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a Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancón, Republic of Panama
b Everglades Division, South Florida Water Management District, 3301 Gun Club Road, West Palm Beach, FL 33406
* Corresponding author (turnerbl{at}si.edu)
Received for publication February 17, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: NMR, nuclear magnetic resonance • WCA, Water Conservation Area
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In terms of their behavior, inositol phosphates react strongly in soil to form insoluble complexes and there is little evidence of their uptake by plants (Turner et al., 2002). Other compounds, such as nucleic acids and their breakdown products, are less stable and may contribute to plant nutrition (Bowman and Cole, 1978). Phosphate is the main inorganic form, although condensed inorganic phosphates (pyro- and polyphosphate) that require hydrolysis before plant uptake may be also present.
Given the diversity of P compounds present in soils, structural information is a fundamental prerequisite to understanding the biogeochemistry of soil P. Yet the P composition of wetland soils is almost unknown. Most studies focus on inorganic phosphate, with organic P typically measured only as part of a sequential fractionation scheme (Ivanoff et al., 1998), although studies from Florida used solution 31P NMR spectroscopy to analyze recently reflooded organic agricultural soils (Robinson et al., 1998) and benthic floc from a constructed wetland (Pant and Reddy, 2001). We addressed the lack of information on organic P in wetland soils by analyzing samples from nutrient-enrichment gradients in two contrasting wetlands in the Florida Everglades. Our aim was to determine the compounds likely to be involved in P cycling in subtropical wetlands.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Samples were taken from two impounded wetlands, termed Water Conservation Areas, in the northern Everglades (Fig. 1) . Both wetlands receive approximately half of their water from rainfall and half from surface discharge via culverts and pump stations (calculated from data in Sklar et al., 2002). Water Conservation Area 1 (WCA 1) is the only remaining soft water portion of the Everglades. Established as a wildlife refuge in 1951, it is enclosed within 90 km of levees and canals and encompasses 59000 ha of the northern-most remnant of Everglades habitat. As a result of canal water input there are distinct P and mineral gradients extending into WCA 1 from the western boundary to the interior of the marsh, although canal-water intrusions are generally restricted to the marsh perimeter (Newman et al., 1997). Soils within WCA 1 are Histosols dominated by Loxahatchee Peat, which is acidic and has the lowest ash content of peats in South Florida (Gleason, 1984). Water leaves WCA 1 primarily through culverts along the southern levee (labeled S10s on Fig. 1).
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In both wetlands, P-enriched areas are characterized by dense mono-specific stands of cattail (Typha spp.), while interior sites are characterized by sawgrass (Cladium jamaicense Crantz) ridges interspersed with open-water sloughs. The periphyton community in WCA 1 is an assemblage of desmids (unicellular green algae) and diatoms adapted to the soft water conditions, whereas the sloughs in WCA 2A are dominated by calcareous periphyton mats comprised of Ca-precipitating cyanobacteria and diatoms (McCormick et al., 2001). Other key distinctions between the soft water and calcareous sloughs are that calcerous sloughs contain a more cohesive benthic mat and fewer emergent macrophytes.
Samples for NMR analysis were all collected on a single day in June 2003. Sites were selected to represent P enriched (F1, X1) and unenriched (U3, X4) sites in both conservation areas. In addition, samples were collected from sites considered to represent the transition between P-enriched and unenriched conditions (X2, F4). The distinct soft water nature of WCA 1 was assessed by collecting samples from an additional site in the heart of the marsh where experimental mesocosms are located (Mesocosm).
At each site, three replicate cores (10-cm diameter) were taken to 10 cm depth in the organic soil layer. Benthic unconsolidated flocculent material (floc), which included plant detritus and algae, was separated from underlying soil in the field. Samples were transported on ice to the laboratory where they were immediately frozen at –80°C to halt possible nutrient transformations. Time from sampling to freezing was 2 d. Frozen samples were lyophilized and ground to pass a 2-mm sieve. Replicate samples from each site were analyzed separately to provide information on field variability.
Determination of Chemical Properties
Total C and N were determined by combustion and gas chromatography using a Flash EA1112 CNH analyzer (CE Elantech, Lakewood, NJ). Soil pH was determined in a 1:20 ratio of lyophilized soil to deionized water (approximate 1:2 on a wet weight basis). Total Al, Ca, and Fe were determined by digestion of a 0.5-g sample in concentrated HNO3 (8 mL) and HClO4 (5 mL) (Olsen and Sommers, 1982), with detection by inductively coupled plasma–optical emission spectrometry (ICP–OES). Total phosphorus was determined by automated molybdate colorimetry following ashing at 550°C for 3 h and digestion in 6 M HCl. Surface water samples were filtered through 0.45-µm polyethersulfone membranes (Environmental Systems, Ann Arbor, MI) and analyzed for total P (EPA 365.4), reactive P (EPA 365.1), nitrate plus nitrite (EPA 353.2), organic carbon (EPA 415.1), and calcium and iron (EPA 200.7) by standard procedures (USEPA, 1983). Surface water chemistry of the sampling sites is shown in Table 1.
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For solution 31P NMR spectroscopy, each freeze-dried extract (approximately 100 mg) was redissolved in 0.1 mL of deuterium oxide and 0.9 mL of a solution containing 1 M NaOH and 0.1 M EDTA, then transferred to a 5-mm NMR tube. The deuterium oxide provided an NMR signal lock and the NaOH raised the pH to >13 to ensure consistent chemical shifts and optimum spectral resolution. Inclusion of EDTA in the NMR tube reduces line broadening by chelating free Fe in solution (Turner and Richardson, 2004).
Solution 31P NMR spectra were obtained using a Bruker (Billerica, MA) Avance DRX 500 MHz spectrometer operating at 202.456 MHz for 31P and 500.134 MHz for 1H. Samples were analyzed using a 6-µs pulse (45°), a delay time of 1.0 s, and an acquisition time of 0.2 s. Between 48000 and 69000 scans were acquired depending on the P concentration of the lyophilized extract, and broadband proton decoupling was used for all samples. Chemical shifts of signals were determined in parts per million (ppm) relative to an external standard of 85% H3PO4. Signals were assigned to individual P compounds or functional groups based on literature reports (Turner et al., 2003b) and signal areas calculated by integration. Spectra were plotted with a line broadening of 8 Hz, although additional spectra were plotted with a line broadening of 1 Hz to preserve fine resolution in the phosphate monoester region.
Statistical Analysis
Data are reported as means ± standard deviation of three replicate cores, which indicates spatial variability at each site. The statistical significance of the difference between means along the nutrient gradients was determined using one-way analysis of variance, with least significant difference of means (5%) shown in Table 2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In WCA 2A, changes in P concentrations along the enrichment gradient were only significant in the benthic floc (p < 0.001). The highest P concentration was detected at the transitional site (1.62 ± 0.13 g P kg–1 dry wt.), decreasing to 0.31 ± 0.13 g P kg–1 dry wt. in the unenriched calcareous slough (Table 2). In the soil of WCA 2A, P concentrations ranged from 0.67 ± 0.52 g P kg–1 dry wt. at the inflow, to 0.43 ± 0.16 g P kg–1 dry wt. in the unenriched slough, but were not significantly different (p = 0.73) due to the marked variability at the enriched site. At all sites in both Water Conservation Areas, P concentrations were smaller in the soil than the benthic floc (p < 0.05), although the exception was the unenriched calcareous slough in WCA 2A.
There were significant differences (p < 0.01) in concentrations of C, N, Al, Ca, and Fe in benthic floc along the enrichment gradients in both Water Conservation Areas. For soils, there were significant differences in all elements along the enrichment gradients except for C (p = 0.08) and Al (p = 0.40) in WCA 1 and Al (p = 0.20) in WCA 2A. Carbon concentrations were relatively similar among sites, although the concentration in the benthic floc from the unenriched slough in WCA 2A was noticeably lowest (Table 2). Nitrogen concentrations in both Water Conservation Areas were greater in unenriched slough sites for both floc and soil, while for benthic floc in WCA 2A the transitional site contained a high total N concentration (42.0 ± 1.6 g N kg–1 dry wt.) compared to the other sites (Table 2). Much of the C in this floc sample almost certainly occurred as CaCO3.
There were significant differences in the C to N and N to P ratios (p < 0.001) in both benthic floc and soil along enrichment gradients in both Water Conservation Areas, with the exception of the N to P ratio for the soil of WCA 2A. The C to N ratios generally decreased with distance from the pollutant inflow, while N to P ratios increased. For example, N to P mass ratios in the benthic floc of WCA 1 increased more than fourfold along the enrichment gradient, from 17 ± 2 at the enriched site, to 73 ± 6 in the unenriched slough in the marsh interior (Mesocosm site). Ratios were similar throughout WCA 2A and were greater in soil than benthic floc, with a maximum value of 137 ± 10 in soil of the unenriched slough (Mesocosm site) in WCA 1.
One of the major chemical differences between the two Water Conservation Areas was Ca concentration. A clear Ca enrichment gradient was evident in benthic floc and soil from WCA 1, with Ca concentrations in benthic floc decreasing from 40.0 ± 2.7 g Ca kg–1 dry wt. in the enriched site, to 11.1 ± 0.2 g Ca kg–1 dry wt. in the unenriched slough (Mesocosm site). The greatest Ca concentration was in the benthic floc of the calcareous slough in WCA 2A (204.6 ± 31.8 g Ca kg–1 dry wt.). Aluminum and Fe showed no clear trends, although a high Fe concentration was detected in soil of the unenriched calcareous slough in WCA 2A.
Phosphorus Composition by Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy
Phosphorus recovery was generally greatest from benthic floc in WCA 1 (55–66%), although the highest value was for benthic floc from the transitional site in WCA 2A (74%) (Table 3). Phosphorus recovery was less efficient from soil, but was relatively consistent, being 47 to 58% for soils from WCA 1 and 37 to 46% for soils from WCA 2A. The lowest P recovery was for benthic floc from the unenriched calcareous slough in WCA 2A (24%).
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Signals between 4 and 6 ppm were assigned to phosphate monoesters, which constituted a large proportion of the extracted P in all samples. Concentrations in benthic floc ranged from 40 mg P kg–1 dry wt. in the unenriched calcareous slough in WCA 2A, to 320 mg P kg–1 dry wt. from the transitional site in WCA 2A. Concentrations were lower in soils (16–93 mg P kg–1 dry wt.), representing between 13 and 36% of the extracted P. Phosphate monoesters were proportionally largest in the unenriched calcareous slough of WCA 2A (62%), although this sample contained a relatively small P concentration.
In all samples, the largest monoester signals were at 5.24 and 4.91 ppm, indicating the presence of phosphatidic acid and ß-glycerophosphate, respectively (e.g., in the extract of benthic floc from the transitional site in WCA 2A; Fig. 4) . These compounds originate from the hydrolysis of phospholipids, notably phosphatidyl choline, in alkaline solution (Turner et al., 2003b). Further phosphate monoester signals appeared close to 4.85, 4.78, 4.69, 4.51, and 4.42 ppm (Fig. 4), which are characteristic of mononucleotide degradation products of RNA in alkaline solution (Turner et al., 2003b). A further signal at 3.5 ppm in some extracts was assigned to glucose 1-phosphate. Signals from phytic acid (myo-inositol hexakisphosphate) were absent in all extracts with the exception of soil from the unenriched sawgrass ridge in WCA 2A, as indicated by a small signal at 5.9 ppm from the phosphate at the C-2 position on the inositol ring (Fig. 3).
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Traces of signals between 0.5 and 2.0 ppm were assigned to phospholipids and RNA that were not degraded during extraction and analysis (see above). Long-chain polyphosphates (signals close to –20 ppm) and phosphonates (signals close to 20 ppm) were not detected in any sample.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phosphate diesters are the main input of organic P to soils, but typically constitute only a small fraction of the soil organic P (Anderson, 1967). However, there is little comparable information on the organic P composition of submerged wetland soils. In recently reflooded (1–5 yr) Histosols with a history of cultivation and pH values close to neutral, the organic P in NaOH–EDTA extracts was mainly phosphate monoesters, with diester-to-monoester ratios between 0.1 and 0.2 (Robinson et al., 1998). Alkaline extracts of benthic floc from a constructed wetland near the current study sites was reported to contain large proportions of phosphoenolpyruvate (Pant and Reddy, 2001), although this was almost certainly a mis-assignment of the DNA signal. Subsequent analysis of the water column of this constructed wetland revealed that >70% of the soluble organic P was hydrolyzed by phosphodiesterase and was therefore in the form of phosphate diesters (Pant et al., 2002).
Clearly, the turnover of phosphate diesters must be important in subtropical wetlands, yet the availability of phosphate diesters to organisms in wetlands is relatively unknown. Published data on phosphodiesterase activity in wetlands is rare, although some information is available from P-limited environments elsewhere. In the English uplands, for example, rates of phosphodiesterase activity in aquatic mosses were greater in streams at higher altitude (Christmas and Whitton, 1998). The authors suggested that this was linked to the greater cover of blanket peat at higher altitude, which is rich in phosphate diesters (Turner et al., 2003a). There is evidence for the expression of root phosphomonoesterase activity in some common wetland macrophytes, including cattail and sawgrass (Kuhn et al., 2002), although rates of phosphodiesterase have not been measured.
The assessment of phosphodiesterase activity is confounded by potential use of phosphate diesters by heterotrophic microbes as a source of energy or N rather than P (Heath, 2005). There is some evidence that this occurs in Everglades communities (Wright and Reddy, 2001b), although it is likely to be most important in nutrient-enriched sites where productivity approaches N limitation.
Information on the origins of the extracted phosphate diesters would be useful to infer their potential bioavailability. Some were almost certainly derived from intact microbial cells, because microbes can contain a considerable proportion of the P in wetland soils (Qualls and Richardson, 1995). However, much of the DNA was probably present as part of the nonliving soil organic P (Makarov et al., 2002). Most phosphate diesters are degraded relatively rapidly after addition to soil (Bowman and Cole, 1978), although they accumulate in acidic soils with high organic matter concentrations (e.g., Cade-Menun et al., 2000) and can be stabilized by sorption to clays (Greaves and Wilson, 1969). As soils in the current study were not strongly acidic and contained little clay, further investigation of the mechanisms involved in stabilizing phosphate diesters in wetland soils is required.
Pyrophosphate was detected in considerable proportions in benthic floc but was relatively absent in underlying soil, suggesting that it decomposes rapidly. Despite its common occurrence in soils from a wide range of environments, the origin and function of pyrophosphate in soil remain unclear. It may have been extracted from live microbes, possibly originating as inorganic or organic polyphosphates (e.g., adenosine 5'-triphosphate). However, polyphosphates were not detected in any samples and their absence is not due to degradation during extraction and analysis (Hupfer et al., 1995; Cade-Menun and Preston, 1996). The presence of pyrophosphate in unenriched samples indicates that it is unlikely to be derived exclusively from anthropogenic sources, as suggested in a study of estuarine sediments (Sundareshwar et al., 2001).
The Mesocosm site in WCA 1 was included in this study to compare a true soft water slough with a hard water equivalent. Differences between the sloughs, including composition of the algal community and the abundance of macrophytes, almost certainly contribute to differences in the forms and stability of organic P. Periphyton in calcareous sloughs mediates the precipitation of insoluble Ca-phosphates or coprecipitation of phosphate with CaCO3. This reduces P availability to organisms (Dodds, 2003) and may intensify the demand for P from organic compounds. Also, the cohesive nature of the periphyton mat in WCA 2A means that nutrients are tightly cycled within the mat structure. Both factors may explain in part the lack of DNA and pyrophosphate in the benthic floc from the calcareous slough, although strong P limitation in the soft water marsh clearly does not preclude the accumulation of large concentrations of DNA.
A large proportion of the total P was recovered from most samples. Values were typical for alkaline extracts of most soils, although high recoveries were reported from high organic matter acidic soils (Cade-Menun et al., 2000; Turner et al., 2003a). For wetland soils, Robinson et al. (1998) reported recovery of between 47 and 64% of the P from recently flooded organic soils in Florida. The NaOH–EDTA extraction procedure is designed to quantitatively recover organic P from soil (Bowman and Moir, 1993; Turner et al., 2005a), so P not extracted in NaOH–EDTA was probably inorganic phosphate, most likely in the form of calcium or magnesium phosphate precipitates (Reddy et al., 1998).
Small amounts of recalcitrant organic P may not have been extracted, although it is impossible to assess this because there is no direct method of determining the total organic P concentrations in soil (Turner et al., 2005a). Total organic P in wetland soils is conventionally assessed by ignition, but this can be inaccurate. Similarly, estimation of organic P by alkaline extraction is compromised by interference in P speciation by molybdate colorimetry, which can markedly overestimate organic P due to the presence of humic–metal–phosphate complexes (Turner et al., 2003a). We therefore consider the combination of NaOH–EDTA extraction with solution 31P NMR spectroscopy to be the optimum method for characterization of organic P in wetland soils.
In summary, phosphate diesters dominated the organic P in alkaline extracts of benthic floc and soils from sites along nutrient gradients in subtropical wetlands. The exception was in benthic floc from a calcareous slough, which contained no detectable DNA. As phosphate diesters are also the dominant organic P compounds in plant and microbial inputs to wetlands, the hydrolysis of phosphate diesters seems likely to be the rate limiting step in soil organic P turnover in the Everglades. This in turn highlights the importance of phosphodiesterase activity in P acquisition by wetland organisms. We recommend that greater emphasis should be placed on understanding phosphodiesterase activity and the turnover of phosphate diesters in wetlands. Emphasis should also be placed on identifying the unextractable fraction of the soil P, which is critical for understanding long-term P sequestration in wetland soils.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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