the composition of the

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27 phytoplankton productivity (Wetzel 2001). Deviations in this ratio can indicate nutrient limitation as well as affect P uptake by microorganisms. Microbial Communities Coupling and feedback between sediment bi ogeochemistry and water column primary productivity often depends on biogeochemical pr ocesses within sediments and associated microbial communities. Heterotrophic bacteria play an important role in C and nutrient cycling in lakes. Phytoplankton and/or heterotrophic bacteria are the ma jor drivers of C and nutrient cycling in the water column, while the hetero trophic bacteria domi nate in sediments. Allochthonous and autochthonous particulate OM in the water column is deposited in the sediment, leading to high concentrations of nut rients and high microbial biomass. Lake depth affects the quality of organic material reaching the sediment. In deep lakes, sedimenting OM undergoes intense decomposition in the water column, due to the prolonged period of settling. Consequently low amounts of la bile organic C reach the sedime nt (Suess 1980; Meyers 1997). In shallow lakes, the supply of labile C and nutrients can be higher than in d eep lakes, and the latter often can have more refractory OM. Organic matter deposition is an important sour ce of C to sediments. Organic compounds and associated nutrients supplied to the sedime nt surface are mineralized through heterotrophic decomposition (Gchter and Meye r 1993; Capone and Kiene 1988; Megonigal et al. 2004) (Figure 1-3). The composition a nd activities of the microbial community are regulated by the quality and availability of C. In high depositional environments such as eutrophic, or deep thermally stratified lakes, organic cont ent in sediments is often high, oxygen (O2) consumption occurs rapidly, and O2 is depleted several millimeters below the sediment water interface (Jrgensen 1983; Jrgensen and Revsbrech 1983) In these systems, facultative and strict anaerobic communities dominate. Complete oxidati on of a broad range of organic compounds in

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28 these systems can occur, especi ally through the seque ntial activity of different groups of anaerobic bacteria (Capone and Kiene 1988). In methanogenic habitats, i.e., in the absen ce of inorganic electron acceptors, different groups of microorganisms participate in d ecomposition of OM as no single anaerobic microorganism can completely degrade organi c polymers (Zinder 1993, Megonigal et al. 2004). Cellulolytic bacteria hydrolyze organic polym ers through extracellular enzyme production and further break down monomers to alc ohols, fatty acids, and hydrogen (H2) through fermentation. Alcohols and fatty acids are degraded by syntrophic bacteria (secondary fermenters) into acetate, H2, and carbon dioxide (CO2), which is used as a substr ate by methanogens (Zinder 1993, Conrad 1999, Megonigal et al. 2004). The structur es and functions of anaerobic microbial communities are therefore strongl y affected by competition for fermentation products such as H2 and acetate. Microorganisms derive energy by transf erring electrons from an external source or donor to an external electron sink or terminal electron acceptor. Organic electron donors vary from monomers that support fermentation to simple compounds such as acetate and methane (CH4). Fermenting, syntrophic, methanogenic bacteria and most other anaerobic microorganisms (e.g., su lfate, iron reducers) are sensitive to the concentrations of substrates and products. Thei r activities can be inhib ited by their end products and are dependent on the end product consump tion by other organisms (Stams 1994; Megonigal et al. 2004). While fermenting bacteria shift th eir product formation to more oxidized products, syntrophic bacteria only metabo lize compounds when methanogens or other anaerobic bacteria consume H2 and formate efficiently (Stams 1994). Microbial functional diversity in cludes a vast range of activit ies. One component of this diversity has been characterized by measuring catabolic response profiles, i.e., short-term

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29 response of microbial communities to addition of a wide variety of C-substrates (Degens and Harris 1997; Degens 1998a). This has been widely applied in soil studies to address differences in microbial communities in different soil types, disturbance, and land use (Degens and Harris 1997; Lu et al 2000; Degens et al. 2000, 2001; Stevenson et al. 2004). Substrate induced respiration is often dependent on the size of the microbial biomass pool, however, response of microbial communities is also related to the catabolic diversity of soil microorganisms (Degens 1998). A greater relative catabolic response to a substrate in one system as compared with another indicates that the microbial community is more functionally adapted to use that resource as well as the presence of enzyme s capable of their util ization, and previous exposure to different C-sources (Degens and Harris 1997; Degens 1998; Baldock et al. 2004; St evenson et al. 2004). Metabolic response of a microbi al community in lake sediment may vary due to several factors that influence either the microbial comm unity or due to physico-chemical characteristics of lakes, which include source and chemi cal composition of particulate matter and biogeochemical processes in the sediment and water column. Eutrophic and hypereutrophic lakes usually receive high external loads of nutrients and displa y high primary productivity and nutrient concentrations in the water column and these nutrients eventually reach the sediment, therefore sediments from eutrophic and hypere utrophic lakes are expected to have high concentrations of OM. Binford and Brenner (1986) and Deevey et al (1986) showed that net accumulation rates of OM and nutrients increase with trophic state for Florida lakes. In contrast small, oligotrophic lakes are expected to have rela tively high proportions of allocht honous C input to their sediments (Gu et al. 1996). Sediments with different C-so urces quality and quantity as well as nutrient concentration, will have different microbial communities. These communities can display

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30 distinct a catabolic response, as the mineralizat ion rates of a microbial community are dependent upon the metabolic capacity for a given s ubstrate (Torien and Cavari 1982). Several factors limit bacterial metabolism in sediments, i.e., temperature, C, and nutrient concentration. Most studies of microbial activ ity in sediments focus on C limitation and the effect of electron donors or acceptors in the production of CO2 and/or CH4 (Capone and Kiene 1988; Schulz and Conrad 1995; Maassen et al. 2003; Thomsen et al. 2004). Little work has been done relating production of CO2 and CH4 with biogeochemical properties of sediments such as nutrient availability. Studies in th e water column of lakes have shown that several factors can limit bacterial metabolism (Gurung and Urabe 1999; Jasson et al. 2006). Although it has been generally accepted that the heterotrophic community is C/energy limited, recent studies have shown that inorganic nut rients, especially P, can be the most limiting nutrient for the bacterial community (Gurung and Urabe 1999; Vadstein 2000; Olsen et al. 2002; Vadstein et. al. 2003; Smith and Prairie 2004; Jasson et al. 2006). Reviewing data from freshwater ecosystems, Vadstein (2000) show ed that P limitation is a common phenomenon. Phosphorus limitation occurred in 86% of the cases, while N or C limitation was identified in 15% and 20%, respectively (percentages add up to more than 100% due to methodological aspects, cf. Vadstein 2000). He terotrophic microbial metabolism can be limited by a single factor or multiple variables. Limitation varies among lakes and depends on lake characteristics and biogeochemical properties of the sediment. Benthic sediments play a critical role in nut rient cycling by acting as sources or sinks for nutrients, and heterotrophic metabol ism dominates in this compartm ent (Figure 1-3). It is, thus, important to study biogeochemical properties of sediments and how they relate to microbial community composition, growth, and activity to better understa nd processes that occur in

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31 sediment. The primary goal of this study was to develop a linkage between the biogeochemical properties of benthic sediments and their bacteria l communities in relation to their activities in sub-tropical lakes of di fferent trophic states. The main fo cus of this study was on P compounds as it is the nutrient that in high concentration is reported to be respons ible for eutrophication of freshwater ecosystems. The centr al hypothesis of this study was that lakes with contrasting trophic states will have sediments with differe nt biogeochemical properties that will have a selection pressure (i.e., C, N and P availability or limitation) on the microbial community that will be reflected by their activities. Site Descriptions Three Florida lakes (USA) were selected for this study base d on water quality variables and trophic status (Figure 1-4) Lake Annie, a small (0.37 km2) oligo-mesotrophic lake, is located in south-central Florid a (Highlands County) at the northern end of the Archbold Biological Station. Lake Annie is characterized by pristine water quality with little surface water input (most is ground water), and low anthropogeni c impact due to the absence of development around the lake (Layne 1979). This lake has no natural surface stream s but two shallow man made ditches allow surface water to flow into the lake and contribute to wa ter and nutrient inputs during high rainfall periods (Ba ttoe 1985). Benthic sediments vary from organic to sand in the littoral zone (Layne 1979) (Figure 1-4). Lake Okeechobee is a large (1800 km2) shallow lake located in south Florida. It is considered to be a eutrophic lake that has e xperienced cultural eutr ophication over the last 50 years (Engstrom et al. 2006). Benthic sediments are characterized as: m ud (representing 44% of the total lake surface area), sand and rock (28%), littoral (19%), dominated by macrophyte growth, and peat (9%) that refers to partially decomposed plan t tissues (Fisher et al. 2001) (Figure 1-4).

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32 Lake Apopka is also a shallow lake with 125 km2 of surface area, located in central Florida. Once a clear-water macrophyte-dominated lake, Lake Apopka has changed to a turbid, algal-dominated lake since 1947 (Clugston 1963). This shift may have been caused by nutrient input from several sources, including agricultura l drainage from adjacent vegetable farms (Baird and Bateman 1987, Schelske et al. 2000), although some suggest that th e proximal trigger for the switch was a hurricane or tornado (Bachmann et al. 1999). Even though these inputs were controlled and regulated to some degree, the eutrophication process continued and Lake Apopka is considered hypereutrophic. Benthic sediments are characterized by unc onsolidated material, which mainly consists of algal deposit s (Reddy and Graetz 19 91) (Figure 1-4). Objectives The specific objectives of this study were to: Determine the biogeochemical properties of sediments and examine relationships among sediment biogeochemical properties (nutrient concentrations and availability) and microbial biomass and activity (Chapter 2). Determine relative distributions of P compounds in sediment profile s using two different techniques, 31P NMR spectroscopy and a P chemical fr actionation scheme. (Chapter 3). Characterize P-related enzyme activities in se diment profiles and determine relationships between different P compounds and enzyme activities (Chapter 4). Determine stratigraphic biogeochemical propert ies in sediment cores and evaluate how they are related to microbial biomass and activity; and establish whether there is nutrient limitation of the microbial community (Chapter 5). Determine the source and long-term accumulation of OM to sediments using 13C and 15N signatures. (Chapter 6). Evaluate the catabolic diversity of microbi al communities in sediments (Chapter 7). Identify microbial communities that utili ze acetate through RNA-st able isotope probing (Chapter 8). A series of field sampling and laboratory studies were conducted to accomplish these objectives. Results of these stud ies provided insights into the re lationships between sediment

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33 biogeochemical properties and the microbial community, how they differ among lakes with different trophic states. Moreove r, it demonstrated the importance of considereing several variables, such as C, N and P, to addre ss questions related to microbial communities. Dissertation Format This dissertation begins with Chapter 1 in which a gene ral introduction, main hypothesis and objectives are presented. Chapter 2 consis ts of a characterization of biogeochemical properties and microbial community activity of sediments (0-10 cm ) from sixteen different sites from the three different lakes. The following four chapters (3, 4, 5 and 6) present data from the studies conducted in deep cores collected from se lected sites. In Chapter 3, organic P compounds were characterized in sediment prof ile using two different techniques, 31P NMR spectroscopy a and chemical P fractionation scheme. Chapter 4 focused on P-related enzyme activities and Chapter 5 focused on vertical distribution of microbial biomass and activity and addressed nutrient limitation in each sediment type. Chap ter 6 investigated the long-term OM accumulation and stable isotope signatures in sediments of the three lakes. Microbial functional diversity of sediments (0-10 cm) of the lakes was investigat ed in Chapter 7 by measuring catabolic response to a wide variety of C-substrates. Chapter 8 presents the study of identification of microorganisms that utilize acetate in these sediments using RNA stable isotope probing. Chapter 9 is the summary and conclusions of the results of the dissertation.

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34 Figure 1-1. Schematic of major pr ocesses occurring in sediment and water column of lakes. Figure 1-2. Schematic showing draw of chemical a nd biological P processe s in lake sediments. Nutrient and OM Sediment Water Column Microbial Community Labile Slowly Available Dissolved Refractory OM Sediment release Dissolved Living Particles Non Living Particles External Organic P Labile Slowly Available Recalcitrant DRP P Mineralization Regulators: Enzyme activity Eh Repress enzyme activity Accumulation of Poly-P Sediment P release Sediment Microbial Activity Inorganic P Chemical

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35 Figure 1-3. Schematic showing mi neralization of organic matter through heterotrophic microbial activities in sediments. Figure 1-4. Map of Lake Annie, Lake Okeechob ee, and Lake Apopka with their location in Florida State. CO2 and CH4 Microbial Community Sediment Carbon Nitrogen Phosphorus C:N:P Dissolved C N P Mineralization Activity Biomass Sediment release -A B C 050100150200250 25Kilometers A Lake Annie C Lake Apopka B Lake Okeechobee km 2 = 1800 km 2 = 0.37 km 2 = 125 Littoral Mud Peat Sand Rock

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36 CHAPTER 2 BIOGEOCHEMICAL PROPERTIES AND MI CROBIAL ACTIVITY OF BENTHIC SEDIMENTS OF SUBTROPICAL LAKES Introduction Particulate matter that enters a lake (all ochthonous) or is produced within a lake (autochthonous) is deposited and becomes an integral part of sediments. Consequently, lakes function as natural traps for particulate matte r and associated nutri ents. Accumulation of particulate matter can alter th e physico-chemical properties of sediments and associated biogeochemical processes in the sediment a nd water column (Rybak 1969). Accumulation and retention of particulate matter and nutrients in sediments depe nds on lake morphometry, water renewal, nutrient loading, edaphic characterist ics of the drainage basin, among others (Bostrm et al. 1988). Lake sediment ch aracteristics can provide eviden ce of anthropogenic impacts through time (Smeltzer and Swain 1985) as lake histories are arch ived in sediments (Smol 1992). Organically bound nutrients in pa rticulate matter supplied to th e sediment are mineralized by heterotrophic decomposition, resultin g in release of nutrients into water column and potential for stimulation of biological pr oductivity (Capone and Kiene 1988; Gchter and Meyer 1993; Brooks and Edgington 1994). Consequently benthic se diments may play a critical role in nutrient cycling by acting as both sour ces and sinks of nutrients. Coupling and feedback between sediment bi ogeochemistry and water column primary productivity often depends on biogeochemical pr ocesses within sediments and associated microbial communities. Oxygen (O2) availability in lake sediments typically is restricted to the uppermost few millimeters below the sedime nt-water interface due to limited O2 diffusion and rapid O2 consumption by the heterotrophic community (Charlton 1980; Bostrm et al. 1982). Facultative and strict anaerobic communities ty pically dominate the sediments. Anoxic sediments can be a good habitat fo r bacterial growth as they usua lly have high concentrations of

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37 organic matter and inorganic nutrients (Fenchel et al. 1990; Pace and Funke 1991; Cole et al. 1993). In methanogenic habitats, i.e., in the ab sence of inorganic electron acceptors, different groups of microorganisms participate in decom position of organic matter as no single anaerobic microorganism can completely degrade organi c polymers (Zinder 1993, Megonigal et al. 2004). Fermenting bacteria hydrolyze organic polymer s through enzyme production and further break down monomers to alcohols, fatty acids and hydrogen (H2). Alcohols and fatty acids are degraded by syntrophic bacteria into acetate, H2 and carbon dioxide (CO2), which are used as substrates by methanogens (Zinder 1993; Conr ad 1999; Megonigal et al. 2004). Consequently, carbon dioxide (CO2) and methane (CH4) are important end products in anaerobic decomposition of organic matter and their conc entration can be used as a measure of microbial activity in sediments. The availability and quality of organic material can influence the microbial community, due to nutrient limitation for bacter ial growth and competition for resources. Several factors limit bacterial metabolism in sediments, i.e., temperature, biodegradable organic carbon, nutrients, and elec tron acceptors. Most studies of microbial activity in sediments focus on carbon (C) limitation and the effect of electron donors or acceptors in CO2 and/or CH4 production (e.g. Capone and Kiene 1988; Schulz a nd Conrad 1995; Thomsen et al 2004). Few studies have related production of CO2 and CH4 with biogeochemical properties of sediments and with nutrient availab ility or limitation. Benthic sediments pl ay a critical role in nutrient cycling by acting as sources or sinks for nutrients, and hetero trophic metabolism dominates in this compartment. Thus, it is important to stud y biogeochemical properties of sediments and how they relate to microbial community compos ition, growth, and activity to better understand processes occurring in lake sediment.

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38 The central hypothesis of this st udy was that lakes with contrast ing trophic states will have sediments with different biogeochemical properties that will have a selection pressure (i.e., C, N and P availability or limitation) on the microbial community; that will be reflected in their activities. The specific objectiv es of this study were to: (i ) determine the biogeochemical properties of benthic sediments in three subtropi cal Florida lakes with di fferent trophic states (Lake Annie oligo-mesotrophic, Lake Okeechobee eutrophic, and Lake Apopka hypereutrophic), and (ii) examine relationshi ps among sediment biogeochemical properties (nutrient concentrations and availability ) and microbial biomass and activity. Materials and Methods Study Sites Three Florida lakes (USA) were selected for this study base d on water quality variables and trophic status (Table 21, Figure 2-1). Lake Annie (F igure 2-1A), a small (0.37 km2) oligomesotrophic lake, is located in south-central Flor ida (Highlands County) at the northern end of the Archbold Biological Station. Lake Annie is characterized by pris tine water quality with little surface water input (most is ground water), and low anthropogenic impact due to the absence of development around the lake (Layne 1979). This lake has no natural su rface streams but two shallow man made ditches allow surface water to flow into the lake and contribute to water and nutrient inputs during high rainfall periods (Bat toe 1985). Benthic sediment s vary from organic to sand in the littoral zone (Layne 1979). La ke Okeechobee (Figure 2-1B) is a large (1800 km2) shallow lake located in south Florida. It is consid ered to be a eutrophic la ke that has experienced cultural eutrophication over the last 50 years (Engstrom et al 2006). Benthic sediments are characterized as: mud (representing 44% of the to tal lake surface area), sand and rock (28%), littoral (19%), dominated by macrophyte growth, and peat (9%) that re fers to partially decomposed plant tissues (Fisher et al. 2001). Lake Apopka (Figure 2-1C) is also a shallow lake

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39 with 125 km2 surface area, located in central Florid a. Once a clear-water macrophyte-dominated lake, Lake Apopka has changed to a turbid, al gal-dominated lake since 1947 (Clugston 1963). This shift may have been caused by nutrient inpu t from several sources, including agricultural drainage from adjacent vegetable farms (Bai rd and Bateman 1987, Schelske et al. 2000), although some suggest that the proximal trigge r for the switch was a hurricane or tornado (Bachmann et al. 1999). Even though these inputs were controlled a nd regulated to some degree, the eutrophication process continued and Lake Apopka is considered hypereutrophic. Benthic sediments are characterized by unconsolidated mate rial, which mainly consists of algal deposits (Reddy and Graetz 1991). Field Sampling Three sites were sampled in Lake Annie on July 18, 2004 (North, South, and Central) (Figure 2-1A, Table 2-2). Nine si tes representing four major sedi ment types (sites: M17 = peat; O11, M9 and K8 = mud; J7, KR and TC = sand, J5 and FC = littoral) in Lake Okeechobee were sampled on May 17 and 18, 2003 (Figure 2-1B, Table 2-2). Four sites were sampled in Lake Apopka on January 19, 2004 (North, South, Cent ral and West) (Figure 2-1C, Table 2-2). Triplicate sediment cores were collected using a piston corer (Fisher et al. 1992) or by SCUBA divers. The topmost 10 cm of sediment were collect ed from each core for analyses. Results of all sediment variables are reported on a dry weight basis (dw). Meas urements of water temperature (C), electrical conductivity (S cm-1) and dissolved oxygen (mg L-1) were taken at 1 m water depth from each site during sampling, with a ha ndheld YSI 85 (YSI Inc., Yellow Springs, OH). Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Before each analysis, samples were homogenized and sub-samples taken. Sediment bulk density (BD) was determined on a dry weight basis (i.e., g of dry/cc wet) at 70 C for 72 hours, and pH was determined on wet

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40 sediments (1:2 sediment-to-water ratio). Orga nic matter content (LOI-loss on ignition) was determined by weight loss at 550C. Total P was measured by ignition method, followed by acid digestion (6 M HCl) and measured colorimetrically with a Bran+Luebbe TechniconTM Autoanalyzer II (Anderson 1976; Method 365.1, EP A 1993). Total carbon (TC) and total nitrogen (TN) were determined on oven-dr ied samples using a Carlo Erba NA-1500 CNS Analyzer (Haal-Buchler Instruments, Saddlebroo k, NJ). Measurements of TP, TC, and TN were conducted on sediment that was previously ovendried (at 70 C for 72 hours), ground in a ball mill, and passed through a # 40 mesh sieve. Sediment Phosphorus Fractionation Organic phosphorus (P) pools were measured using a chemical fractionation scheme described by Ivanoff et al. (1998). The procedur e involved sequential chemical extraction in a 1:50 dry sediment-to-solu tion ratio, with: 1) 0.5 M NaHCO3 (pH = 8.5) representing labile inorganic and organic P; 2) 1 M HCl representing inorganic P bound to Ca, Mg, Fe, and Al; 3) 0.5 M NaOH representing organic P associated with fulvic and humic frac tions (moderately and highly resistant organic P, respect ively). Phosphorus remaining in the residual sediment after the sequential extraction was measured by the igniti on method and is called re sidual P, non-reactive P that includes both organic and inorganic P. Extracts from each of these fractions were centrifuged at 10,000 x g for 10 min and filtere d through a 0.45 m membrane filter, and analyzed for SRP or digested for TP (with sulf uric acid and potassium persulfate). Solutions were analyzed by colorimetry, determined by reaction with molybdate using a Bran+Luebbe TechniconTM Autoanalyzer II (Murphy a nd Riley 1962; Method 365.1, EPA 1993). Residual P was determined using an ignition method (Anders on 1976), and analyzed as described previously for TP.

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41 Microbial Biomass Carbon, Nitrogen, and Phosphorus Microbial biomass carbon (MBC), nitroge n (MBN), and phosphorus (MBP) were measured through the chloroform fumigationextraction method (Hedley and Stewart 1982; Brookes et al. 1985; Vance et al. 1987; Horwath and Paul 1994; Ivanoff et al. 1998). Briefly, sediment samples were split in two: one sample was treated with alcohol-free chloroform (0.5 mL) to lyse microbial cells, placed in a vac uum desiccator, and incubated for 24 hrs. The duplicate sample was left untreated. Bo th sets were extracted with 0.5 M K2SO4 for MBC and MBN, and with 0.5 M NaHCO3 (pH = 8.5) for MBP, using a 1:50 dry sediment-to-solution ratio. Extracts from MBC and MBN samples were cen trifuged at 10,000 x g for 10 min and filtered through Whatman # 42 filter paper, and 5 mL of th e extracts were subjecte d to Kjeldahl nitrogen digestion (for MBN) and analyzed for total Kj eldahl-N colorimetrically using a Bran+Luebbe TechniconTM Autoanalyzer II (Method 351.2, EPA 1993). MBC extracts were acidified (pH < 2) and analyzed in an automated Shim adzu TOC 5050 analyzer (Method 415.1, EPA 1993). Extracts from MBP samples were filtered usi ng a 0.45 m membrane filter and digested for TP with sulfuric acid and potassium persulfate, a nd analyzed as describe d previously. Microbial biomass (C, N and P) was determined by the diffe rence between treated and non-treated samples. Non fumigated controls represent extractable organic carbon (Ext-C ), extractable labile nitrogen (Ext-N), and extractable la bile phosphorus (Ext-P). Microbial Activity Anaerobic microbial respirati on and methanogenesis were qua ntified by incubating an amount of sediment (based on 0.5 g of dry we ight) using methodology described by Wright and Reddy (2001). For microbial respira tion experiments, sediments were incubated anaerobically in the dark at 30 C, and evolved CO2 was trapped in vials containing 0.2 M NaOH. Trapped samples were periodically removed (2, 4, 7, a nd 10 days) and sealed. Samples were acidified

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42 with 3 M HCl and CO2 released was measured by gas chromatography using a Shimadzu 8A GC-TCD equipped with Poropak N co lumn (Supelco Inc., Bellefonte, PA), using He as a carrier gas. For the methanogenesis experiment, samples we re placed in a glass vial, closed with rubber stoppers and aluminum crimp seals, and incuba ted anaerobically at 30 C. Gas samples were obtained at 2, 4, 7, 10 days and analyzed on a Shim adzu gas chromatograph-8A fitted with flame ionization detector (110 C), N2 as the carrier gas and a 0.3 cm by 2 m Carboxen 1000 column (Supelco Inc., Bellefonte, PA) at 160 C. Prior to measuring both CO2 and methane (CH4), headspace pressure was determined with a di gital pressure indicator (DPI 705, Druck, New Fairfield, CT). Concentrations of CO2 and CH4 were determined by comparison with standard concentrations and produc tion rates were calculate d by linear regression ( r2 > 0.95). Methane was not detected during the inc ubation period in Lake Okeechobee samples. Suspecting substrate limitation for methane pr oduction, additional experiments were conducted to evaluate the effect of naturally pr esent electron donors acetate and hydrogen (H2) on methane production in sediments. Wet sediment (based on 0.5 g of dry weight) was added to incubation bottles, sealed, and purged with N2 gas. One control (no substrat e addition) and three treatments were applied to each sediment type: 1) Acetate, 2) H2, and 3) Acetate + H2. Acetate (20 mM or 480 mg C kg-1 on a dry weight basis) wa s added from anaerobic sterile stock solution and H2 addition was done by purging the headspace with 80:20 (vol/vol) H2-CO2 gas at 150 Kpa. Samples were incubated anaerobically in the dark at 30 C. Gas samples were obtained at 2, 4, 6, 8, 10 and 14 days after incubation and analyzed on a Shimadzu gas chromatograph-8A as described above. Statistical Analysis A regression analysis was conducted to co mpare microbial biomass C and anaerobic respiration. A Principal Compone nt Analysis (PCA) was performed to address relationships

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43 between variables. A one-way analysis of variance (ANOVA) was condu cted to compare the effect of electron donors on meth ane production and also to comp are the responses among sites in Lake Okeechobee. Pairwise comparisons of means were conducted using Tukeys HSD. All statistical analyses were conducted with Statistica 7.1 (StatSoft 2006) software. Results Electrical conductivity values re flected the trophic conditions of the three lakes, with lowest values for Lake Annie (43-45 S cm-1), and higher for both Lake Okeechobee (232-603 S cm-1), and Lake Apopka (370-418 S cm-1). Day-time dissolved oxygen concentrations were similar for all lakes (4.9-7.3 mg L-1), with Lake Apopka (8.5-10.6 mg L-1) presenting higher values, which is probably due to high algal bi omass and lower (i.e. winter) temperatures (15.816.6 C). Surface water temperature in Lake Annie (29.9-30.1 C), and in Lake Okeechobee (28.2-30.7 C) were high, reflecting summer temperatures (Table A-1 Appendix). Sediment Properties Sediment pH varied from 5.7 to 8.1. Lake A nnie sediment pH was lower than the other lakes. Both Lake Okeechobee and Lake Apopka se diment pH were around circum-neutral to slightly alkaline, reflecting eu trophic conditions of these lake s. Both Lake Apopka and Lake Annie (south and central) sediments had lower bu lk density than Lake Okeechobee sediments (Table 2-3). Organic matter content (LOI %) a nd total carbon (TC) were highest in sediments from the peat zone in Lake Okeechobee (M17 site ), followed by all sites in Lake Apopka, Lake Annie (south and central) (Table 2-3). The La ke Okeechobee peat zone is characterized by partially decomposed plant tissues (Fisher et al. 2001), with high organic matter content (72% LOI). High organic matter content in Lake Apopka sediments (>60%) was due to its algal origin. Total nitrogen (TN) was highest in Lake Apopka sediments followed by the peat zone in Lake

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44 Okeechobee and Lake Annie (south and central). Lake Annie (south and central) sediments had higher TP concentrations than Lake Okeechobe e and Lake Apopka sediments (Table 2-4). Sediment Phosphorus Forms Relative proportions of P pools varied among lakes and sediment type. Inorganic P (HClPi) extracted with 1 M HCl (apatite and non-apatite P) was the major component of the P pool in all sediment types from Lake Okeechobee (38-91% of total P) (Table 2-4). Labile organic P (labile-Po) was low in all lakes, while labile i norganic P (labile-Pi) was higher in sediments from Lake Okeechobee (2.5-8.9% of total P) and Lake Annie sandy sediments (13% of total P). For Lake Apopka, the major fraction of the P was in microbial biomass (46-62% of total P) followed by HCl-Pi (13-35% of total P). In Lake Annie mud sediments (south and centr al), major P forms included: HCl-Pi (3641% of total P) and moderately and highly resistan t organic P: fulvic acid P (26-28% of total P) and humic acid P (15-16% of total P) (Table 24). Residual P (Res.P) wa s low in Lake Annie mud sediments (0.3-0.6% of total P), with higher values for Lake Apopka (11-15% of total P) and Lake Okeechobee (4.5-18% of total P). Lake Annie sediments contained approximately equal proportions of inorganic and organic P pools, while Lake Okeechobee was dominated by inorganic P in all sediment types ( 46-94% of total P). Organic P was the major component of the TP in Lake Apopka sediments ( 70.5-86% of total P) (Table 2-4). Microbial Biomass Lake Apopka had the highest concentration of MBC, MBN and MBP, followed by Lake Annie (mud sediment) (Table 2-5). All sandy sedi ment types had low microbial biomass. Total C:N ratio (weight basis) was higher in Lake Okeechobee and La ke Annie, while C:P and N:P ratios were higher in Lake Apopka (Table 23, 2-4, A-2 Appendix). Extr actable C:N ratio was similar in all sediments, however extractable C:P and N:P ratios were higher in Lake Apopka

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45 (Table 2-5, A-2 Appendix). Extractab le C:N:P represents the labile forms of these nutrients, and lower ratios could indicate nut rient limitation. Although microbial biomass C:N ratio was also similar among sediments, C:P and N:P ratios showed a different result, with Lake Apopka having the lowest ratios among the se diments (Table 2-5, A-2 Appendix). Microbial Activity Anaerobic respiration (CO2-C mg kg-1d-1) rates were higher in Lake Apopka sediments followed by Lake Annie mud sediments, as comp ared to Lake Okeechobee sediments types. All sandy sediments had low anaerobic respiration ra tes (Table 2-6). Meth ane production rates (CH4C mg kg-1d-1) were higher in Lake Annie central site than south site in Lake Annie and all sites in Lake Apopka. Addition of H2 or acetate + H2 to Lake Okeechobee sediments caused higher methane production rates (Table 2-6). Re sults of one-way ANOVA showed that methane production rates of the electron donor experiment with Lake Ok eechobee sediments were significantly different among treatments (n = 27, df = 3, F-test = 19.70, p < 0.00001). Tukeys pairwise multiple comparison method showed methane production rates were significantly different between control and H2, and control and acetate + H2 addition, but were not significantly different between control and acetate. Results were also significantly different when comparing acetate and H2, and acetate and acetate + H2 addition. However, results were not significantly different when comparing H2 and acetate + H2 addition. One-way ANOVA sh owed that there was significant difference in methane producti on rates among sediment types (n = 12, df = 8, F-test = 5.10, p < 0.00001). Tukeys pairwise multiple co mparison method showed that methane production in sites located in the mud zone of La ke Okeechobee were statistically different from methane production in all other sediment type s, but were not different among each other. Methane production rates were not significantly different among p eat, littoral, and sand deposits.

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46 Because linear regression between MBC w ith MBN and MBP showed a strong positive significant relationship (Figure 22A, B) statistical analyses were performed using MBC as proxy for microbial biomass. Regression analysis of MBC and anaerobic re spiration indicate that over a wide range of MBC repres ented by all three lakes there was a logarithmic relationship (Figure 2-3A). However, in the lower range of MBC, the relationship was linear, showing that anaerobic respiration increases rapidly with MBC (Figure2-3B). This regr ession analysis showed that the three lakes fall into distinct groups (Figure 2-3A). Although significant, the linear regression between anaerobic respirati on and methanogenesis was weak (n = 47, r2 = 0.30, p = 0.0039). The first Principal Component Analysis (PCA-1 ) was performed using data from the three lakes to address relationships among biogeochemi cal properties. The second (PCA-2) used only Lake Okeechobee data and was conducted to veri fy how the results from the electron donor experiment relate to the biogeochemical data The PCA-1 had 60.2% of the data variability explained by Axis 1 while Axis 2 explained 18.9 % (Figure 2-4A). Inorga nic P forms (labile-Pi and HCl-Pi) were the variables se lected by Axis 2 while most variables were selected by Axis 1 (excluding CH4, Res.P, extractable C:N, labile-Pi and HCl-Pi) and were plot ted opposite to bulk density, showing an inverse relationship. Micr obial biomass C was grouped with anaerobic respiration and ratios of extr actable C:P and extractable N:P ratios. Methane production rates were plotted with most P forms measured in this study. The position of the sites in relation to the variables loadings in the first PCA showed that the three lakes are separated into different groups. Lake Apopka (all sites) placed in the pos ition of microbial biomass, extractable C:P and extractable N:P ratios and anaerobic respiration. Lake Annie mud sediment type was placed in the position of methane production and P forms. Lake Okeechobee placed in a different position

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47 from the other two lakes, and also displayed a separation of its sediment types. Mud sediment types (M9, O11, K8 sites) of Lake Okeechobee were placed closer to both forms of inorganic P (labile-Pi and HCl-Pi) with a gradient in relation to the three mud sites that were related to the KR site (sand sediment). The peat zone (M17) wa s placed in a different position with extractable C:N ratio, and was unrelated to any other site sampled. Sa ndy sediments from both Lake Okeechobee and Lake Annie were placed with bulk density (Figure 2-4B). The PCA-2, using only Lake Okeechobee, corr oborates the results from Pearsons correlation (Figure 2-5A, Appendix A-3). Methane production rates were placed with microbial biomass, showing that the stimulation of me thane production was dependent on the original microbial biomass (MBC). Again, highest methane production rates were placed with P forms. Axis 1 explained 60.6% of the variability of the da ta and the variables selected were BD and in an opposite position all P forms, anaerobic respir ation, methane production with electron donor addition, LOI and MBC. Axis 2 with 20.1% of the da ta variability explaine d selected extractable C:N, C:P and N:P ratios. The same distributi on of Lake Okeechobee sites seen in PCA-1 was repeated in PCA-2. Peat zone position showed that this site had the high est concentration of the variables selected by Axis 2. Sandy sediments were placed with the bulk density and opposite to the other sites and parameters. Again the same distribution of the mud sediments with the KR site is seen and they were placed with P forms and microbial biomass and activity (Figure 2-5B). Discussion In this study commonly applied methods in so il science were used to measure microbial biomass in lake sediments. The chloroform fu migation-extraction method is a quick and simple procedure that has been used widely to measur e microbial biomass in soils (e.g. Jenkinson et al. 2004). Soil microbial C, N, and P extraction by this method is largely dependent on soil characteristics and microbial community com position (Jenkinson et al 2004). Therefore,

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48 extraction efficiency is corrected by the kec factor. Reported kec values can vary from 0.2 to 0.45 for C, N and P (Bailey et al 2002). In this study, kec factor was not used, to avoid overestimation of the microbial biomass. For example, La ke Apopka sediments had MBP concentrations varying from 561 to 1031 mg kg-1. If the reported kec factor of 0.37 for MBP (Hedley and Stewart 1982) was applied, the final MBP concen tration in Lake Apopka sediments would be higher than the TP (1650-2786 mg kg-1). These kec factors were determined for soils with lower microbial biomass than sediments like Lake Apopka. The efficiency of P extraction from samples with high microbial biomass is probably higher, thus resulting in low kec factors. Therefore the kec factors reported for typical soil samp les are probably not suitable for use in samples containing high labile P in the microbial biomass. Several studies, however, reported that MBC measured through the chloroform fumigation-extraction method (not corrected with the kec factor) yields similar results when compared with other alternative methods to meas ure microbial biomass in soils. Leckie et al. (2004), using humic soils, reported a strong positive linear relationship ( r2 = 0.96, p = 0.007) between microbial biomass C measured with chloroform fumigation-extraction (with no correction factor) and total phospholipids fatty acid analysis, a more accurate methodology to measure microbial biomass. Baile y et al. (2002), using mineral so ils, also reported a strong linear relationship between these two measurements. Microbial biomass C c oncentration in eight different soils, including sewage sl udge, were also strongly correlated ( r2 = 0.96) with DNA measurements (Marstorp et al. 2000). The use of microbial biomass concentrations not corrected by the kec factor is, therefore, a good measure of mi crobial biomass present in the samples. Although the chloroform fumigationextraction has not been used widely in sediment studies

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49 (Mcdowell 2003), the data enable comparisons among lakes in this study, and provide a good proxy for microbial biomass (Marstorp et al. 2000; Bailey et al. 2002; Leckie et al. 2004). It is reasonable to expect that near-surf ace sediment variables will reflect recent lake trophic state conditions. Eutrophic and hypereutrophic lakes usually receive high external loads of nutrients. Eutrophic and hypere utrophic lakes also display high primary productivity and nutrient concentrations in the water column and these nutrients will eventually reach the sediment. Sediments from eutr ophic and hypereutrophic lakes might be expected to have high concentrations of organic matter and nutrients. Binford and Brenner (198 6) and Deevey et al. (1986) showed that net accumulation rates of or ganic matter and nutrients increase with trophic state for Florida lakes. Severa l other studies also have show n that there is a significant correlation between trophic condition (bas ed on water measurements) and nutrient concentrations in sediments (Rybak 1969; Flan ery et al. 1982; Wisnie wski and Planter 1985; Maassen et al. 2003), while others have shown this is not always true, especially for P content (Brenner and Binford 1988; Lopez and Morgui 1993; Gonsiorczyk et al. 1998). The results from this study showed that organic matter, N and P c oncentrations were high in sediments with lower bulk density, and that trophic st ate conditions were not related to nutrient content of sediments. For example, Lake Annie, an oligo-mesotrophi c lake, had higher sediment TP concentration compared to the other two lakes studied. Organi c matter, TC, and TN in Lake Annie deposits were similar to values in Lake Okeechobee and Lake Apopka sediments (Table 2-3). Sediment composition reflects an integrative effect of troph ic state conditions and diagenesis over a long period of time relative to wate r column physico-chemical variab les. Moreover, the relative importance of P forms in sediments is more impo rtant than total P concentration and will depend

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50 on sediment composition, sedimentation rate and physicochemical conditions (Lopez and Morgui 1993; Gonsiorczyk et al 1998; Kaiserli et al. 2002). Lake Annie organic sediments contain high TP concentrations (south and central sites), with up to 45% of TP in moderate to highly re sistant organic P pools (N aOH soluble), suggesting that organic P in this lake is old and stable (Table 2-4). The ot her major fraction is HCl-Pi, which makes up 40% of the total P, and represents to tal inorganic P bound to Ca Mg, Fe and Al. Its solubility is controlled by e ither pH or redox potential (Moore and Reddy 1994). Being a deep lake that is thermically stratified from Febr uary through November or December (Battoe 1985), Lake Annie sediment P has little effect on P co ncentration of the water column during most of the year. In Lake Apopka, > 50% of the total P is in the microbial biomass in most of the sampled sites. This P form is highly available and P storage within microbial cells has been reported to contribute signifi cantly to P release from sedime nts (Davelaar 1993; Gchter and Meyer 1993; Hupfer et al. 2004). Lake Apopka is sh allow, and benthic sediments are subject to resuspension into the water column, potentially releasing soluble P (Reddy et al. 1991). Kenney et al. (2001) showed that polyphospha te (P storage within microbial cells) played an important role in the TP of Lake Apopka sediments, and suggested that betw een 25 and 90% of the sediment TP may be sequestered as polyphospha te. Lake Okeechobee is also shallow, with sediments frequently resuspended into the water column. In Lake Okeechobee, HCl-Pi constitutes approximately 60-91% of the total P, similar to values reported in other studies of Lake Okeechobee (Olila et al. 1995 ; Brezonik and Engstrom 1998). Total C:N ratios (by weight) ra nged from 6 to 19, similar to results reported by Brenner and Binford (1988). In both Lake Apopka and Lake Okeechobee sediment total C may include inorganic C (i.e. carbonates). Sediment C:N ratio can reflec t varying contributions of

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51 allochthonous (high ratio) versus autochthonous (low ratio) organic matter (Hutchinson 1957; Mackereth 1966). Terrestr ial autotrophs have higher C:P a nd C:N ratios than does lacustrine particulate organic matter (Elser et al. 2000). Autochthonous organic matter has a C:N ratio around 12:1 (Wetzel 2001). Among all sediment type s in this study, peat zone deposits from Lake Okeechobee had the highest total weight C: N and C:P ratios reflecting its higher plant origin. Deposits from other sediment types, es pecially Lake Apopka, with lower C:N, reflect algal origin. Extractable nutrient ratios were low for Lake Annie, reflecting high concentrations of extractable labile nutrients relative to C. High availability of N and P may indicate C limitation in Lake Annie sediments. Carbon limitation may re flect the recalcitrant na ture of C entering the lake and physical characteristics of this lake. La ke Annie has experienced an increase in color during the past decades, probabl y from high dissolved organic carbon (DOC) input to the lake from adjacent land (Swain and Gaiser 2005). Ba ttoe (1985) reported high input of surface waters enriched in humic content to Lake Annie duri ng high rainfall periods. This allochthonous DOC, of humic origin, will be utilized in the water column. Because La ke Annie is deep, the DOC will be mineralized during its descent to the sediment (Suess 1980). Consequently lower concentrations of DOC will reach the sediment (also being highly refractory) leading to low C:nutrient ratios. Carbon and N limitation was observed in most Lake Okeechobee sediments, especially in the mud zone. Hence, there is low microbial biom ass and activity. Crisman et al. (1995) reported that temperature and trophic st ate variables Secchi, total P, and total N, showed a weak correlation with bacterioplankt on abundance (number of cells mL-1) in a seasonal study in Lake Okeechobee. They concluded that the factors cont rolling bacterioplankton communities could be

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52 related to grazing and/or C and nutrient ava ilability. Work et al (2005) reported high bacterioplankton production (mg L-1 h-1) in Lake Okeechobee during summer. Also, several studies have shown that bacteri oplankton is an important source of C to the food web in Lake Okeechobee (Havens and East 1997; Work and Have ns 2003; Work et al. 2005). However, to my knowledge, there is no study addressing C or nutrient limitation of the bacterioplankton community in Lake Okeechobee. Nevertheless, Phlip s et al. (1997) showed that in the central region of Lake Okeechobee (mud zone), phytoplankton abundance was high in the summer. Light is the most limiting factor of the phytoplankton community durin g most of the year in this area, however, during summer m onths, light limitation is relaxed and N becomes the limiting factor of the phytoplankton community (Aldri dge et al. 1995). High labile inorganic P availability in mud zone sediments causes a high demand for C and N that is not met. The opposite is seen for Lake Apopka with highe r ratios for extractable C:P and N:P, but lower ratios in microbial bioma ss. Low nutrient ratios in microb ial biomass strongly indicate P accumulation in cells. The bacterial community can have low C:P and N:P ratios by accumulating polyphosphate and reducing C and N content (Makino and Cotner 2004). Lake Apopka has high primary productivity (Carrick et al. 1993) and algae accumulate in surface sediments leading to high concentrations of labile C (Gale et al. 1992; Gale and Reddy 1994). The primary productivity of Lake Apopka is domi nated by cyanobacteria and the dominant taxa are Synechococcus sp., Synechocystis sp. and Microcystis incerta (Carrick et al. 1993; Carrick and Schelske 1997). Approximately 1034 g C m-2 yr-1 from primary production is deposited in surface sediments of Lake Apopka (Gale and Reddy 1994). Schulz and Conrad (1995) showed that acetate concentrations incr ease drastically, from 100 M to 1300 M, in sediments of Lake Constance (Germany) after stimulation by great er algal deposition. High primary production in

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53 Lake Apopka, with consequent sedimentation, is leading to high C concen tration in sediments that is supporting high micr obial biomass and activity. The positions of extractable C:P and N:P ratio s and microbial biomass or activity in PCA-1 (Figure 2-4), support the idea that P availabilit y, more than any other nutrient, influences microbial community in these sediments. High P availability accompanied by relatively low C and N limits microbial community biomass and ac tivity in these sediments. Both anaerobic respiration and CH4 production rates reflect microbial activit y in these sediments and C, N, P are required for microbial metabolism and growth. Hi gh availability of C and nutrients can support a larger microbial community that will be reflec ted in a higher turnover of organic substrates. Other studies have found the same relationshi p between nutrients and microbial activity. Drabkova (1990), in a study of bact erial production and respiration in lakes with different trophic conditions, reported that bacterial production correlates with P con centration, and that respiration increases with trophic state, but to some lim it. Anaerobic respiration appears to approach an asymptote with increasing micr obial biomass (Figure 2-3A). Other studies in the water column of lakes have shown that CO2 concentrations correlate positively with P and N concentrations (Kortelainen et al. 2000; Huttunen et al. 2003). Kortelainen et al. (2006) showed that highest CO2 emissions from sediments to the water column were found in small shallow lakes with high total P and N and organic C. del Giorgio and Peters (1994) concluded that CO2 flux from Quebec lakes was associated with TP concentration in the water column. Despite the fact that most investig ators accept the idea that C availability is the major factor limiting heterotrophic microbial pr ocesses, in both aquatic and terrestrial ecosystems, nutrients other than C are likely li miting where detrital organic matter is nutrient poor (Grimm et al. 2003). Cimbleris and Kalff (1998) showed that for planktonic bacterial

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54 respiration, the best predictor was TP, but al so that higher respira tion was observed with increasing C:N and C:P ratios, similar to the findings in my study. Phosphorus control of microbial ac tivity seems to be stronger for CH4 production. In both statistical analyses (two PCAs ), methane production had a strong relationship with P forms (Figures 2-4 and 2-5). Several studies have show n that methane production rates were higher in eutrophic than oligotrophic lakes. (Casper 1992; Rothfuss et al. 1997; Falz et al. 1999; Nsslein and Conrad 2000; Huttunen et al. 2003; Dan et al 2004). Other than these studies that reported higher CH4 production in eutrophic lakes, there is no clear indication of how P availability affects methane production. Methane was not detected in Lake Okeechobee sediments without the addition of electron donors. However, Fisher et al. (2005) reported CH4 in sediment porewater of sites M9 and M17 in Lake Okeechobee. They also reported sulfate (SO4 -2) in these sediment porewaters, and its decline with sediment depth was related to the use of SO4 -2 as a terminal electron acceptor in the oxidation of sediment organic matter. Iron (Fe) is important in controlling P solubility in Lake Okeechobee sediments (Moore and Reddy 1994) and Fe-reducers might also be present. Structure and function of anaerobic microbial communities are strongly affected by competition for fermentation products such as H2 and acetate (e.g., Megonigal et al. 2004). Ironand SO4 -2reducers outcompete methanogens for H2/CO2 and acetate, due to higher substrate affinities, and higher energy and growth yield (Lovley and Klug 1983; Lovley a nd Phillips 1986; Conrad et al. 1987; Bond and Lovley 2002), however, both proce sses can coexist (Mountfort and Asher 1981; Holmer and Kristensen 1994; Roy et al. 1997; Holmer et al. 2003; Roden and Wetzel 2003; Wand et al. 2006). Coexistence occurs because of spatial variation in the abundance of terminal electron acceptors or because the supply of electron donors is non-limiting (Roy et al. 1997;

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55 Megonigal et al. 2004). Consequently low C availa bility with concomitant presence of Feand SO4 -2-reducers is the probable explanation for la ck of methanogenesis in Lake Okeechobee sediments. Methanogens (archaebacteria) are obligate anaer obes and can be divided into two groups: H2/CO2 (hydrogenotrophic) and acetate (acetoclastic) consumers, CH4 being the final product of both metabolisms (Deppenmeier 1996). Methanogens use a limited number of substrates, mainly acetate or H2/CO2. Theoretically H2/CO2 should account for 33% of total methanogenesis, although much higher contributions have been f ound (Conrad 1999). A ratio of 2:1 or higher of acetate and H2/CO2 contribution for methane production is usually expected (Conrad 1999). Although it has been reported that acetoclastic me thanogenesis dominates freshwater ecosystems while hydrogenotrophic dominates marine syst ems (Whiticar 1999), results from the electron donor experiment in Lake Okeechobee show that in this freshwater ecosystem H2/CO2 is the major substrate for methane production. Other studies have reporte d that hydrogenotrophic methanogenesis can be dominant in freshwater ecosystems (Chauhan et al. 2004; Banning et al. 2005; Castro et al. 2005; Wand et al. 2006). One explanation for higher methane production with H2/CO2 than acetate is temperature. Some studies in lakes have shown that aceto clastic methanogenesis is dominant at low temperatures, 10 C. Higher temperatures lead to an increased contribution of other fermentation pathways and H2/CO2-dependent methanogenesis (Sch ulz and Conrad 1996; Falz et al. 1999; Glissmann et al. 2004). In a study of ri ce paddy soil, Chin and Conrad (1995) reported that low temperatures led to a decrease in H2-dependent methanogenesis that was caused by inhibition of H2-production reactions (i.e. synt rophic bacteria) that seem to be sensitive to low temperatures. Lake Okeechobee lies in south-central Florida. It is subject to subtropical climate,

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56 and the annual water column temperature ra nges from 15-31 C (Rodusky et al. 2001). During sampling for this study, water temperature in Lake Okeechobee was around 28-31 C. Because the lake is shallow, sediment temperature is pr obably in this range. Another explanation for low methane production from acetate is the fact that high P availability inhibits acetotrophic methanogenesis (Conrad et al. 2000 ). Lake Okeechobee had high labi le inorganic P availability (Table 2-4). Conrad et al. (2000) reported that high phosphate availability led to a 60% contribution of total methane production from H2/CO2. In Figure 2-6, the major characteristics of sediments from the different lakes are summarized. Sediments from the central site were selected to represent Lake Annie data, while sediments from the mud zone were selected to represent Lake Okeechobee data. The three lakes, ranging in trophic state, had di stinct sediment biogeochemical properties, however some similarities were present, such as high TP con centration in sediments fr om the different lakes. Sediments from the oligo-mesotrophic Lake Annie had the major P forms as HAP, FAP and HCl-Pi. Low extractable C:P and N:P ratios resulted from a high extractable labile P concentration (Figure 2-6). Lake Okeechobee mud sediments had similarities with Lake Annie sediments, such as low extractable C:P and N:P ratios due to a high extractable labile P concentration, and HCl-Pi as the major P form. Di fferences in sediments from this eutrophic lake included low microbial activity (CO2 and CH4 production rates), and hi gh concentrations of labile Pi (Figure 2-6). The hypereutrophic Lake Apopka had high concentr ations of microbial biomass P, N and C, as well as high extractabl e C:P and N:P ratios, a nd high microbial activity (CO2 and CH4 production rates) (Figure 2-6). Conclusions Eutrophic and hypereutrophic lakes usually rece ive high external loads of nutrients, and display high primary productivity and nutrient concentrations. Consequently, sediments from

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57 these lakes might be expected to have higher concentra tions of organic matte r and nutrients than oligo-mesotrophic lakes. The results from this study, however, showed that trophic state conditions were not related to the nutrient content of sedime nts. Organic matter, N and P concentrations were higher in sediments with lower bulk density, independent of the trophic state of the lake. Sediment composition therefore refl ects an integrative effect of trophic state conditions and diagenesis over a long period of time, relative to water column physico-chemical variables. The relative importance of P forms present in the sediments seemed to be more important than total P concentration in characterizing the se diment of each of the studied lakes. The oligomesotrophic Lake Annie organic sediments contai ned major P forms in moderate to highly resistant organic P (NaOH soluble) and HCl-Pi, suggesting P in this lake is old and stable. The Lake Okeechobee sediment major P form was HC l-Pi, which constituted approximately 60-91% of the total P, while hypereutr ophic Lake Apopka sediment had > 50% of the total P in the microbial biomass. Extractable nutrient ratios seemed to have stronger influence on sediment microbial communities than total concentrations. Extractabl e nutrient ratios were low for Lake Annie, reflecting high concentrations of extractable labile nutrients rela tive to C, indicating C limitation in these sediments. High labile inorganic P ava ilability resulted in low extractable C:P and N:P ratios, and C and N limitation in most Lake Okee chobee sediments, especially in the mud zone, followed by low microbial biomass and activity. Mo reover, low C availability with concomitant presence of FeSO4 -2-reducers appears to be inhibiting th e methanogenic community in Lake Okeechobee sediments. Limitation of the methanogenic community in these sediments is supported by the positive effect of the addi tion of electron donors on methane production. The

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58 results of electron donor addi tion also indicated that H2/CO2 is the major substrate for methane production in Lake Okeechobee sediments. Hypereutrophic Lake Apopka sediments had high er ratios for extractable C:P and N:P, and the high C concentration in sediments is suppor ting high microbial biomass and activity. Lake Apopka sediments are highly influenced by the de position of the primary production in the water column. The results from this study suggest th at although the microbial community is C/energy limited, C, coupled with N and P availability has a strong influence in microbial communities in these lakes sediments. Therefore, studies of sediment heterotrophic microbial communities should take into account C as well as N and P availability.

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59 Table 2-1. Morphometric and limnological vari ables of the three subtropical lakes. Lake Anniea,b Okeechobeec Apopkac Surface Area (km2) 0.366 1800 125 Mean depth (m) 9.1 2.7 1.6 Maximum depth (m) 20.7 Electrical Conductivity (S cm-1) 43.7 447.7 384 Chlorophyll-a (g L-1) 3.6 26 90 Total Nitrogen (g L-1) 373 1510 4890 Total Phosphorus (g L-1) 5.0 100 190 Secchi Transparency (m) 3.4 0.5 0.23 Trophic Classification Oligomesotrophic Eutrophic Hypereutrophic a Florida Lake Watch (2001), b Archbold Station (2005), c Havens et al. (1999) Table 2-2. Location and sediment type of the sites sampled in the three different lakes. Lake Date Sediment Type Site Latitude Longitude Mud/Clay South 27 81 Mud/Clay Central 27 81 Annie July/04 Sand North 27 81 Peat M17 26.4 80.8 Mud O11 26.8 80.8 Mud M9 26.6 80.4 Mud K8 27.6 80.1 Littoral/Sand FC 26.5 80.8 Littoral/Sand J5 27.1 80.8 Sand TC 27 80 Sand KR 27.5 80.8 Okeechobee May/03 Sand J7 27 80.8 Organic South 28 81 Organic Central 28 81 Organic West 28 81 Apopka Jan/04 Organic North 28 81

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60Table 2-3. pH, bulk density (BD), organic matter content (LOI loss on ignition), total nitrogen, and total carbon concentrati on in sediments from three subtropical lakes. (mean standard deviation). Sediment depth 0-10 cm. Total Nitrogen Total Carbon Lake Site pH BD (g of dry cm-3 of wet) LOI (%) (g kg-1 dw) South 5.7 0.1 0.024 0.003 53.8 0.8 19.1 1.6 263 11 Central 5.8 0.01 0.026 0.005 54.9 0.5 20.2 0.7 265 10 Annie North 6.0 0.1 1.64 0.11 0.45 0.3 0.26 0.0 1.6 0.1 M17 7.4 0.2 0.19 0.02 72.2 5.3 21.5 2.8 403 36 O11 7.5 0.03 0.16 0.04 40.2 2.6 11.9 0.6 186 6.5 M9 7.6 0.03 0.26 0.02 28.5 2.0 8.0 0.6 146 8.3 K8 7.5 0.02 0.16 0.04 36.5 1.7 11.4 0.6 175 3.2 FC 7.1 0.2 1.50 0.07 2.6 0.6 0.2 0.1 1.3 0.7 J5 7.6 0.1 1.43 0.16 1.6 0.3 0.3 0.1 3.7 1.7 TC 7.2 0.4 1.35 0.12 2.4 0.0 0.4 0.0 5.1 0.2 KR 7.5 0.1 0.47 0.06 23.5 3.5 6.4 1.2 97 15 Okeechobee J7 8.1 0.2 1.60 0.14 2.2 0.8 0.3 0.0 4.4 1.3 South 7.5 0.2 0.022 0.005 64.2 2.9 29.7 1.6 335 12 Central 7.4 0.2 0.016 0.003 67.8 1.9 31.5 1.1 349 1.7 West 7.7 0.1 0.016 0.001 69.4 2.7 30.5 0.4 356 5.1 Apopka North 7.6 0.03 0.015 0.003 69.2 0.2 32.9 0.04 356 6.1

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61Table 2-4. Phosphorus fractionation in sediments from Lake Annie, Lake Okeechobee, and Lake Apopka. Percentage (%) Total Phosphorus Labile P Lake Site Total P (mg kg-1) Microbial Biomass P Organic Inorganic Inorganic P Moderately Available Fulvic Acid-P Highly Resistant Humic Acid-P Residual P South 1428 2.8 2.9 4.5 36.1 28.1 15.2 0.3 Central 1435 3.7 2.4 5.5 41.4 26.5 16.6 0.6 Annie North 7.4 11.7 3.5 13.5 10.6 16.2 9.4 13.2 M17 374 1.3 2.0 4.9 59.9 4.9 4.8 8.8 O11 1166 1.8 1.7 7.7 66.0 9.5 3.0 17.8 M9 922 0.4 1.1 8.4 79.6 2.2 0.6 15.0 K8 1200 1.4 1.3 8.1 71.8 7.8 2.9 18.2 FC 67 1.2 0.8 3.9 83.1 2.0 1.4 4.5 J5 30 4.6 3.3 7.6 38.5 11.0 1.3 14.3 TC 110 1.6 1.2 5.5 86.6 4.5 1.8 9.2 KR 814 0.1 1.0 2.5 91.1 7.7 2.7 13.2 Okeechobee J7 60 1.6 2.1 8.9 62.2 3.4 0.0 13.2 South 1221 45.9 2.3 0.4 16.9 12.4 9.9 13.5 Central 1417 52.6 2.5 0.2 20.3 15.0 9.1 13.4 West 1215 51.9 3.2 0.7 35.2 15.5 14.6 15.3 Apopka North 1635 61.9 1.6 0.1 12.9 15.4 6.9 11.3

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62Table 2-5. Extractable and microbial biomass C, N, and P concentrations in sediments from three subtropical lakes. (mean stan dard deviation). Extractable C and N non-fumigated 0.5 M K2SO4, Extractable P non-fumigated digested 0.5 M NaHCO3. Extractable (mg kg-1 dw) Microbial Biomass (mg kg-1 dw) Lake Site Carbon Nitrogen Phosphorus Carbon Nitrogen Phosphorus South 1642 224 670 84 107 23 1526 187 305 41 40 10 Central 2619 603 780 139 108 2 1705 145 299 24 53 7.4 Annie North 51 5 3 2 1.3 0.1 42 7 3.7 2 0.8 0.2 M17 1645 221 179 19 26 7 249 25 39 6 4.9 1.6 O11 887 99 196 13 111 37 655 71 90 26 21.2 7.7 M9 482 44 104 9 87 7 338 22 80 18 3.7 2.3 K8 945 171 156 32 113 20 579 104 130 20 17.0 6 FC 18 9 10 1 3.1 0.1 18 8 2.2 2 0.8 0.4 J5 115 41 21 3 3.3 0.8 56 9 8 7 1.4 1.4 TC 101 5 24 1 7.4 1.3 44 13 8 3 1.8 1.7 KR 228 59 71 13 29 6 125 31 7 6 0.9 0.9 Okeechobee J7 66 9 20 2 6.7 0.2 34 14 6 4 0.9 0.7 South 3827 827 1035 224 33 5 13182 1524 2378 278 561 59 Central 4169 711 1331 163 38 1 20771 2342 3516 292 746 48 West 3711 347 1070 256 43 5 18742 830 2977 130 632 149 Apopka North 4316 655 1660 309 29 0.4 23244 1327 4068 254 1031 83

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63Table 2-6. Anaerobic respiration and methane production rates in sediments from subtropi cal lakes. Control are values for basal methane production without substrate addition, and acetate *, hydrogen* and acetate + hydrogen* results from electron donor addition experiment only for Lake Okeechobee sediments. (mean standard deviation). Methane Production (CH4-C mg kg-1d-1 dw) Lake Site Anaerobic Respiration (CO2-C mg kg-1d-1 dw) Control Acetate* Hydrogen* Acetate + Hydrogen* South 362 48 48 10 Central 283 32 118 17 Annie North 3.8 1.2 0.15 0.02 M17 76 17 N.D. 5.0 4.5 3.3 2.6 98 42 O11 117 26 N.D. 27.3 5.6 217 64 127 45 M9 54 14 N.D. 11.6 5.2 130 56 122 7.1 K8 98 11 N.D. 24.8 6.9 204 60 230 30 FC 5.6 0.5 N.D. 1.6 1.5 41.6 25 23.7 14 J5 15 2.8 0.26 12.1 10 21.0 17 33.2 13 TC 13 1.7 N.D. 0.6 0.4 35.4 10 43.5 3.5 KR 78 19 N.D. 3.4 1.0 38.6 20 76.2 22 Okeechobee J7 11 3.3 N.D. 0.5 0.2 48.4 7.8 55.2 6.5 South 563 28 31 4.5 Central 654 87 40 13 West 455 54 34 24 Apopka North 1170 47 52 19 N.D. = Not Detected.

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64 Figure 2-1. Map of the three subtro pical lakes with sampled sites and their locati on in Florida State: A) Lake Annie (with wate r column depth in meters, modified from Layne 1979), B) Lake Okeechobee with different sedi ment types, and C) Lake Apopka. -A B C 050100150200250 25Kilometers 0 0.1 0.2 0.3Kilometers A) Lake Annie Florida

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65 Figure 2-1. continued B) Lake Okeechobee . 0246 1Kilometers-West North Central South West North South CentralC) Lake Apopka

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66 0400080001200016000200002400028000 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 200 400 600 800 1000 1200 MBN = 8.28+ 0.17 MBC r2=0.996, p < 0.00001 MBP = -4.26+ 0.04 MBC r2=0.973, p < 0.00001 040080012001600200024002800320036004000 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 MBN = -1.44 + 0.18 MBC r2=0.95, p < 0.00001 MBP = -0.78 + 0.03 MBC r2=0.89, p < 0.00001 Figure 2-2. Linear regressions between 1) microbial biomass carbon and microbial biomass nitrogen, and 2) microbial biomass car bon and microbial biomass phosphorus of sediments from A) all lakes and B) data from Lake Annie and Lake Okeechobee only. A B Microbial Biomass Nitrogen (mg kg-1) Microbial Biomass Phosphorus (mg kg-1) Microbial Biomass Carbon (mg kg-1)

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67 026005200780010400130001560018200208002340026000 0 200 400 600 800 1000 1200 1400 1600 CO2 = -425 + 241 Log(MBC)r2 = 0.75 0200400600800100012001400160018002000 0 100 200 300 400 500 CO2 = 8.27 + 0.19 MBCr2 = 0.96, p < 0.0001 Microbial Biomass Carbon (mg kg-1) Figure 2-3. Relationship between anaerobic resp iration and microbial biomass carbon of sediments from A) Lake Annie (blue circ les), Lake Okeechobee (red squares), and Lake Apopka (green triangles) and B) da ta from Lake Annie and Lake Okeechobee only. A B Anaerobic respiration (mgCO2-C kg-1 d-1)

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68 BD IP Ext-C:N -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (60.2%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (18.9%) LabPi LabPo ResP CH4CO2Ext-C:P Ext-N:P Ext-N Ext-C TC MBC TN LOI TP FAP HAP -2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5 Axis 1 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Axis 2 Central South South West North M17 M9 K8 TC J7 J5 FC KR O11 North Central Figure 2-4. Results of the Principa l Component Analysis (PCA-1), a) loadings (n = 47), and B) the plot of the scores of the sites from Lake Annie (blue circles), Lake Okeechobee (red squares), and Lake Apopka (green triangles). BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon, TN: total nitrogen, ExtN: extractable labile nitrogen, TP: total phosphorus, La bPi: labile inorganic phosphorus, LabPo: labile organic phosphorus, IP: HCl-Pi inorganic phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resistant organic phosphorus, ResP: residual phosphorus, Ext-P: extractable labile phosphorus, MBC: microbial biomass carbon, CO2: basal anaerobic respiration, CH4: basal methane production rates. B A

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69 BD Ext-N Ext-C:N Ext-C:P Ext-N:P -1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2 Axis 1 (60.6) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (20.1%) Basal (CH4) H2Acetate + H2Acetate CO2TC HAP FAP IP TP LabPo LabPi ResP MBC TN LOI Ext-C -2.0-1.5-1.0-0.50.00.51.01.52.0 Axis 1 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Axis 2 M17 KR M9 O11 J5 FC J7 TC K8 Figure 2-5. Results of the Principa l Component Analysis (PCA-2), A) loadings of (n =27), and B) the plot of the scores of the sites of Lake Okeechobee. BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon, TN: total nitrogen, ExtN: extractable labile nitrogen, TP: total phosphorus, La bPi: labile inorganic phosphorus, LabPo: labile organic phosphorus, IP: HCl-Pi inorganic phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resistant organic phosphorus, ResP: residual phosphorus, Ext-P: extractable labile phosphorus, MBC: microbial biomass carbon, CO2: basal anaerobic respiration, Basal (CH4): basal methane produc tion rates, Acetate, H2, and Acetate + H2, methane production rates from electron donor addition. B A

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70 Figure 2-6. Graphic representation of sediment characteristics of th ree lakes in relation to their trophic state. Ext-C: extractable organic ca rbon, Ext-N: extractable labile nitrogen, TP: total phosphorus, Inorgani c-P: HCl-Pi, FAP: moderate labile organic phosphorus, HAP: highly resistant organic phosphorus Res-P: residual phosphorus, Ext-P: extractable labile phosphorus, MBC: mi crobial biomass carbon, MBP: microbial biomass phosphorus, MBN: microbial bioma ss nitrogen, and microbial activity: CO2 and CH4 production rates. Trophic State High Low Lake Annie Central Lake Okeechobee Mud Zone Lake Apopka TP HAP FAP Inorganic-P Ext-P TP Labile-Pi Inorganic-P Ext-P Medium Ext-C:P Ext-N:P Res-P TP Ext-C, Ext-N Ext-C:P Ext-N:P Microbial Activity MBC, MBP, MBN Microbial Activity MBC Ext-N:P Ext-C:P Microbial Activity MBC Ext-P

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71 CHAPTER 3 SEDIMENT PHOSPHORUS FORMS IN SUBTROPICAL LAKES Introduction Phosphorus (P) is often the limiting nutrient for primary productivity in freshwater ecosystems. Sources of P to lakes can be extern al (allochthonous) or in ternal (autochthonous). Allochthonous P input originates in the drainage basin, while autochthonous P originates from primary and secondary productivity within lakes. A major portion of P from these sources added to the water column accumulates in sediments. Sediment P is present in both inorganic and organic forms. Organic P and cellular constituents of the biota represent 90% of total P (TP) in freshwater ecosystems (Wetzel 1999), and in se diments 30-80% of TP is typically in organic form (Williams and Mayer 1972; Bostrm et al. 1982). Although organic P is an important component of sediment P, it has been relatively understudied as compared with the fate of inorganic P (Turner et al. 2005). The reason for this is that there is no direct way to measure organic P. It is usually estimat ed by difference (before and after ignition at high temperature) (Saunders and Williams 1955), or by se quential extraction or chemical fractionation (Condron et al. 2005; McKelvie 2005). These chemical fractionations are based on different solubilities of P forms in al kaline and acid extractio ns with different pH. Turner et al. (2006) compared two methodolog ies, chemical fractionation and phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy, to measure organic P, and showed that for wetland soils, alkaline extraction with molybdate colorimetry overestimated organic P (between 30-54%). They concluded that alkaline extraction with 31P NMR spectroscopy is a more accurate method to quantify organic P. In recent year s there have been many studies using this methodology to distinguish differe nt organic P forms in lake sediments (Hupfer et al. 1995, 2004; Carman et al. 2002; Ahlgre n et al. 2005; Ahlgren et al. 2006 a b ; Reitzel et. al 2006 a b

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72 2007). Phosphorus-31 NMR spectroscopy can identif y different P compounds, based on their binding properties, as orthophosphate, pyrophos phate (pyro-P), polyphosphate (poly-P), phosphate monoester, phosphate diester (e .g., DNA, lipids), and phosphonates (Newman and Tate 1980; Turner et al. 2003). These different P compounds present in the sedi ment will be released to the water column (internal load) due to chemical, physical and bi ological processes. Therefore benthic sediments may play a critical role in P cycling by acting as sources, or as sinks for P. With the reduction and control of external nutrient load, the internal load can become a major issue in regulating the trophic state and the time lag for recovery of lakes (Petterson 1998). Determination of the relative abundance of different P forms in sediments is important to understand sediment P processes and internal loading. In this study I characterized phosphorus compounds as a function of sediment depth using two different techniques, 31P NMR spectroscopy and conventional organic P fractionation method. I hypothesized that surface sediments repres ent material accreted in recent years and chemically it will have diffe rent characteristics compared to subsurface older sediments. The specific objectives of this study were to: (i) to characterize organic P compounds in vertical sediment profiles using two different techniques, 31P NMR spectroscopy and P fractionation extraction, (ii) address factors c ontrolling P solubility in these sediments. Materials and Methods Study Sites Three Florida (USA) lakes, ra nging in trophic state, were se lected. Lake characteristics were described in Chapter 2 (Table 3-1, Figure 3-1). Field Sampling Sediment sampling sites were selected base d on previous spatial study conducted in all lakes (Chapter 2). Sediment water interface core s of variable lengths were collected using a

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73 piston corer (Fisher et al. 1992) or by SCUBA divers. One central site (80-cm core) was sampled in Lake Annie on June 25, 2005 (Figure 3-1A, Table 3-1). Cores were collected at three sites in Lake Okeechobee on July 16, 2005: M17 = peat (40-cm core), M9 = mud (70-cm core) and KR = sand (40-cm core) (Figs. 3-1B, Table 3-1). A we stern site (98-cm core) was sampled in Lake Apopka on May 28, 2005 (Figure 3-1C, Table 3-1). Co res were sectioned in the field at the following intervals: 0-5, 5-10, 10-15, 1520, 20-30, 30-45, 45-60, 60-80, 80-100 cm. Samples were placed in plastic bags, seal ed, and kept on ice. Nine cores were collected from each site. Three cores were used to make a composite core to obtain sufficient material for all measurements. The nine cores yielded three rep licates of composite sediments from each site. All measured sediment variables are reported on a dry weight basis (dw). Water quality variables were described in Chapter 4, and in the present stu dy values were used to characterize the lakes. Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Before each analysis, samples were homogenized and sub-samples taken. Sediment bulk density (g dry cm-3 wet) was determined on a dry weight basis at 70 C for 72 hours, and pH was determined on wet sediments (1:2 sediment-to-water ratio). Sediment samples were ground in a ball mill and passed through a # 40 mesh sieve. Organic matter content (LOI-los s on ignition) was determined by weight loss at 550C. Total P was measured by ignition method, followed by acid digestion (6 M HCl) and measured colorimetrically with a Bran+Luebbe TechniconTM Autoanalyzer II (Anderson 1976; Method -365.1, EPA 1993). Sediment Phosphorus Fractionation Due to high water content of Lake Annie and Lake Apopka sediments, pore water was extracted (centrifuged at 10,000 x g for 10 min) prior to P fractionati on. Pore water TP was measured after digestion with 11N H2SO4 and potassium persulfate (Method 365.1, EPA 1993).

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74 Organic P pools were measured using a chemical fractionation scheme described by Ivanoff et al. (1998). This procedure involved sequential ch emical extraction in a 1:50 dry sediment-tosolution ratio, with (F igure 3-2): 1) 0.5 M NaHCO3 (pH = 8.5) representing labile inorganic and organic P; 2) 1 M HCl representing inorganic P bound to Ca, Mg, Fe, and Al; 3) 0.5 M NaOH representing organic P associated with fulvic and humic fractions (moderately and highly resistant organic P, respectively). Phosphorus re maining in residual sediment after sequential extraction was measured by ignition method and is called residual P, non-r eactive P that includes both organic and inorganic P. Extracts from each of these fractions were centrifuged at 10,000 x g for 10 min and filtered through a 0.45 m membrane filter, and analyzed for SRP or digested for TP (with sulfuric acid and potassium persul fate). Solutions were analyzed by colorimetry, determined by reaction with molybd ate using a Bran+Luebbe TechniconTM Autoanalyzer II (Murphy and Riley 1962; Met hod 365.1, EPA 1993). Residual P was determined using an ignition method (Anderson 1976), and analyzed as described previously for TP. Microbial biomass P (MBP) was measured by the chloroform fumigation-extraction method (Hedley and Stewart 1982; Horwath a nd Paul 1994; Ivanoff et al. 1998). Briefly, sediment samples were split into duplicates One sample was treated with alcohol-free chloroform (0.5 mL) to lyse microbial cells, plac ed in a vacuum desiccator and incubated for 24 hrs. The other sample was left untreated. Bo th sample sets were extracted with 0.5 M NaHCO3 (pH = 8.5) in a 1:50 dry sediment-t o-solution ratio. Extracts from both sets were filtered through a 0.45 m membrane filter and digested for TP with sulfuric acid and potassium persulfate, and analyzed as describe previous ly. Microbial biomass was calcu lated by the difference between treated (with chloroform) and non-treated samples.

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7531P Nuclear Magnetic Resonance Samples from each depth interv al in triplicate cores from each site were combined and extracted using the methods described by Hupfer et al. (1995, 2004) and Tu rner et al. (2006). A pre-extraction with 100 mL of 0.067 M EDTA (1 hour shaking, centrifuged at 10,000 x g for 30 min) was conducted to reduce the influence of iron and calcium that can interfere with 31P NMR spectroscopy (Hupfer et al. 1995, 2004). Samples were then extracted with 40 ml of 0.2 M NaOH/0.067 M EDTA (1:20 dry sediment-t o-solution ratio), shaken for 2 hours and centrifuged at 10,000 x g for 30 min. A small aliquot of each ex tract (2 mL) was used to determine total P (NaOH-EDTA TP), digested and measured as described for organic P forms. The remaining sample was frozen immediately after centrifugation at -80 C and later l yophilized. Samples were analyzed by 31P NMR spectroscopy as described by Turner et al. (2006). Each lyophilized extract (approx. 100 mg) was redissolved in 0.1 mL deut erium oxide (to provide a NMR signal lock) and 0.9 mL of a solution containing 1.0 M NaOH (to raise the pH to > 13 to ensure consistent chemical shifts and optimum spectral resolution ) and 0.1 mL EDTA, and transferred to a 5 mm NMR tube. Solution 31P NMR spectra were determined using a 6 s pulse (45), a delay time of 1.0 s and acquisition time of 0.2 s, with a Bruker Avance DRX 500 MHz spectrometer operating at 202.456 MHz for 31P. Chemical shifts of signals were expressed in parts per million (ppm) relative to an external standard of 85% H3PO4. Signals were assigned to individual P compounds or functional groups based on literature (M akarov et al. 2002; Turner et al. 2003). Statistical Analysis A Pearson correlation and a Principal Compone nt Analysis (PCA) were performed to determine relations among P forms measured with different methods. All statistical analyses were conducted with Statisti ca 7.1 (StatSoft 2006) software.

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76 Results Sediment Properties Acidic pH conditions were observed in Lake Annie sediments and neutral to alkaline values in Lake Okeechobee and Lake Apopka deposits (Table 3-2). Sediment bulk density values were lowest in Lake Apopka, followed by Lake Anni e reflecting their high fluid content relative to Lake Okeechobee sediments. Bulk density incr eased with depth in lakes Apopka, Annie and at Okeechobee site M9. Lake Okeechobee M17 and KR sediments, showed no clear trend. Organic matter content was highest at Lake Okeechobee M17 reflecting its high peat content, followed by Lake Apopka, Lake Annie, and Lake Okeechobee sites M9 and sandy KR (Table 3-2). Sediment Phosphorus Forms Total P decreased with depth in all lake cores (Table 3-3). Among surface sediment samples from all cores, Lake Annie had the high est TP concentrations as compared with other lakes. In Lake Okeechobee sites M9 and M17, and Lake Apopka sediments, the deepest layer had about half the TP concentration measured in surface sediments. The KR site in Lake Okeechobee displayed the most dramatic decr ease in TP with depth (Table 3-3). Surface sediment labile inorganic P (labilePi) concentrations were highest in Lake Okeechobee site M9 followed by Lake Annie (Tab le 3-3). Approximately 5% of the TP was present as labile-Pi in sediments of most sites. In sediments at site M9 in Lake Okeechobee, 715% of TP was present as labile-Pi. Labile-Pi de creased with sediment depth in Lake Annie and Lake Okeechobee sediments. Labile Pi increased with depth (0.1-9% of TP) in Lake Apopka. Inorganic P (HCl-Pi) was highe st at M9 in Lake Okeechobee, followed by Lake Annie and Lake Apopka sediments. There was no clear tren d with depth in Lakes Annie and Apopka, but a slight decrease with depth in mud sediment s of Lake Okeechobee (M9-site) was seen. Lake Okeechobee sites M17 and KR sediments showed a pronounced decrease of HCl-Pi with depth.

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77 HCl-Pi accounted for 26-56 % of TP in Lake A popka, 26-49% of TP in Lake Annie sediments, while in Lake Okeechobee site M9 sediments 64-89% of TP was present in inorganic P pool. In sediments of M17 and KR sites HCl-Pi cont ribution was 37-79% and 20-94% respectively, and decreased with depth. Labile organic P (labile-Po) was highest in Lake Annie and Lake Apopka with lower concentrations in all Lake Ok eechobee stations. Approximately 0.13% of the TP was present as labile-Po in sediments of most sites, with a general decrease w ith depth (Table 3-3). Microbial biomass P (MBP) decreased with depth in all co res except KR, where values were consistently low throughout. Highest MBP values were detect ed in Lake Apopka, where 47% of the TP was present as MBP in surface sediments. Moderately available organic P (FAP) was higher in Lake Annie and Lake Apopka, with lower values in Lake Okeechobee sediments. FAP displayed a general decrease with depth in the cores (Table 3-3). Similar results we re detected for highly resistant organic P (HAP). Lake Annie had th e highest values, followed by Lake Apopka, then Lake Okeechobee. Fulvic acid-P and HAP account ed for 17-26% and 12-19% of TP in Lake Annie, while in Lake Apopka was 5-17% and 4-10%, respectively. Residual P (Res-P) was higher in Lake Apopka and site M9 in Lake Ok eechobee, and decreased with depth. In sediments at site M9 6-20% of TP was present as Res-P wi th a decreased with depth. In the other Lake Okeechobee sites, M17 and KR, 6-15% and 3-46% of TP was present as Res-P, respectively, and increased with depth. Lake Apopka Res-P (19-25% of TP) did not present a clear depth trend. In Lake Annie, the concentration of Res-P and its contribution to TP (0-0.5%) were low. 31P Nuclear Magnetic Resonance 31P NMR spectroscopy enabled the identification of discrete pools of organic P (Figure 33A, B, C, Table 3-4). Orthophosphate was the major P compound in sediments of Lake Okeechobee and none of the organic compounds were detected with this technique. Results of

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7831P NMR spectroscopy were in agreement with the results of chemical P fractionation (Tables 33, 3-4). NMR analyses show that Lake Okeechobee sediments are dominated by inorganic P as orthophosphate (M9: 68-100%, M 17: 100% and KR 100%), alt hough in upper layers of mud sediments (site-M9) phosphate monoester (2427%), and DNA-P (7-9%) were also detected (Figure 3-3B, Table 3-4). In Lake Annie, thr ee P compounds were detected in all sediment depths: orthophosphate (51-71%), phosphate monoe ster (23-36%), and DNA-P (6-10%), but no clear trend was observed with de pth (Figure 3-3A, Table 3-4). In Lake Apopka sediments, six different P compounds were dete cted: orthophosphate (28-85%), phosphate monoester (12-28%), DNA-P (15-31%), lipid-P (3-4%), pyro-P (3-10 %), and poly-P (8-11%) (Figure 3-3C, Table 34). There was a general decrease in orthophospha te and organic P forms (phosphate monoester, lipid-P, DNA-P) with depth in Lake Apopka sediments. Comparisons of P forms determined by th e two different methods showed that orthophosphate (NMR) was co rrelated with HCl-Pi ( r = 0.68), labile-Po ( r = 0.73), FAP ( r = 0.82), and HAP ( r = 0.80) (chemical fractionation). Phos phate monoester (NMR) was strongly correlated with FAP ( r = 0.94) and HAP ( r = 0.68) (chemical fractionation). Lipid-P ( r = 0.88) and DNA-P ( r = 0.76) (NMR) showed positive correlation ( r > 0.7) with MBP (chemical fractionation). To address relations between di fferent P forms in sediments from the three different lakes a Principal Component Analys is (PCA) was conducted (Figure 3-4). The PCA had 40.5% of the data variability explained by Axis 1. Axis 2 explained 29.4% of the data variability and the selected variables were Lipid-P, MB P and Res-P (Figure 3-4A). Orthophosphate, phosphate monoester, DNA, labile -Po, FAP, HAP and TP were the variables selected by Axis 1. The position of the sites and sediment depth in relation to the variables loadings in the PCA showed that the three lake s are separated into di fferent groups (Figure 3-

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79 4B). Lake Apopka placed in the position of the pa rameters selected by Axis 2 and Lake Annie in the position of variables select ed by Axis 1. Lake Okeechobee was placed in the position of inorganic P forms, i.e., HCl-Pi and labile-Pi (Figure 3-4B). Discussion Although an oligo-mesotrophic lake, Lake Anni e contained more TP in sediments than both eutrophic Lake Okeechobee and hypereutroph ic Lake Apopka. Lake Annie water inputs are from ground water (90%) and direct rainfall (10%), with negligible surface runoff. Anthropogenic impact is low (Swain and Gaiser 2 005) and high TP concentration at all sediment depths is natural, not induced by anthropogenic activities. Schottler and Engstrom (2006) dated sediment cores from Lake Annie by 210Pb and 137Cs and reported that sediments at ~ 80 cm depth were approximately 125 years old. The results showed higher concentrations of TP at that depth in Lake Annie than in Lake Okeechobee and Lake Apopka sediments. Several studies in Lake Apopka and Lake Okeechobee indicate that the in crease in TP concentration in upper sediment layers was due to cultural eutrophication (Br ezonik and Engstrom 1998; Schelske et al. 2000; Kenney et al. 2002; Waters et al. 2005; Schottler and Engstr om 2006; Engstrom et al. 2006). Nevertheless, a decrease in TP with sedime nt depth has been observed in many lakes (Sndergaard et al. 1996; Gonsiorczyk et al 1998; Ahlgren 2005; Reitzel et al. 2006 a 2007). The relative abundance of P forms in sedime nts is more important than the total concentration with respect to sediment P proces ses and internal loading, and was quite different among the study lakes. Also, concentrations of various P compounds changed with sediment depth, indicating different processe s were controlling P reactivity and mobility in these lakes. The intrinsic difference of these P compounds in different sediments is highlighted by the PCA (Figure 3-4A, B).

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80 Lake Annie had more stable P compounds with greater sediment depth. Dominant P forms were HCl-Pi, FAP, and HAP, as determined by chemical fractionation, and orthophosphate and phosphate monoester as determined by 31PNMR. Inorganic P represents P bound to Ca, Mg, Fe, and Al, and its solubility is controlled by pH and/or redox potential. Lake Annie sediments (central site) were characteri zed as having high Fe (3640 mg kg-1) and Al (34640 mg kg-1) concentration, and its mineral particle size co mposition was clay (48%), silt (49%), and sand (2%) (Thompson 1981). Lake Annie sediment pH is low, and decreased with depth. Although redox potential was not measured in this study, th ese sediments are apparently highly reduced as they are under persistent anaerob ic conditions. Consequently the influence of redox potential and pH in P solubility in this lake must be mini mal, as physical and chemical conditions in Lake Annie already favor solubilization of inorganic P. There is no incr ease in labile-Pi with greater sediment depth, but there is an increase in HClPi contribution to TP. T hus, it seems that total inorganic P is present in stable forms in deep er sediments. Also, i norganic P can be bound to clay minerals, in a stable form, as protonation of surface Fe and Al functional groups in clays increase the P binding capacity of non calcareous sediments (Edz wald et al. 1976). High labile-Pi in surface sediment of Lake Annie is probabl y caused by mineralization of organic P through enzyme activity (Chapter 4). High enzyme and mi crobial activities in Lake Annie, along with lake physico-chemical characteristics, and the major P forms found in the sediments, strongly indicate that biotic processes pl ay an important role in P solu bility in these mud sediments. Lake Okeechobee sediments were dominated by HCl-Pi (chemical fractionation) and orthophosphate (31P NMR). In Lake Okeechobee mud sediment s, Fe-P precipitation controls the behavior of P under oxidizing cond itions while Ca-P mineral pr ecipitation governs P solubility under reducing conditions (Moor e and Reddy 1994). Moore and Re ddy (1994) reported that the

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81 concentrations of soluble reactive P (SRP) in pore water increased under reduced conditions, and were low near neutral (pH 6.5 a nd 7.5), but higher under slightly acidic (pH 5.5) or basic (pH 8.5) conditions. Furthermore, Olila and Reddy (1997) reported that SRP in creases exponentially with a decrease in redox poten tial in sediments from the mud zone of Lake Okeechobee. Consequently, P solubility in Lake Okeec hobee mud sediments is controlled by abiotic processes, either pH, redox potential, or bot h (Moore and Reddy 1994; Olila and Reddy 1997). The control of P solubility in other sedi ment types of Lake Okeechobee has not been studied. Nevertheless, the dominance of inorgani c P in all Lake Okeechobee sites and lack of organic P found in 31P NMR, suggests that pH and redox poten tial also regulate P solubility in M17 and KR sediments. Labile Pi follows the HC l-Pi distribution in si tes of Lake Okeechobee (especially M9 and KR). Consid ering that the major P forms in Lake Okeechobee are HCl-Pi, as well as the fact that these sediments had low enzy me and microbial activities (Chapter 4), it is reasonable to speculate that abiotic processe s control P solubility in these sediments. In contrast to Lake Annie and Lake Okeechobee, in which either biotic or abiotic processes alone control P solubility respectively, in Lake Apopka sediments, P solubility is controlled by a combination of biotic and abiotic processes. Do minant P forms were MBP and HCl-Pi (chemical fractionation), and orthophosphate, phosphate monoester and DNA-P (31P NMR). The high contribution of organic P forms in relation to to tal P in Lake Apopka results from deposition of algal primary producers to the sediment. Ga le and Reddy (1994) reported gross primary productivity in Lake Apopka of 1400 g C m-2 yr-1 of which approximately 1034 g C m-2 yr-1 is deposited in sediments. The Lake Apopka phytoplankton community is dominated by cyanobacteria, ( Synechococcus sp., Synechocystis sp., and Microcystis incerta ), with little variation throughout the year (Carrick 1993; Carrick and Sc helske 1997). Brunberg (1995)

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82 simulated a Microcystis sedimentation event in a eutrophic lake deposit and found that organic P was the dominant fraction in the cells (74% of to tal P). After 15 days of incubation, most of the TP was transformed into total lab ile P and organic P (NaOH soluble). The dominant P forms in Lake Apopka sediment reflect the high contribution of primary producers to sediment P. In Lake Apopkas calc areous sediments, abio tic phosphate uptake and solubility are controlled by pH (Olila and Reddy 1995, 1997). Phosphorus release is associated with dissolution of Ca-P, and a six-fold increase in pore water SRP concentration occurred with a 0.5 decrease in pH (Olila and Reddy 1995, 1997). Bio tic P control is also important in Lake Apopka. Olila and Reddy (1997) reported a large increase in labile-Pi with highly reducing conditions and suggested it was caused by lysed micr obial cells or degrada tion of stored poly-P. If the downcore decline in concentr ation of P forms measured with 31P NMR is indicative of P degradation (Reitzel et al. 2007) then biotic processes are im portant in Lake Apopka. Almost 50% of the total P is in microbial biomass in surface sediments (Table 3-3). Presence of poly-P and pyro-P in sediments also indicates high activ ity of microorganisms involved in biological P cycling (Hupfer et al. 2004; Ahlgren et al. 2005; Reitzel et al. 2006a, 2007). High enzymatic activities found in Lake Apopka se diments strongly support the biolog ical control of P solubility in these sediments (Chapter 4). Low concentratio ns of labile-Pi and its low percent contribution to total P in surface sediments in hypereutrophi c Lake Apopka probably reflects a high P demand by the microbial community. Some studies found a significant correlation between bacterial biomass and organic P extracted with NaOH, and suggested that organi c P extracted with NaOH can be used as a proxy measure of bacterial P (polyP) (Uhlman and Bauer 1988; Waara et al. 1993; Goedkoop and Petterson 2000). My results do no t support this sugges tion as Lake Annie had the highest

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83 concentration and contribution to TP, of orga nic P extracted with NaOH (FAP and HAP), but poly-P was not detected with 31P NMR (Tables 3-3 and 3-4). Moreover, pyro-P that can include degradation products of poly-P (Hup fer et al. 1995), was absent in Lake Annie. Several studies showed that microbial biomass co rrelates with enzyme activity (D avis and Goulder 1993; Massik and Cotello 1995; Barik et al. 2001). Since a high correlation between both FAP and HAP and PMEase activity (Chapter 4) exists, it seems th at the correlation between NaOH-P and microbial biomass reflects the fact that these fractions are used as a P source by microorganisms through enzyme activity. Lake Apopka was the only lake where polyP was detected. Gchter and Meyer (1993) postulate that if suffici ent organic carbon and PO4 -3 are available under aerobic conditions, bacteria can store poly-p. The occurrence of poly-P and the identif ication of phosphateaccumulating organisms come from studies in wa stewater treatment plants with enhanced biological P removal (Seviour 2003). In lakes, the mechanism of poly-P formation is poorly understood, although poly-P has been detected in several recent studies (Hupfer et al. 1995, 2004; Carman et al. 200 2; Reitzel et al. 2006 a b 2007). Alternation of aerobic/anaerobic conditions, combined with availa ble carbon and phosphate, leads to dominance of bacteria that can store poly-P (Mino et al. 1998; Seviour 2003). Khoshmanesh et al. (2001) used 31P NMR and transmission electron microscopy, on a sediment spiked with acetate, and concluded that under aerobic conditions when acetate was available, microorganisms accumulated phosphate as polyP. Some sediments have ideal conditions fo r poly-P formation, such as oscillating aerobic/anaerobic conditions, labile dissolved organic carbon and labi le phosphorus. These conditions exist in Lake Apopka (Chapter 4 a nd 5). Lake Annie, has available DOC, but low

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84 DOC:P ratios (Chapter 4, Table 4-3). Perhap s more important its sediments are always anaerobic. Carman et al. (2002) reported si milar results. They found high poly-P in Lake Gmmaren, with well-oxidized se diments and high organic matter content, some poly-P in Lake Lngsjn, with oscillating oxic/a noxic conditions, and no poly-P in Lake Flaten, with constant anoxic conditions. Lake Okeechobee, though shallo w, and probably subject to oscillating aerobic/anaerobic conditions, seems to be carbon lim ited for poly-P formation, with low ratios of DOC:P (Chapter 4 and 5). Perhaps more important is the high availability of labile-Pi in Lake Okeechobee. Poly-P does not accumulate under c onstant P-sufficient conditions (Vadstein 2000) as the enzyme responsible for poly-P formation (polyphosphate kinase) is a repressible enzyme that is derepressed under P starvation (Harold 1966). Absence of poly-P in the uppermost 5 cm of sedi ment is surprising as Hupfer et al. (2004) reported poly-P in the top 0.5 cm of sediment fo r 22 European lakes, and Ahlgren et al. (2006 b ) found poly-P in the top 1 cm of sediment in tw o oligotrophic mountain lakes in Sweden. Hupfer et al. (2004) explained the presence of poly-P in surface sediments as coming from poly-P formed in the water column then deposited on the lake bottom. I found poly-P at 10, 15 and 20 cm depth in the sediment, and other recent studies reported similar results. Carman et al. (2002) reported distinct signals of polyP in 0-7 and 8-16 cm in Lake Gmmaren. Reitzel et al. (2007) found poly-P in 0-10 cm sections of sediment from mesotrophic Lake Erken. In hypereutrophic Lake Snderby (Denmark), Reitzel et al. (2006 a ) found poly-P in anoxic sediment layers up to 24 cm deep. Absence of poly-P in the uppermost fi rst 5 cm of sediment in Lake Apopka may be related to the relatively lower DOC :P ratio, as the highest ratios were found in the sections where poly-P was detected (Chapter 4, Ta ble 4-3). Also, presence of polyP in deeper sections of the

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85 sediment is a strong indication th at the sediment microbial commun ity is producing poly-P rather than its being produced in the water column and deposited in sediment. Kenney et al. (2001), however, reported that 25-90% of the sediment TP in Lake Apopka may be sequestered in intact phytoplankton cells as poly-P, and found poly-P at 50 cm depth. Moreover Kenney et al. (2001) stated that poly-P c ould be used as an indicator of eutrophication, and suggested that poly-P is chemically inert. They used heat extraction and colorimetry, which may have overestimated poly-P. Phosphorus -31 NMR spectroscopy is a more accurate methodology, and chemical extraction and colorimetr y have been shown to overestimate organic P compounds (Turner et al 2006). My results contradict the findings of Kenney et al. (2001). Reitzel et al. (2007) also disagree with respect to the inert na ture of poly-P. Several studies have shown that poly-P is highly labile and plays an important role in P release from sediment (Trnbon and Rydin 1998; Petterson 2001; Hupfer et al. 2004; Ahlgre n et al. 2005; Re itzel et al. 2007). Conclusions All lakes had a decrease in TP concentra tion with sediment depth, and although oligomesotrophic, Lake Annie contained more TP in sediments than both eutrophic Lake Okeechobee and hypereutrophic Lake Apopka. The relative abunda nce of P forms in sediments, however, is more important than the total concentration with respect to sediment P processes and internal loading, and was quite different among the study lakes. Also, concentrations of various P compounds changed with sediment depth, indicating that different processes were controlling P reactivity and mobility in these lakes. Lake A nnie had more stable compounds with greater sediment depth. Dominant forms of TP were inorganic P (HCl-Pi), FAP, and HAP, as determined by chemical fractionation, and orthophosphate and phosphate monoester as determined by 31P NMR. Lake Annie physico-chemical char acteristics, as well as the major P

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86 forms found in the sediment, strongl y indicated that biotic processe s play an important role in P solubility in these mud sediments. Lake Ok eechobee sediments were dominated by inorganic P (HCl-Pi) (chemical fracti onation) and orthophosphate (31P NMR), indicating abiotic processes control P solubility in these sediments. Domi nant P forms in Lake Apopka were MBP and HClPi (chemical fractionation), and orth ophosphate, phosphate m onoester and DNA-P (31P NMR). Almost 50% of the total P was in microbial biom ass in surface sediments. The presence of polyP and pyro-P in these sediments also indicate d high activity of micr oorganisms involved in biological P cycling. Low concentra tions of labile-Pi, and its low percent contribution to total P in surface sediments in hypereutrophic Lake Apopka, probably reflects a high P demand by the microbial community. This study also showed that the results of 31P NMR spectroscopy were in agreement with the results of ch emical P fractionation, and that th e determination of the relative abundance of different P forms in sediments is important to understand sediment P processes.

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87Table 3-1. Characteristics of sampled sites in the three different lake s with sampling date, location, sediment type and water quality parameters (measured at 1 m). Lake Anniea Okeechobeeb Apopkab Parameters Central M9 M17 KR West Sampling Date June/2005 July/2005 May/2005 Sediment Type Mud/Clay Mud Peat Sand Organic Water Column Depth (m) 20 4.0 2.5 3.1 2.0 Secchi (m) 2.0 0.08 0.15 0.5 0.3 Latitude 27 26.6 26.427.5 28 Longitude 81 80.4 80.881 51.8 81 Temperature (C) 30.2 29.5 28.8 30.8 26.6 Electrical Conductivity (S cm-1) 41.9 385 320 143 443 pH 5.1 7.8 7.6 6.0 7.6 Dissolved Oxygen (mg L-1) 6.4 6.5 6.3 1.8 8.7 Dissolved Organic Carbon (g L-1)* 13.8 14.5 17.9 19.8 31.1 Total Phosphorus (g L-1)* 33.2 255.9 263.2 146.4 69.7 Soluble Reactive Phosphorus (g L-1)* 7.4 90.4 113.1 62.5 11.1 Total Nitrogen (g L-1)* 1807 3439 3362 2957 11149 Ammonium NH4-N (g L-1)* 181.6 103.0 60.4 83.6 119.6 Mean concentration in the water column. a Average depth: 0.51-2-5-10-20 (m) and b Average depth: 0.5-1-2 (m).

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88 Table 3-2. pH, bulk density (BD), organic matter content (LOI loss on ignition) in sediment profiles of the three lakes. (mean standard deviation SD). ** No replicates for SD calculation. Lake Site Depth (cm) pH BD (g of dry cm-3 of wet) LOI (%) 5 5.4 0.15 0.04 0.006 58 0.4 10 5.2 0.05 0.04 0.003 57 1.2 15 5.3 0.09 0.06 0.003 55 1.0 20 5.3 0.11 0.07 0.003 54 1.6 30 5.4 0.03 0.07 0.003 52 0.8 45 5.5 0.09 0.08 0.002 52 0.1 60 5.5 0.06 0.10 0.006 50 0.9 Annie Central 80 5.7 0.06 0.11 0.007 50 1.0 5 7.7 0.16 0.11 0.005 36 1.7 10 7.7 0.03 0.16 0.003 37 1.2 15 7.8 0.04 0.20 0.020 21 1.3 20 7.8 0.04 0.23 0.020 26 6.9 30 7.9 0.03 0.26 0.056 16 3.8 45 7.9 0.05 0.33 0.040 25 6.5 60 8.0 0.01 0.30 0.009 29 4.7 M9 70 8.0 0.08 0.33 0.043 35 3.5 5 7.6 0.20 0.14 0.003 83 0.9 10 7.5 0.08 0.13 0.011 88 0.9 15 7.4 0.06 0.13 0.002 89 0.3 20 7.4 0.10 0.12 0.005 89 0.5 30 7.3 0.15 0.12 0.010 89 0.2 M17 40 7.4 0.17 0.13 0.023 88 0.4 5 7.4 0.19 1.22 0.314 1.9 2.8 10 7.5 0.19 1.13 0.272 3.9 2.9 15 7.7 0.46 1.15 0.323 4.9 3.5 20 7.4 0.41 0.51 0.085 18.1 4.2 30 7.2 0.27 0.55 0.096 16.6 8.8 Okeechobee KR 40 6.9 ** 1.07 ** 6.4 ** 5 7.5 0.07 0.01 0.001 69 0.7 10 7.3 0.06 0.02 0.001 67 3.2 15 7.2 0.02 0.02 0.004 67 1.1 20 7.2 0.06 0.03 0.006 65 2.7 30 7.3 0.04 0.03 0.009 64 1.0 45 7.2 0.05 0.04 0.014 66 1.9 60 7.1 0.10 0.06 0.010 68 1.3 80 7.0 0.06 0.07 0.005 69 0.8 Apopka West 98 7.0 ** 0.07 ** 71 **

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89Table 3-3. Phosphorus fraction concentrati ons in sediment profiles. (mean SD). ** No replicates for SD calculation. Labile P Total P TP Pore Water MBP Inorg. Org. HCl-Pi FAP HAP Res. P Lake Site Depth (cm) (mg kg-1 dw) 5 1439 35 12 3 78 8 72 13 50 9 368 61 371 71268 71 6 6 10 1423 28 7 3 64 18 67 12 43 7 444 13 341 37255 28 4 4 15 1459 67 9 3 44 3 55 6 40 5 548 58 378 17185 18 0.0 0 20 1531 80 15 3 37 5 52 9 35 6 600 73 378 7 183 41 3 3 30 1484 191 25 8 30 7 42 8 28 2 726 78 338 33213 17 0.5 0.8 45 1513 258 24 9 24 10 34 10 22 3 665 116 285 29180 13 1 1 60 1133 32 18 4 22 10 22 0.3 19 2 544 33 195 30184 64 0.0 0 Annie Central 80 1149 51 8 1 18 6 15 3.8 16 1 540 33 206 1 153 20 0.0 0 5 1051 39 ND 50 2 110 7 6 4 670 40 72 8 34 4 213 12 10 924 24 ND 34 5 87 11 8 1 582 11 64 3 35 3 194 9 15 835 ND 28 8 127 337 4 607 35 2 3 8 7 229 53 20 732 83 ND 18 2 55 7 4 1 575 7 4 3 4 3 127 35 30 644 57 ND 11 2 80 6 2 0 496 16 0 0 0 0 132 29 45 575 ND 9 1 55 8 3 1 510 42 0 0 0 0 108 10 60 590 3 ND 8 0.4 46 3 2 1 458 66 1 1 2 3 67 10 Okeechobee M9 70 497 155 ND 2 1 36 2 1 1 423 35 4 3 5 2 50 5

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90Table 3-3. continued Labile P TP TP Pore Water MBP Inorg. Org. HCl-Pi FAP HAP Res-P Lake Site Depth (cm) (mg kg-1 dw) 5 270 13 ND 6 0.6 14 6 5 0.5 213 40 5 2 14 2 16 1 10 138 7 ND 5 0.3 4 0.3 4 0.4 56 3 8 1 15 2 16 2 15 129 16 ND 3 1 3 1 3 0.5 55 28 5 1 15 4 17 6 20 121 9 ND 3 0.8 3 0.4 4 0.7 44 3 5 0.5 17 3 18 4 30 112 14 ND 2 0.8 4 0.3 4 0.8 42 3 5 1 15 2 16 2 M17 40 144 27 ND 2 1 3 0.4 3 0.6 70 12 4 1 16 7 17 7 5 263 20 ND 1 0.5 4 1 0.4 0.3234 24 4 2 1 0.2 8 6 10 280 50 ND 1 0.2 7 2 1 0.5 263 31 3 2 1 2 14 6 15 258 83 ND 1 0.6 3 2 0.4 0.3211 45 2 2 1 1 10 2 20 131 29 ND 2 0.3 3 1 1 0.4 85 40 7 1.6 2 0.5 19 2 30 55 12 ND 1 0.9 2 1 1 0.2 22 7 11 6 3 1 18 2 Okeechobee KR 40 16 ** ND 1 ** 1 ** 0.3 ** 3 ** 2 ** 1 ** 7 ** 5 1264 53 10 3 598 17 2 0.4 15 8 325 13 214 6 125 12240 30 10 1303 73 7 1 596 85 1 0.3 19 7 347 40 200 36118 29257 10 15 1350 37 5 1 616 13 19 2 25 7 382 48 219 20133 32262 32 20 1275 122 5 1 523 11520 5 24 13 361 35 148 2695 34 270 8 30 1234 153 6 2 399 16328 8 15 5 418 6 120 3093 38 284 47 45 990 296 10 2 267 22437 3 11 7 340 31 93 46 65 30 198 43 60 795 156 12 4 106 76 34 15 16 8 356 71 63 20 48 18 188 31 80 615 7.3 16 4 31 11 42 8 7 2 351 44 37 3.3 29 7 154 8 Apopka West 98 694 ** 7 ** 52 ** 61 ** 1 ** 386 ** 34 ** 27 ** 139 ** Total P: total phosphorus, MBP: microbial bi omass phosphorus, Inorg.: inorganic, Org.: organic, HCl-Pi: inorganic phosphorus, F AP: moderate labile organic phosphorus, HAP: hi ghly resistant organic phosphorus, Res-P: residual phosphorus. ND = not determined

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91Table 3-4. Phosphorus composition of the sediment depth profile determined by 31P NMR spectroscopy. (Percentage in relation to total extracted phosphorus with NaOH/EDTA in parenthesis) TP (NaOH/EDTA) Phosphate Monoester Lipid DNA Pyro-P Poly-P Lake Site Depth (cm) mg P kg-1 dw (%) 5 1559 788 (51) 552 (35) TR 219 (10) ND ND 10 1525 828 (54) 551 (36) TR 146 (9) TR ND 15 1468 852 (58) 483 (33) TR 132 (9) ND ND 20 1498 866 (58) 505 (34) TR 127 (6) ND ND 30 1584 1127 (71) 367 (23) TR 94 (6) TR ND 45 1296 777 (60) 438 (34) ND 81 (8) ND ND 60 1041 636 (61) 321 (31) ND 82 (10) ND ND Annie Central 80 854 518 (61) 248 (29) ND 88 (8) ND ND 5 896 604 (68) 210 (24) ND 82 (9) TR ND 10 547 363 (67) 147 (27) ND 36 (7) TR ND 15 405 405 (100) ND ND ND ND ND 30 174 174 (100) ND ND ND ND ND Okeechobee M9 60 228 228 (100) ND ND ND ND ND 5 1139 313 (28) 320 (28) 37.2 (3)355 (31) 113 (10) ND 10 824 263 (32) 181 (22) 32.6 (4)237 (29) 17 (2) 93 (11) 15 670 231 (35) 136 (20) 20.1 (3)204 (31) 18 (3) 61 (9) 20 858 290 (33) 191 (22) 30.4 (4)261 (30) 21 (3) 64 (8) 30 349 165 (47) 87 (25) TR 97 (28) ND ND 45 224 141 (63) 31 (14) TR 52 (23) ND ND 60 150 95 (64) 18 (12) TR 37 (25) ND ND 80 135 107 (80) ND ND 28 (21) ND ND Apopka West 98 102 86 (85) ND ND 16(15) ND ND ND: not detected, TR: trace (i.e., not quantifiable). TP NaOH /EDTA: total phosphorus in NaOH/EDTA extracts, Phosphate: orthophosphate, Monoester: phosphate monoe ster, Lipid: phospholipids, Pyro-P : pyrophosphate, and Poly-P: polyphosphate.

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92 Figure 3-1. Map of the three subtro pical lakes with sampled sites and their locati on in Florida State: A) Lake Annie (with wate r column depth in meters, modified from Layne 1979), B) Lake Okeechobee with different sedi ment types, and C) Lake Apopka. -A B C 050100150200250 25Kilometers 0 0.1 0.2 0.3Kilometers A) Lake Annie Florida

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93 Figure 3-1. continued . 0246 1Kilometers-West North Central South West B) Lake Okeechobee A) Lake Apopka

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94 Figure 3-2. Fractionating scheme fo r the characterization of P orga nic forms (based on Ivanoff et al. 1998). Sediment Acidified Not Digested 0.5M NaHCO3 ( 16h ) centrifugation, filtration Total Labile P Labile Inorganic P Fumigation with CHCl3 (24h) 0.5M NaHCO3 (16h) 1N HCL (3h) Pellet III Pellet II Acidified Digested for TP 0.5M NaOH (17h) Pellet IV centrifugation, filtration Digested for TP centrifugation, filtration Inorganic P Ignition method, digested with 6M Residual P centrifugation, filtration Digested for TP TP Fumigated Labile Organic P Difference Moderate Resistance Or g anic P Fulvic Acid P Difference Humic Acid P Digested for TP Difference MBP

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95 Figure 3-3. 31PNMR spectra of the NAOH/EDTA extracts of sediment depth pr ofile in Lake: A) Annie, B) Okeechobee M9, and C) A popka. 1 Orthophosphate, 2 Phosphate monoester, 3 Lipids, 4 DNA, 5 Pyrophosphate and 6 Polyphosphate 20 10 0 -10 -20 Chemical shift (ppm) 0-5 cm 5-10 cm 10-15 cm 15-20 cm 20-30 cm 30-45 cm 45-60 cm 60-80 cm 1 2 4 A) Lake Annie

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96 Figure 3-3B 0-5 cm 10-15 cm 5-10 cm 20-30 cm 45-60 cm 20 10 0 -10 -20 Chemical shift (ppm) 1 2 4 B) Lake Okeechobee M9

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97 Figure 3-3C 2 1 0 -10 -20 Chemical shift 0-5 cm 5-10 cm 10-15 cm 15-20 c m 20-30 cm 30-45 c m 45-60 cm 60-80 c m 80-100 cm 1 2 4 5 6 3 C) Lake Apopka

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98 LOI BD Orthophosphate DNA-P Pyrophosphate Labile Inorganic P Inorganic P Residual P -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (40.5%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (29.4%) Polyphosphate Lipid-P Total Phosphorus HAP FAP Labile Organic P P-monoester MBP 5 10 30 45 60 80 5 10 15 5 10 5 5 15 30 45 60 98 -2.0-1.5-1.0-0.50.00.51.01.52.0 Axis 1 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Axis 2 15 10 20 80 20 30 60 Figure 3-4. Results of the Prin cipal Component Analysis, A) loadings of the different phosphorus compounds measured by 31P NMR and P fractionati on (n = 25), and B) the plot of the scores of the sites and se diment depth (numbers cm) from Lake Annie (blue circles), Lake Okeechobee: M9 (re d squares), M17 (brown diamonds), KR (orange crosses), and Lake Apopka (green triangles). B A

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99 CHAPTER 4 ENZYME ACTIVITIES IN SEDIMENTS OF SUBTROPICAL LAKES Introduction Sediment phosphorus (P) is present in both i norganic and organic forms. Organic P and cellular constituents of the biota represent 90% of total phosphorus (TP) in freshwater ecosystems (Wetzel 1999). These organic P compounds present in sediments must be hydrolyzed before their uptake by microorganisms (Chros t 1991; Sinsabaugh et al. 1991). Organic P is hydrolyzed by enzymes produced by microbial communities (Gchter et al. 1988; Davelaar 1993; Gchter and Meyer 1993), and the product of this enzymatic hydrolysis is orthophosphate which is readily used by microorganisms (Barik et al. 2001). Conseque ntly the breakdown of organic P compounds through enzyme activity and release of labile inorganic P is an important component of P processing in sediments. Enzyme production can be induced by the presence of organic P and low levels of bi oavailable inorganic P (Kuenzler 1965; Aaronson and Patni 1976). On the other hand, high levels of inorganic P in hibit the synthesis of enzymes (Torriani 1960; Lien and Knutsen 1973; Elser and Kimmel 1986; Jasson et. al. 1988; Ba rik et al. 2001). Three main groups of hydrolytic enzymes are responsible for phosphate release: non specific and/or partially specific phosphoesterases (mono and diesterase), nucleotidases (mainly 5-nucleotidase), and nucl eases (exo and endonucleases ) (Chrost and Siuda 2002). Phosphomonoesterases (PMEase) are nonspecifi c enzymes that hydrolyze phosphate monoester, and are reported to be produced by several microorganisms (e.g., bacteria, algae, fungi, and protozoan) that are found in the water column a nd sediment of lakes. Nonspecific PMEases are divided into two groups, depending on the pH at which they exhi bit maximum activity, alkaline (pH 7.6-10) and acid (pH 2.6-6.8) (Si uda 1984). Both can be found inside or outside the cell, and the same cell can produce both alkaline and acid PMEase (Siuda 1984). Although both PMEase

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100 activities have been repo rted to be regulated by availability of orthophosphate, acid PMEase is usually regarded as a constitutive enzyme (Siuda 1984; Jasson et al. 1988). The production of constitutive enzymes is neith er repressed nor stimulated by high or low orthophosphate availability in th e environment. Its production is related to P concentration and demand inside the cell (Siuda 1984, Jasson et al. 1988). Jasson et al. (198 1), however, suggested that in acidified lakes, acid PMEa se may have a similar role to that of alkaline PMEase in neutral systems, as its production is also inhibited by orthophosphate. In aquatic systems, alkaline PMEase is by far the most studied enzyme, pr obably due to the high num ber of systems with neutral pH, that are inappropriate for preserva tion of extracellular acid PMEase (Siuda 1984). Another important phosphatase is phosphodiesterase (PDEase) that hydrolyzes phosphate diester and is known to degrade phospholipids and nucleic acids (Hino 1989; Tabatabai 1994; Pant and Warman 2000). It is the least studied enzyme in freshwater ecosystems. Few studies have reported on the occurrence and distribution of phosphatases or other organic P hydrolyzing enzymes in sediments or their association with sediment bacteria (Wetzel 1991; Chrost and Siuda 2002). As sediment P is important in P cycling in lakes, and it is well known that microorganisms can influence the organic P dynam ics in sediments, the study of different P compounds and associated enzymes is important for understanding P cycling in sediments. The primary hypothesis of this study is that enzyme activities will be higher in recently accreted sediments (surface) as compared to olde r sediments (sub-surface) and will be related to P forms and availability, as well as to microbial community activ ity. The specific objectives of this study were: (i) measure ver tical distribution of PMEase a nd PDEase activities and relate them to microbial activity in sediments; and (i i) to explore relations hips between different phosphorus compounds and enzyme activity.

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101 Materials and Methods Study Sites Three Florida (USA) lakes ranging in trophic state were selecte d. Lake characteristics were described in Chapter 2. The characteristics a nd location of sampled s ites and field sampling procedures were described in Ch apter 3 (Table 3-1, Figure 3-1). Water Characteristics Water temperature (C), electr ical conductivity (EC), pH, a nd dissolved oxygen (DO) were measured with a YSI 556 Multi-Probe Sensor (YSI Environmental, Yellow Springs OH) at different depths (Table 4-2). Greater depths in the Lake A nnie water column, C, EC, and DO were measured using a handheld YSI 85 (YSI Inc., Yellow Springs, OH). Water samples were collected from various depths at each site using a Van Dorn bottle. Water column nutrient concentrations were measured using U.S. EPA methods (EPA 1993). Total Kjeldahl nitrogen (TN) was measured by digestion with concentrated sulfuric acid (H2SO4) and Kjeldahl salt catalyst, and determin ed colorimetrically (Method 351.2). Total phosphorus (TP) was digested with 11N H2SO4 and potassium persulfate (Method 365.1). Water samples were filtered through a 0.45 m me mbrane filter and filtrate was analyzed for dissolved reactive phosphorus (DRP ) (Method 365.1), ammonium-N (NH4-N) (Method 351.2), and dissolved organic carbon (DOC) (a utomated Shimadzu TOC 5050 analyzer (Method 415.1). Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Before each analysis, samples were homogenized and sub-samples taken. Sediment bulk density, pH, organic matter (LOI-loss on ignition), and tota l phosphorus were measured and described in a previous study (Chapter 3).

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102 Water extracts were centrifuged at 10,000 x g for 10 min, filtered through a 0.45 m membrane filter, and analyzed for dissolved reactive P (DRP) and DOC with the same method used for water samples. Enzyme Activity Enzyme activities including PMEase and PDEase were determined colorimetrically using as substrate p -nitrophenyl phosphate and bisp -nitrophenyl phosphate, respectively (Tabatabai 1994; Alef et al. 1995), both from Sigma Chemi cal Co (St Louis, MO). Assays were conducted using three replicates and a control for each sample to account for non-enzymatic color development. As PMEase activity depends on pH range (Tabatabai 1994 ; Alef et al. 1995), alkaline phosphatase activity was measured in Lake Okeechobee and Lake Apopka; while the acid phosphatase activity was measured in Lake Annie sediments (see Table 3-2). A known amount of wet sample, 0.5 g for high organic sedi ment, and 1 g for mineral sediment, was added to polypropylene centrifuge bot tles with the artificial substrate (1 ml of 0.05 M p -nitrophenyl phosphate for PMEase, and bisp -nitrophenyl phosphate for PDEase), toluene (to inhibit microbial growth during measurement), a pH buf fer (pH = 11 for alkaline, pH = 6.5 for acid phosphatase, and pH = 8 for PDEase) and incubate d at 37 C for 1 hour. Enzymatic activity was stopped after incubation by addi tion of 1 mL of 0.5 M CaCl2 and 4 mL 0.5 M NaOH (for PMEase) and 0.1 M /0.5 M THAM/NaOH (THAM: tris(hyd roxymethyl)aminomethane) extractant solution (for PDEase). Samples we re centrifuged and filtered through a Whatman # 1 paper filter and analyzed at 420 m using a UV-VIS spectrophotometer (Shimadzu Model UV 160) (Tabatabai 1994; Alef et al. 1995). Absorbance was compar ed with standards. Control values were subtracted from sa mple values to account for non-en zymatic substrate hydrolysis.

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103 Statistical Analysis Sediment P compounds and anaerobic microbial respiration (microbial activity) methods and data was reported in other studies (Chapter 3 and 5 respectively). The combined data was used to explore relationships between these diffe rent variables and enzyme activity. A Pearson correlation analysis was performed to determin e relations among P forms and enzyme activity. Regression analyses were conducted to compare se diment P forms and activities of enzymes and microbes. A Principal Component Analysis (P CA) was performed to address relations among variables, and how they relate to each lake and sediment dept h. All statistical analyses were conducted with Statistica 7.1 (StatSoft 2006) software. Results Water Characteristics Lake Annie displayed strong summer therma l stratification (Tab le 4-1). Electrical conductivity reflected trophic state conditions of the lakes, with higher values in Lake Apopka, followed by Lake Okeechobee and Lake Annie. Di ssolved oxygen was highest in Lake Apopka, followed by Lake Annie and Lake Okeechobee s ites M9 and M17. Lake Okeechobee site KR had the lowest values. Lake A nnies water column was anoxic below 5 m depth (Table 4-1). Lake Annie water column pH was lower than pH in the other lakes, and decreased with depth. Both Lake Okeechobee (except site KR) and La ke Apopka had pH values near neutral or alkaline (Table 4-1). Highest DOC values were found at the sediment surface in Lake Apopka (53.9 mg L-1), while all other DOC values in the lakes ranged from 12.3 to 25.1 mg L-1. Surface water TP and DRP were highest in Lake Okeechobee, while TN was highest in Lake Apopka. Although Lake Annie displayed generally low TP, TN and NH4-N concentrations in the water column, high values were registered at 20 m depth, just above the sediment surface (Table 4-2).

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104 Sediment Properties Sediment properties (i.e., pH bulk density, and organic matte r) and concentrations of TP and different P compounds were reported in a pr evious study (Chapter 3). Acidic pH conditions were observed in Lake Annie se diments and neutral to alkaline values in Lake Okeechobee and Lake Apopka deposits (Chapter 3). Water extractable organic C (expressed as DOC) displayed different distributions with se diment depth among lakes. In Lake Annie, sediment DOC increased with depth. A similar trend was also observed in Lake Okeechobee M17 sediments. However, DOC distribution in La ke Apopka sediments decreased with depth and no clear trend was noted at sites M9 and KR (Table 4-3). C oncentration of water extractable DRP did not present a clear pattern with depth in the profile at any of the site s, although deeper sections in Lake Annie and Lake Apopka had higher concen trations than upper sections (Table 4-3). Enzyme Activity Lake Okeechobee sediments had very low enzy me activities for both PMEase (0.3-4.5 mg p -nitrophenol g-1 dw h-1) and PDEase (0.4-5.7 mg bisp -nitrophenol g-1 dw h-1) (Figure 4-1A, B). Phosphomonoesterase (PMEase) and phosphodiestera se (PDEase) activit ies decreased with sediment depth at all sites (Figure 4-1A, B). Lake Annie sediments had the highest PMEase activity compared with sediment s of the other lakes, while PD Ease was higher in Lake Apopka sediments. Lake Okeechobee sediments had higher PDEase activity than PMEase, while Lake Annie and Lake Apopka had higher PMEase activity. Phosphomonoest erase activity was strongly correlated ( r > 0.7) with phosphate monoester, lab ile organic P, FAP, and HAP, and increased linearly with phosphate monoester concentration (Figure 4-2). Phosphodiesterase activity showed strong correlation ( r > 0.7) with MBP, lipid-P, DNA-P, and pyrophosphate, and increased linearly with phosphate diester (i.e ., Lipid-P, DNA-P) concentration (Figure 4-3).

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105 Pore water DOC and DRP concentrations in Lake Annie and Apopka were measured and described in another study (Chapter 5, Table 5-2), and the data were used to verify if there was a relation between enzyme production and DOC and DRP pore water concentration. In Lake Annie, there was no relationship between either acid PMEase or PDEase activities and pore water DOC and DRP. For Lake Apopka, however, there was an inverse relationship between DRP pore water concentration and both PMEase and PDEase (Figure 4-4A). Enzyme activities in Lake Apopka showed a strong linear relati onship with pore water DOC (Figure 4-4B). Anaerobic respiration (CO2 production rates) data was desc ribed elsewhere (Chapter 5), and the values were used to address relations between anaerobic respiration and enzyme activity. Microbial activity had a positive relationship with both PMEase and PDEase activities. In Lake Annie, Lake Apopka, and Lake Okeechobee site-M9 sediments, microbial activity had a significant linear relationship with both enzyme activities (Figure 4-5A, B, E). The same relationship was observed between microbial activity and PDEase activity in site KR sediments, although no relationship was observed for PMEase activity (Figure 4-5D). In peat sediments (site-M17) no relationship between enzyme and microbial activities was observed (Figure 4-5C). Two Principal Component Analyses (PCA) were conducted. One analysis used data from 31P NMR, chemical fractionation (Chapter 3), and microbial (Chapter 5) and enzyme activities ( n = 25) (Figure 4-6). The other used data from ch emical P fractionation (Cha pter 3), and microbial (Chapter 5) and enzyme activities ( n = 107) (Figure 4-7). The firs t PCA had 38.6% of the data variability explained by Axis 1. Axis 2 explaine d 30.3% of the data vari ability (Figure 4-6A). Lipid-P, DNA-P, anaerobic resp iration, microbial biomass P (MBP), fulvic acid P (FAP), PMEase and PDEase activity were the variab les selected by Axis 1, while orthophosphate, phosphate monoester and residual P (Res. P) were selected by Axis 2. Phosphomonoesterase

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106 activity was placed with labile organic P (lab ile-Po), FAP, humic acid P (HAP), and phosphate monoester. Phosphodiesterase activ ity was placed with anaerobi c respiration, MBP, DNA-P and lipid-P. The position of the sites and sediment dept h in relation to the variable loadings in the PCA showed that the three lakes are separated into different gr oups (Figure 4-6B). Lake Apopka placed in the PDEase cluster and Lake Annie in the PMEase cluster. Lake Okeechobee was further from any of these clusters (Figure 4-6B). In the second PCA, using chemical P fracti onation, Axis 1 explained 41.1% of the data variability and the variables selected were la bile-Po, FAP, HAP, anaer obic respiration, PMEase, and PDEase activity. Axis 2, with 21.1% of the da ta variability explaine d, selected MBP (Figure 4-7A). Again FAP, HAP and labile-Po were placed with PMEase activity, while MBP was placed with PDEase activity and anaerobic respir ation. The position of the sites and sediment depth in relation to the variables loadings in PCA-2 showed results similar to the first PCA-1 (Figure 4-7B). Samples from the three lakes we re separated into diffe rent groups, and Lake Apopka placed in the PDEase cluster a nd Lake Annie in the PMEase cluster. Discussion There are few studies reporting on P related enzy me activity in freshwater sediments, and they focus on PMEase activity. The PMEase values found in the present study in Lake Annie and Lake Apopka are much higher than those obser ved in other freshwater systems. Lake Okeechobee sediment PMEase values although being much smaller th an the other lakes of this study, had similar or higher values than the one s reported for other freshwater sediments. In shallow eutrophic Lake Donghu (China), PMEase activity in surface sediments was much lower than the values detected in Lake Annie and Lake Apopka, but higher than the values detected in Lake Okeechobee sediments (17.6-44.05 mg p -nitrophenol g-1 dw h-1) (Yiyong et al. 2001). Wobus et al. (2003) in a study of sediments of rese rvoirs of different troph ic states in Germany

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107 reported higher values for PMEase in oligotrophic Muldenberg, (17.2 mg p -nitrophenol g-1 dw h1), than mesotrophic Saidenbach (0.8 mg p -nitrophenol g-1 dw h-1) and eutrophic Quitzdorf (0.17 mg p -nitrophenol g-1 dw h-1). In a study of shallow, nutrient rich, freshwater sediments Boon and Sorrell (1991) reported values of PMEase that ranged from 1.35-1.75 mg p -nitrophenol g-1 dw h1). Small values of PMEase were reported by Ba rik et al. (2001) for 12 different nutrient rich fishpond sediments (2559 g p -nitrophenol g-1 dw h-1). Wright and Reddy (2001) reported PMEase values in soils of a freshwater wetland (Florida Everglades) ranging from 5.0 mg p nitrophenol g-1 dw h-1 in P-impacted sites to 25 mg p -nitrophenol g-1 dw h-1 in non-impacted sites. Enzyme activity decreased with greater sediment depth in all lakes, a re sult also reported in other studies. Wobus et al. (2003) found that PMEase activity declin ed with sediment depth in a mesotrophic reservoir in Germany. The same resu lt was reported by Sinke et al. (1991) for Loosdretch Lake in the Netherlands. The decrease of enzyme activity with depth reflects lower microbial biomass (MBP) (Chapter 3), and is accompanied by decreased anaerobic respiration (Chapter 5). A positive correlation between PMEase activity and microbial density was found in several ecosystems (Reichart 1978; Kobori and Taga 1979; Davi s and Goulder 1993; Massik and Cotello 1995; Barik et al. 2001). Enzymes can be produced by several organisms. Enzymes produced by algae predominate in the water column, while in sediments bacterial enzymes are dominant (Siuda 1984). Strong corr elations between enzyme activit ies and anaerobic respiration indicate (PMEase r = 0.65, PDEase r = 0.91) that bacterial enzyme s dominate these sediments (Figure 4-5). Only at sites M17 and KR was PMEa se activity not related to anaerobic respiration (Figure 4-5C, D), indicating that in these sites other organism s (i.e., algae) are producing

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108 hydrolytic enzymes. Moreover, the demand for av ailable P can be low in these sediments, resulting in low enzyme activity. Both enzyme activities, however, are related not only to microbial biomass and activity, but rather, they reflect different phosphorus com position and availability in these lakes. Wobus et al. (2003) reported higher activ ities of PMEase in an oligotr ophic reservoir than in meso and eutrophic reservoirs. In my study, highest PMEa se activity was found in the oligo-mesotrophic lake (Lake Annie). Phosphomonoest erase activity was lowest in eutrophic Lake Okeechobee, but displayed intermediate activity in hypereutrophic Lake Apopka. Lake Okeechobee had high concentrations of labile inor ganic P (Chapter 3) and lowest activities for both PMEase and PDEase. Lake Annie had high concentrations of labile-Po, FAP and HAP fractions and phosphate monoester, and had high PMEase activit y. Lake Apopka had high concentrations of MBP and phosphate diester (lipids and DNA), as we ll as PDEase activity. Statistical analyses support these results. There were higher correl ation coefficients between PMEase and phosphate monoester ( r = 0.86), labile-Po ( r = 0.83), FAP ( r = 0.86) and HAP( r = 0.89), while PDEase had high correlations with ph osphate diester (lipid-P r = 0.89, DNA-P r = 0.93) and MBP ( r = 0.88). Linear regression analyses show ed strong significant relations between PMEase and phosphate monoester, and between PDEase and phosphate dies ter concentrations (Fig ures 4-2, 4-3). These results were corroborated by the two PCAs positioning these P form s and their respective related enzymes as clusters (Figure 4-7, 4-8). Also in relation to the PCAs, if enzyme production were only a reflection of microbial biom ass and activity, both enzymes, CO2 production and MBP would all cluster together, but ther e is clear separation of these vari ables. These results show that although microbial activity (CO2), microbial biomass (MBP) and enzyme activities are related, as expected, different P forms in sedime nts strongly influence enzyme production.

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109 The lack of relationship between pore water SR P and enzyme activities in Lake Annie can be attributed to different mechanisms of en zyme production and sedime nt properties. Acid PMEase is usually regarded as a constitutive en zyme, and its production is not repressed by high orthophosphate availability (Siuda 1984; Jasson et al. 1988). In acidified lakes, however, acid PMEase seem to have a similar role to alkaline PMEase in neutral syst ems (Jasson et al. 1981). I measured acid instead of alkaline PMEase in Lake Annie sediments to evaluate the maximum potential enzyme activity. Measurement of alkaline PMEase activity would probably be underestimated in Lake Annie, as it would be influenced by pH rather than P availability (Chapter 3). In a study of acid PMEase in the wa ter column of acid Lake Grdsjn, Sweden, with high aluminum (Al) and iron concentration, Jasson (1981) showed that high acid PEMase activity was induced as a response of the plankt on community to high Al concentration that blocks substrates by reacting with phosphate. Lake Annie se diments (central site) were characterized as having high Fe (3640 mg kg-1) and Al (34640 mg kg-1) concentration (Thompson 1981). In Lake Annie, high PMEase activity, unrelated to P availability, can be a result of several factors: 1) high Al and Fe concentration in it s sediment, 2) high P demand inside microorganism cells, 3) or presence of more stable phospha te monoester (i.e., inositol phosphate). Some phosphate monoesters (e.g., inositol phosphate) are more resistant to degradation than phosphate diester (Makarov et al. 2002), probably due to higher charge density, which enables the phosphate monoester to form strong complexes wi th cations, protecting them from degradation (Celi et al. 1999). Inositol phosphate which is considered to be st able in soils, was present in Lake Annie spectra (Chapter 3, Figure 3-3A). Moreover, according to Turner and Haygarth (2005), both PDEase and PMEase are necessary for release of free phosphate from phosphate

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110 diester. Initial hydrolysis by PDEase releases phosphate monoester and stimulates the production of PMEase. Lake Apopkas PMEase production seems to be controlled by other mechanisms, perhaps related to both DOC and DRP av ailability. In a stu dy of alkaline phosphatase activity in Lake Apopka sediments (topmost 30 cm, n = 6) Newman and Reddy (1993) reported different results. Phosphomonoesterase activity had an inverse correlation with labile-Po and HAP, and no correlation with DRP. My study f ound that there is an inverse relation between pore water DRP and PMEase activity, and a positiv e correlation with organic P form s (including HAP). I used the same method used by Newman and Reddy (1993) but my sample size was larger. Several studies have shown that there is an inverse correlation between PMEase activity and DRP in sediments (Jasson et. al. 1988; Barik et al. 2001; Wobus et al. 2003; Jin et al 2006; Rejmankova and Sirova 2007). However, Siuda and Chrost (2001) conclude d from controlled experiments that even during periods of high concentration of orthophospha te in lake water, PM Ease is still produced, and exhibits activity. They suggest ed that PMEase activity of b acteria is used for organic P hydrolysis and uptake of associated organic C moieties, concluding that bacterial PMEase contributes substantially to DOC decomposition in lake water. This seems to be the case of PMEase production in Lake Apopka, as I found a high correlation between enzyme activity and DOC concentration. Conclusions This study showed that PMEase and PDEase act ivities were related to sediment microbial biomass and activity, as well as to the different P composition a nd availability. Enzyme activity decreased with greater depth in all lakes, reflecting lower microbi al biomass and activity. Strong correlations between enzyme activities and anaerobi c respiration indicated that bacterial enzymes dominate these sediments. Differe nt P forms in sediments were also affecting enzyme activity.

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111 Highest PMEase activity was found in the o ligo-mesotrophic lake (Lake Annie) with high concentrations of labile-Po, FAP and HAP. Lake Okeechobee had high concen trations of labilePi and lowest activities of both PMEase and PD Ease. Lake Apopka had high concentrations of MBP and phosphate diester (lipids and DNA), as well as PDEase activity. The mechanisms controlling PMEase activity, however, seemed to vary according to the difference in lake sediment. In Lake Annie, high PMEase activity was unrelated to DRP availability, and probably was c ontrolled by factors such as high Al and Fe concentrations, high P demand inside microorganism cells, and/or pres ence of more stable phosphate monoester (i.e., inositol phosphate) in th e sediment. Lake Apopkas PMEase production seemed to be controlled by both DOC and DRP availabilit y. There was an inverse relati on between pore water DRP and PMEase activity, and a positive relation between pore water DOC and PMEase activity. In Lake Apopka sediments production of PMEase by the mi crobial community was related to organic P hydrolysis, and uptake of asso ciated organic C moieties.

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112 Table 4-1. Measured parameters in the water column of Lake Annie, Lake Okeechobee, and Lake Apopka. C: water temperature in Ce lsius, EC: electri cal conductivity, DO: dissolved oxygen. DO Lake Site Depth (m) C EC (S cm-1) pH (mg L1) 0.5 30.4 42.0 6.2 6.3 1 30.2 41.9 5.1 6.4 2 29.2 40.3 4.8 4.9 3 28.8 40.7 4.7 4.3 4 27.2 40.3 4.6 2.4 5 25.8 40.3 4.6 0.3 6 22.3 38.6 4.4 0.2 7 20.9 37.3 4.4 0.2 8 19.4 36.3 4.2 0.2 9 18.7 35.6 3.9 0.1 10 18.2 35.3 3.7 0.1 11 17.9 35.1 NM 0.1 12 17.6 36.5 NM 0.1 13 17.4 37.2 NM 0.1 Annie Central 14 17.3 37.8 NM 0.1 0.5 29.8 385 7.9 6.4 1 29.5 385 7.8 6.5 1.5 29.1 385 7.8 6.6 2 29.1 385 7.8 6.5 3 29.1 385 7.7 6.4 M9 4 29.0 385 7.6 6.3 0.5 28.8 320 7.5 6.1 1 28.8 320 7.6 6.3 1.5 28.7 320 7.6 6.3 M17 2.5 28.7 320 7.6 5.1 0.5 31.0 144 6.0 1.8 1 30.8 143 6.0 1.8 1.5 30.7 143 5.9 1.9 2 30.7 143 5.9 1.9 Okeechobee KR 3 30.7 142 5.9 1.7 0.5 27.8 455 7.6 9.2 1 26.6 443 7.6 8.7 1.5 26.4 471 6.7 7.4 Apopka West 2 26.3 652 6.4 3.0 NM: not measured

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113 Table 4-2. Concentration of TP: total phosphorus DRP: soluble reactiv e phosphorus, TN: total nitrogen, NH4-N: ammonium-N and DOC: dissolv ed organic carbon in the water column of Lake Annie, Lake Okeechobee, and Lake Apopka. TP DRP TN NH4-N DOC Lake Site Depth (m) (g L1) (mg L1) 0.5 22.8 9.5 1484 51.8 14.3 1 22.0 7.8 1374 102.5 15.2 2 16.1 5.5 1264 66.7 15.3 5 10.6 6.5 1154 48.2 12.3 10 7.8 5.2 1099 111.3 12.4 15 8.8 5.5 1319 183.4 13.1 Annie Central 20 144.2 11.8 4955 707.6 14.1 0.5 211.3 90.5 3192 92.3 16.1 1 258.3 93.4 3192 130.9 13.5 M9 4 298.0 87.4 3934 85.8 13.8 0.5 224.6 121.5 2938 53.5 20.2 1 247.4 107.3 2883 69.4 16.0 M17 2 317.7 110.3 4266 58.3 17.6 0.5 113.9 64.3 2717 80.7 20.2 1 118.4 59.9 2717 79.5 18.8 Okeechobee KR 3 206.9 63.5 3436 90.8 20.4 0.5 60.0 15.3 5505 233.9 14.5 1 72.6 10.2 6056 74.9 25.1 Apopka West 2 76.3 7.8 21884 50.0 53.9

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114 Table 4-3. Water extractable dissolved organi c carbon (DOC), and dissolved reactive phosphorus (DRP). (mean SD). **No replicates for SD calculation. Water Extractable DOC DRP DOC:DRP Lake Site Depth (cm) mg kg1 dw (Weight) 5 499 136 1.40 0.5 378 10 430 284 0.66 0.3 618 15 1022 413 1.5 0.5 665 20 1264 441 2.9 0.53 417 30 3298 1676 7.7 2.8 411 45 4268 1105 10.0 3.7 438 60 4693 617 7.6 0.3 617 Annie Central 80 4332 930 9.1 3.0 488 5 424 276 2.4 1.72 187 10 262 21 0.78 0.1 351 15 278 86 0.78 0.4 393 20 204 29 0.82 0.4 306 30 201 52 0.52 0.1 407 45 279 77 0.56 0.4 615 60 275 138 1.4 0.4 221 M9 70 447 124 4.9 0.2 91 5 459 59 0.88 0.3 605 10 854 86 0.25 0.09 3843 15 1219 115 0.24 0.04 5156 20 1559 249 0.31 0.08 5275 30 1648 129 0.28 0.02 5950 M17 40 2010 895 0.34 0.10 5878 5 34 34 0.12 0.03 288 10 29 18 0.07 0.03 570 15 53 33 0.14 0.08 586 20 212 4.3 0.06 0.00 3364 30 244 65 0.05 .03 5652 Okeechobee KR 40 109 ** 0.01 ** 10105 5 2020 199 1.3 0.1 1624 10 1422 209 0.63 0.02 2258 15 1171 240 0.49 0.2 2628 20 1072 38 0.38 0.2 3750 30 1003 240 2.8 0.8 408 45 819 7.2 8.2 5.4 127 60 642 65 12.2 2.7 54 80 733 13 13.3 0.8 55 Apopka West 98 684 ** 7.7 ** 88

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115 020406080100120140160 020406080100120140160 (mg p-nitrophenol g-1 dw h-1) 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 80-100 05101520253035 05101520253035 (mg bis-p-nitrophenol g-1 dw h-1) 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 80-100 Figure 4-1. Enzyme activity of sediment de pth profile in Lake Annie, Lake Okeechob ee: M9, M17 and, KR, and Lake Apopka. A) Phosphomonoesterase and B) phosphodiesterase. B A Depth (cm) Enzyme Activity Annie M9 M17 KR Apopka Annie M9 M17 KR Apopka

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116 0100200300400500600 Phosphate Monoester (mg kg-1 dw) 0 20 40 60 80 100 120 140 Phosphomonoesterase Activity (mg p -nitrophenol g-1 dw h-1) PMEase = 2.04 +0.14 P-Monoesterr2 = 0.75, p < 0.0001 Figure 4-2. Relationship between phosphate m onoester concentration and phosphomonoesterase activity in sediments from Lake Annie (blue circles), Lake Okeechobee M9 (red squares), and Lake Apopka (green triangles). 050100150200250300350400450 Orthophosphate Diester (mg kg-1 dw) 0 5 10 15 20 25 30 Phosphodiesterase Activity (mg bisp -nitrophenol g-1 dw h-1) PDEase = 0.73 + 0.066 P-Diesterr2 = 0.89 p < 0.00001 Figure 4-3. Relationship between phosphate diester concentrati on and phosphodiesterase activity in sediments from Lake Annie (blue circ les), Lake Okeechobee M9 (red squares), and Lake Apopka (green triangles).

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117 048121620 0 20 40 60 80 100 PMEase = 24.51 DRP-0.40 r2 = 0.50 PDEase = 12.6 DRP-0.59 r2 = 0.74 0400800120016002000 0 20 40 60 80 100 PDEase = -6.51 + 0.024 DOC r2 = 0.89, p < 0.00001 PMEase = -17.7 + 0.055 DOC r2 = 0.91, p < 0.00001 Pore water concentration (mg kg-1) Figure 4-4. Relationship between enzyme activity, phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) and A) pore wa ter dissolved reactive phosphorus (DRP) and B) dissolved organic carbon (DOC) c oncentration in sediments from Lake Apopka. A Enzyme Activity (mg bisp or p -nitrophenol g-1 dw h-1) B

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118 020406080100120140160 0 20 40 60 80 100 120 140 160 180 200 220 CO2 vs. PMEase r2 = 0.91, p < 0.00001 CO2 vs. PDEase r2 = 0.93, p < 0.00001 01234567 0 2 4 6 8 10 12 14 16 18 20 22 CO2 vs. PMEase r2 = 0.76, p < 0.00001 CO2 vs. PDEase r2 = 0.88, p < 0.00001 0123456 0 5 10 15 20 25 30 35 40 CO2 vs. PDEase r2 = 0.09, p = 0.208 CO2 vs. PMEase r2 = 0.23, p = 0.043 0.00.20.40.60.81.01.21.41.61.82.0 0 2 4 6 8 10 12 14 CO2 vs. PMEase r2 = 0.19, p = 0.093 CO2 vs PDEase r2 = 0.78, p < 0.00001 0102030405060708090100 50 100 150 200 250 300 350 400 CO2 vs. PDEase r2 = 0.78, p < 0.00001 CO2 vs. PMEase r2 = 0.86, p < 0.00001 Figure 4-5. Relationship of different microbial activities: 1) anaerobic respiration and phosphomonoesterase activity (PMEase), and 2) anaerobic respiration and phosphodiesterase (PDEase) in sediments from A) Lake Annie, Lake Okeechobee sites: B) M9, C) M17, D) KR, and E) Lake Apopka. A B C E Enzyme Activity (mg bisp or p -nitrophenol g-1 dw h-1) Anaerobic respiration (mgCO2-C kg-1 d-1) D

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119 Orthophosphate Inorganic P Residual P Water Ext-DOC Water Ext-P PMEase Water Ext-DOC:P -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (38.6%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (30.3%) CO2PDEase DNA-P Lipids-P Polyphosphate Pyrophosphate MBP Labile Inorganic P Labile Organic P FAP HAP P-monoester 5 10 15 20 30 45 60 80 5 5 10 5 5 10 15 20 30 45 60 -2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5 Axis 1 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Axis 2 10 15 60 80 98 30 Figure 4-6. Results of the Princi pal Component Analysis, A) load ings of different phosphorus compounds measured by 31P NMR and P fractionation, enzymes and microbial activities (n = 25), and B) the plot of th e scores of the sites and sediment depth (numbers cm) from Lake Annie (circles ), Lake Okeechobee: M9 (squares), M17 (diamonds), KR (crosses), and Lake Apopka (triangles). B A

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120 Labile Inorganic P Inorganic P MBP Residual P Water Ext-DOC Water Ext-DOC:P -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (41.1%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (21.1%) CO2PDEase Water Ext-P FAP HAP Labile Organic P PMEase 5 10 20 45 5 45 5 10 20 45 5 30 10 15 20 5 10 -2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5 Axis 1 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Axis 2 10 15 30 80 60 60 45 30 20 15 5 15 20 80 98 60 5 40 30 20 5 15 30-70 10 20 20 30 30 5-15 10-404510 30 45 60 60 60 80 80 20 30 30 15 15 Figure 4-7. Results of the Princi pal Component Analysis, A) load ings of different phosphorus compounds measured by P fractionation, enzy mes and microbial activities (n = 107), and B) the plot of scores for the sites and sediment depth (numbers cm) from Lake Annie (circles), Lake Okeechobee: M9 (squa res), M17 (diamonds), KR (crosses), and Lake Apopka (triangles). B A

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121 CHAPTER 5 MICROBIAL BIOMASS AND ACTIVITY IN SEDIMENTS OF SUBTROPICAL LAKES Introduction Phytoplankton and/or heterotr ophic bacteria are the major drivers of carbon (C) and nutrient cycling in the water co lumn of lakes, while the heterotrophic bacteria dominate in sediments. Allochthonous and autochthonous partic ulate organic matter in the water column is deposited in the sediment. Water column depth af fects the quality of or ganic material reaching the sediment. In deep lakes, detrital organic matter undergoes intense decomposition in the water column, due to the prolonged period of settling. Consequently low amount s of labile organic C reach the sediment (Suess 1980; Meyers 1997). In shallow lakes, the supply of labile C and nutrients can be higher than in deep lakes, and th e latter often can have more refractory organic matter (Suess 1980; Meyers 1997). As bacteria are the dominant group in sedi ments, organic compounds and associated nutrients supplied to the sediment surface ar e mineralized through heterotrophic decomposition (Gchter and Meyer 1993; Capone and Kiene 1988). Complete oxi dation of a broad range of organic compounds occurs through the sequential activity of a variety of anaerobic bacteria (Capone and Kiene 1988). In high depositional environments, such as eutrophic, or deep thermally stratified lakes, organic cont ent in sediments is often high, oxygen (O2) consumption occurs rapidly, and O2 is depleted several millimeters below the sediment water interface (Jrgensen 1983; Jrgensen and Revsbrech 1983). In these systems, facultative and obligate anaerobic communities dominate. In methanogenic habitats, i.e., in the absence of inorganic electron acceptors, different gr oups of microorganisms participat e in decomposition of organic matter as no single anaerobic microorganism can completely degrade organic polymers (Zinder 1993, Megonigal et al. 2004). Fermenting bacteria hydrolyze organic polymers through enzyme

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122 production and further break down monomers to alcohols, fatty acids, and hydrogen (H2). Alcohols and fatty acids are degraded by syntrophic bacteria into acetate, H2, and carbon dioxide (CO2), which is used as substrate by methanoge ns (Zinder 1993, Conrad 1999). Consequently, CO2 and methane (CH4) are important end products of an aerobic organic matter decomposition and such gas production can be used as a m easure of microbial activity in sediments. Several factors limit bacterial metabolism in sediments, i.e., temperature, biodegradable organic C, nutrients, and electr on acceptors. Most studies of microbial activity in sediments focus on C limitation and the effect of electr on donors or acceptors in production of CO2 and/or CH4 (Capone and Kiene 1988; Schulz and Conrad 1995; Maassen et al. 2003; Thomsen et al. 2004). Little work has been done relating production of CO2 and CH4 with biogeochemical properties of sediments such as nutrient availabil ity. Studies in the water column of lakes have shown that several factors can limit bacteria l metabolism (Gurung and Urabe 1999; Jasson et al. 2006). Although it has been generally accepted th at the heterotrophic community is C/energy limited, recent studies have shown that inorganic nutrients, especially pho sphorus (P) can be the most limiting nutrient for the bacterial comm unity (Gurung and Urabe 1999; Vadstein 2000; Olsen et al. 2002; Vadstein et. al. 2003; Smith and Prairie 2004; Jasson et al. 2006). Reviewing data from freshwater ecosystems, Vadstein (2000) showed that P limitation is a common phenomenon. Phosphorus limitation occurred in 86% of the cases, while nitrogen or C limitation was identified in 15% and 20%, respectively (p ercentages add up to more than 100% due to methodological aspects, Vadstein 2000). Hetero trophic microbial metabolism can be limited by a single factor or multiple variables. Limita tion varies among lakes and depends on lake characteristics and biogeochemical properties of sediments.

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123 The primary hypothesis of this study is that mi crobial activities will be higher in recently accreted sediments (surface) as compared to older sediments (sub-surface). The second hypothesis is that nutrient limitation will vary am ong sediments from different lakes, and will be related to sediment biogeochemical properties (i .e., nutrient concentration and availability). The objectives of this study were to: (i) determine stratigraphi c biogeochemical properties in sediments from three subtropical lakes of differe nt trophic status and evaluate how they are related to microbial biomass and activity; (ii) m easure microbial biomass at different depths in the sediment from the three different lakes, and te st whether there is nutr ient limitation; and, (iii) measure sediment microbial activity and establis h relationships with nut rient concentration and availability. Materials and Methods Study Sites Three Florida (USA) lakes ranging in trophic state were selecte d. Lake characteristics were described in Chapter 2. The characteristics a nd location of sampled s ites and field sampling procedures were described in Ch apter 3 (Table 3-1, Figure 3-1). Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Before each analysis, samples were homogenized and sub-samples taken. Sediment bulk density, pH, organic matter (LOI-loss on ignition), and total phosphorus (TP) were measured and described in a previous study (Chapter 3). Total carbon (TC) and total nitr ogen (TN) were determined using a Flash EA1121 NC soil analyzer (Therm o Electron Corporation). Extractable C, N and P Due to high water content of Lake Annie and Lake Apopka sediments, pore water was extracted (centrifuged at 10,000 x g for 10 min) pr ior to C and nutrient extractions. Pore water

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124 TP, dissolved reactive P (DRP), ammonium-N (NH4-N), total nitrogen (TN), and dissolved organic carbon (DOC) were measured using U. S. EPA methods (EPA 1993). Total Kjeldahl nitrogen (TN) was measured by digesti on with concentrated sulfuric acid (H2SO4) and Kjeldahl salt catalyst, and determined co lorimetrically (Method 351.2). Total P was digested with 11N H2SO4 and potassium persulfate (Method 365.1). Wa ter samples were filtered through a 0.45 m membrane filter and filtrate wa s analyzed for DRP (Method 365.1), NH4-N (Method 351.2), and DOC (automated Shimadzu TOC 5050 analyzer (Method 415.1). Microbial Biomass C, N and P Microbial biomass carbon (MBC), nitroge n (MBN), and phosphorus (MBP) were measured with the chloroform fumigation-extr action method (Hedley and Stewart 1982; Brookes et al. 1985; Vance et al. 1987; Horwath and Paul 1994; Ivanoff et al. 1998). Briefly, sediment samples were split in two: one sample was treate d with alcohol-free chloroform (0.5 mL) to lyse microbial cells, placed in a vacuum desiccator, and incubated for 24 hrs. The duplicate sample was left untreated. Both sets were extracted with 0.5 M K2SO4 for MBC and MBN, and with 0.5 M NaHCO3 (pH = 8.5) for MBP, using a 1:50 dry se diment-to-solution ratio. Extracts from MBC and MBN samples were centrifuged at 10,000 x g for 10 min and filtered through Whatman # 42 filter paper, and 5 mL of the extracts were subjected to Kjeldahl nitrogen digestion (for MBN) and analyzed for total Kj eldahl-N colorimetrically using a Bran+Luebbe TechniconTM Autoanalyzer II (Method 351.2, EPA 1993). MBC extracts were acidified (pH < 2) and analyzed in an automated Shimadzu TOC 5050 analyzer (Method 415.1, EPA 1993). Extracts from MBP samples were filtered usi ng a 0.45 m membrane filter and digested for TP with sulfuric acid and potassium persulfate. Solu tions were analyzed by colorimetry, determined by reaction with molybdate us ing a Bran+Luebbe TechniconTM Autoanalyzer II (Murphy and Riley 1962; Method 365.1, EPA 1993) Microbial biomass (C, N and P) was determined by the

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125 difference between treated (with chloroform) and non-treated samples. Untreated samples represent extractable orga nic carbon (Ext-C), extract able labile nitrogen (E xt-N), and extractable labile phosphorus (Ext-P). Undigested N extracts were analyzed for ammonium-N (NH4-N) (Method 351.2, EPA 1993), and represent extractable ammonium-N (Ext-NH4-N). The difference between Ext-N and Ext-NH4-N represents extractabl e labile organic nitrogen (Ext-ON) (Mulvaney 1996). Undigested P extracts were analyzed for solubl e P as described previ ously, and this fraction represents labile inorganic P (E xt-Pi). The difference between ExtP and Ext-Pi, represents labile organic phosphorus (Ext-Po) (Ivanoff et al. 1998). Microbial Activity Anaerobic microbial respiration (CO2) and methanogenesis (CH4) were quantified by incubating a known amount of wet sediment equivale nt to 0.5 g of dry weight, in 5 mL of DI water at 30 C under anaerobic condi tions. Samples were placed in a glass vial and closed with rubber stoppers and aluminum crimp s eals. Samples were purged with N2 gas to achieve anaerobic conditions. Gas samples were obtained at 2, 4, 7, and 10 days and CO2 released was measured by gas chromatography using a Shim adzu 8A GC-TCD equipped with Poropak N column (Supelco Inc., Bellefonte, PA), using He as a carrier gas. Methane was measured with the Shimadzu gas chromatograph-8A fitted w ith a flame ionization detector (110 C), N2 as the carrier gas and a 0.3 cm by 2 m Carboxen 1000 column (Supelco Inc., Bellefonte, PA) at 160 C. Headspace pressure was determined with a di gital pressure indicator (DPI 705, Druck, New Fairfield, CT). Concentrations of CO2 and CH4 were determined by comparison with standard concentrations and produc tion rates were calculate d by linear regression ( r2 > 0.95). At time zero of the incubation experiment a sub-sample of sediment was extracted with 25 mL of DI water, shaken for 1 hour, centr ifuged at 10,000 x g, and passed through a 0.45 m

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126 membrane filter. After the 10-day incubation period using the same extraction procedure, sediments were extracted. Disso lved organic carbon (DOC), NH4-N and DRP were measured as described above. Statistical Analysis A t test was performed to evaluate whethe r there was a statis tical difference in concentration (increase or decrease) of DOC, NH4-N and DRP during in cubation (time zero versus time ten) for each site and sediment depth. A Principal Component Analysis (PCA) was performed to address relations between the variab les and in each lake and how they vary with sediment depth. All statistical analyses were conducted with Statisti ca 7.1 (StatSoft 2006) software. Results Sediment Properties Sediment properties (i.e., pH bulk density, and organic matte r) and TP concentration were reported in a previous study (C hapter 3). Sediment TC was hi ghest in Lake Okeechobee site M17, reflecting its peat nature. Next highest in TC was Lake Apopka, followed by Lake Annie, and sites M9 and KR in Lake Okeechobee (Table 51). Lake Annie alone di splayed a decrease in TC with greater sediment depth. Total N also de clined with depth in Lake Annie sediments and showed a generally similar trend to the core fr om Lake Okeechobee site M9. Total N was highest in Lake Apopka sediments, followed by Lake Ok eechobee site M17. Neither core showed a clear trend in TN concentration with depth (Table 5-1) Total C:N ratios were similar in all sediments. Total C:P ratios were highest in M17 sediments. There was no clear trend in total C:P and N:P ratios in Lake Annie and Lake Okeechobee mud sediments (M9-site). Sites M17 and KR (Lake Okeechobee) and Lake Apopka showed an increase in total C:P and N:P rati os with depth (Table 5-1).Extractable and Micr obial Biomass C, N and P

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127 Lake Apopka had the highest concentration of DOC, NH4-N and TN pore water concentration (Table 5-2). Lake Annie had lo w DRP pore water concen tration (Table 5-2). Extractable organic C was highe st in Lake Apopka and decreased with sediment depth, followed by site M17 in Lake Okeechobee and Lake Annie (Table 5-3). In site M17 there was a general trend of increase of ExtOC with sediment depth. The ot her sediments (Lake Annie, and Lake Okeechobee sites M9 and KR) showed no clear trend of Ext-OC distribution with sediment depth. Lake Apopka also had the highest concentration of Ext-NH4-N and Ext-ON. There was a general increase in Ext-NH4-N concentration with depth in Lake Apopka, Lake Annie and M17 site in Lake Okeechobee. Lake Okeechobee sand (KR and mud (M9) sediments were characterized by low Ext-NH4 +-N concentration. While there wa s a trend of decrease in Ext-ON with depth in Lake Apopka and Lake Annie se diments, the same did not occur in Lake Okeechobee peat sediments (M17) where a general increase with depth was present (Table 5-3). In Lake Okeechobee mud and sand sediments there was no clear trend with depth. Surface sediment labile inorganic P (lab ile-Pi) concentrations were high est in Lake Okeechobee site M9 followed by Lake Annie (Table 5-3). There was a general decrease with sediment depth for Lake Annie and Lake Okeechobee mud sediments. Labile Pi increased with depth in Lake Apopka. Labile organic P (labile-Po) was highest in Lake Annie and Lake Apopka with lower concentrations in all Lake Ok eechobee stations. There was a gene ral decrease in labile-Po with depth in Lake Annie and mud sediments of Lake Okeechobee, while the other sediments did not present a clear trend (Table 5-3). Lake Apopka had the highest concentration of MBC, MBN and MBP, followed by Lake Annie. Among Lake Ok eechobee sites, M9 had higher MBN and MBP, and M17 had the highest MBC. There was a general decrease in microbial biomass with depth in all sediments (Table 5-4).

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128 Microbial Activity Lake Apopka (Figure 5-1E) ha d the highest anaerobic CO2 and CH4 production rates, followed by Lake Annie (Figure 5-1A), and sites M17 (Figure 5-1C), M9 (Figure 5-1B) and KR (Figure 5-2D) in Lake Okeechobee. Both CO2 and CH4 production rates gene rally decrease with sediment depth at all sites except KR (Figure 51D). In KR sediments, there was a peak of CO2 production at 15-20 cm of depth that coincided with an incr ease in organic matter content (Figure 5-1D, Table 3-2). In Lake Annie ther e was a sharp decrease in methane production below 10 cm depth, and CH4 production was low in all sites from Lake Okeechobee. Water extractable DOC, NH4-N, and DRP concentrations before and after 10-day anaerobic incubation were different in the three lakes (Table 5-5). For each depth at each site, a ttest was run for each variable to test if th ere was a significant st atistical difference in concentration with incubation time, and significant differences ( p < 0.05) are in bold (Table 5-5). Dissolved organic C concentration increased dur ing 10-day incubation at all depths of Lake Apopka and in the topmost 10 cm in Lake A nnie (Table 5-5). In Lake Okeechobee M17 sediments, DOC decreased with incubation at all depths (Table 5-5). Sediments from sites M9 and KR showed variable effects of incubati on on DOC concentration at different depths. Concentrations of NH4-N were also higher after incubation in Lake Apopka sediments, and Lake Okeechobee M17 sediments (Table 5-5). The other sediments had variable trends for each depth although there was a general decrease in NH4-N in M9 with time. Dissolved reactive P increased in site M17 and in some depths at site M9 in Lake Okeechobee. In Lake Annie there was a decrease with incubation time in deeper sedi ments (Table 5-5). In Lake Apopka sediments soluble P increased with in cubation time in deeper sediments (Table 5-5).

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129 As MBC, MBN and MBP are hi ghly correlated (MBC x MBN r = 0.97, MBC x MBP r = 0.96, and MBN x MBP r = 0.97), MBC was chosen as a proxy of microbial bioma ss in statistical analyses. High correlations were seen between MBC and anaerobic CO2 ( r = 0.88) and CH4 ( r = 0.85) production rates. High correlations were seen between MBC and extractable N:P ( r = 0.74), and extractable NH4-N:Pi ratios ( r = 0.73), while the other ratios had either low or not significant correlations with MBC. The same re sults were observed for microbial activity. Anaerobic CO2 ( r = 0.63) and CH4 ( r = 0.77) had significant correla tions with extractable NH4N:Pi, and with extractable N:P ratios ( r = 0.62 for both CO2 and CH4). Principal Component Analysis was conducted to see how nutrient availability relates to microbial biomass and activities. Results show ed that 48.6% of the data variability was explained by Axis 1 while Axis 2 explained 22.8% (Figure 5-2A). LOI, TN, Ext-C, Ext-NH4-N, Ext-ON, Ext-N:Ext-P, MBC, CO2, and CH4, were the variables selected by Axis 1. While TP in the negative axis, and Ext-C:Ext-P and Ext-C:Ext-N in the positive axis were selected by Axis 2. The positions of the sites and sedi ment depths in relation to variab le loadings in the PCA showed that the three lakes are sepa rated into different groups (Figure 5-2B). Lake Apopka was positioned with variables selected by Axis 1 a nd had clear separation by sediment depth. Site M17 in Lake Okeechobee also presented some se paration of sediment depth, but to a lower degree, and was placed in the positive axis of variables selected by Axis 2. Lake Annie sediments and site M9 were positioned with la bile-Pi and KR with bul k density, and did not show clear separation by sedi ment depth (Figure 5-2B). Discussion Hypereutrophic Lake Apopka had the highest microbial biomass and activity (both CO2 and CH4) among the study lakes. Other studies that co mpared lakes of different trophic state conditions found that both bacteria l production and respiration, and CH4 production increased

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130 with trophic state (Drabk ova 1990, Casper 1992, Huttunen et al. 2003, Massen et al. 2003, Wobus et al. 2003). In this study, however, o ligo-mesotrophic Lake Annie had higher biomass and activity than eutrophic Lake Okeechobee. Although Lake Annie is oligo-mesotrophic based on water column variables, organic matter conten t and nutrient concentrations are high in the sediment (Table 5-3, Chapter 3). High nutrient concentrations in sedi ments of this oligomesotrophic lake lead to high microbial biomass and activity. There was a decrease in microbial activity with sediment depth which also has been reported in other studies and is related to the decrease in easily degraded organic matter with se diment depth (Rothfuss et al. 1997, Falz et al. 1999, Kostka et al. 2002, Roden a nd Wetzel 2003; Dan et al. 2004). Statistical analysis showed that nutrient (C N and P) concentrations and nutrient ratios influenced the microbial community. This suggests that C, coupled with N and P availability has a strong influence in microbial communities in these lakes sediments. When sediments were incubated, there was a general increase in DOC a nd with time in Lake Apopka (all depths), and Lake Annie surface sediments (0-10 cm). Ammo nium accumulation with time was detected in Lake Apopka and Lake Okeechobee peat sedime nts (site M17). Accumulation of DOC and NH4N can indicate high microbial activity. Maasen et al. (2003) and Wobus et al. (2003) studied sediments from reservoirs of different troph ic states and concluded that high DOC and NH4 + in pore water was related to high microbial activity because both are end products of microbial decomposition. Falz et al. (1999) used NH4 + concentrations as evidence of high microbial activity that correlated with high CH4 production in Lake Rotsee (Switzerland) sediments. Lake Annie sediments had higher microbial activitie s than peat sediments in Lake Okeechobee. Consequently, if DOC and NH4-N accumulation were only a reflection of microbial activities they should have been higher in Lake Annie. In Lake Annie there is no accumulation of NH4-N

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131 and deeper sediment layers had a significant decrease in DOC concentration with time. This suggests that other factors are in fluencing accumulation of DOC and NH4-N in these sediments. Several studies have indicated that DOC accu mulation is a reflection of P limitation in freshwater ecosystems. Gurung and Urabe (1999) concluded from controlled experiments on the bacterial planktonic community from eutrophic Lake Biwa (Japan) that DOC accumulation in surface water during summer is induced by the high bacterial growth rate and P limitation. Also, Olsen et al. (2002) studied nutrient limitation of aquatic food webs and showed that DOC accumulated in experiments where P was limiting, i.e., with high C:P ratios. Other studies also reported that in lakes where there is P limita tion of heterotrophic bacteria, labile DOC accumulates (Vadstein et al. 2003). Jasson et al (2006) did controlled experiments with bacterioplankton in subarctic Lake Diktar Er ik, Sweden, and showed that growth of the heterotrophic community was controlled by DOC a nd inorganic nutrients. In their experiments, bacterial production was stimulated by the DOC supply, but the use of DOC for growth was dependent on the DOC:Pi ratio. Furthermore, DOC was used for growth under C-limited conditions, but used for respirat ion under Pi limitation, when b acterioplankton communities tend to respire large portions of assimilated C. The increase in NH4-N at the end of incubation is another indication of P limitation. Bacteria preferably utilize N in the form of amino acids over NH4 + (Kirchman 1990), and this preference has been reported to be stronger when bacterial growth is Plimited (Schweitzer and Simon 1995, Gurung and Urabe 1999). This can lead to an accumulation of NH4 + and other inorganic N forms in P limited systems (Gur ung and Urabe 1999). In Lake Apopka sediments there was a general increase in DOC and NH4-N with time, strongly in dicating that there is P limitation during summer.

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132 There is high P hydrolytic enzyme activity in surface sediments of Lake Apopka indicating high demand for labile inorganic P (Chapter 4) Lake Apopka sediments have high extractable C:P and N:P ratios, and the decrease of these ratios with depth is related to an increase in labile inorganic P concentration (Tab le 5-3). Although hypereutrophic lakes are characterized by high P concentration, P limitation can occur during summe r due to high demand and competition for P (Gurung and Urabe 1999; Vadstein et al 2003; Vrede 2005; Jasson et al. 2006). Nitrogen is the primary limiting nutrient in Lake Apopka although co-l imitation with P can occur (Aldridge et al. 1993). Phosphorus limitati on of primary productivity, however, has been detected during summer in Lake Apopka (Newma n et al. 1994). Even though this P limitation has been established for the phytoplankton comm unity in the water column, Lake Apopka is shallow and there is high inte raction between the water colu mn and sediments. Moreover, bacteria have three times higher P requirements than do typical algae (Vadstein 2000). If the demand for P in the water column is high during summer, less P will reach the sediment, and there will be low labile P concentration in the sediments. Summer temperatures in Lake Apopka sedi ments can be high, which could stimulate microbial activity and demand for P. High primar y production and high labile C sedimentation (Gale et al. 1992, Gale and Reddy 1994) will lead to high demand for labile P in surface sediment that was reflected in high C:labile nutr ient ratios. With decomposition and utilization of C by the heterotrophic community, C becomes increas ingly refractory and ther e is a decrease in microbial biomass and activity with sediment depth, reducing the demand for P. Consequently labile P will accumulate in deeper sediments, yi elding lower C:P ratios as seen for Lake Apopka deeper sediments.

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133 The M17 sediment from Lake Okeechobee also had an increase in NH4-N concentration during incubation and highest C:nut rient ratios. These sediments al so seem to be P limited. The primary productivity in the southern region (peat zone) of Lake Okeechobee was reported to be limited by N and a high frequency of co-limitatio n by N + P occurs (Aldridge et al. 1995). A dual limitation by P and C, however, seems to be occurring in M17 sediments. M17 sediments are characterized by peat deposits formed by in complete decomposition of higher plants (Reddy et al. 1991). Extractable C is probably rich in humic substa nces known to be refractory. Consequently, although the C:nutrient ratios are high for this site, available C is probably low. If DOC is not easily available, C can limit hetero trophic bacteria (Vadstei n et al. 2003). Vrede (2005) showed that lakes with high concentratio ns of humic substances are usually limited by both C and P. The incubation experiment strongly i ndicates the refractory nature of C in these sediments. There was a decrease in DOC con centration during incuba tion, probably reflecting high demand for labile C by the microbial commun ity. This site was the only one that showed a significant accumulation of DRP, and NH4-N following incubation. The decrease in C and simultaneous increase in inor ganic nutrients, N and P, indicates a high demand of these sediments for labile C. High availability of P in Lake Okeechobee si tes M9 and KR surface sediments is causing C limitation in the system. Moreover, in M9 sediments, low values of Ext-N:Ext-P indicate that N can be also limiting in this system. Crisman et al. (1995) reported that temperature and trophic state variables Secchi disk depth, total P, and total N, had a weak correlation with bacterioplankton abundance (number of cells mL-1) in a seasonal study of Lake Okeechobee. They concluded bacterioplankton communities we re probably controlled by grazing and/or C and nutrient availability. Work et al. (2005) reported high ba cterioplankton production (mg L-1 h-

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1341) in Lake Okeechobee during summer. Also, several studies have shown that bacterioplankton is an important source of C to the food web in La ke Okeechobee (Havens and East 1997; Work and Havens 2003; Work et al. 2005). However, to my knowledge, there is no study addressing C or nutrient limitation of the bacterioplan kton community in Lake Okeechobee. Nevertheless, Phlips et al. (1997) showed that in the central, mud zone region of Lake Okeechobee phytoplankton was dominated by smal l-celled species of cyanobacteria and diatoms. Light is the most limiting factor of the phytoplankton community during most of the year in this area, however, dur ing summer months, light limitation is relaxed and N becomes the limiting factor of the phytoplankton community (A ldridge et al. 1995). Se veral studies have reported low chlorophylla and primary productivity in the mud zone is caused by light limitation (Aldridge et al. 1995, Phlips et al. 1993, 1995 c ; Gu et al. 1997). There are, however, no data for Lake Okeechobee reporting the contri bution of primary productiv ity to sediment C. Also, although sites M9 and KR showed simila r values of DOC in the water column, water extractable DOC was low in M9 sediments and ev en lower in KR deposits (Table 5-5). These low-DOC sediments had the lowest anaerobic re spiration. There was no accumulation of DOC and NH4-N, suggesting consumption of C and N with time. Also, site M9 had high labile inorganic P concentration, resul ting in high demand for C and N. It is clear that C and N limit microbial biomass and activity at sites M9 and KR. Lake Annie sediments appear to be C-limited, with low ratios of extractable C:P and N:P. Carbon limitation is probably a cons equence of the C sources and phys ical characteristics of this lake. Lake Annie has experienced an increase in color in the water column during the past decade probably caused by recent high DOC input to the lake from the watershed (Swain and Gaiser 2005). Battoe (1985) repor ted high inputs of humic content to Lake Annie in surficial

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135 runoff during high rainfall periods. This allochthonous DOC, from humic origin, is utilized in the water column. Because Lake Annie is deep, the DOC is highly mineralized before reaching the sediment (Meyer 1997). Thus low amounts of DOC r each the sediment and are highly refractory (Suess 1980), leading to low C:nut rient ratios and C limitation. In Lake Annie, 36-56% of the total sediment P is bond to humic materials (Chapter 3), so much of the C in the sediments is probably in humic forms. High demand for C will lead to inorganic nutrient accumulation in this system, with consequently low C:nutrient ratios. Carbon limitation may be the main reason why there is a sharp decline in methane production with depth. Humic substances play an important role as electron sinks for anaerobic and fermentative bacteria, and high concentrati ons can inhibit methanogenesis as these compounds are used by better energetically competing organisms (Coates et al. 2002; Kappler et al. 2004; Karakashev et al. 2005). The lack of methanogenesis in deeper sections of the sediments can also be due to competition between iron (Fe) or sulfate (SO4 -2) -reducers for labile C. Lake Annie sediments were characterized by high Fe (3640 mg kg-1) concentration (Thompson 1981), and dissolved SO4 -2 concentration (7.2 mg L-1) in the water column (Swain and Gaiser 2005). High SO4 -2 reduction has also been reported to occur in the water column (Swain and Gaiser 2005). Sulfate reduction can be important in oligotrophic lake sediment where low organic matter input allows SO4 -2-reducers to contribute to organic C oxidati on (Lovley and Klug 1983). Although Fe oxides and SO4 -2 concentrations were not meas ured in this study it is probabl y safe to assume that both Feand SO4 -2-reducers are active in the Lake Annie sediments. Structure and function of anaerobic microbial communities are strongly af fected by competition for fermentation products such as H2 and acetate (e.g., Megonigal et al. 2004). Iron and SO4 -2-reducers outcompete

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136 methanogens for H2/CO2 and aceteate, due to higher substrate affinities, and higher energy and growth yield (Lovley and Klug 1983; Lovley a nd Phillips 1986; Conrad et al. 1987; Bond and Lovley 2002), however, both processes can co exist (Mountfort and As her 1981; Holmer and Kristensen 1994; Roy et al. 1997; Holmer et al. 2003; Roden and Wetzel 2003; Wand et al. 2006). Coexistence occurs because of spatial va riation in the abundance of terminal electron acceptors or because the supply of electron dono rs is non-limiting (Roy et al. 1997; Megonigal et al. 2004). During the incubation experiment, DOC accumulated in Lake Annie surface sediments (Table 5-5). In the upper 10 cm of Lake Anni e sediment, the concentration of electron donors must be sufficient for both methanogenesis and other anaerobic metabolic pathways to occur. Lake Apopka displays high C availability a nd can thus support a more diverse community as reflected by anaerobic respiration and metha nogenesis in the sediments. Algal deposition has been shown to increase acetate concentr ation, with a consequent increase in CH4 production in sediments (Schulz and Conrad 1995). Several studi es have shown that methane production rates are higher in eutrophic than oligotrophic lake s. (Casper 1992; Rothfuss et al. 1997; Falz et al. 1999; Nusslein and Conrad 2000; Dan et al. 2004). The negligible CH4 production in Lake Okeechobee is clearly a consequence of electron donor limitation (Chapter 2). However, Fisher et al. (2005) reported CH4 in sediment porewater of sites M9 and M17 in Lake Ok eechobee. They also reported SO4 -2 in these sediment porewaters, and its decline with sedime nt depth was related to the use of SO4 -2 as a terminal electron acceptor in the oxidation of sediment organic matter. Iron is important in controlling P solubility in Lake Okeechobee sediments (Moo re and Reddy 1994) and Fe -reducers might also be present. As discussed before, Feand SO4 -2-reducers outcompete methanogens for substrates,

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137 consequently low C availability with concomitant presence of Fe and SO4 -2-reducers is the probable explanation for lack of metha nogenesis in Lake Okeechobee sediments. Conclusions The results from this study showed that hypereutrophic Lake Apopka had the highest microbial biomass and activity (both CO2 and CH4) followed by oligo-mesotrophic Lake Annie. Microbial activity decreased w ith sediment depth and was related to decrease in easily degradable OM. Carbon, N and P concentrations, and especially nutrient ratios, had a strong influence on microbial communities in these sediments. The sediment microbial community in each lake, or site, was limited by different variables. The Lake Apopka surface sediments appear to be P-limited. High primary production and high labile C sedimentation resulted in high demand fo r labile P in surface sediment, as reflected in high C:P ratio. Peat sediments of Lake Okeech obee were limited by both C and P. Nitrogen and C limitation were observed in mud and sand sedime nts of Lake Okeechobee. High availability of P in Lake Okeechobee mud and sand surface sedi ments resulted in C and N limitation. Lake Annie sediments seem to be C-limited, with lo w ratios of extractable nutrient ratios. Carbon limitation was probably a consequence of C s ources (high humic content) and physical characteristics (deep) of this lake. The results showed that heterotrophic microbial metabolism can be limited by a single factor or multiple variables, and limitation varies among lakes depending on lake characteristics and bi ogeochemical properties of sediments.

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138 Table 5-1. Total carbon (TC), total nitrogen (TN), and C:N:P ratios (weight) in sediment profiles of the three lakes. (mean standard deviat ion). ** No replicates for SD calculation. TC TN Ratios (weight) Lake Site Depth (cm) (g kg-1) C:N C:P N:P 5 289 9 21.6 0.4 13 201 15 10 280 10 21.3 0.8 13 197 15 15 271 6 20.5 0.7 13 186 14 20 266 2 20.0 0.2 13 174 13 30 257 5 18.4 0.5 14 175 12 45 257 1 18.3 0.4 14 173 12 60 251 3 16.6 0.2 15 221 15 Annie Central 80 245 6 16.3 0.3 15 213 14 5 187 3 12.1 0.2 15 178 11 10 194 1 12.6 0.2 15 203 13 15 131 6 6.6 0.4 20 157 8 20 157 4 8.5 0.2 19 217 12 30 113 13 4.6 1.1 25 176 7 45 130 8 5.0 0.9 26 227 9 60 155 8 7.3 0.7 21 263 12 M9 70 192 9 10.6 0.5 18 419 23 5 467 13 26.7 1.1 17 1735 99 10 493 7 27.7 0.6 17 3589 201 15 499 3 27.4 0.2 18 3913 215 20 498 3 26.7 0.9 18 4144 222 30 496 2 25.5 1.6 19 4488 229 M17 40 496 5 24.9 0.6 20 3526 176 5 11 16 0.8 0.9 10 44 3 10 19 18 1.2 1.1 14 73 5 15 31 24 2.0 1.4 14 130 8 20 101 27 6.8 2.0 15 800 54 30 106 25 8.4 1.8 13 1905 151 Okeechobee KR 40 35 ** 2.6 ** 13 2187 162 5 353 3 28.1 0.6 13 280 22 10 349 6 28.0 0.9 12 268 21 15 349 3 28.7 1.0 12 258 21 20 342 1 28.1 0.6 12 270 22 30 343 1 29.0 0.8 12 281 24 45 353 11 28.8 0.8 12 382 31 60 357 8 28.5 0.2 13 461 36 80 371 5 29.4 0.6 13 602 48 Apopka West 98 379 ** 28.8 ** 13 542 41

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139Table 5-2. Pore water dissolved orga nic carbon (DOC), ammonium-N (NH4-N), and dissolved reactiv e phosphorus (DRP), total nitrogen (TN), and total phosphorus (TP). (mean SD). **No replicates for SD calculation. Pore water (mg kg1 dw) Lake Site Depth (cm) DOC NH4-N DRP TN TP 5 268 163 62 9 0.4 0.1 384 81 12 3 10 96 11 94 17 0.1 0.03 374 89 7 3 15 73 24 107 25 0.1 0.05 402 98 9 3 20 73 4 114 25 0.1 0.03 423 156 15 3 30 209 168 83 18 0.2 0.1 506 211 25 8 45 161 44 77 26 0.3 0.1 484 98 24 9 60 242 135 76 34 0.7 0.1 437 90 18 4 Annie Central 80 232 163 116 71 0.8 0.1 382 108 8 1 5 1586 189 323 140 0.7 0.1 885 303 10 3 10 1160 201 541 232 0.5 .2 1353 406 7 1 15 892 96 617 271 0.4 0.1 1294 535 5 1 20 842 124 647 248 0.3 0.1 1581 595 5 1 30 658 149 617 236 4 2 1418 536 6 2 45 557 88 585 191 8 3 1280 374 10 2 60 478 45 460 36 11 5 1017 44 12 4 80 479 35 456 31 13 1 1020 61 16 4 Apopka West 98 432 ** 513 ** 5 ** 1093 ** 7 **

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140 Table 5-3. Extractable orga nic carbon, ammonium (NH4-N), labile organic nitrogen (ON), labile inorganic phosphorus (LabilePi) and la bile organic phosphorus (LabilePo) concentrations in sediment pr ofiles of the three lakes. Extractable (mg kg-1 dw) Lake Site Depth (cm) Carbon NH4-N ON LabilePi LabilePo 5 784 111 66 15 112 3 72 13 50 9 10 808 133 108 8 120 22 67 12 43 7 15 779 76 169 42 102 13 55 6 40 5 20 646 51 200 67 89 17 52 9 35 6 30 803 66 262 109 113 9 42 8 28 2 45 722 58 284 111 106 23 34 10 22 3 60 768 119 363 160 82 22 22 0.3 19 2 Annie Central 80 771 66 485 245 88 12 15 3.8 16 1 5 322 46 20 11 92 17 110 7 6 4 10 241 11 71 11 75 7 87 11 8 1 15 296 24 86 5 91 21 127 33 7 4 20 236 39 78 13 68 2 55 7 4 1 30 249 52 63 7 86 17 80 6 2 0 45 259 64 5 84 14 55 8 3 1 60 355 21 66 3 85 16 46 3 2 1 M9 70 355 84 51 12 74 9 36 2 1 1 5 714 91 14 2 118 10 14 6 5 0.5 10 1193 199 27 4 163 16 4 0.3 4 0.4 15 1337 35 4 170 25 3 1 3 0.5 20 1620 12 38 2 187 9 3 0.4 4 0.7 30 1613 231 48 4 199 18 4 0.3 4 0.8 M17 40 1657 257 49 2 194 19 3 0.4 3 0.6 5 52 27 6 5 20 9 4 1 0.4 0.3 10 73 22 10 18 6 7 2 1 0.5 15 79 8 4 14 2 3 2 0.4 0.3 20 232 27 28 34 3 3 1 1 0.4 30 206 26 43 7 40 5 2 1 1 0.2 Okeechobee KR 40 89 ** 14 ** 15 ** 1 ** 0.3 ** 5 2670 118 104 41 419 74 2 0.4 15 8 10 2243 311 234 73 419 106 1 0.3 19 7 15 2024 267 340 89 384 65 19 2 25 7 20 1638 164 377 97 351 58 20 5 24 13 30 1804 228 427 74 349 45 28 8 15 5 45 1505 216 449 28 275 29 37 3 11 7 60 1587 82 464 15 243 59 34 15 16 8 80 1675 188 534 68 214 22 42 8 7 2 Apopka West 98 1300 ** 665 ** 228 ** 61 ** 1 **

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141 Table 5-4. Microbial biomass car bon, nitrogen and phosphorus c oncentrations in sediment profiles of the three lakes. (mean SD). ** No replicates for SD calculation. Microbial Biomass (mg kg-1 dw) Lake Site Depth (cm) Carbon Nitrogen Phosphorus 5 5419 195 282 12 78 9 10 5089 166 283 38 64 18 15 4387 218 172 7 44 3 20 4205 177 106 19 37 5 30 3919 405 65 13 30 7 45 3652 692 60 3 24 10 60 3401 624 41 2 22 10 Annie Central 80 2740 422 20 7 18 6 5 3821 479 122 2 50 2 10 3672 187 115 11 34 5 15 3465 231 89 14 28 8 20 2773 205 64 22 18 2 30 2616 283 55 10 11 2 45 2361 164 42 5 9 1 60 2177 92 35 8 8 0.4 M9 70 1983 437 33 6 2 1 5 3800 437 75 23 6 0.6 10 3811 370 72 5 5 0.3 15 3746 873 66 11 3 1 20 4667 461 97 21 3 0.8 30 5128 447 100 25 2 0.8 M17 40 3354 988 43 11 2 1 5 574 249 4 4 1 0.5 10 653 227 12 4 1 0.2 15 644 220 15 7 1 0.6 20 1446 335 39 14 2 0.3 30 1296 113 26 11 1 0.9 Okeechobee KR 40 638 ** 20 ** 1 ** 5 36617 3193 2630 294 598 17 10 32926 5437 2469 278 596 85 15 30486 3924 2284 366 616 13 20 22265 5640 1863 55 523 115 30 19355 4608 1731 63 399 163 45 14725 4586 804 91 267 224 60 11037 4291 479 72 106 76 80 9584 1273 85 18 31 11 Apopka West 98 8011 ** 67 ** 52 **

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142 Table 5-5. Water extractable dissolved organic carbon (DOC), dissolved reactive P (DRP), and ammonium-N (NH4 +) concentrations at time 0 (befor e incubation) and time 10 (after incubation). Results of t test of incubation experime nt, significant differences ( p < 0.05) between T = 0 vs. T = 10 are in bold ( n = 3 and df = 2 for all analysis). Water Extractable (mg kg-1) DOC DRP NH4-N Lake Site Depth (cm) T=0 T=10 T=0 T=10 T=0 T=10 5 499 1225 1.4 1.1 80 134 10 430 875 0.7 1.5 111 119 15 1022 370 1.5 1.0 135 137 20 1264 238 2.9 1.4 141 154 30 3298 1916 7.7 6.9 106 108 45 4268 1473 10.0 2.4 100 93 60 4693 1545 7.5 1.6 87 79 Annie Central 80 4332 382 9.1 2.0 159 109 5 424 229 2.35 1.8 28 43 10 262 258 0.8 1.2 40 40 15 278 316 0.8 1.4 42 38 20 204 291 0.8 1.7 35 27 30 201 154 0.5 2.5 28 25 45 279 225 0.6 1.0 29 25 60 275 607 1.4 2.1 27 21 M9 70 447 637 4.9 4.3 26 12 5 459 334 0.9 2.5 9 19 10 854 458 0.3 1.4 10 17 15 1219 723 0.2 1.1 12 16 20 1559 955 0.3 1.1 15 16 30 1648 1376 0.3 1.3 16 21 M17 40 2010 1193 0.3 0.8 17 20 5 34 26 0.03 0.09 1 9 10 29 35 0.03 0.07 3 3 15 53 49 0.08 0.07 4 6 20 212 208 0.01 0.07 10 10 Okeechobee KR 30 244 178 0.03 0.06 9 5 5 2020 2963 1.3 2.4 378 1042 10 1422 2267 0.6 1.3 548 1159 15 1171 1822 0.5 0.9 731 1178 20 1072 1679 0.4 0.7 788 1120 30 1003 1438 2.8 2.2 762 1041 45 819 1347 8.2 10.4 636 865 60 642 1412 12.2 14.4 457 667 Apopka West 80 733 1509 13.3 25.0 466 666

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143 020406080100120140160180200 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 02468101214 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 0246810121416182022 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 050100150200250300350 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 80-100 0510152025303540 0-5 5-10 10-15 15-20 20-30 30-45 45-60 60-80 Figure 5-1. Microbial activity (CO2 and CH4 production rates) in sediments from: A) Lake Annie, B) Lake Ok eechobee: M9, C) M17, D) KR, and E) Lake Apopka. Bars represent standard errors. Microbial Activity (mg C kg-1 d-1) De p th ( cm ) A B C D E De p th ( cm ) CO2 CH4 CO2 CH4 CO2 CH4 CO2 CH4 CO2 CH4

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144 BD TC TP Labile-Pi Labile-Po Ext-C:Ext-P MBC -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (48.6%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (22.8%) Ext-C:Ex-N Ext-ON Ext-N:Ex-P TN Ext-NH4:Ext-Pi Ext-NH4CH4CO2Ext-C LOI -3.5-3.0-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.53.03.5 Axis 1 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Axis 2 5 5 5 5 10 10 15 20 45 60 30 70 80 60 5 5 5 5 5 10 10 10 10 20 20 20 15 15 15 15 45 45 60 80 98 30 30 30 10 15 20 30 40 15 20 30 40 45 30 20 Figure 5-2. Results of the Principa l Component Analysis: A) loadings of sediment variables (n = 107), and B) the plot of the scores of the sites and sediment depth (numbers cm) from Lake Annie (blue circles), Lake Oke echobee: M9 (red squares), M17 (brown diamonds), KR (orange crosses), and Lake Apopka (green triangles).BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon, TN: total nitrogen, Ext-N: extractable labile nitrogen, Ext-NH4N: extractable ammonium, Ext-ON: extractable labile organic nitrogen, TP: total phosphorus, Ext-P: extractable labile phosphorus, Ext-Pi: labile inorganic phosphorus, Ext-Po: labile organic phosphorus, MBC: microbial biomass carbon, CO2: anaerobic respiration, and CH4: methane production rates. A B Lake Okeechobee Lake Okeechobee Peat Lake Annie Lake Apopka

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145 CHAPTER 6 NUTRIENT ACCUMULATION AND STABLE ISOTOPE SIGNATURES IN SEDIMENTS OF SUBTROPICAL LAKES Introduction Organic matter (OM) that enters a lake from the watershed (allochthonous) or is produced within the lake (autochthonous) itself will be de posited on the lake bottom and incorporated into the sediments. Lakes function as natural traps fo r OM and associated nutrients. Lake sediments contain an archive of past environmental conditions in and around the water body (Smol 1992) and can be used to document anthropogenic im pacts through time (Smeltzer and Swain 1985). Sediment OM provides informa tion about past impacts and bioge ochemical processes within lakes, and has been studied extensively usi ng paleolimnological methods (Meyers 1997). The timing of past events in a basi n is based on reliable dating of sediment cores. Sediment dating provides an age/depth relation from which bulk sediment accumulation rates can be calculated (Smeltzer and Swain 1985). The lead-210 (210Pb) technique is used routinely to provide age/depth relations for the last 100-150 years (Appleby et al. 1986), and has been used widely in studies of Florida lake sediment cores (e.g., Binford and Brenner 1986; Brezonik and Engstrom 1998; Whitmore et al. 1996; Brenner et al. 2006; Schottler and Engstrom 2006). Bulk sediment accumulation rates in combination with analyses of sediment composition, can be used to calculate accumulation rates of se diment constituents such as OM and nutrients. Such measures provide insights into past change s in productivity and human imp acts on the aquatic ecosystem. Nutrient and OM accumulation rates in sedime nt have been studied in conjunction with stable isotope analyses ( 13C and 15N) to infer past environmental impacts in marine (e.g., Gearing et al. 1991; Savage et al. 2004), lacustrine (e.g., Schels ke and Hodell 1991; Gu et al. 1996; Bernasconi et al. 1997; Hodell and Schels ke 1998; Ostrom et al. 1998; Brenner et al. 1999), and riverine ecosystems (e.g., McCallister et al. 2004; Anderson and Cabana 2004; Brunet

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146 et al. 2005). Measurements of 13C and 15N in several lake compartm ents, (i.e., dissolved and particulate matter in the water column and sediment s) have been used to identify the origin of lacustrine OM (Filley et al. 2001; Griffths et al. 2002), infer pa st primary productivity (Schelske and Hodell 1991; Hodell and Schelske 1998; Be rnasconi et al. 1997), document historical eutrophication (Gu et al. 1996; Ostrom et al. 1998; Brenner et al. 1999), elucidate biogeochemical cycles (Terranes and Bernasconi 2000; Jonsson et al. 2001; Lehmann et al. 2004), and shed light on microbial activity (Hol lander and Smith 2001; Lehmann et al. 2002; Gu et al. 2004; Terranes and Bernasco ni 2005; Kankaala et al. 2006). Allochthonous OM usually has more negative 13C values than does autochthonous OM. Values of 13C can also be used to distinguish periods of high versus low primary productivity. Algae fractionate against the heavier isotope, 13C. Consequently, under conditions of low to moderate primary productivity autoch thonous OM displays very negative 13C. During periods of very high primary productivity the preferred 12C in the water column is exhausted and fractionation is diminishe d, yielding OM with higher 13C (Mizutani and Wada 1982; Raul et al. 1990). Hypereutrophic lakes with high rates of primary productivit y have low concentrations of carbon dioxide (CO2) in the water column. Moreover, in alkaline (high-pH) waters bicarbonate (HCO3 -) dominates the dissolved inorganic C, and has a 13C that is 8 heavier than dissolved CO2 (Fogel et al. 1992). High demand for inorganic C and low free CO2 leads to utilization of HCO3 as a C source resulting in heavier 13C of OM (Goericke et al. 1994). Stable isotope signatures of sediment OM can sometimes be used to identify impacts of anthropogenic activities. Wastewater and agricultural runoff can be identified because they yield OM depleted in 13C and enriched in 15N (Gearing et al. 1991; Burnett and Schaffer 1980; Savage et al. 2004). Stable isotope 15N has also been used to study the nitrogen (N)

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147 biogeochemical cycle. Measurement of 15N in suspended and sedimented OM was used to address the source of N, as well as N limita tion of, and utilization by the phytoplankton community in Lake Lugano (Terranes and Bernasconi 2000). In summary, sediment OM a nd nutrient content, along with 13C and 15N signatures, have proven useful to identify the origin of OM infer past lake productivity, and understand mineralization processes in lakes. The objectives of this study were to: (i) determine sediment accumulation rates, and (ii) determine 13C and 15N signatures of OM in sediment cores from subtropical lakes of di fferent trophic states. Material and Methods Study Sites Three Florida (USA) lakes ranging in trophic state were selecte d. Lake characteristics were described in Chapter 2. The characteristics a nd location of sampled s ites and field sampling procedures were described in Chapter 3 (Table 31, Figure 3-1). Sectioning of the cores (Set # 1) was described previously (Chapter 3). One additional core was collected at each site for isotope analyses and 210Pb dating (Set # 2). Sediment cores from Lake Annie (60 cm maximum depth), Lake Apopka (72 cm maximum depth) and Lake Okeechobee site M9 (73 cm maximu m depth) were sectioned at 4-cm intervals. Sediment cores from Lake Okeechobee sites M1 7 (36 cm maximum depth) and KR (16 cm maximum depth) were sectioned at 2-cm interval s. All sediment variables are reported on a dry weight basis (dw). Water quality variables were described in a previ ous study (Chapter 4). Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Total phosphorus (TP) total carbon (TC), and total nitrogen (TN) of core Set # 1 were measur ed and described in previous studies (Chapter 3, 5).

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148 Sediment samples (Set # 2) for 210Pb dating and isotopic analyses were dried in a Virtis Unitrap II freeze drier. Sediment bulk density (g dry cm-3 wet) was determined on a dry weight basis (weight before and after freeze-drying). Drie d samples were ground in a mortar and pestle and passed through a 2.0-mm mesh sieve. Organi c matter content (LOI-l oss on ignition) was determined by weight loss at 550C. Isotopic Analyses Sediment samples for organic C isotope analys is were pretreated with acid to remove inorganic C (carbonates) (Harris et al. 2001). Samples were weighed in silver capsules, placed in the wells of a microtiter plate, and 50 L of DI water was added to moisten the sediment. Plates were placed in a vacuum desiccator with 100 mL of concentrated HCl, and exposed to HCl vapor for 24 hours. Samples were dried at 60 C for 4 hours to remove any remaining HCl. Carbon (organic) and nitrogen (total) isotope values were determined using methodology described by Inglett and Reddy (2006). Isotope analyses were conducted using a Costech Model 4010 Elemental Analyzer (Costech Analytical Industr ies, Inc., Valencia, CA) coupled to a Finnigan MAT DeltaPlusXL Mass Spectrometer (CF-IRMS, Thermo Finnigan) via a Finnigan Conflo II interface. Stable isotope results are expressed in standard delta notation, with samples measured relative to the Pee Dee Belemnite for C and atmospheric N2 for N. Analytical accuracy and precision were established using known isotopic standards (wheat flour, 13C = -26.43 15N = 2.55 Iso-Analytical; IAEA-N1, 15N = 0.4 ; ANU-Sucrose, 13C = -10.5 ). Analytical precision for standards was less than 0.1 for 13C and 0.3 for 15N. 210Pb Dating Lead-210 dating was done by gamma counting (Appleby et al. 1986; Schelske et al. 1994). Samples were placed in plastic SarstedtTM tubes to a height of ~ 30 cm. Sample mass was determined and tubes were sealed with e poxy glue and set aside for 3 weeks to allow 214Bi and

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149214Pb to equilibrate with in situ 226Ra. Radioisotope activities were measured using ORTECTM Intrinsic Germanium Detectors connected to a 4096 channel, multichannel analyzer. Total 210Pb activity was obtained from the photopeak at 46.5 kilo electron volts (keV). Supported 210Pb activity, expressed as 226Radium activity, was estimate d by averaging activities of 214Pb (295.1 keV and 351.9 keV) and 214Bi (609.3 keV). Cesium-137 activit y was determined from the 662 keV photopeak. Unsupported 210Pb activity was estimated by s ubtraction of supported activity from the total activity measured at each level. Activ ities are expressed as decays per minute per gram of dry sediment (dpm g-1). Sediment ages and bulk sediment accumulation were calculated using the constant rate of supply (CRS) model (Appleby a nd Oldfield 1978, 1983). Lead-210 dates correspond to the base of each sediment sec tion. In all cores but the one from Lake Annie, radioisotope activity was measured in all sample s from the sediment water interface to the base of the section. The 2-4 cm portion of the Lake Annie core was lost during extrusion so interpolated values for bulk dens ity and activities were used to compute dates. This is thought to have introduced negligible error as the topmost 12 cm have near ly identical bulk densities and activity values. Results and Discussion Core Chronology Lake Annie In Lake Annie, total 210Pb activity declined with increasing sediment depth. 226Ra activity, i.e., supported 210Pb activity, varied from 2.9 0.5 dpm g-1 in surface sediments to 2.1 0.6 dpm g-1 in deeper sediments. Cesium-137 activity declined with sediment depth and showed no distinct peak (Figure 6-1A). Chronologies de termined with the CRS model yield reasonably precise dates from c. 1900 (Figure 6-1B). The 210Pb results of the current study are similar to those reported by Schottler and Engstrom (2006) fo r Lake Annie. The average sedimentation rate

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150 (since c.1900) was 36.8 mg cm-2 yr-1 while Schottler and Engstrom (2006) reported a value of 34 mg cm-2 yr-1. Lake Annies sedimentation rate and organic matter accumulation rates varied slightly through time (Figure 6-1C). Sediment accumulation generally increased from late 1800 until ~ 1940, then decreased through 1970s. Over the past several decades, the sedimentation rate has increased. Lake Annie water input s are from ground water (90%) and atmospheric deposition (10%) (Swain and Gaiser 2005). This lake has no natural su rface streams but two shallow man made ditches flow into the lake al ong the south and southeas t sides. Surface runoff from these ditches was reported to contribute to water and nutrie nt input to the lake during high rainfall periods (Battoe 1985). Shif ts in historic sedimentation rates may reflect changing inputs of allochthonous OM and nutrients from the surrounding landscape. Water column characteristics in Lake Annie have experienced profound changes in the la st 10 years. The lake has transformed from a cl ear-water system to a water body with appreciable dissolved color. The increase in color was probably due to high di ssolved organic carbon ( DOC) input to the lake from adjacent land (Swain and Gaiser 2005). Most of the water input to the lake is from groundwater, and the source of DOC to this lake is allochthonous. The increase in color has been accompanied by a decrease in Secchi disk depth and dissolved oxygen, while pH has increased. No changes were recorded in electrical conductivity, and slight increases in N and P as well as chlorophylla have been detected in the past 20 years (Swain and Gleiser 2005). The incr ease of Lake Annies se dimentation rate and OM accumulation rates are probably related to the increase in allochthonous DOC input. Lake Okeechobee In Lake Okeechobee site M9, activities of 210Pb, 226Ra, and 137Cs could only be detected in near-surface sediments (Figure 6-2A). In sedime nts at sites M17 and KR activity values were below detection limits suggesting the sites we re non-depositional, which precluded dating.

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151 Brezonik and Engstrom (1998) dated 11 cores from the Lake Okeechobee mud zone and two cores from the peat zone collected in 1998. In 2003, Schottler and Engstrom (2006) and Engstrom et al. (2006) re-sampled three sites in the mud zone that were previously dated (Brezonik and Engstrom 1998). Using 210Pb dating models and 137Cs, the authors concluded that Lake Okeechobee sediments preserve a reliable stat igraphic history of th e lake. Schottler and Engstrom (2006) concluded, however, that210Pb dating of Okeechobee sediments is problematic, not very precise, with error terms for the last half century ca. 10 years. Furthermore, in September 2004, the eyes of two hurricanes, Frances and Jeanne, passed to the north of lake. Strong winds generated a large su rface seiche in the lake (Chi mmey 2005). Lake stage rose abruptly, by about 3.06 m during hu rricane Frances, and 4.91 m dur ing hurricane Jeanne. Peak wind velocity reached hurricane strength at pl atforms over the center of the lake (144 km h-1) (Chimmey 2005). Because Lake Okeechobee is large and shallow, its sediments are easily disturbed by wind, especially in the mud zone (Hav ens et al. 2007). Havens et al. (2007) reported the impact of Hurricane Irene on Lake Okeechobee. The storm pa ssed 80 km south of the lake in October 1999 and produced maximal winds of 90 km h-1 over the center of the lake. Mean pelagic TP increased from 88 to 222 g L-1, and it is estimated that more than 10,000 metric tons of fine-grained mud sediment was resuspended during the storm (H avens et al. 2007). Considering that the 2004 hurricanes passed closer to the la ke, and had higher winds than Hu rricane Irene, it is very likely the storms caused substantial sediment resuspen sion and deposition. I sampled just 10 months after the storms. Extensive sediment transloc ation may explain why the cores I took were undatable. Alternatively, the sites I selected fo r samples may simply be inappropriate. Lake

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152 Okeechobee displays heterogeneous sediment distribution, and not all locations yield datable cores (Schottler and Engstrom 2006). Lake Apopka Total 210Pb activity remained relatively constant over the topmost 44 cm of the Lake Apopka core (Figure 6-2B). Furthermore, total 210Pb values exceeded 226Ra activities in the base of the section, suggesting there was still unsupported 210Pb at the bottom of the core. The fairly constant total 210Pb activity in the upper 44 cm of the co re may be explained by two processes: 1) incoming unsupported 210Pb is being diluted by higher and higher sediment accumulation rates, the increase in depositio n keeping pace with the 210Pb decay, or 2) uppermost sediments are mixed by physical or biological action, yielding fairly constant activities throughout the section. Waters et al. (2005) also repor ted failure to date Lake Apopka sediments. The CRS model assumes constant input of excess of 210Pb through time. In some large, shallow lakes in Florida, resuspension and focusing of organic sediments may lead to violations of the dating model assumptions (Whitmore et al. 1996). Furthermor e, land use and hydrological changes, including reduction of lake surface area as surrounding we tlands were cleared for agriculture, and construction of the Apopka-Beauclair Canal, both lowered lake stage by ~ 1 m and established a permanent outflow, which caused conditions th at violated the assumptions of the CRS 210Pb dating model (Schelske et al. 2005; Waters et al 2005). Cesium-137 showed a slight peak at 4852 cm depth (Figure 6-4B). A 137Cs peak can sometimes be used to identify the period of maximum cesium fallout, from atomic bomb testing around 1963 (Krishnaswami and Lal 1978) and may be used to verify 210Pb dates (Schelske and Hodell 1995). Neither 210Pb nor 137Cs yielded a reliable chronology. The 1963 137Cs peak is absent or pres erved poorly in many Florida lakes as Cs is poorly bound and may move in the sediment column (B renner et al. 1994, 2004; Schelske et al. 1994).

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153 Carbon ( 13C) and Nitrogen ( 15N) Isotope Signatures Lake Annie Lake Annie sediment TC:TN ratios increased with sediment depth, while TN:TP decreased to ~ 50 cm and then increased again (Figure 6-3B, C). Both 13C and 15N values are depleted towards the sediment surface, varying from -28.2 to -29.4 and 2.0 to 0.9 (Figure 63D, E). Surface sediments are depleted relative to basal deposits by -1.24 ( 13C) and -1.13 ( 15N). Organic matter content (LOI %) decreased with sediment depth (Figure 6-3F). Statigraphic isotopic signat ures of Lake Annie sediments indicat e that this lake is going through changes in recent years. Statig raphic decrease in TC:TN ratios towards the sediment surface indicates contribution of autochthonous OM to sediments in Lake Annie. However, in recent years, lake productivity has slightly changed, but other changes, such as increase in DOC and color, occurred (Swain and Gleiser 2005). Isotopic signatures of 13C and 15N of Lake Annie sediments probably resulted from a combination of several factors such as allo chthonous OM input, primary productivity, and microbial biomass and activity. Small, oligotrophic lakes are expected to have relatively a high proportion of allochthonous C i nput to their sediments (Gu et al. 1996). Terrestrial C3 plants discriminate against 13C, and organic matter derived fr om land plants typically have 13C values of -27 to -29 (Bird et al. 1994, Meyers 1997). Hammarlund et al. (1997) rela ted successive depletion of 13C in Lake Tibetanus (Sweden) with changes in the input of allochthonous material from surrounding vegetation. Moreover, Jonsson et al. (2001) reported very negative 13C values of dissolved C in a humic lake (Lake rtrsket, Sweden), resulting from the mine ralization of allochthonous organic matter. Phytoplankton also discriminate against 13C in the water column when CO2 concentration is high, which is expected in this lake with low pH. Consequently, autochthonous organic matter is

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154 also expected to be depleted in 13C. The heterotrophic microbial community can also contribute to depleted values of 13C. Heterotrophic uptake of DOC preserves the C is otopic signature of the source, and biomass associated with chemoautrotrophic and methanotr ophic microorganisms is generally depleted in 13C (Conway et al. 1994; Kelley et al. 1998). Th e isotopic study on sediments from Lake Mendota (Wisconsin) illustrated that mineralization of C by the heterotrophic microbial community associated with expansion of anoxic conditions in the water column resulted in low 13C values in sediments (Hollander and Smith 2001). Moreover, seasonal and long term increases in contribution of deplet ed microbial biomass to sediments results in depleted values in the 13C (Hollander and Smith 2001). Lehma nn et al. (2002) reported that 13C depleted OM of sinking particles and sediments resulted from anaerobic decomposition in Lake Lugano (SwissItalian border). Terranes and Be rnasconi (2005) associated the 13C of sedimentary OM in Lake Baldeggersee (Switzerland) to variation of rela tive inputs of eukaryotic biomass, which is enriched in 13C and the contribution of micr obial biomass, depleted in 13C, which is produced in the expanding anoxic bottom waters. In Lake Anni e the thermocline has been detected to be moving to shallower depths (i.e., higher) in the water column during thermal stratification, with anoxia below 5 m depth (Swain a nd Gaiser 2005). High sulfate redu ction has been reported to occur in the anoxic layers of the water column (Swain and Gaiser 2005). Increased anoxia in the water column can be leading to increased anaerobic decomposition of already depleted suspended OM, and both depleted microbial biomass and OM will eventually reach the sediment. The same processes are affecting N isotopic si gnatures. Plants tend to fractionate against 15N during inorganic N uptake (Han dley and Raven 1992). Allocht honous organic matter derived

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155 from C3 land plants has 15N is around +0.4 (Peterson and Howarth 1987). Autochthonous OM of aquatic ecosystems not do minated by cyanobacteria has a 15N signature of +8.6 (Peterson and Howarth 1987), while N2 fixation by N-fixing algae yields values near 0 (Meyers 1997). Nitrogen fixation is also found in several microorganisms such as aerobic and anaerobic heterotrophic bacteria, methane oxidizing bacteria sulfate reducers, among others (Siegee 2004). Nitrogen-fixing algae have not be en detected in Lake Annie (Gaiser personal communication), however, N2-fixation can be present and ca rried out by the heterotrophic microbial community. The N transformations can substantially modify organic matter signatures (Meyer 1997). High N availability in the Lake Annie water column (Table 4-2) can lead to autochthonous organic matter with depleted 15N as it will allow greater algae discrimination against 15N (Meyers 1997). Nitrogen availability is al so high in these sediments (Chapter 5). Allochthonous OM, primary productivity as well as heterotrophic microbial community in this lake can produce the 13C and 15N isotopic signatures seen in these sediments (Figure 6-9A). The data presented here do not allow a clear separation of the main factors influencing isotopic signatures in Lake A nnie sediments. Depleted alloch thonous particulate and dissolved OM can be the major source of C in the lake, and is mineralized and utilized by the microbial community that will have depleted microbial biomass. Depleted end products, such as CO2, NH4 + resulting from heterotrophic metabolism will be utilized by primary producers that will also produce a depleted autochthonou s OM. A more detailed study of 13C and 15N isotopes in several compartments, i.e., disso lved C, different N compounds, phytoplankton biomass, bacteria biomass, particulate OM in the water column a nd sediment, of this lake, can elucidate major processes affecting the isotopic sign atures in Lake Annie sediments.

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156 Lake Okeechobee Mud zone sediments (site M9) in Lake Okeech obee showed variable TC:TN ratios over the length of the core. From 70 cm depth TC:TN rati os rose until 50 cm, suggesting increased input of allochthonous material, or N loss to mineraliz ation (Figure 6-4B). At 50 and 30 cm the ratio was similar, and from 30 cm to the surface th ere was a general decrease in TC:TN. Organic matter content was highly variable over the length of the core, however, there was an increase in OM content in surface sediments (Figure 6-4F). TN:TP showed a general decrease from the bottom of the core to the surface, reflecting more rapid increase of TP than TN concentration (Figure 6-4C). The same trend was reported by Engstrom et al. (2006) for the mud zone and was attributed to increase in TP content of these uppermost sediments, as a result of the eutrophication process. The 13C sediment profile showed a si milar pattern of TC:TN ratios (Figure 6-4B, D). Delta 13C values varied from -26.0 to -29.9 with surface sediments only slightly depleted (0.15) relative to bottom deposits (Figure 6-4D). Delta 15N varied from 2.6 to 3.9 and showed ~ 1.3 enrichment in surface deposits relative to bottom deposits (Figure 6-4E). A similar patter n, i.e., depletion of 13C and enrichment of 15N, was reported by Rosenmeier et al. (2004) in a study of recent eutr ophication of Lake Petn Itz, Guatemala, in which changes were related to sewage input (depleted in 13C and enriched in 15N) and increased presence of cyanobacteria. Engstrom et al. (2006) also found 15N enrichment (1) in the mud zone, but did not discuss the mechan isms responsible.Stratigraphic changes in 13C and 15N in the mud zone are probably controlled by autochthonous OM, availability and demand for C and N and varying intensities of mineraliza tion. Lake Okeechobee mud zone sediments are probably C and N limited and N demand is high in th ese sediments (Chapter 5). In the mud zone of Lake Okeechobee, light is the most limiting factor of the phytoplankton community during most of the year (Aldridge et al. 1995). During summer months, light lim itation is relaxed and N

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157 becomes the limiting factor for the phytoplankt on community (Aldridge et al. 1995). The 15N of sediments in eutrophic and hypereutr ophic lakes can be influenced by N2-fixation by cyanobacteria (Gu et al. 1996; Rosenmeier et al. 2004). These cyanobacter ia do not fractionate against 15N and have 15N similar to atmospheric N (~0) (Peterson and Fry 1987). Non-N2 fixing cyanobacteria, however, typically dominate the phytoplankton community and the N2 fixation rate is low in the central area of La ke Okeechobee (Cichra et al 1995; Gu et al. 1997; Phlips et al. 1997). Nitrogen limitation can lead to autochthonous organi c matter with enriched 15N as algae discrimination against 15N will be diminished (Meyers 1997). As a consequence autochthonous OM is expect ed to have an enriched 15N signature (Peterson and Howarth 1987). Although some studies indicate that the isotopic signature of OM is re sistant to alteration during water-column or post-burial diagenesis (Meyers and Eadie 1993; Schelske and Hodell 1995; Hodell and Schelske 1998; Terranes and Bernasconi 2000), others have shown that selective degradation of OM fractions change isotopic signatures (Ber nasconi et al. 1997; Meyers 1997; Lehmann et al. 2002, 2004). Labile carbohydrates, pr oteins and amino acids are generally more enriched in 13C, while lipids and cellulose ar e lighter (Meyers 1997). Selective loss of heavy amino acids, proteins and carbohydr ates, which are particularly susceptible to microbial degradation, leaves residual (substrat e) OM isotopically lighter, with respect to 13C, than the original material (Hedges et al. 1988). Loss of high 15N compounds (e.g., amino acids) can also occur, lowering the 15N in residual material. Nevertheless, decomposition of OM is generally thought to increase 15N through preferential loss of 14N (Nadelhoffer and Fry 1988). Bern asconi et al. (1997) reported shifts in the 13C (depletion) and 15N (enrichment) of sinking OM in Lake Lugano, which they attributed to selective rem oval of C and N compounds during mineralization. Additionally,

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158 sediment 13C in Lake Lugano indicates overall isot opic depletion during early sedimentary diagenesis (Lehmann et al. 2002). In the Lake Okeechobee mud zone, phytoplankton community and N limitation, high demand for C and N in sedi ments, and selective mineralization of OM probably influence 13C and 15N values in the sediments (Figure 6-9B). Sediment OM content was highest in Lake Ok eechobee site M17, reflecting its peat nature (Figure 6-5F). Sediment OM content decreased towards the sediment surface. Peat zone (site M17) TC:TN ratios increase downcore and are the hi ghest values reported from sediment at all sites, reflecting their higher plant origin (Fi gure 6-5B). The TN:TP ratio declines above 30cm, reaching the lowest values at the sediment surf ace (Figure 6-5C). This pattern is driven by the high TP concentration in surface sediments (Figure 6-5A, C). The 13C of OM varied little in the core from site M17, from -26.7 to 26.3 (Figure 6-5D). With respect to 15N, from 36 cm to 22 cm, values decline by about -0.6, but are followed by a period of enrichment of ~1.13 up to the sediment surface. Stratigraphic changes in sediment in 13C and 15N from the peat zone probably reflect selective mineralization of OM. Small shifts in 13C of peat zone sediment may result from shifting intensity of mineraliza tion. Sediments of the peat zone are probably C and P limited (Chapter 5). The demand for labile C is high in surface sediments (Chapter 5), and mineralization of C is reflected in the isotopic signature as well as in lower TC:TN ratios in surface sediments (Figure 6-5B, D). Values of 15N declined from about 1.4 to 0.7 from 36 to 22 cm, but rose again to a hi gh of a little more than 1.8 at th e surface. Contra ry to the mud zone, the phytoplankton communities in the peat zone at the south end of the lake are dominated by large N2-fixing cyanobacteria (Phlips et al. 1997). Nitrogen fixation rates can be high (Gu et al. 1997; Phlips et al. 1997), although NH4 + is the most important N source for phytoplankton

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159 uptake (Gu et al. 1997). The depletion in 15N from 36 up to 22 cm may indicate high deposition of N2-fixer biomass with low 15N. With selective degradation of labile autochthonous OM, isotopically light 15N is removed and remaining material is enriched in 15N. Ammonium-N concentration increases during anaerobic decom position of OM (Chapter 5). Ammonium derived from OM decomposition is usually relatively depleted in 15N (Terranes and Bernasconi 2000), while residual material is left rela tively enriched. Isotopic signatures ( 13C and 15N) of sediment OM are related to several factors, including sedi ment origin (i.e., plant tissue), intensities of primary productivity and diag enesis (Figure 6-9C). Sand zone (site KR) OM content and TC:TN ratios were low (< 16) (Figure 6-6B, F). Similar to the peat zone, TN:TP ratio increase with greater depth reflec ting the decline in TP with depth in the core (Figure 6-6A, C). Sediment 13C values varied throughout the profile (25.63 to -26.36) and were most depleted in 13C near the sediment surface (Figure 6-6D). Nitrogen isotope values ( 15N) varied from ~ 0.63 to 4.22 and show general enrichment towards the sediment surface (Figure 6-6E). There are two periods where 15N declined (from 16-12 cm, and 4-0 cm), and where 15N increased (from 10-4 cm). Organic N mineralization is an important source of inorganic N in these sediments (Fisher et al. 2005). The KR site differs from ot her sites in receiving greater influence from allochthonous material. The KR site is located near the loca tion where the Kissimmee River flows into the lake. It is the largest inflow to La ke Okeechobee (31% of inflow), a nd carries a substantial nutrient load (Frederico et al. 1981; Au men 1995). Agricultural activities, ma inly dairies, are a principal non-point source of nutrients to the Kissimmee River, and are responsible for the nutrient enrichment of this lake (Aumen 1995; Re ddy et al. 1995; Havens and Gawlik 2005). Other nutrient sources are sewage from treatment plants septic tanks, urban runoff, and industrial

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160 pollution (Aumen 1995; Whalen et al. 2002). Th e northern area of Lake Okeechobee is characterized by high intra-annu al variability in chlorophylla concentration (Havens 1994; Phlips et al. 1995). Nitrogen limits primary productivity (Aldridge et al. 1995), and the phytoplankton community is dominated by large N2-fixing cyanobacteria (Phlips et al. 1997), however, N2-fixation is low (Gu et al. 1997; Phlips et al. 1997). Organic matter content in these sediments is low, and the contri bution of autochthonous OM to isot opic signatures does not seem to be great at this site. Wastewater and agricultural r unoff are usually enriched in 15N (Bedard-Haughn et al. 2003; Anderson and Cabana 2005), and se wage effluents are depleted in 13C (Gearing et al. 1991). Other studies relate d the enrichment of 15N in Florida lakes sediments to agricultural runoff (Riedinger-Whitmore et al. 2005; Whitmore et al. 2006). Depletion of 13C and enrichment of 15N, in sediments of Lake Petn Itz (Guatemala) were related to sewage input (Rosenme ier et al. 2004). In a study of 13C distribution in sediments and food webs of estuaries, Gearing et al. (1991) reported that sewa ge C accumulated in sediments, and 13C value of impacted sites (-24.2) was significantly lower than the values from nonimpacted sites (-21.6). In a study of a sewage dumpsite in the New York Bight, Burnett and Schaffer (1980) showed that OM from wastew ater (-26.2) and from marine origin (-22.0) had distinct 13C signatures. Seasonality also can affect particulate organic carbon 13C values in rivers. During periods of high disc harge in Sanaga River (Cameroon) 13C values are high, caused by an increase in the proportion of contribution of OM derived from C4 plants from the further savanna region transporte d overland by wet season rains (B ird et al. 1994; Bird et al. 1998). In the dry season, when discharge is low, 13C values are low, reflecting OM derived primarily from C3 plants growing close to the river ba nk (Bird et al. 1994; Bird et al. 1998).

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161 Stratigraphic variation in 13C and 15N at the KR site probably reflects multiple factors as input of wastewater from anthropogenic activities, and variable contribu tions of river-borne allochthonous input, related to inter-annual rainfall va riations (Figure 6-9D). Lake Apopka The Lake Apopka core displayed a narrow ra nge of TC:TN values (~11.8-13.1). There was a general decrease from about 98-30 cm of depth, and then increase to the sediment surface (Figure 6-7B). The TN:TP ratio decreased toward the sediment surface is related to increase in TP concentration (Figure 6-7C). Organic matter 13C showed a general in crease upward over the length of the core from -22.6 to -18.4 (Figure 67D). Nitrogen isotopic values also displayed an increasing trend upcore, from 3.9 (lower depths) to 4.7 (surface sediments) (Figure 67E). Organic matter content increased towa rds the sediment surface (Figure 6-7F). Nitrogen availability is high in these sedime nts (Chapter 5). Nitrogen transformations in sediments of Lake Apopka include mineralization of organic N, NH4 + adsorption on sediment, nitrification, denitrification, and dissimilatory nitrate (NO3 -) reduction (DAngelo and Reddy 1993). In Lake Apopka sediments, nitrification is high in surface sediments with aerobic conditions, and dissimilatory NO3 reduction to NH4 +, a respiratory proces s used by facultative and obligate anaerobic bacteria, is high in anae robic sediments when the ratio of C/electron acceptors is high (DAngelo and Reddy 1993). Lake Apopka had the highest 13C values and showed the greatest enrichment am ong studied sites. In a study of 83 Florida lakes that ranged in trophic state, Lake Apopka plankton had the highest 13C (Gu et al. 1996). Greater 13C towards the surface probably indicates in creased in primary productivity reflecting greater nutrient concentration, i.e., eutrophi cation (Brenner et al. 1999). The C isotopic signature of autochthonous OM is influenced by the 13C of the dissolved inorganic C pool in lake water. Hodell and Sc helske (1998) related th e seasonal pattern of 13Corg

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162 in Lake Ontario to seasonality of prim ary productivity. Gu et al. (2006) reported 13C enrichment of particulate OM in Lake Wauberg (Florida) re sulted from reduced isotopic fractionation due to C limitation and use of isotopically heavy inorganic C. Lehmann et al. (2004) concluded that the most important process controlling the C-isotopic signature of susp ended particulate OM in Lake Lugano (Swiss-Italian border) is the concentration of CO2 in surface water, which is a function of phytoplankton photosynthesis. Algae fractionate against 13C, so autochthonous OM is depleted in 13C. During periods of high primary pr oductivity, however, this fractionation diminishes and more 13C is incorporated into primary pr oducer biomass (Mizutani and Wada 1982; Raul et al 1990). Hypereutrophic lakes with high rates of primary productivity have depleted CO2 (aq) concentrations in the water column. Moreove r, in alkaline waters bicarbonate (HCO3 -) is the dominant form of inorganic C, and is 8 heavier than C in dissolved CO2 (Fogel et al. 1992). High demand for inorganic C and low free CO2 leads to utilization of HCO3 as a C source resulting in heavier 13C (Goericke et al. 1994). Gu et al. (2004) reported high 13C of inorganic C in the water column of Lake Apopka. The authors showed that heavy 13C DIC in the water column was a result of isotopic fractionati on from methanogenesis in the sediments. Methanogenesis produces 13C-rich CO2 and 13C-poor methane (CH4) (Games and Hayes 1976). Lake Apopka has low CO2 partial pressure, high pH, and strong buffering capacity. Consequently isotopically heavy CO2 is transferred from the sediments to the DIC of the water column (Gu et al. 2004). Lake Apopka sediments display high CH4 production rates (Chapter 2 and 5). Furthermore, most of the primary productivit y in this lake is depo sited in its sediments (Gale and Reddy 1994). Primary productivity is dominated by cyanobacteria ( Synechococcus sp., Synechocystis sp. and Microcystis incerta ) (Carrick et al. 1993; Ca rrick and Schelske 1997).

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163 Cyanobacteria are capable of active CO2 transport (Miller et al. 1991) or utilizing HCO3 (Epsie et al. 1991), and both can result in enriched 13C in phytoplankton biomass. Jones et al. (2001) reported enrichment of phytoplankton 13C resulted from 13C DIC enrichment in Loch Ness (Scotland). Similar results we re found in urban Lake Jyv skyl (Finland), where heavy 13C DIC resulted in enriched 13C of phytoplankton and zooplankton biomass (Syvranta et al. 2006). Heavy 13C DIC in the water column, with high de mand for inorganic C due to high primary productivity, will produce autochthonous OM with enriched 13C, which is then deposited in the sediments. Recent 15N enrichment in Lake Apopka sediment s was surprising as it is generally expected that eutrophic and hypereut rophic lakes will have depleted 15N, as a consequence of high rates of N2 fixation (Fogel and Cifuentes 1993). Gu et al. (1996) also reported enriched 15N in Lake Apopka sediments. In this lake, N fo r phytoplankton assimilation is primarily supplied by transformation of organic N to NH4 + and then to NO3 by nitrification (DAngelo and Reddy 1993). Although the phytoplankton community is do minated (> 90%) by cyanobacteria (Carrick et al. 1993), N2 fixation is relatively unimportant in N dynamics (Schelske et al. 1992). High NO3 availability can lead to au tochthonous OM with depleted 15N (Meyers 1997). However, if N incorporation uses a significant amount of the lakes NO3 pool, the residual NO3 will become enriched, ultimately leading to an increase in the 15N of newly produced OM (Terranes and Bernasconi 2000; Syvranta et al. 2006). Jones et al. (2004) reported heavier sediment 15N when inorganic N was low in the water colum n, reflecting reduced isotopic fractionation under N limitation. Nitrogen is the primary limiting nutri ent in Lake Apopka a lthough co-limitation with P can occur (Aldridge et al. 1993) Periods of N limitation in the water column can lead to

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164 enrichment of autochthonous OM a nd stratigraphic variation in the 15N signature of sediments in Lake Apopka may indicate periods of N limitation. Other mechanisms may also influence 15N of Lake Apopka sediments. The N isotopic signature of sediment integrates multiple fractionation processes that occur in the sediment and water column (Lehmann et al. 2004). A mmonium production through organic matter mineralization is high in these se diments (Chapter 5). Such minera lization processes can lead to isotopic enrichment of the remaini ng OM. Inglett et al. (2007) related 15N enrichment in the Everglades soil that is highly impacted with P, to an increase in microbial processes (i.e., respiration, mineralization rates). Moreover, denitrification discri minates against heavy 15N and increases in denitrification rates have been related to enriched 15N signatures in sediments (Terranes and Bernasconi 2000; Savage et al. 2004). The N isotope sign ature in Lake Apopka sediments is generated by multiple factors incl uding the isotopic signature of autochthonous N sources, the primary producer community, and N related processes in the water column and sediments (Figure 6-9E). Samples from each core were plotted in isotope space, i.e., 13C vs. 15N (Figure 6-8). Each core occupies a distinct region in isotope space. Lake Apopka is relatively enriched in both 13C and 15N. Mud zone site M9 in Lake Okeechobee displays intermediate values for both isotopes. Lake Annie is most depleted in 13C, but similar in 15N to sites M17 and KR. Excluding the highly different sedi ment types, peat (M17) and sa nd (KR) from the figure, it seems that the remaining sediments s how a gradient in relation to both 13C and 15N. Oligomesotrophic Lake Annie is at the bottom of th e graph with low values, followed by eutrophic Lake Okeechobee (mud sediments M9) with interm ediate values, and then hypereutrophic Lake

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165 Apopka with enriched values for both 13C and 15N. This shows a trend of enrichment in 13C and 15N with increasing trophic state. However, sediment 13C and 15N signatures in each site result from different mechanisms, and they might be more important in interpreting the data than solely tr ophic state (Figure 6-9). Difference in sediment isotope C and N can i ndicate variability on seas onal, inter-annual and century long-time scales of fractionation f actors associated with allochthonous and autochthonous organic matter as well as mine ralization processes o ccurring within lakes (Lehmann et al. 2004) (Figure 6-9). Conclusions In this study, the 210Pb dating technique was used to pr ovide an age/depth relation in the sampled sediments. Lake Annie sediments were the only datable samples, while sediments collected from Lake Okeechobee could not be dated re liably due to low or variable activities of 210Pb and 226Ra. In Lake Apopka, it is possible that uppermost sediments were mixed and it appears that the supported/unsupported boundary was not reached in the core. In Lake Annie, the bottom sediment layer of the core was esti mated to date to the 1800s and the average sedimentation rate (since c.1900) was determined to be 36.8 mg cm-2 yr-1. Isotopic signatures in Lake Annie sediments, depleted in 13C and 15N, probably resulted from a combination of several factors such as allochthonous OM input OM from primary productivity, and microbial biomass and activity. In the Lake Okeechobee mud zone, 13C values were slightly depleted while 15N values were enriched towards the sediment surface. These isotopic signatures resulted from several factors such as the phytopl ankton community, high demand for C and N in sediments, and selective mineralization of OM. In the peat zone of Lake Okeechobee, the isotopic signatures (enrichment of 13C and 15N towards the sediment surface) of sediment OM

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166 were related to several factors, including sediment origin (i.e., pl ant tissue), intensities of primary productivity and diagenesis. Stratigraphic variation in 13C and 15N at the KR site probably reflects an input of wastewater from anthropogenic activities, and variable contributions of riverborne allochthonous input, relate d to inter-annual rainfall vari ations. In Lake Apopka, heavy 13C DIC in the water column, with high de mand for inorganic C due to high primary productivity, produced autochthonous OM with enriched 13C. The enriched 15N signature in Lake Apopka sediments was generated by multiple factors including the isotopic signature of autochthonous N sources, the primary producer co mmunity, and N related processes in the water column and sediments. A more detailed study of 13C and 15N isotopes in several compartments, i.e., dissolved C, different N compounds, phytoplankton biomass, bacteria biomass, particulate OM in the water column and sediment, can confirm the major processes affecting the isotopic signatures of these sediments.

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167 051015202530 Activity (dpm g-1) 0 10 20 30 40 50 60 70 Depth (cm) Cs 137 Ra 226 Pb 210 020406080100 Accumulation Rates (mg cm-1 yr-1) 2005 1998 1988 1976 1966 1957 1947 1940 1931 1920 1904 1890 1884 1880 1875 1870210Pb Age (yr) 2005 1998 1988 1976 1966 1957 1947 1940 1931 1920 1904 1890 1884 1880 1875 1870Depth (cm) Dry Mass Organic Matter 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 1860 1880 1900 1920 1940 1960 1980 2000210Pb Age (yr) 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60Depth (cm) Figure 6-1. Results of 210Pb dating of Lake Annie sediments: A) Radioisotope activities (total 210Pb, 226Ra, and 137Cs) versus depth, B) sediment dept h vs. age/date, and C) sediment and organic matter accumulation rates vs. age/year. A B C

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168 051015202530 Activity (dpm g-1) 0 10 20 30 40 50 60 70 80 Depth (cm) Cs 137 Ra 226 Pb 210 02468101214 Activity (dpm g-1) 0 10 20 30 40 50 60 70 80 Depth (cm) Cs 137 Ra 266 Pb 210 Figure 6-2. Radioisotope activities (total 210Pb, 226Ra, and 137Cs) versus depth, in A) Lake Okeechobee, site M9 and, B) Lake Apopka. A B

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169 2004006008001000120014001600 TP (mg kg-1) 0 10 20 30 40 50 60 70 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 801998 2005 1988 1976 1966 1957 1947 1930 1920 1904 1884 1889 1940 -30.0-29.5-29.0-28.5-28.0-27.5-27.0 13Corg () 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 601998 2005 1988 1976 1966 1957 1947 1940 1930 1920 1904 1889 12.012.513.013.514.014.515.015.516.0 TC:TN 0 10 20 30 40 50 60 70 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 801998 2005 1988 1976 1966 1957 1947 1940 1930 1920 1904 1889 1884 0.81.01.21.41.61.82.02.22.4 15N () 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 1998 2005 1988 1976 1966 1957 1947 1940 1930 1920 1904 1889 1884 10111213141516 TN:TP 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 802005 1998 1976 1966 1957 1947 1940 1930 1920 1904 1889 1884 1988 495051525354555657 LOI (%) 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 1998 2005 1988 1976 1966 1957 1947 1940 1930 1920 1904 1889 1884 Figure 6-3. Lake Annie sediment dept h profile of: A) Total phosphorus, B) TC:TN ratio, C) TN:TP ratio, D) 13Corg of sediment organic carbon, E) sediment 15N, and F) organic matter content (LOI %). De p th ( cm ) De p th ( cm ) A B C D E F

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170 020040060080010001200 Total Phosphorus (mg kg-1) 0 10 20 30 40 50 60 70 -26.4-26.2-26.0-25.8-25.6-25.4-25.2-25.0-24.8-24.6 13Corg () 0 10 20 30 40 50 60 70 80 141618202224262830 TC:TN 0 10 20 30 40 50 60 70 2.42.62.83.03.23.43.63.84.0 15N () 0 10 20 30 40 50 60 70 80 4681012141618202224262830 TN:TP 0 10 20 30 40 50 60 70 1618202224262830323436384042 LOI (%) 0 10 20 30 40 50 60 70 80 Figure 6-4. Lake Okeechobee mud zone (site M9 ) sediment depth profile of: A) Total phosphorus, B) TC:TN ratio, C) TN:TP ratio, D) 13Corg of sediment organic carbon, E) sediment 15N, and F) organic matter content (LOI %).De p th ( cm ) De p th ( cm ) A B C D E F

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171 050100150200250300 Total Phosphorus (mg kg-1) 0 10 20 30 40 -27.4-27.2-27.0-26.8-26.6-26.4-26.2-26.0 13Corg () 0 5 10 15 20 25 30 35 40 17.017.518.018.519.019.520.020.5 TC:TN 0 10 20 30 40 0.60.81.01.21.41.61.82.0 15N () 0 5 10 15 20 25 30 35 40 80100120140160180200220240260 TN:TP 0 10 20 30 40 838485868788899091 LOI (%) 0 5 10 15 20 25 30 35 40 Figure 6-5. Lake Okeechobee peat zone (site M17) sediment depth profile of: A) Tota l phosphorus, B) TC:TN ratio, C) TN:TP ratio D) 13Corg of sediment organic carbon, E) sediment 15N, and F) organic matter content (LOI %). De p th ( cm ) De p th ( cm ) A B C D E F

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172 050100150200250300350 Total Phosphorus (mg kg-1) 0 10 20 30 40 -26.4-26.2-26.0-25.8-25.6-25.4 13Corg () 0 2 4 6 8 10 12 14 16 18 20 68101214161820 TC:TN 0 10 20 30 40 0.00.51.01.52.02.53.03.54.04.5 15N () 0 2 4 6 8 10 12 14 16 18 20 020406080100120140160180 TN:TP 0 10 20 30 40 0.00.20.40.60.81.01.2 LOI (%) 0 2 4 6 8 10 12 14 16 18 20 Figure 6-6. Lake Okeechobee sand zone (site KR ) sediment depth profile A) Total phosphor us, B) TC:TN ratio, C) TN:TP ratio, D) 13Corg of sediment organic carbon, E) sediment 15N, and F) organic matter content (LOI %). De p th ( cm ) De p th ( cm ) A B C D E F

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173 0200400600800100012001400 Total Phosphorus (mg kg-1) 0 10 20 30 40 50 60 70 80 90 100 -23-22-21-20-19-18 13Corg () 0 10 20 30 40 50 60 70 80 11.011.512.012.513.013.514.0 TC:TN 0 10 20 30 40 50 60 70 80 90 100 3.63.84.04.24.44.64.85.0 15N () 0 10 20 30 40 50 60 70 80 1520253035404550 TN:TP 0 10 20 30 40 50 60 70 80 90 100 606264666870 LOI (%) 0 10 20 30 40 50 60 70 80 Figure 6-7. Lake Apopka sediment de pth profile A) Total phosphorus, B) TC:TN ratio, C) TN:TP ratio, D) 13Corg of sediment organic carbon, E) sediment 15N, and F) organic matter content (LOI %). De p th ( cm ) De p th ( cm ) A B C D E F

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174 -30-28-26-24-22-20-18-16 13Corg () 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 15N () Annie Apopka M9 M17 KR Figure 6-8. Carbon vs. nitrogen isotopic values of sediments in Lake Annie, Lake Okeechobee (sites M9, M17, and KR), and Lake Apopka.

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175 Figure 6-9. Major mechanisms affecting the sediment 13C and 15N signatures in: A) Lake Annie, Lake Okeechobee B) site M9, C) site M17 and D) site KR, and E) Lake Apopka. Allochthonous ? Agricultural runoff 13C 15N Phytoplankton Non N2-Fixer ?13C 15N 13C 15N S edimen t C and N limitation B) Lake Okeechobee M9 Mineralization Water Column [N] N2 Fixation ? A) Lake Annie 13C 15N S edimen t 13C 15N Phytoplankton 13C 15N Allochthonous C3 Plants OM 13C 15N Bacteria 13C 15N [NH4 +] 15N [CO2] 13C OM 13C 15N Mineralization Mineralization Biomass Mineralization Biomass Groundwater Anoxia Water Column

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176 Figure 6-10. continued Figure 6-9. continued Seasonal Allochthonous Input Agricultural runoff, sewage 13C 15N 13C 15N D ) Lake Okeechobee K R S edimen t Water Column E) Lake Apopka NH4 + NO3 N2 15N Sediment Methanogenesis Phytoplankton 13C 15N N2 Fixation CO2 13C OM 13C 15N DIC CO2/HCO3 13C [NO3 -] 15N Mineralization 13C 15N Water Column Mineralization Phytoplankton N2-Fixer ?13C 15N 13C 15N C limitation Peat 13C 15N D ) Lake Okeechobee M17 S edimen t N2 Fixation NH4 + 15N Water Column

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177 CHAPTER 7 HETEROTROPHIC MICROBIAL ACTIVITY IN SEDIMENTS: EFFECTS OF ORGANIC ELECTRON DONORS Introduction Organic matter deposition is an important sour ce of carbon (C) in lake sediments. Organic compounds and associated nutrients supplied to sediments are mineralized through heterotrophic decomposition (Gchter and Meyer 1993; Capone and Kiene 1988; Megonigal et al. 2004). The composition and activities of microbial communities are regulated by the quality and availability of carbon. In high depositional environments such as eutrophic or deep thermally stratified lakes, organic content in sediments is often high, such that oxygen (O2) is rapidly consumed, and is depleted within several millimeters below the sediment water interface (Jrgensen 1983). In these systems, facultative and strict anaerobic communities dominate. Complete oxidation of a broad range of organic compounds in these syst ems can occur through the sequential activity of different groups of anaerobic ba cteria (Capone and Kiene 1988). In methanogenic habitats, i.e., in the absen ce of inorganic electron acceptors, different groups of microorganisms participate in decom position of organic matter as no single anaerobic microorganism can completely degrade organi c polymers (Zinder 1993, Megonigal et al. 2004). Cellulolytic bacteria hydrolyze organic polym ers through extracellular enzyme production and further break down monomers to alc ohols, fatty acids, and hydrogen (H2) through fermentation. Alcohols and fatty acids are degraded by syntrophic bacteria (secondary fermenters) into acetate, H2, and carbon dioxide (CO2), which is used as substrate by methanogens (Zinder 1993, Conrad 1999, Megonigal et al. 2004). The structures and functions of anaerobic microbial communities are therefore strongly affected by compe tition for fermentation products such as H2 and acetate. Microorganisms derive energy by transferring electr ons from an external source or donor to an external electron sink or terminal electron acceptor. Organic electron donors vary from

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178 monomers that support fermentation to simple compounds such as acetate and methane (CH4). Fermenting, syntrophic, methanogenic bacteria and most other anaer obic microorganisms (e.g., sulfate, iron reducers) are sensitive to the c oncentrations of substr ates and products. Their activities can be inhibited by their end products and are dependent on the end product consumption by other organisms (Sta ms 1994; Megonigal et al. 2004). Microbial functional diversity in cludes a vast range of activit ies. One component of this diversity has been characterized by measuring catabolic response profile, i.e., short-term response of microbial communities to addition of a wide variety of C-substrates (Degens and Harris 1997; Degens 1998a). This has been widely applied in soil studies to address differences in microbial communities in different soil types, disturbance, and land use (Degens and Harris 1997; Lu et al. 2000; Degens et al. 2000, 2001; Stevenson et al. 2004). Substrate induced respiration is often dependent on the size of the microbial biomass poo l; however, response of microbial communities is also related to the catabolic diversity of soil microorganisms (Degens 1998). A greater relative catabolic response to a substrate in one system as compared with another indicates that the microbial community is more functionally adapted to use that resource, and may indicate previous exposure to those Csources (Degens and Harris 1997; Degens 1998; Baldock et al. 2004; St evenson et al. 2004). The metabolic response of microbial commun ities in lake sediments may vary due to several factors that either in fluence the microbial community or are due to physico-chemical characteristics of lakes, such as the source a nd chemical composition of particulate matter and biogeochemical processes in the sediment and water column. Accumula tion and retention of particulate matter and nutrients in sediments depends on lake morphometry, water renewal, nutrient loading, edaphic characte ristics of the drainage basi n, among other factors (Bostrm et

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179 al. 1988). Eutrophic and hypereutrophic lakes typically receive high external loads of nutrients and display high primary productivity and nutrient concentrations in the water column and these nutrients eventually reach the sediment, theref ore sediments from eutrophic and hypereutrophic lakes are expected to have hi gh concentrations of organic ma tter. Binford and Brenner (1986) and Deevey et al. (1986) showed that net accu mulation rates of organic matter and nutrients increase with trophic state for Flor ida lakes. In contrast, small olig otrophic lakes are expected to exhibit a relatively high proportion of allochthon ous carbon input to their sediments (Gu et al. 1996). Lake depth can also affect th e quality of organic material reaching the sediment. In deep lakes, sedimenting organic matter undergoes inte nse decomposition in the water column due to the prolonged period of settling. Consequently, low amounts of la bile organic carbon reach the sediment (Suess 1980; Meyers 1997) In shallow lakes, however, the supply of labile carbon and nutrients can be higher in sediments than in de ep lakes, and the latter often can have more refractory organic matter. Sediments with differe nt C-sources and with different quality and quantity as well as nutrient concentration wi ll have different microbial communities. These communities can display distinct catabolic respon ses as the mineralization rates of a microbial community are dependent upon the metabolic capac ity for a given substrate (Torien and Cavari 1982). The objective of this study was to evaluate the short term catabolic response of the microbial communities in sediments of three subtropical lakes characterized by different trophic states. The central hypothesis is that sediments with higher C availability will have higher catabolic diversity.

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180 Materials and Methods Study Sites Three Florida (USA) lakes ranging in trophic state were selecte d. Lake characteristics were described in Chapter 2. The characteristics and location of sampled site s were described in Chapter 3 (Table 3-1, Figure 3-1). Field Sampling Triplicate sediment cores were collected usi ng a piston corer (Fishe r et al. 1992) or by SCUBA divers. The topmost 10 cm of sediment we re collected from one central site in Lake Annie on June 25, 2005 and a western site in Lake Apopka on May 28, 2005 (Figure 3-1C, Table 3-1). Cores were collected at three sites in Lake Okeechobee on July 16, 2005: M17 = peat, M9 = mud and KR = sand (Figure 3-1B, Table 3-1). Samples were placed in plastic bags, sealed, and kept on ice. All measured sediment variables are reported on a dry wei ght basis (dw). Water quality variables were described in a previous study (Chapter 4). Sediment Properties Samples were transported on ice and stored in the dark at 4 C. Before each analysis, samples were homogenized and sub-samples taken. Sediment bulk density (g dry wt cm-3 wet) was determined on a dry weight basis at 70 C for 72 hours, and pH was determined on wet sediments (1:2 sediment-to-water ratio). Sediment samples were ground in a ball mill and passed through a # 40 mesh sieve. Organic matter cont ent (LOI-loss on ignition) was determined by weight loss at 550C. Total P was measured by ignition method, followed by acid digestion (6 M HCl) and measured colorimetrica lly with a Bran+Luebbe TechniconTM Autoanalyzer II (Anderson 1976; Method 365.1, EPA 1993). Total ca rbon (TC) and total nitrogen (TN) were determined using a Flash EA-1121 NC soil analyzer (Thermo Electron Corporation).

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181 Extractable Carbon (C), Nitrogen (N), and Phosphorus (P) Sediment samples were extracted with 0.5 M K2SO4 for extractable N and with 0.5 M NaHCO3 (pH = 8.5) for extractable P, using a 1: 50 dry sediment-to-solution ratio (Mulvaney 1996; Ivanoff et al. 1998). Extracts from sample s were centrifuged at 10,000 x g for 10 min and filtered through Whatman # 42 filter paper. For N an alysis, 5 mL of the extracts were subjected to Kjeldahl nitrogen digesti on and analyzed for total Kjelda hl-N colorimetrically using a Bran+Luebbe TechniconTM Autoanalyzer II (Method 351.2, EP A 1993). Undigested N extracts were analyzed for ammonium (NH4-N) (Method 351.2, EPA 1993), a nd represent extractable ammonium (Ext-NH4-N). The difference between Ext-N and Ext-NH4-N represents extractable labile organic nitrogen (Ext-ON) Extracts from P samples were centrifuged at 10,000 x g for 10 min and filtered through a 0.45 m membrane filte r, and analyzed for soluble reactive P or digested for TP (with sulfuric acid and potassium persulfate). Solutions were analyzed by colorimetry, determined by reaction with molybdate using a Bran+Luebbe TechniconTM Autoanalyzer II (Murphy and Riley 1962; Me thod 365.1, EPA 1993). Undigested P extracts represents labile inorganic P (Ext-Pi). The difference between total extracted P and Ext-Pi represents labile organic phosphorus (Ext-Po). Microbial Biomass Carbon Microbial biomass carbon (MBC) was meas ured with the chloroform fumigationextraction method (Hedley and Stewart 1982; Vance et al. 1987). Briefly, sediment samples were split in two: one sample was tr eated with alcohol-free chloroform (0.5 mL) to lyse microbial cells, placed in a vacuum desiccator, and incubated for 24 hrs. The duplicate sample was left untreated. Both sets were extracted with 0.5 M K2SO4 centrifuged at 10,000 x g for 10 min and filtered through Whatman # 42 filter paper. Carbon extracts were aci dified (pH < 2) and analyzed in an automated Shimadzu TOC 5050 analyzer (Method 415.1, EPA 1993). Microbial biomass

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182 C was calculated by the difference between treated and non-treated samples. Extracts from the untreated samples represent extr actable organic carbon (Ext-C). Electron Donors Basic catabolic response was characterized by increasing CO2 and CH4 production rates from sediment samples by addition of different electron donors. Eight diffe rent simple organic compounds (electron donors) were added to each se diment sample. They consisted of two amino acids (alanine and arginine), four carboxylic acid s (acetate, formate, butyrate, and propionate), one polysaccharide (glucose), a nd lake suspended solids (Lake-SS). Wet sediment (based on 0.5 g of dry weight) was added to incubation bottle s, sealed with rubber stoppers and aluminum crimp seals, and purged with N2 gas. Alanine, arginine, acetate, formate, butyrate, propionate, and glucose were added from an aerobic sterile stock solutions to sediments on a C-equivalent basis, reaching a final concentra tion of 42 mM C (504 g of C g-1 on a dry weight basis) (Degens 1998 a ). All stock solutions were ad justed to pH around 7.0 using eith er HCl or NaOH at the time of preparation to avoid any substrat e-pH effects on microbial communities. Lake-SS was obtained by centr ifugation (10,000 x g for 30 min) of water samples collected at approximately 50 cm depth in the water colu mn of each lake. Lake-SS was characterized for LOI, TC, TN, and TP as describe d previously, and was added on th e same C-equivalent basis as the other electron donors. A sample from each Lake-SS was incubated to account for CO2 and CH4 production of the material. Values obtained were subtracted from CO2 and CH4 production rates of the Lake-SS treatment for each lake. Sedi ments from each site were also incubated with no substrate addition (control) to ob tain values of basal anaerobic CO2 and CH4 production rates. Samples were incubated anaerobically at 30 C in the dark. Gas samples were taken at 1, 2, 4, 7, 10, and 14 days and quantified for CO2 and CH4. Gas samples from Lake Annie and Lake Okeechobee sediments were also taken at 20 days of incubation due to low CH4 production

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183 detected with some treatments at 14 days of the experiment. CO2 was measured by gas chromatography using a Schimadzu (8A GC-T CD) and Poropak N column (Supelco Inc., Bellefonte, PA) with He as a carrier gas and CH4 was analyzed on a Shimadzu gas chromatograph-8A fitted with flam e ionization detector (110 C), N2 as the carrier gas and a 0.3 cm by 2 m Carboxyn 1000 column (Supelco Inc ., Bellefonte, PA) at 160 C. Prior to measurements of both CO2 and CH4, headspace pressure was determined with a digital pressure indicator (DPI 705, Druck, New Fairfi eld, CT). Concentrations of CO2 and CH4 were determined by comparison with standard concentrations a nd production rates were calculated by linear regression ( r2 > 0.95). Final production rates were determ ined after removing the lag phase (the time between substrate addition a nd quantifiable gas production) in each site. Turnover rates (d-1) were determined by di viding the sum of CO2 and CH4 production rates by the amount of C added. Anaerobic CO2 and CH4 production rates were standard ized by microbial biomass carbon of each sediment sample (CO2 or CH4 production divided by MBC). Statistical Analysis All statistical analyses were conducted w ith standardized values of anaerobic CO2 and CH4 production rates. One-way analysis of variance (ANOVA) with pairwise multiple comparisons Tukeys HSD test was used to assess the eff ect of different electr on donor additions on CO2 and CH4 production and turnover rates. One-way ANOVA was performed separately on each site. A Principal Component Analysis (PCA) was pe rformed to determine major patterns of CO2 and CH4 production rates with the addition different el ectron donors. All statis tical analyses were conducted with Statistica 7.1 (StatSoft 2006) software.

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184 Results Sediment Properties Sediment pH ranged from 5.9 to 7.8 (Table 7-1) Lake Annie sediment pH was lower than other lakes. Both Lake Okeec hobee and Lake Apopka sediment pH were circum-neutral to alkaline (Table 7-1). Surface sediment bulk dens ities were lowest in Lake Apopka, followed by Lake Annie, and then Lake Okeechobee sites M9, M17, and KR, respectively (Table 7-1). Sediment organic matter content was highest at Lake Okeechobee site M17, reflecting high peat content. Next in order were Lake Apopka, Lake Annie, followed by site M9, and sandy KR in Lake Okeechobee (Table 7-1). Total C was highest in peat zone sediments of Lake Okeechobee, followed by sediments from Lake Apopka and Lake Annie. Lake Apopka and peat zone sediments of Lake Okeechobee exhibited simila r values of TN (Table 7-1). Lake Annie sediments exhibited higher TP concentra tions than Lake Okeechobee and Lake Apopka sediments (Table 7-1). Extractable organic C and MBC were highest in Lake Apopka sediments (Table 7-1). Lake Apopka sediments also had the highest concentrations of Ext-ON and Ext-NH4-N (Table 7-1). Lake Annie sediments, however, ha d the highest concentrations of labile inorganic P and labile organic P (Table 7-1). Sediment Ext-C:Ext-N ratio was similar in all lake sediments (Table 7-1). Lake Annie and sites M9 and KR in Lake Okeechobee exhibited low Ext-C:Ext-P and ExtN:Ext-P ratios. Electron Donors Dry suspended material content of three la kes (Lake-SS) was characterized as: 34.2 % C and 2.9% N from Lake Annie; 15.6 % C and 1.5% N from Lake Okeechobee; and 33.6 % C and 3.7% N from Lake Apopka. Addition of electro n donors to sediment microcosms stimulated heterotrophic microbial activity (Figures 7-1, 7-2, 7-3, 7-4, 7-5, Table 7-3). All sediments

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185 showed a quick response by increasing CO2 production after addition of electron donors (Figures 7-1, 7-2, 7-3, 7-4, 7-5B, C). Basal CO2 and CH4 production rates were highest in Lake Apopka sediments (Table 7-3). Sediments from Lake Okeechobee had the longest lag phase in CH4 production (Figures 7-1, 7-2, 7-3, 7-4, 7-5D, E, F). Addition of different electron donors produced different response in each lake sedi ment (Table 7-3). Results from one-way ANOVAs were significantly different ( p < 0.05, Table 7-2). Lake Annie sediments had the highest CO2 production rates with both amino acids and form ate addition. All Lake Okeechobee sediments had the highest CO2 production rates with both amino aci ds and glucose addition. Lake Apopka sediments had the highest CO2 production rates w ith alanine and formate (Table 7-3). Higher CH4 production rates in Lake Annie sediment s were detected with the addition of both amino acids and acetate. In Lake Okeechob ee mud (site M9) and sand (site KR) sediments, higher CH4 production rates were detected in alanine, butyrate, and glucose treatments. In the peat zone sediments (site M17) of Lake Okeech obee, addition of the two amino acids, acetate, and butyrate produced higher CH4 than other substrates. In Lake Apopka, highest CH4 production rates were detected with the addition of alanine, gluc ose, and Lake-SS (Table 7-3). Turnover rates were highest for alanine, arginine and acetate in Lake Annie sediments. In Lake Okeechobee M9 and KR sediments, turnover rates were higher for alanine and glucose. In the peat sediments the highest turnover rates were for alanine, arginine, and glucose. Lake Apopka had similar values of turnover rates for differe nt C-sources and the highest was detected for alanine (Table 7-3). Lake Apopka sediments had th e highest turnover rates of all C-sources when compared to the other sediments (Two-way ANOVA, F = 3.88, d.f. = 28, p < 0.00001).

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186 The magnitude of CO2 and CH4 production following addition of different electron donors was strongly related to microbial biomass at each site. There was a strong significant positive correlation between MBC and CO2, and MBC and CH4 production rates (Figure 7-6A, B). Two Principal Component Analyses (PCA) were conducted, PCA-1 was performed using the effect of electr on donor additions on CO2 production rates, and PCA-2 on CH4 production rates. The PCA-1 indicated that 40.7% of the data variability was explained by Axis 1 while Axis 2 explained 20.1% (Figure 7-7A). Anaerobic respira tion with the additions of acetate, butyrate, formate, and Lake-SS were the variable s selected by Axis 1. Basal anaerobic CO2 production was selected by Axis 2. The position of sites in relation to variable load ings in PCA-1 showed that sediments from each lake and site are sepa rated into different groups (Figure 7-7B). Lake Annie sediments were plotte d in the position of basal CO2 production (Figure 7-7B). Lake Apopka sediments with Lake-SS cluster (Lake-SS butyrate, acetate, formate, and propionate) opposite from Lake Annie. Lake Okeechobee si te M17 was plotted close to Lake Apopka sediments, while the KR site was in the posi tion with glucose and alanine additions. Lake Okeechobee mud zone (site M9) was not placed with any specific carbon addition (Figure 7-7B). The PCA-2 had 33.6% of the data variability explained by Axis 1 while Axis 2 explained 27.4% (Figure 7-8A). Methane prod uction rates with addi tions of alanine, butyrate, and glucose were the variables selected by Axis 1. Meth ane production rates from arginine, and basal production rate were selected by Axis 2. The posi tion of the sites in re lation to the variable loadings in PCA-2 showed a separation of sediment s from each lake and site (Figure 7-8B). Lake Annie sediment was placed with the basal produ ction, arginine, and acetate cluster. Lake Okeechobee M9 site was plotted in the position of propionate and formate and close to the KR

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187 site that was positioned with glucose, alanine, and butyrate. Site M17 was plotted in opposite position of all C-sources. Lake Apopka sediments were placed with Lake-SS (Figure 7-8B). Discussion Addition of organic electron donors to sedime nt microcosms stimulated heterotrophic activity. Findlay et al. (2003) showed that the ad dition of different carbon sources, i.e., glucose, bovine serum albumin and natural leaf leachat e to hyporheic biofilms enhanced microbial activities. Wang et al. (2007) showed that addi tion of electron donors (glucose, sucrose, potato starch, and sodium acetate) stimulated denitrific ation in Lake Taihu (China) sediments. In the study of benthic microbial response to the deposit ion of natural seston in Lake Erken (Sweden), Trnblom and Rydin (1998) showed that seston addition caused an immediate increase in bacterial production, activity, a nd total sediment metabolism. The extent of response to electron donor addition was strongly related to microbial biomass. Most sediments responded rapidly to ad dition of most of electron donors by increasing their CO2 production rates. Sediments from site KR in Lake Okeechobee with the lowest microbial biomass showed the longest lag phas e before responding to electron donor addition (Figure 7-4A, B, C, Table 7-3). The turnover rates were also related to microbial biomass. Lake Apopka sediments with the largest microbial bioma ss exhibited the highest turnover rates (Table 7-1, 7-3). Statistical co rrelations suggest that observed ra tes of carbon source consumption are strongly a function of microbial biomass at each site (Figure 7-6A, B). These results are in accordance with other studies (Lu et al. 2000) that have shown that the response of soils to the addition of C sources is depende nt on microbial biomass. Although the magnitude of response to electron donor additions was related to microbial biomass, different responses in each sediment were related to the catabolic diversity of microorganisms. Principal Component Analysis 1 results showed that Lake Apopka had the

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188 highest respiration per microbia l biomass with most of the el ectron donor additions (propionate, Lake-SS, butyrate, acetate, and, fo rmate), indicating that these sedi ments respired most of the C added (Figure 7-7A, B). This suggests that the ca tabolic diversity and activity in these sediments is higher than other sediments. Increased biogeochemical diversity can be present in environments with high organic matter content, with diversity in organic compounds as well as increased by-products diversity (Odum 1969). As an example, Castro et al. (2005) studied the distribution of sulfate (SO4 -2)-reducing prokaryotic assemblages in soils of the nutrient impacted regions of the Florida Everglad es. The authors reported that co mplete oxidizing species, which are able to use a broader array of electron donor s were dominant in eutrophic and transitional sites while incomplete oxidizers, which are more efficient at taking up lo w concentrations of a few substrates, were present in oligotrophic regions. The authors concluded that eutrophic regions with greater amount of car bon may select for generalists ca pable of taking advantage of a greater diversity of carbon substrates. Lake Apopka exhibits high primary production and high labile C sedimentation (Gale et al. 1992, Ga le and Reddy 1994), supporting higher catabolic diversity. Others studies, however, have reported that under P limitation hetero trophic bacteria tend to respire added C. In controlled experiments with bacterioplankton in subarctic Lake Diktar Erik, Sweden, Jasson et al. (2006) showed that addition of C was used for growth under Climited conditions, but used for respiration under Pi limitation. They concluded that bacterioplankton communities tend to respire large portions of added C under P limitation, and high respiration rates of exce ss C was partly used to support growth and not only for maintenance. Lake Apopka exhibited the highest ex tractable C:P and N:P ra tios (Table 7-1). In another study, I found that microorganisms in Lake Apopka surface sediments are P limited,

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189 while the other sediments are limited by C alone (Lake Annie) or co-limited by C and N (Lake Okeechobee sites M9 and KR) or C and P (Lake Ok eechobee site M17) (Chapter 5). The results from PCA-1 also placed peat sediments (site M1 7), which are also P limited, close to Lake Apopka sediments. These results indicated that sediments limited by P respired the C-added, while sediments limited by C might have used the C-added for growth (Jasson et al. 2006). Addition of some electron donors did not stimulat e heterotrophic microbi al respiration, and with others the stimulation was not significantly different from basal activities (Table 7-3). This could indicate lack of organisms able to use th e substrate as well as th e assimilation of added C into microbial biomass rather than being released as CO2 via respiratory pathways (Bremer and van Kessel 1990; Degens 1998a). Trnblom and R ydin (1998) found that af ter seston addition to sediment, bacterial biomass doubled indicating assimilation of C into microbial biomass. For forested soils, the partitioning between bi omass-C incorporation and respiratory CO2-C was determined to be substraterather than soildependent. van Hees et al. (2005) reported for forested soils that 60-90% of organic acid, 20 -60% of monosaccharide and 10-30% of amino acid is evolved as CO2. Studies with different microorganisms reported that between 30 and 40% of glucose and up to 80% of formate of the C s ource supplied is immediatel y used for respiration and the remaining for biomass growth (Stout hamer 1976). King and Klug (1982) reported that the addition of glucose into microbial biomass was low (20%) in a eutrophic lake sediment (Wintergreen Lake). In this study, amino acids, glucose and formate were the C-sources that were used through respiratory pathways rather then added into biomass. Lake Annie was positioned with basal CO2 production rates indicating that this site had the highest anaerobic respiration per microbial biomass (Figure 7-7A, B). Lake Apopka was positioned on the opposite side, indicating the lowe st basal anaerobic respiration per microbial

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190 biomass. The metabolic quotient ( q CO2; proportion of basal respira tion per microbial biomass) has been used in soil studies to indicate ecol ogical efficiency of the soil microbial community (Anderson and Domsch 1990; Degens 1998b). This index is based on Odums theory of ecosystem succession (1969), where during ecosyst em succession towards maturity there is a trend of increasing efficiency in energy utiliza tion concomitant with an increase in diversity. High q CO2 indicates inefficient use of energy, while low q CO2 indicates high efficiency and more carbon is utilized for biomass produc tion (Anderson and Domsch 1990; Degens 1998b; Anderson 2003; Francaviglia 2004). Moreover, if the progression of lakes in time from less productive (oligotrophic) to more productive (e utrophic-hypereutrophic) can be viewed as a natural succession, higher q CO2 should be detected in oligotrophi c lakes. The trend of decreasing q CO2 with increasing trophic state is clearly presented in Axis 2 of the PCA-1 (Figure 7-7A, and B). The same results were reported by Smith and Prairie (2004) in the st udy of bacterioplankton of lakes of different trophic states. These authors concluded that oligotrophy places high respiratory demands on bacterioplan kton, with greater DOC flow to CO2 rather than to biomass. In Lake Annie sediments addition of pr opionate inhibited microbial activity (CO2 and CH4 production rates) (Table 7-3). La ke Annie sediments are charact erized by high Fe (3640 mg kg-1) (Thompson 1981), and dissolved SO4 -2concentration (7.2 mg L-1) in the water column (Swain and Gaiser 2005). High SO4 -2 reduction has also been reported to occur in the water column (Swain and Gaiser 2005). Although Fe oxides and SO4 -2 concentrations were not measured in this study it is probably safe to assume that both Feand SO4 -2-reducers are presen t and/or active in the Lake Annie sediments. Sulfate reducers are able to utilize a variety of organic compounds, including propionate and butyrate (Widell 1988). Propionate can also be oxidized by syntrophic and acetogenic

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191 bacteria (Stams 1994; Schink 1997). These conve rsions, however, are often energetically unfavorable, and continuous removal of their pro ducts by methanogens is required so that these conversions become exergonic (Stams 1994; Schink 1997; Kleerebezem and Stams 2000). Presence of Feand SO4 -2-reducers is thought to limit syntrophic bacteria as these are able to use products of primary fermentation more effici ently (Stams 1994; Schink 1997). In marine sediments, however, it has been shown that syntrophy occurs in sediments with high SO4 -2 concentration (Kendall et al. 2006 ). The oxidation of butyrate has a mechanism similar to that one described for propionate, a lthough different syntrophic specie s are usually involved (Schink 1997; Kleebrebezem and Stams 2000). Addition of but yrate did not inhibit anaerobic respiration; however, anaerobic CO2 production was not statistic ally different from basal respiration (Table 7-3). Holmer and Kristensen (1994) in the study of fish farm sediments amended with labile organic matter, reported accumulation of propionate due to SO4 -2 reducers inhibition. They concluded that this was an indication of suppression of H2-sensitive fermentation reactions, as the formation of H2 and acetate from propionate is thermodynamically more sensitive to H2 inhibition than other reactions as with butyrate and ethanol. In the study of intermediary metabolism of organic matter in sediments of Wintergreen Lake (USA), Lovley and Klug (1982) reported that addition of H2 inhibited the metabolism of propionate whereas the butyrate metabolism was only partially inhibited. The mech anism for inhibition of anaerobic respiration with propionate addition cannot be determined with the present data. However it can be speculated that it could have re sulted from the absence of species able to use propionate, or H2 was not efficiently removed by methanogens. Basal CH4 production rates were highest in hyper eutrophic Lake Apopka. Several studies have shown similar results where methane pr oduction rates were highe r in eutrophic than

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192 oligotrophic lakes (Casper 1992; Rothfuss et al. 1997; Falz et al. 1999; Nsslein and Conrad 2000; Huttunen et al. 2003; Dan et al. 2004). Extremely low basal CH4 production rates in eutrophic Lake Okeechobee sediments may be explained by electron donor limitation. In a previous study (Chapter 2), basal CH4 production was not detected, but was stimulated after the addition of acetate and/or H2 in sediments of Lake Okeechobee. Although a lag phase for CH4 production was observed in all sediments, CH4 production was much dela yed in sediments from sites M17 and KR in Lake Ok eechobee (Figures 7-1, 7-2, 7-3, 7-4, 7-5D, E, F). Methanogens (Archaea) are obligate anaerobes and use a li mited number of substrates, including H2 plus CO2, formate, acetate, methanol, and methylated amines (Oreland 1988). The most important substrates for methanogens are H2/CO2 and acetate, and they often depend on other anaerobic bacteria for these substrates (Conrad 1999). Other anaerobic bacteria (i.e., Fe and SO4 -2 reducers) can outcompete methanogens for H2/CO2 and acetate due to higher subs trate affinities and higher energy and growth yields (Lovley and Klug 1983; Lovley and Phillips 1986; Conrad et al. 1987; Bond and Lovley 2002); however, both processes can coexist (Mountfort and Asher 1981; Holmer and Kristensen 1994; Roy et al. 1997; Holmer et al. 2003; Roden and Wetzel 2003; Wand et al. 2006). Coexistence occurs because of spatial variat ion in the abundance of terminal electron acceptors or because the supply of electron donors is non-limiting (Roy et al. 1997; Megonigal et al. 2004). The lag phase observed for CH4 production in all sediments can be explained by two mechanisms. First methanogenic activity was stimulated in the presen ce of their substrates that were produced by fermentative activity. Second, methanogens became active after other electron acceptors (FeIII, SO4 -2) were consumed and depleted in sediment microcosms.

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193 Most methanogenic species use H2/CO2 and a fewer number of species can use acetate (Garcia et al. 2000). The present data do not allow any conclusions about the major pathways of CH4 production in these sediments. Formate can be used as a substitute for H2/CO2, however, only about half of the H2-users are able to use formate for CH4 production (Vogels et al. 1988). Methane production rates from acetate can also result from syntrophic acetate oxidation to CO2 and H2 coupled with methanogenesis from H2/CO2 (Zinder 1994). In lake sediments the dominance of acetocla stic versus hydrogenotrophic methanogenesis has been reported to be related to sediment prope rties (i.e., pH and temperature). In acidic Lake Grosse Fuchskuhle (Germany), with high humic content, acetate users ( Methanosarcinaceae ) were the only detected methanogens (Casper et al. 2003). Phelps and Zeik us (1984) reported that acetoclastic methanogenesis was the major pathway for CH4 production in a mildly acidic (pH 6.2) lake (Knaack Lake, Wisconsin). The increase in pH to neutral values enhanced total CH4 production from H2/CO2, but did not affect the CH4 produced from acetate (Zeikus 1984). Acetoclastic methanogenesis is dominant at low temperatures In mesotrophic Lake Rotsee (Switerland) sediments, it was reported that Methanosaeta (acetoclastic methanogen) was the major methanogenic population (91%), indicating th at in cold sediments acetate is the main CH4 precursor, and hydrogenotrophs were only found in the organic-rich, upper 2 cm of sediment (Falz et al. 1999). Nsslein and Conrad (2000) reported that CH4 was produced from acetate at low temperatures (4 C) but it wa s produced from both acetate and H2/CO2 at higher temperatures (25 C) in sedi ments of eutrophic Lake Plu see (Germany). Schulz and Conrad (1996, 1997) reported a change in the methanogeni c degradation pathway of organic matter in sediments of mesotrophic Lake Constance (Germany). The authors showed that CH4 production in these cold (4 C) sediments was exclusively from acetate, however, an in crease in temperature

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194 (20-25 C) lead to an incr ease in contribution of CH4 production from H2/CO2 and probably from methanol. They hypothesized that at low temper atures hydrogenotrophs were unable to compete with H2-utilizing homoacetogenic bacteria. Moreover, methanogenic degradation of organic matter should be dominated by homoacetogenesis plus acetoclastic methanogenesis at low temperatures versus fermentation, syntrophy, H2 production and hydrogenotrophic methanogenesis at high temperatures. The same results were reported for sediments from eutrophic Lake Dagow (Germany), however, the change of dominance from acetoclastic to hydrogenotrophic methanogenesis with an increase in temperature was not followed by a change in community structure of the major phylogentic groups of methanogens (Glissmann et al. 2004). Lake Annie sediments are acidi c and probably maintain fairly constant low temperatures. Thermal stratification of the water column was detected during sampling in this lake with a temperature of 17.3 C below 14 m water column depth (Chapter 4). Sediment temperature is probably much lower in this deep (20 m) lake. Se diment acidic pH and low temperatures as well as the high CH4 production rate with addition of acetate and the placement of Lake Annie with acetate cluster in the PCA-2 suggests that acetoc lastic methanogenesis may be an important pathway for CH4 production in these sediments (Table 7-3, Figure 7-8A, B). Lake Okeechobee and Lake Apopka had high temperatures at th e sediment-water surface (26.3-30.7 C), and both lakes had circum-neutral to alkaline sedi ment pH (Table 7-1), good conditions for hydrogenotrophic methanogenesis. In Lake Okeechob ee sediments it has been determined that hydrogenotrophic methanogenesis is the main pathway of CH4 production (Chapter 2). The pathway for methane production in Lake Apopka ca nnot be determined with the current data, however, hydrogenotrophic methanogenesis might be an important pathway in this hypereutrophic lake. Algae deposition is an impor tant source of C to methanogenic activity in

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195 Lake Apopka sediments as high stimulation of CH4 production with addition of Lake-SS was detected (Table 7-3, Figure 7-8A, B). Lake A popka has high primary productivity (Carrick et al. 1993) and suspended solids in the water column are mainly composed of phytoplankton biomass (Phlips et al. 1995 c ; Havens et al. 1996). Although most sediments showed an increase in CO2 and CH4 with Lake-SS, in Lake Apopka the increase was highest. CH4 production in Lake Apopka sediments was highly stimulated by the add ition of Lake-SS and stat istically higher than basal productions. This shows how well adapted methanogens in these sediments are to using algae derived-C. Molecular studi es targeting the archaeal commun ity are necessary to elucidate the major pathway for methane produc tion in the present study lakes. Conclusions Addition of organic electron donors to sedime nts stimulated heterotrophic activity. The extent of the response, however, was strongl y related to microbial biomass and catabolic diversity. Although the magnitude of the respons e to electron donor addition was related to microbial biomass, the different response in each sediment was related to the catabolic diversity of the sediment microbial community. The additi on of some electron dono rs did not stimulate heterotrophic microbial respira tion, and probably resulted in the incorporation of C into microbial biomass rather than release via resp iratory pathways. Lake Apopka had the highest respiration per microbial biomass, indicating that these sediments respired most of the C added. This was probably caused by a P limitation. Lake Annie showed the highest q CO2, indicating an inefficient use of energy. The low q CO2 found in Lake Apopkas sediment indicates high efficiency. Lake Apopkas sediment catabolic diversity was higher than in the other sediments. In relation to methane production, acetoclastic methanogenesis is probably more important in Lake Annie sediments. The importance of hydroge ntrophic methanogenesis in Lake Okeechobee sediments was determined in another study. The pathway for methane production in Lake

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196 Apopka cannot be determined with the current da ta. These results showed that the sediments with different biogeochemical properties had di fferent microbial communities with distinct catabolic responses to additions of Csources.

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197 Table 7-1. Sediment biogeochemical properties of Lake Annie, Lake Okeechobee, and Lake Apopka. Lake Annie Okeechobee Apopka Variables Central M9 M17 KR West pH 5.9 0.01 7.8 0.07 7.7 0.02 7.6 0.2 7.5 .06 BD 0.052 0.0020.137 0.060.143 0.0181.183 0.29 0.019 0.003 LOI (%) 55.6 1.0 37.5 0.7 86.6 2.0 4.6 4.3 64.9 1.8 Carbon TC (g kg1) 272 6.2 193 2.2 482 8.9 25 24.0 288 9.2 Ext-C (mg kg1) 946 279 8 894 87 76 19 4029 719 MBC (mg kg1) 12116 4873910 157 4081 157 666 231 42618 6423 Nitrogen TN (g kg1) 20.3 0.9 12.6 0.3 27.7 0.4 1.5 1.5 27.3 1.2 Ext-NH4-N (mg kg1) 226 96 48 4 27 1 8 4 386 32 Ext-Org. N (mg kg1) 147 23 83 7 141 14 17 1 859 89 Phosphorus TP (mg kg1) 1427 34 1018 48 207 12 366 78 1185 74 Lab. Pi (mg kg1) 124 9 99 6 4.8 2 6.5 2 1.8 0.6 Lab. Po (mg kg1) 71 19 8.3 1.6 4.2 1.1 1.2 1.5 32.3 6.5 Ratios Ext-C:Ext-N 3 2 5 3 3 Ext-C:Ext-P 5 3 102 10 119 Ext-N:Ext-P 2 1 19 3 37 BD: bulk density, LOI: loss on ignition, TC: to tal carbon, Ext-C: extractable organic carbon, MBC: microbial biomass carbon, TN : total nitrogen, Ext-N: extrac table labile nitrogen, ExtNH4-N: extractable ammonium, Ext-ON: extracta ble labile organic nitrogen, TP: total phosphorus, Lab. Pi: extractable labile phosphorus Lab. Po: labile inorganic phosphorus, ExtPo: labile organic phosphorus, ExtP: extractable labile phosphorus.

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198 Table 7-2. One-way ANOVA statistics of the effect of the different carbon sources addition to sediment CO2 and CH4 production rates and turnover rates. Lake Annie Okeechobee Apopka ANOVA Central M9 M17 KR West Anaerobic Respiration (mg CO2-C kg-1 d-1) n 27 27 27 27 27 d.f. 8 8 8 8 8 F 63.39 33.83 27.36 9.80 3.68 p <0.00001 <0.00001 <0.00001 0.00003 0.0103 Methanogenesis (mg CH4-C kg-1 d-1) n 27 27 27 27 27 d.f. 8 8 8 8 8 F 91.14 124.30 43.94 4.32 24.34 p <0.00001 <0.00001 <0.00001 0.00471 <0.00001 Turnover Rates (d-1) n 27 27 27 27 27 d.f. 7 7 7 7 7 F 98.09 139.31 14.84 6.02 3.89 p <0.00001 <0.00001 <0.00001 0.0014 0.0115

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199 Table 7-3. Sediment CO2 and CH4 production, and turnover rates, wi th the addition of different carbon sources. Tukeys test was conducted with in sites and different letters indicate significant statistical differences at p <0.05. (mean SD). Lake Annie Okeechobee Apopka Treatment Central M9 M17 KR West Anaerobic Respiration (mg CO2-C kg-1 d-1) Basal 100 9 a 26 1 a 21 4 ae 3 2 ac 217 23 a Alanine 217 9 b 62 7 bcd 75 10 bcd 18 4 bc 874 109 b Arginine 212 10 b 50 6 bc 86 8 bcf 10 4 abc 623 350 ab Acetate 135 16 a 33 1 a 50 10 bde 5 5 ac 500 259 ab Butyrate 87 3 ac 30 2 a 33 6 ade 4 3 ac 307 183 ab Formate 209 22 b 28 2 a 51 4 bde 3 3 ac 773 324 b Propionate 55 13 c 32 1 a 42 11 ade 4 3 ac 629 152 ab Glucose 96 12 a 66 5 bd 104 25 cef 13 3 bc 559 129 ab Lake-SS 101 12 a 29 1 a 33 3 ade 4 2 ac 418 160 ab Methanogenesis (mg CH4-C kg-1 d-1) Basal 37 8 a 0.09 0.0 a 0.16 0.02 a 0.04 0.01 a 80 7 a Alanine 120 15 b 45 4 be 10 4 b 12 5 b 563 20 b Arginine 159 7 c 19 3 cdf 11 3 b 2 2 a 220 24 ace Acetate 155 15 c 26 1 cd 11 1 b 5 9 a 102 23 acd Butyrate 85 4 d 41 3 b 8 1 b 5 2 a 192 16 acd Formate 36 15 a 5 2 a 0.3 0.08 a 3 4 a 44 8 acd Propionate 3 1 e 13 1 ce 0.8 0.4 a 0.4 0.5 a 48 10 acd Glucose 44 8 a 52 5 bf 0.7 0.4 a 13 5 b 374 22 ce Lake-SS 47 8 a 8 2 ae 0.0 0.0 a 2 1 a 355 14 ce Turnover Rates (d-1) Alanine 0.67 ab 0.21 a 0.17 ab 0.06 a 2.8 a Arginine 0.74 ab 0.14 b 0.19 ab 0.03 ab 1.67 ab Acetate 0.57 ac 0.12 b 0.12 acd 0.02 b 1.24 b Butyrate 0.34 d 0.14 b 0.08 cd 0.02 b 0.99 b Formate 0.49 c 0.07 c 0.10 acd 0.01 b 1.62 ab Propionate 0.12 e 0.09 c 0.08 acd 0.01 b 1.34 b Glucose 0.28 d 0.23 a 0.21 b 0.05 a 1.85 ab Lake-SS 0.34 d 0.08 c 0.07 cd 0.01 b 1.81 ab

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200 02468101214161820 0 1000 2000 3000 4000 5000 6000 02468101214161820 0 500 1000 1500 2000 2500 3000 3500 02468101214161820 0 1000 2000 3000 4000 5000 6000 02468101214161820 0 500 1000 1500 2000 2500 3000 3500 02468101214161820 0 1000 2000 3000 4000 5000 6000 02468101214161820 0 500 1000 1500 2000 2500 3000 3500 Figure 7-1. Microbial activity res ponse to the different carbon s ource addition in Lake Annie sedi ments: A, B and C) Anaerobic respiration (mg CO2-C kg-1) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg-1) vs. time (days). (Anaerobic Respiration (mgCO2-C kg-1) Time (days) Methanogenesis (mgCH4-C kg-1) A B C E F D Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal

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201 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 02468101214161820 0 200 400 600 800 1000 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 02468101214161820 0 200 400 600 800 1000 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 02468101214161820 0 200 400 600 800 1000 Figure 7-2. Microbial activity res ponse to the different carbon s ource addition in the mud sediments (site M9) of Lake Okeechob ee: A, B and C) Anaerobic respiration (mg CO2-C kg-1) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg-1) vs. time (days). *Different scales. Time (days) Anaerobic Respiration (mgCO2-C kg-1) Methanogenesis (mgCH4-C kg-1) A D B* C E F Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal

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202 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 02468101214161820 0 20 40 60 80 100 120 140 160 180 200 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 02468101214161820 0 20 40 60 80 100 02468101214161820 0 200 400 600 800 1000 1200 1400 1600 1800 2000 02468101214161820 0 20 40 60 80 100 Figure 7-3. Microbial activity res ponse to the different carbon s ource addition in the peat sedime nts (site M17) of Lake Okeech obee: A, B and C) Anaerobi c respiration (mg CO2-C kg-1) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg-1) vs. time (days). *Different scales. Time (days) Anaerobic Respiration (mgCO2-C kg-1) Methanogenesis (mgCH4-C kg-1) A D* B* C E F Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal

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203 02468101214161820 0 100 200 300 400 500 600 02468101214161820 0 20 40 60 80 100 120 140 160 180 200 02468101214161820 0 100 200 300 400 500 600 02468101214161820 0 20 40 60 80 100 120 140 160 180 200 02468101214161820 0 100 200 300 400 500 600 02468101214161820 0 20 40 60 80 100 Figure 7-4. Microbial activity res ponse to the different carbon s ource addition in the sand sediments (site KR) of Lake Okeecho bee: A, B and C) Anaerobic respiration (mg CO2-C kg-1) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg-1) vs. time (days). *Different scales Time (days) Anaerobic Respiration (mgCO2-C kg-1) Methanogenesis (mgCH4-C kg-1) A D B C E F* Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal

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204 02468101214 0 2000 4000 6000 8000 10000 12000 14000 02468101214 0 1000 2000 3000 4000 5000 6000 02468101214 0 2000 4000 6000 8000 10000 12000 14000 02468101214 0 1000 2000 3000 4000 5000 6000 02468101214 0 2000 4000 6000 8000 10000 12000 14000 02468101214 0 1000 2000 3000 4000 5000 6000 Figure 7-5. Microbial activity res ponse to the different carbon s ource addition in Lake Apopka sedi ments: A, B and C) Anaerobic respiration (mg CO2-C kg-1) vs. time (days), and D, E and F) methanogenesis (mg CH4-C kg-1) vs. time (days). Time (days) Anaerobic Respiration (mgCO2-C kg-1) Methanogenesis (mgCH4-C kg-1) A D B C E F Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal Acetate Alanine Arginine Formate Glucose Propionate Butyrate Lake Basal

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205 050001000015000200002500030000350004000045000 0 100 200 300 400 500 600 700 800 Anaerobic Respiration (mg CO2-C kg-1 d-1) Carboxylic Acids Amino Acids Polysaccharide Lake Material Basalr = 0.91 050001000015000200002500030000350004000045000 0 50 100 150 200 250 300 350 400 450 Methane Production (mg CH4-C kg-1 d-1) Carboxylic Acids Amino Acids Polysaccharide Lake Material Basalr = 0.86 Microbial Biomass Carbon (mg kg-1) Figure 7-6. Relationship between microbi al biomass carbon a nd activity: A) CO2, and B) CH4 production rates, with the different groups of carbon sources added to sediments from different lakes. Lake Okeechobee Lake Okeechobee Lake Annie Lake Apopka Lake Apopka Lake Annie A B

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206 Alanine Arginine Formate Propionate Basal -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (40.7%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (20.1%) Glucose Acetate Lake-SS Butyrate -2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5 Axis 1 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Axis 2 Lake Annie Lake Apopka Lake Okeechobee M9 Lake Okeechobee M17 Lake Okeechobee KR KR Figure 7-7. Results of the Principa l Component Analysis (PCA-1): A) loadings of the effect of different carbon sources addition on sediment CO2 production rates, and B) the plot of the scores of the sites from Lake Annie (blue circles), Lake Okeechobee: M9 (red squares), M17 (brown diamonds), KR (ora nge crosses), and Lake Apopka (green triangles). CO2 production rates were normalized by microbial biomass carbon. A B

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207 Acetate Alanine Butyrate Formate Propionate Lake-SS -1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0 Axis 1 (33.6%) -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0Axis 2 (27.4%) Glucose Arginine Basal -2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5 Axis 1 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Axis 2 Lake Annie Lake Apopka Lake Okeechobee M17 Lake Okeechobee M9 Lake Okeechobee KR KR Figure 7-8. Results of the Principa l Component Analysis (PCA-2): A) loadings of the effect of different carbon sources addition on sediment CH4 production rates, and B) the plot of the scores of the sites from Lake Annie (blue circles), Lake Okeechobee: M9 (red squares), M17 (brown diamonds), KR (ora nge crosses), and Lake Apopka (green triangles). The CH4 production rates were normalized by microbial biomass carbon. A B

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208 CHAPTER 8 RNA-STABLE ISOTOPE PROBING OF ACET ATE-UTILIZING MICROORGANISMS IN SEDIMENTS OF SUBTROPICAL LAKES Introduction In anoxic environments, different groups of microorganisms participate in anaerobic decomposition of organic matter as no single anaerobic microorganism can completely degrade organic polymers (Zinder 1993, Megonigal et al 2004). Bacteria hydrolyze organic polymers through extracellular enzyme production, and under methanogenic conditions, ferment monomers to alcohols, fatty acids, and hydrogen (H2). Alcohols and fatty acids are converted by syntrophic bacteria into acetate, H2, and carbon dioxide (CO2), which is used as substrate by methanogens (Zinder 1993, Conrad 1999, Megonigal et al. 2004). The structures and functions of anaerobic microbial commun ities are strongly affected by competition for fermentation products such as H2 and acetate, and competition favors the following order of reduction processes, based on highest thermodynamic yield: NO3 > Mn(IV) > Fe(III) > SO4 -2 > HCO3 (i.e., methanogenesis) (e. g., Megonigal et al. 2004). Several microorganisms use acetate as a carbon (C) source, making this compound the most important intermediate for microbial co mmunities under anaerobic conditions. Acetate is assimilated into microorganism biom ass and converted to methane (CH4) and/or CO2. In a previous study the addition of acetate enhanced both anaerobic CO2 and CH4 production rates in sediment microcosms of subtropical lakes with different trophic st ates (Chapter 7). However, the acetate-utilizing microorganisms in these la ke sediments are not known. In a recent study, Schwarz et al. (2007) used RNA-based stable isotope probing to iden tify acetate-utilizing Bacteria and Archaea in sediments of Lake Kinnere t (Israel). The authors concluded that acetate was predominantly consumed by acetoclastic meth anogens and was also utilized by a small and heterogeneous community of anaerobic bacteria. In a previous study in Lake Kinneret sediments

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209 Nsslein et al. (2001), using addition labeled acetate, CH4 production measurements and terminal restriction fragment polymorphism (T-RFLP), reported contrasting results from Schwarz et al. (2007). The au thors concluded that hydrogenot rophic methanogenesis was the major pathway for CH4 production, and acetate was used s yntrophically by a consortium of acetate-oxidizing bacteria and hydrogenotrophs. Schwarz et al. (2007) related these different results to changes in biogeochemistry of CH4 formation caused by environmental changes (i.e., unusually heavy rainfall, high input of C, phosph orus, nitrogen, and pollutants, and changes in biological and chemical variables). These different results, however, can be due to the use of a more sensitive technique, such as RNA-SIP, that can identify active microorganisms that constitute only a minor fraction of the total community (Schwarz et al. 2007). Stable isotope probing (SIP) is an important tool to identify organisms utilizing a specific substrate (Radajewski et al. 2003; Whiteley et al 2006; Neufeld et al. 20 07). This procedure is based on the addition of a commercially prepared 13C-labeled substrate into an environmental sample. The microorganisms that actively tr ansform this substrate will incorporate 13C into cellular biomarkers (Radajewski et al 2003; Whitele y et al. 2006; Neufeld et al. 2007). Originally SIP was applied to trace single C compounds into polar-lipid derived fatty acids of active microorganisms in sediments of Lake Loosdrec ht (The Netherlands) (Boschker et al. 1998). Later this technique was extended to the use of DNA (DNA-SIP) (Radajew ski et al. 2000) and RNA (RNA-SIP) (Manefield et al 2002a) as labeled biomarkers. DNAand RNA-SIP are based on the principl e that if an organism consumes the 13Clabeled substrate, cell components will incorp orate the heavy isotope through anabolic processes (Radajewski et al 2003; Whiteley et al. 2006; Neufeld et al. 2007). The separation of labeled and non-labeled nucleic acids is acco mplished by isopycnic centrifugation. In this

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210 density gradient centrifugation, the heavier 13C migrates faster than the light unlabeled 12C nucleic acids. The DNA and RNA sequences present in the heavy gradient fractions must be derived from organisms that have consumed the added 13C labeled substrate. DNA-SIP is the least sensitive approach because it requires cell division to obtain sufficient label into DNA, thus requiring longer incubation times where cross-feed ing and false results can occur (Wellington et al 2003; Neufeld et al. 2007). RNA-SIP has prov en a more sensitive approach, since in active cells RNA synthesis occurs at higher rates, and labeling can o ccur without repl ication of the organism (Manefield et al. 2002a). In the present study, RNA-SIP was used to iden tify microorganisms that utilize acetate in sediments of subtropical lakes with different trophic states. This approach, however, did not work. This chapter was written with the intent to document the methods used at every step and explore the source of error that contribut ed to the failure of the proposed study. Materials and Methods Study Sites and Field Sampling Three Florida (USA) lakes ra nging in trophic state were se lected: Lake Annie (oligomesotrophic), Lake Okeechobee (eutrophic) and Lake Apopka (hypereutrophic). A map of the lakes with sampling locations as well as descripti ons of the three lakes were reported previously (Chapter 2 and 3) Triplicate sedi ment cores were collected usi ng a piston corer (Fisher et al. 1992) or by SCUBA divers. The topmost 10 cm of se diment were collected from one central site in Lake Annie on June 25, 2005 and a western site in Lake Apopka on May 28, 2005. Cores were collected at three sites in Lake Okeechobee on July 16, 2005: M17 = peat, M9 = mud and KR = sand. Samples were transported on ice and stored in the dark at 4 C. Sub-samples were taken and frozen and kept at -80 C. These samples were also used in a previous study in which eight

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211 different C sources were a dded to the sediment and mi crobial activity (anaerobic CO2 and CH4 production) was measur ed (Chapter 7). RNA Extraction Due to the high water content of sediments from Lake Annie and Lake Apopka and site M9 in Lake Okeechobee, pore water was removed ( centrifuged at 10,000 x g for 10 min) prior to RNA extraction. Total RNA was extracted with the RNA PowerSoil isolation kit (Mo Bio Laboratories, Solana Beach, CA) using 1.0 g of sediment. Extracted RNA was evaluated by electrophoresis in agarose gel a nd ethidium bromide staining. Pre-Experiment Samples from sediments with high (site M9) and low (site KR) RNA yield from the first extraction were used to evaluate if the concentration of added [13C]acetate and/or the length of the incubation would affect th e concentration of the RNA extracted. Experiments were conducted in duplicate for both incubation time and concentration of substrate. Sediment (1:2 sediment to medium ratio, i.e., 1.0 g sedimen t:2 ml of medium) was added to anoxic BCYT-R medium (basal carbonate yeast extrac t trypticase-peptone containing 0.01 g L-1 trypticasepeptone) (Touzel and Albagnac 1983; Chauhan and Ogram 2006 a ) under N2 stream to prevent exposure to oxygen and immediately crimped usi ng butyl rubber septa and aluminum. Samples were reduced with cysteine (2%) to a final re dox potential of approxima tely -110 to -200 mV (Chauhan and Ogram 2006 a ) and preincubated at 28 C in the dark for 1 week, prior to acetate addition. 13C-labeled acetate (both carbon atoms labe led; Isotec, Miamisburg, OH) was added from anaerobic sterile stock solutions at two fina l concentrations (1 mM and 5 mM) and kept in the same incubation conditions. After 24 hours and 1 week of incubation, RNA was extracted as described previously.

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212 RNA-SIP Experiment Incubation and RNA extraction Triplicate samples from Lake Apopka (cor e # 93, 94, 95), Lake Annie (core # 120, 121, 122), and Lake Okeechobee sites M9 (core # 147, 148, 149) and M17 (core # 168, 169, 170) were incubated for 1 week, as described previously. 13C-labeled acetate was added from anaerobic sterile stock solutions to a final concentration of 1 mM. After 24 hours, total RNA was extracted as described previously. Escherichia coli RNA Escherichia coli ( E. coli ) RNA was extracted to be used as a RNA control (unlabeled 12C RNA) to evaluate possible mixing of [12C] RNA with 13C labeled bands (Chauhan and Ogram 2006 a ). E. coli (strain TOP10F) was grown in 10 ml Luria-Bertani (LB) medium at 37C for 24h. The culture was then transferred to 260 ml LB medium and incubated for 2 hours in a shaker at 100 rpm at 37 C. Total RNA was extracted from E. coli culture with TRI Reagent (Ambion, Austin, TX) according to the manufacturers instructions. E. coli RNA was resuspended in 1.0 ml of nuclease-free water, and the concentrati on was determined by spectrophotometry (GeneQuant, Biochem Ltd., Cambridge, UK). Isopycnic centrifugation Density gradient centrifugati on was performed as describe d by Manefield et al. (2002 a b ) and Lueders et al. (2004 a ). A total of four different centrif ugations were performed and some modifications were made in each one to improve the density gradient. First centrifugation. The gradient medium consis ted of 2.56 ml of a 2.0 g ml-1 cesium trifluroacetate (CsTFA ) (Amersham Pharmacia Biotech, Buckinghamshire, UK), 410 l of nuclease-free water and 120 l of formamide. Ten microliters of E. coli RNA (100 ng) and 100 l of sample RNA were then added to the gradie nt medium. Gradient solutions were loaded in

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213 Beckman polyallomer bell-top Quick-Seal centrif uge tubes (13 x 32 mm), sealed and centrifuged in a Beckman Coulter Optima TLX ultracentrifuge (TLA100.3 rotor) at 128,000 x g for 36 h at 20 C. Gradient solutions were fractionated via displacement by water (0.1% v/v DEPCdiethylpyrocarbonate treated) at the top of the tube and fractions were collected at the base of the tube. A controlled flow rate (0.2 ml min-1) of water was used (LC-10AS Schimadzu HPLC pump) (Figure 8-1, 8-2). A total of 15 fracti ons of 200 l each were collected from each centrifuge tube. The density of each fraction was determined by weighing 10l of each fraction. RNA from each fraction was isol ated by precipitation with isop ropanol (Whiteley et al. 2007). Presence of RNA in each fraction was confirme d by standard agarose gel electrophoresis and ethidium bromide staining. Second centrifugation A second centrifugation was performed with DNA-free RNA samples as described before. DNA was removed from samples as described below. DNA removal. The triplicate incubations for each sediment core were combined to increase the amount of RNA. All RNA samples (sediment and E. coli ) were re-purified with a PureLink micro-to-mid Total RNA Purification System (Invitrogen, Chicago, IL) according to the manufacturers instructions for optimal DNAse in-column treatment. RNA concentration was determined by spectrophotometry (GeneQuant, Biochem Ltd., Cambridge, UK). The presence of DNA in DNase-treated E. coli RNA samples was verified by PCR. E. coli RNA not treated with DNase (i.e, with DNA presen t) was used as a positive control to assure that the absence of E. coli DNA in cleaned samples resulted from successful removal of the DNA and not from PCR technical is sues. PCR tubes were prepared as follows: 2.0 l of nuclease free water, 1.0 l of each primer, 10 l of HotS tarTaq Master Mix (QIAGEN, Valencia, CA) and 5.0 l of E. coli RNA. Conditions of the PCR used were reported by Uz et al. (2003).

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214 Third centrifugation. I contacted Dr. Andrew Whiteley (CEH Oxford, UK), co-author of Manefield et al. (2002a, b), to discuss potential troubleshooting of my experiments. Following Dr. Whiteleys suggestions and protocols (Whitele y et al. 2007), some m odifications were done. First, two blank density gradie nts (without RNA) were prepared and centrifuged along with experimental samples to verify the distributi on of the density gradients. The new gradient medium consisted of 2.64 mL of a 2.0 g/mL CsTFA (Amersham Pharmacia Biotech, Buckinghamshire, UK), 508 l of nuclease free water, 109 l of formamide. Prior to centrifugation, RNA samples were concentrated by precipitation with isopropanol and resuspended in 10 l of nuclease free water (large volumes of water with RNA can affect the shape of the gradient Whitele y pers. comm.). Then, 1.0 l of E. coli RNA (100 ng) and 9.0 l of sediment sample RNA (or 10 l of nuclease -free water for the blank) were added to the gradient medium. A second modifica tion consisted of preparing the density gradient for all tubes and later loading the specific amount to each centrifuge tube containing an RNA sample. Previous preparations were made separately for each tube and small differences could occur due to pipetting error. Density gradie nts were centrifuged as describe d previously with the following minor modification: an extended period of centr ifugation of 42 h. Gradients were fractionated and a total of 30 fractions of 100 l were collected from each tube. In addition, a loading dye (green) was mixed with the water used for disp lacement of the gradient to facilitate the visualization of mixing betw een water and the gradient solutions (Figure 8-2). Fourth centrifugation. Gradient media were prepared as described in the third centrifugation. Three gradient density blanks and three samples containing different concentrations of E. coli RNA were used (1.0 ng, 10 ng and 100 ng). This new approach was performed to determine if different concentrations of E. coli RNA could improve the separation

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215 of heavy and light RNA. Density gradients we re centrifuged as describe d previously with an extended period of centrifugation of 46 h. RTPCR RTPCRs were conducted using an Access RT-PCR System (Promega, Madison, WI) following manufacturer instructions. E. coli -specific primers ECA75F (5GGAAGAAGCTTGCTT CTTTGCTGAC-3) and ECR619R (5AGCCCGGGGATTTCACATCTGACTTA-3) were used (Sabat et al. 2000). Bacterial genes were amplified with universal bacterial primers 16S rRNA gene sequences 27F (5AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-TACGGYTACCTTGTTACGACTT-3) (Lane 1991). Archaeal 16S rRNA genes were amp lified with the universal primer 1492R and Archaea-specific primer 23F (5-TGCAGAYC TGGTYGATYCTGCC-3) (Burggraf et al. 1991). RTPCRs were performed in an iCycler PCR system (Bio-Rad, Hercules, CA). RT-PCR products were analyzed by agarose electrophoresis. Results RNA Extraction RNA extraction was successful for Lake A nnie, Lake Apopka and Lake Okeechobee site M9 sediments but not for sediments from site s M17 and KR since RNA bands could not be visualized in agarose gels (Figure 8-3). All following experiments were conducted using 1.0 g of sediment from lakes Annie, Apopka and Okeechob ee site M9. For sites KR and M17, the amount of sediment used was increased to 2.0 g to improve RNA extraction yield. Pre-Experiment Neither time nor concentration affected the quality of the RNA extracted from site M9 (Figure 8-4A). No visible RNA was extracted from sediments from site KR (Figure 8-4B).

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216 Sediments from site KR are characterized by havi ng low microbial biomass and activity (Chapter 7) and were excluded from further experiments. RNA-SIP Experiment Both RNA and DNA were observed in triplicat e samples from sediments of Lake Apopka, Lake Annie, and Lake Okeechobee sites M9 and M17 (Figure 8-5). Escherichia coli RNA Concentration of extracted RNA from E. coli culture was 500 g l-1, and DNA was observed in the samples (Figure 8-6). Isopycnic centrifugation First centrifugation. Six RNA samples were used in th e first ultracentrifugation, and two samples were lost due to problems during pierci ng of the Beckman tube. The density of the gradient did not follow the expected lin ear distribution (Manefield et al. 2002 b Whiteley et al. 2007) thus no separation of heavy and light RNA occurred (Figure 8-7, 8-8). RNA from each fraction could not be visual ized in agarose gel electrophoresis. Such phenomena could be due to the low concentration of RNA in each fraction. Reverse transcription polymerase chain reaction (RT-PCR) was conducted to verify if E. coli RNA was present in each fraction. Lake Apopka (core # 93) fractions were chosen as it was the best density distribution obtained for all samples. However, smears visualized in the agarose gel coul d be an indication of either primer contamination or RNA and/or pr imers degradation (Figur e 8-9A). New primers were ordered and RT-PCR was conducted as described previously. Agarose (1%) gel electrophoresis showed the presence of E. coli in all fractions and confirmed that the ultracentrifugation failed to separate h eavy and light RNA (Figure 8-9B). The presence of DNA in both E. coli and sediment RNA samples (Figure 8-5A, B, 8-6), could be affecting the gradient medium a nd may be responsible for the detection of E. coli (DNA

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217 or RNA) in all fractions. Thus, DNA was removed from all samples. E. coli DNA was not present in samples that were treated with DNase indicating a successful removal of DNA (Figure 8-10). Second centrifugation. The results of the second centrifugation from two samples are as follows: there was some improvement of the expe cted linear distributi on the density gradient (Figure 8-11A, B, 8-8A, B). RT-PCR was performed with Lake Apopka fractions to detect E. coli RNA with the minor modification of 35 PCR cycles. E. coli RNA could be detected in all fractions confirming that the ultracentrifugation failed to separate th e heavy and light RNA (Figure 8-12). Third centrifugation. Four samples (two blanks and two sediment samples) were used in this ultracentrifugation. One of the blanks was lost due to problems during piercing of the Beckman tube. The expected linea r distribution of the gradient improved considerably and it was similar to the one reported by Manefield et al. (2002 b ) and Whiteley et al (2007) (Figure 8-13, 8-8A, B), indicating that modifications im proved the methodology considerably. However, plateaus could be observed in all density gradie nt graphs. A slightly longer centrifugation may solve this problem (Whiteley pers.comm.). If the new gradient fraction by density successf ully separated the heavy and light RNA, fully labeled RNA was expected to have densities of approximately 1.79-1.81 g ml-1, therefore around fraction numbers 5-7 (Whiteley pers. co mm.). RTPCR was conducted to check for Bacteria and Archaea RNA present in each fraction of Lake Apopka (core # 94). Expected products of RT-PCR were found in all fractions. The fractionation of heavy and light RNA was not achieved by the modified methodology (F igure 8-14). RT-PCR products were found in all fractions when universal bact erial primers were used, indicati ng the presence of light RNA

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218 from E. coli and/or native bacteria from sediments. Products of RT-PCR Archaea RNA were not found in any fractions (Figure 8-14). Fourth centrifugation. Another attempt was made to verify if a density gradient fractionation with only light RNA could be obt ained. A longer centrifugation was performed in an attempt to remove the plateaus observed in the gradient fractions from the previous ultracentrifugation. One of the blanks and the 1.0 ng E. coli RNA was lost during fractionation due to problems during piercing of the Beckman tube. The distribution of the gradient density improved with longer centrifugati on; however, plateaus could still be observed in some samples (Figure 8-15). RTPCR was done with the 10 ng and 100 ng E. coli RNA fractions. RT-PCR products for 10 ng E. coli RNA fractions were not found in any fraction, likely due to low RNA concentration (Figure 8-16A). RT-PCR products for 100 ng E. coli RNA fractions, however, were found in all fractions (Figure 8-16B). A lthough a linear distribution of density gradient could be observed, still light E. coli RNA could be found throughout the gradient medium. These experiments were conducted from Febr uary-November 2006. Collectively, the data indicated that improvements were still needed to obtain optimal density gradient fractioning of RNA samples. As all sediment samples from La ke Annie and Lake Apopka were used during the trial experiments, RNA-SIP expe riments were discontinued. Discussion, Conclusions and Recommendations RNA-SIP has proven to be a sensitive appr oach to link microorganism function with phylogeny (Lueders et al. 2004 b ; Manefield et al. 2005; Haichar et al. 2007; Hatamoto et al. 2007; Hori et al. 2007; Schwarz et al 2007). RNA-SIP has been successfully applied to study functional diversity in several ecosystems. Manefi eld et al. (2005) used RNA-SIP to identify the

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219 dominant phenol-degrading organisms from an indus trial wastewater treatment plant. Schwarz et al (2007) used RNA-SIP to identif y acetate-utilizing B acteria and Archaea in sediments of Lake Kinneret (Israel). RNA-SIP was also used to id entify acetate-assimilating organisms in an anoxic rice field (Hori et al. 2007) and cellulolytic bact eria in soils (Haichar et al. 2007). Organisms responsible for syntrophic oxida tion in sludge (Hatamoto et al 2007) and flooded soil (Lueders et al. 2004 b ) were also identified thro ugh RNA-SIP. The use of RNA-SIP has proven to be an effective method, because RNA is produced inde pendently of cellular replication, and the activity of slowand non-replic ating cells can be detected (Manefield et al. 2007). Fully labeling the target RNA with 13C is essential to achieve an optimal separation of heavy and light RNA (Whiteley et al. 2005). A substantial amount of stable isotope atoms in the target RNA facilitates the density gradient separation by ultracentrifugation of labeled and unlabeled nucleic acids (Manefield et al. 2002 a 2007). High levels of labeled substrate beyond the naturally occurring concentrations and extend ed periods of incubation, however, can increase the chance of labeling non-target organisms through trophic interactions and cross feeding (Manefield et al. 2007). Thus, the [13C]-acetate concentration and s hort incubation periods were chosen to avoid the above mentioned problems. However, RNA from the lake sediments were probably not fully labeled with 13C. One week pre-incubation was chosen to exhaust naturally occurring C sources. Thus, when the [13C]-acetate was added to the sediment microcosms, the microorganisms would assimilate it faster. To verify if the target RNA is fully labeled, measuring isotope ratio by mass spectrometry ha s been suggested (Manefield et al. 2002a). Different [13C]-acetate concentrations must be run along with different periods with incubation to determine the optimal concentr ation and incubation period.

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220 Another important issue that may have contribut ed to, or might be the main reason for the failure of this study, was the appa ratus for collecting the gradient fractions (Figure 8-1, 8-2). After ultracentrifugation, tubes we re placed in a holder. Then, need les (top and bottom) had to be introduced manually (Figure 8-2). To introduce the needle into the tube, a fair amount of force had to be used and sometimes it would make the needle go too deep in th e tube and may disrupt the density gradient. If not enough force was used the tip of the needle would be located too close to the hole and leaking would occur. The gr adient then would be fractionated by the air, causing an improper fractionation. Of all procedures to fractionate the samples, the introduction of both needles proved to be the most difficult, unreliable, and consequently difficult to reproduce. Therefore, proper fract ionation of the samples must be achieved. Samples were lost with every ultracentrifugation because of problems during puncture of the tube. Manual fractionation for small volume gradients is extrem ely difficult to control accurately (Whiteley et al. 2007). The use of a Beckman Fraction Reco very System, an apparatus developed to fractionate Quick-Seal tube s, is highly recommended (Whiteley et al. 2007). The addition of control E. coli RNA to sediment RNA samples was not the reason for the failure of the experiment, since the density gr adient was achieve in the third and fourth experimental centrifugation. However, it might not be suited for RNA-SIP experiments. Although this procedure has proven to be a successful control for DNA-SIP experiments (Chauhan and Ogram 2006 a b ), for the RNA-SIP experiments it seems not to be adequate. DNA-SIP density gradient medium is prepared with CsCl (cesium chloride) and ethidium bromide and, typically, the labeled and unlabeled DNA can be visualized as two distinct bands, under UV light (Radajeski et al. 2000; Friedrich 2006). Chauhan and Ogram (2006 a ) reported that E. coli DNA was not detected in the denser [13C]DNA fractions, but it was detected in all

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221 lighter[12C]DNA fractions, demonstrating that E. coli DNA was a successful control for the separation of the heavy and light DNA. In th is study, although similar density gradients were obtained (Figure 8-13, 8-15) as desc ribed by (Manefield et al. (2002 b ); Whiteley et al. 2007) (Figure 8-8A, B), E. coli RNA was found throughout the density gradient, demonstrating that even after extended ultracentrifuga tion periods, heavy RNA fracti ons can still contain light RNA (Figure 8-14). Furthermore, the presence of E. coli RNA mixed with the heavy RNA may be a further problem in the experiment. The creati on of a heavy RNA library is the goal of this method, thus the cross-contamination of heavy and light RNA may produce a large number of false-positive clones. Small differences in buoyant densities are usually observed in RNA-SIP experiments. Typically, unlabeled RNA has a buoyant density of 1.755 g ml-1 while labeled RNA has a buoyant density of between 1.795 and 1.80 g ml-1. However, several studies have shown overlapping of these two frac tions (Manefield et al. 2002 a ; Lueders et al. 2004 a ). The detection of heavy and light RNA in the CsTFA densit y gradient fractions can also be caused by interactions of RNA molecules forming s econdary structures (Lueders et al. 2004 a ). Recently, Lueders et al. (2004 a ) demonstrated that DNAand RNASIP methodologies are distinguished in the fractioning of 12C and 13C-containing targets (Figures 817, 8-18). The authors compared the sensitivity of the two SIP methods with labeled (13C) and unlabeled (12C) pure cultures of Methylobacterium extorquens and Methanosarcina barkeri DNA-SIP CsCl dens ity gradient was effective to separate heavy a nd light DNA, either when samp les were centrifuged separately (Figure 8-17A) or simultaneously in the same tube (Figure 8-17B). However, separation of heavy and light RNA using RNA-SIP CsTF A density gradient could only be achieved when they were centrifuge d separately (Figure 8-18A). An incomplete

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222 separation was observed when samples were cen trifuged in the same tube (Figure 8-18B). Several studies have shown overlap between heavy and light RNA in CsTFA density gradients (Manefield et al. 2002 a ; Lueders et al. 2004 a b ; Haichar et al. 2007; Hatamoto et al. 2007; Schwarz et al. 2007). Although using E. coli RNA as control is a va lid approach, it should not be added to the tube with the heavy RNA sample, but centrif uged in a separate tube. Then, the density gradient of the E. coli RNA can be compared with the density gradient of the experimental sample. Considering the problems that occurred during the RNA-SIP experiment several suggestions can be made. The first recommendati on is to conduct several experiments with different concentrations and maybe pulses of [13C]-acetate, with different incubation periods. Second, samples should be checked for amount of labeling by mass spec trometry, since it is necessary to assure that a suffici ent amount of labeled RNA is present. Once this is determined, samples should be incubated in two different sets: one with [13C]-acetate and another with [12C]acetate. RT-PCR of [12C]-acetate density gradients can be used to compare with [13C]-acetate density gradients. E. coli RNA can be used as a control as long as it is not added in the same tube of labeled target RNA. Blank density gradient s should be used along with other samples during centrifugation, so the linear distri bution of blank density gradient s should be verified before fractionation of the samples. Finally, proper equipment to fractionate the samples after ultracentrifugation, such as a Beckman Fraction Recovery System, should be used for a more precise fractioning of the samples.

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223 Figure 8-1. Picture of the apparatu s for fractionating the gradients. Figure 8-2. Photograph of gradient fractionation by displacement with stained water (green). CsTFA gradient Stained water

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224 Figure 8-3. Agarose (2%) gel electrophoresis of R NA extracted from the three lakes sediments. Figure 8-4. Agarose (2%) gel electrophoresis of RNA extracted from sediments of Lake Okeechobee sites M9 (A) and KR (B). DNA 23S rRNA 16S rRNA Lake Annie Lake Okeechobee M9 M17 KR Lake Apopka 1 mmol 24hrs 1 mmol 1 week 5 mmol 24hrs 5 mmol 1 week 1 mmol 24hrs 1 mmol 1 week 5 mmol 1 week 5 mmol 24hrs A 1 mmol 24hrs 1 mmol 1 week 5 mmol 24hrs 5 mmol 1 week 1 mmol 24hrs 1 mmol 1 week 5 mmol 1 week 5 mmol 24hrs B DNA 23S rRNA 16S rRNA

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225 Figure 8-5. Agarose (2%) gel electrophoresis of RNA extracted of samples from A) Lake Annie, Lake Apopka, and B) Lake Okeechobee sites M9 and M17. Figure 8-6. Agarose (2%) gel electr ophoresis of RNA extracted from E. coli culture. DNA 23S rRNA 16S rRNA DNA 23S rRNA 16S rRNALake Apopka Lake Annie Lake Okeechobee M9M17 B 23S rRNA DNA A 16S rRNA

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226 0246810121416 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 1.92 1.94 1.96 1.98 Gradient Bouyant Density (g ml-1) 0246810121416 Fraction Number 1.50 1.52 1.54 1.56 1.58 1.60 1.62 1.64 1.66 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 1.92 1.94 1.96 Gradient Buoyant Density (g ml-1) Figure 8-7. Graph illustrating the buoyant density of gradient fr actions: (A) Lake Annie (core #120); (B) Lake Apopka (core # 93). Figure 8-8. Graph illustrating the buoyant density of gradient fr actions: (A) Manefield et al. (2002 b ); (B) Whiteley et al. (2007). A B A B

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227 Figure 8-9. Agarose (2%) gel elec trophoresis of RT-PCR of the E.coli added to Lake Apopka samples (core # 93). (A) old primer s; (B) new primers. Figure 8-10. Agarose gel (1%) electrophoresis of PCR of E. coli RNA samples treated with DNase and not treated with DNase. Treated Treated Non-treated PCR Products Smears Smears A Fraction numbers 2 3 4 5 6 7 8 9 + Control B

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228 0246810121416 Fraction Number 1.60 1.62 1.64 1.66 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 Gradient Buoyant Density (g ml-1) 0246810121416 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) Figure 8-11. Graph illustrating th e buoyant density of gradient fractions. (A) Lake Apopka (core # 95); (B) Lake Okeechobee-M9 (core # 148). Figure 8-12. Agarose (2%) gel electrophoresis of RT-PCR of RNA extracted from Lake Apopka fractions (core # 95). E. coli specific primers were used. A B Fraction numbers 1 2 3 4 5 6 7 8 9 10 + Control

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229 051015202530 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) 024681012141618202224262830 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) 024681012141618202224262830 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) Figure 8-13. Graph illustrating th e buoyant density of gradient fractions. (A) Blank (no RNA), (B) Lake Apopka (core #94) a nd (C) Lake Apopka (core # 95). A B C Plateau Plateaus

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230 Figure 8-14. Agarose gel electrophoresis of RT -PCR of RNA extracted from Lake Apopka fractions (core # 94). Universal bacteria a nd Archaea primers were used. Fraction numbers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + Control Bacteria Primers Archaea Primers + Control

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231 024681012141618202224262830 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) 024681012141618202224262830 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) 024681012141618202224262830 Fraction Number 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 Gradient Buoyant Density (g ml-1) Figure 8-15. Graph illustrating th e buoyant density of gradient fractions. (A) Blank (no RNA), (B) E. coli 10 ng and (C) E. coli 100 ng. A B C Plateaus

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232 Figure 8-16. Agarose gel electrophoresis of RT-PCR of E. coli RNA extracted from gradient fractions. E. coli -specific primers were used. (A) E. coli 10 ng fractions and (B) E. coli 100 ng fractions. Fraction numbers 1 2 3 4 5 6 7 8 9 10 10ng + Control 100ng Fraction numbers 1 2 3 4 5 6 7 8 9 10

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233 Figure 8-17. CsCl density grad ient centrifugation of isotopi cally distinct DNA species and quantitative evaluation of nucleic acid distribution within gradient fractions. 12Cand 13C-DNA was centrifuged individually (A) or simultaneously (B) and detected fluorometrically (full symbols) or vi a domain-specific real-time PCR (empty symbols). Figures and captions are from Lueders et al. (2004 a ). Figure 8-18. CsTFA density gradient centrifugati on of isotopically distinct rRNA species and quantitative evaluation of nucleic acid distribution within gradient fractions. 12Cand 13C-rRNA was centrifuged individually (A) or simultaneously (B) and detected fluorometrically (full symbols) or via domain-specific real-time RT-PCR (empty symbols). Figures and captions are from Lueders et al. (2004 a ).

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234 CHAPTER 9 SUMMARY AND CONCLUSIONS In lakes, allochthonous and autochthonous par ticulate matter is deposited and becomes an integral part of sediments. Accumulation of particulate matte r can alter the physico-chemical properties of sediments and associated biogeoc hemical processes in the sediment and water column. Coupling and feedback between sediment biogeochemistry and water column primary productivity often depends on biogeochemical pr ocesses within sediments and associated microbial communities. Benthic sediments may play a critical role in nutrient cycling by acting as sources or sinks for nutrients, and hetero trophic metabolism typically dominates in this compartment. The primary goal of this study was to deve lop a linkage between the biogeochemical properties related to organic phosphorus dynamics of benthic sediments and the bacterial community in relation to their activities in sub-tropical lakes of different trophic states. The main focus of this study was on phosphorus (P) comp ounds as it is the nutrient that in high concentration is reported to be responsibl e for eutrophication of fr eshwater ecosystems. Eutrophic and hypereutrophic lakes us ually receive high external lo ads of nutrients, display high primary productivity and nutrient co ncentrations, consequently sediments from these lakes might be expected to have higher c oncentrations of organic matter (OM) and nutrients than oligomesotrophic lakes. To accomplish the main goal of this research, a series of laboratory experiments were performed with sediments from three subtropical Flor ida lakes (Lake Annie oligo-mesotrophic, Lake Okeechobee eutrophic and Lake Apopka hypereutrophic) with different trophic states. The specifi c objectives of this study were to: Determine the biogeochemical properties of surficial benthic sediments and examine relationships among sediment biogeochemical properties (nutrient concentrations and availability) and microbial biomass and activity.

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235 Determine relative distributions of P compounds in sediment profile using two different techniques, 31P NMR spectroscopy and a P chemical fractionation scheme. Characterize P-related enzyme activities in se diment profiles and determine relationships between different P compounds and enzyme activity. Determine stratigraphic biogeochemical propert ies in sediment cores and evaluate how they are related to microbial biomass and activity; and establish whether there is nutrient limitation of the microbial community. Determine the source and long-term accumulation of OM and explore how they relate to sediment 13C and 15N signatures. Evaluate the short term catabolic response to the addition of different carbon (C) sources to existing microbial communities in sediments. Identity microorganisms that utilize acetate through RNA-stable isotope probing. Key findings related to the above stat ed objectives are summarized below. Biogeochemical properties and microbial activity of sediments (Objective 1) This study consisted of a spatial study in which sediment was sampled from sixteen different sites from the three different lakes. This study revealed that trophic state conditions were not related to the nutrient content of sediments. Organic matter, nitrogen (N) and P concentrations were higher in sediments with lower bulk density, independent of the trophic state of the lake. The relative importa nce of P forms present in sediments seemed to be more important than total P concentration in characte rizing and understanding the processes occurring in the sediment compartment of each of the st udied lakes. The oligo-mesotrophic Lake Annie organic sediments contained P in moderate to hi ghly resistant organic P forms (NaOH soluble), and inorganic P (HCl-Pi, Fe, Al, Ca and Mg bound-P) suggesting P in this lake is old and stable. In eutrophic Lake Okeechobee sediments, the major P form was HCl-Pi, which constituted approximately 60-91% of the total P, while hypere utrophic Lake Apopka sediment had > 50% of the total P in the microbial biomass (MBP).

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236 Extractable nutrient ratios also seemed to have stronger in fluence on sediment microbial communities than total concentrations. Extractabl e C:P ratio was low for Lake Annie, reflecting high concentrations of extractabl e labile nutrients relative to C, indicating a C limitation in these sediments. High labile inorganic P availability resulted in low extractable C:P and N:P ratios, and C and N limitation in most Lake Okeechobee se diments, especially in the mud zone, along with low microbial biomass and activity. Moreover low C availability appears to be inhibiting the methanogenic community in Lake Okeechobee sediments. Limitation of the methanogenic community in these sediments is supported by th e positive effect of th e addition of electron donors on methane (CH4) production, which indicated that H2/CO2 is the major substrate for methane production in Lake Okeechobee sediments. Hypereutrophic Lake Apopka sediments had high er ratios for extractable C:P and N:P, and the high C concentration in sediments is suppor ting high microbial biomass and activity. Lake Apopka sediments are highly influenced by the de position of primary producers from the water column. The results from this study suggest th at although the microbial community is C/energy limited, C, coupled with N and P availability has a strong influence in microbial communities in these lake sediments. Sediment phosphorus forms (Objective 2) Organic P compounds were characterized in sediment profiles using two different techniques, 31P NMR spectroscopy and a chemical P frac tionation scheme. In all study lakes TP concentration decreased with sediment dept h, and although an oligo-mesotrophic lake, Lake Annie contained more TP in sediments than both eutrophic Lake Okeechobee and hypereutrophic Lake Apopka. This study showed th at the concentrations of various P compounds changed with sediment depth, indicating that di fferent processes were controlling P reactivity and mobility in these lakes.

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237 Lake Annie had more stable compounds with greater sediment depth. Dominant forms of TP were HCl-Pi, fulvic acid P (FAP), and hum ic acid P (HAP), as determined by chemical fractionation, and orthophosphate and phosphate monoester as determined by 31P NMR. Lake Annie physico-chemical characteristics, as well as the major P forms found in the sediment, strongly indicated that biotic processes play an important role in P solubility in these mud sediments. Lake Okeechobee sediments were dominated by HCl-Pi (chemical fractionation) and orthophosphate (31P NMR), indicating abiotic processes cont rol P solubility in these sediments. Dominant P forms in Lake Apopka were MB P and HCl-Pi (chemical fractionation), and orthophosphate, phosphate monoester and DNA-P (31P NMR). Almost 50% of the total P was in microbial biomass in surface sediments. The pr esence of poly-P and pyro-P in these sediments also indicated high activity of microorganisms involved in biologi cal P cycling. This study also showed that the results of 31P NMR spectroscopy were in agreemen t with the results of chemical P fractionation, and that the determination of th e relative abundance of different P forms in sediments is important to understand sediment P processes. Enzyme activities in sediments (Objective 3) This study showed that phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities were related to sedime nt microbial biomass and activity, as well as to the different P composition and availability. Enzyme activity decr eased with sediment depth, reflecting lower microbial biomass and activity. Strong correlations between enzy me activities and anaerobic respiration indicated that bacterial enzymes do minate these sediments. Different P forms in sediments were also affecting enzyme activity. Highest PMEase activity was found in the oligomesotrophic lake (Lake Annie) with high con centrations of labile -Po, FAP and HAP. Lake Okeechobee had high concentrations of labile-P i and lowest activities of both PMEase and

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238 PDEase. Lake Apopka had high concentrations of MBP and phosphate diester (lipids and DNA), as well as PDEase activity. The mechanisms controlling PMEase activity, however, seemed to vary among studied lakes. In Lake Annie, high PMEase activity wa s unrelated to dissolved reactive P (DRP) and dissolved organic C (DOC) concentration, and pr obably was controlled by factors such as high Al and Fe concentrations, high P demand inside microorganism cells, and/or presence of more stable phosphate monoester (i.e ., inositol phosphate) in the se diment. Lake Apopkas PMEase production seemed to be controlled by both DOC and DRP availability. There was an inverse relation between pore water DRP and PMEase activity, and a positive relation between pore water DOC and PMEase activity. In Lake A popka sediments production of PMEase by the microbial community was related to organic P hydrolysis, and upt ake of associated organic C moieties. Microbial biomass and activity in sediments (Objective 4) The results from this study showed that hypereutrophic Lake Apopka had the highest microbial biomass and activity (both CO2 and CH4) followed by oligo-mesotrophic Lake Annie. Microbial activity decreased w ith sediment depth and was related to decrease in easily degradable OM. Carbon, N and P concentrations, and especially nutrient ratios, had a strong influence on microbial communities in these sediments. The sediment microbial community in each lake, or site, was limited by different variables. The Lake Apopkas surface sediment heterotrop hic community appears to be P-limited. High primary production and high labile C sedimentation resulted in high demand for labile P in surface sediment, as reflected in high C:P rati o. Peat sediments of Lake Okeechobee were limited by both C and P. Nitrogen and C limitation was observed in mud and sand sediments of Lake Okeechobee. High availability of P in La ke Okeechobee mud and sand surface sediments

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239 resulted in C and N limitation. Lake Annie sedime nts seem to be C-limited, with low ratios of extractable nutrient ratios. Carbon limitation wa s probably a consequence of C sources (high humic content) and physical char acteristics (deep) of this la ke. The results showed that heterotrophic microbial metabolism can be limited by a single factor or multiple variables, and limitation varies among lakes depending on lake ch aracteristics and biogeochemical properties of sediments. Long-term OM accumulation and stable isot ope signatures in sediments (Objective 5) In this study, the 210Pb technique was used to provide an age/depth relation in the sampled sediments. Lake Annie sediments were the only datable samples, while sediments collected from Lake Okeechobee could not be dated reliably due to low or vari able activities of 210Pb, and 226Ra. Lake Apopka deposits were undatable due to possi ble mixing of the upper sediments and failure to reach the unsupported/supported 210Pb boundary. In Lake Annie, the bottom sediment layer of the core was estimated to date to the 1800s a nd the average sedimenta tion rate (since c.1900) was determined to be 36.8 mg cm-2 yr-1. Lake Annie sediments were depleted in 13C and 15N, probably due to a combination of several factors such as allochthonous OM input OM from primary productivity, and microbial biomass and activity. In mud se diments of Lake Okeechobee, 13C values were slightly depleted while 15N were enriched towards the sediment surface. These isotopic signatures resulted from several factors such as the phytoplankton community, high demand for C and N in sediments, and selective mineralization of OM In the peat zone of Lake Okeechobee, the isotopic signatures of sediment OM (enriched in 13C and 15N towards the sediment surface) were related to several factors, including sediment origin (i.e., plant tissue), intensities of primary productivity, and diagenesis. Stratigraphic variation in 13C and 15N at the KR site probably reflects an input of

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240 wastewater from anthropogenic activities, a nd variable contributi ons of river-borne allochthonous input, related to in ter-annual rainfall variations. In Lake Apopka, heavy 13C DIC in the water column, w ith high demand for inorganic C due to high primary productivity, produced autochthonous OM with enriched 13C. The enriched 15N signature in Lake Apopka sediments was generated by multiple factors including the isotopic signature of autochthonous N sources, the primary producer community, and N related processes in the water column and se diments. A more detailed study of 13C and 15N isotopes in several compartments, i.e., dissolved carbon, different N co mpounds, phytoplankton biomass, bacteria biomass, particulate OM in the wate r column and sediment, can confirm the major processes affecting the isotopic si gnatures of these sediments. Microbial activity in sediments: effects of organic electron donors (Objective 6) Microbial functional diversity of surface sediments of the subtropical lakes was investigated by measuring catabo lic response to a wide variety of C-substrates. Addition of organic electron donors to sediment microcosms from all lakes s timulated heterotrophic activity, however the extent of the response was strongl y related to microbial biomass and catabolic diversity. Although the magnitude of the respons e to electron donor addition was related to microbial biomass, the different response in each sediment was related to the catabolic diversity of sediment microorganisms. The addition of some electron donors did not stimulate heterotrophic microbial respira tion, and probably resulted in th e addition of C into microbial biomass rather than release via respiratory pathways. Lake Apopka had the highest respiration per microbial biomass, i ndicating that these sediments respired most of the C added, as a c onsequence of a P limitation. Lake Annie showed the highest metabolic quotient ( q CO2; proportion of basal respira tion per microbial biomass) indicating inefficient use of energy. The low q CO2 found in Lake Apopkas sediment indicates

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241 high efficiency. Lake Apopkas sediment catabo lic diversity was higher than in the other sediments. In relation to methane production, acet oclastic methanogenesis is probably important in Lake Annie sediments. Lake Okeechobee sediments were characterized by lower CO2 production rates that the other sediments. The dominance of hydrogentrophic methanogenesis in Lake Okeechobee sediments was determined in another study (Chapter 2). The pathway for methane production in Lake Apopka cannot be de termined with the current data. Molecular studies targeting the archaeal community are n ecessary to elucidate th e major pathway for CH4 production in these lakes sediment. These results showed that the sediments with different biogeochemical properties had diffe rent microbial communities with distinct catabolic responses to addition of the C sources. RNA-stable isotope probing of acetateutilizing microorgani sms (Objective 7) An attempt was made to identity microorgani sms that utilize acetate in these sediments using RNA stable isotope probing. This approach, however, did not work. In this chapter the methods used at every step were documented and s ources of error that cont ributed to the failure of the proposed study were discussed. Synthesis In Figure 9-1, the major characteristics of surface sediments (0-15 cm) in the different studies from the lakes are summarized. Sediments fr om the central site were selected to represent Lake Annie data, while sediments from the m ud zone were selected to represent Lake Okeechobee data. The three lakes, ranging in trophi c state, had distinct sediment biogeochemical properties despite some similarities were present. All sediments (mud sediments from Lake Annie, Lake Okeechobee mud sites, and all sites of Lake Apopka) had high TP concentration. Se diments from the oligo-mesotrophic Lake Annie had the major P forms as HAP, FAP and HCl-Pi. These sediments were also characterized by

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242 high PMEase activity and q CO2. Low extractable C:P and N:P ratios resulted from a high availability of P. Isotope signatures of these sediments revealed low values of 13C and 15N. Lake Okeechobee mud sediments had similarities with Lake Annie sediments, such as low extractable C:P and N:P ratios due to a high extractable labile-P i concentration, and HCl-Pi as the major P form. Differences in sediments from this eutrophic lake included low microbial activity (CO2 and CH4 production rates), and enzyme ac tivities. Metabolic quotient ( q CO2) and 13C and 15N values were placed between the other lakes values (Figure 9-1). Hypereutrophic Lake Apopka had high concentra tions of microbial biomass P, N and C, as well as high extractable C:P and N:P ra tios, and high microbial activity (CO2 and CH4 production rates). These sediments were also ch aracterized by having hi gh PDEase activity and high values of 13C and 15N. Metabolic quotient ( q CO2) and labile-Pi concentrations were low in this lake (Figure 91). Among all variables, 13C and 15N values and q CO2 were the ones that presented a gradient in relation to the trophic st ate of the lakes. Metabolic quotient was high in the oligo-mesotrophic lake and decreased with increasing trophic stat e. Isotopic signatures increased from the oligo-mesotrophic lake to the hypereutrophic lake (Figure 9-1). Although sharing some similarities, each lake had dist inct sediment biogeochemical properties, and sediment processes which were a reflection of an integrative effect of trophic state conditions and diagenesis over a long period of time. Lake Annie Oligo-mesotrophic acidic Lake Annie, with high allochthonous OM input, had high TP concentration in its sediments, which is probably naturally occurring as th e decrease of TP with sediment depth is not accentuated. The TP mainly consisted of organic bound-P with consequently high PMEase activity that indicates th at P solubility in these sediments is mainly controlled by biotic processes (Figure 9-2). Th e production of PMEase is not controlled by P

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243 availability in the sediments, rather it resulted from a combination of fact ors such as high Al and Fe concentrations, high P demand inside microorga nism cells, and/or pres ence of more stable phosphate monoester in the sediment. High labile-P concentration in thes e sediments resulted in low extractable C:P and N:P ratios and a C limita tion of the microbial heterotrophic community. Carbon limitation probably causes inefficient us e of energy by the heterotrophic microbial community, where there is high respiration pe r microbial biomass. Heterotrophic microbial communities in these sediments probably have high respiratory demands, with greater C flow to CO2 rather than to biomass. Lake Okeechobee Eutrophic Lake Okeechobee mud sediment had its TP pool dominated by inorganic P (HCl-Pi) (Figure 9-3). Sediments were character ized by having high lab ile-Pi concentration and low enzyme activity. High P availability in th ese sediments is repres sing the production of P related enzyme activities. P solubility in these sediments is controlled by abiotic processes (Figure 9-3). High labile-Pi concen tration in these sediments resulted in low extractable C:P and N:P ratios, and a C and N limitation of the mi crobial heterotrophic community. Carbon and N limitation is causing low microbial activities in th ese sediments. Methanogenesis was inhibited due to low electron donor availabi lity with concomitant presence of iron and sulfate reducers. Moreover, it was established for these sediments that H2/CO2 is the major substrate for methane production. Lake Apopka Hypereutrophic Lake Apopka, with high au tochthonous OM input and highly organic sediments, had the sediment TP pool dominated by diester P (i.e., MBP, DNA-P, Lipid-P) followed by inorganic P (HCl-Pi), orthophospha te, FAP/HAP and phosphate monoester. An intrinsic characteristic of these sediments was the presence of polyphosphate in some of the

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244 sediment layers. P solubility in these sediments is controlled by a combination of abiotic (pH) and biotic processes (Figure 94). High concentration of diester P resulted in high PDEase activity. The activity of PMEase was also hi gh and its production re pressed by inorganic P availability, however, it seems that in these sedi ments PMEase is also related to C acquisition by the heterotrophic microbial community. Sediment s were characterized by high extractable C and labile-N and low labile-Pi concentration, whic h resulted in high C:P and N:P ratios, and indicated P limitation in these sediments. Micr obial biomass and activity were high in these sediments. High C availability in these sedime nts probably accounts for efficient use of energy that it is used for biomass (growth) as we ll as respiration. The he terotrophic microbial community in these sediments has high catabolic diversity. Results from these studies demonstrated the mutual dependency of C, N and P transformations in regulating the sediment micr obial community and nutrient bioavailability, especially P. Activity of the heterotrophic mi crobial community can be limited by a range of properties and will depend on limnological characteri stics of lakes and sediment biogeochemical properties. The results also highlighted the si gnificance of the relationships between sediment biogeochemical properties and microbial community activities in lakes with different trophic states, and showed how the physico-chemical cond itions of lakes affect sediment properties and microbial mediated processes. Moreover, it il lustrated the importance of measuring several variables, such as C, N and P, to addre ss questions related to microbial communities. Future studies should focus on identifying co mmunities that regulate the OM turnover and nutrient mobilization. Controlled experiments addr essing the effect of C, N and P addition to sediment microbial biomass and activity can stre ngthen the conclusions about nutrient limitation in each of these lake sediments. The study of other enzyme activities, such as C (i.e.,

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245 glucosidase) and N (i.e. proteas e) related enzymes, would incr ease the knowledge of nutrient dynamics and microbial communities in these se diments. One important point that was not covered by the current study is the seasonal variat ion of nutrient limitation. Seasonal variation of nutrient availability occurs in the water column of lakes and can occur in sediments of shallow lakes like Lake Okeechobee and Lake Apopka. A study encompassing sampling of surficial sediments in different seasons (i.e. wint er and summer) should also be conducted.

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246 Figure 9-1. Graphic representation of main sediment characteristics of three lakes in relation to their trophic state. Ext-C: extractable organic carbon, Ext-N: extractable labile nitrogen, TP: total phosphorus, Inorganic-P: HCl-Pi, FAP: moderate labile organic phosphorus, HAP: highly resistant organic phosphorus, Res-P: residual phosphorus, Ext-P: extractable labile phosphorus, MBC: microbial biomass carbon, MBP: microbial biomass phosphorus, MBN: microbial biomass nitrogen, and microbial activity: CO2 and CH4 production rates. High Low Lake Annie Central Lake Okeechobee Mud Zone Lake Apopka Trophic State TP HAP/FAP Inorganic-P Ortho-P P-Monoester PMEase q CO2 TP Labile-Pi Inorganic-P Ortho-P Medium Ext-C:P Ext-N:P Res-P TP Poly-P DNA-P Ext-C, Ext-N Ext-C:P Ext-N:P Microbial Biomass Microbial Activity PDEase Labile-Pi Microbial Biomass Microbial Activity PDEase Ext-C:P Ext-N:P Microbial Biomass Microbial Activity PMEase/PDEase Labile-Pi q CO2 q CO2 13C 15N 13C 15N 13C 15N PMEase

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247 Figure 9-2. Summary of the main biogeochemical properties and processes occurring in Lake Annie water column and sediments. Numbers are mean of 0-15 cm sediment depth. EC: electrical conductivity (S cm-1); SRP: soluble reactive P (g L-1); NH4 +: ammonium (g L-1); DOC: dissolved organic carbon (mg L-1); C:N:P: ratios of extractable carbon, labile nitrogen and phosphorus. P forms % in relation to total phosphorus (P); HCl-Pi: inorganic P; FAP: moderate labile organic P; HAP: highly resistant organic P, Labile-Pi: extractable labile P; Ortho-P: orthophosphate (31P NMR); P-Mono: phosphate monoester, Poly-P: polyphosphate; PMEase: phosphomonoesterase activity (mg g-1 dw h-1); PDEase: phosphodiesterase activity(mg g-1 dw h-1), MBC: microbial biomass carbon(mg kg-1), q CO2: metabolic quotient (basal respiration/microbial biomass); CO2: anaerobic respiration(mg kg-1 dw d-1); CH4: methane production rates (mg kg-1 dw d-1). Dissolved P Biotic control of P Mineralization Sediment Microbial Community Lake Annie Living/Non Living Particles Allochthonous OM Anoxia At 1 m pH= 5.1 EC = 42 SRP = 8 NH4 + = 102 DOC = 15 16 m 4 m Living/Non Living Particles MBC = 4965 CO2 = 136 q CO2 = 0.0083 CH4 = 43 At 20 m SRP = 12 NH4+ = 708 DOC = 14 Labile-P C:P = 8 N:P = 4 C Limitation Enzyme activity PMEase = 97 PDEase = 11 Enzyme activity Not Re p ressed Organic P FAP/HAP 42% P-Mono 35% OM 5 8% Carbon CO2 C-Biomass

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248 Figure 9-3. Summary of the main biogeochemical properties and processes occurring in the Lake Okeechobee site M9 water column and sedi ments. Numbers are mean of 0-15 cm sediment depth. EC: electrical conductivity (S cm-1); SRP: soluble reactive P (g L-1); NH4 +: ammonium (g L-1); DOC: dissolved organic carbon (mg L-1); C:N:P: ratios of extractable carbon, labile nitrogen and phosphorus. P forms % in relatio n to total phosphorus (P); HCl-Pi: inorganic P; FAP: moderate labile organic P; HAP: highly resi stant organic P, Labile-Pi: extractable labile P; Ortho-P: orthophosphate (31P NMR); P-Mono: phosphate monoeste r, Poly-P: polyphosphate; PMEase: phosphomonoesterase activity (mg g-1 dw h-1); PDEase: phosphodiesterase activity(mg g-1 dw h-1), MBC: microbial biomass carbon(mg kg-1), q CO2: metabolic quotient (basal respiration/microbial biomass); CO2: anaerobic respiration(mg kg-1 dw d-1); CH4: methane production rates (mg kg-1 dw d-1). Dissolved P Abiotic control of P Mineralization pH and/or Eh Microbial Community MBC = 3653 CO2 = 17 q CO2 = 0.0067 CH4 = 0.1 Labile-Pi C:P = 3 N:P = 1 C/N Limitation Enzyme activity PMEase = 4.5 PDEase = 4.7 Enzyme activity Repressed Inorganic P HCl-Pi 66% Labile-Pi 11% Ortho-P 78% Lake Okeechobee Mud Zone (site M9) Living/Non Living Particles Water Column At 1 m pH= 7.8 EC = 385 SRP = 93 NH4 + = 130 DOC = 13 At 4 m SRP = 87 NH4 + = 86 DOC = 14 4m Sediment OM 31 % Carbon CO2 C-Biomass

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249 Figure 9-4. Summary of the main biogeochemical properties and processes occurring in the Lake Apopka water column and sediments. Number s are mean of 0-15 cm sediment depth. EC: electrical conductivity (S cm-1); SRP: soluble reactive P (g L-1); NH4 +: ammonium (g L-1); DOC: dissolved organic carbon (mg L-1); C:N:P: ratios of extractable carbon, labile nitrogen and phosphorus. P forms % in relation to total phosphorus (P); HCl-Pi: inorganic P; FAP: moderate labile organic P; HAP: highly resistant organic P, Labile-Pi: extractable labile P; MBP: microbial biomass P; Ortho-P: orthophosphate (31P NMR); P-Mono: phosphate monoeste r, Poly-P: polyphosphate; PMEase: phosphomonoesterase activity (mg g-1 dw h-1); PDEase: phosphodiesterase activity(mg g-1 dw h-1), MBC: microbial biomass carbon(mg kg-1), q CO2: metabolic quotient (basal respiration/microbial biomass); CO2: anaerobic respiration(mg kg-1 dw d-1); CH4: methane production rates (mg kg-1 dw d-1). Biotic control of P Mineralization Microbial Community MBC = 33343 CO2 = 240 q CO2 = 0.0057 CH4 = 117 Labile-Pi C:P = 120 N:P = 58 P Limitation Enzyme activity PMEase = 47 PDEase = 23 Enzyme activity Repressed P Diester MBP 47% DNA-P 31% Organic P FAP/HAP = 26% P-Mono = 23% Lake Apopka Living/Non Living Particles Water Column At 1 m pH= 7.6 EC = 443 SRP = 10 NH4 + = 75 DOC = 25 At 2 m SRP = 8 NH4 + = 50 DOC = 53 2m Sediment Dissolved P Abiotic control of P Mineralization: pH Accumulation of Poly-P 10% HCl-Pi 27% Ortho-P 31% OM68% Carbon CO2 C-Biomass

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250 APPENDIX A SUPPLEMENTAL TABLES Table A-1. Water variables from Lake Annie, Lake Okeechobee and Lake Apopka (samples taken at 1 m depth). Lake Site Temperature (C) Electrical Conductivity (S cm-1) Dissolved Oxygen (mg L-1) South 29.7 45 6.5 Central 29.9 43 6.7 Annie North 30.1 43 7.0 M17 29.3 465 5.8 O11 30.1 467 6.4 M9 28.7 471 6.4 K8 28.5 512 6.2 FC 30.7 393 1.2 J5 28.2 603 0.3 TC 29.6 362 7.3 KR 29.1 232 4.9 Okeechobee J7 29.3 524 6.3 South 15.9 366 8.5 Central 16.0 370 10.6 West 15.8 418 9.7 Apopka North 16.6 382 10.2

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251 Table A-2 Total, extractable and microbial bi omass carbon, nitrogen and phosphorus ratio (weight basis) measured in sediments from Lake Annie, Lake Okeechobee, and Lake Apopka. Total Extractable Microbial Biomass Lake Site C:N C:P N:PC:NC:PN:P C:N C:PN:P South 14 185 13 2 16 8 5 40 8 Central 13 185 14 3 24 6 6 33 6 Annie North 6 230 37 18 40 7 13 51 45 M17 19 1079 58 9 68 3 6 55 9 O11 16 160 10 5 9 7 8 33 4 M9 18 159 9 5 6 2 4 12828 K8 15 147 10 6 8 1 5 35 8 FC 7 19 3 2 6 1 9 23 3 J5 12 123 10 5 35 3 11 76 7 TC 13 46 1 4 14 6 6 49 8 KR 15 123 1 3 8 3 26 25017 Okeechobee J7 16 72 1 3 10 3 6 12225 South 11 275 24 4 11832 6 23 4 Central 11 247 22 3 11035 6 28 5 West 12 293 25 4 87 25 6 31 5 Apopka North 11 218 20 3 15158 6 23 4

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252Table A-3. Pearson correlation coe fficients of sediment biogeoche mical properties significant at p < 0.05. Carbon Nitrogen Phosphorus BD LOI TC ExtC TN ExtN TP Pi TIP Po FAP HAPRes LOI -0.91 TC -0.88 1.00 Ext-C -0.71 0.86 0.85 TN -0.80 0.91 0.91 0.92 Ext-N -0.65 0.78 0.77 0.96 0.87 TP -0.92 0.80 0.76 0.74 0.75 0.75 Lab.Pi -0.47 0.17*0.13* -0.11* 0.00* -0.14*0.47 IP -0.67 0.34 0.29 0.08* 0.21* -0.04*0.64 0.80 Lab.Po -0.77 0.77 0.74 0.77 0.76 0.78 0.85 0.29*0.36 FAP -0.67 0.65 0.61 0.64 0.63 0.70 0.81 0.34 0.35 0.85 HAP -0.67 0.68 0.64 0.68 0.67 0.71 0.75 0.19* 0.29*0.84 0.90 Res.P -0.64 0.52 0.50 0.52 0.52 0.47 0.68 0.28*0.55 0.41 0.14* 0.12* Ratios Ext-C:Ext-N 0.30 -0.22*-0.25*-0.25* -0.25* 0.31 -0.38 -0.10*-0.22*-0.34 -0.34 -0.32-0.19* Ext-C:Ex-tP -0.39 0.64 0.67 0.86 0.78 0.82 0.39 -0.47 -0.27*0.41 0.29*0.35 0.40 Ext-N:Ext-P -0.43 0.62 0.63 0.87 0.78 0.91 0.52 -0.40 -0.19*0.50 0.41 0.42 0.47 BD: bulk density, LOI: loss on ignition, TC : total carbon, Ext-C: extrac table organic carbon, TN: tota l nitrogen, Ext-N: extra ctable labile nitrogen, TP: total phosphorus, Lab.Pi: labile inorgani c phosphorus, Lab.Po: labile orga nic phosphorus, IP: inorganic phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resist ant organic phosphorus, Res.P: residual phosphorus, ExtP: extractable labile phosphorus. *Not significant at p < 0.05.

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253Table A-4. Pearsons correlation coefficients of biogeochemical properties and microbi al biomass and activity significant at p < 0.05. Microbial Biomass Methane Production Carbon NitrogenPhosphorus Anaerobic Respiration Control Acetate*Hydrogen*Acetate + Hydrogen* BD -0.49 -0.49 -0.47 -0.59 -0.50 -0.59 -0.53 -0.69 LOI 0.65 0.66 0.64 0.71 0.53 0.36** 0.25** 0.52 TC 0.65 0.66 0.64 0.70 0.50 0.30** 0.18** 0.49 Ext-C 0.89 0.89 0.87 0.89 0.61 0.42 0.25** 0.57 TN 0.80 0.80 0.78 0.81 0.55 0.38 0.26** 0.40 Ext-N 0.91 0.92 0.90 0.95 0.66 0.67 0.55 0.68 TP 0.57 0.58 0.56 0.71 0.63 0.71 0.78 0.77 Lab.Pi -0.36 -0.36 -0.38 -0.17** 0.19** 0.80 0.89 0.80 IP -0.09** -0.10**-0.12** -0.01** -0.15** 0.62 0.68 0.71 Lab.Po 0.60 0.60 0.56 0.67 0.70 0.71 0.67 0.66 FAP 0.42 0.42 0.41 0.61 0.83 0.74 0.76 0.65 HAP 0.48 0.48 0.46 0.59 0.86 0.68 0.59 0.64 Res.P 0.57 0.57 0.55 0.46 0.79 0.89 0.79 MBC 1.00 0.99 0.88 0.38 0.86 0.81 0.78 MBN 1.00 0.99 0.90 0.39 0.83 0.78 0.84 MBP 0.99 0.99 0.90 0.36 0.84 0.76 0.64 Ratios Ext-C:Ext-N -0.25** -0.25** -0.24** -0.30 -0.27** 0.27** -0.01** 0.39 Ext-C:Ext-P 0.87 0.88 0.88 0.83 0.31 -0.16**-0.44 -0.01** Ext-N:Ext-P 0.93 0.95 0.95 0.94 0.40 -0.36**-0.63 -0.33** BD: bulk density, LOI: loss on ignition, TC : total carbon, Ext-C: extrac table organic carbon, TN: tota l nitrogen, Ext-N: extra ctable labile nitrogen, TP: total phosphorus, Lab.Pi: labile inorgani c phosphorus, Lab.Po: labile orga nic phosphorus, IP: inorganic phosphorus, FAP: moderate labile organic phosphorus, HAP: highly resist ant organic phosphorus, Res.P: residual phosphorus, ExtP: extractable labile phosphorus. Data from La ke Okeechobee sediments only. **Not significant at p < 0.05.

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279 BIOGRAPHICAL SKETCH Isabela Claret Trres was born in Belo Horizo nte (Minas Gerais State, Brazil). She grew up surrounded by mountains, water falls, Cerrado, a nd the remains of Atlantic Forest. She was always taken by her family to explore natural hab itats, and taught to value and protect nature and animals. Before going to college she lived fo r one and a half years in Israel where she experienced the life of two diffe rent Kibbutzim near Haifa. In 1991, she started her studies in biology at the Federal University of Minas Gerais (UFMG). Her first experience with science was working with freshwater zooplankton specie s, and later she joined the Population Ecology Laboratory where she conducted research on a spid er population in the beau tiful mountain fields of Serra do Cip (M.G.). During this study she had a unique opportunity to work with Dr. Jos E. C. Figueira, her advisor, a gr eat ecologist who taught her a passion for ecology, science, and statistics. She graduated in 1995 with a B.S. degree in biology (major in ecology), and, knowing that lack of water would be the major issue th at humanity would face in the near future, she decided to return to water research. In 1997, she joined the masters program in ecology, conservation and management of wildlife, at UFMG, where she studied mass balance of nutrients of a eutrophic reser voir, and graduated in 1999. In 2002 she joined the graduate program in soil and water science to pursue her P h.D. degree. In this program, she felt she was becoming a more complete limnologist, as she was studying sediment processes. Studying lake sediment made her realize that she was following in the steps of her uncle and godfather, Dr. Geraldo Eustquio Trres ( in memoriam ); a mud limnologist and be nthos ecologist who left this world too soon and is truly missed by her. Af ter graduating, she wants to become a professor so she can pass on to the next generation the sa me passion for nature, science, biology, ecology, and limnology that were passed to her by so me of her professors and her family.