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Journal of Environmental Quality 31:1748-1756 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Wetlands and Aquatic Processes

Bioavailability of Organic Phosphorus in a Submerged Aquatic Vegetation–Dominated Treatment Wetland

H. K. Panta, K. R. Reddy*,a and F. E. Dierbergb

a University of Florida, Soil & Water Science Dep., Institute of Food and Agricultural Sciences, P.O. Box 110510, Gainesville, FL 32611-0510
b DB Environmental Inc., Rockledge, FL 32955

* Corresponding author (krr{at}ufl.edu)

Received for publication November 7, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Enzymatic hydrolysis and mineralization of organic phosphorus (P) were determined in surface water samples collected from inflow and outflow of a submerged aquatic vegetation (SAV)–dominated treatment wetland of the Florida Everglades. Water samples were fractionated into three size fractions (>0.4 µm, <0.4 to >0.05 µm, and <0.05 µm) with a sequential flow filtration technique. The fractionated water samples were incubated to hydrolyze with alkaline phosphatase (APase) and phosphodiesterase (PDEase), and to mineralize at different redox and pH. Unlike APase, which hydrolyzed <=10% of organic P, PDEase hydrolyzed >=71% of organic P in unfiltered water from both inflow and outflow waters, suggesting the domination of bioavailable diester P in the water. Phosphodiesterase completely hydrolyzed organic P in the <0.4- to >0.05-µm and <0.05-µm fractions, as compared with <=35% in the >0.4-µm fraction. However, the P mineralization in inflow and outflow waters at different redox and pH showed that P associated with particulate > 0.4 µm had been mineralized the most. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy showed that surficial sediments from the inflow region contained a high proportion of polynucleotides, nucleoside monophosphates, and previously unreported glycerophosphoethanolamine and phosphoenolpyruvates. However, at the outflow, the relative proportion of polynucleotides and nucleoside monophosphates was reduced substantially. This suggests that the SAV wetland may sequester P via accretion of organic matter.

Abbreviations: APase, alkaline phosphatase • ATPase, adenosine triphosphatase • PDEase, phosphodiesterase • SAV, submerged aquatic vegetation • SRP, soluble reactive phosphorus • STA, stormwater treatment area • TP, total phosphorus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
MICROORGANISMS MINERALIZE organic P through the secretion of various phosphatases (Hino, 1989; Newman and Reddy, 1993; Pant and Warman, 2000). Similarly, plants can use organic P fractions by means of phosphatase activity (Beck et al., 1989; Pant et al., 1994a, b). Extracellular phosphatase activity in soils and waters could originate from plant roots (Bergstrom and McGill, 1981), fungi (Tarafdar and Junk, 1987), ectomycorrhizae (Bergstrom and McGill, 1981), or from bacteria (Tarafdar and Claassen, 1988). Regardless of the source of phosphatases, their activities are affected by biochemical changes (Burns, 1986). The prediction of P availability in the surface water cannot be solely based on bioassay or chemical tests (Williams et al., 1980). However, the potential availability can be estimated if incubated with sufficient phosphatases (Hayes et al., 2000). Different types of phosphatases and phosphate esters are known to occur in surface waters and soils and/or sediments. Alkaline phosphatase (APase) hydrolyzes phosphomonoester, while phosphodiesterase (PDEase) hydrolyzes phosphodiester (Hino, 1989; Pant and Warman, 2000), and adenosine triphosphatase (ATPase) hydrolyzes adenosine triphosphate (ATP). Thus, determining different phosphatase-induced hydrolyses would provide potential bioavailability of P compounds in the water column of stormwater treatment areas (STAs).

The rate-limiting step in mineralization of organic matter could be extracellular enzyme-induced hydrolysis of macromolecules (Pflugmacher et al., 1999). Aerobic decomposition of organic matter involves numerous enzymes, most of which are specific to individual groups of compounds, and each compound can be rapidly and completely metabolized by a single group of microorganisms (Canfield, 1994). Anaerobic microorganisms, on the other hand, are unable to degrade most high molecular weight organic compounds (Jorgensen and Bak, 1991) and may depend on fermentative microorganisms for the supply of metabolizable low molecular weight compounds (Kristensen et al., 1995). The components of organic matter of different complexities degrade at different rates, and, in general, the aerobic rates are greater than the anaerobic (Westrich and Berner, 1984; Lee, 1992; Sun et al., 1993a,b). Moreover, diel variations in surface water pH levels due to the photosynthesis–respiration mechanism of wetland plant communities may induce alteration in the solubility of P associated with Ca and Mg.

Eutrophication is a major problem in both freshwater and coastal water systems, which has led to numerous investigations on nutrient limitation of primary production in aquatic ecosystems. Data on freshwaters, including the Florida Everglades and some coastal systems, indicate that P is usually a primary limiting nutrient. Stormwater treatment areas (STAs) are constructed wetlands that have been built to reduce P loading to the northern Everglades to meet an interim standard of 50 µg P L-1. The STAs tend to accumulate organic matter because of the production of detrital materials from biota and their suppressed rates of degradation due to anoxic conditions. With the possibility that a 50 µg P L-1 effluent standard may be insufficient to protect the Everglades, the Everglades Forever Act (EFA) mandates the South Florida Water Management District to evaluate a series of supplemental technologies to achieve P reduction to as low as 10 µg L-1.

The study of the potential hydrolysis and mineralization of organic P in the STAs, especially in the outflow water, may provide vital information for the further removal of P and determination of the ecological significance of the released water to the Everglades. The extent of dissolved and particulate P mineralization in inflow and outflow waters could influence overall P removal efficiency of treatment wetlands. Therefore, this study was conducted to determine the potential (i) APase- and PDEase-induced hydrolysis of organic P and (ii) mineralization of P under different redox and pH conditions in the inflow and outflow waters of a treatment wetland in south Florida.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Site Description
The Everglades Forever Act (EFA Section 373.4592, Florida statutes) was enacted by the Florida Legislature in 1994 for the ecological restoration of the Everglades, an internationally known subtropical wetland ecosystem that covers about 0.8 million ha. This initiated a number of measures, including the construction of several STAs to treat the drainage water discharged from the Everglades Agricultural Area (EAA). The EAA is a large (240 000 ha) productive drainage basin in southern Florida with sugarcane (Saccharum officinarum L.) as a major crop. The STA-1 West (1545 ha) is one of the several STAs built to reduce P loading to the northern Everglades. The STA-1 West is built on agricultural land previously used to grow sugarcane, and is located in southern Florida at 26°38' N and 80°25' W (Fig. 1) . Water from the S-5A drainage basin, which drains the northeastern part of EAA, is distributed to two independent parallel treatment trains (Cells 1 and 3; Cells 2 and 4) separated by a levee. Treatment Cells 1 and 2 may remove the bulk of the P while Treatment Cells 3 and 4 provide final removal of P (polishing of the water; receives partially treated agricultural drainage waters) from the outflow water. This study was conducted in Cell 4, which occupies 146 ha and is maintained as a periphyton–submerged macrophyte community dominated by coontail (Ceratophyllum demersum L.) and southern naiad [Najas guadalupensis (Spreng.) Magnus]. Periphyton (green filamentous algae) also inhabits the cell at various densities throughout the year. Cell 4 receives water form nine culverts at the inflow and discharges water by five culverts at the outflow. The average hydraulic retention time in Cell 4 is approximately 5 d.



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  		Fig. 1. A schematic orientation of Cell 4 in the Stormwater Treatment Area (STA)-1 West of the Everglades.

 
Sample Collection
Thirteen liters of inflow water from each of the three representative culverts of the inflow of Cell 4 of the STA-1 West were obtained. Similarly, 57.5 L of outflow water was sampled from a representative culvert (situated in the middle) of the outflow of Cell 4. To obtain fractionated samples from the composite inflow water (iron = 14 µg L-1; total suspended solids = 2.0 mg L-1) and outflow water (iron = 7 µg L-1; total suspended solids = 0.3 mg L-1), a sequential tangential-flow filtration device was used (Fig. 2) . About 31.3 L of inflow and 49.5 L of outflow waters were used for the fractionation. From the unfiltered inflow and outflow waters, fractionated water samples of >0.4-, <=0.4- to >0.05-, and <=0.05-µm-sized particulate suspensions were obtained.



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  		Fig. 2. Sequential tangential-flow filtration procedure used in fractionations of the inflow and outflow waters.

 
In a sequential tangential-flow filtration device, the unfiltered water is first filtered through a 0.4-µm-pore-size polycarbonate filter (upper filter cassette in Fig. 2). Fluids and particles <0.4 µm penetrate the filter and are collected in a separate container. Particles >0.4 µm (0.4-µm retentate) are excluded from passing through the filter, and are therefore constantly recirculated through the outer chamber of the device until enough <0.4-µm particles and fluid have been collected. The <0.4-µm particles and fluid are then filtered with a 0.05-µm-pore-size polycarbonate filter in the device whereby particles >0.05 µm and <0.4 µm are excluded from passing through the filter, and are thus recirculated until enough volume of the permeate (i.e., <0.05 µm) has been collected. Since flow is parallel to the filter surface, unlike perpendicular as in most dead-end filtration devices, artifacts from concentration polarization that are typical of most dead-end stirred-cell filtration devices are minimized in the tangential-flow filtration device. Size ranges were selected based on general consensus for particulate, colloidal, and dissolved. Both 0.4- and 0.05-µm filter retentates of the inflow waters were concentrated by a factor of 6. For the outflow waters, the 0.4- and 0.05-µm filter retentates were concentrated by factors of 6.2 and 7.3, respectively. The selected properties of the water samples are given in Table 1. The potential enzymatic hydrolysis of organic P by different phosphatases, and the mineralization of organic P at varying pH under aerobic and anaerobic conditions, were determined. Multiple representative surficial sediments (0–2 cm) samples were also collected from both inflow and outflow regions of Cell 4. The samples were homogenized and composite samples were used to identify P compounds in the sediments that could be potential P sources to the water column.


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  		Table 1. Selected chemical characteristics of inflow and outflow waters of Cell 4 of the Stormwater Treatment Area (STA)-1 West of the Everglades.

 
Enzyme Incubation
Alkaline phosphatase, PDEase, and ATPase (obtained from Sigma–Aldrich, St. Louis, MO) were evaluated for their capacity to hydrolyze organic P in the fractionated outflow and inflow waters. Alkaline phosphatase (Type VII-T) is prepared from bovine (Bos taurus) intestinal mucosa suspended in 3.0 M NaCl containing 1 mM MgCl2, and 0.1 mM ZnCl2 and 30 mM triethanolamine at pH 7.6. It had 2000 to 4000 units of activity per milligram enzyme protein. One unit hydrolyzes 1.0 µmole of p-nitrophenyl phosphate per minute at pH 9.8 at 37°C. Similarly, PDEase is prepared from eastern diamondback rattlesnake (Crotalus adamanteus) venom, and is a lyophilized powder containing approximately 35% tris buffer salts with 0.2 to 0.4 units of activity per milligram solid at pH 8.8 at 37°C. One unit hydrolyzes 1.0 µmole of bis(p-nitrophenyl) phosphate per minute at pH 8.8 at 37°C. The ATPase is prepared from rabbit (Sylvilagus spp.) kidney, and is a lyophilized powder containing approximately 10% protein. It is balanced primarily in sucrose, and had 0.3 to 1.0 units of activity per milligram protein. One unit liberates 1.0 µmole of inorganic P from ATP per minute at pH 7.4 at 37°C in the presence of Na+, K+, and Mg2+.

Unless otherwise stated, all the buffer solutions were prepared from tris-HCl. All the enzymes were prepared in pH 8.0 buffer one hour before the experiment was performed. The PDEase and ATPase solutions contained 0.5 units enzyme activity mL-1. Similarly, APase containing 5 units enzyme activity mL-1 was prepared in pH 7.5, 8.0, and 9.0 buffers. Because of the high background soluble reactive phosphorus (SRP), both the PDEase and ATPase were dialyzed against pH 8.0 buffer for 10 h at 4°C to remove background SRP, using dialysis sacks prior to their use for the incubation (Pant et al., 1994a).

To inhibit microbial activities during the incubation, 50 µL of 5.2% sodium azide (NaN3) solution was added to the 10-mL triplicate samples (unfiltered water, 0.4-µm retentate, and retentate and permeate of a 0.05-µm filter of inflow and outflow waters) to yield a final concentration of 4 mmol L-1. Thereafter, 1 mL of enzyme solutions (APase, PDEase, and ATPase) were added to each vial separately and in combination, and incubated for 6 h at 25°C. The activity of each enzyme required for individual incubation was predetermined by measuring the organic P concentration in the samples, and adding the sufficient unit of enzymes. For the controls, 10-mL samples were spiked with 50 µL NaN3 and 1 mL buffer (pH 8.0), and incubated for 6 h at 25°C. To determine the chemical hydrolysis of organic P by buffer (pH 8.0) and the NaN3 separately and in combination, 10-mL samples were spiked with 50 µL NaN3 and 1 mL deionized water, and with 1.05 mL deionized water only, and incubated for 6 h at 25°C.

For the outflow water only, the samples (unfiltered water, 0.4-µm filter retentate, and retentate and permeate of the 0.05-µm filter) were also incubated at pH 7.5 and 9.0 to determine if organic P hydrolysis by APase at different pH values affected the bioavailability. Control experiments were also performed at the same pH values.

The amount of enzyme-hydrolyzed organic P during the 6-h incubation was calculated by subtracting SRP measured in the sample incubated without enzymes from the sample incubated with enzymes. This represented the potentially hydrolyzable amount of organic P in the samples.

Influence of Redox and pH on Organic Phosphorus Mineralization
Four hundred milliliters of each size fraction (unfiltered water, 0.4-µm filter retentate, and retentate and permeate of the 0.05-µm filter) of inflow and outflow surface waters were placed in duplicate reactors and incubated in the dark with frequent stirring on a magnetic stirrer for 30 d at 25 ± 2°C. The pH of the suspension was maintained at neutrality (approximately 7.5) by bubbling air containing 0.3% CO2. The pH and Eh of the incubated water samples under a given treatment are provided in Table 2. An aliquot of 25 mL water was sampled to measure SRP and pH levels in the waters at 0- and 8-h and 1-, 2-, 4-, 10-, 20-, and 30-d intervals. A similar set of samples (control) was also incubated as described above, but no attempt was made to adjust pH so that mineralization of P at ambient pH of the water (approximately 8.5) could be determined. However, air was bubbled frequently through the controls to maintain aerobic conditions, and the changes in pH as well as in SRP content were measured.


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  		Table 2. The pH and Eh of the samples incubated under different conditions.{dagger}

 
A similar set of experiments was also conducted under anoxic conditions by bubbling N2 + 0.3% CO2 intermittently, and the pH of the suspension was maintained near neutrality (approximately 7.5). An aliquot (25 mL) of water was sampled for pH and SRP determinations at 0- and 8-h and 1-, 2-, 4-, 10-, 20-, and 30-d intervals. As a control, N2 only was bubbled instead of N2 + 0.3% CO2, and the pH of the suspension was maintained at approximately 9.

For both the pH and redox treatments, and the controls, the potential mineralization rate of organic P was calculated by plotting the SRP release in the water vs. time. The measured SRP in the water represented net (resultant) increase in the concentrations over the period of incubations. The cumulative SRP released over the 30-d incubation period divided by total organic P (total phosphorus [TP] - SRP; operationally defined and may include molybdate-unreactive inorganic P) present in the sample provided the potentially mineralizable fraction of the organic P under the incubation conditions.

Phosphorus-31 Nuclear Magnetic Resonance Spectroscopic Analysis
To extract organic P, surficial sediments (40 g on a wet-weight basis) from both inflow and outflow were extracted twice with 80 mL 0.4 M NaOH, for four hours each, by shaking in an end-over-end shaker at 20 ± 2°C. After each extraction, the suspensions were centrifuged for 20 min at 5000 x g. The supernatants were combined and subjected to gel filtration. The extracts were fractionated with a G-25 Sephadex column (with a fractionation range of 100 to 5000 molecular weight; dry bead diameter = 20 to 80 µm; bed volume = 4 to 6 mL g-1; column volume = 75 mL; Amersham Pharmacia Biotech, Piscataway, NJ) as described by Pant et al. (1999) and Pant and Reddy (2001). The extract (20 mL) was pipetted onto the top of the column and eluted with demineralized water by pumping at a rate of 0.6 mL min-1. Eighty, 3-mL fractions were collected with a fraction collector. The fractions containing NaOH were separated with a litmus paper test. To check for loss of any forms of P, the NaOH fractions were tested for the presence of P. No NaOH was found up to the 49th fraction and no P was found after the 49th fraction. The fractions free from NaOH were combined and concentrated (10 times) in vacuum rotatory evaporator at 35°C.

Four milliliters of the concentrated extract was scanned in a 12-mm tube at 121.4688 MHz on a NT 300 31P nuclear magnetic resonance (NMR) spectrometer (Buszko et al., 1998) with a 90° pulse with a 5.0-s delay and sampling interval of 0.0000622 s. To obtain better signal to noise ratio, 10 000 scans were collected. To inhibit any possible microbial activities during the scan, 0.25 mL toluene was mixed with the sample prior to collection of the scans. The chemical shifts were determined with respect to an external standard of 85% phosphoric acid. The identification of peaks of different P compounds in the NMR spectra was done by comparing chemical shifts of the peaks with those of the references reported by Gadian et al. (1979) and Pant et al. (1999). Moreover, since the resonances are sensitive to pH (Navon et al., 1977; Preston, 1996) and salt concentration (Gadian, 1982), samples were spiked with 0.1 mL (10 mg P mL-1) of pyrophosphate (Na4HP2O7) as an internal standard.

Phosphorus Analysis
Soluble reactive P in all the samples was determined with an automated ascorbic acid method (Method 365.1; USEPA, 1993). Total P in the water samples and the sediment extracts were also determined by the above method after persulfate digestion (Method 365.1; USEPA, 1993). The difference between total phosphorus (TP) and SRP, occasionally referred as unreactive P, is considered as organic P in this study. Unless otherwise stated, all the experiments were performed in triplicates, and the data analyzed by two-way analysis of variance (ANOVA) with SAS (SAS Institute, 1996). The mean comparisons were done by least significant difference (LSD) at the p < 0.05 level.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Enzymatic Hydrolysis
After dialysis, the background SRP concentration of the commercially prepared PDEase was reduced to less than 1 µg L-1 from its initial 129 µg L-1. The proportionate removal of background SRP from ATPase (from 298 to 81 µg L-1) was not as high as from PDEase. Substantial increase in the release of SRP apparently from ATPase was obtained in incubation with the water samples, indicating instability of ATPase. Thus, experiments containing ATPase were discarded to avoid overestimation of bioavailability of organic P. Though adenosine triphosphate (ATP) could be derived into the surface water from decomposing aquatic plants or organisms, it is unlikely that the waters used for this study contained nondegraded ATP because of its unstable nature. Based on the control incubations, chemical hydrolysis of organic P in the inflow and outflow waters was not attributable to effects from the buffer or sodium azide (NaN3) that were used in incubation of size-fractionated surface waters with the enzymes.

Alkaline phosphatase hydrolyzed less than 10% of the total organic P in the unfiltered inflow water, while PDEase hydrolyzed 71% of the total organic P (Fig. 3) . A maximum hydrolysis by PDEase of only 27% of the organic P in the 0.4 µm retentate may suggest that relatively stable or unhydrolyzable P are mainly associated with particulates > 0.4 µm in diameter. Alkaline phosphatase did not hydrolyze organic P in the 0.05-µm retentate and permeate of inflow water, whereas PDEase hydrolyzed the P completely. This may indicate the lack of monoesterase-hydrolyzable P, and the domination of diesterase-hydrolyzable P in the retentate and permeate from the 0.05-µm filter. Since Cell 4 is a polishing cell (receiving partially treated agricultural drainage waters), the low APase-induced hydrolysis of the organic P in the inflow indicates that more labile P compounds were hydrolyzed in the treatment cells upstream of Cell 4, leaving only relatively stable P compounds entering Cell 4. The amount (76 µg L-1) and the potential hydrolysis (71% by PDEase) of organic P in the unfiltered water from the inflow of Cell 4 indicate that polishing cell could play a crucial role in removing P from the drainage water prior to its release into the Everglades.



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  		Fig. 3. Enzymatic hydrolysis of organic P in waters of Cell 4 of the Stormwater Treatment Area (STA)-1 West. The numbers in parentheses indicate the pH of the enzyme incubations. The term retn. is retentate and perm. is permeate. Bars indicate standard errors of the means.

 
Overall, organic P hydrolysis in the unfiltered outflow water by APase was about 8% of the total organic P (Fig. 3). The inability of APase to hydrolyze organic P in the unfiltered water of both inflow and outflow is not known; however, this may indicate the lack of monoester P, which is the substrate for APase. The effect of pH (7.5, 8.0, and 9.0) was not apparent in the hydrolysis of organic P by APase, probably due to the overall low hydrolysis. The complete hydrolysis of organic P in unfiltered, 0.05-µm retentate, and 0.05-µm permeate of outflow water by PDEase mimics the results for the inflow water, indicating that the organic P in both waters was dominated by diesterase-hydrolyzable compounds. As in the inflow water, only 35% of the organic P found in the 0.4-µm outflow retentate was hydrolyzed by PDEase. Considering the extent of the hydrolysis of organic P in unfiltered waters from both inflow and outflow, it appears that substantially low P hydrolysis even by PDEase occurred in the 0.4-µm retentate. The reason is not known; however, Harris and Farve (2001) reported the domination of diatoms and algae in 0.4-µm retentate, which are not directly accessible to the enzymes. Moreover, an increase in concentrations of inhibitory species including SRP could also suppress the enzyme activities (Rai et al., 1998; Kang and Freeman, 1999; Treseder and Vitousek, 2001). The complete hydrolysis of both the retentate and permeate of the 0.05-µm filter of the inflow and outflow surface waters by PDEase may suggest the domination of highly soluble diesterase-hydrolyzable P. Moreover, this also suggests that PDEase activity under field conditions is low in Cell 4.

Mineralization of Organic Phosphorus at Different Redox and pH
Under anaerobic high pH (approximately 9.2) conditions, overall organic P mineralization was low (<=10% of total organic P) in all size fractions of the inflow waters, though the mineralization was substantial in the incubated outflow waters (Fig. 4) . The elevated pH (approximately 9) could have led to an increase in precipitation of Ca-P (Diaz et al., 1993), and decrease in microbial activities (Kuehn et al., 2000). Under aerobic ambient pH (8.3) conditions, the P mineralization was low (<=9% of the total organic P) in the unfiltered water, 0.05-µm retentate, and 0.05-µm permeate of the inflow, but the mineralization was 39% of the total organic P in 0.4-µm retentate. This suggests that P associated with particulates >0.4 µm is relatively unstable compared with the other smaller size fractions including permeate of the 0.05-µm filter (dissolved P), because of the active nature of particulates to microbial decomposition (Stevenson, 1986; Correll, 1998).



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  		Fig. 4. Cumulative P mineralization in waters of Cell 4 of the Stormwater Treatment Area (STA)-1 West under different redox and pH conditions during 30 d of incubations. Bars indicate standard errors of the means.

 
In general, the cumulative organic P mineralization under aerobic or anaerobic conditions that were maintained at pH around neutral (approximately 7.4) was significantly higher compared with those maintained at higher pH conditions (ambient, approximately 8.4 and anaerobic, approximately 9.2) for both inflow and outflow waters (Fig. 4). Perhaps an increase of microbial activities with decreased pH (from 8.4 to 7.4) may have stimulated the P accumulation by microorganisms under aerobic conditions, while under anaerobic conditions, precipitation of Ca-P may have occurred due to an increase in pH to 9.2. In general, no significant difference (p <= 0.05) in the cumulative P mineralization was observed in 0.05-µm permeates of the inflow and outflow waters under any treatment conditions, probably because of the very low average mineralization rates (<0.4 µg L-1 d-1).

A higher cumulative P mineralization was expected at pH 7.4 under aerobic than anaerobic conditions because of the expected higher microbial activity under aerobic conditions (Pettersson, 1980; Newman and Reddy, 1993). However, no significant difference was observed between aerobic and anaerobic incubations at pH 7.4 in either source of water (Fig. 4). Both conditions (air + CO2 and N2 + CO2) yielded similar pH (approximately 7.5) as well as the percent P mineralization. This may indicate that pH and/or anaerobic respiration were more important than aerobic respiration in affecting the P mineralization in these water samples; otherwise, greater P mineralization was expected under aerobic conditions.

An overall higher percent of organic P mineralization occurred in 0.4-µm retentate than the other size fractions for both inflow and outflow waters (Fig. 4). This suggests that P associated with particulates > 0.4 µm is not as stable as the P associated with smaller size fractions, in contrast to the findings of enzymatic hydrolysis experiments. The discrepancy between the mineralization and enzymatic hydrolysis of P associated with particulate >0.4 µm may have been due to the supply of the energy sources to bacterial communities. According to the carbon (C) limitation theory (Currie and Kalff, 1984a,b,c), the supply of C apparently controls the bacterial mineralization of organic P, especially in C limiting systems. Since C is more abundant in particulate than dissolved fractions, the consequent preferential utilization of particulates by bacteria induces P mineralization.

In general, the 24-h (potential) P mineralization rates were significantly greater than the 30-d (average) P mineralization rates both in the inflow and outflow waters (Table 3), indicating that readily degradable P compounds were a more significant factor in the shorter incubation period. The 30-d incubation period represents a closer approximation to the hydraulic retention time of the STAs, and even though the P mineralization rates were generally low, there would still be a net removal of P as the mineralized P is biologically and chemically removed.


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  		Table 3. Phosphorus mineralization rates in inflow and outflow waters of the submerged aquatic vegetation (SAV)–dominated Cell 4 in the Stormwater Treatment Area (STA)-1 West under different incubation times and conditions.{dagger}

 
Phosphorus Distributions in Surficial Sediments
Stability of P in surficial sediments would be crucial because it can be a potential source of P to the water column of the STA in an event of resuspension, diffusion, bioturbation, or desiccation. The 31P nuclear magnetic resonance analysis of NaOH extracts of the surficial sediments (P in the extracts, 1178 [84% of TP] and 540 [92% of TP] mg kg-1 dry sediments from inflow and outflow, respectively) revealed relatively high proportions of active forms of organic P, including previously unreported phosphoenolpyruvates (PEP) and glycerophosphoethanolamines (GPEA) (Fig. 5) . The surficial sediments from the inflow region of the cell contained substantial proportions of glycerophosphates (glyP), nucleoside monophosphates (NMP), polynucleotides (polyN), GPEA, and PEP, though polyN, GPEA, and PEP were the dominant forms of organic P. Similarly, the sediments from the outflow region contained glyP, NMP, polyN, GPEA, and PEP as the major forms of organic P; however, the relative proportions of polyN and GPEA were substantially lower, unlike those in the sediments from the inflow region, suggesting their comparatively low stability. The accretion of the surficial sediments into organic matter in STAs could effectively sequester P. Dierberg et al. (2002) reported a consistent removal of P by Cell 4 during the period of May 1995 to April 1999, supporting the notion that SAV-dominated systems can have high efficiency for P removal from drainage water.



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  		Fig. 5. Phosphorus distributions in surficial sediments from Cell 4 of the Stormwater Treatment Area (STA)-1 West. PEP, phosphoenolpyruvates; glyP, glycerophosphates; NMP, nucleoside monophosphates; polyN, polynucleotides; and GPEA, glycerophosphoethanolamines.

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The hydrolysis of organic P of the inflow and the outflow water of Cell 4 of STA-1 West of the Everglades can be high, if optimal supply of appropriate enzymes is present under field conditions. Although laboratory studies have shown complete hydrolysis of organic P, it is very unlikely high activity of enzymes exists under field conditions, resulting in export of dissolved organic P. The occurrence of a significant amount of organic P in particulates >0.4 mm of the inflow water, and its bioavailability, may indicate the potential of further P removal in the SAV-dominated Cell 4 of STA-1 West. However, about 63% of the organic P in the outflow water was associated with particulates that had low potential for enzymatic hydrolysis. Complete removal of organic P does not occur within Cell 4 because organic P is continuously generated within the cell by the resident biological communities. These results, coupled with lower mineralization of organic P in the outflow water under anaerobic conditions at higher pH, suggest that organic P exported from Cell 4 may not be readily available under field conditions.


   ACKNOWLEDGMENTS
 
The authors are thankful to J.E. Colson, I.C. Torres, and H.M. Spencer (University of Florida, Soil and Water Science Dep.) and T.A. DeBusk (DB Environmental Inc.) for their invaluable help in experimental work. This research was supported by the Florida Agricultural Experiment Station, and partial grants from the South Florida Water Management District, the Florida Dep. of Environmental Protection, and the U.S. Environmental Protection Agency. Florida Agricultural Experiment Station Journal Series no. R-08509.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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