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Linkage between Biogeochemical Properties and Microbial Activities in Lake Sediments

Permanent Link: http://ufdc.ufl.edu/UFE0021592/00001

Material Information

Title: Linkage between Biogeochemical Properties and Microbial Activities in Lake Sediments Biotic Control of Organic Phosphorus Dynamics
Physical Description: 1 online resource (279 p.)
Language: english
Creator: Torres, Isabela Claret
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biogeochemistry, carbon, eutrophication, isotopes, lake, methanogenesis, microorganisms, nitrogen, phosphodiesterase, phosphomonoesterase, phosphorus, respiration, sediment
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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.
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.
Statement of Responsibility: by Isabela Claret Torres.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Reddy, Konda R.
Local: Co-adviser: Ogram, Andrew V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021592:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021592/00001

Material Information

Title: Linkage between Biogeochemical Properties and Microbial Activities in Lake Sediments Biotic Control of Organic Phosphorus Dynamics
Physical Description: 1 online resource (279 p.)
Language: english
Creator: Torres, Isabela Claret
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biogeochemistry, carbon, eutrophication, isotopes, lake, methanogenesis, microorganisms, nitrogen, phosphodiesterase, phosphomonoesterase, phosphorus, respiration, sediment
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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.
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.
Statement of Responsibility: by Isabela Claret Torres.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Reddy, Konda R.
Local: Co-adviser: Ogram, Andrew V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021592:00001


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LINKAGE BETWEEN BIOGEOCHEMICAL PROPERTIES AND MICROBIAL ACTIVITIES
IN LAKE SEDIMENTS: BIOTIC CONTROL OF ORGANIC PHOSPHORUS DYNAMICS























By

ISABELA CLARET TORRES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007




































O 2007 Isabela Claret Torres





























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.









ACKNOWLEDGMENTS

During my j ourney down this "road" towards completing my graduate studies a number of

people participated in this process in different ways. Some participated directly helping me with

field and laboratory procedures, others indirectly 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 financial 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. Brenner's support to

my proj ect, our talks about science, politics and life, data discussion, and his help with dating

sediments. To William Kenney, (Geology Department/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, especially 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 and laboratory assistance. Also, Gavin Wilson was

always prompt to help solve difficulties and taught me about equipment and analysis, and Xiao

Wei Gu, Xian Ying Tian, and Hui X Lu for their help. My deepest thanks to Ron Elliot (in

memoriamn), for teaching me to use the Autoanalyzer, and for his friendship, he is truly missed.

To my colleague Matt Fisher, without whom I would not be able to get my samples, for his









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.










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.


























"We wrest secrets from nature by most unlikely routes. Societies will, of course wish to exercise

prudence in deciding which applications of science are to be pursued and which not. But without

funding of basic research, without supporting acquisition of knowledge for its own sake, our

options become dangerously limited ... Without vigorous, farsighted and continuing

encouragement of fundamental scientific 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)












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ........._.___..... .__. ...............12....


LIST OF FIGURES .............. ...............14....


AB S TRAC T ............._. .......... ..............._ 18...


CHAPTER


1 INTRODUCTION ................. ...............20.......... ......


Sediment Or ganic Matter............... ...............20.
Sediment Phosphorus ................. ...............23.................
Microbial Communities ................. ...............27.......... ......
Site Descriptions ................. ...............3.. 1..............
Obj ectives ................. ...............32.......... .....
Dissertation Format .............. ...............33....


2 BIOGEOCHEMICAL PROPERTIES AND MICROBIAL ACTIVITY OF BENTHIC
SEDIMENT S OF SUBTROPICAL LAKES................ ...............36.


Introducti on ................. ...............36.................
Materials and Methods .............. ...............38....

Study Sites ................. ...............38.................
Field Sampling............... ...............39
Sediment Properties ................. ........... ...............39.......
Sediment Phosphorus Fractionation ................. ........... ........_.. ......... 4
Microbial Biomass Carbon, Nitrogen, and Phosphorus ................. .................4
M icrobial Activity .............. ...............41....
Statistical Analysis .............. ...............42....
Re sults........._.._... ......_ __ ...............43.....
Sediment Properties ............._. ...._... ...............43....
Sediment Phosphorus Forms .............. ...............44....
Microbial Biomass............... ...............44
M icrobial Activity .............. ...............45....
Discussion ............._. ...._... ...............47....
Conclusions............... ..............5


3 SEDIMENT PHOSPHORUS FORMS IN SUBTROPICAL LAKES ................. ...............71


Introducti on ........._.. ... ..._.. ...............71.....
Materials and Methods .............. ...............72....

Study Sites ............._. ...._... ...............72....












Field Sampling............... ...............72
Sediment Properties ................. ......___ ...............73.......
Sediment Phosphorus Fractionation ................. ...............73......._.. ....
31P Nuclear Magnetic Resonance .............. ...............75....
Statistical Analysis .............. ...............75....
Re sults ................ .......___ ...............76.......

Sediment Properties ................. ...............76......... ......
Sediment Phosphorus Forms .............. ...............76....
31P Nuclear Magnetic Resonance .............. ...............77....
Discussion ............._. ...._... ...............79....
Conclusions............... ..............8


4 ENZYME ACTIVITIES INT SEDIMENTS OF SUBTROPICAL LAKES............................99


Introducti on ................. ...............99.................
Materials and Methods .............. ...............101....

Study Sites ................ ...............101......... ......
Water Characteristics............... ............10

Sediment Properties ................. ...............101................
Enzyme Activity ................. ...............102......... ......
Statistical Analysis .............. ...............103....
Re sults ................ ........... .................103.....
Water Characteristics............... ............10
Sediment Properties ................. ...............104................

Enzyme Activity ................. ...............104......... ......
Discussion ................. ...............106................
Conclusions............... ..............11


5 MICROBIAL BIOMASS AND ACTIVITY INT SEDIMENTS OF SUBTROPICAL
LAKES .............. ...............121....


Introducti on ................. ...............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....
Re sults ................ ........... ...............126......

Sediment Properties ............... ... .... ........ ... ...............126....
Extractable and Microbial Biomass C, N and P ................ ............... ......... ...126
Microbial Activity .............. ...............128....
Discussion ................. ...............129................
Conclusions............... ..............13












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












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....










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....










7-1 Sediment biogeochemical properties 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, with 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 Pearson' s correlation coefficients of biogeochemical properties and microbial
biomass and activity............... ...............25











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......... ......










4-4 Relationship between enzyme activity, phosphomonoesterase and phosphodiesterase
and pore water dissolved reactive phosphorus and dissolved organic carbon
concentration in sediments from Lake Apopka. ................ ................. ......... ..11

4-5 Relationship of different microbial activities. ................ ...............118..............

4-6 Results of the Principal Component Analysis 1 ................ .......... ............... ...11

4-7 Results of the Principal Component Analysis 2. ................ ...............120.............

5-1 Microbial activity in sediments from Lake Annie, Lake Okeechobee and Lake
Apopka ................. ...............143................

5-2 Results of the Principal Component Analysis. ................ ...............144........... ..

6-1 Results of 210Pb dating of Lake Annie sediments ................. ...._.._ .................167

6-2 Radioisotope activities versus depth, in Lake Okeechobee and Lake Apopka. ...............168

6-3 Lake Annie sediment depth profile. .............. ...............169....___ ....

6-4 Lake Okeechobee mud zone (site M9) sediment depth profile. ............. ....................170

6-5 Lake Okeechobee peat zone (site M17) sediment depth profile ................. .................1 71

6-6 Lake Okeechobee sand zone (site KR) sediment depth profile. ............. ....................172

6-7 Lake Apopka sediment depth profile. .....__.....___ .........._ ............7

6-8 Carbon vs nitrogen isotopic values of sediments .............. ...............174....

6-9 Maj or mechanisms affecting the sediment 813C and 6 "N signatures. ............................175

7-1 Microbial activity response to the different carbon source addition in Lake Annie
sediments ................. ...............200................

7-2 Microbial activity response to the different carbon source addition in the mud
sediments (site M9) of Lake Okeechobee. ....__ ......_____ .......___ ..........20

7-3 Microbial activity response to the different carbon source addition in the peat
sediments (site M17) of Lake Okeechobee. ...._._._._ .... ... .___ ....._.. .........20

7-4 Microbial activity response 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 source addition in Lake Apopka
sediments ................. ...............204....._._. .....

7-6 Relationship between microbial biomass carbon and activity. .............. ...................205










7-7 Results of the Principal Component Analysis 1. ............. ...............206....

7-8 Results of the Principal Component Analysis 2. ................ ..............................207

8-1 Picture of the apparatus for fractionating the gradients ................. ......__ ............223

8-2 Photograph of gradient fractionation. ................ .............. ......... ........ .....223

8-3 Agarose gel electrophoresis of RNA extracted from the three lakes sediments. .............224

8-4 Agarose gel electrophoresis of RNA extracted from sediments of Lake Okeechobee
sites M9 (A) and KR (B) ................. ...............224..............

8-5 Agarose gel electrophoresis of RNA extracted of samples from A) Lake Annie, Lake
Apopka, and B) Lake Okeechobee sites M9 and M17. ............. ....................25

8-6 Agarose gel electrophoresis of RNA extracted from E. cobi culture. ............. ...... ........._225

8-7 Graph illustrating the buoyant density of gradient fractions. ...........__.. .............. ....226

8-8 Buoyant density of gradient fractions: (A) Manefield et al. (2002b); (B) Whiteley et
al. (2007). .............. ...............226....

8-9 Agarose gel electrophoresis of RT-PCR of the E. cobi added to Lake Apopka samples
(A) old primers; (B) new primers. ............. ...............227....

8-10 Agarose gel electrophoresis of PCR of E. cobi 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 from Lake Apopka
fractions ................. ...............228................

8-13 Buoyant density of gradient fractions. ................ ........................ ..............229

8-14 Agarose gel electrophoresis of RT-PCR of RNA extracted from Lake Apopka
fractions ................. ...............230................

8-15 Graph illustrating the buoyant density of gradient fractions. ................ ................ ...23 1

8-16 Agarose gel electrophoresis of RT-PCR of E. cobi RNA extracted from gradient
fraction s ................ ...............232................

8-17 CsCl density gradient centrifugation of isotopically distinct DNA species and
quantitative evaluation of nucleic acid distribution within gradient fractions. ................233

8-18 CsTFA density gradient centrifugation of isotopically distinct rRNA species and
quantitative evaluation of nucleic acid distribution within gradient fractions ................ .233











9-1 Graphic representation of main sediment characteristics of three lakes in relation to
their trophic state............... ...............246.

9-2 Summary of the main biogeochemical properties and processes occurring in Lake
Annie water column and sediments. ............. ...............247....

9-3 Summary of the main biogeochemical properties and processes occurring in the Lake
Okeechobee site M9 water column and sediments. ............. ...............248....

9-4 Summary of the main biogeochemical properties and processes occurring in the Lake
Apopka water column and sediments. ............. ...............249....









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,









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 813C and 6 "N 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 613C

and 6 "N with increasing trophic state. Oligo-mesotrophic Lake Annie sediment had the lowest

values of 613C and 6 5N. Eutrophic Lake Okeechobee mud sediments displayed intermediate

values for both isotopes. And hypereutrophic Lake Apopka had the highest values for both 813C

and 6 "N. 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.









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









biogeochemical processes (Rybak 1969). Organically bound nutrients in particulate matter

supplied to the sediment are mineralized by heterotrophic decomposition, resulting in release

of nutrients into the water column and stimulation of biological productivity (Capone and

Kiene 1988; Gachter and Meyerl993; 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 matter (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

et al. 2005). Measurements of 613C and 6 "N in several lake compartments, (i.e., dissolved and

particulate matter in the water column and sediments) have been used to identify the origin of

lacustrine OM (Filley et al. 2001; Griffths et al. 2002), infer past primary productivity (Schelske

and Hodell 1991; Hodell and Schelske 1998; Bernasconi 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 (Hollander and Smith 2001; Lehmann et al. 2002; Gu

et al. 2004; Terranes and Bernasconi 2005; Kankaala et al. 2006).

Allochthonous OM usually has more negative 613C ValUeS than does autochthonous OM.

Values of 613C 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 autochthonous OM displays high negative 613C. During periods

of very high primary productivity the preferred 12C in the water column is exhausted and

fractionation is diminished, yielding OM with higher 613C (Mizutani and Wada 1982; Raul et al.

1990). Hypereutrophic lakes with high rates of primary productivity 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 613C that is 8%o 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 613C Of OM (Goericke et al. 1994).

Stable isotope signatures of sediment OM can be used to identify impacts of anthropogenic

activities. Sources of OM from wastewater and agricultural runoff can be identified because they









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









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).









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

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 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

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 not 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; 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









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, energetic and

function of all life forms. The degradation of organic P is closely related to organic C

degradation, as both are constituents of the OM. As an example, Siuda and Chrost (2001)

demonstrated from controlled experiments that PMEase 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 constituent 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 (Sarndergaard 1984, Azam 1998). As C is

the maj or driver and basic constituent in all living forms, its cycle is strongly linked to the P

cycle. As a result C:P ratios of the sediment-water column can influence P uptake by the bacteria

community .

Nitrogen is also one of the maj or 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 cycles 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 plankton, stated that there is a constancy in the molar C:N:P ratio =

106: 16: 1 (by weight 41:7.2:1). This ratio can be applied to different ecosystems and to processes

such as decomposition of OM. The C:N:P ratio of materials is reflected in the composition of the










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









these systems can occur, especially through the sequential activity of different groups of

anaerobic bacteria (Capone and Kiene 1988).

In methanogenic habitats, i.e., in the absence of inorganic electron acceptors, different

groups of microorganisms participate in decomposition of OM as no single anaerobic

microorganism can completely degrade organic polymers (Zinder 1993, Megonigal et al. 2004).

Cellulolytic bacteria hydrolyze organic polymers through extracellular enzyme production and

further break down monomers to alcohols, 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 substrate by methanogens (Zinder 1993,

Conrad 1999, Megonigal et al. 2004). The structures and functions of anaerobic microbial

communities are therefore strongly affected by competition for fermentation products such as H2

and acetate. Microorganisms derive energy by transferring 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., sulfate, iron reducers) are sensitive to the

concentrations of substrates and products. Their activities can be inhibited by their end products

and are dependent on the end product consumption by other organisms (Stams 1994; Megonigal

et al. 2004). While fermenting bacteria shift their product formation to more oxidized products,

syntrophic bacteria only metabolize compounds when methanogens or other anaerobic bacteria

consume H2 and format efficiently (Stams 1994).

Microbial functional diversity includes a vast range of activities. One component of this

diversity has been characterized by measuring catabolic response profiles, 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 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









distinct a catabolic response, as the mineralization rates of a microbial community are dependent

upon the metabolic capacity for a given substrate (Torien and Cavari 1982).

Several factors limit bacterial metabolism in sediments, i.e., temperature, C, and nutrient

concentration. Most studies of microbial activity 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 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 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 N or C limitation was identified in

15% and 20%, respectively (percentages add up to more than 100% due to methodological

aspects, of. 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 the sediment.

Benthic sediments play a critical role in nutrient cycling by acting as sources or sinks for

nutrients, and heterotrophic metabolism dominates in this compartment (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 understand processes that occur in









sediment. The primary goal of this study was to develop a linkage between the biogeochemical

properties of benthic sediments and their bacterial communities in relation to their activities in

sub-tropical lakes of different trophic states. The main focus of this study was on P compounds

as it is the nutrient that in high concentration is reported to be responsible for eutrophication of

freshwater ecosystems. 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 by their activities.

Site Descriptions

Three Florida lakes (USA) were selected for this study based 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 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) (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 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)

(Figure 1-4).









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









biogeochemical properties and the microbial community, how they differ among lakes with

different trophic states. Moreover, it demonstrated the importance of considering several

variables, such as C, N and P, to address questions related to microbial communities.

Dissertation Format

This dissertation begins with Chapter 1 in which a general introduction, main hypothesis

and obj ectives are presented. Chapter 2 consists 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 selected sites. In Chapter 3, organic P compounds

were characterized in sediment profile 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. Chapter 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 investigated 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.








































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.





Mineralization


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











C km2 = 1800
Lake Apopka
km2 = 125








Lake Annie
km2 = 0.37 C-u Mud
4~ ~ -* eat
I Sand
I Littoral
I Rock


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


Sediment
Release


Sediment


Dissolved
C. N. P


CO2 and CH4




P hnus Act vity
Biomass
Microbial
Community









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










organic matter and inorganic nutrients (Fenchel et al. 1990; Pace and Funke 1991; Cole et al.

1993). 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 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; Conrad 1999; Megonigal et al. 2004). Consequently,

carbon dioxide (CO2) and methane (CH4) are important end products in anaerobic decomposition

of organic matter and their concentration 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 bacterial growth and competition for resources.

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

organic carbon, nutrients, and electron 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 and Conrad 1995; Thomsen et al 2004). Few

studies have related production of CO2 and CH4 with biogeochemical properties of sediments

and with nutrient availability or limitation. Benthic sediments play a critical role in nutrient

cycling by acting as sources or sinks for nutrients, and heterotrophic metabolism dominates in

this compartment. Thus, it is important to study biogeochemical properties of sediments and how

they relate to microbial community composition, growth, and activity to better understand

processes occurring in lake sediment.









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









with 125 km2 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 adjacent 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).

Field Sampling

Three sites were sampled in Lake Annie on July 18, 2004 (North, South, and Central)

(Figure 2-1A, Table 2-2). Nine sites representing four maj or sediment types (sites: M17 = peat;

011, 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, Central 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 collected from each core for analyses. Results of all

sediment variables are reported on a dry weight basis (dw). Measurements of water temperature

(oC), electrical conductivity (CIS cm- ) and dissolved oxygen (mg L^1) were taken at 1 m water

depth from each site during sampling, with a handheld YSI 85 (YSI Inc., Yellow Springs, OH).

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 (BD) was determined

on a dry weight basis (i.e., g of dry/cc wet) at 70 oC for 72 hours, and pH was determined on wet









sediments (1:2 sediment-to-water ratio). Organic matter content (LOI-loss on ignition) was

determined by weight loss at 550oC. Total P was measured by ignition method, followed by acid

digestion (6 M HC1) and measured colorimetrically with a Bran+Luebbe TechniconTM

Autoanalyzer II (Anderson 1976; Method 365.1, EPA 1993). Total carbon (TC) and total

nitrogen (TN) were determined on oven-dried samples using a Carlo Erba NA-1500 CNS

Analyzer (Haal-Buchler Instruments, Saddlebrook, NJ). Measurements of TP, TC, and TN were

conducted on sediment that was previously oven-dried (at 70 oC 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 procedure involved sequential chemical extraction in a

1:50 dry sediment-to-solution ratio, with: 1) 0.5 M NaHCO3 (pH = 8.5) representing labile

inorganic and organic P; 2) 1 M HCI 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 remaining in the residual sediment after the

sequential extraction was measured by the ignition method and is called residual 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 filtered through a 0.45 Clm membrane filter, and

analyzed for SRP 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; Method 365.1, EPA 1993). Residual P

was determined using an ignition method (Anderson 1976), and analyzed as described previously

for TP.









Microbial Biomass Carbon, Nitrogen, and Phosphorus

Microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) were

measured through the chloroform fumigation-extraction 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 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 sediment-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 Kj eldahl 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 using a 0.45 Clm membrane fi1ter and digested for TP

with sulfuric acid and potassium persulfate, and analyzed as described previously. Microbial

biomass (C, N and P) was determined by the difference between treated and non-treated samples.

Non fumigated controls represent extractable organic carbon (Ext-C), extractable labile nitrogen

(Ext-N), and extractable labile phosphorus (Ext-P).

Microbial Activity

Anaerobic microbial respiration and methanogenesis were quantified by incubating an

amount of sediment (based on 0.5 g of dry weight) using methodology described by Wright and

Reddy (2001). For microbial respiration experiments, sediments were incubated anaerobically in

the dark at 30 oC, and evolved CO2 WAS trapped in vials containing 0.2 M NaOH. Trapped

samples were periodically removed (2, 4, 7, and 10 days) and sealed. Samples were acidified









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









between variables. A one-way analysis of variance (ANOVA) was conducted to compare the

effect of electron donors on methane production and also to compare the responses among sites

in Lake Okeechobee. Pairwise comparisons of means were conducted using Tukey's HSD. All

statistical analyses were conducted with Statistica 7.1 (StatSoft 2006) software.

Results

Electrical conductivity values reflected the trophic conditions of the three lakes, with

lowest values for Lake Annie (43-45 CIS cm- ), and higher for both Lake Okeechobee (232-603

CIS cm- ), and Lake Apopka (370-418 CIS cm )~. Day-time dissolved oxygen concentrations were

similar for all lakes (4.9-7.3 mg L^)~, with Lake Apopka (8.5-10.6 mg L^1) presenting higher

values, which is probably due to high algal biomass and lower (i.e. winter) temperatures (15.8-

16.6 oC). Surface water temperature in Lake Annie (29.9-30.1 oC), and in Lake Okeechobee

(28.2-30.7 oC) were high, reflecting summer temperatures (Table A-1 Appendix).

Sediment Properties

Sediment pH varied from 5.7 to 8.1. Lake Annie sediment pH was lower than the other

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

slightly alkaline, reflecting eutrophic conditions of these lakes. Both Lake Apopka and Lake

Annie (south and central) sediments had lower bulk density than Lake Okeechobee sediments

(Table 2-3). Organic matter content (LOI %) and 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 Lake 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









Okeechobee and Lake Annie (south and central). Lake Annie (south and central) sediments had

higher TP concentrations than Lake Okeechobee and Lake Apopka sediments (Table 2-4).

Sediment Phosphorus Forms

Relative proportions of P pools varied among lakes and sediment type. Inorganic P (HCl-

Pi) extracted with 1 M HCI (apatite and non-apatite P) was the maj or component of the P pool in

all sediment types from Lake Okeechobee (3 8-91% of total P) (Table 2-4). Labile organic P

(labile-Po) was low in all lakes, while labile inorganic 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 maj or 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 central), maj or P forms included: HCl-Pi (36-

41% of total P) and moderately and highly resistant organic P: fulvic acid P (26-28% of total P)

and humic acid P (15-16% of total P) (Table 2-4). Residual P (Res.P) was low in Lake Annie

mud sediments (0.3-0.6% of total P), with higher values for Lake Apopka (1 1-15% 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 sediment types had low microbial biomass. Total

C:N ratio (weight basis) was higher in Lake Okeechobee and Lake Annie, while C:P and N:P

ratios were higher in Lake Apopka (Table 2-3, 2-4, A-2 Appendix). Extractable C:N ratio was

similar in all sediments, however extractable C:P and N:P ratios were higher in Lake Apopka









(Table 2-5, A-2 Appendix). Extractable C:N:P represents the labile forms of these nutrients, and

lower ratios could indicate nutrient 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 sediments (Table 2-5, A-2 Appendix).

Microbial Activity

Anaerobic respiration (CO2-C mg kg- d- ) rates were higher in Lake Apopka sediments

followed by Lake Annie mud sediments, as compared to Lake Okeechobee sediments types. All

sandy sediments had low anaerobic respiration rates (Table 2-6). Methane production rates (CH4-

C mg kg- d l) 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). Results of one-way ANOVA showed that methane production rates

of the electron donor experiment with Lake Okeechobee sediments were significantly different

among treatments (n = 27, df = 3, F-test = 1 9.70, p < 0.00001i). Tukey's 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 showed that there was

significant difference in methane production rates among sediment types (n = 12, df = 8, F-test =

5.10, p < 0.00001). Tukey's pairwise multiple comparison method showed that methane

production in sites located in the mud zone of Lake Okeechobee were statistically different from

methane production in all other sediment types, but were not different among each other.

Methane production rates were not significantly different among peat, littoral, and sand deposits.









Because linear regression between MBC with MBN and MBP showed a strong positive

significant relationship (Figure 2-2A, B) statistical analyses were performed using MBC as

proxy for microbial biomass. Regression analysis of MBC and anaerobic respiration indicate that

over a wide range of MBC represented 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 regression analysis showed

that the three lakes fall into distinct groups (Figure 2-3A). Although significant, the linear

regression between anaerobic respiration 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 biogeochemical properties. The second (PCA-2) used only

Lake Okeechobee data and was conducted to verify 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). Inorganic P forms (labile-Pi

and HCl-Pi) were the variables selected 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 plotted opposite to bulk

density, showing an inverse relationship. Microbial biomass C was grouped with anaerobic

respiration and ratios of extractable 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 position 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









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,









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










(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-surface sediment variables will reflect recent lake

trophic state conditions. Eutrophic and hypereutrophic lakes usually receive high external loads

of nutrients. Eutrophic and hypereutrophic lakes also display high primary productivity and

nutrient concentrations in the water column and these nutrients will eventually reach the

sediment. Sediments from eutrophic and hypereutrophic lakes might be expected to have high

concentrations of organic matter and nutrients. 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. Several other studies also have shown that there is a significant

correlation between trophic condition (based on water measurements) and nutrient

concentrations in sediments (Rybak 1969; Flanery et al. 1982; Wisniewski 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 concentrations were high in sediments with lower

bulk density, and that trophic state conditions were not related to nutrient content of sediments.

For example, Lake Annie, an oligo-mesotrophic lake, had higher sediment TP concentration

compared to the other two lakes studied. Organic 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 trophic state conditions and diagenesis over a long

period of time relative to water column physico-chemical variables. Moreover, the relative

importance of P forms in sediments is more important than total P concentration and will depend









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 resistant organic P pools (NaOH soluble), suggesting

that organic P in this lake is old and stable (Table 2-4). The other maj or fraction is HCl-Pi, which

makes up 40% of the total P, and represents total inorganic P bound to Ca, Mg, Fe and Al. Its

solubility is controlled by either pH or redox potential (Moore and Reddy 1994). Being a deep

lake that is thermically stratified from February through November or December (Battoe 1985),

Lake Annie sediment P has little effect on P concentration 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 significantly to P release from sediments (Davelaar 1993; Gachter and

Meyer 1993; Hupfer et al. 2004). Lake Apopka is shallow, and benthic sediments are subj ect to

resuspension into the water column, potentially releasing soluble P (Reddy et al. 1991). Kenney

et al. (2001) showed that polyphosphate (P storage within microbial cells) played an important

role in the TP of Lake Apopka sediments, and suggested that between 25 and 90% of the

sediment TP may be sequestered as polyphosphate. 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) ranged 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 reflect varying contributions of









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









related to grazing and/or C and nutrient availability. Work et al. (2005) reported high

bacterioplankton production (mg L-1 h- ) in Lake Okeechobee during summer. Also, several

studies have shown that bacterioplankton is an important source of C to the food web in Lake

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 bacterioplankton

community in Lake Okeechobee. Nevertheless, Phlips 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 during most of the year in this

area, however, during summer months, light limitation is relaxed and N becomes the limiting

factor of the phytoplankton community (Aldridge 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 higher ratios for extractable C:P and N:P, but

lower ratios in microbial biomass. Low nutrient ratios in microbial 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 dominated by cyanobacteria and the dominant taxa

are Synechococcus sp., Synechocystis sp. and M~icrocystis incerta (Carrick et al. 1993; Carrick

and Schelske 1997). Approximately 1034 g C m-2 -1l fTOm primary production is deposited in

surface sediments of Lake Apopka (Gale and Reddy 1994). Schulz and Conrad (1995) showed

that acetate concentrations increase drastically, from 100 CIM to 1300 CIM, in sediments of Lake

Constance (Germany) after stimulation by greater algal deposition. High primary production in









Lake Apopka, with consequent sedimentation, is leading to high C concentration in sediments

that is supporting high microbial biomass and activity.

The positions of extractable C:P and N:P ratios and microbial biomass or activity in PCA-1

(Figure 2-4), support the idea that P availability, 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 activity in these sediments. Both anaerobic

respiration and CH4 prOduction rates reflect microbial activity in these sediments and C, N, P are

required for microbial metabolism and growth. High availability of C and nutrients can support a

larger microbial community that will be reflected in a higher turnover of organic substrates.

Other studies have found the same relationship between nutrients and microbial activity.

Drabkova (1990), in a study of bacterial production and respiration in lakes with different trophic

conditions, reported that bacterial production correlates with P concentration, and that respiration

increases with trophic state, but to some limit. Anaerobic respiration appears to approach an

asymptote with increasing microbial 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 fTOm Quebec lakes was associated with TP concentration in the

water column. Despite the fact that most investigators accept the idea that C availability is the

maj or factor limiting heterotrophic microbial processes, in both aquatic and terrestrial

ecosystems, nutrients other than C are likely limiting where detrital organic matter is nutrient

poor (Grimm et al. 2003). Cimbleris and Kalff (1998) showed that for planktonic bacterial









respiration, the best predictor was TP, but also that higher respiration was observed with

increasing C:N and C:P ratios, similar to the findings in my study.

Phosphorus control of microbial activity 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 shown that methane production rates were higher in

eutrophic than oligotrophic lakes. (Casper 1992; Rothfuss et al. 1997; Falz et al. 1999; Niisslein

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). Iron- and SO42-2

reducers outcompete methanogens for H2/CO2 and acetate, 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). Consequently low C availability with concomitant presence of Fe- and

SO4-2-reducers is the probable explanation for lack of methanogenesis in Lake Okeechobee

sediments .

Methanogens (archaebacteria) are obligate anaerobes 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 found (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 methanogenesis dominates freshwater ecosystems

while hydrogenotrophic dominates marine systems (Whiticar 1999), results from the electron

donor experiment in Lake Okeechobee show that in this freshwater ecosystem H2/CO2 is the

maj or substrate for methane production. Other studies have reported 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 acetoclastic methanogenesis is dominant at low

temperatures, 10 oC. Higher temperatures lead to an increased contribution of other

fermentation pathways and H2/CO2-dependent methanogenesis (Schulz and Conrad 1996; Falz et

al. 1999; Glissmann et al. 2004). In a study of rice 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. syntrophic bacteria) that seem to be sensitive to low

temperatures. Lake Okeechobee lies in south-central Florida. It is subj ect to subtropical climate,









and the annual water column temperature ranges from 15-31 oC (Rodusky et al. 2001). During

sampling for this study, water temperature in Lake Okeechobee was around 28-31 oC. Because

the lake is shallow, sediment temperature is probably 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 labile 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 maj or 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 distinct sediment biogeochemical properties, however some

similarities were present, such as high TP concentration in sediments from 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 maj or P form. Differences in sediments from this eutrophic lake

included low microbial activity (CO2 and CH4 prOduction rates), and high concentrations of

labile Pi (Figure 2-6). The hypereutrophic Lake Apopka had high concentrations of microbial

biomass P, N and C, as well as high extractable C:P and N:P ratios, and high microbial activity

(CO2 and CH4 prOduction rates) (Figure 2-6).

Conclusions

Eutrophic and hypereutrophic lakes usually receive high external loads of nutrients, and

display high primary productivity and nutrient concentrations. Consequently, sediments from









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









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.









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"










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










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










Table 2-5. Extractable and microbial biomass C, N, and P concentrations in sediments from three subtropical lakes. (mean a standard
deviation). Extractable C and N non-fumigated 0.5 M K2SO4, Extractable P non-fumigated digested 0.5 M NaHCO3.

Extractable (mg kg-l dw) Microbial Biomass (mg kg-l dw)
Lake Site
Carbon Nitrogen Phosphorus Carbon Nitrogen Phosphorus


670 184

780 & 139

3 &2

179 & 19

196 & 13

104 & 9

156 & 32

10 &1

21 &3

24 & 1

71 +13

20 & 2

1035 1224

1331 +163

1070 1256

1660 & 309


107 123

108 & 2

1.3 & 0.1

26 & 7

111 & 37

87 & 7

113 & 20

3.1 & 0.1

3.3 & 0.8

7.4 & 1.3

29 & 6

6.7 & 0.2

33 15

38 &1

43 5

29 & 0.4


305 141

299 & 24

3.7 &2

39 &6

90 & 26

80 & 18

130 & 20

2.2 & 2

8 &7

8 &3

7 &6

6 &4

2378 1278

3516 & 292

2977 1 130

4068 & 254


40 + 10

53 & 7.4

0.8 & 0.2

4.9 & 1.6

21.2 & 7.7

3.7 & 2.3

17.0 & 6

0.8 & 0.4

1.4 & 1.4

1.8 &1.7

0.9 & 0.9

0.9 & 0.7

5611 59

746 & 48

632 1 149

1031 & 83


South

Central

North

M17

011

M9

K8

FC

J5

TC

KR

J7

South

Central

West

North


1642 1224

2619 & 603

51 &5

1645 & 221

887 & 99

482 & 44

945 A 171

18 &9

115 & 41

101 &5

228 & 59

66 & 9

3827 1827

4169 & 711

3711 1347

4316 & 655


1526 1 187

1705 A 145

42 & 7

249 & 25

655 & 71

338 & 22

579 & 104

18 &8

56 & 9

44 & 13

125 & 31

34 & 14

131821 1524

20771 & 2342

18742 1830

23244 & 1327


Annie










Okeechobee











Apopka










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.












A) Lake Annie


Florida


NOT



5.4NOT




C~~2. 16.4 1i2. 9.1 7. 1.8


18.2


0 25 50 100 150 200 250 Kilometers A6 1.9









0 0.1 0.2 0 3 Kilometers
1111I


Figure 2-1. Map of the three subtropical lakes with sampled sites and their location in Florida State: A) Lake Annie (with water
column depth in meters, modified from Layne 1979), B) Lake Okeechobee with different sediment types, and C) Lake
Apopka.

















C) Lake Apopka


0 2.F 5; ]0 15 20 25; 30 35 Kilometrers


0 1 2 4 6 Kilometers
I I 1 I I I I I I I I I


Figure 2-1. continued

























































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










1600


1400

1200 A

CO2 = -425 + 241 Log(MBC)
1000
r2 = 0.75
800

600




ON200


0 2600 5200 7800 10400 13000 15600 18200 20800 23400 26000



.8 00


LO O
3Y 00




200







0 200 400 600 800 1000 1200 1400 1600 1800 2000

Microbial Biomass Carbon (mg kg l)

Figure 2-3. Relationship between anaerobic respiration and microbial biomass carbon of
sediments from A) Lake Annie (blue circles), Lake Okeechobee (red squares), and
Lake Apopka (green triangles) and B) data from Lake Annie and Lake Okeechobee
only.












IP *
eLabPi


TP
*FAP
Lab o


*) C IRBeP
TN
Ext-C
E t-N

MBC
E ~t-N:P
Ext-C:P


S0.2

S0.0

S-0.2


Ext-C:N


-0.4

-0.6


-0.8 t


-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2


0.4 0.6


0.8 1.0


Axis 1 (60.2%)


/Centmal

South O


KR
O,


O


WYest ~


Centmal ~aA
ASouth


Nodth AA


riC
FCa
JT7


Nodth


-0.5


-1.0


-1.5


-2.5 -2.0 -1.5 -1.0 -0.5 0.0
Axis 1


0.5 1.0 1.5 2.0 2.5


Figure 2-4. Results of the Principal 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, 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, CH4: basal methane production rates.





















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.















































Trophic State


TP
Labile-Pi
Inorganic-P
Ext-P


TP
Ext-C, Ext-N
Ext-C :P
Ext-N:P
Microbial Activity
MBC, MBP, MBN


TP
HAP
FAP
Inorganic-P
Ext-P


High


Medium














Low


Microbial Activity
MBC


Ext-N:P
Ext-C :P
Microbial Activity
MBC

Lake Okeechobee
Mud Zone


Ext-C: P
Ext-N:P
Res-P


Ext-P


Lake Annie
Central


Lake Apopka


Figure 2-6. Graphic representation of 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.









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,









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. 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 represent material accreted

in recent years and chemically it will have different 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 controlling P solubility in these sediments.

Materials and Methods

Study Sites

Three Florida (USA) lakes, ranging in trophic state, were selected. Lake characteristics

were described in Chapter 2 (Table 3-1, Figure 3-1).

Field Sampling

Sediment sampling sites were selected based on previous spatial study conducted in all

lakes (Chapter 2). Sediment -water interface cores of variable lengths were collected using a









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).









Organic P pools were measured using a chemical fractionation scheme described by Ivanoff et

al. (1998). This procedure involved sequential chemical extraction in a 1:50 dry sediment-to-

solution ratio, with (Figure 3-2): 1) 0.5 M NaHCO3 (pH = 8.5) representing labile inorganic and

organic P; 2) 1 M HCI 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 remaining in residual sediment after sequential

extraction was measured by ignition method and is called residual 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 filtered through a 0.45 Clm membrane filter, and analyzed for SRP or digested

for TP (with sulfuric acid and potassium persulfate). Solutions were analyzed by colorimetry,

determined by reaction with molybdate using a Bran+Luebbe TechniconThl Autoanalyzer II

(Murphy and Riley 1962; Method 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 and 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, placed in a vacuum desiccator and incubated for 24

hrs. The other sam le was left untreated. Both sam le sets were extracted with 0.5 M NaHCO3

(pH = 8.5) in a 1:50 dry sediment-to-solution ratio. Extracts from both sets were filtered through

a 0.45 Clm membrane filter and digested for TP with sulfuric acid and potassium persulfate, and

analyzed as describe previously. Microbial biomass was calculated by the difference between

treated (with chloroform) and non-treated samples.









3P Nuclear Magnetic Resonance

Samples from each depth interval in triplicate cores from each site were combined and

extracted using the methods described by Hupfer et al. (1995, 2004) and Turner 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-to-solution ratio), shaken for 2 hours and centrifuged

at 10,000 x g for 30 min. A small aliquot of each extract (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 oC and later lyophilized. 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 deuterium 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 Cls pulse (45o), 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 (Makarov et al. 2002; Turner et al. 2003).

Statistical Analysis

A Pearson correlation and a Principal Component Analysis (PCA) were performed to

determine relations among P forms measured with different methods. All statistical analyses

were conducted with Statistica 7.1 (StatSoft 2006) software.












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 Annie reflecting their high fluid content relative

to Lake Okeechobee sediments. Bulk density increased 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 highest 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 decrease in TP with depth (Table 3-3).

Surface sediment labile inorganic P (labile-Pi) concentrations were highest in Lake

Okeechobee site M9 followed by Lake Annie (Table 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, 7-

15% of TP was present as labile-Pi. Labile-Pi decreased 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 highest at M9 in Lake Okeechobee, followed by Lake Annie and

Lake Apopka sediments. There was no clear trend with depth in Lakes Annie and Apopka, but a

slight decrease with depth in mud sediments of Lake Okeechobee (M9-site) was seen. Lake

Okeechobee sites M17 and KR sediments showed a pronounced decrease of HCl-Pi with depth.


Results









HCl-Pi accounted for 26-56 % of TP in Lake Apopka, 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 contribution 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 Okeechobee stations. Approximately 0. 1-3% of the TP was present as

labile-Po in sediments of most sites, with a general decrease with depth (Table 3-3). Microbial

biomass P (MBP) decreased with depth in all cores except KR, where values were consistently

low throughout. Highest MBP values were detected 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 were detected for highly

resistant organic P (HAP). Lake Annie had the highest values, followed by Lake Apopka, then

Lake Okeechobee. Fulvic acid-P and HAP accounted 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 Okeechobee, and decreased with depth. In sediments

at site M9 6-20% of TP was present as Res-P with 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.

3P Nuclear Magnetic Resonance

31P NMR spectroscopy enabled the identification of discrete pools of organic P (Figure 3-

3A, B, C, Table 3-4). Orthophosphate was the maj or P compound in sediments of Lake

Okeechobee and none of the organic compounds were detected with this technique. Results of









31P NMR spectroscopy were in agreement with the results of chemical P fractionation (Tables 3-

3, 3-4). NMR analyses show that Lake Okeechobee sediments are dominated by inorganic P as

orthophosphate (M9: 68-100%, M17: 100% and KR 100%), although in upper layers of mud

sediments (site-M9) phosphate monoester (24-27%), and DNA-P (7-9%) were also detected

(Figure 3-3B, Table 3-4). In Lake Annie, three P compounds were detected in all sediment

depths: orthophosphate (5 1-71%), phosphate monoester (23 -3 6%), and DNA-P (6-10%), but no

clear trend was observed with depth (Figure 3-3A, Table 3-4). In Lake Apopka sediments, six

different P compounds were detected: 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 3-

4). There was a general decrease in orthophosphate and organic P forms (phosphate monoester,

lipid-P, DNA-P) with depth in Lake Apopka sediments.

Comparisons of P forms determined by the two different methods showed that

orthophosphate (NMR) was correlated with HCl-Pi (r = 0.68), labile-Po (r = 0.73), FAP (r =

0.82), and HAP (r = 0.80) (chemical fractionation). Phosphate 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 different P forms in sediments from the three

different lakes a Principal Component Analysis (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, MBP 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 lakes are separated into different groups (Figure 3-









4B). Lake Apopka placed in the position of the parameters selected by Axis 2 and Lake Annie in

the position of variables selected 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 Annie contained more TP in sediments than

both eutrophic Lake Okeechobee and hypereutrophic 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 2005) 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 increase in TP concentration in upper sediment

layers was due to cultural eutrophication (Brezonik and Engstrom 1998; Schelske et al. 2000;

Kenney et al. 2002; Waters et al. 2005; Schottler and Engstrom 2006; Engstrom et al. 2006).

Nevertheless, a decrease in TP with sediment depth has been observed in many lakes

(Sarndergaard et al. 1996; Gonsiorczyk et al 1998; Ahlgren 2005; Reitzel et al. 2006a, 2007).

The relative abundance of P forms in sediments 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 different processes 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).









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









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)









simulated a M~icrocystis sedimentation event in a eutrophic lake deposit and found that organic P

was the dominant fraction in the cells (74% of total P). After 15 days of incubation, most of the

TP was transformed into total labile 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 Apopka' s calcareous sediments, abiotic 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). Biotic 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 microbial cells or degradation of stored poly-P.

If the downcore decline in concentration of P forms measured with 31P NMR is indicative of P

degradation (Reitzel et al. 2007), then biotic processes are important 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 activity 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 sediments strongly support the biological control of P solubility

in these sediments (Chapter 4). 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.

Some studies found a significant correlation between bacterial biomass and organic P

extracted with NaOH, and suggested that organic P extracted with NaOH can be used as a proxy

measure of bacterial P (poly-P) (Uhlman and Bauer 1988; Waara et al. 1993; Goedkoop and

Petterson 2000). My results do not support this suggestion as Lake Annie had the highest









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









DOC:P ratios (Chapter 4, Table 4-3). Perhaps more important its sediments are always

anaerobic. Carman et al. (2002) reported similar results. They found high poly-P in Lake

Giimmaren, with well-oxidized sediments and high organic matter content, some poly-P in Lake

Lingsjiin, with oscillating oxic/anoxic conditions, and no poly-P in Lake Flaten, with constant

anoxic conditions. Lake Okeechobee, though shallow, and probably subj ect to oscillating

aerobic/anaerobic conditions, seems to be carbon limited 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 constant P-sufficient conditions (Vadstein 2000)

as the enzyme responsible for poly-P formation polyphosphatee kinase) is a repressible enzyme

that is derepressed under P starvation (Harold 1966).

Absence of poly-P in the uppermost 5 cm of sediment is surprising as Hupfer et al. (2004)

reported poly-P in the top 0.5 cm of sediment for 22 European lakes, and Ahlgren et al. (2006b)

found poly-P in the top 1 cm of sediment in two 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 poly-P in 0-7 and 8-16 cm in Lake Giimmaren. Reitzel et al. (2007)

found poly-P in 0-10 cm sections of sediment from mesotrophic Lake Erken. In hypereutrophic

Lake Sarnderby (Denmark), Reitzel et al. (2006a) found poly-P in anoxic sediment layers up to

24 cm deep. Absence of poly-P in the uppermost first 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, Table 4-3). Also, presence of poly-P in deeper sections of the









sediment is a strong indication that the sediment microbial community 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 could 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 colorimetry 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 nature of poly-P. Several studies have

shown that poly-P is highly labile and plays an important role in P release from sediment

(Toirnbon and Rydin 1998; Petterson 2001; Hupfer et al. 2004; Ahlgren et al. 2005; Reitzel et al.

2007).

Conclusions

All lakes had a decrease in TP concentration with sediment depth, and although oligo-

mesotrophic, Lake Annie contained more TP in sediments than both eutrophic Lake Okeechobee

and hypereutrophic Lake Apopka. The relative abundance 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 Annie 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 characteristics, as well as the maj or 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 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.










Table 3-1. Characteristics of sampled sites in the three different lakes with sampling date, location, sediment type and water quality
parameters (measured at 1 m).


Lake

Okeechobeeb

M9 M17 KR

July/2005
Mud Peat Sand

4.0 2.5 3.1

0.08 0.15 0.5

26058'17.6" 26o45'24.4" 27o58'll.5"

80o45'38.4" 80o46'36.8" 81000'51.8"

29.5 28.8 30.8

385 320 143

7.8 7.6 6.0

6.5 6.3 1.8

14.5 17.9 19.8

255.9 263.2 146.4

90.4 113.1 62.5

3439 3362 2957

103.0 60.4 83.6
0.5-1-2-5-10-20 (m) and bAverage depth: 0.5-1-2 (m).


Apopkab
West

May/2005

Organic
2.0

0.3

28o38' 01"

81o39'36"

26.6

443

7.6

8.7

31.1

69.7

11.1

11149

119.6


Parameters Annica

Central

Sampling Date June/2005
Sediment Type Mud/Clay

Water Column Depth (m) 20

Secchi (m) 2.0
Latitude 27ol2'27"

Longitude 81o21'44"

Temperature (oC) 30.2
Electrical Conductivity (CIS cm l) 41.9

pH 5.1
Dissolved Oxygen (mg L^1) 6.4

Dissolved Organic Carbon (Clg L^)*" 13.8

Total Phosphorus (Clg L^)*" 33.2

Soluble Reactive Phosphorus (Clg L^)*" 7.4

Total Nitrogen (Clg L^)*" 1807

Ammonium NH4-N (Clg L^)*" 181.6
* Mean concentration in the water column. a Average depth:










Table 3-2. pH, bulk density (BD), organic matter content (LOI loss on ignition) in sediment
profiles of the three lakes. (mean a standard deviation SD). ** No replicates for SD
calculation.


Depth
(cm)
5
10
15
20
30
45
60
80
5
10
15
20
30
45
60
70
5
10
15
20
30
40
5
10
15
20
30
40
5
10
15
20
30
45
60
80
98


BD
(g of dry cm3 of wet)
0.04 & 0.006
0.04 & 0.003
0.06 & 0.003
0.07 & 0.003
0.07 & 0.003
0.08 & 0.002
0.10 + 0.006
0.11 & 0.007
0.11 & 0.005
0.16 & 0.003
0.20 + 0.020
0.23 & 0.020
0.26 & 0.056
0.33 & 0.040
0.30 + 0.009
0.33 & 0.043
0.14 & 0.003
0.13 & 0.011
0.13 & 0.002
0.12 & 0.005
0.12 & 0.010
0.13 & 0.023
1.22 & 0.314
1.13 & 0.272
1.15 & 0.323
0.51 & 0.085
0.55 & 0.096
1.07 & **
0.01 & 0.001
0.02 & 0.001
0.02 & 0.004
0.03 & 0.006
0.03 & 0.009
0.04 & 0.014
0.06 & 0.010
0.07 & 0.005
0.07 & **


LOI
(%)
58 & 0.4
57 & 1.2
55 A 1.0
54 & 1.6
52 & 0.8
52 & 0.1
50 + 0.9
50 & 1.0
36 & 1.7
37 & 1.2
21 +1.3
26 & 6.9
16 & 3.8
25 & 6.5
29 & 4.7
35 & 3.5
83 & 0.9
88 & 0.9
89 & 0.3
89 & 0.5
89 & 0.2
88 & 0.4
1.9 & 2.8
3.9 & 2.9
4.9 & 3.5
18.1 & 4.2
16.6 & 8.8
6.4 & **
69 & 0.7
67 & 3.2
67 & 1.1
65 & 2.7
64 & 1.0
66 & 1.9
68 & 1.3
69 & 0.8
71 & *


Lake


Site


pH
5.4 & 0.15
5.2 & 0.05
5.3 & 0.09
5.3 & 0. 11
5.4 & 0.03
5.5 & 0.09
5.5 & 0.06
5.7 & 0.06
7.7 & 0.16
7.7 & 0.03
7.8 & 0.04
7.8 & 0.04
7.9 & 0.03
7.9 & 0.05
8.0 + 0.01
8.0 + 0.08
7.6 & 0.20
7.5 & 0.08
7.4 & 0.06
7.4 & 0.10
7.3 & 0.15
7.4 & 0.17
7.4 & 0.19
7.5 & 0.19
7.7 & 0.46
7.4 & 0.41
7.2 & 0.27
6.9 & **
7.5 & 0.07
7.3 & 0.06
7.2 & 0.02
7.2 & 0.06
7.3 & 0.04
7.2 & 0.05
7.1 & 0.10
7.0 + 0.06
7.0~ +**


Annie


Central


Okeechobee


M17






KR


Apopka


West










Table 3-3. Phosphorus fraction concentrations in sediment profiles. (mean & SD). ** No replicates for SD calculation.
TP Labile P
Depth Total P Pore IVBP HCl-Pi FAP HAP Res. P
Lake Site (c)WtrInorg. Org.

(mg kg' dw)


50 19

43 17

40 15

3516

28 12

22 13

1912

161 1

614

81 1

714

41 1

210

3 A1

21 1

11 1


368 161

444 1 13

548 158

600 173

726 178

665 1 116

544 133

540 133

670 140

582 1 11

607 135

575 17

496 1 16

510 142

458 166

423 135


5

10

15

20
Annie Central
30

45

60

80

5

10

15

20
Okeechobee 149
30

45

60

70


1439 135

1423 128

1459 167

1531 +80

1484 1 191

1513 1258

1133 132

1149 151

1051 139

924 124

835 116

732 183

644 1 57

575 148

590 13

497 1 155


12 13

713

913

1513

25 18

24 19

1814

81 1

NU)

NU)

ND)

NU)

ND)

NU)

NH)

NH)


78 18

64 1 18

44 13

37 15

30 17

24 1 10

22 1 10

1816

50 12

34 15

281 8

1812

1112

91 1

8 10.4

21 1


72 1 13

67 1 12

55 16

52 19

42 18

34 1 10

22 10.3

151 3.8

11017

87 1 11

127 133

55 17

80 16

55 18

46 13

36 12


3711 71

341 137

378 1 17

378 17

338 133

285 129

195 130

206 1 1

72 18

64 13

213

413

010

010

11 1

413


268 171

255 128

185 1 18

183 141

213 1 17

180+ 13

184 164

153 120

34 14

35 13

817

413

010

010

213

512


616

414

0.)0

313

0.5 10.8

1~11

0.)0

0.)0

213 A 12

194 19

229 1 53

127 135

132 129

108 1 10

67 1 10

50 15










Table 3-3. continued
TP Labile P
Depth TP Pore MBP HCl-Pi FAP HAP Res-P
Lake Site (c)WtrInorg. Org.

(mg kgl dw)


5 270 113


ND 6 10.6 14 16 5 10.5 213 140 5 12


14 12 16~11


13817
129 116
12119
112 114
144 & 27
263 & 20
280 150


ND
ND
ND
ND
ND
ND
ND


5 10.3
3~11
3 10.8
2 10.8
2 &1
1 & 0.5
1 10.2


4 10.3
3~11
3 10.4
4 10.3
3 & 0.4
4 &1
712
3 &2
3 A1
2~11
1 &**
2 10.4
1 & 0.3
1912
20 15
28 & 8
37 &3
34 1 15
42 & 8
611**


4 10.4
3 10.5
4 10.7
4 10.8
3 & 0.6
0.4 & 0.3
1 10.5
0.4 & 0.3
1 & 0.4
1 10.2
0.3 & **
1518
19 &7
25 17
24 1 13
15 &5
11 &7
1618
7 &2
11**


56 13
55 128
44 13
42 13
70 & 12
234 & 24
263 +31
211 & 45
85 & 40
22 17
3 & **
325 1 13
347 & 40
382 148
3611 35
418 &6
340 & 31
356 171
351 & 44
386 1**


8~11
5~11
5 10.5
5~11
4 &1
4 &2
312
2 &2
7 &1.6
11+6
2 & **
21416
200 & 36
219 120
148 126
120 & 30
93 & 46
63 120
37 & 3.3
34 1**


1512
1514
1713
1512
16 &7
1 & 0.2
112


1612
1716
1814
1612
17 &7
8 &6
1416


M17


Okeechobee


15
KR
20
30
40
5
10
15
20
West 30
45
60
80
98


258 & 83
131 & 29
55 112
16 & **
1264 153
1303 & 73
1350 137
1275 1 122
1234 & 153
990 & 296
795 1 156
615 & 7.3
694 1**


ND
ND
ND
ND
1013
7 &1
51 1
51 1
6 &2
10 &2
12 14
16 &4
71**


1 & 0.6
2 & 0.3
1 10.9
1 &**
598 1 17
596 & 85
616 13
523 1 115
399 & 163
267 & 224
106 176
31 +11
52 1**


1 +1
2 & 0.5
3~11
1 &**
125 1 12
118 & 29
133 132
95 134
93 & 38
65 & 30
48 1 18
29 & 7
27 1**


10 &2
19 &2
1812
7 & *
240 130
257 & 10
262 132
270 18
284 & 47
198 & 43
188 131
154 & 8
1391**


Apopka


Total P: total phosphorus, MBP: microbial biomass phosphorus, Inorg.: inorganic, Org.: organic, HCl-Pi: inorganic phosphorus, FAP:
moderate labile organic phosphorus, HAP: highly resistant organic phosphorus, Res-P: residual phosphorus. ND = not determined









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.








































I I I I


Florida


A) Lake Annie


Ni





0 25 50 100 150 200 250 Kilometers
'IIIIIIIIIII


0.3 Kilometers


0 0.1
I I


Figure 3-1. Map of the three subtropical lakes with sampled sites and their location in Florida State: A) Lake Annie (with water
column depth in meters, modified from Layne 1979), B) Lake Okeechobee with different sediment types, and C) Lake
Apopka.














































a 2 5 s 1o IS ao 25 3o as KiC.ceters


0 1 2 4 6 Kilometers
I I I I I I


Figure 3-1. continued


B) Lake Okeechobee


A) Lake Apopka































1N HCL (3h)
centrifugation, filtration


Ignition method, >Rsda
digested with 6M


Acidified


0.5M NaHCO3 (16h)

centrifugation, filtration


Fumigation
with CHCl3
(24h)


0.5M NaHCO3 (16h)
centrifugation, filtration


Pellet II


Pell~t III


Acidified
Digested for TP


0.5M NaOH (17h)


centrifugation, filtration


Digested for TP


Pellet IV


Figure 3-2. Fractionating scheme for the characterization of P organic forms (based on Ivanoff et
al. 1998).


MBP
I MBP I


IInorganicP P


















































IIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIII IIIIIIIIIII


20 10 0 -10 -20

Chemical shift (ppm)

Figure 3-3. 31PNMR spectra of the NAOH/EDTA extracts of sediment depth profile in Lake: A)
Annie, B) Okeechobee M9, and C) Apopka. 1 Orthophosphate, 2 Phosphate
monoester, 3 Lipids, 4 DNA, 5 Pyrophosphate and 6 Polyphosphate











95











B) Lake Okeechobee M9













10-15 cm




45-60 cm









20 10 0 -10 -20

Chemical shift (ppm)

Figure 3-3B






















































l" "'' I 'I 'I''''"" I''' I''' I'' '
2 1 0 -10 -20
Chemical shift

Figure 3-3C


C) Lake Apopka





































































-0.5 0.0 0.5 1.0 1.5 2.0


Axis 1

Figure 3-4. Results of the Principal Component Analysis, A) loadings of the different
phosphorus compounds measured by 31P NMR and P fractionation (n 25), and B)
the plot of the scores of the sites and sediment depth (numbers cm) from Lake Annie
(blue circles), Lake Okeechobee: M9 (red squares), M17 (brown diamonds), KR
(orange crosses), and Lake Apopka (green triangles).


* *


0.8 t


O~hpopae InorganidcP Labile Inrganic P

P-Inonoester

IIAP BD
Labile Organic P
FAP

Total Phosphorus



LOI
DNA-P
Pymophosphate
Re s dual P
Polyphos phate
Lipid-P 8
MBP


0.6 C


-0.2

-0.4

-0.6

-0.8

-1.0
-1.


0


-0.8 -0.6 -0.4 -0.2


0.0 0.2 0.4 0.6 0.8 1.0


Axis 1 (40.5%)


-0.5

-1.0

-1.5

-2.0

-2.5
-2.0


-1.5 -1.0









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









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.












Study Sites

Three Florida (USA) lakes ranging in trophic state were selected. Lake characteristics were

described in Chapter 2. The characteristics and location of sampled sites and Hield sampling

procedures were described in Chapter 3 (Table 3-1, Figure 3-1).

Water Characteristics

Water temperature (oC), electrical conductivity (EC), pH, and 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 Annie water column, oC, 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 Kj eldahl nitrogen (TN) was measured by digestion with concentrated sulfuric acid

(H2SO4) and Kjeldahl salt catalyst, and determined 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 Clm membrane fi1ter and fi1trate was analyzed for

dissolved reactive phosphorus (DRP) (Method 365.1), ammonium-N (NH4-N) (Method -

351.2), and dissolved organic carbon (DOC) (automated Shimadzu TOC 5050 analyzer (Method

- 415.1).

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, pH, organic matter

(LOI-loss on ignition), and total phosphorus were measured and described in a previous study

(Chapter 3).


Materials and Methods









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.









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 different variables and enzyme activity. A Pearson

correlation analysis was performed to determine relations among P forms and enzyme activity.

Regression analyses were conducted to compare sediment P forms and activities of enzymes and

microbes. A Principal Component Analysis (PCA) was performed to address relations among

variables, and how they relate to each lake and sediment depth. All statistical analyses were

conducted with Statistica 7.1 (StatSoft 2006) software.

Results

Water Characteristics

Lake Annie displayed strong summer thermal stratification (Table 4-1). Electrical

conductivity reflected trophic state conditions of the lakes, with higher values in Lake Apopka,

followed by Lake Okeechobee and Lake Annie. Dissolved oxygen was highest in Lake Apopka,

followed by Lake Annie and Lake Okeechobee sites M9 and M17. Lake Okeechobee site KR

had the lowest values. Lake Annie's 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 Lake 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^)~,

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).









Sediment Properties

Sediment properties (i.e., pH, bulk density, and organic matter) and concentrations of TP

and different P compounds were reported in a previous study (Chapter 3). Acidic pH conditions

were observed in Lake Annie sediments 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 sediment 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 Lake Apopka sediments decreased with depth and no clear trend

was noted at sites M9 and KR (Table 4-3). Concentration of water extractable DRP did not

present a clear pattern with depth in the profile at any of the sites, although deeper sections in

Lake Annie and Lake Apopka had higher concentrations than upper sections (Table 4-3).

Enzyme Activity

Lake Okeechobee sediments had very low enzyme activities for both PMEase (0.3-4.5 mg

p-nitrophenol g-l dw h- ) and PDEase (0.4-5.7 mg bis-p-nitrophenol g-l dw h- ) (Figure 4-1A, B).

Phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities decreased with

sediment depth at all sites (Figure 4-1A, B). Lake Annie sediments had the highest PMEase

activity compared with sediments of the other lakes, while PDEase 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. Phosphomonoesterase activity was

strongly correlated (r > 0.7) with phosphate monoester, labile 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).









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









activity was placed with labile organic P (labile-Po), FAP, humic acid P (HAP), and phosphate

monoester. Phosphodiesterase activity was placed with anaerobic respiration, MBP, DNA-P and

lipid-P. The position of the sites and sediment depth in relation to the variable loadings in the

PCA showed that the three lakes are separated into different groups (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 fractionation, Axis 1 explained 41.1% of the data

variability and the variables selected were labile-Po, FAP, HAP, anaerobic respiration, PMEase,

and PDEase activity. Axis 2, with 21.1% of the data variability explained, 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 respiration. 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 were separated into different groups, and Lake

Apopka placed in the PDEase cluster and Lake Annie in the PMEase cluster.

Discussion

There are few studies reporting on P related enzyme 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 observed in other freshwater systems. Lake

Okeechobee sediment PMEase values, although being much smaller than the other lakes of this

study, had similar or higher values than the ones 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-l dw h- ) (Yiyong et al. 2001).

Wobus et al. (2003) in a study of sediments of reservoirs of different trophic states in Germany









reported higher values for PMEase in oligotrophic Muldenberg, (17.2 mg p-nitrophenol g-l dw h-

1), than mesotrophic Saidenbach (0.8 mg p-nitrophenol g-l dw h- ) and eutrophic Quitzdorf (0. 17

mg p-nitrophenol g-l dw h- ). In a study of shallow, nutrient rich, freshwater sediments Boon and

Sorrell (1991) reported values of PMEase that ranged from 1.3 5-1.75 mg p-nitrophenol g-l dw h-

1). Small values of PMEase were reported by Barik et al. (2001) for 12 different nutrient rich

fishpond sediments (25- 59 lg p-nitrophenol g-l dw h- ). Wright and Reddy (2001) reported

PMEase values in soils of a freshwater wetland (Florida Everglades) ranging from I 5.0 mg p-

nitrophenol g-l dw h-l in P-impacted sites to 25 mg p-nitrophenol g-l dw h-l in non-impacted

sites.

Enzyme activity decreased with greater sediment depth in all lakes, a result also reported in

other studies. Wobus et al. (2003) found that PMEase activity declined with sediment depth in a

mesotrophic reservoir in Germany. The same result 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; Davis 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 correlations between enzyme activities and anaerobic respiration

indicate (PMEase r = 0.65, PDEase r = 0.91) that bacterial enzymes dominate these sediments

(Figure 4-5). Only at sites M17 and KR was PMEase activity not related to anaerobic respiration

(Figure 4-5C, D), indicating that in these sites other organisms (i.e., algae) are producing










hydrolytic enzymes. Moreover, the demand for available 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 composition and availability in these lakes. Wobus

et al. (2003) reported higher activities of PMEase in an oligotrophic reservoir than in meso and

eutrophic reservoirs. In my study, highest PMEase activity was found in the oligo-mesotrophic

lake (Lake Annie). Phosphomonoesterase activity was lowest in eutrophic Lake Okeechobee, but

displayed intermediate activity in hypereutrophic Lake Apopka. Lake Okeechobee had high

concentrations of labile inorganic 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 activity. Lake Apopka had high concentrations of

MBP and phosphate diester (lipids and DNA), as well as PDEase activity. Statistical analyses

support these results. There were higher correlation coeffieients 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 phosphate diester (lipid-P r = 0.89, DNA-P r = 0.93) and MBP (r = 0.88).

Linear regression analyses showed strong significant relations between PMEase and phosphate

monoester, and between PDEase and phosphate diester concentrations (Figures 4-2, 4-3). These

results were corroborated by the two PCAs positioning these P forms 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 biomass and activity, both enzymes, CO2 prOduction and MBP

would all cluster together, but there is clear separation of these variables. These results show that

although microbial activity (CO2), miCTObial biomass (MBP) and enzyme activities are related,

as expected, different P forms in sediments strongly influence enzyme production.









The lack of relationship between pore water SRP and enzyme activities in Lake Annie can

be attributed to different mechanisms of enzyme production and sediment properties. Acid

PMEase is usually regarded as a constitutive enzyme, 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 systems (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 water column of acid Lake Girdsjoin, Sweden, with

high aluminum (Al) and iron concentration, Jasson (1981) showed that high acid PEMase

activity was induced as a response of the plankton community to high Al concentration that

blocks substrates by reacting with phosphate. Lake Annie sediments (central site) were

characterized as having high Fe (3640 mg kg- ) and Al (34640 mg kg- ) 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 its sediment, 2) high P demand inside microorganism

cells, 3) or presence of more stable phosphate 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 with cations, protecting them from degradation

(Celi et al. 1999). Inositol phosphate, which is considered to be stable 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









diester. Initial hydrolysis by PDEase releases phosphate monoester and stimulates the production

of PMEase.

Lake Apopka's PMEase production seems to be controlled by other mechanisms, perhaps

related to both DOC and DRP availability. In a study 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 found that there is an inverse relation between pore water DRP

and PMEase activity, and a positive correlation with organic P forms (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) concluded from controlled experiments that even

during periods of high concentration of orthophosphate in lake water, PMEase is still produced,

and exhibits activity. They suggested that PMEase activity of bacteria 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 activities were related to sediment microbial

biomass and activity, as well as to the different P composition and availability. Enzyme activity

decreased with greater depth in all lakes, reflecting lower microbial biomass and activity. Strong

correlations between enzyme activities and anaerobic respiration indicated that bacterial enzymes

dominate these sediments. Different P forms in sediments were also affecting enzyme activity.









Highest PMEase activity was found in the oligo-mesotrophic lake (Lake Annie) with high

concentrations of labile-Po, FAP and HAP. Lake Okeechobee had high concentrations of labile-

Pi and lowest activities of both PMEase and 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 according to the

difference in lake sediment. In Lake Annie, high PMEase activity was unrelated to DRP

availability, 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.















)i


NM: not measured


Table 4-1. Measured parameters in the water column of Lake Annie, Lake Okeechobee, and
Lake Apopka. oC: water temperature in Celsius, EC: electrical conductivity, DO:
dissolved oxygen.
Depth EC DO
Lake Site oC .1pH
(m) (pS cm ') (mg L
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
Annie Central 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
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
M9
2 29.1 385 7.8 6.5
3 29.1 385 7.7 6.4
4 29.0 385 7.6 6.3
0.5 28.8 320 7.5 6.1
Okeechobee 1 28.8 320 7.6 6.3
M17
1.5 28.7 320 7.6 6.3
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
KR 1.5 30.7 143 5.9 1.9
2 30.7 143 5.9 1.9
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
Apopka West
1.5 26.4 471 6.7 7.4
2 26.3 652 6.4 3.0










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











Table 4-3. Water extractable dissolved organic carbon (DOC), and dissolved reactive phosphorus
(DRP). (mean & SD). **No replicates for SD calculation.
Water Extractable
Lake Site Depth (cm) DOC DRP DOC:DRP
mg kgl dw (Weight)


499 1 136
430 1284
1022 & 413
1264 & 441
3298 1 1676
4268 & 1105
4693 & 617
4332 1930
424 1276
262 & 21
278 & 86
204 & 29
201 & 52
279 & 77
275 1 138
447 & 124
459 & 59
854 186
12191 115
1559 1249
1648 1 129
2010 +895
34 & 34
29 1 18
53 1 33
212 & 4.3
244 & 65
109 1**
2020 + 199
1422 1209
1171 1240
1072 & 38
1003 1240
819 & 7.2
642 165
733 1 13
684 ** "


1.40 10.5
0.66 10.3
1.5 & 0.5
2.9 & 0.53
7.7 12.8
10.0 & 3.7
7.6 & 0.3
9.1 13.0
2.4 1 1.72
0.78 & 0.1
0.78 & 0.4
0.82 & 0.4
0.52 & 0.1
0.56 & 0.4
1.4 10.4
4.9 & 0.2
0.88 & 0.3
0.25 +0.09
0124 10.04
0.31 10.08
0128 10.02
0.34 10.10
0.12 & 0.03
0.07 10.03
0.14 10.08
0.06 & 0.00
0.05 10.03
0.011**
1.3 10.1
0.63 10.02
0.49 10.21
0.38 & 0.2
2.8 +011
8.2 & 5.4
12.2 12.7
13.3 10.8
7.7 ** "


378
618
665
417
411
438
617
488
187
351
393
306
407
615
221
91
605
3843
5156
5275
5950
5878
288
570
586
3364
5652
10105
1624
2258
2628
3750
408
127
54
55
88


Annie


Central


Okeechobee


1417







KR


Apopka


West












Enzyme Activity


(mgp-nitrophenol g' dwh-')
0 20 40 60 80 100 120 140


(mg bis-p-nitmophenol g d h l)
160 0 5 10 15 20 25


30 35


0-5

5-10

10-15

E`15-20

Er20-30

ul 30-45

45-60

60-80

80-100


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 depth profile in Lake Annie, Lake Okeechobee: M9, M17 and, KR, and Lake Apopka. A)
Phosphomonoesterase and B) phosphodiesterase.














h h


$%
~ 'pa

9
8-
88
L,
o
~pa
~O ~f
a


100 200 300 400 500


Phos phate Monoe ster (mg kgl dw)
Figure 4-2. Relationship between phosphate monoester concentration and phosphomonoesterase
activity in sediments from Lake Annie (blue circles), Lake Okeechobee M9 (red
squares), and Lake Apopka (green triangles).

30 ....

PDEase = 0.73 + 0.066 P-Diester
25 12 = 0.89 p < 0.00001






OO

0 = ** **
0 0 10 15 0 50 30 30 0 5




and Lak ppa(re rage)


































' A PM~ase = -17.7 + 0.055 DOC
r T = 0.91, p < 0.00001
'A PD~ase = -6.51 + 0.024 DOC
r2 = 0.89, p < 0.00001



4t


100


80


S60


S40









60


4 8 12 16


800


1200


1600


2000


Pore water concentration (mg kg l)


Figure 4-4. Relationship between enzyme activity, phosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) and A) pore water dissolved reactive phosphorus (DRP)
and B) dissolved organic carbon (DOC) concentration in sediments from Lake
Apopka. .














8~ ,



\6 + m,~ .P M C~a s e
4~c 2= 0.19, p = 0.093
2~ co, PD~ase
p /=0.78,p<0.00001

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0


0 1 2 3 4 5 6 7 0 10 20 30 40 50 60 70 80 90 100


C + O + Figure 4-5. Relationship of different microbial
~O activities: 1) anaerobic respiration
o ~ 1and phosphomonoesterase activity
oO~ (PMEase), and 2) anaerobic
o $ respiration and phosphodiesterase
o (PDEase) in sediments from A)
Lake Annie, Lake Okeechobee
sites: B) M9, C) M17, D) KR, and
O o~ .rmease + C0 ms.PD~ase E) Lake Apopka.
Y= 0.23, p = 0.043 / =0.09, p= 0.208
0 1 2 3 4 5 6


0 20 40 60 80 100 120 140 160


Enzyme Activity (mg bis-p or p-nitrophenol gl dw h l)






































































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










































B 45
30 O O


Inorganic P
*Labile Inorganic P

Water Ext-P
FAP Water Ext-DOC
* HAP

Labile Organic P
PMEase


0.4 .

0.2 .

0.0 -

-0.2

-0.4 '


ResidualP


Water Ext-D OC:P


-0.6

-0.8


MBP
PDEase*


-1.0 L
-1.0


-0.8 -0.6 -0.4 -0.2 0.0 0.2

Axis 1 (41.1%/)


0.4 0.6 0.8 1.0


,80
80




60 21
45A

30 10-40
20 3 30
j +40


45

303o200456
20CY 20

015


30



30 a 10


5a 10 02


-2.5
-2.5


-2.0 -1.5 -1.0 -0.5


0.0 0.5 1.0 1.5 2.0 2.5
Axis 1


Figure 4-7. Results of the Principal Component Analysis, A) loadings of different phosphorus
compounds measured by P fractionation, enzymes 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 (squares), M17 (diamonds), KR (crosses), and
Lake Apopka (triangles).









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









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.









The primary hypothesis of this study is that microbial 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 among sediments from different lakes, and will be

related to sediment biogeochemical properties (i.e., nutrient concentration and availability). The

obj ectives of this study were to: (i) determine stratigraphic biogeochemical properties in

sediments from three subtropical lakes of different trophic status and evaluate how they are

related to microbial biomass and activity; (ii) measure microbial biomass at different depths in

the sediment from the three different lakes, and test whether there is nutrient limitation; and, (iii)

measure sediment microbial activity and establish relationships with nutrient concentration and

availability.

Materials and Methods

Study Sites

Three Florida (USA) lakes ranging in trophic state were selected. Lake characteristics were

described in Chapter 2. The characteristics and location of sampled sites and field sampling

procedures were described in Chapter 3 (Table 3-1, Figure 3-1).

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, 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 nitrogen (TN) were determined using a Flash EA-

1121 NC soil analyzer (Thermo 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) prior to C and nutrient extractions. Pore water









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 Kj eldahl

nitrogen (TN) was measured by digestion with concentrated sulfuric acid (H2SO4) and Kj eldahl

salt catalyst, and determined colorimetrically (Method 351.2). Total P was digested with 11N_

H2SO4 and potassium persulfate (Method 365.1). Water samples were filtered through a 0.45

Clm membrane fi1ter and fi1trate was 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), nitrogen (MBN), and phosphorus (MBP) were

measured with the chloroform fumigation-extraction 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 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 (H = 8.5) for MBP, using a 1:50 dr sediment-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 subj ected to Kj eldahl nitrogen

digestion (for MBN) and analyzed for total Kj eldahl-N colorimetrically using a Bran+Luebbe

TechniconThl 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 using a 0.45 Clm membrane fi1ter and digested for TP

with sulfuric acid and potassium persulfate. Solutions were analyzed by colorimetry, determined

by reaction with molybdate using a Bran+Luebbe TechniconThl Autoanalyzer II (Murphy and

Riley 1962; Method 365.1, EPA 1993). Microbial biomass (C, N and P) was determined by the









difference between treated (with chloroform) and non-treated samples. Untreated samples

represent extractable organic carbon (Ext-C), extractable labile nitrogen (Ext-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 extractable labile organic nitrogen (Ext-ON) (Mulvaney 1996).

Undigested P extracts were analyzed for soluble P as described previously, and this fraction

represents labile inorganic P (Ext-Pi). The difference between Ext-P 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 equivalent to 0.5 g of dry weight, in 5 mL of DI

water at 30 oC under anaerobic conditions. Samples were placed in a glass vial and closed with

rubber stoppers and aluminum crimp seals. Samples were purged with N2 gaS to achieve

anaerobic conditions. Gas samples were obtained at 2, 4, 7, and 10 days 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. Methane was measured with

the Shimadzu gas chromatograph-8A fitted with a 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.

Headspace 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).

At time zero of the incubation experiment a sub-sample of sediment was extracted with 25

mL of DI water, shaken for 1 hour, centrifuged at 10,000 x g, and passed through a 0.45 Clm









membrane filter. After the 10-day incubation period using the same extraction procedure,

sediments were extracted. Dissolved organic carbon (DOC), NH4-N and DRP were measured as

described above.

Statistical Analysis

A t test was performed to evaluate whether there was a statistical difference in

concentration (increase or decrease) of DOC, NH4-N and DRP during incubation (time zero

versus time ten) for each site and sediment depth. A Principal Component Analysis (PCA) was

performed to address relations between the variables and in each lake and how they vary with

sediment depth. All statistical analyses were conducted with Statistica 7.1 (StatSoft 2006)

software.

Results

Sediment Properties

Sediment properties (i.e., pH, bulk density, and organic matter) and TP concentration were

reported in a previous study (Chapter 3). Sediment TC was highest 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 5-1). Lake Annie alone displayed a decrease in

TC with greater sediment depth. Total N also declined with depth in Lake Annie sediments and

showed a generally similar trend to the core from Lake Okeechobee site M9. Total N was highest

in Lake Apopka sediments, followed by Lake Okeechobee 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 ratios with depth (Table

5-1).Extractable and Microbial Biomass C, N and P









Lake Apopka had the highest concentration of DOC, NH4-N and TN pore water

concentration (Table 5-2). Lake Annie had low DRP pore water concentration (Table 5-2).

Extractable organic C was highest 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 Ext-OC with sediment depth. The other 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 was a trend of decrease in Ext-ON

with depth in Lake Apopka and Lake Annie sediments, 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 (labile-Pi) concentrations were highest 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 Okeechobee stations. There was a general 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 Okeechobee 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).










Microbial Activity

Lake Apopka (Figure 5-1E) had 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 generally decrease with

sediment depth at all sites except KR (Figure 5-1D). In KR sediments, there was a peak of CO2

production at 15-20 cm of depth that coincided with an increase in organic matter content

(Figure 5-1D, Table 3-2). In Lake Annie there 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

t-test was run for each variable to test if there was a significant statistical difference in

concentration with incubation time, and significant differences (p < 0.05) are in bold (Table 5-5).

Dissolved organic C concentration increased during 10-day incubation at all depths of Lake

Apopka and in the topmost 10 cm in Lake Annie (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 incubation 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 sediments (Table 5-5). In Lake Apopka sediments

soluble P increased with incubation time in deeper sediments (Table 5-5).









As MBC, MBN and MBP are highly 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 biomass 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 results were observed for microbial activity.

Anaerobic CO2 (r = 0.63) and CH4 (r = 0.77) had significant correlations with extractable NH4-

N: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 showed 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 sediment depths in relation to variable loadings in the PCA showed

that the three lakes are separated into different groups (Figure 5-2B). Lake Apopka was

positioned with variables selected by Axis 1 and had clear separation by sediment depth. Site

M17 in Lake Okeechobee also presented some separation 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 labile-Pi and KR with bulk density, and did not

show clear separation by sediment 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 compared lakes of different trophic state

conditions found that both bacterial production and respiration, and CH4 prOduction increased









with trophic state (Drabkova 1990, Casper 1992, Huttunen et al. 2003, Massen et al. 2003,

Wobus et al. 2003). In this study, however, oligo-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 content and nutrient concentrations are high in the

sediment (Table 5-3, Chapter 3). High nutrient concentrations in sediments of this oligo-

mesotrophic 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 sediment depth (Rothfuss et al. 1997, Falz et al.

1999, Kostka et al. 2002, Roden and 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 and with time in Lake Apopka (all depths), and

Lake Annie surface sediments (0-10 cm). Ammonium accumulation with time was detected in

Lake Apopka and Lake Okeechobee peat sediments (site M17). Accumulation of DOC and NH4-

N can indicate high microbial activity. Maasen et al. (2003) and Wobus et al. (2003) studied

sediments from reservoirs of different trophic states and concluded that high DOC and NH4+ i

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 activities 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









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.









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 (Table 5-3). Although hypereutrophic lakes are characterized by high

P concentration, P limitation can occur during summer 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-limitation with P can

occur (Aldridge et al. 1993). Phosphorus limitation of primary productivity, however, has been

detected during summer in Lake Apopka (Newman et al. 1994). Even though this P limitation

has been established for the phytoplankton community in the water column, Lake Apopka is

shallow and there is high interaction between the water column 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 sediments can be high, which could stimulate

microbial activity and demand for P. High primary 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:1abile nutrient ratios. With decomposition and utilization of

C by the heterotrophic community, C becomes increasingly refractory and there is a decrease in

microbial biomass and activity with sediment depth, reducing the demand for P. Consequently

labile P will accumulate in deeper sediments, yielding lower C:P ratios as seen for Lake Apopka

deeper sediments.









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-









1) in Lake Okeechobee during summer. Also, several studies have shown that bacterioplankton is

an important source of C to the food web in Lake 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 bacterioplankton community in Lake Okeechobee.

Nevertheless, Phlips et al. (1997) showed that in the central, mud zone region of Lake

Okeechobee phytoplankton was dominated by small-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, during summer months, light limitation is relaxed and N becomes the

limiting factor of the phytoplankton community (Aldridge et al. 1995). Several studies have

reported low chlorophyll-a and primary productivity in the mud zone is caused by light

limitation (Aldridge et al. 1995, Phlips et al. 1993, 1995c; Gu et al. 1997). There are, however,

no data for Lake Okeechobee reporting the contribution of primary productivity to sediment C.

Also, although sites M9 and KR showed similar values of DOC in the water column, water

extractable DOC was low in M9 sediments and even lower in KR deposits (Table 5-5). These

low-DOC sediments had the lowest anaerobic respiration. 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, resulting 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 consequence of the C sources and physical 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) reported high inputs of humic content to Lake Annie in surficial









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 reach the sediment and are highly refractory

(Suess 1980), leading to low C:nutrient 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 concentrations can inhibit methanogenesis as these compounds are used by

better energetically competing organisms (Coates et al. 2002; Kappler et al. 2004; Karakashey 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- ) 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 oxidation (Lovley and Klug 1983). Although Fe oxides

and SO4-2 COncentrations were not measured in this study it is probably safe to assume that both

Fe- and SO4-2-reducers are active in the Lake Annie sediments. 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). Iron and SO4-2-reducers outcompete









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,









consequently low C availability with concomitant presence of Fe and SO4-2-reducers is the

probable explanation for lack of methanogenesis 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 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 surface sediments appear 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 were observed in mud and sand sediments of Lake Okeechobee. High availability of

P in Lake Okeechobee mud and sand surface sediments resulted in C and N limitation. Lake

Annie sediments seem to be C-limited, with low ratios of extractable nutrient ratios. Carbon

limitation was probably a consequence of C sources (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 biogeochemical properties of sediments.























































Apopka


West


Depth TC TN
Lake Site 1
(cm) (g kg- )


Table 5-1. Total carbon (TC), total nitrogen (TN), and C:N:P ratios (weight) in sediment profiles
of the three lakes. (mean a standard deviation). **" No replicates for SD calculation.


Ratios (weight)
C:N C:P N:P
13 201 15
13 197 15
13 186 14
13 174 13
14 175 12
14 173 12
15 221 15
15 213 14
15 178 11
15 203 13
20 157 8
19 217 12
25 176 7
26 227 9
21 263 12
18 419 23
17 1735 99
17 3589 201
18 3913 215
18 4144 222
19 4488 229
20 3526 176
10 44 3
14 73 5
14 130 8
15 800 54
13 1905 151
13 2187 162
13 280 22
12 268 21
12 258 21
12 270 22
12 281 24
12 382 31
13 461 36
13 602 48
13 542 41


289 & 9
280 & 10
271 &6
266 & 2
257 & 5
257 & 1
251 &3
245 & 6
187 & 3
194 & 1
131 &6
157 & 4
113 A13
130 + 8
155 & 8
192 & 9
467 & 13
493 & 7
499 & 3
498 & 3
496 & 2
496 & 5
11 +16
19 &18
31 & 24
101 & 27
106 & 25
35 & **
353 & 3
349 & 6
349 & 3
342 & 1
343 A 1
353 A 11
357 & 8
371 &5
379 ** "


21.6 & 0.4
21.3 & 0.8
20.5 & 0.7
20.0 + 0.2
18.4 & 0.5
18.3 & 0.4
16.6 & 0.2
16.3 & 0.3
12.1 & 0.2
12.6 & 0.2
6.6 & 0.4
8.5 & 0.2
4.6 & 1.1
5.0 + 0.9
7.3 & 0.7
10.6 & 0.5
26.7 & 1.1
27.7 & 0.6
27.4 & 0.2
26.7 & 0.9
25.5 A 1.6
24.9 & 0.6
0.8 & 0.9
1.2 &1.1
2.0 & 1.4
6.8 & 2.0
8.4 & 1.8
2.6 & **
28.1 & 0.6
28.0 + 0.9
28.7 & 1.0
28.1 & 0.6
29.0 + 0.8
28.8 & 0.8
28.5 & 0.2
29.4 & 0.6
28.8 ** "


Annie Central









M9


Okeechobee


M17






KR



















Aumie










Apopka


Central










West


45 161 144
60 242 1 135
80 232 1 163
5 15861 189
10 11601201
15 892 196
20 842 1 124
30 658 1 149
45 557 188
60 478 145
80 479 135
98 432 +**


Table 5-2. Pore water dissolved organic carbon (DOC), ammonium-N (NH4-N), and dissolved reactive phosphorus (DRP), total
nitrogen (TN), and total phosphorus (TP). (mean & SD). **No replicates for SD calculation.


Lake


Site Depth
(cm)
5


Pore water (mg kg' dw)
DRP
0.4 10.1
0.1 10.03
0.1 10.05
0.1 10.03
0.2 10.1
0.3 10.1
0.7 10.1
0.8 10.1
0.7 10.1
0.5 10.21
0.4 10.1
0.3 10.1
412
813
1115
131 1
51**


DOC
268 1 163
96 1 11
73 124
73 14
209 1 168


NH4-N
62 19
94 1 17
107 125
114 125
83 1 18
77 126
76 134
116171
323 1 140
541 1232
617 1271
647 1248
617 1236
585 1 191
460 136
456 131
513+**


TN
384 181
374 189
402 198
423 1 156
506 1211
484 198
437 190
382 1 108
885 1 303
1353 1406
1294 1535
15811 595
1418 1536
1280 1374
1017 144
1020 +61
1093 +**


TP
12 13
713
913
1513
25 18
24 19
1814
8~11
1013
7~11
5~11
5~11
612
1012
12 14
1614
71**










Table 5-3. Extractable organic carbon, ammonium (NH4-N), labile organic nitrogen (ON), labile
inorganic phosphorus (LabilePi) and labile organic phosphorus (LabilePo)
concentrations in sediment profiles of the three lakes.


Depth
Lake Site
(cm)
5
10
15
20
Annie Central
30
45
60
80
5
10
15
20
349
30
45
60
70
5
10
keechobee
15
1417
20
30
40
5
10
15
KR
20
30
40
5
10
15
20
Apopka West 30
45
60
80
98


Carbon
784 1 111
808 1 133
779 176
646 151
803 166
722 158
768 1 119
771 166
322 146
241 11
296 124
236 139
249 152
259 136
355 121
355 184
7141 91
1193 1 199
1337 180
1620 + 12
1613 1231
1657 1257
52 & 27
73 122
79 +31
232 127
206 126
89 1**
2670 + 118
2243 1311
2024 1267
1638 1 164
1804 1228
1505 1216
1587 182
1675 1 188
1300 +**


NH4-N
66 1 15
108 18
169 142
200 167
262 1 109
284 1 111
363 1 160
485 1245
20 + 11
711 11
86 15
78 1 13
631 7
64 15
66 13
51 12
1412
27 14
3514
3812
48 14
49 12
6 &5
1015
8 &4
28113
43 17
14 1**
104 141
234 173
340 189
377 197
427 174
449 128
464 1 15
534 168
6651* "


ON
11213
120 122
102 1 13
89 1 17
11319
106 123
82 122
88 112
92 1 17
75 17
91 21
68 12
86 1 17
84 1 14
85 1 16
74 19
118 10
163 1 16
170 125
18719
199 1 18
194 1 19
20 + 9
1816
14 & 2
34 13
40 15
151**
419 174
4191 106
384 165
351 158
349 145
275 129
243 159
214 122
228 1**


LabilePi
72 1 13
67 1 12
55 16
52 19
42 18
34 1 10
22 10.3
151 3.8
11017
87 1 11
127 133
55 17
80 16
55 18
46 13
36 12
14 16
4 10.3
31 1
3 10.4
4 10.3
3 10.4
4 &1
712
3 &2
31 1
21 1
li**
2 10.4
1 10.3
1912
20 15
28 18
37 13
34 1 15
42 18
611**


LabilePo
50 19
43 17
40 15
35 16
28 12
22 13
1912
1611
614
8~11
714
4~11
210
3~11
2~11
1~11
5 10.5
4 10.4
3 10.5
4 10.7
4 10.8
3 10.6
0.4 & 0.3
1 10.5
0.4 & 0.3
1 10.4
1 10.2
0.3 +**
1518
1917
25 17
24 1 13
1515
11+7
1618
712
li**


D:


Extractable (mg kgl dw)






























$49


Dkeechobee


&417






KR


Apopka


West


Depth Microbial Biomass (mg kg~ dw)
Lake Site


~c(m)


Table 5-4. Microbial biomass carbon, nitrogen and phosphorus concentrations in sediment
profiles of the three lakes. (mean & SD). ** No replicates for SD calculation.


Carbon
54191 195
5089 1 166
4387 1218
4205 1 177
3919 1405
3652 1692
3401 1624
2740 1422
3821 1479
3672 1 187
3465 1 231
27731 205
26161 283
23611 164
21771 92
1983 1437
3800 1437
3811 +370
3746 1873
4667 1461
5128 1447
3354 1988
574 & 249
653 & 227
644 & 220
1446 1335
1296 1 113
638 1**
36617 13193
32926 15437
30486 13924
22265 15640
19355 14608
14725 14586
11037 14291
9584 1 1273
8011+**


Nitrogen
282 1 12
283 138
172 17
106 1 19
65 1 13
60 13
4112
20 17
122 12
115 11
89 1 14
64 122
55 1 10
42 15
35 & 8
33 16
75 123
72 15
66 1 11
97 121
100 125
43 1 11
4 &4
12 & 4
15 &7
39 14
26 1 11
20 +**
2630 1294
2469 1278
2284 1366
1863 155
1731 +63
804 191
479 172
85 1 18
67 1**


Phosphorus
78 19
64 1 18
44 13
3715
3017
24 1 10
22 1 10
1816
50 12
34 15
28 18
1812
1112
9~11
8 & 0.4
2~11
6 10.6
5 10.3
3~11
3 10.8
2 10.8
2~11
1 & 0.5
1 & 0.2
1 & 0.6
2 10.3
1 10.9
li**
598 1 17
596 185
616 13
523 1 115
399 1 163
267 1224
106 176
31 11
52 +**


Annie


Central

















T=0
1.4
0.7
1.5
2.9
7.7
10.0
7.5
9.1
2.35
0.8
0.8
0.8
0.5
0.6
1.4
4.9
0.9
0.3
0.2
0.3
0.3
0.3
0.03
0.03
0.08
0.01
0.03
1.3
0.6
0.5
0.4
2.8
8.2
12.2
13.3


Annie


Central


Okeechobee


M17






KR


Apopka


West


DOC


Lake Site up
(cm)


Table 5-5. Water extractable dissolved organic carbon (DOC), dissolved reactive P (DRP), and
ammonium-N (NH4 ) COncentrations at time 0 (before incubation) and time 10 (after
incubation). Results of t test of incubation experiment, significant differences (p <
0.05) between T = 0 vs. T = 10 are in bold (n = 3 and df = 2 for all analysis).
_,1_ Water Extractable (mg kg l)


DRP
T=10
1.1
1.5
1.0
1.4
6.9
2.4
1.6
2.0
1.8
1.2
1.4
1.7
2.5
1.0
2.1
4.3
2.5
1.4
1.1
1.1
1.3
0.8
0.09
0.07
0.07
0.07
0.06
2.4
1.3
0.9
0.7
2.2
10.4
14.4
25.0


NH4-N
T=-0 T=-10
80 134
111 119
135 137
141 154
106 108
100 93
87 79
159 109
28 43
40 40
42 38
35 27
28 25
29 25
27 21
26 12
9 19
10 17
12 16
15 16
16 21
17 20
1 9
3 3
4 6
10 10
9 5
378 1042
548 1159
731 1178
788 1120
762 1041
636 865
457 667
466 666


T=-0
499
430
1022
1264
3298
4268
4693
4332
424
262
278
204
201
279
275
447
459
854
1219
1559
1648
2010
34
29
53
212
244
2020
1422
1171
1072
1003
819
642
733


T=-10
1225
875
370
238
1916
1473
1545
382
229
258
316
291
154
225
607
637
334
458
723
955
1376
1193
26
35
49
208
178
2963
2267
1822
1679
1438
1347
1412
1509













0 20 40 60 80 100 120 140 160 180 200


Microbial Activity (mg C kg' d )
0 2 4 6 8 10 12 14 16 18 20 22


0 5 10 15 20 25 30 35 40


0 2 4 6 8 10 12 1-


\~+ CO2



















D





0-5


5-10


S10-15


15-20


S20-30

30-45


45-60


60-80


0-5


5-10


10-15


15-20


20Jo .


30-45


45-60


60-80







5-10

10-15

15-20

20-30

30-45

45-60

60-80

so-too


O CO2
# CH4


O CO2
SCH4


O CO2
SCH4


0 50 100 150 200 250 300 350


-I





-r C




-l


- A


a CO2
A CH4


Figure 5-1. Microbial activity (CO2 and CH4 prOduction rates) in sediments from: A) Lake

D) KR, and E) Lake Apopka. Bars represent standard errors.


Annie, B) Lake Okeechobee: M9, C) M17,
































































Axis 1

Figure 5-2. Results of the Principal 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 Okeechobee: 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-NH4-N: 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, COz: anaerobic respiration, and CH4: methane production rates.


* *


~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~


Ext-C:Ext-P *
Ext-C:Ex-N A

TC
LO *

T~g Ext-N:Ex-P
'Ext-C
BD





-CO2 Ext-NH4~
Labile-Po
a Labile-Pi

TP


0.8 ~


-0.6

-0.8

1 0


-1.0


-0.8 -0.6 -0.4 -0.2


0.0 0.2 0.4 0.6 0.8


Axis 1 (48.6%)


Lake Okeechobee Peat


-1.5

-2.0

-2.5

-3.0i
-3.5


Lake Annie




-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5


Lake Okeechobee


1.0 1.5 2.0 2.5 3.0 3.5









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









et al. 2005). Measurements of 613C and 6 "N in several lake compartments, (i.e., dissolved and

particulate matter in the water column and sediments) have been used to identify the origin of

lacustrine OM (Filley et al. 2001; Griffths et al. 2002), infer past primary productivity (Schelske

and Hodell 1991; Hodell and Schelske 1998; Bernasconi 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 (Hollander and Smith 2001; Lehmann et al. 2002; Gu

et al. 2004; Terranes and Bernasconi 2005; Kankaala et al. 2006).

Allochthonous OM usually has more negative 613C ValUeS than does autochthonous OM.

Values of 613C 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 autochthonous OM displays very negative 613C. During periods

of very high primary productivity the preferred 12C in the water column is exhausted and

fractionation is diminished, yielding OM with higher 613C (Mizutani and Wada 1982; Raul et al.

1990). Hypereutrophic lakes with high rates of primary productivity 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 613C that is 8%o heavier than dissolved

CO2 (Fogel et al. 1992). High demand for inorganic C and low free CO2 leads to utilization of

HCO3~ aS aC source resulting in heavier 613C 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 613C and enriched in 61 N (Gearing et al. 1991; Burnett 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).

In summary, sediment OM and nutrient content, along with 813C and 6 "N 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 613C and 6 "N signatures of OM in sediment cores from

subtropical lakes of different trophic states.

Material and Methods

Study Sites

Three Florida (USA) lakes ranging in trophic state were selected. Lake characteristics were

described in Chapter 2. The characteristics and location of sampled sites and field sampling

procedures were described in Chapter 3 (Table 3-1, 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 maximum depth) were sectioned at 4-cm intervals.

Sediment cores from Lake Okeechobee sites M17 (36 cm maximum depth) and KR (16 cm

maximum depth) were sectioned at 2-cm intervals. All sediment variables are reported on a dry

weight 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 oC. Total phosphorus (TP) total

carbon (TC), and total nitrogen (TN) of core Set # 1 were measured and described in previous

studies (Chapter 3, 5).









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). Dried samples were ground in a mortar and pestle

and passed through a 2.0-mm mesh sieve. Organic matter content (LOI-loss on ignition) was

determined by weight loss at 550oC.

Isotopic Analyses

Sediment samples for organic C isotope analysis were pretreated with acid to remove

inorganic C carbonatess) (Harris et al. 2001). Samples were weighed in silver capsules, placed in

the wells of a microtiter plate, and 50 CIL of DI water was added to moisten the sediment. Plates

were placed in a vacuum desiccator with 100 mL of concentrated HC1, and exposed to HCI vapor

for 24 hours. Samples were dried at 60 oC for 4 hours to remove any remaining HC1. 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 Industries, 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, 613C = -26.43 %o, 615N =

2.55 %o, Iso-Analytical; IAEA-N1, 65N = 0.4 %o; ANU-Sucrose, 613C = -10.5 %o). Analytical

precision for standards was less than f 0.1%o for 613C and f 0.3%o for 615N.

21oPb Dating

Lead-210 dating was done by gamma counting (Appleby et al. 1986; Schelske et al. 1994).

Samples were placed in plastic SarstedtTIL tubes to a height of ~ 30 cm. Sample mass was

determined and tubes were sealed with epoxy glue and set aside for 3 weeks to allow 214Bi and









214Pb 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 estimated by averaging activities of 214Pb (295.1

keV and 351.9 keV) and 214Bi (609.3 keV). Cesium-137 activity was determined from the 662

keV photopeak. Unsupported 210Pb activity was estimated by subtraction of supported activity

from the total activity measured at each level. Activities are expressed as decays per minute per

gram of dry sediment (dpm g- ). Sediment ages and bulk sediment accumulation were calculated

using the constant rate of supply (CRS) model (Appleby and Oldfield 1978, 1983). Lead-210

dates correspond to the base of each sediment section. In all cores but the one from Lake Annie,

radioisotope activity was measured in all samples 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 density and activities were used to compute dates. This is thought to

have introduced negligible error as the topmost 12 cm have nearly 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-l in surface sediments to 2. 1 & 0.6 dpm

g-l in deeper sediments. Cesium-137 activity declined with sediment depth and showed no

distinct peak (Figure 6-1A). Chronologies determined 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) for Lake Annie. The average sedimentation rate









(since c. 1900) was 36.8 mg cm-2 -1l while Schottler and Engstrom (2006) reported a value of 34

mg cm-2 -1l. Lake Annie's 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 1970's. Over the past several decades, the sedimentation

rate has increased. Lake Annie water inputs are from ground water (90%) and atmospheric

deposition (10%) (Swain and Gaiser 2005). This lake has no natural surface streams but two

shallow man made ditches flow into the lake along the south and southeast sides. Surface runoff

from these ditches was reported to contribute to water and nutrient input to the lake during high

rainfall periods (Battoe 1985). Shifts 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 last 10 years. The lake

has transformed from a clear-water system to a water body with appreciable dissolved color. The

increase in color was probably due to high dissolved organic carbon (DOC) input to the lake

from adj acent 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 chlorophyll-a have been detected in the

past 20 years (Swain and Gleiser 2005). The increase of Lake Annie's sedimentation 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, 226Ral, and 137CS could only be detected in

near-surface sediments (Figure 6-2A). In sediments at sites M17 and KR, activity values were

below detection limits suggesting the sites were non-depositional, which precluded dating.









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 statigraphic history of the 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 surface seiche in the lake (Chimmey 2005). Lake stage rose

abruptly, by about 3.06 m during hurricane Frances, and 4.91 m during hurricane Jeanne. Peak

wind velocity reached hurricane strength at platforms over the center of the lake (144 km h- )

(Chimmey 2005).

Because Lake Okeechobee is large and shallow, its sediments are easily disturbed by wind,

especially in the mud zone (Havens et al. 2007). Havens et al. (2007) reported the impact of

Hurricane Irene on Lake Okeechobee. The storm passed 80 km south of the lake in October 1999

and produced maximal winds of 90 km h-l over the center of the lake. Mean pelagic TP increased

from 88 to 222 Clg L^1, and it is estimated that more than 10,000 metric tons of fine-grained mud

sediment was resuspended during the storm (Havens et al. 2007). Considering that the 2004

hurricanes passed closer to the lake, and had higher winds than Hurricane Irene, it is very likely

the storms caused substantial sediment resuspension and deposition. I sampled just 10 months

after the storms. Extensive sediment translocation may explain why the cores I took were

undatable. Alternatively, the sites I selected for samples may simply be inappropriate. Lake









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 aIt the bottom of the core. The fairly

constant total 210Pb activity in the upper 44 cm of the core may be explained by two processes: 1)

incoming unsupported 210Pb is being diluted by higher and higher sediment accumulation rates,

the increase in deposition 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 reported 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). Furthermore, land use and hydrological changes, including

reduction of lake surface area as surrounding wetlands 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 that violated the assumptions of the CRS 210Pb

dating model (Schelske et al. 2005; Waters et al. 2005). Cesium-137 showed a slight peak at 48-

52 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 preserved poorly in many Florida

lakes as Cs is poorly bound and may move in the sediment column (Brenner et al. 1994, 2004;

Schelske et al. 1994).









Carbon (61 C) and Nitrogen (61sN) 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 813C and 6 "N values are depleted

towards the sediment surface, varying from -28.2 %o to -29.4 %o and 2.0%o to 0.9%o (Figure 6-

3D, E). Surface sediments are depleted relative to basal deposits by -1.24 %o (613C) and -1.13%o

(6 5N). Organic matter content (LOI %) decreased with sediment depth (Figure 6-3F).

Statigraphic isotopic signatures of Lake Annie sediments indicate that this lake is going through

changes in recent years. Statigraphic 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 613C and 6 5N of Lake Annie sediments probably resulted from a

combination of several factors such as allochthonous OM input, primary productivity, and

microbial biomass and activity. Small, oligotrophic lakes are expected to have relatively a high

proportion of allochthonous C input to their sediments (Gu et al. 1996). Terrestrial C3 plants

discriminate against 13C, and organic matter derived from land plants typically have 613C ValUeS

of -27%o to -29%o (Bird et al. 1994, Meyers 1997).

Hammarlund et al. (1997) related 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 613C ValUeS of dissolved C in a humic lake (Lake

Ojrtrasket, Sweden), resulting from the mineralization 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









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









from C3 land plants has 6 5N is around +0.4%o (Peterson and Howarth 1987). Autochthonous

OM of aquatic ecosystems not dominated by cyanobacteria has a 6 5N signature of +8.6%o

(Peterson and Howarth 1987), while N2 Eixation by N-fixing algae yields values near 0%o

(Meyers 1997). Nitrogen Eixation 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 been detected in Lake Annie (Gaiser personal

communication), however, N2-fixation can be present and carried 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 6 "N a1s it will allow greater algae discrimination

against 15N (Meyers 1997). Nitrogen availability is also high in these sediments (Chapter 5).

Allochthonous OM, primary productivity as well as heterotrophic microbial community in this

lake can produce the 613C and 6 "N 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 Annie sediments. Depleted allochthonous particulate and dissolved

OM can be the maj or 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' TOSulting from heterotrophic metabolism will be utilized by primary producers that will

also produce a depleted autochthonous OM. A more detailed study of 613C and 6 "N isotopes in

several compartments, i.e., dissolved C, different N compounds, phytoplankton biomass, bacteria

biomass, particulate OM in the water column and sediment, of this lake, can elucidate maj or

processes affecting the isotopic signatures in Lake Annie sediments.









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









becomes the limiting factor for the phytoplankton community (Aldridge et al. 1995). The 6 5N of

sediments in eutrophic and hypereutrophic lakes can be influenced by N2-Hixation by

cyanobacteria (Gu et al. 1996; Rosenmeier et al. 2004). These cyanobacteria do not fractionate

against 15N and have 6 5N similar to atmospheric N (~0%o) (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 Lake Okeechobee (Cichra et al. 1995; Gu et al. 1997;

Phlips et al. 1997). Nitrogen limitation can lead to autochthonous organic matter with enriched

6 5N as algae discrimination against 15N will be diminished (Meyers 1997). As a consequence

autochthonous OM is expected to have an enriched 6 "N signature (Peterson and Howarth 1987).

Although some studies indicate that the isotopic signature of OM is resistant 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 (Bernasconi et al. 1997;

Meyers 1997; Lehmann et al. 2002, 2004). Labile carbohydrates, proteins and amino acids are

generally more enriched in 13C, while lipids and cellulose are lighter (Meyers 1997). Selective

loss of "heavy" amino acids, proteins and carbohydrates, which are particularly susceptible to

microbial degradation, leaves residual (substrate) OM isotopically lighter, with respect to 813C

than the original material (Hedges et al. 1988).

Loss of high 8 "N compounds (e.g., amino acids) can also occur, lowering the 6 5N in

residual material. Nevertheless, decomposition of OM is generally thought to increase 6 5N

through preferential loss of 14N (Nadelhoffer and Fry 1988). Bernasconi et al. (1997) reported

shifts in the 613C (depletion) and 6 "N (enrichment) of sinking OM in Lake Lugano, which they

attributed to selective removal of C and N compounds during mineralization. Additionally,









sediment 813C in Lake Lugano indicates overall isotopic 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 sediments, and selective mineralization of OM

probably influence 613C and 6 "N values in the sediments (Figure 6-9B).

Sediment OM content was highest in Lake Okeechobee 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 highest values reported from sediment at all

sites, reflecting their higher plant origin (Figure 6-5B). The TN:TP ratio declines above 30cm,

reaching the lowest values at the sediment surface (Figure 6-5C). This pattern is driven by the

high TP concentration in surface sediments (Figure 6-5A, C). The 613C Of OM varied little in the

core from site M17, from -26.7%o to -26.3%o (Figure 6-5D). With respect to 8 "N, from 36 cm to

22 cm, values decline by about -0.6%o, but are followed by a period of enrichment of ~1. 13%o up

to the sediment surface.

Stratigraphic changes in sediment in 613C and 6 "N from the peat zone probably reflect

selective mineralization of OM. Small shifts in 613C Of peat zone sediment may result from

shifting intensity of mineralization. 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 6 5N declined from about 1.4%o to 0.7%o from 36

to 22 cm, but rose again to a high of a little more than 1.8%o at the surface. Contrary 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









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









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).









Stratigraphic variation in 613C and 6 "N at the KR site probably reflects multiple factors as input

of wastewater from anthropogenic activities, and variable contributions of river-borne

allochthonous input, related to inter-annual rainfall variations (Figure 6-9D).

Lake Apopka

The Lake Apopka core displayed a narrow range 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 613C Showed a general increase upward over the

length of the core from -22.6%o to -18.4%o (Figure 6-7D). Nitrogen isotopic values also displayed

an increasing trend upcore, from 3.9%o (lower depths) to 4.7%o (surface sediments) (Figure 6-

7E). Organic matter content increased towards the sediment surface (Figure 6-7F).

Nitrogen availability is high in these sediments (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 (D'Angelo and Reddy

1993). In Lake Apopka sediments, nitrification is high in surface sediments with aerobic

conditions, and dissimilatory NO3- reduction to NH4 a TOSpiratory process used by facultative

and obligate anaerobic bacteria, is high in anaerobic sediments when the ratio of C/electron

acceptors is high (D'Angelo and Reddy 1993). Lake Apopka had the highest 813C ValUeS and

showed the greatest enrichment among studied sites. In a study of 83 Florida lakes that ranged in

trophic state, Lake Apopka plankton had the highest 813C (Gu et al. 1996). Greater 613C towards

the surface probably indicates increased in primary productivity reflecting greater nutrient

concentration, i.e., eutrophication (Brenner et al. 1999).

The C isotopic signature of autochthonous OM is influenced by the 613C Of the dissolved

inorganic C pool in lake water. Hodell and Schelske (1998) related the seasonal pattern of 613 org









in Lake Ontario to seasonality of primary productivity. Gu et al. (2006) reported 13C enrichment

of particulate OM in Lake Wauberg (Florida) resulted 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 suspended 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 613C. During periods of high primary productivity, however, this fractionation

diminishes and more 13C iS incorporated into primary producer biomass (Mizutani and Wada

1982; Raul et al 1990).

Hypereutrophic lakes with high rates of primary productivity have depleted CO2 tag)

concentrations in the water column. Moreover, in alkaline waters bicarbonate (HCO3 ) is the

dominant form of inorganic C, and is 8%o 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 613C (Goericke et al. 1994). Gu et al. (2004) reported high 813C Of inorganic

C in the water column of Lake Apopka. The authors showed that heavy 613C DIC in the water

column was a result of isotopic fractionation 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 productivity in this lake is deposited in its sediments

(Gale and Reddy 1994). Primary productivity is dominated by cyanobacteria (Synechococcus sp.,

Synechocystis sp. and M~icrocystis incerta) (Carrick et al. 1993; Carrick and Schelske 1997).









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









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









Apopka with enriched values for both 813C and 6 5N. This shows a trend of enrichment in 613C

and 6 "N with increasing trophic state.


However, sediment 813C and 6 "N signatures in each site result from different mechanisms,

and they might be more important in interpreting the data than solely trophic state (Figure 6-9).

Difference in sediment isotope C and N can indicate variability on seasonal, inter-annual and

century long-time scales of fractionation factors associated with allochthonous and

autochthonous organic matter as well as mineralization processes occurring within lakes

(Lehmann et al. 2004) (Figure 6-9).

Conclusions

In this study, the 210Pb dating 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 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 estimated to date to the 1800s and the average

sedimentation rate (since c. 1900) was determined to be 36.8 mg cm-2 -1l. ISotopic signatures

in Lake Annie sediments, depleted in 613C and 6 "N, 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, 613C ValUeS were slightly depleted

while 6 5N values 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 (enrichment of 613C and 6 "N towards the sediment surface) of sediment OM









were related to several factors, including sediment origin (i.e., plant tissue), intensities of primary

productivity and diagenesis. Stratigraphic variation in 613C and 6 "N at the KR site probably

reflects an input of 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 C, 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.

















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












































** *


TP (mg kg )
200 400 600 800 1000 1200 1400 1600
0~ = = =42005


TC:TN
13.0 13.5 14.0 14.5 15.0 15.5


TN:TP
11 12 13 14 15 16


12.0 12.5
0


10


20


30


40


50


16.0
-2005
1998
1988
1976
1966
1957
1947
1940
1930
1920
1904
1889
1884


10


S20

30


Q)40


50


60


70


80


8 13Corg (%)
-29.5 -29.0 -28.5 -28.0 -27.5


6 15N (%o)
1.2 1.4 1.6 1.8


LoI~/o)
49 50 51 52 53 54


-27.0 0.8 1.0
- nna 0


2.0 2.2


2.4
-2005
1998
1988
1976
1966
1957
1947
1940
1930
1920
S1904
1889
1884


55 56 57


-30.0
0 -
4
8
12
16
20
24
28
32
36
40
44
48
52
56
60D


20 p


C) TN:TP ratio, D) 613Corg Of sediment


Figure 6-3. Lake Annie sediment depth profile of: A) Total phosphorus, B) TC:TN ratio,

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











































































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





























































Figure 6-5. Lake Okeechobee peat zone (site M17) 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 %).


u>o~/o)
83 84 85 86 87 88 89 90








40 I


Total Phosphorus (mg kg )
0 50 100 150 200


TC:TN
17.5 18.0 18.5 19.0 19.5 20.0 20.5


TN:TP
80 100 120 140 160 180 200 220 240 2
0)' ''


250 300 17.0


8 13Corg (%)
-26.8 -26.6


6 15N (%o)
1.2 1.4 1.6 1.8


-26.4


26.2 -26.0 06
0,

I
i 5C


0.8 1.0


-27.4 -27.2 -27.0


i I


D










































O








211


Total Phosphorus (mg kg )
0 50 100 150 200 250 300


TC:TN
8 10 12 14


TN:TP
0 20 40 60 80 100 120 140 160 180


16 18 20


8 13Corg (%)
-26.2 -26.0 -25.8


8 15N (%o)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0


OI (o/o)
0.0 0.2 0.4 0.6


0.8 1.0 1.2


-25.6 -25.4 0.0





S248


Figure 6-6. Lake Okeechobee sand zone (site KR) sediment depth profile A) Total phosphorus, B) TC:TN ratio, C) TN:TP ratio, D)

813 org Of sediment organic carbon, E) sediment 8 "N, and F) organic matter content (LOI %).









































513Corg(%o)
23 -22 -21 -20 -19 -18


uIDI(o/o)
4.6 4.8 5.0 60 62 64 66 68 70


80

TN:TP ratio, D) 613 org Of sediment organic


Total Phosphorus (mg kg )
0 200 400 600 800 1000 1200 1400


TC:TN
12.0 12.5 13.0 13.5 14.0


TN:TP
30 35 40 45 50


11.0 11.5


15 20 25


6 '5N (%o)
3.8 4.0 4.2 4.4


3.6


VI


10



"20


30


Q)40


50


60


70


E


Figure 6-7. Lake Apopka sediment depth profile A) Total phosphorus, B) TC:TN ratio, C)
carbon, E) sediment 8 "N, and F) organic matter content (LOI %).


80l


80









J.U

4.5

4.0

3.5

S 3.0 t"

2.5

2.0 Anne Apopka

1.5 O

1.0 O ~K

0.5

0.0
-30 -28 -26 -24 -22 -20 -18 -16

6 1Cjorg (%o)

Figure 6-8. Carbon vs. nitrogen isotopic values of sediments in Lake Annie, Lake Okeechobee
(sites M9, M17, and KR), and Lake Apopka.
































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.










N2 Fixation


D) Lake Okeechobee M17


Water Column Phytoplankton
N2-Fixer

I NH4+ \L15N


Sediment


C limitation
Peat 813C Mineralization
\6 "N


\ l/t63C /15N


Seasonal Allochthonous Input
Agricultural runoff, sewage
\13C /\15


D) Lake Okeechobee KR


Water Column


Sediment


813C /\15N


\N2 Fixation


/ Phytoplankton
S1613C 15'N [J/ N O3- 15N1


OM
/\13C /\15N


N4


Methanogenesis


Sediment


Mineralization NO3~ N2
8t\13C /15N
/t61N


Figure 6-9. continued


E) Lake Apopka

Water Column


DIC CO2/HCO3~



CO2 /\13









CHAPTER 7
HETEROTROPHIC MICROBIAL ACTIVITY INT SEDIMENTS: EFFECTS OF ORGANIC
ELECTRON DONORS

Introduction

Organic matter deposition is an important source of carbon (C) in lake sediments. Organic

compounds and associated nutrients supplied to sediments are mineralized through heterotrophic

decomposition (Gachter 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 (Oz) is rapidly consumed, and is

depleted within several millimeters below the sediment water interface (Jarrgensen 1983). In

these systems, facultative and strict anaerobic communities dominate. Complete oxidation of a

broad range of organic compounds in these systems can occur through the sequential activity of

different groups of anaerobic bacteria (Capone and Kiene 1988).

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).

Cellulolytic bacteria hydrolyze organic polymers through extracellular enzyme production and

further break down monomers to alcohols, 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 competition for fermentation products such as H2 and acetate.

Microorganisms derive energy by transferring 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.,

sulfate, iron reducers) are sensitive to the concentrations of substrates and products. Their

activities can be inhibited by their end products and are dependent on the end product

consumption by other organisms (Stams 1994; Megonigal et al. 2004).

Microbial functional diversity includes a vast range of activities. 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 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,

and may indicate previous exposure to those C-sources (Degens and Harris 1997; Degens 1998;

Baldock et al. 2004; Stevenson et al. 2004).

The metabolic response of microbial communities in lake sediments may vary due to

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

characteristics of lakes, such as the source and chemical composition of particulate matter and

biogeochemical processes in the sediment and water column. 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 other factors (Bostroim et









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.












Study Sites

Three Florida (USA) lakes ranging in trophic state were selected. Lake characteristics were

described in Chapter 2. The characteristics and location of sampled sites were described in

Chapter 3 (Table 3-1, Figure 3-1).

Field Sampling

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 (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 weight 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 oC. Before each analysis,

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

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

wei ht loss at 550oC. Total P was measured by inition method, followed by acid di estion (6 M

HC1) and measured colorimetrically with a Bran+Luebbe TechniconThl Autoanalyzer II

(Anderson 1976; Method 365.1, EPA 1993). Total carbon (TC) and total nitrogen (TN) were

determined using a Flash EA-1121 NC soil analyzer (Thermo Electron Corporation).


Materials and Methods









Extractable Carbon (C), Nitrogen (N), and Phosphorus (P)

Sediment samples were extracted with 0.5 M K2SO4 for extractable N and with 0.5 M1

NaHCO3 (pH = 8.5) for extractable P, using a 1:50 dry sediment-to-solution ratio (Mulvaney

1996; Ivanoff et al. 1998). Extracts from samples were centrifuged at 10,000 x g for 10 min and

filtered through Whatman # 42 fi1ter paper. For N analysis, 5 mL of the extracts were subj ected

to Kj eldahl nitrogen digestion and analyzed for total Kj eldahl-N colorimetrically using a

Bran+Luebbe TechniconTM Autoanalyzer II (Method 351.2, EPA 1993). Undigested N extracts

were analyzed for ammonium (NH4-N) (Method 351.2, EPA 1993), and 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 Clm membrane fi1ter, 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; Method 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 measured with the chloroform fumigation-

extraction method (Hedley and Stewart 1982; Vance et al. 1987). 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 vacuum desiccator, and incubated for 24 hrs. The duplicate sample was left

untreated. Both sets were extracted with 0.5 M K2SO4 centrifu ed at 10,000 x for 10 min and

filtered through Whatman # 42 fi1ter paper. Carbon extracts were acidified (pH < 2) and analyzed

in an automated Shimadzu TOC 5050 analyzer (Method 415.1, EPA 1993). Microbial biomass









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









detected with some treatments at 14 days of the experiment. CO2 WAS measured by gas

chromatography using a Schimadzu (8A GC-TCD) 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 flame ionization detector (110 oC), N2 aS the carrier gas and a 0.3

cm by 2 m Carboxyn 1000 column (Supelco Inc., Bellefonte, PA) at 160 oC. Prior to

measurements of both CO2 and CH4, headspace 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). Final production rates were determined after removing the lag phase (the

time between substrate addition and quantifiable gas production) in each site. Turnover rates (d l)

were determined by dividing the sum of CO2 and CH4 prOduction rates by the amount of C

added. Anaerobic CO2 and CH4 prOduction rates were standardized by microbial biomass carbon

of each sediment sample (CO2 Or CH4 prOduction divided by MBC).

Statistical Analysis

All statistical analyses were conducted with standardized values of anaerobic CO2 and CH4

production rates. One-way analysis of variance (ANOVA) with pairwise multiple comparisons

Tukey's HSD test was used to assess the effect of different electron 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 performed to determine maj or patterns of CO2 and

CH4 prOduction rates with the addition different electron donors. All statistical analyses were

conducted with Statistica 7.1 (StatSoft 2006) software.












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









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 sediment (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 format addition. All Lake Okeechobee sediments

had the highest CO2 prOduction rates with both amino acids and glucose addition. Lake Apopka

sediments had the highest CO2 prOduction rates with alanine and format (Table 7-3).

Higher CH4 prOduction rates in Lake Annie sediments were detected with the addition of

both amino acids and acetate. In Lake Okeechobee 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 Okeechobee, 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, glucose, 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 different C-sources and the highest was detected for

alanine (Table 7-3). Lake Apopka sediments had the highest turnover rates of all C-sources when

compared to the other sediments (Two-way ANOVA, F = 3.88, dJf = 28, p < 0.00001).









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









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 sediment microcosms stimulated heterotrophic

activity. Findlay et al. (2003) showed that the addition of different carbon sources, i.e., glucose,

bovine serum albumin and natural leaf leachate to hyporheic biofilms enhanced microbial

activities. Wang et al. (2007) showed that addition of electron donors (glucose, sucrose, potato

starch, and sodium acetate) stimulated denitrification in Lake Taihu (China) sediments. In the

study of benthic microbial response to the deposition of natural seston in Lake Erken (Sweden),

Tornblom and Rydin (1998) showed that seston addition caused an immediate increase in

bacterial production, activity, and total sediment metabolism.

The extent of response to electron donor addition was strongly related to microbial

biomass. Most sediments responded rapidly to addition 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 phase 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 biomass exhibited the highest turnover rates (Table

7-1, 7-3). Statistical correlations suggest that observed rates 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 dependent 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









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,









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 Okeechobee site M17) (Chapter 5). The results

from PCA-1 also placed peat sediments (site M17), 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 stimulate heterotrophic microbial 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 the substrate as well as the assimilation of added C

into microbial biomass rather than being released as CO2 Via TOSpiratory pathways (Bremer and

van Kessel 1990; Degens 1998a). Toirnblom and Rydin (1998) found that after seston addition to

sediment, bacterial biomass doubled indicating assimilation of C into microbial biomass. For

forested soils, the partitioning between biomass-C incorporation and respiratory CO2-C WAS

determined to be substrate- rather than soil-dependent. 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 format of the C source supplied is immediately used for respiration

and the remaining for biomass growth (Stouthamer 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 format 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 lowest basal anaerobic respiration per microbial









biomass. The metabolic quotient (qCO2; prOportion of basal respiration per microbial biomass)

has been used in soil studies to indicate ecological efficiency of the soil microbial community

(Anderson and Domsch 1990; Degens 1998b). This index is based on Odum's theory of

ecosystem succession (1969), where during ecosystem succession towards maturity there is a

trend of increasing efficiency in energy utilization concomitant with an increase in diversity.

High qCO2 indicates inefficient use of energy, while low qCO2 indicates high efficiency and

more carbon is utilized for biomass production (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 (eutrophic-hypereutrophic) can be viewed as a

natural succession, higher qCO2 Should be detected in oligotrophic lakes. The trend of decreasing

qCO2 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 study of bacterioplankton

of lakes of different trophic states. These authors concluded that oligotrophy places high

respiratory demands on bacterioplankton, with greater DOC flow to CO2 rather than to biomass.

In Lake Annie sediments addition of propionate inhibited microbial activity (CO2 and CH4

production rates) (Table 7-3). Lake Annie sediments are characterized by high Fe (3640 mg kg- )

(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 Fe- and SO4-2-reducers are present 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









bacteria (Stams 1994; Schink 1997). These conversions, however, are often energetically

unfavorable, and continuous removal of their products by methanogens is required so that these

conversions become exergonic (Stams 1994; Schink 1997; Kleerebezem and Stams 2000).

Presence of Fe- and SO4-2-reducers is thought to limit syntrophic bacteria as these are able to use

products of primary fermentation more efficiently (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, although different syntrophic species are usually involved (Schink

1997; Kleebrebezem and Stams 2000). Addition of butyrate did not inhibit anaerobic respiration;

however, anaerobic CO2 prOduction was not statistically 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 mechanism for inhibition of anaerobic respiration

with propionate addition cannot be determined with the present data. However it can be

speculated that it could have resulted from the absence of species able to use propionate, or H2

was not efficiently removed by methanogens.

Basal CH4 prOduction rates were highest in hypereutrophic Lake Apopka. Several studies

have shown similar results where methane production rates were higher in eutrophic than









oligotrophic lakes (Casper 1992; Rothfuss et al. 1997; Falz et al. 1999; Niisslein 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 delayed in sediments from

sites M17 and KR in Lake Okeechobee (Figures 7-1, 7-2, 7-3, 7-4, 7-5D, E, F). Methanogens

(Archaea) are obligate anaerobes and use a limited number of substrates, including H2 plus CO2,

format, 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 substrate 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 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). The lag phase

observed for CH4 prOduction in all sediments can be explained by two mechanisms. First

methanogenic activity was stimulated in the presence 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.









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










(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









Lake Apopka sediments as high stimulation of CH4 prOduction with addition of Lake-SS was

detected (Table 7-3, Figure 7-8A, B). Lake Apopka has high primary productivity (Carrick et al.

1993) and suspended solids in the water column are mainly composed of phytoplankton biomass

(Phlips et al. 1995c; 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 addition of Lake-SS and statistically higher than

basal productions. This shows how well adapted methanogens in these sediments are to using

algae derived-C. Molecular studies targeting the archaeal community are necessary to elucidate

the maj or pathway for methane production in the present study lakes.

Conclusions

Addition of organic electron donors to sediments stimulated heterotrophic activity. The

extent of the response, however, 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 the sediment microbial community. The addition of some electron donors did not stimulate

heterotrophic microbial respiration, and probably resulted in the incorporation 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.

This was probably caused by a P limitation. Lake Annie showed the highest qCO2, indicating an

inefficient use of energy. The low qCO2 found in Lake Apopka' s sediment indicates high

efficiency. Lake Apopka' s 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 hydrogentrophic methanogenesis in Lake Okeechobee

sediments was determined in another study. The pathway for methane production in Lake










Apopka cannot be determined with the current data. These results showed that the sediments

with different biogeochemical properties had different microbial communities with distinct

catabolic responses to additions of C- sources.










Table 7-1. Sediment biogeochemical properties of Lake Annie, Lake Okeechobee, and Lake
Apopka.
Lake

Variables Annie Okeechobee Apopka
Central M9 M17 KR West


5.9 & 0.01 7.8 & 0.07 7.7 & 0.02 7.6 & 0.2


7.5 10.06


0.052 & 0.002 0.137 & 0.06 0.143 & 0.018


1.183 & 0.29


0.019 & 0.003


LOI (%)

Carbon

TC (g kg )


55.6 & 1.0 37.5 & 0.7 86.6 & 2.0 4.6 & 4.3



272 & 6.2 193 & 2.2 482 & 8.9 25 & 24.0


64.9 & 1.8


288 & 9.2

4029 & 719


Ext-C (mg kg )

MBC (mg kg )


946 1142


279 & 8


894 & 87


76 & 19


12116 & 487


3910 & 157


4081 + 157


666 & 231 42618 & 6423


Nitrogen


TN (g kg )

Ext-NH4-N (mg kg )

Ext-Org. N (mg kg )


20.3 & 0.9 12.6 & 0.3 27.7 & 0.4


1.5 A 1.5

8 &4

17 &1


27.3 A 1.2

386 & 32

859 & 89


226 & 96

147 & 23


48 & 4

83 & 7


27 & 1

141 +14


Phosphorus

TP (mg kg )


1427 & 34 1018 & 48 207 & 12


366 & 78

6.5 & 2

1.2 & 1.5


1185 & 74

1.8 & 0.6

32.3 & 6.5


Lab. Pi (mg kg )

Lab. Po (mg kg )

Ratios

Ext-C:Ext-N

Ext-C: Ext-P

Ext-N:Ext-P


124 & 9

71 +19


99 & 6

8.3 A 1.6


4.8 &2

4.2 & 1.1


BD: bulk density, LOI: loss on ignition, TC: total carbon, Ext-C: extractable organic carbon,
MBC: microbial biomass carbon, TN: total nitrogen, Ext-N: extractable labile nitrogen, Ext-
NH4-N: extractable ammonium, Ext-ON: extractable labile organic nitrogen, TP: total
phosphorus, Lab. Pi: extractable labile phosphorus, Lab. Po: labile inorganic phosphorus, Ext-
Po: labile organic phosphorus, Ext-P: extractable labile phosphorus.









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

ANOVA Annie Okeechobee Apopka

Central M9 M17 KR West

Anaerobic Respiration (mg CO2-C kgl d- )

n 27 27 27 27 27

df8 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-l d- )

n 27 27 27 27 27

df8 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- )

n 27 27 27 27 27

df7 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










Table 7-3. Sediment CO2 and CH4 prOduction, and turnover rates, with the addition of different
carbon sources. Tukey' s test was conducted within sites and different letters indicate
significant statistical differences at p<0.05. (mean & SD).
Lake
Treatment Annie Okeechobee Apopka
Central M9 M17 KR West
Anaerobic Respiration (mg CO2-C kg-l d l)


3 & 2 ac
18 & 4 bc
10 & 4 abc
5 & 5 ac
4 & 3 ac
3 & 3 ac
4 & 3 ac
13 & 3 bc
4 & 2 ac


0.04 & 0.01a
12 &5 b
2 &2 a
5 &9 a
5 &2 a
3 &4 a
0.4 & 0.5 a
13 & 5 b
2 &1 a


0.06 a
0.03 ab
0.02 b
0.02 b
0.01 b
0.01 b
0.05 a
0.01 b


21 & 4 ae
75 A 10 bed
86 & 8 bef
50 & 10 bde
33 & 6 ade
51 & 4 bde
42 & 11 ade
104 & 25 cef
33 & 3 ade

mg CH4-C kg-' d
0. 16 & 0.02a
10 &4 b
11 &3 b
11 1b
8&1 b
0.3 & 0.08 a
0.8 & 0.4 a
0.7 & 0.4 a
0.0 + 0.0 a
Rates (d- )
0.17 ab
0.19 ab
0.12 acd
0.08 ed
0.10 acd
0.08 acd
0.21 b
0.07 ed


217 & 23 a
874 & 109 b
623 & 350 ab
500 & 259 ab
307 & 183 ab
773 & 324 b
629 & 152 ab
559 & 129 ab
418 & 160 ab


80 & 7 a
563 & 20 b
220 & 24 ace
102 & 23 acd
192 & 16 acd
44 & 8 acd
48 & 10 acd
374 & 22 ce
355 A 14 ce


2.8 a
1.67 ab
1.24 b
0.99 b
1.62 ab
1.34 b
1.85 ab
1.81 ab


Basal
Alanine

Arginine
Acetate

Butyrate
Form ate

Propionate
Glucose
Lake-S S


Basal
Alanine

Arginine
Acetate

Butyrate
Form ate

Propionate
Glucose
Lake-S S


Alanine

Arginine
Acetate

Butyrate
Formate

Propionate
Glucose
Lake-S S


100 + 9 a 26 & 1 a
217 & 9 b 62 & 7 bed
212 & 10 b 50 & 6 bc
135 A 16 a 33 A 1 a
87 & 3 ac 30 & 2 a
209 & 22 b 28 & 2 a
55 A 13 c 32 & 1 a
96 & 12 a 66 & 5 bd
101 + 12 a 29 & 1 a
Methanogenesis (1
37 & 8 a 0.09 & 0.0 a
120 & 15 b 45 & 4 be
159 &7 c 19 &3 edf
155 A 15 c 26 & 1 ed
85 & 4 d 41 & 3 b
36 & 15 a 5 & 2 a
3 A 1 e 13 A 1 ce
44 & 8 a 52 & 5 bf
47 & 8 a 8 & 2 ae
Turnover
0.67 ab 0.21 a
0.74 ab 0.14 b
0.57 ac 0.12 b
0.34 d 0.14 b
0.49 c 0.07 c
0.12 e 0.09 c
0.28 d 0.23 a
0.34 d 0.08 c































0 2 4 6 8 10 12 14 16 18 20


4a 000



2r 000






3 000


25 00

S2000

S1500
ooo



3500


690


sooo


4000


3000


2000


1000


0


3500

3000

2500

2000

1500

looo

500

0


6900


sooo


4000


3000


2000


Butyrate C
O Lake
J4 Basal











,li ....
l~


0 2 4 6 8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20


3500

3000


Butyrate F
.O Lake
A Basal







-.-

... ..,,-'":


2500

2000

1500

looo


0 2 4 6 8 10 12 14 16 18 20


0 2 46


8 10 12 14 16 18 20


0 2 4 6 8 10 12 14 16 18 20
Time (days)


Figure 7-1. Microbial activity response to the different carbon source addition in Lake Annie sediments: 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).































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.











2000
-4
1800

~1600

1400

S1200



.b 800

o 600

0y 400


d0


2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0


100 r


2000

1800

1600

1400

1200

1000


13utvrate (
'O Lal e
JNt Basal









,Q-
...- -f
.- -.-$ ~


800~


0 2 4 6 8 10 12 14 16 18 20



Butyrate F
<> Ladae
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.





























0 2 4 6 8 10 12 14 16 18 20


Butyrate F*
O Lake
&A Basal














0 2 4 6 8 10 12 14 16 18 20


S600

soo



o0 400


0p 300


S200


100





200


Butyrate
O Lae
J4 Basal


soo


400


200


100


0 2 4 6 8 10 12 14 16 18 20


A Acetate D
O Alanine
O Arginine














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)
Figure 7-4. Microbial activity response to the different carbon source addition in the sand sediments (site KR) 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





























































0 2 4 6 8 10 12 14


h
r(
oa
re
U

O
U
oa
E
V
E
.P
r
L
a
o

.Y
a

o
E




~3 oa
oSC
P
U

X
U
oa
E
V

o
E
o
oa
o
E
ed
E

E


14000


12000










4000



2000


0


6000 -


5000 -


4000 -


3000 -


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1000


14000


12000


loooo


8900





4000


2000


Butyrate C
O Lake
J Basal













...4-


0 24 6 8


0 2 4


6 8 10 12 14


0 2 4 6 8 10 12 14


10 12 14


6000


5000


4000


3000


2000


1000


A Acetate D
O Alanine
0 Arginine I/ I..









I


/ -


Butyrate F
O Lake
4A Basal













a'


5000


4000


3000


2000


1000


0 2 4 6 8 10 12 14

Time (days)


0 24 6 8


10 12 14


Figure 7-5. Microbial activity response to the different carbon source addition in Lake Apopka sediments: 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).


































o +nY;take Ok~eehobee
0 "5000 10000 15000 20000 25000 30000 35000 40000 45000


U "-* *' *
O 5000 10000 15000 20000 25000 30000 35000 40000 45000

Microbial Biomass Carbon (mg kg l)


Figure 7-6. Relationship between microbial biomass carbon and activity: A) CO2, and B) CH4
production rates, with the different groups of carbon sources added to sediments from
different lakes.


Ih
I








\I


r .1


O Carboxylic Acids
A Amino Acids
O Polysaccharide
O Lake Material
+ Basal










1, A Lake Annie


700 p


Lake Apopka I


400


300


200


C
'~ ~CJ\


450-

400

350


O Carboxylic Acids
A Ami~no Acids
O Polysaccharide
+ Lake Material
+ Basal


' s





SO




I


I
+/


Lake Apopkal


200


\ ae




Lake Okeechobee


:Annie


,'











1.0 ....
A
0.8 C Basal

0.6

0.4

S0.2

SFonnate
0 .0
Acetate Agnn
-0. *Butyrate

-0.4 Lake-SS
Alanine

-0.6~ Pop onate Gluco e

-0.8

-1.0 **
-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

Axis 1 (40.7%)

2.0 ....


1.5 C O
O Lake Annie
O
1.0
O
O Lake Okeechobee M9
0.5 C O
SKR




-0.5
Lake Okeechobee M17

-1.0Lake Okeechole KR
a Lake Apopka
-1.5


-2.0 ****
-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

Axis 1


Figure 7-7. Results of the Principal 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 (orange crosses), and Lake Apopka (green
triangles). CO2 prOduction rates were normalized by microbial biomass carbon.



206








































* ** **I I I


0.5 1.0 1.5 2.0 2.5


Pmopionate

Fonnate
e Glucose *

Butyrate

Acetate Alanine

Lake-SS



Basal


0.8


0.6


0.4


0.2


-0.2


-0.4


-0.6


-0.8


-1.0


-1~ 0


-0.8 -0.6 -0.4 -0.2


0.0 0.2 0.4 0.6 0.8 1.0


Axis 1 (33.6%)


SLake Okeeclibee M17




Lake Okeechobee M9



i................ ...La~e.ke.e.OkaeechabeR.- KR



Lake Apopka






Lake Annie


1.5


1.0


0.5


-0.5


-1.0


-1.5


-2.0 *
-2.5 -2.0 -1.5 -1.0 -0.5 0.0


Axis 1


Figure 7-8. Results of the Principal 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 (orange crosses), and Lake Apopka (green
triangles). The CH4 prOduction rates were normalized by microbial biomass carbon.









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









Niisslein 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 authors concluded that hydrogenotrophic methanogenesis was the

maj or pathway for CH4 prOduction, and acetate was used syntrophically 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, phosphorus, 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. 2007). This procedure is

based on the addition of a commercially prepared 13C-labeled substrate into an environmental

sample. The microorganisms that actively transform this substrate will incorporate 13C into

cellular biomarkers (Radaj ewski et al 2003; Whiteley 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 Loosdrecht (The Netherlands) (Boschker et al. 1998).

Later this technique was extended to the use of DNA (DNA-SIP) (Radaj ewski et al. 2000) and

RNA (RNA-SIP) (Manefield et al. 2002a) as labeled biomarkers.

DNA- and RNA-SIP are based on the principle that if an organism consumes the 13C

labeled substrate, cell components will incorporate 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 accomplished by isopycnic centrifugation. In this









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









different C sources were added to the sediment and microbial activity (anaerobic CO2 and CH4

production) was measured (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 Power Soil isolation kit (Mo Bio

Laboratories, Solana Beach, CA) using 1.0 g of sediment. Extracted RNA was evaluated by

electrophoresis in agarose gel and 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 the 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 sediment:2 ml of medium) was added to anoxic BCYT-R

medium (basal carbonate yeast extract trypticase-peptone containing 0.01 g L-1 trypticase-

peptone) (Touzel and Albagnac 1983; Chauhan and Ogram 2006a) under N2 Stream to prevent

exposure to oxygen and immediately crimped using butyl rubber septa and aluminum. Samples

were reduced with cysteine (2%) to a final redox potential of approximately -1 10 to -200 mV

(Chauhan and Ogram 2006a) and preincubated at 28 oC in the dark for 1 week, prior to acetate

addition. 13C-labeled acetate (both carbon atoms labeled; Isotec, Miamisburg, OH) was added

from anaerobic sterile stock solutions at two final 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.









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









Beckman polyallomer bell-top Quick-Seal centrifuge 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 oC. Gradient solutions were fractionated via displacement by water (0.1% v/v DEPC-

diethylpyrocarbonate 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 ) of water was used (LC-10AS Schimadzu HPLC

pump) (Figure 8-1, 8-2). A total of 15 fractions of 200 Cll each were collected from each

centrifuge tube. The density of each fraction was determined by weighing 10C1l of each fraction.

RNA from each fraction was isolated by precipitation with isopropanol (Whiteley et al. 2007).

Presence of RNA in each fraction was confirmed 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 manufacturer's 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 present) 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 issues. PCR tubes were prepared as follows: 2.0 Cll of nuclease

free water, 1.0 Cll of each primer, 10 Cll of HotStarTaq Master Mix (QIAGEN, Valencia, CA) and

5.0 Cll of E. coli RNA. Conditions of the PCR used were reported by Uz et al. (2003).









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. Whiteley's suggestions and protocols (Whiteley et al. 2007), some modifications were done.

First, two blank density gradients (without RNA) were prepared and centrifuged along with

experimental samples to verify the distribution 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 Cll of nuclease free water, 109 Cll of formamide. Prior to

centrifugation, RNA samples were concentrated by precipitation with isopropanol and

resuspended in 10 Cll of nuclease free water (large volumes of water with RNA can affect the

shape of the gradient Whiteley pers. comm.). Then, 1.0 Cll of E. coli RNA (100 ng) and 9.0 Cll

of sediment sample RNA (or 10 Cll of nuclease-free water for the blank) were added to the

gradient medium. A second modification 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 gradients were centrifuged as described previously with the following

minor modification: an extended period of centrifugation of 42 h. Gradients were fractionated

and a total of 30 fractions of 100 Cll were collected from each tube. In addition, a loading dye

(green) was mixed with the water used for displacement of the gradient to facilitate the

visualization of mixing between 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









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).









Sediments from site KR are characterized by having low microbial biomass and activity (Chapter

7) and were excluded from further experiments.

RNA-SIP Experiment

Both RNA and DNA were observed in triplicate 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. cobi culture was 500 Clg CIl and DNA was

observed in the samples (Figure 8-6).

Isopycnic centrifugation

First centrifugation. Six RNA samples were used in the first ultracentrifugation, and two

samples were lost due to problems during piercing of the Beckman tube. The density of the

gradient did not follow the expected linear distribution (Manefield et al. 2002b, 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 visualized 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 cobi 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 could be an indication of

either primer contamination or RNA and/or primers degradation (Figure 8-9A). New primers

were ordered and RT-PCR was conducted as described previously. Agarose (1%) gel

electrophoresis showed the presence ofE. cobi in all fractions and confirmed that the

ultracentrifugation failed to separate 'heavy' and 'light' RNA (Figure 8-9B).

The presence of DNA in both E. cobi and sediment RNA samples (Figure 8-5A, B, 8-6),

could be affecting the gradient medium and may be responsible for the detection of E cobi (DNA









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 expected linear distribution 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 the '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 linear distribution of the gradient improved considerably and it was

similar to the one reported by Manefield et al. (2002b) and Whiteley et al. (2007) (Figure 8-13,

8-8A, B), indicating that modifications improved the methodology considerably. However,

plateaus could be observed in all density gradient graphs. A slightly longer centrifugation may

solve this problem (Whiteley pers.comm.).

If the new gradient fraction by density successfully separated the 'heavy' and 'light' RNA,

fully labeled RNA was expected to have densities of approximately 1.79-1.81 g ml l, therefore

around fraction numbers 5-7 (Whiteley pers. comm.). RT- PCR 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 (Figure 8-14). RT-PCR products were found in

all fractions when universal bacterial primers were used, indicating the presence of 'light' RNA









from E. cobi 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 obtained. 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. cobi RNA was lost during fractionation

due to problems during piercing of the Beckman tube. The distribution of the gradient density

improved with longer centrifugation; however, plateaus could still be observed in some samples

(Figure 8-15).


RT- PCR was done with the 10 ng and 100 ng E. cobi RNA fractions. RT-PCR products

for 10 ng E. cobi RNA fractions were not found in any fraction, likely due to low RNA

concentration (Figure 8-16A). RT-PCR products for 100 ng E. cobi RNA fractions, however,

were found in all fractions (Figure 8-16B). Although a linear distribution of density gradient

could be observed, still 'light' E. cobi RNA could be found throughout the gradient medium.

These experiments were conducted from February-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 Lake Annie and Lake Apopka were used during the

trial experiments, RNA-SIP experiments were discontinued.

Discussion, Conclusions and Recommendations

RNA-SIP has proven to be a sensitive approach to link microorganism function with

phylogeny (Lueders et al. 2004b; 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. Manefield et al. (2005) used RNA-SIP to identify the









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.









Another important issue that may have contributed to, or might be the main reason for the

failure of this study, was the apparatus for collecting the gradient fractions (Figure 8-1, 8-2).

After ultracentrifugation, tubes were placed in a holder. Then, needles (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 the 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 gradient 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 fractionation 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 extremely difficult to control accurately (Whiteley et

al. 2007). The use of a Beckman Fraction Recovery System, an apparatus developed to

fractionate Quick-Seal tubes, 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 gradient 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 2006a, 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 (2006a) reported

that E. coli DNA was not detected in the denser [13C]DNA fractions, but it was detected in all









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









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.

































Figure 8-1. Picture of the apparatus for fractionating the gradients.















Stained
water
CsTFA
gradient









Figure 8-2. Photograph of gradient fractionation by displacement with stained water (green).











DNA
23S rRNA
16S rRNA














Figure 8-3. Agarose (2%) gel electrophoresis of RNA extracted from the three lakes sediments.





DNA
23S rRNA
16S rRNA











Figure 8-4. Agarose (2%) gel electrophoresis of RNA extracted from sediments of Lake
Okeechobee sites M9 (A) and KR (B).














DNA


DNA


NA
NA


23S rBNA 's. 3Sr
IcS rR1
16S rRNA '~ "_C 1






R Lake Ok~eechlobee


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.


23S rRNA
16S rRNA


Figure 8-6. Agarose (2%) gel electrophoresis of RNA extracted from E. coli culture.
























































~"-r
I



?liibG
A "


1.96-


S1.86
1.84
1.82
1.80
S1.78
1.76
S1.74
S1.72
S1.70
1.68
S1.66
S1.64
S1.62
S1.60
~1.58
1.56
1.54
1.52
1.50
14 16 0


.


*


B



















2 4 6 8 10 12 14 1
Fraction Number


0 2 4 6 8 10 12
Fraction Number


Figure 8-7. Graph illustrating the buoyant density of gradient fractions: (A) Lake Annie (core
#120); (B) Lake Apopka (core # 93).


1.87

~ 185
E
n
E 1.83
e

m
6
~ ,,,


Dislance to Gcadient Base fImnar)


25 30


Fraction number


Figure 8-8. Graph illustrating the buoyant density of gradient fractions: (A) Manefield et al.
(2002b); (B) Whiteley et al. (2007).


1.RI

1.86

j 1~85

LLI~
X
1.1
ir
" 1.78
pi
6
$ 1.3~
P 1.74
e
u 1.31

1.7

1~68
















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.





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.




























































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
*
*




.
















Bacteria Primers




Archaea Primers


Figure 8-14. Agarose gel electrophoresis of RT-PCR of RNA extracted from Lake Apopka
fractions (core # 94). Universal bacteria and Archaea primers were used.




















- A
*.
*
-
*
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


S1.80

~1.78

S1.76

S1.74


-~B





- Plateaus

.



***
-


1.86

1.84

1.82




S1.78

1.76

S1.74


1.84

1.s2

1.80

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





























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.












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).









CHAPTER 9
SUMMARY AND CONCLUSIONS

In lakes, allochthonous and autochthonous particulate matter is deposited and becomes an

integral part of sediments. Accumulation of particulate matter can alter the physico-chemical

properties of sediments and associated biogeochemical processes in the sediment and water

column. Coupling and feedback between sediment biogeochemistry and water column primary

productivity often depends on biogeochemical processes 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 heterotrophic metabolism typically dominates in this

compartment.

The primary goal of this study was to develop 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) compounds as it is the nutrient that in high

concentration is reported to be responsible for eutrophication of freshwater ecosystems.

Eutrophic and hypereutrophic lakes usually receive high external loads of nutrients, display high

primary productivity and nutrient concentrations, consequently sediments from these lakes might

be expected to have higher concentrations of organic matter (OM) and nutrients than oligo-

mesotrophic lakes. To accomplish the main goal of this research, a series of laboratory

experiments were performed with sediments from three subtropical Florida lakes (Lake Annie -

oligo-mesotrophic, Lake Okeechobee eutrophic and Lake Apopka hypereutrophic) with

different trophic states. The specific obj ectives 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.









* 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 sediment profiles and determine relationships
between different P compounds and enzyme activity.

* 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.

* Determine the source and long-term accumulation of OM and explore how they relate to
sediment 813C and 6 "N 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 stated obj ectives 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 importance of P forms present in sediments seemed to be more

important than total P concentration in characterizing and understanding the processes occurring

in the sediment compartment of each of the studied lakes. The oligo-mesotrophic Lake Annie

organic sediments contained P in moderate to highly 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 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 (MBP).









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.









Lake Annie had more stable compounds with greater sediment depth. Dominant forms of

TP were HCl-Pi, fulvic acid P (FAP), and humic 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 maj or 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 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. 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.

Enzyme activities in sediments (Objective 3)

This study showed that phosphomonoesterase (PMEase) and phosphodiesterase (PDEase)

activities were related to sediment microbial biomass and activity, as well as to the different P

composition and availability. Enzyme activity decreased with sediment depth, reflecting lower

microbial biomass and activity. Strong correlations between enzyme activities and anaerobic

respiration indicated that bacterial enzymes dominate these sediments. Different P forms in

sediments were also affecting enzyme activity. Highest PMEase activity was found in the oligo-

mesotrophic lake (Lake Annie) with high concentrations of labile-Po, FAP and HAP. Lake

Okeechobee had high concentrations of labile-Pi and lowest activities of both PMEase and









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









resulted in C and N limitation. Lake Annie sediments seem to be C-limited, with low ratios of

extractable nutrient ratios. Carbon limitation was probably a consequence of C sources (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 biogeochemical properties of

sediments .

Long-term OM accumulation and stable isotope 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 variable activities of 210Pb, and 226Ra.

Lake Apopka deposits were undatable due to possible 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 and the average sedimentation rate (since c. 1900)

was determined to be 36.8 mg cm-2 -1l

Lake Annie sediments were depleted in 613C and 6 "N, probably due to a combination of

several factors such as allochthonous OM input, OM from primary productivity, and microbial

biomass and activity. In mud sediments of Lake Okeechobee, 613C ValUeS were slightly depleted

while 6 5N 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 613C and 6 "N 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 613C and 6 "N at the KR site probably reflects an input of









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










high efficiency. Lake Apopka' s sediment catabolic diversity was higher than in the other

sediments. In relation to methane production, acetoclastic 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 determined with the current data. Molecular

studies targeting the archaeal community are necessary to elucidate the maj or pathway for CH4

production in these lakes sediment. These results showed that the sediments with different

biogeochemical properties had different microbial communities with distinct catabolic responses

to addition of the C sources.

RNA-stable isotope probing of acetate-utilizing microorganisms (Objective 7)

An attempt was made to identity microorganisms 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 sources of error that contributed to the failure

of the proposed study were discussed.

Synthesis

In Figure 9-1, the maj or characteristics of surface sediments (0-15 cm) in the different

studies from the 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 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. Sediments from the oligo-mesotrophic Lake Annie

had the maj or P forms as HAP, FAP and HCl-Pi. These sediments were also characterized by









high PMEase activity and qCO2. Low extractable C:P and N:P ratios resulted from a high

availability of P. Isotope signatures of these sediments revealed low values of 613C and 6 "N.

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-Pi concentration, and HCl-Pi as

the maj or P form. Differences in sediments from this eutrophic lake included low microbial

activity (CO2 and CH4 prOduction rates), and enzyme activities. Metabolic quotient (qCO2) and

813C and 6 "N values were placed between the other lakes values (Figure 9-1).

Hypereutrophic Lake Apopka had high concentrations of microbial biomass P, N and C, as

well as high extractable C:P and N:P ratios, and high microbial activity (CO2 and CH4

production rates). These sediments were also characterized by having high PDEase activity and

high values of 613C and 6 5N. Metabolic quotient (qCO2) and labile-Pi concentrations were low

in this lake (Figure 9-1). Among all variables, 613C and 6 "N values and qCO2 were the ones that

presented a gradient in relation to the trophic state of the lakes. Metabolic quotient was high in

the oligo-mesotrophic lake and decreased with increasing trophic state. Isotopic signatures

increased from the oligo-mesotrophic lake to the hypereutrophic lake (Figure 9-1). Although

sharing some similarities, each lake had distinct 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 the decrease of TP with

sediment depth is not accentuated. The TP mainly consisted of organic bound-P with

consequently high PMEase activity that indicates that P solubility in these sediments is mainly

controlled by biotic processes (Figure 9-2). The production of PMEase is not controlled by P









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









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.,










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.

















































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










































/ Labile-P Lru L 2=VVb ims
\LC:P = 8 CH4 = 43
\LN:P = 4
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 (pLS cm ); SRP: soluble reactive P (pLg L '); NH4 : ammonium (pLg L ');
DOC: dissolved organic carbon (mg L '); 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
( 'P NMR): P-Mono: phosphate monoester, Poly-P: polyphosphate: PMEase: phosphomonoesterase
activity (mg g' dw h'): PDEase: phosphodiesterase activitv(mg g' dw h ), MBC: microbial biomass
carbon(mg kg '), qCO,: metabolic quotient (basal respiration/microbial biomass): CO,: anaerobic
respiration(mg kg dw d '): CH,: methane production rates (mg kg dw d ').


At 1 m
pH= 5.1
EC = 42 Living/Non Living
SRP = 8 Particles
NH4 = 102
DOC =15
Living/Non Living
Anoxia Particles






Sediment +


Lake Annie


All chthonous OM


4 m





16 m


At 20 m
SRP = 12
NH4+ = 708
DOC = 14


Biotic control of P Mineralization Dissolved P

COz
activity 2Enzyme activity Not
DEase = 11 Repressed

Microbial Community
MBC = 4965 *.z
n CO2 = 36 **4,


OMI 58%

~Organic P
FAP/HAP 42
P-Mono 35% /1Enzyme a
PMEase = 97 P




C Limitation
























VUUIIIIUIIC I I


Abiotic control of P Mineralization
pH and/or Eh


Lake Okeechobee Mud Zone (site M9)


Water Column

pH=t I7.8 Living/Non Living Particles
EC = 385
SRP = 93
4m
NH4+ = 130 At 4 m
DOC = 13 SRP = 87


1I


NH4+ 86
DOC = 14


Sediment


9 Dissolved P


OMI 31%

/ Inorganic P
HCI-Pi 66%
Labile-Pi 11%/,
Ortho-P 78% ''***..~

PME;






+ Labile-Pi
J, C:P = 3
\L N:P = 1


Enzyme activity Repressed


~~Enzyme activity JCO2
ase = 4.5 PDEase = 4.7


Microbial Community
MBC = 3653 *.
Limitation CO
C~~4 1


\C-Biomass


qCO2 = 0.0067
CH4 0.1


Carbon


Figure 9-3. Summary of the main biogeochemical properties and processes occurring in the Lake
Okeechobee site M9 water column and sediments. Numbers are mean of 0-15 cm
sediment depth. EC: electrical conductivity (pLS cm ); SRP: soluble reactive P (pLg L '); NH4 :
ammonium (pLg L '): DOC: dissolved organic carbon (mg L '): 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 ( 'P NMR): P-Mono: phosphate monoester, Poly-P: polyphosphate: PMEase:
phosphomonoesterase activity (mg g dw h '); PDEase: phosphodiesterase activitv(mg g dw h ),
MBC: microbial biomass carbon(mg kg '), qC02: metabolic quotient (basal respiration/microbial
biomass): C02: anaerobic respiration(mg kg' dw d '): CH4: methane production rates (mg kg' dw d ').
























OMI 68%


Abiotic control of P Mineralization: pH


Lake Apopka

Water Column
At 1 m
pH= 7.6
EC = 443
SRP = 10
NH4+ = 75
DOC = 25


SLiving/Non Living
Particles


2m


At 2 m
SRP = 8
NH4+ = 50
DOC = 53


Dissolved P


Ortho-P 31%

+ P Diester
MBP 47%
DNA-P 31%
Organic P
FAP/HAP = 26%
P-Mono = 23%


Enzme activity Re dressed


Biotic control of P Mineralization


Enzyme activity CO2 IY 1~~II JI~lU
PMEase = 47 PDEase = 23Acu ltino
r J Pol -P 10%
Microbial Community'
MBC = 33343
1 ~CO2 = 240
P Limitation qCO2 = 0.0057
Carbon CH4 = 117 C-Biomass


JLabile-Pi
/IC:P = 120

Figure 9-4. Summary of the main biogeochemical properties and processes occurring in the Lake
Apopka water column and sediments. Numbers are mean of 0-15 cm sediment depth.
EC: electrical conductivity (pLS cm ); SRP: soluble reactive P (pLg L '); NH4 : ammOnium (pLg L ');
DOC: dissolved organic carbon (mg L '); 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 ( 'P NMR): P-Mono: phosphate monoester, Poly-P: polyphosphate: PMEase:
phosphomonoesterase activity (mg g' dw h '); PDEase: phosphodiesterase activity(mg g' dw h ),
MBC: microbial biomass carbon(mg kg '), qCO,: metabolic quotient (basal respiration/microbial
biomass): CO,: anaerobic respiration(mg kg dw d '): CH4: methane production rates (mg kg dw d ').

















Site


South

Central

North

M17

011

M9

K8

FC

J5

TC

KR

J7

South

Central

West

North


Annie










Okeechobee











Apopka


APPENDIX A
SUPPLEMENTAL TABLES


Table A-1. Water variables from Lake Annie, Lake
taken at 1 m depth).


Okeechobee and Lake


Apopka (samples


Electrical
Conductivity
(pS cm')
45

43

43

465

467

471

512

393

603

362

232

524

366

370

418

382


Dissolved
Oxygen
(mg L^')
6.5

6.7

7.0

5.8

6.4

6.4

6.2

1.2

0.3

7.3

4.9

6.3

8.5

10.6

9.7

10.2


Temperature
o"C)

29.7

29.9

30.1

29.3

30.1

28.7

28.5

30.7

28.2

29.6

29.1

29.3

15.9

16.0

15.8

16.6


Lake










Table A-2. Total, extractable and microbial biomass carbon, nitrogen and phosphorus ratio
weightt basis) measured in sediments frornLake Annie, Lake Okeechobee, and Lake
Apopka.
Microbial
Total Extractable.
Biomass
Lake Site
C:N C:P N:P C:N C:P N:P C:N C:P N:P

South 14 185 13 2 16 8 5 40 8

Annie Central 13 185 14 3 24 6 6 33 6

North 6 230 37 18 40 7 13 51 45

1417 19 1079 58 9 68 3 6 55 9

011 16 160 10 5 9 7 8 33 4

\49 18 159 9 5 6 2 4 128 28

K8 15 147 10 6 8 1 5 35 8

Okeechobee FT 7 19 3 2 6 1 9 23 3

J5 12 123 10 5 35 3 11 76 7

Ill 13 46 1 4 14 6 6 49 8

KR 15 123 1 3 8 3 26 250 17

J7 16 72 1 3 10 3 6 122 25

South 11 275 24 4 118 32 6 23 4

Central 11 247 22 3 110 35 6 28 5
Apopka
West 12 293 25 4 87 25 6 31 5

North 11 218 20 3 151 58 6 23 4










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.










Table A-4. Pearson' s correlation coefficients of biogeochemical properties and microbial biomass and activity significant at p< 0.05.


Microbial Biomass


Anaerobic
Respiration
-0.59
0.71
0.70
0.89
0.81
0.95
0.71
-0.17**
-0.01**
0.67
0.61
0.59
0.46
0.88
0.90
0.90

-0.30
0.83
0.94


Methane Production
Control Acetate* Hydrogen* Acetate + Hydrogen*
-0.50 -0.59 -0.53 -0.69
0.53 0.36** 0.25** 0.52
0.50 0.30** 0.18** 0.49
0.61 0.42 0.25** 0.57
0.55 0.38 0.26** 0.40
0.66 0.67 0.55 0.68
0.63 0.71 0.78 0.77
0.19** 0.80 0.89 0.80
-0.15** 0.62 0.68 0.71
0.70 0.71 0.67 0.66
0.83 0.74 0.76 0.65
0.86 0.68 0.59 0.64
0.79 0.89 0.79
0.38 0.86 0.81 0.78
0.39 0.83 0.78 0.84
0.36 0.84 0.76 0.64


Carbon
-0.49
0.65
0.65
0.89
0.80
0.91
0.57
-0.36
-0.09**
0.60
0.42
0.48
0.57

1.00
0.99

-0.25**
0.87
0.93


Nitrogen
-0.49
0.66
0.66
0.89
0.80
0.92
0.58
-0.36
-0.10**
0.60
0.42
0.48
0.57
1.00


Phosphorus
-0.47
0.64
0.64
0.87
0.78
0.90
0.56
-0.38
-0.12**
0.56
0.41
0.46
0.55
0.99
0.99


BD
LOI
TC
Ext-C
TN
Ext-N
TP
Lab.Pi
IP
Lab .Po
FAP
HAP
Res.P
MBC
MBN
MBP
Ratios
Ext-C:Ext-N
Ext-C:Ext-P
Ext-N:Ext-P


0.99


-0.25**
0.88
0.95


-0.24**
0.88
0.95


-0.27**
0.31
0.40


0.27**
-0.16**
-0.36**


-0.01**
-0.44
-0.63


0.39
-0.01**
-0.33**


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. Data from Lake Okeechobee sediments only. **Not significant at p< 0.05.










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BIOGRAPHICAL SKETCH

Isabela Claret T8rres was born in Belo Horizonte (Minas Gerais State, Brazil). She grew

up surrounded by mountains, water falls, Cerrado, and the remains of Atlantic Forest. She was

always taken by her family to explore natural habitats, and taught to value and protect nature and

animals. Before going to college she lived for one and a half years in Israel where she

experienced the life of two different "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 species, and later she joined the Population Ecology

Laboratory where she conducted research on a spider population in the beautiful mountain fields

of Serra do Cip6 (M.G.). During this study she had a unique opportunity to work with Dr. Jose E.

C. Figueira, her advisor, a great ecologist who taught her a passion for ecology, science, and

statistics. She graduated in 1995 with a B.S. degree in biology (maj or in ecology), and, knowing

that lack of water would be the maj or issue that humanity would face in the near future, she

decided to return to water research. In 1997, she joined the master' s program in ecology,

conservation and management of wildlife, at UFMG, where she studied mass balance of

nutrients of a eutrophic reservoir, and graduated in 1999. In 2002 she joined the graduate

program in soil and water science to pursue her Ph.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 Eustaquio T8rres (in mentoria~n); a "mud" limnologist and benthos ecologist who left

this world too soon and is truly missed by her. After graduating, she wants to become a professor

so she can pass on to the next generation the same passion for nature, science, biology, ecology,

and limnology that were passed to her by some of her professors and her family.





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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

PAGE 45

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 yiel