Effect of phosphorus fertilization on the microbial phosphorus pool in a spodosol under a slash pine plantation

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Effect of phosphorus fertilization on the microbial phosphorus pool in a spodosol under a slash pine plantation
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Thesis (Ph.D.)--University of Florida, 2003.
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Includes bibliographical references.
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by Christine Marie Bliss.
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THE EFFECT OF PHOSPHORUS FERTILIZATION ON THE MICROBIAL
PHOSPHORUS POOL IN A SPODOSOL UNDER A SLASH PINE PLANTATION















By

CHRISTINE MARIE BLISS


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


2003















ACKNOWLEDGMENTS

I would first and foremost like to acknowledge and thank my major advisor, Dr.

Nick Comerford. His support, dedication, patience, and wisdom helped me to improve

both my professional and personal potential. He offered encouragement, advice, and

belief in my abilities that helped me to achieve my goals. I would also like to

acknowledge my committee members, Drs. E.J. Jokela, R.M. Muchovej, K.R. Reddy, and

R.D. Rhue. I greatly appreciate their help and suggestions for improving my research.

I will always be grateful for all the help I received from Mary Mcleod. Her

knowledge, support, and friendship motivated me through frustrating times in the

laboratory. Miranda Lucas was an invaluable asset to my research. I cannot thank her

enough for all of the help and support she provided me. I would also like to thank Nick

Comerford for his help in the laboratory. Adam Glassman and Marinela Capanu very

generously provided me with statistical help.

I would like to thank my family, especially my parents, for the endless

encouragement and support. Lastly, I would like to thank my friends. I cannot thank

them enough for their support, encouragement, and patience. I am most thankful to Dr.

Traci Ness and Jennifer Koski for listening and offering invaluable advice.















TABLE OF CONTENTS
Page

ACKNOW LEDGMENTS................... .............. ......... ..................

LIST OF TABLES ...................... .......................... ......- v

LIST OF FIGURES.............................. ................................... vii

ABSTRA CT .......................... .... .. .................... ......................ix

CHAPTER

1 INTRODUCTION ........................... ..... .... .. ........... 1

2 DETERMINATION OF MICROBIAL PHOSPHORUS Kp FACTORS:
INFLUENCE OF HORIZON AND WATER POTENTIAL................................ 9

Introduction .................................... ....................... 9
M methods ....... ......................... ....................... 12
Study Area and Field Sampling ....... ................ ....... .. ................... 12
Growth and Culture of Microbes .................... ... ................ ...... 12
K, Determination .............. ........... .........................14
Extractants .. ...................... ................... .................................... 15
Evaluation of Water Potential and Soil Characteristics on the Kp Method.... 16
Statistical A analysis ................... ........ ................... 16
R results ............................ ....... .. ...... ......... ............. 17
Soil Characterization................... ............. ................ ... 17
Recovery of P Spike .................................................... ...................... 17
Kp Factors by Extractant ................................................. 17
K, Factors by W ater Potential................................... ........................... 19
Kp Factors by Horizon................................ ................. ............ 20
Comparison of Inorganic P vs. Total P Kp Factors .................................... 20
Evaluation of Water Potential and Soil Characteristics on the Kp Method.... 20
Comparison of K, Factors with the Literature Kp Factor........................... 21
Regression Equations.............................. ..... ... .... .... ............ 21
D discussion ..................................... .. .............. ....................... 2 1
Objective 1: The Most Efficient Extractant............................ ........... 21
Objective 2: Effect of Water Potential on K, Factors................................ 23
Objective 3: Influence of Horizon on Kp Factors..................................... 26
Conclusion ................................. ...... ................... ....................... 28









3 SEQUESTERING OF PHOSPHORUS FERTILIZER IN MICROBIAL BIOMASS
OF A COASTAL PLAIN SPODOSOL ....................................... 45

Introduction ............................................................................ 45
M methods ................................................................................................ 46
Study Area and Field Sam pling ....................................... ....................... 46
Statistical A analysis ................................ ............... ................................ 48
Results and Discussion................................ ........ .................................. 49
Objective 1: Investigate the Influence of P Bioavailabilty on the Microbial
Biom ass ......................................... ...................... 49
Objective 2: Estimate the Immobilization and Retention of P Fertilizer by the
Soil M icrobial Biom ass ............................................ 51
Objective 3: Investigate How Soil Water Potential Affects Microbial Uptake
and P Fertilizer Retention....................... .............. 56
C conclusion ......................................... .................. ..................... 58

4 SUMMARY AND CONCLUSIONS.............................................................. 72

Objective 1: Determine the Superior Extractant for the Determination of Kp Factors
and Estimating Microbial Phosphorus on Acidic Spodosols of the
Coastal Plain.................................. .............................................. 73
Objective 2: Determine of K, Changes with Water Potential and Soil Horizon
Characteristics ........................................... ............................. 73
Objective 3: Determine of Microbial Biomass and Microbial Phosphorus Respond
to Phosphorus Fertilizer and How Those Responses Develop Over
Tim e........................................................ ............................ 74
Objective 4: Determine if Differing Water Potentials Affects Immobilization and
Retention of Phosphorus Fertilizer.................. ...... ............. 75
Research Conclusions......................................................... ............... ...... 75
Future R research ................................................ .... .......... ...................... 78

APENDIX A: K, FACTOR FIGURES............................................ ..................... 79

APPENDIX B: ANALYSIS OF VARIANCE TABLES........................ .............. 97

APPENDIX C: FERTILIZATION STUDY FIGURES....................... ...................... 124

LIST OF REFERENCES .............................................................. ............. 144

BIOGRAPHICAL SKETCH ............................................................. 154















LIST OF TABLES


Table page

2-1. Soil characterization for each horizon ..................... ........... ................... 29

2-2. Percent recovery of P, added as a spike................................... ......................29

2-3. Comparison of inorganic Kp factors by extractant within each water potential and by
water potential within each extractant in the A, E, and Bh horizons. ................... 30

2-4. Comparison of total Kp factors by extractant within each water potential and by
water potential within each extractant for the A and Bh horizons ......................... 32

2-5. Comparison of inorganic Kp factors by horizon within each water potential for each
extractant ....................................................... ..................................... 3 5

2-6. Comparison of total Kp factors by horizon within each water potential for each
extractant ................................................................................. ...................... 37

2-7. Significant differences (P<0.05) between the inorganic P and total P
Kp factors for the A and Bh horizons......................... ................... 38

2-8. Inorganic Kp factors measured in the "test" horizons.............................................. 39

2-9. Comparison of microbial P concentrations with determined inorganic Kp factors from
0.5 MNaHCO3 and aKp factor of 0.40 ..................... ............ ............ ... 40

2-10. Regression equations for inorganic Kp factors for each extractant and horizon...... 41

3-1. Comparison of microbial C by treatment within each day and by day within each
treatment for each water potential ................................................... 59

3-2. Comparison of microbial C between the control, 100 kg P ha-1, and DAP fertilizer
treatments within each day for each water potential......................................62

3-3. Comparison of microbial P by P fertilizer treatment within each day and by day
within each P fertilizer treatment for each water potential....................................... 63

3-4. Percent of P fertilizer immobilized and retained on day 77 by the microbial biomass...........66

3-5. Comparison of microbial P between the control, 100 kg P ha ', and DAP fertilizer
treatments within each day for each water potential .....................................67






vi





3-6. Comparison of microbial C:P ratios...................... ............................................ 68

3-7. Comparison of microbial P by water potential within each day for each treatment
............................................................................................. ....................... ......................... 6 9

3-8. Comparison of microbial C by water potential within each day for each treatment
...................................................... .... ................................... ................................ .............. 7 1

B-1. Analysis of variance table for inorganic Kp factors........................ .....................97

B-2. Analysis of variance table for microbial P from the P-only fertilizer treatments.....101

B-3. Analysis of variance table for microbial P from all fertilizer treatments, including
D A P ..................... ................................................................ ...... .......... 107

B-4. Analysis of variance for microbial C from all P-only fertilizer treatments............... 114

B-5. Analysis of variance table for microbial C from all fertilizer treatments, including
D A P ...................................................................................... ........................... ........... 119















LIST OF FIGURES


Figure page

1-1. Phosphorus cycle ........................................................ ........................................... 8

2-1. Inorganic K, factors by extractant within each water potential in the A horizon...... 31

2-2. Total Kp factors by extractant within each water potential in the A horizon............. 33

2-3. Inorganic Kp factors by water potential within each extractant for the A horizon .... 34

2-4. Inorganic Kp factors for the A, E, and Bh horizon within each water potential using 3
m M oxalate ........................................ ..................................... .................... 36

2-5. Inorganic Kp regression curves for the A, E, and Bh horizon.................................. 42

3-1. Solution inorganic P in the control at each water potential................................... 60

3-2. Microbial C for each day in the -3 kPa soil at each P fertilizer treatment ............... 61

3-3. Microbial P by P fertilizer treatments over time in the -0.1 kPa soil.................... 64

3-4. Microbial P over time for each water potential in the 30 kg P ha-' treatment........... 65

3-5. Microbial P by water potential in the control ......................... ............. .......... 70

A-1. Inorganic Kp factors by extractant within each water potential ............................. 79

A-2. Total K, factors by extractant within each water potential .................................. 81

A-3. Inorganic Kp factors by water potential within each extractant ............................ 82

A-4. Total K, factors by water potential within each extactant ..................................... 84

A-5. Inorganic Kp factors by horizon within each water potential................................ 85

A-6. Regression curves for inorganic K, factors in the A horizon.................................. 88

A-7. Regression curves for inorganic K, factors in the E horizon.............................. .... 91

A-8. Regression curves for inorganic Kp factors in the Bh horizon................................ 94









C-1. Microbial C by day at each P fertilizer treatment ....................................... 124

C-2. Microbial P by P fertilizer treatment for each day................... ................... 128

C-3. Microbial P by water potential for each day .............................. .................... 131

C-4. Microbial P by water potential in each P fertilizer treatment .............................. 133

C-5. Microbial C by water potential for each day......................................... 136

C-6. Microbial C by water potential for each treatment ...................... .................... 138

C-7. Microbial P by day for each P fertilizer treatment............................................ 141














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

EFFECTS OF PHOSPHORUS FERTILIZATION ON THE MICROBIAL
PHOSPHORUS POOL IN A SPODOSOL UNDER A SLASH PINE PLANTATION

By

Christine Marie Bliss

August 2003

Chair: Dr. N.B. Comerford
Major Department: Soil and Water Science Department

The microbial biomass is significant in nutrient cycling and nutrient bioavailability.

The acidic, sandy soils in the southeastern U.S. are phosphorus (P) limited for forest

production and commonly fertilized with P. The surface horizon has no P retention

capacity, resulting in possible leaching of P fertilizer, so the microbial biomass may be a

significant sink and source of labile P for plant uptake in these soils. The ability of the

microbial biomass to immobilize and retain P fertilizer before it is leached below

seedling rooting depth would determine how important it might be as a source of

bioavailable P. Spodosols in the southeastern U.S. have a fluctuating water table that

influences the water potential of the surface horizon. Microbial communities are linked

to water potential and so may influence microbial immobilization of P fertilizer. An

accurate estimate of microbial P is dependent upon a correct Kp factor that may also be

affected by water potential.








The extractant, 0.5 MNaHCO3 (pH 8.5), used to measure microbial P was

questioned for use on these acidic soils. K, factors were measured using six extractants,

0.5 MNaHCO3, Mehlich 1, Bray and Kurtz, and 1 mM, 2 mM, and 3 mMoxalate, at five

water potentials ranging from -0.1 to -1000 kPa in the A, E, and Bh horizons. The

superior extractant for estimating microbial P in this soil was determined to be 3 mM

oxalate. Kp factors were determined to change with soil horizon and soil water potential.

Five treatments, control, 30, 60, and 100 kg P ha', and a DAP treatment, were

added to the A horizon at five water potentials. Microbial P and microbial biomass were

measured over time until 77 days after fertilizer addition. The microbial biomass in this

soil is not P limited but is N limited. After fertilization, up to three or more times the P

than the annual demand by pine plantations was sequestered and held for several weeks.

Water potential did not affect P immobilization. Microbial P would become slowly

available to plants over time, possibly after fertilizer is leached from the rooting zone.














CHAPTER 1
INTRODUCTION



Soil microbial biomass has been defined as the soil's living component (Sparling,

1985) and consists mainly of bacteria and fungi. It holds approximately 2-3 percent of

the total soil organic carbon. The microbial biomass has been shown to be a factor in

nutrient cycling and soil fertility (Cosgrove, 1977; Smith and Paul, 1991) by controlling

mineralization and immobilization. Soil microorganisms are both an important sink and

source of available essential nutrients (Jenkinson and Ladd, 1981; Tate and Salcedo,

1988). The size and turnover of the microbial biomass influence the supply of labile

nutrients to plants (Anderson and Domsch, 1980). Since the microbial biomass relies on

organic matter as an energy source, it has been proposed as an indicator of soil organic

matter change (Anderson and Domsch, 1989; Insam et al., 1989; Wolters and Joergensen,

1991; Sparling, 1992) and soil fertility status (Marumoto, 1984; Hassink et al., 1991).

The phosphorus (P) content of the microbial biomass can be a significant

component of bioavailable P. Microbial P reportedly varies from 6 to 106 jig P g- soil

(Brookes et al., 1984; Diaz-Ravifia et al., 1995; Joergensen et al., 1995). Microbial

biomass uptake ofP may be three to five times greater than plant uptake in semiarid

grasslands (Cole et al., 1977) and the flux of P through the microbial biomass may also

be three to five times greater than the quantity removed by annual harvesting of crops

(Chen and He, 2002).









The P cycle (Figure 1-1) is influenced by biological, chemical, and physical soil

properties. The original source of P in the soil is primary minerals, such as the various

forms of apatite. Through weathering, soluble P is released and made available to plant

or microbial uptake, leaching, or sorbed onto secondary minerals (Smeck, 1985).

Equilibrium is maintained between labile P and soluble P, with rapid exchange between

the pools (Olsen and Khasawneh, 1980). Removal of soluble P is buffered by the labile P

pool, but as both are depleted, these pools are replenished by both primary and secondary

P and mineralization of organic P (Po) (Smeck, 1985).

Secondary minerals, as sources of P, include minerals with P chemisorbed to the

surface, such as iron (Fe) and aluminum (Al) oxides and carbonates, and minerals derived

by crystallization at low temperatures with P as a structural component, such as variscite,

strengite, brushite, monetite, and Al, Fe, and calcium (Ca) phosphates (Smeck, 1985).

Secondary minerals may also physically occlude P when they form. Occluded P may

occur as Fe oxides, hematite, and goethite. Secondary minerals slowly release P into the

soil solution (Olsen and Khasawneh, 1980) and seldom achieve equilibrium with the

labile P pool (Murrmann and Peech, 1969; Olsen and Khasawneh, 1980) due to the

constant flux ofP in the soil (Olsen and Khasawneh, 1980). However, the long-term

tendency of P is to form secondary minerals (Smeck, 1985).

In soils with little or no ability to sorb P, the microbial biomass must be the

controlling factor in P cycling. The biota is decomposed into organic matter with various

forms of Po through microbial synthesis (Smeck, 1985). Microbial synthesis is believed

to be the origin of Po (Cosgrove, 1977; Anderson, 1980). Organic P in soil exists mainly

as ester linkages to inositols with small quantities of nucleic acids and phospholipids









(Stewart and McKercher, 1982). Some Po is transferred into a stable Po pool, which is

slow to hydrolyze. The small quantities of nucleic acids and phh...ph,-.ipid.. jr I,.

hydrolyzed into soluble and labile forms while the inositols tend to accumulate (Halstead

and McKercher, 1975). Transformation of Po occurs through plant uptake of soluble P,

some of which returns to the soil in plant litter with decomposition and accumulation of

Po, and microbial mineralization and return to soluble P pool.

Phosphorus mineralization is controlled by inorganic P (Pi) availability and P

demand by microorganisms and plants (Stewart and Tiessen, 1987; Walbridge, 1991).

When the labile P, pool is high, Po mineralization is repressed causing Po to accumulate

(Spiers and McGill, 1979; Smeck, 1985). Equilibrium occurs between Po and non-

occluded Pi (Williams and Walker, 1969). Unlike nitrogen (N) and carbon (C),

mineralization of Po may occur independently from C and is therefore labeled

biochemical mineralization (McGill and Cole, 1981). Phosphorus mineralization is

catalyzed by the enzyme, phosphohydrolase, which may be produced by plant roots,

mycorrhizae, or soil microbes (Gould et al., 1979; Smeck, 1985). Phosphohydrolase is

repressed when labile P, is high, causing the soil C:P ratio to decrease due to the

accumulation of Po (Smeck, 1985). This occurs when the C:P ratio is generally less than

100, but if the ratio exceeds 200, then phosphohydrolase is induced (Smeck, 1985).

The microbial community structure is affected by several factors, including soil

water potential (Sheilds et al., 1973; Diaz-Ravina et al., 1993). Seasonal community

structure changes have been linked to water potential (Smit et al, 2001) as have both

microbial C and microbial P contents (McLaughlin et al., 1988; Ross, 1987; Srivastava,

1992; Ghoshal and Singh, 1995). In addition of community structure, microbial activity









has historically been associated with soil water potential with changes in activity

decreasing with both excessive and deficient water conditions (Wilson and Griffin, 1975;

Orchard and Cook, 1983; Ross, 1987; Skopp et al., 1990).

Phosphorus fertilization, along with weed control, is a common management

practice in souther pine plantations due to P limiting forest production on Spodosols in

the Coastal Plain of the southeastern United States (Colbert et al., 1990). The sandy

surface horizons have a limited P retention capacity (Humphreys and Pritchett, 1971;

Ballard and Fiskell, 1974; Fox et al., 1990a; Harris et al., 1996; Zhou et al., 1997) which

allows for P mobility. Therefore, if sufficient rainfall events occur, P fertilizer not

absorbed by plants or microorganisms may be leached below the A horizon (Harris et al.,

1996; Nair et al., 1999). Since the surface horizon has a low P retention and the

associated understory vegetation is removed or reduced, the question is whether the

microbial population would be a significant sink for P fertilizer.

Earlier methods for determining microbial P employed C:P ratios to estimate

microbial P from microbial C (Anderson and Domsch, 1980; Chauhan et al., 1981).

Subsequently, microbial P was measured by a fumigation method developed by Hedley

and Stewart (1982) which was adapted from microbial C methodology and revised by

others (Brookes et al., 1982; McLaughlin et al., 1986; Walbridge and Vitousek, 1987;

Walbridge, 1991).

Hedley and Stewart (1982) measured microbial P by incubating air-dried soil at pH

7.4, removing Pi from the soil with an anion-exchange resin after which liquid

chloroform was added and the soil extracted with 0.5 MNaHCO3 (8.5 pH) following

chloroform evaporation. The authors calibrated their method by measuring a Kp factor









(the fraction of P recovered from the microbial biomass) using commonly found soil

bacteria and fungi and reported that the Kp factor was 0.37.

Brookes et al. (1982) tried to improve this method by using fresh soil, pH 5.6 to 7.3

and a correction for sorption of P. The authors used chloroform vapor and extracted the

samples with 0.5 M NaHCO3 (8.5 pH) along with fresh samples that had been incubated

for the same length of time as the samples under chloroform. Kp factors were measured

by spiking soils with several different common soil microorganisms and adding a spike

of P to another set of samples in order to determine the amount of P adsorbed by the soil.

Brookes et al. (1982) concluded the Kp factor to be 0.40, similar to that determined by

Hedley and Stewart (1982).

McLaughlin et al. (1986) used native microorganisms grown from their study soil,

pH 6.0 to 8.5, to derive Kp factors based on microbial assemblages more closely

associated with their study soils. The authors tested several lysing agents and extractants

and determined that using hexanol and 0.5 MNaHCO3 was best for their system. The Kp

factors ranged from 0.33 to 0.57, and like Hedley and Stewart (1982), Brookes et al.

(1982) and McLaughlin et al. (1986) concluded that Kp can differ according to soil type.

Walbridge and Vitousek (1987) developed yet another method for measuring

microbial P on a soil at pH 3.7. The authors used a different extractant, dilute acid-

fluoride (Bray and Kurtz extractant), which is widely used as an index ofP availability in

acid soils. Walbridge (1991) followed this same procedure on soils with a pH ranging

from 3.7 to 3.9. The author used labeled 32P043 to measure Kp factors and the recovery

of the inorganic spike by incubating the soil with the labeled P for 4 days to allow for









microbial immobilization and adsorption. Walbridge's research indicated that K, factors

ranged from 0.37 to 0.46.

Due to the limited P retention of the surface soils in the southeastern U.S. and the

potential for P fertilizer to leach quickly below the range of seedling root depth, the

microbial P pool may be a significant source of labile P in the surface horizon. If the

microbial biomass were able to immobilize P within hours, as was determined in

laboratory studies by Walbridge and Vitousek (1987), then the microbial biomass would

be able to immobilize a portion of the P fertilizer before it is leached. How long the

microbial biomass is able to retain immobilized P fertilizer and whether increasing the

level of P fertilizer also increases the level of microbial P and microbial C in these soils

was questioned. How different water potentials affect the uptake and retention of P

fertilizer is an additional concern due to the water table fluctuating significantly in

Flatwood soils. An accurate estimate of microbial P is dependent on measuring an

appropriate Kp factor. Microbial populations are sensitive to changes in water potential.

Therefore, one might suspect that Kp factors also are a function of water potential. The

use of the extractant, 0.5 MNaHCO3, also needs to be further questioned for acidic soils

as was done by Waldbridge and Vitousek (1987) and Waldbridge (1991).

Given the above discussion, the objectives of this study were to: 1) determine if

0.5 MNaHCO3 (pH 8.5) is a suitable extractant for microbial P on acidic Spodosols of

the Coastal Plain with the initial hypothesis that 0.5 MNaHCO3 is not a suitable

extractant for microbial P on acidic soils in Spodosols; 2) determine ifKp factors change

with water potential and soil horizon characteristics with the hypothesis that Kp factors do

not change with water potential and that Kp is a constant; 3) determine if microbial






7


biomass and microbial P increase with P fertilization, testing the hypothesis that

microbial C and microbial P increase with fertilizer addition; and 4) determine if water

potential affects immobilization and retention of P fertilizer, addressing the hypothesis

that water potential does not affect P fertilizer immobilization and retention.

Chapter 2 describes the testing of the basic standard microbial P extractant against

several acidic extractants. K,p factors were developed for the A, E, and Bh horizons at

five water potentials for each extractant (Objectives 1 and 2). Chapter 3 describes the

response of microbial biomass and microbial P response to P fertilizer over time at

differing water potentials (Objectives 3 and 4). Chapter 4 is a summary and discussion of

this dissertation and also includes relevant topics for future research.















Plants Plant
residues


\ \


Biological
Transformations



Pedologic
Transformations


Occluded P


Figure 1-1. Phosphorus cycle. Dashed arrows indicate microbial mediated processes.














CHAPTER 2
DETERMINATION OF MICROBIAL PHOSPHORUS Kp FACTORS: INFLUENCE
OF WATER POTENTIAL AND SOIL HORIZON

Introduction

Microbial biomass, which is composed of bacteria, fungi, and other microbiota, is

a major controlling component of nutrient transformations and cycling in soils.

Phosphorus (P) transformation and cycling through the microbial biomass have been

recently studied (Seeling and Zasoski, 1993; Gijsman et al., 1997; He et al., 1997b;

Grierson et al., 1998) under a variety of soil conditions. Brookes et al. (1984) estimated

that the P held in the microbial biomass ranges from 5 to 106 u.g P g' soil. Subsequent

studies fall within this estimated range of microbial P (Diaz-Ravifia et al., 1995;

Joergensen et al., 1995).

In the Coastal Plain of the southeastern U.S., P limits forest production on

Spodosols (Colbert et al., 1990). The sandy nature of the surface horizons limits P

retention capacity (Humphreys and Pritchett, 1971; Ballard and Fiskell, 1974; Fox et al.,

1990a; Harris et al., 1996; Zhou et al., 1997), resulting in P mobility. Therefore, P that is

not absorbed by plants or microorganisms may be leached below the A horizon (Harris et

al., 1996; Nair et al., 1999). Due to the low native bioavailability of P in these

Spodosols, P fertilization is commonly applied in combination with weed control during

a pine plantation's rotation. Since the surface horizon has a low P retention and the

associated understory vegetation is removed or reduced, the microbial population could

be a significant sink for P fertilizer.









Microbial P estimates, as defined by Brookes et al. (1982), Hedley and Stewart

( i" i and McLaughlin et al. (1986), are based on extracting soil with 0.5 MNaHCO3 at

pH 8.5. McLaughlin et al. (1986) investigated both basic and acidic soil extracting

solutions and concluded that 0.5 MNaHCO3 at pH 8.5 removed the most P from the

microbial biomass. It is important to note that the pH of the soils they tested was 6.0 and

higher. However, forested soils in the southeastern Coastal Plain are acidic, with the pH

often ranging between 3.8 to 4.5. Inorganic P (P,) chemistry is dominated by aluminum

oxides where present, such as in the spodic and argillic horizons. The appropriateness of

using a high pH extractant under these conditions is therefore questionable for this type

of soil. Other extractants, such as low molecular weight organic acids that promote

ligand exchange and aluminum oxide dissolution (Fox et al., 1990b; Lan et al., 1995), are

known to better remove P from acidic soils and may recover more P when measuring

microbial P.

The fumigation-extraction method underestimates microbial P due to the

incomplete release of P from the microbial cells during fumigation and due to subsequent

adsorption of the released Pi onto the mineral soil surface (Brookes et al., 1982; Hedley

and Stewart, 1982; McLaughlin et al., 1986). A correction factor, Kp, is used to

accurately estimate the total quantity of P held in the microbial biomass (Brookes et al.,

1982; Hedley and Stewart, 1982; McLaughlin et al., 1986). Kp factors may vary with soil

condition, due to either changes in microbial communities or sorption of P (Brookes et

al., 1982; Hedley and Stewart, 1982; McLaughlin et al., 1986). Brookes et al. (1982)

determined that inorganic Kp (Kp,) factors ranged from 0.32 to 0.38 and total Kp (K,,)

factors ranged from 0.44 to 0.49, while Hedley and Stewart (1982) reported a similar









range of 0.32 to 0.47 for Kp, factors. Brookes et al. (1982) and Hedley and Stewart

(1982) concluded that a Kof 0.40 provided a good estimate for soils with a basic pH, but

suggested that K, factors may vary with soil conditions.

Many studies have used 0.40 as the correction factor in microbial P estimation

(Perrott et al., 1990; Tate et al., 1991; Clarholm, 1993; Gijsman et al., 1997; Lukito et al.,

1998). McLaughlin et al. (1986) determined that Kp factors ranged from 0.33 to 0.57 for

a range of soils and suggested that Kp factors must be calibrated for each soil because of

different microbial populations present in each environment. Since microbial

communities also vary with soil water potential (Wilson and Griffin, 1975; Lund and

Goksoyr, 1980; Orchard and Cook, 1983; Skopp et al., 1990), it seems reasonable to

suggest that K, factors may also vary with this soil variable.

This study's overall purpose was to examine the fumigation-extraction method for

measuring microbial P in acidic, forested, sandy soils. There were three specific

objectives. The first objective was to determine which extractant was most efficient at

removing microbial P by comparing the standard basic extractant, 0.5 MNaHCO3 at pH

8.5, against several acidic and oxalate extractants with the hypothesis that 0.5 MNaHCO3

is not the most efficient extractant. Since soil water potential controls microbial

communities and population levels, the second objective was to evaluate whether the K,

factor was influenced by the soil water potential. The null hypothesis was that K, factors

are not affected by water potential. The third objective was to test whether the K, factor

differed by soil horizon within a profile of a representative Flatwoods Spodosol. The

hypothesis for the third objective was that Kp factors do not differ by soil horizon.









Methods

Study Area and Field Sampling

The study area was located 33 km northeast of Gainesville, FL. The site was a 16

ha managed slash pine plantation (Pinus elliottii var. Elliottii Engelm.) that was clearcut

in April 1994, bedded in September and November 1994, and planted with slash pine

seedlings in January 1995. In February 1995, 85 g ha-1 ofimazypyr was applied as

Arsenal in 1.5 m wide bands down the beds for weed control. The plantation is underlain

by a sandy, siliceous, thermic Ultic Alaquod, which is primarily Pomona fine sand and

sand. The average annual temperature is 21 C, and the long-term average annual rainfall

is approximately 1330 mm (Soil Conservation Service, 1995). Table 2-1 provides

chemical and physical characteristics of the A, E, and Bh soil horizons.

Soil was sampled in July 1996 from a subsection of the study area, approximately

2 ha. Approximately 10 kg of soil from each horizon was collected from 20 random

points with a 7.5 cm diameter soil auger. The samples were combined to form one

combined sample for each horizon. The soil was sieved to pass a 2 mm screen and air-

dried.

Growth and Culture of Microbes

Five water potentials were selected to represent a dry soil (-1000 kPa), near field

capacity (-15 kPa and -8 kPa), an unsaturated condition that represents a high water table

(-3 kPa), and near saturation (-0.1 kPa). These water potentials were based on results

from P mineralization studies that evaluated mineralization rate versus soil water

potential in a similar soil (Grierson et al., 1999).

In order to culture native microbial populations representative of the different water

potentials, soil samples from each horizon were incubated at each water potential for a









minimum of 10 days at 28 C. Water potential was kept constant throughout the

incubation period by weighing the samples and adding double deionized water when

required. The soil sample was then diluted with double deionized water to a soil to

solution ratio of 1:10 and 1:1000 for fungal and bacterial growth, respectively. The

native fungal populations were grown by adding 1 ml of the 1:10 dilution to a growth

medium containing 660 mg NaNO3, 330 mg KH2PO4, 265 mg KC1, 165 mg MgSO4

7H20, 6.6 mg FeSO4, 165 mg yeast extract, 10 g sucrose, 100 mg streptomycin sulphate,

and 5 mg tetracycline hydrochloride in one L of double deionized water (McLaughlin et

al., 1986). Native bacterial populations were grown by adding 1 ml of the 1:1000

solution to a 0.3 percent typic soy broth in double deionized water containing 100 mg

cycloheximide L-' (McLaughlin et al., 1986). The microbes were grown in the dark at 28

C for approximately 7 days. The fungal population was filtered from the nutrient

solution, rinsed in double deionized water, blotted dry, and subsampled before adding

back into soil samples for determination of Kp factors. Bacterial populations were grown

at the same temperature and time length as fungal populations. The bacterial suspension

was centrifuged, rinsed with double deionized water, and resuspended in double

deionized water before addition back into soil samples.

Total P (Pt) was measured in the fungal and bacterial populations following a

modified method of Smethurst and Comerford (1993). A known amount of sample

(approximately 300 mg of fungi and 5 mL of bacterial suspension) was dried in a muffle

furnace at 104 C overnight. The samples were then ashed at 500 C for 4 hours. When

cool, 7 mL of 40 percent HCI were added to samples and evaporated to dryness on a hot

plate. Five mL of concentrated HCI were added and again evaporated until dry. Ten mL









of 0.1 N HCI were added to samples and allowed to stand overnight. A known quantity

of the sample in 0.1 N HCI, depending upon the P concentration, was diluted to 25 mL

and analyzed for P using the Murphy and Riley (1962).

Kp Determination

Approximately 80 mg of the fungi and 1 ml of the bacterial suspension were

added to soil samples at the same water potential and horizon from which the

microorganisms were grown. The samples were then fumigated with 2 mL of liquid

chloroform for 24 hours. The chloroform was allowed to evaporate, and the soil was

extracted for P. The Kp factor, or percentage recovery of microbial P, was calculated by

using the equation:

Kp =(m-n)/(xy) (2-1)

from Brookes et al. (1982), where m is the amount of P, measured in the samples that

contained the added microbial biomass, n is the amount of Pi measured in fumigated

samples without additional microbes added, x is the percent recovery of Pi determined

from a P spike, and y is the amount of Pt in the microbial biomass added to the sample.

This equation is the same when measuring Kp factors for microbial Pt, except that m

becomes the amount of P, measured in the samples with added microbes and n becomes

the P, measured in sample without the added microbes. Total P was determined by

digestion of extractant as described above.

In order to determine the percent recovery of Pi for the variable x above, a range of

P, concentrations from 40 to 100 gg P g' soil were added to a set of soil samples in the

Bh horizon. This range was used because of the variable P contained in the added

microbes and to determine if P adsorption was linear. Although P adsorption was









assumed to be negligible in the A and E horizons (Harris et al., 1996; Zhou et al., 1997),

a spike of 40 ig P g' soil was added. This concentration of P was used because added P

in the microbes was generally 40 pg P g-' soil. Phosphorus recovery is presented in

Table 2-2. Inorganic P was used as the spike for both Kp, and K,, on account of most P

released from the microbial cells is P, (Brookes et al., 1982) and sorption of P, and Po was

assumed to be similar.

Extractants

The following solutions were used to extract P from the soil samples: 0.5 M

NaHCO3 at pH 8.5 (Olsen et al., 1954), 0.03N NH4F and 0.25 NHCI (Bray and Kurtz,

1945), 0.05 N HCI and 0.025 N H2SO4, or Mehlich 1, (Nelson etal., 1953), and 1mM, 2

mM, and 3 mMoxalate at pH 3.2, 2.9, and 2.7, respectively. The Bray and Kurtz

extractant has been used in acidic soils as an index of P availability. It removes easily

acid-soluble forms of P from calcium phosphates and a portion of aluminum and iron

phosphates (Bray and Kurtz, 1945). Mehlich 1 was used as another measure of P

availability in acidic soils because of its ability to dissolve aluminum and iron phosphates

(Nelson et al., 1953). Oxalate is a naturally occurring, low molecular weight organic acid

whose presence in the soil solution is due to microbial activity and root exudation.

Oxalate increases P solubility through formation of stable complexes with aluminum

(Martell et al., 1988) in solution, ligand exchange (Stumm, 1986; Fox et al., 1990a), and

dissolution of metal-oxide surfaces (Stumm, 1986).

The NaHCO3 extract was used with a 1:10 soil to solution ratio and shaken for one

hour (Grierson et al., 1998). A 1:7 soil to solution ratio with a 1 minute shaking time and

a 1:4 soil to solution ratio with a 5 minute shaking time was used for the Bray and Kurtz









extract and Mehlich I, respectively (Olsen and Sommers, 1982). A 1:10 soil to solution

ratio and 10 minute shaking time was used with the oxalate solutions. Ten minute

shaking time was determined with initial studies to be the shaking time that extracted the

most P from the soil sample.

After extraction and filtration, Pi was measured using the Murphy and Riley (1962)

method. When required, filtered samples were treated with HCI to lower the pH for

analysis by this method. The NaHCO3 samples had to be refiltered after adding the acid

because acidification caused precipitation of organic compounds. Total P was also

measured for each sample as described previously.

Evaluation of Water Potential and Soil Characteristics on the Kp Method

Bacterial and fungal populations were grown from each horizon at -8 kPa using

the same methods described above. These microbial populations were then added into

soil samples maintained at each of the five different water potentials. Microbes were also

added into soil samples from the other horizons maintained at -8 kPa. For instance,

microbes grown from the A horizon at -8 kPa were added to soil samples from the A

horizon at each water potential and into the E horizon and Bh horizon at -8 kPa. Samples

were then extracted with 3 mM oxalate and analyzed for P,.

Statistical Analysis

Significant differences were determined using SAS, version 8.01 (SAS Institute,

Inc 2001) using a mixed ANOVA model. The least-squared means were analyzed for

significant differences, (P<0.05). Kp factors were analyzed within the main effects

(horizon, extractant, and water potential) and between main effects. Regression

equations for Kp, factors within each water potential, extract, and horizon were developed

using Statistica, 1999 edition (StatSoft Inc., 1999).









Results

Soil Characterization

Soil characteristics varied by horizon (Table 2-1). Organic carbon (C) was

significantly different by horizon, with the most organic C in the Bh and the least in the E

horizon. The greatest concentration of sand was in the E horizon, and the most silt and

clay was in the Bh horizon. Aluminum concentration was highest in the Bh horizon.

Recovery of P Spike

In the Bh horizon, more P was recovered with the 2 mM oxalate, 3 mM oxalate, and

Mehlich 1 extractants (Table 2-1). Between 80 to 89, 82 to 86, and 79 to 90 percent were

recovered in 2 mM oxalate, 3 mMoxalate, and Mehlich 1, respectively. Less P was

recovered with 1 mM oxalate and even less with NaHCO3. Only approximately 50

percent of the P spike were recovered when NaHCO3 was used. In the A and E horizons,

3 mM oxalate recovered all of the P spike while NaHCO3 recovered 94 and 90 percent in

the A and E horizon, respectively. The Bray and Kurtz extractant and Mehlich 1

recovered 96 and 95 and 96 and 94 percent in the A and E horizons, respectively.

K, Factors by Extractant

Inorganic Kp factors ranged from 0.14 to 0.72 in the A horizon, 0.19 to 0.99 in the

E horizon, and 0.11 to 0.45 in the Bh horizon (Table 2-3; Appendix A, Figure A-1). The

K,, factors for each extractant within each water potential were compared by horizon.

The A horizon provides an example of the differences in extraction efficiency (Figure 2-

1). The low concentrations of oxalate provided similar K,, factors to the standard

extractant, 0.5 MNaHCO3 (pH 8.5), in all three horizons. However, within the different

concentrations of oxalate, 3 mM oxalate had a tendency to be more efficient in removing

Pi that was attributable to the microbial biomass. The Bray and Kurtz extractant









removed less P, from the microbial biomass, but it was not always significantly lower

than the oxalate concentrations or 0.5 MNaHCO3. Mehlich 1 consistently extracted less

P than all other extractants. It extracted significantly less P, than both oxalate and 0.5 M

NaHCO3 but was not always significantly lower than Bray and Kurtz.

In the E horizon, the solutions extracted similarly to the A horizon. The oxalate

concentrations extracted similar quantities of P from the microbial biomass as 0.5 M

NaHCO3. Less P, was extracted when using Bray and Kurtz, but it was not always

significantly less than the oxalate concentrations or 0.5 MNaHCO3. Mehlich 1 again

consistently extracted the least quantity of P,. It was significantly lower than both oxalate

and 0.5 MNaHCO3 but not always significantly different from Bray and Kurtz.

Within the Bh horizon, the solutions did not extract P similarly as in the A and E

horizons. Sodium bicarbonate was similar to 2 mM and 3 mM oxalate, but the lowest

concentration of oxalate was clearly less able to extract P. The extraction efficiency of 3

mMoxalate was clearly shown in the Bh horizon, in which the different oxalate

concentrations displayed increasing P extraction with increasing concentration. The

Mehlich 1 solution extracted the least P, from the microbial biomass, but it was not

significantly lower than the other extractants in all cases.

Total Kp factors ranged from 0.22 to 0.97 in the A horizon and 0.10 to 0.89 in the

Bh horizon (Table 2-4; Appendix A, Figure A-2). There was not sufficient sample to

evaluate K,, for the E horizon. As with the Kp,, the K,, factors showed similar trends. In

the A horizon, the K,, factors for the three oxalate concentrations were mostly similar to

0.5 MNaHCO3, with Bray and Kurtz and Mehlich 1 extracting less Pt from the microbial









biomass (Figure 2-2). The Bh horizon provided similar trends. However, 2 mMoxalate

was able to remove more P, from the added microbes overall.

K, Factors by Water Potential

When comparing K,, factors by water potential within extractants (Table 2-3;

Appendix A, Figure A-3), the most Pi was extracted from the microbial biomass when the

soil was near saturation (-0.1 kPa). The A horizon provides an example of the

differences in P extracted from the microbial biomass between the different water

potentials within each extractant (Figure 2-3). Soil at saturation had the significantly

highest K,, factor or was similar to the highest K,, factor for the majority of comparisons.

In the A and E horizons, soil with the water potential closest to field capacity (-8 kPa)

yielded the least quantity of P from the microbial biomass. The K,, factors had a trend of

decreasing as the soil became drier but then increasing after -8 kPa with drier soil in both

the A and E horizons. In the Bh horizon, the lowest K,, occurred in soil at -3 kPa, after

which the K,, increased.

When comparing the amount of P, extracted (Table 2-4; Appendix A, Figure A-4),

the Kp,, factors followed the same trend as the K,, factors with more P extracted in soil at

saturated and dry conditions and least when the soil was at an optimum water potential,

near field capacity. Again, more P was extracted from the microorganisms in the

saturated soil with a decrease in extractability with decreasing water potential.

Extractable P increased again in the drier soils. In the A horizon, the least P was

extracted from the microbial biomass in soil at -8 kPa whereas in the Bh horizon, the

least P was extracted from microbes at -3 kPa.









K, Factors by Soil Horizon

When comparing the K,, factors by horizon, the highest K,, factors tended to be in

the E horizon (Table 2-5; Appendix A, Figure A-5). The A horizon also provided high

Kp, factors, while the Bh horizon usually had the lowest values. An example is provided

comparing the horizons using 3 mM oxalate (Figure 2-4). When comparing horizons

within each extractant, the E horizon provided the highest Kpi factor for all extractants

except for the Mehlich 1 extract; however the value was not always statistically higher.

The K,, factors were significantly higher for the A horizon (Table 2-6). This occurred for

all extractants except Mehlich 1, in which no significant differences were obtained.

Comparison of Inorganic P vs. Total P Kp factors

Significant differences were detected between the inorganic P and total P Kp

factors (Table 2-7). Approximately one half of the K,, factors were significantly different

from the K,, factors. Sodium bicarbonate and 2 mM and 3 mM oxalate concentrations

were significantly different the majority of the time. Bray and Kurtz and Mehlich 1 had

the least differences. When comparing the differences between water potentials, -15 kPa

in the A horizon, and -1000 kPa in the Bh horizon had the most significant differences,

but there did not seem to be a trend across the water potentials that was the same for each

horizon.

Evaluation of Water Potential and Soil Characteristics on the Kp Method

When comparing the Kp, factors measured within the original horizon, there was

no significant difference when microbes from one water potential were added to other

water potentials (Table 2-8). This result was consistent in all three horizons. But when

comparing the Kpi factors measured when the microbes were added to different horizons,

significant differences were determined. When testing microbes from the A horizon in









the E horizon and microbes from the E horizon into the A horizon, there were no

significant differences. Significant differences were detected when A and E microbes

were added into the Bh horizon and when Bh microbes were added into the E horizon.

Comparison of Kp Factors with the Literature Kp Factor

Since the K,, factors measured were determined to be significantly different by soil

water potential and horizon, the differences in microbial P were calculated using the

commonly used literature value of 0.40 (Brookes et al., 1982; Hedley and Stewart, 1982)

and the current study's values (Table 2-9). Assuming a Kp factor from the literature

resulted in estimates of microbial P that were 0 to 160% different from the estimates

using Kp values from this study.

Regression Equations

Regression equations were developed for the Kp, factors for each extractant and

horizon (Table 2-10; Appendix A, Figure 2-6). The R2 values ranged from 0.02 to 0.95.

One regression equation was applicable to 0.5 MNaHCO3, but with oxalate, there were

two distinct curves, one for the -0.1 to -8 kPa soils, or the "wet to moist" soil, and

another for the -8 to -1000 kPa soils, or the "moist to dry" soil (Figure 2-5). This

occurred in all horizons except the Bh horizon with 3 mM oxalate. The Bray and Kurtz

and Mehlich 1 extractants varied with horizon. Many of the equations in the wet to moist

soils had less variability than those for the moist to dry soil. Regression equations for the

Kp, factors were not developed due to the lack of data.

Discussion

Objective 1: The Most Efficient Extractant

There are two main factors that define an efficient extractant for removing P from

the microbial biomass. The extractant should consistently recover the highest percentage









of P from the microbial biomass and should not produce interference with colorimetric P

measurement. In Spodosols, sorption is not a factor in either the A or E horizons due to

the acidic, sandy nature of these soils. Therefore, in these surface soils, extraction should

only involve P released from the microbial biomass. The Bh horizon has the ability to

sorb released microbial P. Due to this sorption capacity, the extractant must have the

ability to re-extract this sorbed microbial P for measurement. Interference with

colorimetric procedures caused increased labor by increasing procedure steps and

diminishing accuracy and precision.

When comparing the ability and suitability of extracts for removing P, from the

microbial biomass, oxalate performed more consistently than the other extractants,

including NaHCO3. In Spodosols of the southeastern U.S., the Bh horizon is dominated

by amorphous Al-oxides (Ballard and Fiskell, 1974; Lee et al., 1988). Oxalate has an

effect on both P and Al release from the Bh horizon because of its ability to replace P

through ligand exchange and dissolution of Al-oxide surfaces (Fox and Comerford,

1992). The A horizon in this soil has little Al (Fox and Comerford, 1992) beyond what is

found on the meager cation exchange capacity, and the Pi in this horizon was nearly all

water soluble (Fox et al., 1990a). Under these conditions, oxalate and NaHCO3 would

extract similar amounts of P,. As shown in the recovery of the P spike in the A and E

horizon, a high percentage was recovered with all extractants, but 3 mM oxalate

recovered all of the spike. In acidic soils, HC03 ions replace P absorbed on the soil

surface (Olsen et al., 1954), but the high pH of NaHCO3 also dissolves some organic

compounds. The dissolution of organic matter results in precipitates when the sample is









acidified for the measurement of P, by the method of Murphy and Riley (1962).

Therefore extra filtration is required.

The Bray and Kurtz extractant was developed to remove acid-soluble forms of P,

mostly calcium phosphates, and some Al and Fe phosphates (Bray and Kurtz, 1945).

While it is a commonly used extractant for P on acidic soils, it did not perform well with

extraction of microbial P. Mehlich 1 uses the same concept of the Bray and Kurtz

extract, with the release of P from Fe phosphates due to the action of acids (Nelson et al.,

1953). Although Mehlich 1 recovered a high percentage of the P spike in the Bh

horizon, it did not perform well in extraction of P from the microbial cells.

For this type of soil, due to the better extraction of P from the microbial biomass

and the ease of analysis, oxalate is recommended. Of the oxalate concentrations tested, 3

mM oxalate was chosen due to its ability to recover more Pi and the ease of use when

compared with NaHCO3. As with Kp,, 2 mM oxalate and 3 mM oxalate were not

significantly different the majority of the time with respect to extraction of P, from the

microbial biomass. Two mM oxalate was determined to be the superior extractant, but

this may be due to missing values, especially in the Bh horizon, from 3 mM oxalate.

Objective 2: Effect of Water Potential on Kp Factors

The Kp equation, equation 2-1, cancels background P associated with the soil and

with the native microorganisms. Existing microbial P in the soil samples is subtracted

from the Kp equation through (m n). Both m and n are fumigated, which eliminates

existing microbial P and also native soil P, providing the quantity of P that is extracted

from the added microbial fungal and bacterial populations. Variable x, the percentage of

P recovery, corrects for any P that may be sorbed by the soil. Therefore, the Kp equation









calculates the fraction of P that is extracted from a microbial population with a known P

concentration.

Soil moisture content is a critical factor affecting microbial activity. Microbial

populations have been shown to increase activity with increasing water potential (Wilson

and Griffin, 1975; Orchard and Cook, 1983). Changes in fungal and bacterial ratios with

water content (Anderson and Domsch, 1975; Wilson and Griffin, 1975; Faegri et al.,

1977) suggest that dominant microbial assemblages change with water contents. Diaz-

Ravifa et al. (1995) determined that differences in nutrient concentrations in the

microbial biomass are significantly related to soil characteristics, including soil moisture.

Soil moisture content has also been shown to affect microbial community diversity

(Brockman et al., 1992). Furthermore, Sparling and West (1989) have already suggested

that the extractability of P from the microbial biomass is a function of soil water content.

When comparing our NaHCO3 study results with other studies (Brookes et al.,

1982; McLaughlin et al., 1986), Kp, factors for the A horizon were consistent with the

published range (0.36 to 0.42) when the soil was -3 kPa or drier. When the soil was near

saturation, Kp, increased to 0.61. This is in contrast to studies that determined the

microbial Kc factor (the fraction of microbial C that decomposes into C02-C in 10 days)

decreased with increasing water content (Ross, 1987; Wardle and Parkinson, 1990).

Wardle and Parkinson (1990) also suggested that water content significantly influenced

K, factors.

Since the methodology used cancels all background factors, there are only two

other factors affecting Kp. Those factors are microbial community changes and water

potential influence on P extraction from the microbial biomass. In order to test the effect









of water potential on the extraction of P, microbes grown at a specific water potential

were added to the same soil at different water potentials. The only factor changing was

water potential because the added microbial population was the same in each sample.

Therefore, the extraction procedure was not being affected by water potential. This

provides evidence that the extraction of P from the microbial biomass is not affected by

water potential as suggested by Sparling and West (1989). Therefore, the only other

factor changing was the microbial community.

Microbial communities have been documented to change with season (Smit et al.,

2001), temperature (Dalias et al., 2001), pH and substrate (Yan et al., 2000), and water

potential (Nazih et al., 2001; Marschner et al., 2002; Treves et al., 2003). Since water

potential has been shown to affect the diversity of microbial communities, it seems

reasonable to assume that this diversity may be influencing the Kp factors measured at

different water potentials.

Brookes et al. (1982) and Hedley and Stewart (1982) measured the Kp factor of

individual microbial species and determined that different species of microorganisms had

different Kp factors. Several studies have determined that Kc factors are also dependent

upon the diversity of organism assemblages (Jenkinson, 1976; Anderson and Domsch,

1978; Nicolardot et al., 1984). Thus, it seems reasonable to conclude that K, factors in

this study differed with water potential because of changes in microbial communities

caused by the change in water potential.

Although regression equations were created, use of these equations to predict Kp

factors should be restricted to soil very similar to this study. As discussed, differences in

soil properties such as organic C cause changes in microbial diversity, which in turn may









cause differences in K, factors. The percentage of variability in these equations indicated

the equations fit the data for many cases. However, with some equations, the predicted

values did not fit observed data, especially in the drier soils. For example, in the E

horizon, the predicted values provided Kp, factors well above 1, which is not possible.

This is most likely due to the design of the experiment that was set up for contrasting

different water potential, not for regressing Kp against water potential. The large gap

between -15 kPa and -1000 kPa does not provide a well-balanced regression curve.

Objective 3: Influence of Soil Horizon on Kp Factors

Microbial population diversity should also occur in different horizons within a soil

profile. Nutrient concentrations, water regime, quantity of and type of organic carbon as

a food source, soil texture, and possibly pH change with horizon. Both organic C and the

water regime in the three soil horizons are considerably different. Organic C, which was

discussed previously, is significantly different by horizon. Organic C is leached from the

A horizon, through the E horizon, and collects in the Bh horizon. Organic C provides

needed energy for microorganisms while also creating community diversity.

Permeability is rapid in the surface horizons (A and E horizons), but the subsurface

horizons have low permeability. In an average year, the shallow water table may be less

than 25 cm from the soil surface for one to three months, between 25 and 100 cm for six

months, and exceeding 100 cm during the dry season (Soil Conservation Service, 1985).

Soil water potential was found to be different between the A horizon and the lower

horizons with the lower horizons being similar when the water table was approximately

96 cm below the soil surface (Phillips, 1987). As mentioned previously, microbial

communities are affected by water potential (Diaz-Ravifia et al., 1995). Because the









horizons have different water regimes, it seems reasonable that microbial diversity may

also change with horizon due to the difference in water potential.

Microbial activity (Bauhus et al., 1998) and microbial community composition

(Sessitsch et al., 2001) also differ with soil texture. When comparing this study's A

horizon K,, factors using NaHCO3 with the K,, factors in similar textured soils by

Brookes et al. (1982) and McLaughlin et al. (1986), the K,, factors were similar; 0.39

(this study at -8 kPa), 0.32 (Brookes et al., 1982), and 0.33 (McLaughlin et al., 1986).

McLaughlin et al. (1986) measured Kp factors on three sites with different soil textures,

finding that K, differed by site. These data support the premise of McLaughlin et al.

(1986) by providing additional examples that soil texture does play a role in microbial

diversity.

Changes in the soil pH cause changes in the microbial community structure

(Frostegard et al., 1993; Degens et al., 2001; Yan et al., 2000) that affect microbial Kc

factors (Vance et al., 1987). Consequently, pH should also affect Kp factors since both

are determined by soil microorganisms. Another factor affecting microbial diversity

may be the diversity of plant roots. Differences in the compounds exuded by plant roots

result in microbial community diversity (Grayston and Campbell, 1996).

As discussed previously, microbial diversity has been linked to the value of

correction factors used in calculating microbial C. This study has shown that soil

physical and chemical properties influence the value of microbial Kp factors, which in

turn appear linked to changes in the microbial community. For example, the P removed

by from the microbial population by extractants was more easily removed from the E









horizon than the A and Bh horizons. Based on the above discussion, this suggests that

the microbial community in the E horizon more easily releases P following fumigation.

This study has clearly shown that K, factors are a function of soil water potential

and soil characteristics and should not be generalized for different soils. Since Kp is

clearly influenced by soil characteristics, using literature values for a K, factor will cause

a significant error, as much as 62, 145, and 48 percent in the A, E, and Bh horizons,

respectively, when using NaHCO3 as an extractant, in microbial P estimates.

Conclusion

A revised extraction method was more useful for measuring microbial P in acidic

sandy horizons with spodic horizons. For the soil used in these studies, oxalate was

determined to extract more or similar amounts of P from the microbial biomass as the

standard 0.5 MNaHCO3 Using 0.5 MNaHCO3 is still appropriate but difficult to apply

to spodic horizons due to the analytical interference. Therefore, it was concluded that

oxalate was a more efficient extractant for Spodosols. Kp factors were determined to be a

function of water potential and other soil characteristics due to the changes in microbial

diversity. It was concluded that a standard Kp for many soil conditions is not appropriate

and may cause significant errors of microbial P. A Kp factor should be assessed for each

soil condition to get the best estimate of microbial P.









Table 2-1. Soil characterization for each horizon
--. -----%- ...------ -------------------cmolc/kg--------------
Horizon pH Sand Silt Clay Organic C Al Fe Ca K Mg Na
A 4.1 94 5 1 0.69 (b)a 0.15 0.01 0.14 0.33 0.27 0.01
E 4.0 96 3 1 0.16(c) 0.00 0.01 0.04 0.12 0.16 0.01
Bh 4.3 90 8 2 1.23 (a) 2.24 0.02 0.04 0.17 0.28 0.03
aSignificant differences (P<0.05) between horizons within organic C




Table 2-2. Percent recovery of P, added as a spike to soil samples for each extractant
------------------% recovery of P added-------------
8.5 M 1 mM 2mM 3 mM Bray &
Horizon NaHCO3 Oxalate Oxalate Oxalate Kurtz Mehlich 1
Bh 40 pg g' soil 50 63 82 85 NDa 79
Bh 60 Lg g- soil 52 61 84 82 ND 83
Bh 80 .g g-' soil 52 72 89 82 ND 82
Bh 100 pg g-' soil 48 75 80 86 ND 90
Bh average 50 68 84 84 ND 83
A 40 plg g-' soil 94 95 97 100 96 96
E 40 pg g' soil 90 96 97 100 95 94
aND data not determined









Table 2-3. Comparison of inorganic Kp factors by extractant within each water potential
and by water potential within each extractant in the A, E, and Bh horizons
------------------------------Water Potential----------------------
-0.1 kPa -3 kPa -8 kPa -15 kPa -1000 kPa
Extractant A horizon
0.5 MNaHCO3 0.65 (Aaab) 0.42 (BCb) 0.35 (Ab) 0.45 (Ab) 0.38 (Bb)
1 mMoxalate 0.72 (Aa) 0.53 (ABb) 0.30 (ABc) 0.41 (Abc) 0.51 (ABb)
2 mM oxalate 0.71 (Aa) 0.47 (Bbc) 0.28 (ABc) 0.48 (Ab) 0.60 (Aab)
3 mMoxalate 0.67 (Aa) 0.65 (Aa) 0.31 (ABc) 0.49 (Ab) 0.56 (Aab)
Bray & Kurtz 0.39 (Bb) 0.29 (CDc) 0.34 (Abc) 0.51 (Aa) 0.28 (BCc)
Mehlich 1 0.22 (Ca) 0.21 (Da) 0.19 (Bab) 0.18 (Bb) 0.14 (Cb)

E horizon
0.5 MNaHCO3 0.99 (Aa) 0.75 (Bb) 0.60 (Ac) 0.73 (Abc) 0.73 (ABbc)
1 mMoxalate 0.66 (Bb) 0.95 (Aa) 0.53 (Ab) 0.70 (Aab) 0.71 (ABab)
2 mMoxalate 0.85 (ABab) 0.90 (Aa) 0.58 (Ac) 0.68 (Abc) 0.86 (Aab)
3 mMoxalate 0.74 (Bab) 0.91 (Aa) 0.48 (Ac) 0.64 (Abc) 0.83 (Aa)
Bray & Kurtz 0.79 (Ba) 0.55 (Cb) 0.30 (Bc) 0.42 (Bbc) 0.59 (Bb)
Mehlich 1 0.43 (Ca) 0.35 (Dab) 0.19 (Bc) 0.28 (Bbc) 0.28 (Cbc)

Bh Horizon
0.5 MNaHCO3 0.36 (ABb) 0.27 (Ab) 0.33 (Ab) NA' 0.72 (Aa)
1 mMoxalate 0.15 (Ca) 0.13 (Ba) 0.17 (Ba) NA 0.13 (Ca)
2 mMoxalate 0.25 (BCa) 0.17 (Bc) 0.23 (ABa) NA 0.18 (BCbc)
3 mMoxalate 0.45 (Aa) 0.28 (Ab) 0.30 (Aab) NA 0.22 (Bb)
Bray & Kurtz NDd ND ND ND ND
Mehlich 1 0.16 (Cab) 0.11 (Bb) 0.14 (Bb) NA 0.21 (BCa)
aSignificant differences (P<0.05) between extractants within each water potential and
horizon (upper case letters down columns)
bSignificant differences (P<0.05) between water potentials within each extractant and
horizon (lower case letters across rows)
'NA missing data
dND data not determined




















S0.5 MNaHCO3
1.0 [ 1 mMoxalate

S2 mMoxalate
0.8 3 mMoxalate

S0.6- Bray & Kurtz
S l i Mehlich I
0.4




0.0
-0.1 -3 -8 -15 -1000
Water potential (kPa)


Figure 2-1. Inorganic Kp factors by extractant within each water potential in the A
horizon









Table 2-4. Comparison of total K, factors by extractant within each water potential and
by water potential within each extractant for the A and Bh horizons. K,
factors were not determined for the E horizon.

--------- ---------Water Potential---- ---------
-0.1 kPa -3kPa -8 kPa -15 kPa -1000 kPa
Extractant A horizon
0.5MNaHCO3 0.91 (Aaab) 0.74 (Aab) 0.63 (Abc) 0.53 (Bc) NAc
1 mMoxalate NA 0.59 (Ba) 0.38 (Bbc) NA 0.51 (Bab)
2 mM oxalate 0.97 (Aa) 0.76 (Aab) 0.44 (Bb) 0.60 (Bb) 0.82 (Aab)
3 mMoxalate 0.74 (Ba) 0.77 (Aa) 0.43 (Bc) 0.59 (Bb) NA
Bray & Kurtz 0.41 (Cb) 0.34 (Cb) 0.38 (Bb) 0.86 (Aa) 0.44 (BCb)
Mehlich 1 0.22 (Da) 0.23 (Ca) 0.22 (Ca) 0.29 (Ca) 0.26 (Ca)

Bh horizon
0.5 MNaHCO3 0.43 (Ab) 0.55 (Ab) 0.40 (Ab) NA 0.78 (Ba)
1 mMoxalate 0.23 (Ba) 0.17 (Ca) 0.23 (Ba) NA 0.21 (Ca)
2 mMoxalate 0.55 (Ab) 0.33 (Bc) 0.43 (Abc) NA 0.89 (Aa)
3 mM oxalate 0.46 (Aa) NA NA NA 0.39 (Cab)
Mehlich I NA 0.16 (Cb) 0.17 (Bb) NA 0.33 (Ca)
aSignificant differences (P<0.05) between extractants within each water potential and
horizon (upper case letters down columns)
bSignificant differences (P<0.05) between water potentials within each extractant and
horizon (lower case letters across rows)
cNA missing data




















S0.5 MNaHCO3
1.2 H 1 mMoxalate
2 mMoxalate
1.0
13 mM oxalate
0.8 T Bray & Kurtz

I 0.6 Mehlich 1

0.4

0.2

0.0
-0.1 -3 -8 -15 -1000
Water Potential (kPa)


Figure 2-2. Total Kp factors by extractant within each water potential in the A horizon























-0.1 kPa

0.8 -3 kPa
-. -8 kPa

0.6 -15 kPa


0.4


0.2
0I -1000 kPa








NaHCO3 Oxl Ox2 Ox3 BK Mehlich I















Figure 2-3. Inorganic Kp factors by water potential within each extractant for the A
horizon. NaHCO3 = 0.5 MNaHCO3, Ox1 = 1 mMoxalate, 0x2 = 2 mM
oxalate, 0x3 = 3 mM oxalate, and BK = Bray and Kurtz










Table 2-5. Comparison of inorganic K, factors by horizon within each water potential for
each extractant

---------------Water potential -----------
-0.1 kPa -3kPa -8 kPa -15 kPa -1000 kPa
Horizon 0.5 MNaHCO3
A 0.65 (ba) 0.42 (b) 0.35 (b) 0.45 (b) 0.38 (b)
E 0.99 (a) 0.75 (a) 0.60 (a) 0.73 (a) 0.73 (a)
Bh 0.36 (c) 0.27 (c) 0.33 (b) NAb 0.72 (a)

1 mM oxalate
A 0.72 (a) 0.53 (b) 0.30(b) 0.41 (b) 0.51 (a)
E 0.66 (a) 0.95 (a) 0.53 (a) 0.70 (a) 0.71 (a)
Bh 0.15 (b) 0.13 (c) 0.17 (b) NA 0.13 (b)

2 mM oxalate
A 0.71 (a) 0.47 (b) 0.28 (b) 0.48 (b) 0.60 (b)
E 0.85 (a) 0.90 (a) 0.58 (a) 0.68 (a) 0.86 (a)
Bh 0.25 (b) 0.17 (c) 0.23 (b) NA 0.18 (c)

3 mM oxalate
A 0.67 (a) 0.65 (b) 0.31 (b) 0.49 (a) 0.56(b)
E 0.74 (a) 0.91 (a) 0.48 (a) 0.64 (a) 0.83 (a)
Bh 0.45 (b) 0.28 (c) 0.30 (b) NA 0.22 (c)

Bray and Kurtz
A 0.39 (b) 0.29 (b) 0.34 (a) 0.51 (a) 0.28 (b)
E 0.79 (a) 0.55 (a) 0.30 (b) 0.42 (b) 0.59 (a)
Bh NDC ND ND ND ND

Mehlich 1
A 0.22(b) 0.21 (b) 0.34(a) 0.18(b) 0.14(b)
E 0.43 (a) 0.35 (a) 0.30 (a) 0.28 (a) 0.28 (a)
Bh 0.16 (b) 0.11 (c) 0.14 (b) NA 0.21 (ab)
aSignificant differences (P<0.05) between horizons within each water potential and
extractant
bNA missing data
'ND data not determined


























0.8 -0 A horizon

[ M E horizon
S0.6 -
S6 Bhhorizon

0.4


0.2


0.0
-0.1 -3 -8 -15 -1000
Water potential (kPa)


Figure 2-4. Inorganic Kp factors for the A, E, and Bh horizons within each water potential
using 3 mM oxalate as an extractant











Table 2-6. Comparison of total Kp factors by horizon within each water potential for each
extractant


--------------Water potential---- -----
-0.1 kPa -3kPa -8 kPa -15 kPa -1000 kPa
Horizon 0.5 MNaHCO3
A 0.91 (aa) 0.74 (a) 0.63 (a) 0.53 (a) NAb
Bh 0.43 (b) 0.55 (b) 0.40 (b) NA 0.78

1 mM oxalate
A NA 0.59 (a) 0.38 (a) NA 0.51 (a)
Bh 0.23 0.17(b) 0.17(b) NA 0.21 (b)

2 mM oxalate
A 0.97 (a) 0.76 (a) 0.44 (a) 0.60 0.82 (a)
Bh 0.55 (b) 0.33 (b) 0.33 (a) NA 0.89 (a)

3 mM oxalate
A 0.74 (a) 0.77 (a) 0.43 (a) 0.59 NA
Bh 0.45 (b) 0.26 (b) 0.10(b) NA 0.39

Bray & Kurtz
A 0.41 0.34 0.38 0.86 0.44
Bh NDc ND ND ND ND

Mehlich I
A 0.22 0.23 (a) 0.22 (a) 0.29 0.26 (a)
Bh NA 0.16(a) 0.17(a) NA 0.33 (a)


aSignificant differences (P<0.05) between horizons within each water potential and
extractant
'NA missing data
'ND data not determined






38


Table 2-7. Significant differences (P<0.05) between the inorganic P and total P Kp factors
for the A and Bh horizons by extractant and water potential. Significant
differences indicated by an "*".
----------------- Water potential-------
-0.1 kPa -3 kPa -8 kPa -15 kPa -1000 kPa
Extract A horizon
0.5MNaHCO3 *
1 mM oxalate *
2 mM oxalate *
3 mM oxalate *
Bray & Kurtz *
Mehlich I *

Bh horizon
0.5 MNaHCO3 NDa
1 mM oxalate ND *
2 mM oxalate ND *
3 mM oxalate ND
Mehlich I ND *
aND not determined









Table 2-8. Inorganic Kp factors measured in the "test" horizons. Microbes were grown from the original horizon, added to the
test horizon, and the Kp factor was measured in the test horizon. For example, microbes were grown from the A horizon at -8
kPa and added to samples in each of the test horizons.


Original "Test" Kp Original "Test" Kp Original "Test" K,
horizon horizon factor horizon horizon factor horizon horizon factor
A (-0.1 kPa) 0.35 E (-0.1 kPa) 0.83 Bh (-0.1 kPa) 0.26
A (-3 kPa) 0.30* E (-3 kPa) 0.92 Bh (-3 kPa) 0.22*
A (-8 kPa) A (-8 kPa) 0.36 E (-8 kPa) E (-8 kPa) 0.94 Bh (-8 kPa) Bh (-8 kPa) 0.28
A (-15 kPa) 0.37 E (-15 kPa) 0.90 Bh (-15 kPa) 0.23
A (-1000 kPa) 0.37 E (-1000 kPa) 0.85 Bh (-1000 kPa) 0.25
E (-8 kPa) 0.42* A (-8 kPa) 0.85 A (-8 kPa) 0.34
Bh (-8 kPa) 0.46* Bh (-8 kPa) 0.65* E (-8 kPa) 0.35*
*inlilri.:nl,. different (P<0.05) from the original horizon value










Table 2-9. Comparison of microbial P concentrations with determined inorganic Kp
factors using 0.5 MNaHCO3 and a Kp factor of 0.40. For comparison purposes, 5 kg P
ha-' of microbial P is used.
-----------.------------------kg P ha ----- -----
A horizon
% Over % Under
Water potential with measured Kp, with K, = 0.40 estimation estimation
-0.1 kPa 8 13 63
-3 kPa 12 13 8
-8 kPa 14 13 -- 7
-15 kPa 11 13 18 --
-1000 kPa 13 13 0 0

E horizon
-0.1 kPa 5 13 160 --
-3 kPa 7 13 86
-8 kPa 8 13 63
-15 kPa 7 13 86
-1000 kPa 7 13 86

Bh horizon
-0.1 kPa 14 13 -- 7
-3 kPa 19 13 -- 32
-8 kPa 15 13 -- 7
-15 kPa NAa 13 --
-1000 kPa 7 13 86 --








Table 2-10. Regression equations for inorganic K, factors for each extractant and horizon
Extractant R2 Regression equation
A horizon
0.5 MNaHCO3 0.65 Kp= 0.489 -0.127*log() + 0.038*log(Y)2
1 mMoxalate (W <= -8 kPa) 0.92 K,p = 0.696 0.243*log(y) 0.219*log(W)2
1 mMoxalate ( >= -8 kPa) 0.63 K = 0.346*log(W)359
2 mMoxalate (W <= -8 kPa) 0.85 Kp = 0.681 0.052* (y)
2 mMoxalate ( >= -8 kPa) 0.70 K, =0.370*log(W)0461
3 mMoxalate ( <= -8 kPa) 0.92 Kp= 0.664 -0.008* (Y)2
3 mMoxalate (W >= -8 kPa) 0.64 K,= 0.389*log(Y)034'
Bray & Kurtz ( <= -15 kPa) 0.91 K,= 0.396 0.282*log(w) 0.008*log (W)2
+ 0.283* log(y)3
Bray & Kurtz (y >= -15 kPa) 0.95 K,= 0.667 0.130*log(y)
Mehlich 1 0.55 K,,= 0.198- 0.00005* (W)

E horizon
0.5 MNaHCO3 0.74 K,= 0.788 0.158*log(y) + 0.046*log(Y)2
1 mMoxalate (y <= -8 kPa) 0.85 K,= 1.158 0.131*log(y)- 0.629*log(Y)2
1 mM oxalate (W >= -8 kPa) 0.13 K = 0.520 + 0.60*log(y)
2 mM oxalate (W <= -8 kPa) 0.74 K,= 0.913 -0.037(y)
2 mMoxalate ( >= -8 kPa) 0.86 K = 0.500 + 0.121*log(y)
3 mM oxalate ( <= -8 kPa) 0.87 K, = 1.138 0.190*log(y) 0.592*log(w)2
3 mM oxalate (y >= -8 kPa) 0.78 Kp = -0.287 + 1.059*log(w) 0.228*log(y)2
Bray & Kurtz (W <= -8 kPa) 0.98 K,= 0.735 0.280*log() 0.228*log(Y)2
Bray & Kurtz (y >= -8 kPa) 0.67 K,= 0.224 + 0.125*log(y)
Mehlich 1 (W <= -8 kPa) 0.87 K, = 0.431 0.024(y) 0.228*log(Y)2
Mehlich 1 (W >= -8 kPa) 0.12 Kp = 0.202 + 0.026*log(y)

Bh horizon
0.5 MNaHCO3 0.92 K, = 0.285 0.016*log(w) + 0.053*log(Y)2
1 mMoxalate ( <= -8 kPa) 0.02 K,= 0.105 + 0.016*log(w) + 0.061*log(Y)2
1 mMoxalate (y >= -8 kPa) 0.45 Kp = 0.188 0.019*log(y)
2 mM oxalate ( <= -8 kPa) 0.67 K,= 0.147 + 0.001*log(y) + 0.106*log(y)2
2 mMoxalate ( >= -8 kPa) 0.54 K, = 0.253 -0.22*log(p)
3 mMoxalate 0.64 K, = 0.361 0.056*log(y)
Mehlich 1 0.73 K, = 0.127 -0.017*log(w) + 0.015*log(y)2














K,= 0.489 0.127*log(y) + 0.038*log(y)2
R2 = 0.65


S _

S* -
*
*
*


Log water potential (-kPa)


.-


-- S


K, = 0.664 0.008* (Y)2
R2 = 0.92 I


$


K, = 0.389*log(Y)0 341
R2 = 0.64


Log water potential (-kPa)


Figure 2-5. Inorganic Kp regression equations in the A horizon for a) 0.5 MNaHCO3 and
b) 3 mMoxalate, E horizon for c) 0.5 M NaHCO3 and d) 3 mM oxalate, and
Bh horizon for e) 0.5 MNaHCO3 and f) 3 mMoxalate


0.7

0.6

0.5

0.4

0.3


0.1


0.0














Kp = 0.788 0.158*log(y) + 0.046*log(y)2
R2= 0.74


S. ..
-. < .- 0
*


0.0 -----
0.1 1 10
Log water potential (-kPa)


100


0.0 -


0 \
*
,
0'


Kp =1.138-0.190*log(y) ,
-.592*log(Y)2
R2 = 0.87


*
A K= -0.287+1.059*log(y)
, 0 -0.228*log(Y)2
R2 = 0.78


10
Log water potential (-kPa)


Figure 2-5 continued


S0.6

0.4

0.2











0.9

0.8

0.7

0.6

0.5

0.4

0.3 -
0.2

0.1
0.0


K, = 0.285 0.016*log(y) + 0.053+log(Y)2
R2 = 0.92


1 10
Log water potential (-kPa)


0.9
0.8

0.7

0.6 K = 0.361 -0.056*log(
SR2 = 0.64
0.5

0.4 .

0.3 *- ..

0.2

0.1

0.0 --
0.1 1 10


Wv)


100


Log water potential (-kPa)


Figure 2-5 continued














CHAPTER 3
SEQUESTERING OF PHOSPHORUS FERTILIZER IN THE MICROBIAL BIOMASS
OF A COASTAL PLAIN SPODOSOL

Introduction

Forest productivity in much of the southeastern U.S. is phosphorus (P) limited

(Pritchett and Smith, 1975). The sandy, quartzic, surface horizons of Coastal Plain

Spodosols limit P retention (Zhou et al., 1997; Harris et al., 1996; Fox et al., 1990a),

resulting in P mobility. Therefore, P that is not absorbed by plants or microorganisms

may be leached below the A horizon (Harris et al., 1996; Nair et al., 1998; Nair et al.,

1999) and beyond newly planted seedling root depth. Since soil P bioavailability is

inherently low, P fertilization is a common practice due to the significant growth

response it elicits in southern pines (Pritchett and Smith, 1975; Prichett and Comerford,

1982). Likewise, the application of nitrogen (N) in addition to P has a significant effect

on southern pine growth (Jokela and Stears-Smith, 1993). While root density at time of

planting is low and early uptake rates do not become significant until about three months

after planting (Smethurst et al., 1993), the microbial biomass may have the potential to

sequester P, maintain it in the soil horizon, and eventually make it available over time.

Microbial cycling and associated P transformations have been well studied

(Seeling and Zasoski, 1993; Gijsman et al., 1997; He et al., 1997b; Grierson et al., 1998)

and have been shown to be dependent upon the concentration of P in the soil (Brookes et

al., 1984). It has also been shown that microbial P changes with water potential (Sparling

et al., 1986; Skopp et al., 1990; Grierson et al., 1999), nutrient bioavailability (Clarholm,









1993; He et al., 1997b; Dilly and Nannipieri, 2001) and microbial biomass (Walbridge,

1991).

The purpose of this study was to determine the interaction between P fertilization

and soil water potential in determining microbial biomass and the sequestering of P as

microbial P. More specifically, the first objective was to investigate the influence of P

bioavailability on microbial biomass and to determine whether microbial biomass growth

in a Spodosol of the southeastern U.S. is P limited. The hypothesis was that P is limiting

to soil microbial growth. The second objective was to estimate the immobilization and

retention of P fertilizer by the microbial biomass and test the hypothesis that microbial

biomass could immobilize and retain significant quantities of P fertilizer. The third

objective was to investigate how soil water potential affected microbial uptake and P

fertilizer retention. The hypothesis tested here is two fold. It first states that microbial

biomass at water potentials promoting poor aeration will immobilize less fertilizer P than

those near field capacity. Secondly, it states that microbial biomass at field capacity will

immobilize more fertilizer P than that at drier water potentials (i.e. -1000kPa).

Methods

Study Area and Field Sampling

The study area was located 33 km northeast of Gainesville, FL. The site was a 16

ha managed slash pine plantation (Pinus elliottii var. elliottii Engelm.) that was clearcut

April 1994, bedded in September and November 1994, and replanted to slash pine in

January 1995. In February 1995, 85 g ha' ofimazypyr was applied as Arsenal in 1.5 m

wide bands on the planted beds for weed control. The soil is a sandy, siliceous, thermic

Ultic Alaquod, mapped primarily as Pomona fine sand and sand. The average annual









temperature is 21 OC, and the long-term average annual rainfall is approximately 1330

mm (Soil Conservation Service, 1995).

Soil was randomly collected from the 15 cm thick A horizon across a subsection

(approximately 2 ha) of the study area in July 1996 with a 7.5 cm diameter soil auger.

Approximately 3 kg of soil were collected from randomly selected points from

approximately 20 auger holes. The soil was sieved to pass a 2 mm screen and air-dried.

Sub-samples containing 800 g of soil were incubated in triplicate bags at 28 OC at five

water potential treatments of-0.1, -3, -8, -15, and -1000 kPa for 10 days. Water potential

was kept constant throughout the incubation period by weighing the samples and adding

double deionized water when required. These water potentials were based on results

from P mineralization studies that evaluated mineralization rate versus soil water

potential for a similar soil (Grierson et al., 1999).

Phosphorus fertilizer treatments of 0 kg P ha-', 30 kg P ha', 60 kg P ha1', and 100

kg P ha-' (or 0, 80, 120, and 230 pg P g-' soil) as triple superphosphate granules were

added to each water potential treatment. An additional fertilizer treatment of

diammonium phosphate (DAP) was added to the study, equivalent to 640 tg P and 590

lg N g-' soil. This high fertilizer treatment was added to determine the quantity of P that

could be incorporated when N is not limiting microbial growth. Although carbon may

also be limiting in this system, it was not considered as a factor for this experiment. The

DAP treatment does not reflect a commercial application of fertilizer at this stage of stand

development which would be in the range of 40 to 50 kg P and N ha' (Kidder et al.,

1987).









Triplicate samples from each bag were taken at 0, 3, 7, 14, 28, 49, and 77 days after

fertilizer addition and analyzed for microbial P, microbial C, soil moisture content, and

pH. Moisture content was measured after drying the samples for 24 hours at 105 oC ,

and pH was measured in a 2:1 water:soil solution. Triplicate samples for microbial P

analysis and for microbial C analysis were removed from each treatment bag for

fumigation. Liquid chloroform was added to each sample, sealed for 24 hours, after

which the chloroform was allowed to evaporate. Microbial P was measured with 3 mM

oxalate extraction at a 1:10 soil:solution ratio with a shaking time of 10 minutes (Chapter

2). Microbial C was measured using the ninhydrin-reactive nitrogen method (Amato and

Ladd, 1988). Unfumigated triplicate samples were also taken at the same time as the

fumigated and immediately measured for both microbial P and microbial C. Microbial P,

the quantity of P in the microbial biomass, was defined as:

[P extracted from fumigated soil P extracted from unfumigated soil]/Kp (Eq. 3-1)

by Brookes et al. (1982). The Kp was determined in a previous study and is specific for

each water potential (Chapter 2). Microbial C, the quantity of carbon in the microbial

biomass, was determined by the method from Amato and Ladd (1988) and is defined as:

[ninhydrin-reactive N in fumigated soil ninhydrin-reactive N in unfumigated

soil]*21 (Eq. 3-2)

Statistical Analysis

Statistical differences were determined using SAS version 8.1 (The SAS Institute,

2001) using a three factor mixed ANOVA model. For each sample bag, the least-squared

means were analyzed for significant differences (P<0.05) by repeated measures analysis

of variance with a first order autoregressive covariance structure. Logarithmic

transformations were applied to microbial P and microbial C values in order to normalize









the variance. Microbial P, microbial C, and microbial C:P ratios were analyzed for

significant differences, (P<0.05), within the main effects (water potential, treatment, and

time) and among effects (Appendix B).

Results and Discussion

Objective 1: Investigate the Influence of P Bioavailabilty on the Microbial Biomass

Microbial C ranged from 18 to 302 [lg C g-' soil in the control (no P added) (Table

3-1). This range was similar to other studies reporting microbial C (Chauhan et al., 1981;

Saggar et al., 1998). Extracted labile inorganic P (P,) for the control ranged from 3.9 to

4.8 (-0.1 kPa), 4.5 to 5.5 (-3 kPa), 3.7 to 5.0 (-8 kPa), 0.7 to 2.8 (-15 kPa), and 2.2 to 3.6

(-1000 kPa) gg g-' soil (Figure 3-1). In a study using similar soil and water potentials,

Grierson et al. (1999) reported comparable solution Pi concentrations using KCI as the

extractant. Brookes et al. (1984) reported solution P, ranging from 3.1 to 84.8 .tg P g'

soil in a variety of soils. Some of the highest P, values were reported by Chauhan et al.

(1981) who measured 285 [tg P g-' in an Ap horizon from a sandy loam. Comparatively,

this soil was low in solution P,.

Microbial C was not affected by the P-only fertilizer treatments at any sampling

time when evaluated within a soil water potential, except in the driest soil at the end of

the experiment (Table 3-1; Appendix C, Figure C-1). Soil at -3 kPa provided an example

of the effect of P fertilization on microbial C (Figure 3-2). After a few days, the fertilizer

treatment had significantly affected microbial C, but there was no trend with time as

some days were significantly higher and some significantly lower than the control. The

overall trend of these data indicated that the microbial biomass was not P limited even









though the soil had low labile P, levels. Therefore, the first hypothesis, which stated that

P was limiting microbial growth, was negated.

The microbial population immobilizes phosphorus released through microbial

mineralization and turnover, and little may be available to plants (Tate et al., 1991).

Therefore, in soils with low Pi, like this Spodosol, the microbial biomass may be

immobilizing enough P to sustain its activity but not initially supply P to the pine

regenerating on the site.

Others have also confirmed that microbial C was not P limited on different sites

(Chauhan et al., 1981; Prescott et al., 1992; Bauhus and Khanna, 1994; Joergensen and

Scheu, 1999). Bauhus and Khanna (1984) added 100 kg P ha'- as superphosphate with

no change in microbial C on an acidic, sandy soil with low total P (P,), but the authors

determined that microbial carbon dioxide (C02) release (i.e. microbial activity) increased.

Parfitt et al. (1994) reported that microbial C in another sandy soil was not influenced by

P fertilizer even when it was added at rates up to 100 kg P ha'. Bolan et al. (1996) also

reported that microbial C was not affected by a range of single superphosphate

applications (15-60 kg P ha 1) in laboratory experiments.

However, microbial C increased in the Bolan et al. (1996) 8-year field study due to

increases in soil organic matter, which resulted from increased plant growth promoted by

fertilization. In contrast, Saggar et al. (1998) determined that microbial C decreased after

P-only fertilizer addition. The current study showed a similar response over the first 28

days when the water potential was maintained at -15 kPa. The large increase observed

towards the end of this study within the two driest soils indicated a delayed response to P

only fertilization. Why this occurred is not known; nevertheless the response is too large









to ignore. Because this response was observed in the two dry soil conditions, it would

not seem to be experimental error. A possible explanation is that the increase is due to

slow growing microorganisms.

In contrast to the P-only fertilizer treatments, the DAP treatment increased

microbial C (Table 3-2), presumably in response to the N addition. This increase

continued to be significantly higher than the 100 kg P ha' treatment and the control

throughout the experiment's duration and ranged between a low of a 291% increase (day

3, -1000 kPa) to a high of 36,887% increase (day 49, -8 kPa). Other studies reported that

additions of N increased microbial C when added along with P (Ghosal and Singh, 1995;

Hossain et al., 1995). The rate of DAP was extremely high and meant to give a glimpse

of how high microbial C could be increased. These soils support extremely high levels of

microbial biomass under high nutrient regimes. Microbial biomass, under the right

moisture regime exceeded 3 mg g-' soil (0.3%). While one would not expect to reach

these levels in sandy Spodosols under normal management regimes, they do provide a

sense of what this soil is capable of supporting if used under bio-solid management with

high rates of N and P.

Objective 2: Estimate the Immobilization and Retention of P Fertilizer by the Soil
Microbial Biomass

Microbial response to P fertilization may be different for growth and P uptake,

since P uptake does not require microbial biomass increase. In this study, microbial P

response to P-only fertilization was in fact unrelated to the microbial C response.

Microbial P did respond to treatment.

While microbial P in the control did not change significantly with time, microbial P

increased in response to rates of P addition (Table 3-3; Appendix C, Figure C-2).









Microbial P at -0.1 kPa water potential illustrates this response (Figure 3-3). Microbial P

peaked within one week of P-only fertilizer additions (Appendix C, Figure C-3).

The 30 kg P ha-' treatment peaked on day 3 for all levels of water potential (Figure

3-4). In the higher P-only treatments, microbial P peaked on day 3 in the three wetter

water potentials (-0.01 to -8 kPa) and on day 7 in the two drier water potentials (-15 and

-1000 kPa). After this initial peak, microbial P tended to decrease over time. In the two

wettest soils (-0.1 and -3 kPa), the unfertilized condition continued to be significantly

lower than the fertilizer treatments. In the other water potentials, the control was similar

to either the 30 or 60 kg P ha' treatments after the initial peak. The 100 kg P ha'

treatment, while significantly different from the control, was often similar to the other P

treatments, yet the tendency was for this treatment to have the highest quantity of

microbial P. This may have been due to the high variability, which may have been due to

the use of fertilizer granules instead of ground fertilizer. Many samples had to be

declared missing due to extremely high P concentrations, which was assumed to be due

to fertilizer granule remnants.

A diminishing returns pattern for microbial P was demonstrated as the P addition

rate increased (Table 3-4), suggesting that the microbial biomass in this soil can only

immobilize a limited concentration of P under these conditions. This level was estimated

to be between 27 and 52 mg P kg-' soil or 45 to 86 kg P ha-'. This may also indicate a

limiting factor, such as N or C, limits additional P immobilization. The decrease in

microbial P over time is consistent with the results of Clarholm (1993) and Grierson et al.

(1998) and may reflect the limited microbial P storage in this surface soil when P is non-

limiting.









Other studies have documented microbial P increases with P fertilizer addition

(Chauhan et al., 1981; Haynes and Swift, 1988; Parfitt et al., 1994; He et al., 1997a;

Quails and Richardson, 2000; Schneider et al., 2001). Haynes and Swift (1988) added

500 mg P g-i soil as Ca(H2P04)2 to an acidic silt loam and reported that microbial P

increased to twice the quantity determined in the control and continued at these levels

throughout the experiment. He et al. (1997a) measured microbial P increases of over

three times the magnitude in the control, from approximately 3 to 10 mg P kg" soil, after

adding nearly 400 mg P kg-' soil of carbonate-substituted phosphate rock to an acid loam.

Microbial P was still increasing at the end of their study (60 days). After adding 50 mg

kg-' soil of triple superphosphate (100 kg ha '), Schneider et al. (2001) determined that

microbial P doubled, from 49 to 104 mg P g-i soil, in soils with a 50 percent or higher

adsorption capacity. In this study, microbial P increased from six to 18 times after the

230 mg P kg-' soil (100 kg ha-' of triple superphosphate) was added and was still three to

six times higher than the control at the end of the study. Luxury uptake and retention of

P by microbes has been documented (Fuhs and Chen, 1975; Khoshmanesh et al., 2002).

The microbial biomass in this study also demonstrated the same capability.

In soils with very little or no P sorption capacity, such as this study's soil,

biological control of P leaching may be a factor in retaining P within the root zone

following fertilization and rainfall leaching events. The microbial biomass in the control

immobilized from 3 to 13 kg P ha'1, with the wetter soils containing the lowest quantity

of P. The annual aboveground uptake of a young slash pine plantation is approximately

1.5 kg P ha (Adegbidi et al., in review), and a semi-mature slash pine plantation's

annual aboveground uptake of P is approximately 4 kg P ha-' (Polglase et al., 1992). The









microbial biomass without fertilization holds enough P to supply the P nutrient demands

of a slash pine stand, especially in the drier soils. The quantity of P sequestered would be

sufficient to supply the needs of a pine plantation if it was slowly released over time due

to microbial turnover and if microbial turnover was timed with plant P demand.

One contribution of the microbial biomass may be controlling the loss of P

fertilizer due to leaching. Within a week, the microbial biomass immobilized from 17 to

52 mg P kg-', equivalent to 9 to 29 kg P ha', in soil for a range of water potentials. The

microbial biomass was able to retain more than enough P for a growing pine plantation,

even in the wettest conditions. Even after 77 days, the microbial biomass still held from

2 to 11 kg P ha'-. The percentage of P fertilizer still sequestered on day 77 was 4 to 17

%, 3 to 9 %, and 2 to 9 % of the 30, 60, and 100 kg P ha-' treatments, respectively (Table

3-5). Given these results, the second hypothesis, which stated that microbial biomass

could retain significant amounts of fertilizer P, was confirmed.

Phosphorus mineralization has been reported to increase with fertilization on

similar soils (Polglase et al., 1992; Grierson et al., 1998; Grierson et al., 1999). In these

studies, P mineralization was related to soil organic matter P, not microbial P (Grierson et

al., 1998). However, the P released from the microbial biomass via turnover, and the

increased mineralization due to fertilization, could combine to provide increased P

bioavailability. A significant quantity of this P may be preserved from leaching due to

biomass immobilization.

With the high DAP treatment, microbial P was significantly increased throughout

the study (Table 3-5). DAP elicited a continued increase in microbial P until days 14 to

28, after which it continued to be significantly higher throughout the experimental









duration. Microbial P increased up to 80 mg P kgl equivalent to 44 kg P ha-', at the

highest quantity and still retained 39 to 45 mg P kg ', equivalent to 21 to 25 kg P ha ', at

the end of the study.

The lack of response in microbial C, combined with the significant effect of P in

increasing microbial P, is further supported by the microbial C:P ratios (Table 3-6). As

soon as fertilizer was added, the microbial C:P ratio decreased in all fertilizer treatments

although they were only significantly different for the two wettest soils (-0.1 and -3 kPa).

Lack of significant differences may be due to the high variability of the ratios. The ratios

ranged from 19:1 to 202:1 before fertilizer addition. Immediately after fertilizer addition,

the microbial C:P ratios dropped dramatically, especially in the wetter soils, from 2:1 to

30:1 in the P-only fertilizer treatments and from 13:1 to 59:1 in the DAP treatments. The

ratios in the P-only fertilizer treatments continued to be much lower than the control, due

to the immobilization of P by the microbial biomass without the increase in microbial

population. The significant decrease in the microbial C:P ratios after fertilization

indicates that microbial P may be easily mineralized for increased P cycling (Parfitt et al.,

1994) due to microbial turnover (Cole et al., 1978). The DAP ratios were higher than the

P-only ratios, but that would likely be due to the increase in microbial biomass along with

the immobilization of P.

Although the microbial biomass in the surface horizon of this Spodosol was not P

limited, a significant quantity of P fertilizer was immobilized and sequestered under

laboratory conditions for more than 77 days. Particularly in the absence of understory

vegetation that is characteristically reduced in intensive forest management, microbial

biomass should be further investigated for its ability to sequester and eventually release









P. The pattern of release will determine how useful this is in increasing the efficiency of

P fertilization.

Objective 3: Investigate How Soil Water Potential Affects Microbial Uptake and P
Fertilizer Retention

In the control treatment, microbial P at the -15 kPa water potential was higher than

the other water potentials, while microbial P in the -0.1 kPa soil was usually lower than

the other water potentials (Table 3-7; Figure 3-5). The two drier soils (-15 and -1000

kPa) were significantly higher than the two wettest soils (-0.1 and -3 kPa) at nearly every

sampling date. With decreasing water potential, the microbial community changes from

an aerobic population to a population dominated by anaerobic and facultative

microorganisms. Microbial activity has been reported to be higher in aerobic soils

(Orchard and Cook, 1983; Lee et al., 1990), which may explain the higher microbial P in

the control at -15 kPa.

Prior to P fertilization, microbial P exhibited significant differences due to water

potential. After fertilization, few significant differences were observed. These

differences did not depict a consistent pattern (Appendix C, Figure C-4). It was

consistent that, after P fertilizer addition, microbial P in all water potentials increased

significantly. More P was taken up by the microbial biomass in the soil at -0.1 kPa,

increasing 20 to 32 times, while the drier condition promoted only a four to nine times

increase. Even with such a large increase, microbial P in the saturated water potential was

similar to microbial P in the other water potentials after day 3. These results confirm that

the last two hypotheses are incorrect when stating that microbial immobilization of P is

less in saturated soils and more in drier soils.









The surficial water table in Spodosols fluctuates widely during the year. In an

average year, the shallow water table may be less than 25 cm from the soil surface for

one to three months, between 25 and 100 cm for six months, and exceeding 100 cm

during the dry season (Soil Conservation Service, 1985). Microbial P is significantly

different between water potentials before fertilization; however, microbial P becomes

similar after fertilization due to the greater immobilization of P in the higher water

potentials. The differences in microbial P before and after fertilization may be due to

changes in microbial activity after addition of fertilizer. Therefore, on systems with

fluctuating water tables, the amount of P fertilizer immobilized is dependent upon the

water table depth. When the water table is high, the microbial biomass should

immobilize more P fertilizer. But when the water table is low, and P may be leached

more quickly from the rooting zone due to high permeability, less P fertilizer is

immobilized.

Why microorganisms in the wetter soil with P fertilizer treatments immobilized

more P is not known, but must be due to changes in the microbial community. Soil water

potential has been shown to affect microbial community diversity (Brockman et al.,

1992). As soil water potential changes, so do fungal and bacterial ratios (Faegri et al.,

1977; Anderson and Domsch, 1975; Wilson and Griffin, 1975). Nutrient concentrations

in the microbial biomass are related to soil characteristics, including soil water potential

(Diaz-Ravifia et al., 1995).

There were significant differences between the microbial biomass at different water

potentials, even after fertilization (Table 3-8; Appendix C, Figure C-5). But again, like

microbial P after fertilization, there were no consistent trends. Studies have reported that









the influence of water potential on microbial C is variable, both affecting (Sarathchandra

et al., 1988) and not affecting (Zaman et al., 1999) microbial C. This study used similar

water potentials to those of Grierson et al. (1999) on a similar soil, with similar results.

Conclusion

The microbial biomass was not P limited for growth, even though pine growth on

similar soils is naturally P limited. However, whereas increasing rates of triple

superphosphate did not increase microbial C, immobilization ofP was significant.

Microbial P increased between six and 18 times within three to seven days following

fertilizer additions. In the wetter soils, more P was taken up after fertilization, such that

microbial P was equivalent to the drier soils. Both microbial C and microbial P increased

dramatically when a large quantity of both N and P were added, demonstrating that this

system was N limited and the microbial biomass was capable of increasing significantly

and immobilizing a large quantity of P. Due to the inability of this soil to retain P in the

surface horizon, the microbial biomass would play a significant role in retention and

supply of P to plants through immobilization, along with the combination of

mineralization and microbial turnover. The question is how microbial turnover occurs

over time, and how is subsequent P mobilization timed with plant demand and the

development of an expanding southern pine root system.






Table 3-1. Comparison of microbial C by treatment within each day and by day within each treatment for each water potential
Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
-------------------------- -----g C g' soil--------------------------------
Treatment -0.1 kPa
Control 105 (A'b') 184 (Aab) 427 (Aa) 105 (Bb) 213 (Ab) 180 (Aab) 171 (Aab)
30 kg P ha- 161 (Aa) 173 (Aa) 62 (Cb) 180 (Aa) 261 (Aa) 164 (Aa) 175 (ABa)
60 kg P ha" 209 (Aa) 174 (Aa) 172 (ABa) 170 (ABa) 209 (Aa) 179 (Aa) 126 (ABa)
100 kg P ha' 46 (Bc) 162 (Aa) 142 (Ba) 102 (Ba) 426 (Aa) 108 (Ab) 92 (Bbc)

-3 kPa
Control 200 (Aa) 128 (ABab) 302 (Aa) 209 (Aa) 61 (Bbc) 46 (Be) 83 (Abc)
30 kg P ha- 109 (Ab) 90 (Bb) 137 (Aa) 217 (Aa) 67 (Bb) 76 (Bb) 54 (Ab)
60 kg P ha' 129 (Ab) 98 (ABb) 181 (Aab) 258 (Aa) Ill (ABb) 243 (Aab) 62 (Ab)
100 kg P ha-' 198 (Aa) 199 (Aa) 138 (Aa) 183 (Aa) 137 (Aa) 139 (ABa) 64 (Aa)

-8 kPa
Control 122 (Aab) 53 (ABb) 136 (Aa) 32 (Ac) 140 (Aa) 18 (Bc) 75 (Aab)
30 kg P ha-' 87 (ABa) 150 (Aa) 82 (Aa) 18 (Ab) 59 (Ba) 69 (Aa) 71 (Aa)
60 kg P ha' 88 (Aa) 33 (Bbc) 83 (Aa) 80 (Aab) 76 (ABab) 27 (Bc) 29 (Bc)
100 kg P ha' 72 (Bb) 67 (ABb) 116 (Aa) NAC 98 (ABa) 157 (Aa) NA

-15 kPa
Control 245 (Aa) 208 (Aa) NA NA 96 (Ab) NA 250 (ABa)
30 kg P ha-' 231 (Aa) 148 (Aab) 61 (Ab) NA 55 (Ab) NA 156 (Bab)
60 kg P ha' 237 (Aa) 155 (Aa) 112(Bb) NA 113(Ab) NA 217 (Ba)
100 kg P ha' 223 (Aa) 212 (Aa) 100 (ABb) NA 17 (Ab) NA 285 (Aa)

-1000 kPa
Control 112 (Aab) 284 (Aa) 56 (Bb) 171 (Aa) NA NA 77 (Bb)
30 kg P ha' 63 (Bc) 267 (Ab) 183 (Ab) 144 (Ab) 76 (Bbc) NA 682 (Aa)
60 kg P ha-' 113 (Ab) 266 (Aa) 93 (Bb) 109 (Ab) 107 (Ab) NA 475 (Aa)
100 kg P ha-' 66 (ABb) 332 (Aa) 70 (Bb) 95 (Ab) 88 (Bb) NA 533 (Aa)
aSignificant differences (P<0.05) between treatments within each day and water potential (upper case letters down columns)
bSignificant differences (P<0.05) between days within each treatment and water potential (lower case letters across rows)
cNA missing data





















*-0.1 kPa
0 -3 kPa
1-8kPa
0-15 kPa
6 -1000 kPa



S4

B 3

2

n 1

0
0 3 7 14 28 49 77
Days from beginning of study


Figure 3-1. Solution inorganic P in the control at each water potential






















400



0 300
3- day0

0 -a -- -day3
O 200 -- -- --day7
S-- o _.-- --day 14
S--- day 28
S100 ---day 77


0 -
0 20 40 60 80 100

P fertilizer added (kg ha-')


Figure 3-2. Microbial C for each day in the -3 kPa soil at each P fertilizer treatment







Table 3-2. Comparison of microbial C between the control, 100 kg P ha-1, and DAP fertilizer treatments within each day for
each water potential
Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
------- ------------------------ g soil------------- ---------------
Treatment -0.1 kPa


Control 105 (B") 184 (B) 427 (B) 105(B) 213 (B) 180(B) 171 (B)
100 kg P ha' 46 (C) 162 (B) 142 (C) 102 (B) 426 (B) 108(B) 92 (C)
DAPb 348 (Acc) 1338 (Aab) 1759 (Aab) 1255 (Ab) 1555 (Aab) 2908 (Aa) 891 (Ab)

-3 kPa
Control 200 (A) 128 (B) 302 (B) 209 (B) 61 (C) 46 (C) 83 (A)
100 kg P ha- 198 (A) 199 (B) 138 (B) 183 (B) 137 (B) 139 (B) 64 (A)
DAP 161 (Ac) 1716 (Ab) 1031 (Ab) 3433 (Aa) 766 (Ab) 942 (Ab) 316 (Abc)

-8 kPa
Control 122 (A) 53 (B) 136 (B) 32 140 (B) 18 (C) 75 (A)
100 kg P ha-' 72 (B) 67 (B) 116(B) NA" 98 (B) 157(B) NA
DAP 104 (Ac) 2122(Aab) 1106(Aab) NA 919(Ab) 5918 (Aa) 786 (Ab)

-15 Kpa
Control 245 (A) 208 (B) ND NA 96 (B) NA 250 (B)
100 kg P ha' 223 (A) 212 (B) 100 (B) NA 17 (C) NA 285 (B)
DAP 207 (Ac) 9470 (Aa) 783 (Ab) NA 549 (Abc) NA 1720 (Ab)

-1000 kPa
Control 112 (A) 284 (B) 56 (B) 171 (B) NA NA 77 (C)
100 kg P ha 66 (B) 332 (B)) 70 (B) 95 (B) 88 (B) NA 533 (B)
DAP 157 (Ac) 1120 (Aab) 942 (Aab) 1206 (Aab) 768 (Ab) NA 2953 (Aa)
aSignificant differences (P<0.05) between treatments within each day and water potential (upper case letters down colums)
bDAP = 350 kg P and 352 kgN ha'
'Significant differences (P<0.05) between days within the DAP treatment for each water potential (lower case letters across rows)
dNA missing data






Table 3-3. Comparison of microbial P by P fertilizer treatment within each day and by day within each P fertilizer treatment
fnr oeilrh nwatPr nntential


Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
------- ----------------- -------- g soil------------------------------------


Treatment -0.1 kPa
Control 1.0 (Ab) 1.2 (Cab) 1.2 (Bab) 2.5 (Ca) 1.7 (Ba) 1.8 (Ba) 2.4 (Ca)
30 kg P ha'' 0.9 (Ab) 17.7 (Ba) 8.9 (Aa) 8.2 (Ba) 15.6 (Aa) 9.0 (Aa) 11.5 (ABa)
60 kg P ha-' 1.0 (Ac) 32.5 (Aa) 9.7 (Aab) 19.7 (ABab) 27.4 (Aa) 28.4 (Aa) 6.6 (BCb)
100 kg P ha' 1.3 (Ab) 24.0 (ABa) 40.4 (Aa) 23.6 (Aa) 37.7 (Aa) 22.4 (Aa) 14.3 (ABa)


-3 kPa
Control 1.8 (Aaab) 1.5 (Ba) 1.4 (Ca) 1.6 (Ba) 1.7 (Ba) 1.7 (Ba) 1.2 (Ba)
30 kg P ha" 3.2 (Ab) 24.4 (Aa) 8.1 (ABab) 5.1 (Bb) 13.3 (Aa) 8.3 (Aa) 7.4 (Ab)
60 kg P ha' 1.5 (Ac) 16.7 (Aa) 7.5 (BCbc) 11.6 (Aa) 25.9 (Aa) 13.6 (Aa) 9.1 (Ab)
100 kg P ha- 1.4 (Ac) 27.1 (Aa) 25.2 (Aa) 20.3 (Aa) 17.7 (Aa) NAC 4.1 (Ab)

-8 kPa
Control 3.6 (Aa) 4.9 (Ca) 5.2 (Ba) 5.3 (Ba) 3.5 (Ba) 4.3 (Aa) 2.9 (Ba)
30 kg P ha' 1.5 (Ac) 21.3 (Ba) 16.7 (Ab) 14.8 (Ab) 11.6 (Ab) 5.2 (Ac) 5.0 (Bc)
60 kg P ha-' 2.9 (Ab) 21.7 (ABa) 11.3 (ABa) 12.1 (ABa) 6.0 (Bb) 12.6 (Aa) 8.6 (ABab)
100 kg P ha' 2.7 (Ac) 33.6 (Aa) 30.1 (Aa) 16.7 (Ab) 14.4 (Ab) 16.2 (Ab) 19.8 (Aa)

-15 kPa
Control 8.1 (Aa) 5.9 (Ca) 7.2 (Da) 5.9 (Ba) 4.7 (Aa) NA 4.7 (Ba)
30 kg P ha' 7.9 (Ab) 30.7 (Ba) 22.5 (Ca) 8.9 (Bab) 9.3 (Aab) NA 13.0 (ABab)
60 kg P ha' 9.6 (Abc) 16.6 (Cab) 37.1 (Ba) 26.0 (Aab) 7.5 (Ac) NA 14.5 (Abc)
100 kg P ha' 7.8 (Ac) 37.1 (Aab) 52.2 (Aa) 21.3 (Ab) 16.8 (Ab) NA 14.4 (Ab)

-1000 kPa
Control 4.0 (Aa) 3.5 (Ba) 5.0 (Ca) 4.5 (Ba) 4.2 (Aa) NA 3.3 (Ca)
30 kg P ha' 3.7 (Ab) 19.2 (Aa) 13.9 (Ba) 14.6 (ABa) 19.1 (Aa) NA 11.1 (ABa)
60 kg P ha' 3.3 (Ad) 11.2 (ABbc) 30.9 (Aa) 18.0 (Aab) 10.0 (Abc) NA 6.2 (BCc)
100 kg P ha' 3.7 (Ac) 23.4 (Ab) 20.0 (ABab) 30.1 (Aa) 9.6 (Ab) NA 13.8 (ABab)
"Significant differences (P<0.05) between treatments within each day and water potential (upper case letters down columns)
bSignificant differences (P<0.05) between days within each treatment and water potential (lower case letters across rows)
'NA data missing


~





















70

60

50
S40 Control
40
SI 30 kg P ha
30 0 60 kg P ha
. 0 100 kg P ha

10

0
0 3 7 14 28 49 77
Days from beginning of study


Figure 3-3. Microbial P by P fertilizer treatments over time in the -0.1 kPa soil
























---0.1 kPa
-- -3 kPa
-- -8 kPa
-- 15 kPa
- 1--1000 kPa


20 40 60 80

Days from beginning of study


Figure 3-4. Microbial P over time for each water potential in the 30 kg P ha' treatment


60

- 50

S40

. 30

. 20

10

0






66



Table 3-4. Percent of P fertilizer immobilized and retained on day 77 by the microbial
biomass
-0.1 kPa -3 kPa -8 kPa -15 kPa -1000 kPa
--------------------% P immobilized-----------
P fertilizer Peak day
30 kg P ha"' 30 42 30 45 29
60 kg P ha-' 29 14 15 27 24
100 kg P ha'1 13 14 16 25 14

Day 77
30 kg P ha 17 11 4 15 14
60 kg P ha' 4 7 5 9 3
100 kg P ha-' 7 2 9 5 6






Table 3-5. Comparison of microbial P between the control, 100 kg P ha ', and DAP fertilizer treatments within each day for
ePn-h wtepr nnotentil


Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
--------------------------------------------- (g P g- soil)--------------------------------
Treatment -0.1 kPa
Control 1.0 (Aa) 1.2 (C) 1.2 (C) 2.5 (C) 1.7 (C) 1.8 (C) 2.4 (C)
100 kg P ha-' 1.3 (A) 24.0 (B) 40.4 (B) 23.6 (B) 37.7 (B) 22.4 (B) 14.3 (B)
DAPb 0.5 (AdC) 64.2 (Abc) 56.7 (Abc) 33.4 (Ac) 203.2 (Aa) 111.2 (Ab) 94.2 (Ab)

-3 kPa
Control 1.8 (A) 1.5 (C) 1.4 (C) 1.6 (C) 1.7 (B) 1.7 1.2 (C)
100 kg P ha' 1.4 (A) 27.1 (B) 25.2 (B) 20.3 (B) 17.7 (A) NAd 4.1 (B)
DAP 1.9 (Ac) 49.5 (Ab) 37.1 (Ab) 122.5 (Aa) 16.1 (Ab) NA 32.2 (Ab)

-8 kPa
Control 3.6 (A) 4.9 (C) 5.2 (C) 5.3 (B) 3.5 (C) 4.3 (B) 2.9 (C)
100 kg P ha-1 2.7 (A) 33.6 (B) 30.1 (B) 16.7 (B) 14.4 (B) 16.2 (B) 19.8 (B)
DAP 2.0 (Ac) 190.3 (Aa) 245.5 (Aa) 314.9 (Aa) 49.8 (Ab) 64.8 (Aab) 62.7 (Ab)

-15 kPa
Control 8.1 (A) 5.9 (C) 7.2 (C) 5.9 (C) 4.7 (B) NA 4.7 (C)
100 kg P ha' 7.8 (A) 37.1 (B) 52.2 (B) 21.3 (B) 16.8 (B) NA 14.4 (B)
DAP 7.8 (Ab) 193.7 (Aa) 266.2 (Aa) 273.1 (Aa) 82.2 (Aa) NA 186.4 (Aa)

-1000 kPa
Control 4.0 (A) 3.5 (C) 5.0 (C) 4.5 (C) 4.2 (B) NA 3.3 (C)
100 kg P ha' 3.7 (A) 23.4 (B) 20.0 (B) 30.1 (B) 9.6 (B) NA 13.8 (B)
DAP 2.2 (Ac) 107.9 (Aab) 102.0 (Aab) 207.8 (Aa) 77.0 (Ab) NA 130.9 (Aab)


aSignificant differences (P<0.05) between treatments within days and water potentials (upper case letters down columns)
bDAP = 350 kg P and 352 kg N ha
'Significant differences (P<0.05) between days within the DAP treatment within each water potential (lower case letters across
rows)
dNA missing data






Table 3-6. Comparison of microbial C:P ratios by treatment within each day for each water potential
Day Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
Treatment -0.1 kPa
Control 193:1 (AVb) 266:1 (Ac) 375:1 (Aa) 50:1 (Ae) 163:1 (Ad) 80:1 (Ae) 84:1 (Ae)
30 kg P ha' 199:1 (Aa) 30:1 (Ba) 14:1 (Ba) 23:1 (Aa) 17:1 (Ba) 11:1 (Ba) 18:1 (ABa)
60 kg P ha-' 202:1 (Aa) 7:1 (Bc) 16:1 (Bc) 13:1 (Ac) 12:1 (Bc) 8:1 (Bc) 20:1 (ABb)
100 kg P ha-' 86:1 (Ba) 7:1 (Bb) 20:1 (Bb) 13:1 (Ab) 10:1 (Bb) 2:1 (Bb) 14:1 (Bb)
DAP 152:1 (Ab) 26:1 (Ba) 31:1 (ABa) 38:1 (Aa) 8:1 (Ba) 38:1 (ABa) 28:1 (ABa)

-3 kPa
Control 112:1 (Ba) 100:1 (Aa) 46:1 (Aa) 140:1 (Aa) 31:1 (Aa) 34:1 (Aa) 76:1 (Aa)
30 kgP ha' 19:1 (Bb) 6:1 (Bb) 13:1 (Ab) 93:1 (ABa) 5:1 (Ab) 11:1 (Ab) 11:1 (Bb)
60 kg P ha-' 63:1 (Ba) 26:1 (Ba) 13:1 (Aa) 33:1 (BCa) 5:1 (Aa) 101:1 (Aa) 12:1 (Ba)
100 kg P ha1 173:1 (Aa) 10:1 (Bb) 7:1 (Ab) 16:1 (Cb) 9:1 (Ab) NAC 17:1 (ABab)
DAP 61:1 (Ba) 18:1 (Ba) 32:1 (Aa) 30:1 (BCa) 15:1 (Aa) NA 10:1 (ABa)

-8 kPa
Control 33:1 (Aa) 12:1 (Aa) 27:1 (Aa) 9:1 (Aa) 44:1 (Aa) 4:1 (Aa) 30:1 (Aa)
30kg Pha-' 44:1 (Aa) 5:1 (Aa) 7:1 (Aa) 3:1 (Aa) 8:1 (Aa) 27:1 (Aa) 25:1 (Aa)
60 kg P ha-' 31:1 (Aa) 9:1 (Aa) 7:1 (Aa) 7:1 (Aa) 31:1 (Aa) 2:1 (Aa) 3:1 (Aa)
100 kg P ha-' 25:1 (Aa) 2:1 (Aa) 7:1 (Aa) NA 12:1 (Aa) 5:1 (Aa) NA
DAP 48:1 (Aa) 21:1 (Aa) 18:1 (Aa) NA 45:1 (Aa) 27:1 (Aa) 13:1 (Aa)

-15 kPa
Control 30:1 (Aa) 34:1 (Aa) NA NA 23:1 (Aa) NA 53:1 (Aa)
30 kg P ha' 31:1 (Aa) 18:1 (Aa) 6:1 (Aa) NA 7:1 (Aa) NA 15:1 (Aa)
60 kg P ha' 25:1 (Aa) 12:1 (Aa) 3:1 (Aa) NA 14:1 (Aa) NA 15:1 (Aa)
100 kg P ha"' 28:1 (Aa) 5:1 (Aa) 3:1 (Aa) NA NA NA 27:1 (Aa)
DAP 32:1 (Aa) 59:1 (Aa) 5:1 (Aa) NA 7:1 (Aa) NA 3:1 (Aa)

-1000 kPa
Control 32:1 (Aa) 105:1 (Aa) 11:1 (Ab) 40:1 (Aa) NA NA 21:1 (Ba)
30 kg P ha 26:1 (Aab) 15:1 (Bab) 16:1 (Aab) 14:1 (Ab) 4:1 (Ab) NA 63:1 (ABa)
60 kg P ha 34:1 (Ab) 24:1 (Bb) 4:1 (Ab) 12:1 (Ab) 43:1 (Ab) NA 100:1 (Aa)
100 kg P ha' 19:1 (Aa) 15:1 (Ba) 8:1 (Aa) 3:1 (Aa) 8:1 (Aa) NA 38:1 (ABa)
DAP 51:1 (Aa) 13:1 (Ba) 6:1 (Aa) 12:1 (Aa) 27:1 (Aa) NA 14:1 (Ba)
'Significant differences (P<0.05) between treatments within each day and water potential (upper case letters down columns)
bSignificant differences (P<0.05) between days within each treatment and water potential (lower case letters across rows)
CNA- data missing










Table 3-7. Comparison of microbial P by water potential within each day for each
treatment
Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
Water --------------------------------------------pg g'- soil --------------------
Potential Control
-0.1 kPa 1.0 (Da) 1.2 (C) 1.2 (B) 2.5 (B) 1.7 (B) 1.8 (B) 2.4 (A)
-3 kPa 1.8 (CD) 1.5(BC) 1.4 (B) 1.6(B) 1.7(B) 1.7(B) 1.2 (B)
-8 kPa 3.6 (BC) 4.9 (A) 5.2 (A) 5.3 (A) 3.5 (AB) 4.3 (A) 2.9 (A)
-15kPa 8.1 (A) 5.9 (A) 7.2 (A) 5.9 (A) 4.7 (A) NAb 4.7 (A)
-1000 kPa 4.0 (AB) 3.5 (AB) 5.0 (A) 4.5 (A) 4.2 (A) NA 3.3 (A)

30 kg P ha-'
-0.1 kPa 0.9 (D) 17.7 (A) 8.9 (A) 8.2 (AB) 15.6 (A) 9.0 (A) 11.5 (A)
-3 kPa 3.2(B) 24.4 (A) 8.1 (A) 5.1 (B) 13.3 (A) 8.3 (A) 7.4(A)
-8 kPa 1.5 (C) 21.3(A) 16.7 (A) 14.8 (A) 11.6 (A) 5.2 (A) 5.0 (B)
-15 kPa 7.9 (A) 30.7 (A) 22.5 (A) 8.9 (AB) 9.3 (A) NA 13.0 (A)
-1000 kPa 3.7 (AB) 19.2 (A) 13.9 (A) 14.6 (A) 19.1 (A) NA 11.1 (A)

60 kg P ha-'
-0.1 kPa 1.0 (C) 32.5 (A) 9.7 (B) 19.7 (A) 27.4 (A) 28.4 (A) 6.6 (AB)
-3 kPa 1.5 (C) 16.7 (B) 7.5 (B) 11.6(A) 25.9 (AB) 13.6(A) 9.1 (AB)
-8 kPa 2.9 (B) 21.7 (AB) 11.3(B) 12.1 (AB) 6.0 (B) 12.6 (A) 8.6 (AB)
-15 kPa 9.6 (A) 16.6 (AB) 37.1 (A) 26.05 (A) 7.5 (B) NA 14.5 (A)
-1000kPa 3.3 (B) 11.2(AB) 30.9(A) 18.0 (A) 10.0 (AB) NA 6.2 (B)

100 kg P ha"'
-0.1 kPa 13 (D) 24.0 (A) 40.4 (AB) 23.6(AB) 37.7 (A) 22.4 (A) 14.3 (AB)
-3 kPa 1.4 (C) 27.1 (A) 25.2 (B) 20.3 (AB) 17.7 (AB) NA 4.1 (B)
-8 kPa 2.7 (BC) 33.6 (A) 30.1 (B) 16.7 (B) 14.4 (AB) 16.2 (A) 19.8 (A)
-15 kPa 7.8 (A) 37.1 (A) 52.2 (A) 21.3 (AB) 16.8 (AB) NA 14.4 (AB)
-1000 kPa 3.7 (AB) 23.4 (A) 20.0 (B) 30.1 (A) 9.6 (B) NA 13.8 (AB)

DAP


-0.01 kPa 0.5 (C) 64.2 (B) 56.7 (B) 33.4 (B) 203.2 (A) 111.2 (A) 94.2 (AB)
-3 kPa 1.9(B) 49.5 (B) 37.1 (C) 122.5 (A) 16.1 (C) NA 32.2 (B)
-8 kPa 2.0 (B) 190.3 (A) 245.5 (A) 314.9 (A) 49.8 (B) 64.8 (A) 62.7 (AB)
-15 kPa 7.8 (A) 193.7 (A) 266.2 (A) 273.1 (A) 82.2 (AB) NA 186.7 (A)
-1000 kPa 2.2 (AB) 107.9 (A) 102.0 (AB) 207.8 (A) 77.0 (B) NA 130.9 (AB)
'Significant differences (P<0.05) between water potentials within each day and treatment
bNA data missing























9
S8 *-0.1 kPa
0 E -3 kPa
7 7
S-8 kPa
6 l -15 kPa
o 52 -a y I I 4 *-1000O kPa
b4






0 3 7 14 28 49 77
Days from beginning of study


Figure 3-5. Microbial P by water potential in the control









Table 3-8. Comparison of microbial C by water potential within each day for each
treatment
Day 0 Day 3 Day 7 Day 14 Day 28 Day 49 Day 77
Water -------------------------------------g--- g soil------------------ -------
Potential Control
-0.1 kPa 105(Ba) 184(AB) 427(A) 105 (A) 213(A) 180(A) 171 (AB)
-3kPa 200 (AB) 128(BC) 302 (A) 209 (A) 61 (B) 46 (B) 83 (B)
-8 kPa 122 (B) 53 (C) 136 (B) 32 (B) 140 (AB) 18(B) 75 (B)
-15 kPa 245 (A) 208 (AB) NAb NA 96 (B) NA 250 (A)
-1000kPa 112(B) 284 (A) 56 (B) 171 (A) NA NA 77 (B)

30 kg P ha-'
-0.1 kPa 161 (AB) 173 (AB) 62(B) 180 (A) 261 (A) 164 (A) 175(B)
-3kPa 109 (B) 90 (B) 137 (AB) 217 (A) 67 (B) 76 (A) 54 (B)
-8 kPa 87 (B) 150 (B) 82 (AB) 18 (B) 59 (B) 69 (A) 71 (B)
-15kPa 231 (A) 148 (B) 61 (B) NA 55 (B) NA 156 (B)
-1000 kPa 63 (B) 267 (A) 183 (A) 144 (AB) 76 (AB) NA 682 (A)

60 kg P ha'
-0.1 kPa 209 (A) 174 (A) 172 (A) 170(AB) 209 (A) 179 (A) 126 (BC)
-3 kPa 129 (AB) 98 (BC) 181 (A) 258 (A) 111 (AB) 243 (A) 62 (C)
-8 kPa 88 (B) 33 (C) 83 (A) 80 (B) 76 (B) 27 (B) 29 (C)
-15 kPa 237 (A) 155 (B) 112 (A) NA 113(AB) NA 217 (B)
-1000kPa 113(AB) 266 (A) 93 (A) 109 (B) 107 (AB) NA 475 (A)

100 kg P ha-l
-0.1 kPa 46 (B) 162 (B) 142 (A) 102 (A) 426 (A) 108 (A) 92 (C)
-3kPa 198 (A) 199(B) 138 (A) 183 (A) 137(B) 139 (A) 64 (C)
-8 kPa 72 (B) 67 (B) 116 (A) NA 98 (B) 157 (A) NA
-15 kPa 223 (A) 212 (B) 100 (A) NA 17 (B) NA 285 (B)
-1000 kPa 66 (B) 332 (A) 70 (A) 95 (A) 88 (B) NA 533 (A)

DAP
-0.1 kPa 348 (A) 1338 (B) 1759 (A) 1255 (B) 1555 (A) 2908 (B) 891 (B)
-3kPa 161 (AB) 1716(B) 1031(A) 3433 (A) 766 (B) 942 (B) 316(B)
-8kPa 104 (B) 2122 (B) 1106(A) NA 919(AB) 5918(A) 786 (B)
-15kPa 207 (A) 9470 (A) 783 (A) NA 549 (B) NA 1720 (AB)
-1000 kPa 157 (AB) 1120(B) 942 (A) 1206 (B) 768 (B) NA 2953 (A)
aSignificant differences (P<0.05) between water potentials within each treatment and day
bNA data missing














CHAPTER 4
SUMMARY AND CONCLUSIONS

The microbial biomass represents an important component influencing nutrient

cycling and nutrient bioavailability. The acidic, sandy soils in the southeastern U.S. are

phosphorus (P) limited for forest production and hence are commonly fertilized with P.

The surface horizon of these soils have very little or no P retention capacity, resulting in

the unique condition where P fertilizer may be leached from the A horizon. The

microbial biomass may represent a significant sink and, later, a source of labile P for

plant uptake, particularly for soils with little or no P retention capacity. The ability of the

microbial biomass to immobilize and retain P fertilizer before it is leached below

seedling rooting depth would determine how important it might be as a source of

bioavailable P. Spodosols in the southeastern U.S. are defined by a fluctuating water

table that influences, along with rainfall, the water potential of the surface horizon. Since

microbial communities are linked to water potential, it is reasonable to assume that water

potential may also influence microbial immobilization of P fertilizer. To determine a

more accurate estimate of microbial P, a Kp factor, or correction value, is used. Kp factors

are dependent upon microbial communities, so the factors may also be affected by water

potential.

Realizing the interactions between microbial communities, biomass, and water

potential, the six objectives of Chapters 2 and 3 can be discussed under the four more

broadly stated objectives: 1) to determine the most appropriate extractant for the

determination of microbial K, factors and for estimating microbial P on acidic Spodosols









of the Coastal Plain; 2) to determine if Kp changes with water potential and soil horizon

characteristics; 3) to determine if microbial biomass and microbial P respond to P

fertilizer and how those responses progress over time, and 4) to determine if water

potential affects immobilization and retention of P fertilizer.

Objective 1: Determine the Most Appropriate Extractant for the Determination of
K, Factors and Estimating Microbial P on Acidic Spodosols of the Coastal Plain.

The hypothsis that 0.5 MNaHCO3 (pH 8.5) would not be suitable for extracting

microbial P on acidic soils in Spodosols was accepted. Oxalate was found to be more

consistent in removing inorganic P (P,) from the microbial biomass than the other

extractants, including NaHCO3. As the Bh horizon is dominated by amorphous Al-

oxides, oxalate extracted more microbial P and is better suited for extracting P, due to it's

ability to replace P through ligand exchange and dissolution of Al-oxide surfaces. In the

A and E horizon, oxalate performed similarly to NaHCO3 as most of the P, in these

horizons was water-soluble. However, the high pH (8.5) of NaHCO3 dissolves organic

compounds that creates interference problems for colorimetric analysis. Therefore, due

to the improved P extraction and the ease of analysis, oxalate is recommended. Of the

oxalate concentrations tested, 3 mM oxalate was recommended due to its greater ability

to recover P,, especially in the Bh horizon.

Objective 2. Determine if K Changes with Water Potential and Soil Horizon
Characteristics.

K, factors were affected by water potential and other soil horizon characteristics,

nullifying the hypothesis that soil water potential would not cause changes in Kp factors.

The highest K, factor was generally detected under the wettest soil condition, indicating

more P, was extractable from those microbial populations. The lower K, factors occurred

in the A and E horizons near field capacity (-8 kPa), while in the Bh horizon, the lowest









K, factor occurred in the -3 kPa soil. Significant differences in K, factors were evident

between horizons at similar water potentials, with the E horizon providing the highest Kp

factors and the Bh horizon the lowest. Changes in dominant microbial species within the

population, along with changing bacterial to fungal ratios due to differences in water

potential and soil characteristics, are suggested as the cause for the changes in Kp factors.

This study recommends that a Kp factor must be measured for each soil condition to get

the most accurate estimate of microbial P.

Objective 3. Determine if Microbial Biomass and Microbial P Respond to P
Fertilizer and How Those Responses Develop Over Time.

No clear trend of P-only fertilization could be seen on microbial C, suggesting

that microbial biomass was not P limited. In contrast to microbial C, microbial P

responded quickly to the addition of P-only fertilizer. Microbial P increased significantly

three days following fertilization and then decreased over time after the initial

immobilization peak. Microbial P increased from six to 18 times after fertilization and

was three to six times higher than the control at the end of the study, indicating microbial

P would continue to be higher than the control over a longer period of time.

Microbial P increased with increasing P rates, but the lower P rates did not differ

from the control after day 3. The highest P rate was different than the control, but not

from the other P-only treatments. The non-linear increase in microbial P suggested an

upper limit of P that the microbial biomass could immobilize under these conditions,

approximately 45 to 85 kg P ha ', depending on the water potential. When large

quantities of both nitrogen (N) and P were added, microbial C and microbial P responded

dramatically. This not only indicated that microbial biomass was N limited, but also that

the microbial biomass was able to immobilize a large quantity of P when made available









once the limiting factor, N, was removed. The microbial biomass was able to sequester

between 15 and 29 kg P ha in the P-only fertilization and up to 44 kg P ha-' when a

large quantity of both N and P was added.

The quantity of P immobilized by the microbial biomass was considerably more

than the annual demand of pine plantations. These data indicate that the microbial

biomass would be able to take up a significant quantity ofP and retain it over time. This

labile P source would become slowly available to plants over time as microbial turnover

occurred.

Objective 4. Determine if Differing Water Potential Affects Immobilization and
Retention of P Fertilizer

Before fertilization, microbial P was lowest in the wettest soil and highest in the

soil maintained at -15 kPa. After fertilization, microbial P increased dramatically and

was similar among all water potentials. More P was immobilized by the microorganisms

in the wettest soil, with microbial P increasing 11 to 29 times more than the drier soils.

Factors that may account for this occurrence include differences in microbial

populations' metabolic demand for P and sensitivity to limiting factors. Retention of P

in the microbial biomass did not seem to be affected by water potential.

Research Conclusions

This dissertation combined method evaluation and revision with investigations on

the role that microbial biomass has on sequestering P when plant biomass is not sufficient

to capture fertilization. The focus was on P fertilization of newly planted and herbicided

pine plantations developing on a typical flatwoods Spodosol of northern Florida. This

cover type and soil type represent a significant component of northern Florida and









southeastern Georgia where forest industry has a concentrated land base and pulp mills at

their highest density.

Phosphorus deficiencies are common on these soils so P fertilization is also

common. The Forest Soils program at the University of Florida has investigated a variety

of aspects P cycling in forested soils including work on fertilization practices, P uptake

by mature and young pine, organic P mineralization, inorganic P chemistry, and

microbial biomass changes due to long-term changes in forest productivity. This study

compliments those previous efforts and adds to the knowledge of: (1) how to measure

microbial P and (2) what is the potential of microbial P influencing the efficiency of P

fertilization practices.

The most adequate extractant for estimating microbial P in a sandy, acidic

Spodosol was 3 mM oxalate. The Kp factor for the most accurate estimate of microbial P

changed with soil horizon and soil water potential. It is recommended that Kp factors be

specific for changing soil conditions. Using an inaccurate Kp factor may cause significant

under or over estimations of microbial P. For example, if the Kp factor of 0.40 by

Brookes et al. (1982) was used for the A horizon at -0.01 kPa, microbial P would be

overestimated by 4 kg ha' in this soil. The microbial biomass in this soil at this stage of

stand development is not P limited but appears to be N limited. After fertilization, up to

three or more times the P than the annual demand by pine plantations will be sequestered

and held for several weeks. Although the microbial population may change due to

differences in water potential, P immobilization will not be affected. The sequestered P

would become slowly available to plants over time, but the timing of release with respect

to plant demand was not investigated and is an unknown.









Surprisingly, little information on K, factors is available in the literature. While K,

factors have been researched previously in the A horizon of soils having different

textures, prior to this study no information existed on how water potential affects K,

factors or if K, factors were influenced by soil horizons within the same soil profile. K,

factors determined in previous studies were from neutral or alkaline soils. Prior to this

study, no information was available for acidic soils of the southeastern U.S.

This research strengthened and expanded the limited information pertaining to K,

factors by demonstrating how significant differences resulted from varying water

potential and soil horizons in an acidic, sandy Spodosol. A standard method was revised

in order to make it more accurate for measuring microbial P in acidic soils. The method is

now simpler and more efficient in extracting P from the Bh horizon than the standard

extractant, 0.5 MNaHCO3.

Few studies have researched the relationship between P-only fertilization and

microbial P, and even fewer have done so on forest soils; and there is little information

on microbial biomass or microbial P in Flatwoods soils. Prior to this study there was no

information on the influence that water potential has on microbial P sequestration of P-

only fertilization. Prior to this study, it was not known that microbial P was not limited

by P bioavailability in this soil type; and prior to this study it was not clear that microbial

biomass could sequester enough P from P fertilization to supply the demands of a young,

developing pine stand. This study has significantly increased our knowledge of microbial

P in forested soils of the southeastern coastal plain and provided a guide as to what would

be productive avenues to continue this type of research. These are itemized below.









Future Research

The above results do not answer all the potential questions regarding the role that

microbial biomass plays in fertilizer P sequestration and subsequent bioavailability to a

developing pine stand. Future avenues of research that are logical extensions of this

dissertation are suggested below:

1. Extending the results of this study, it seems reasonable to think that K, factors
change with nutrient availability. Some microbial species may become more
dominant when they grow in a nutrient non-limiting environment. Since Kp is
related to microbial communities, this may cause changes in the Kp factor.
Measuring the Kp factors from soil with different rates of P fertilizer plus N and P
fertilizer should provide this information.

2. These data showed that microbial sequestration of fertilizer P was rapid, but the
time frame of these studies was too gross to adequately describe the kinetics of this
P absorption. A more refined time scale that defines these kinetics would better
describe the ability and rapidity of microbial P uptake. These kinetics are crucial if
one is to know if microbial uptake is in a competitive time frame with P leaching
following fertilization. A comparative study of these kinetics with and without
leaching events would further define how important P sequestering by the
microbial biomass can be.

3. Once the comparative kinetics are addressed, field tests of fertilizer events,
microbial sequestering, and the time subsequent release of microbial P to plants is
appropriate. Does microbial P release fertilizer P at a rate and in the appropriate
time frame to make the P available to developing pine. The mechanism of release
over time needs to be elucidated and tested under field conditions.

4. It was clear P did not limit microbial biomass. It was also clear that high rates of
DAP did overcome limitations to microbial biomass development. A factorial
approach to define what nutrient conditions limit microbial biomass development
and how the biomass responds to increasing nutrients would be useful. It could also
be done in conjunction with # 1 above.

5. Information on microbial biomass and microbial P in the spodic horizon is lacking
and should be addressed in both a laboratory and field study.












APPENDIX A
Kp FACTOR FIGURES


0.8

0.6

0.4

0.2

0.0


S-

A 'b
_ll l*h


0 NaHCO3
0 1 mM Oxalate
0 2mMOxalate
* 3 mM Oxalate
O Bray & Kurtz
0 Mehlich 1


-0.1 -3 -8 -15 -1000
Water potential (kPa)










Figure A-1. Inorganic Kp factors by extractant within each water potential for the a) A
horizon, b) E horizon, and c) Bh horizon


0
I
3















0.8


S0.6


0.4


0.2


0.0


-0.1 -3 -8 -15 -1000
Water potential (kPa)


1.0


0.8


S0.6


0.4


0.2 -


0.0
-0.1 -3 -8 -15 -1000
Water potential (kPa)


0 NaHCO3
0 1 mM Oxalate
0 2 mMOxalate
* 3mMOxalate
O Bray & Kurtz
O Mehlich 1


Figure A-1 continued













1.0


0.8


0.6


0.4


0.2


0.0 --
-0.1 -3 -8


Water potential (kPa)


-15


1.0


0.8


S0.6


0.4


0.2


0.0


-1000


0 NaHCO3
I 1 mMOxalate
0 2 mMOxalate
* 3 mMOxalate
O Bray & Kurtz
G Mehlich 1


-0.1 -3 -8 -15
Water potential (kPa)


-1000


Figure A-2. Total Kp factors by extractant within each water potential for the a) A horizon
and b) Bh horizon


a
0.






























NaHCO3 Oxl Ox2


Ox3 BK Mehlich 1


NaHCO3 Oxl Ox2 Ox3 BK Mehlich I


Figure A-3. Inorganic Kp factors by water potential within each extractant in the a) A
horizon, b) E horizon, and c) Bh horizon NaHCO3 = 0.5 MNaHCO3, Oxl = 1
mM oxalate, 0x2 = 2 mM oxalate, 0x3 = 3 mM oxalate, and BK = Bray and
Kurtz


0.2


0.0






b
1.0


0.8


0.6


0.4


0.2


0.0


* -0.1 kPa
0 -3 kPa
S-8 kPa
i-15 kPa
-1000 kPa
























C
1.0


0.8

*-0.1 kPa
0.6 0-3 kPa
[ -8 kPa

S0.4 O0-15 kPa
S-1000 kPa

0.2


0.0o
NaHCO3 Oxl 0x2 0x3 Mehlich 1


Figure A-3 continued














0.8

S0.6

0.4

0.2

0.0


NaHCO3 Oxl





Iit


0x2 0x3 BK Mehlich I


I9-0.1 kPa
0-3 kPa
M-8 kPa
0-15 kPa
S-1000 kPa


1.0

0.8

S0.6

0.4

0.2

0.0


NaHCO3 Oxi 0x2 0x3 Mehlich I



Figure A-4. Total Kp factors by water potential within each extactant for the a) A horizon
and b) Bh horizon NaHCO3 = 0.5 MNaHCO3, Oxl = 1 mMoxalate, 0x2 = 2
mM oxalate, 0x3 = 3 mMoxalate, and BK = Bray and Kurtz











1.0


0.8


c 0.6


0.4


0.2


0.0


-0.1 -3 -8 -15 -1000
Water potential (kPa)


O A Horizon
0 E Horizon
* Bh Horizon


1.0


0.8


i 0.6


0.4


0.2


0.0


-0.1 -3 -8 -15 -1000
Water potential (kPa)


Figure A-5. Inorganic Kp factors by horizon within each water potential in a) 0.5 M
NaHCO3, b) 1 mM oxalate. c) 2 mM oxalate, d) 3 mM oxalate, e) Bray and
Kurtz, and f) Mehlich 1










c
1.0


0.8


I 0.6


0.4


0.2


0.0


-0.1 -3 -8 -15 -1000

Water potential (kPa)


0 A Horizon
I E Horizon
* Bh Horizon


d
1.0


0.8


S0.6
S-s

0.4


0.2


0.0


Figure A-5 continued


-0.1 -3 -8 -15 -1000

Water potential (kPa)










1.0


0.8


0.6


0.4


0.2


0.0--
-0.1 -3 -8 -15 -1000
Water potential (kPa)


O A Horizon
I E Horizon
Bh Horizon
1.0


0.8


0.6


0.4


0.2


0.0 -. r
-0.1 -3 -8 -15 -1000
Water potential (kPa)


Figure A-5 continued












0.7 I

0.6 *.,

S 0.5 J

0.4

0.3

0.2

0.1

0.0
0.1


K, =0.489-
R2 = 0.65


0.127*log(y) + 0.038*log(Y)2


S-=-


Log water potential (-kPa)


I--- ----


'0*


K,= 0.696- 0.243*log(y)
0.219*log(Y)2 *
R2 = 0.92


K, = 0.346*log(y)0359
R2 = 0.63


Log water potential (-kPa)


Figure A-6. Regression curves for inorganic Kp factors in the A horizon for a) 0.5 M
NaHCO3, b) 1 mM oxalate, c) 2 mM oxalate, d) 3 mM oxalate, e) Bray and
Kurtz, and f) Mehlich I


w











0.8

0.7

0.6 "

0.5

0.4 -
Kp,=0.681 -0.052* (y)
S0.3 R2 = 0.85

0.2


0.0 -


K, =0.370*log(q)0 46'
R2 = 0.70


10
Log water potential (-kPa)


Kp= 0.664 0.008* (Y)2
R2 = 0.92


2
* .


100 1000


- 3 *


K, =0.389*log()( )341
R2 = 0.64


10
Log water potential (-kPa)


Figure A-6 continued










0.8

0.7
K,= 0.396 0.282*log(y) 0.008*log (Y)2
0.6 + 0.283* log(Y)3

0.5 R2 = 0.91 ,.
0.4 5 -


0 .3 -' "- "
SK, = 0.667

0.2 R2 = 0.95

0.1

0.0
0.1 1 10


100


Log water potential (-kPa)


J.8

0.7

0.6

0.5

Q 0.4

~0.3

0.2

0.1

0.0
0.1


K,= 0.198 0.00005* (y)
R2 = 0.55


-- --.----- .-.....
*


10
Log water potential (-kPa)


Figure A-6 continued


- 0.130*log(y)




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