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Soil Science Society of America Journal 65:588-595 (2001)
© 2001 Soil Science Society of America

DIVISION S-10-WETLAND SOILS

Phosphorus Loading Effects on Extracellular Enzyme Activity in Everglades Wetland Soils

A.L. Wright and K.R. Reddy

Wetland Biogeochemistry Lab., Univ. of Florida, 106 Newell Hall, P.O. Box 110510, Gainesville, FL 32611

Corresponding author (krr{at}ufl.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The impact of P loading to the northern Florida Everglades has been implicated in changing vegetation patterns, peat accretion rates, and other soil physico-chemical properties. This investigation focused on determining the influence of P loading on the activities of various extracellular enzymes along a P-enrichment gradient and relating measured enzyme activities to soil physico-chemical parameters. Alkaline phosphatase activity (APA) was the only enzyme affected by P loading and was negatively related to soil P concentrations and microbial biomass C and P. Arylsulfatase, ß-d-glucosidase, protease, and phenol oxidase were not affected by P loading and were not related to measured soil C, N, S, and P physical and chemical parameters. All enzyme activities were highest in the surface detritus layer and decreased with soil depth. Due to significant relationships between APA and soil and microbial P parameters, APA appears to be a useful indicator for assessing impacts of P enrichment in wetland soils.

Abbreviations: APA, alkaline phosphatase activity • diqc, dihydroindole–quinone–carboxylate • DOC, dissolved organic C • DRP, dissolved reactive P • EAA, Everglades Agricultural Area • WCA, Water Conservation Area


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
WETLAND SOILS support a large diversity of microbial communities that play important roles in the decomposition of organic matter, nutrient cycling, and the abatement of the toxic levels of contaminants. A majority of organic matter in wetlands and aquatic systems is composed of high molecular weight polymeric compounds, of which only a small portion is readily available to microbial communities (Benner et al., 1984; Chrost, 1991). Complex structural compounds must be first hydrolyzed through the activity of extracellular enzymes into low molecular weight compounds (Chrost, 1991; Sinsabaugh et al., 1991). These low molecular weight compounds can be directly transferred to cells, oxidized, and used as an energy source (Chrost, 1991). Nutrient loading can potentially alter the activity of extracellular enzymes, which is critical to the first step in the degradation of soil organic matter and detrital plant tissue (Halemejko and Chrost, 1984; Sinsabaugh et al., 1992).

Several enzymes are known to be involved in the cycling of nutrients and can be used as potential indicators of nutrient cycling processes (McLatchey and Reddy, 1998). In a P-limited wetland or an aquatic ecosystem, alkaline phosphatase activity (APA) plays an important role in the regeneration of inorganic P through its catalysis of the breakdown of organic P esters to inorganic P (Chrost, 1991). Since up to 90% of organic P may be in monoester form (Condron et al., 1985), the role of APA in P regeneration from soils is important. Alkaline phosphatase activity is often repressed by high dissolved reactive P (DRP) concentrations in a process referred to as feedback inhibition (Cembella et al., 1984; Chrost, 1991). Similarly, arylsulfatase and protease enzymes function in nutrient cycling by regenerating inorganic SO2-4 and NH+4 from organic matter. Arylsulfatase catalyzes the hydrolysis of sulfate esters resulting in the release of SO2-4 (Tabatabai and Bremner, 1970); thus, it is likely important to S cycling processes in wetland soils. Protease enzymes are important in the wetland N cycle and function in the breakdown of proteins, resulting in the release of NH4–N (Ladd and Butler, 1972). Glucosidase catalyzes the hydrolysis of glycosides, resulting in the release of a ß-linked monosaccharide (Eivazi and Tabatabai, 1988). Phenol oxidase is important in the breakdown of lignin-containing compounds and depends on O2 availability (Pulford and Tabatabai, 1988; Pind et al., 1994; McLatchey and Reddy, 1998). Cellulose and lignin-degrading enzyme activities have been correlated with degradation rates of detritus (Sinsabaugh et al., 1994; McLatchey and Reddy, 1998).

Microbiological properties, including enzyme activities, can be potentially useful as indicators of soil and water quality. Many of these enzymes are affected by nutrient loading, since bioavailable nutrients can potentially decrease their activity (Chrost, 1991; Wetzel, 1991). Thus, measurement of extracellular enzyme activities may be useful for predicting the impacts of P loading (Gage and Gorham, 1985; Wetzel, 1991; Whitton, 1991; Newman and Reddy, 1993) since they are important to organic-matter degradation, nutrient regeneration, and various elemental cycles.

We measured the activity of various extracellular enzymes in detritus and soil samples collected along a P gradient located in Water Conservation Area 2A (WCA-2A) of the Florida Everglades. The extracellular enzymes assayed were alkaline phosphatase (APA), arylsulfatase, ß-D-glucosidase, protease, and phenol oxidase. The objectives of the study were to determine (i) the influence of P loading to a wetland on activities of various extracellular enzymes in detritus and soil along a P gradient; (ii) the relationship between extracellular enzyme activities and selected microbial and soil physico-chemical parameters; and (iii) whether these enzyme activities are useful as sensitive indicators of P impacts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Site Description
The study site is WCA-2A of the Florida Everglades. This 447-km2 impounded wetland has received P-laden drainage waters for the past several decades from the adjacent Everglades Agricultural Area (EAA). Inflow of these waters has been implicated in contributing to increased P concentrations in the soil and water column (Reddy et al., 1993; DeBusk et al., 1994). Historically, this system was P-limited and contained a mixture of sawgrass (Cladium spp.) and slough communities (Davis, 1991). The addition of P-laden waters and altered hydrology have been implicated as key factors in a vegetation shift from the indigenous sawgrass/slough communities to cattail (Typha spp.)-dominated areas, resulting in increased peat accumulation and additional soil and water alterations (Davis, 1991; Craft and Richardson, 1993; Reddy et al., 1993). Soils near the inflow have the highest P concentrations, and P concentrations decrease with increasing distance from the inflow. Corresponding to changes in soil P concentrations, a gradient in vegetative type exists as well. Cattail is the dominant vegetative type in areas impacted by P, while sawgrass is prominent in unimpacted areas (Davis, 1991; DeBusk et al., 1994).

Detritus and Soil Sampling and Characterization
Detritus and soil samples were collected from eight stations along the P gradient, 1.4, 2.3, 3.3, 4.2, 5.1, 7.0, 8.4, and 10.1 km south from the point of inflow (S10-C) in WCA-2A of the Everglades (Table 1). Sampling sites encompassed different levels of P enrichment ({approx}2000–400 mg of P kg soil-1) and different vegetative types (cattails, mixed area, sawgrass/sloughs). Samples were collected in February, May, and August 1996, and in March 1997 to determine seasonal fluctuations in measured parameters. Samples consisted of recently deposited, distinguishable surface plant detritus and two soil-depth intervals. Detritus was collected by hand at each of the sampling stations from above the cored soil. Soil cores were obtained by driving an aluminum corer (i.d. = 14.6 cm) to a depth of {approx}40 cm. At each sampling station, four soil cores were obtained {approx}1 to 2 m apart. The only exception was that only one core was taken during the May sampling period. The May sampling period was utilized only for enzyme activity measurements and not for detailed soil characterization, thus only one core was taken. Soil cores were sectioned into 0- to 10-cm and 10- to 30-cm layers. Respective layers of all four cores were combined into one bulk sample and homogenized for use in experiments. Since we were interested in sampling along the P gradient, no attempt was made to determine spatial variability at each of the eight stations. Samples were placed in airtight bags and stored on ice for transport back to the laboratory. Subsequently, all samples were stored in a refrigerator at 4°C until analysis.


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  		Table 1. Station locations along the P loading gradient in WCA-2A

 
Various soil and microbial parameters were determined on well-mixed subsamples and are presented in Tables 2 and 3. Soil and microbial parameters were expressed on a dry-weight basis. Total C and N on detritus and soil were determined on oven-dried (70°C) ground samples using a Carlo-Erba NA 1500 CNS Analyzer (Haak-Buchler Instruments, Saddlebrook, NJ). Microbial biomass C was determined using a fumigation-extraction procedure (Vance et al., 1987) with 0.5 M K2SO4 extracts being analyzed using a Dohrman total C analyzer (Rosemount Analytical, Santa Clara, CA). Extractable NH+4 was determined by the method of Mulvaney (1996), using an automated colorimetric procedure (Method 350.1, USEPA, 1993b). Sulfate concentrations were determined by ion chromatography (Method 300.0, USEPA, 1993a). Soil total P was determined by nitric-perchloric acid digestion (Kuo, 1996) and analyzed using an automated ascorbic acid colorimetric procedure (Method 365.4, USEPA, 1993c). Microbial biomass P was also determined by a fumigation-extraction method (Hedley and Stewart, 1982). The NaHCO3–Pi fraction represented the non-fumigated 0.5 M NaHCO3 extractable P.


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  		Table 2. Soil physico-chemical properties in WCA-2A averaged from February and August 1996 and March 1997 sampling times with (standard error)

 

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  		Table 3. Selected forms of P in detritus and soils in WCA-2A averaged from February and August 1996 and March 1997 sampling times with (standard error)

 
Sample Preparation and Enzyme Assays
In the laboratory, subsamples were taken from bulk samples and were further homogenized. Large roots or shells were removed since they were deemed inappropriate for analysis. Approximately 0.5- to 0.75-g wet samples were added to polypropylene centrifuge tubes for analysis of most enzyme activities. Assays were performed using either three or four replications and appropriate controls to account for nonenzymatic color production. Colorimetric methods used in the enzyme assays were as follows: alkaline phosphatase (Tabatabai and Bremner, 1969; Eivazi and Tabatabai, 1977), arylsulfatase, ß-D-glucosidase (Eivazi and Tabatabai, 1988), protease (Ladd and Butler, 1972), and phenol oxidase (Pind et al., 1994). All enzyme activities were expressed on a dry-weight basis.

Alkaline phosphatase, arylsulfatase, and ß-D-glucosidase were analyzed using similar substrates and methodology. The base substrate used was p-nitrophenol bound with phosphate, SO2-4, or glucose (Sigma Chemical, St. Louis, MO). The artificial substrate (1 mL, 0.05 M), toluene to inhibit microbial growth during incubation, a pH buffer (pH = 11 for APA, 5.8 for arylsulfatase, 6.0 for glucosidase), and 0.50- to 0.75-g wet samples were incubated in closed polypropylene centrifuge tubes at 37°C for 1 h. At the end of incubation, enzyme activity was stopped by addition of 4 mL of 0.5 M NaOH (APA and arylsulfatase) or 4 mL of 0.5 M THAM (glucosidase) with 1 mL of 0.5 M CaCl2; the mixture was filtered and the extract analyzed using a UV-VIS spectrophotometer (Shimadzu Model UV-160) at 420 {eta}m. Absorbance of filtrates was compared with p-nitrophenol standards. To account for nonenzymatic substrate hydrolysis, values for controls were subtracted from sample replicates.

Protease activity was determined using 0.5 to 0.75 g of wet soil and 2.5 mL of casein (10 mg mL-1) in 0.1 M THAM buffer at pH = 8.1. This mixture was incubated at 37°C for 1 h. Enzyme activity was stopped by adding 1 mL of 17.5% trichloroacetic acid. After centrifugation, 2 mL of supernatant was mixed with 3 mL of 1.4 M Na2CO3 and 1 mL of Folin reagent. The absorbance of the resulting solution was measured at 700 {eta}m and compared with tyrosine standards.

Phenol oxidase activity was measured using a slurry of 0.1- to 0.3-g wet samples and 5 mL of water mixed with 10 mM of L-DOPA (dihydroxyphenylalanine) solution. Incubation time varied between 1 and 3 min, and the difference in absorbance between incubation times was used to calculate phenol oxidase activity. After incubation, mixtures were filtered and analyzed at 400 {eta}m. Calculation of the quantity of diqc (dihydroindole–quinone–carboxylate) released was based on a 1 cm light path and Beers Law using a molar absorbance coefficient of 3.7 x 104 (Mason, 1948).

Data Analysis
Enzyme activities were compared to determine differences in soil depth, station, and season using ANOVA and Fishers LSD at P < 0.05 (CoStat, Minneapolis, MN). Linear correlation coefficients were also determined between enzyme activity and various soil physico-chemical properties. To eliminate seasonal variability, APA was expressed as a percentage of maximum activity for a given season and soil depth. To determine field variability for ANOVA, eight sampling stations along the P gradient were grouped into three separate units based on vegetative type and total soil P concentrations. The three groupings included Stations 1, 2, and 3 (Typha growing in P impacted areas), Stations 4, 5, and 6 (mixed vegetational areas in transitional zone), and Stations 7 and 8 (Cladium growing in P-unimpacted areas) for expression of arylsulfatase, glucosidase, protease, and phenol oxidase activities. Statistical differences from ANOVA at P < 0.05 were compared between the impacted, transitional, and unimpacted areas.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Soil bulk density increased with soil depth but was not affected by P loading (Table 2). Soil pH generally ranged from 7 to 8 (data not shown). Total C and N were not affected by P loading or soil depth. Total P, NaHCO3–Pi, microbial biomass C, and microbial biomass P were highest in surface detritus and decreased both with soil depth and distance along the P gradient (Tables 2 and 3).

Alkaline Phosphatase
For all sampling times, APA was highest in the surface detrital layer and significantly decreased (P < 0.05) with depth in the soil profile (Fig. 1) . Similar decreases in phosphatase activity with depth have been observed in other aquatic systems (DeGobbis et al., 1984; Newman and Reddy, 1992). Alkaline phosphatase activity was {approx}10- to 15-fold higher in detritus than in underlying soil at unimpacted sites. The detritus consisted of partially degraded, recently deposited plant material, either from Typha or Cladium. Substrate quality and perhaps aerobic conditions in surface detritus make this material more usable for microorganisms than underlying soils (DeBusk, 1996), thus APA would be higher in detritus. Therefore, a decrease in APA with depth would correspond to a decrease in substrate quality with depth.



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  		Fig. 1. Alkaline phosphatase activity (mg of p-nitrophenol g soil-1 h-1) in detritus and two soil depths along the P gradient in WCA-2A for four sampling times. Error bars represent standard error

 
Since APA varied seasonally, activities were normalized to maximum activity measured during that particular season and expressed as a percentage of the maximum rate at a given soil depth and season. The maximum APA at each particular sampling time and soil depth are provided in Table 4. Data from all sampling times were combined for each depth to provide an indication of P loading effects for the various seasons (Fig. 2) . Alkaline phosphatase activity in the detritus and 0- to 10-cm soil layer significantly increased (P < 0.05) with increasing distance from the inflow and was highest at sites >5 km from the inflow. The APA levels were generally constant up to 5 km from the inflow but then increased along the P gradient as P levels in the detritus and 0- to 10-cm soil depth decreased. However, APA activity remained fairly constant in the 10- to 30-cm soil depth along the gradient. The significant response of APA to P loading in detritus and 0- to 10-cm soil may be the result of increased microbial activity in surface soils due to the greater availability of labile C than in deeper soils. The response of the microbial community to P loading would be more dynamic and sensitive in detritus and surface soil than in deeper soil, where lower C bioavailability rather than P concentrations may regulate microbial activity and APA. At lower depths, much of the organic matter is well decomposed, resulting in high lignin/(lignin + cellulose) ratios and low microbial activity (DeBusk and Reddy, 1998). This results in minimal demand for bioavailable P. These conditions resulted in very little or no trend in APA along the P gradient in 10- to 30-cm soil, even though there existed a wide range in soil P concentrations from impacted to unimpacted sites ({approx}600–200 mg of P kg soil-1). In addition, water-table depth was near the soil surface at most sampling times, likely exposing detritus and surface soil to aerobic conditions and increasing microbial activity. Lower water-table depths increased the decomposition process (DeBusk, 1996) and placed higher demand on bioavailable P.


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  		Table 4. Alkaline phosphatase activity expressed at the maximum rate at each soil depth and sampling time. The site and (standard error) are included

 


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  		Fig. 2. Alkaline phosphatase activity in detritus and two soil depths along the P gradient in WCA-2A expressed as a percentage of maximum APA for each sampling time. Data for each depth represent an average of four sampling times. Error bars represent standard error

 
Alkaline phosphatase activity in detritus (February and August 1996, and March 1997 sampling times) was significantly (P < 0.05) negatively correlated with total P (r = -0.75), labile P (r = -0.53), NaHCO3–Pi (SRP) (r = -0.51), and microbial biomass P (r = -0.41). Inverse relationships between soil P parameters and APA in soil are often observed (Cotner and Wetzel, 1991; Newman and Reddy, 1993). However, relationships between APA and soil P parameters may be attributed to other factors. Alkaline phosphatase production may be regulated by the microbial internal P pool, which may not accurately reflect the P pool outside of microbial cells (Chrost, 1991). Mineral inorganic P additions also have been reported to have stimulatory, inhibitory, and no effect on phosphatase activity in soils (Speir and Ross, 1978).

A nonlinear relationship between (APA/APA maximum) and NaHCO3 extractable inorganic P was observed for detritus (Fig. 3) . Alkaline phosphatase activity appears to be stimulated when NaHCO3–P decreases below 40 mg P kg-1. In the 0- to 10-cm depth interval, APA was only significantly (P < 0.05) correlated with soil total P (r = -0.48), although negative relationships with other soil P parameters were observed but were insignificant. Changes in APA along the gradient corresponded to changes in both soil P concentrations and vegetation type (Typha to Cladium). However, changes in APA along the P gradient were not due to differences in vegetation between the Typha and Cladium areas. In a separate study using constructed mesocosms (built in an unimpacted open slough) that received various rates of P loading over several years, similar responses of APA to P loading were observed (Newman et al., 2001). Thus, differences in APA along the P gradient were due to P loading rather than vegetation or substrate quality.



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  		Fig. 3. Alkaline phosphatase activity in detritus expressed as a percentage of maximum plotted against bicarbonate-extractable P (NaHCO3–Pi). Data represent an average of the February and August 1996 and March 1997 sampling times. Error bars represent standard error

 
Alkaline phosphatase activities measured in our study were generally higher than those observed in other aquatic systems and much greater than in agricultural soils. For example, APA values of up to 38000 mg of p-nitrophenol kg-1 h-1 were observed for stream sediment, 1700 mg of p-nitrophenol kg-1 h-1 for suspended particulates in stream, and 2600 mg of p-nitrophenol kg-1 h-1 for fine particulate organic matter (Saylor et al., 1979). Activities in shallow, nutrient-rich, freshwater surface sediments ranged from 1350 mg of p-nitrophenol kg-1 h-1 in unvegetated sediments to 1775 mg of p-nitrophenol kg-1 h-1 in vegetated sediments (Boon and Sorrell, 1991). In a reclaimed wetland, APA values ranged from 12 to 24 mg of p-nitrophenol kg-1 h-1 (McLatchey and Reddy, 1998). Rates of APA in many agricultural surface soils ranged from 20 to 235 mg of p-nitrophenol kg-1 h-1 (Juma and Tabatabai, 1978) but in one soil, up to 5000 mg of p-nitrophenol kg-1 h-1 (Nannipieri et al., 1979). The high organic matter content in Everglades soil likely enhances microbial and enzymatic activity.

Arylsulfatase
Arylsulfatase activity in detritus and soil was not affected by P loading but significantly (P < 0.05) decreased with depth (Fig. 4) . However, arylsulfatase activity was not significantly related to floodwater SO2-4 concentrations. Although arylsulfatase catalyzes the release of SO2-4 from organic ester S, it has been suggested that arylsulfatase may also be produced for the breakdown of carbon-bonded organic S (Oshrain and Wiebe, 1979). Similar to APA, arylsulfatase activity was significantly (P < 0.05) affected by soil depth, with higher activity in the detrital layer. Similar trends in arylsulfatase activity with depth have been reported for other systems (King and Klug, 1980). Detrital arylsulfatase activity was approximately twofold higher than in soils from the 0- to 10-cm depth, and threefold higher than in soils from the 10- to 30-cm depth.



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  		Fig. 4. Arylsulfatase activity (mg of p-nitrophenol g soil-1 h-1) in detritus and two soil depths at the February, May, and August 1996 sampling times in P-impacted, transitional, and unimpacted areas. Error bars represent standard error

 
Arylsulfatase activity along the P gradient in WCA-2A was greater than most values reported in the literature for wetland soils and much greater than for agricultural soils, likely due to the higher organic matter and microbial biomass content of Everglades soils. Ranges of sulfatase activity in wetland peat were 550 to 850 µg g-1 h-1 (Sarathchandra and Perrott, 1981; Press et al., 1985). Sulfatase activity in marine sediments was highest at the sediment surface (80 µg of p-nitrophenol mL-1 h-1) and decreased with depth (10 µg of p-nitrophenol g-1 h-1) (Oshrain and Wiebe, 1979). Arylsulfatase activity in freshwater lake sediments was highest at the surface (12 mg of p-nitrophenol L-1 h-1) and decreased with depth (King and Klug, 1980), corresponding to decreases in O2 content. Additions of inorganic phosphate, NO-3, and SO2-4 have been found to depress sulfatase activity in peat (Press et al., 1985). Sulfatase activity in coastal sands was reported to be higher in vegetated areas (8.5 µg of p-nitrophenol g-1 h-1) than unvegetated areas (1.7 µg of p-nitrophenol g-1 h-1) due to input of plant sulfatases (Skiba and Wainwright, 1983).

It has been suggested that sulfatases are not effective indicators of organic S mineralization since there are many enzymes involved in these processes (Ladd and Jackson, 1982). Thus, these enzymes would not adequately reflect changes in soil SO2-4 concentrations. In addition, sulfatases may be produced for use in supplying SO2-4 for sulfate reduction, which would further limit the role of sulfatase for estimating organic S mineralization (King and Klug, 1980).

ß-D-Glucosidase
Phosphorus loading had no significant effects on glucosidase activity of detritus and soil (Fig. 5) . Glucosidase is an important extracellular enzyme involved in organic matter degradation, since it is responsible for conversion of cellobiose to glucose monomers (Eivazi and Tabatabai, 1988). Glucosidase activity in nutrient-enriched mesocosms was correlated with microbial production (Chrost and Rai, 1993), possibly due to increased organic matter inputs resulting from nutrient enrichment. In a related study on Everglades soils, microbial respiration rates were generally lowest at unimpacted sites compared with impacted and transitional sites (DeBusk and Reddy, 1998). Glucosidase activity was positively related to dissolved organic C (DOC) in nutrient-enriched soil but not in nutrient-impoverished soil (Chrost and Rai, 1993). Thus, greater DOC content generally implies increased availability of substrates for microbial growth. Bacteria appeared to be limited by the supply of readily utilizable monomers under nutrient-enriched soil but the supply of substrates was not limited in nutrient-impoverished soil (Chrost and Rai, 1993). In a nutrient nonlimited wetland soil, McLatchey and Reddy (1998) reported that glucosidase activity provided a good indication of C mineralization rates.



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  		Fig. 5. ß-D-glucosidase (mg of p-nitrophenol g soil-1 h-1) activity in detritus and two soil depths at the February, May, and August 1996 sampling times in P-impacted, transitional, and unimpacted areas. Error bars represent standard error

 
As with APA and arylsulfatase, glucosidase activity was significantly (P < 0.05) highest in the detritus layer and generally decreased with depth. This again may be due to decreases in substrate quality with depth (DeBusk, 1996) or to O2 decrease with depth. Glucosidase activity was inversely related to redox potential. On the contrary, flooding of a dry soil enhanced glucosidase activity (Pulford and Tabatabai, 1988). Glucosidase activity in a northern Typha marsh receiving farmland drainage ranged from 200 µg g-1 h-1 at the inflow site to 5000 µg g-1 h-1 at an outflow site, corresponding to increases in particle size (Jackson et al., 1995).

Protease
Protease activity was not affected by P loading since there were no statistically significant trends along the gradient in detritus or soil for either the May or August sampling times (Fig. 6) . Protease activity was highest (P < 0.05) in the detritus layer and significantly decreased with depth, similar to other studies (Speir and Ross, 1978). Protease activity decreased with depth, likely due to decreases in substrate quality, bacterial populations, and O2 concentrations (McLatchey and Reddy, 1998).



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  		Fig. 6. Protease activity (mg of tyrosine g soil-1 h-1) in detritus and two soil depths at the May and August 1996 sampling times in P-impacted, transitional, and unimpacted areas. Error bars represent standard error

 
Protease activity varied seasonally and was significantly higher (P < 0.05) in August than in May. Protease activity was significantly negatively correlated with soil NH+4 concentrations in the 0- to 10-cm soil depth (r = -0.84; P < 0.05). At high substrate concentrations, some hydrolysis products such as NH+4 are not assimilated by microorganisms and are released to soil solution; thus an excess of NH+4 in solution at high substrate concentrations may repress some enzyme activities (Hoppe et al., 1988).

Phenol Oxidase
Phenol oxidase activity was not affected by P loading in detritus and soil, with levels remaining constant along the P gradient in detritus and soil depths (Fig. 7) . Phenol oxidase activity was highest in the detritus layer and decreased with depth at both sampling times (P < 0.05). Phenol oxidase often decreases with depth due to O2 limitations (Pind et al., 1994; McLatchey and Reddy, 1998). There was no seasonal variability in phenol oxidase activity. The presence of water on the soil surface may limit phenol oxidase activity (Benner et al., 1984). Phenol oxidase activity increased as particle size decreased or as organic matter became more degraded (Jackson et al., 1995). Phenol oxidase activity in reclaimed wetlands was only detected under aerobic soil conditions at a rate of 1.3 mmol diqc kg-1 min-1 (McLatchey and Reddy, 1998). Phenol oxidase activity in northern peat soils ranged from 0.24 mmol diqc kg-1 min-1 at the soil surface to 0.05 mmol diqc kg-1 min-1 at 40 cm below surface (Pind et al., 1994).



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  		Fig. 7. Phenol oxidase activity (µmol diqc g-1 min-1) in detritus and two soil depths at the May and August 1996 sampling times in P-impacted, transitional, and unimpacted areas. Error bars represent standard error

 
Relationships between microbial parameters and extracellular enzyme activities are often contradictory because of a wide-range of factors that may influence enzyme activities including redox conditions, nutrients, substrates, and other physico-chemical properties. Due to the complexity of enzyme and substrate interactions and the complimentary functions of many of these enzymes, relationships between enzyme activity and soil and microbial parameters are often not clearly observed in the field (Marsden and Gray, 1986). In our study, sulfatase, glucosidase, and phenol oxidase activity were not related to soil physico-chemical parameters. Protease activity in soil appeared to be regulated, in part, by feedback inhibition and presence of high NH+4 concentrations. Phosphatase activity, however, was sensitive to changes in soil and microbial P factors, indicating that these factors were in part controlling APA activity.

Rates of extracellular enzyme activity in WCA-2A were often much greater than values reported in the literature for other systems. This difference suggests that detritus and soil in WCA-2A are quite productive and biologically active, and hence enzyme activity may respond differently in this system than in others.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Phosphorus loading to an oligotrophic, P-limited wetland had a significant influence on APA in detritus and soil but not on other measured extracellular enzyme activities. Extracellular enzyme activity markedly decreased with depth in the soil profile, and detritus was most responsive to changes in P concentrations. The APA appeared to be regulated by specific soil and microbial P parameters in detritus and the upper soil depths. However, relationships between soil and microbial physico-chemical properties and other measured extracellular enzyme activities seldom produced significant relationships. Alkaline phosphatase activity appears to be suitable for use as an indicator of P eutrophication.


   ACKNOWLEDGMENTS
 
This research was supported by the Florida Agricultural Experiment Station and a grant from the South Florida Water Management District, and approved for publication as Journal Series no. R-07810.

Received for publication January 3, 2000.


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




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Phosphorus Cycling in Wetland Soils: The Importance of Phosphate Diesters
J. Environ. Qual., September 8, 2005; 34(5): 1921 - 1929.
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 J. Environ. Qual.Home page
M. J. Cohen, J. P. Prenger, and W. F. DeBusk
Visible-Near Infrared Reflectance Spectroscopy for Rapid, Nondestructive Assessment of Wetland Soil Quality
J. Environ. Qual., July 5, 2005; 34(4): 1422 - 1434.
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 Appl. Environ. Microbiol.Home page
H. Castro, S. Newman, K. R. Reddy, and A. Ogram
Distribution and Stability of Sulfate-Reducing Prokaryotic and Hydrogenotrophic Methanogenic Assemblages in Nutrient-Impacted Regions of the Florida Everglades
Appl. Envir. Microbiol., May 1, 2005; 71(5): 2695 - 2704.
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R. Corstanje and K. R. Reddy
Response of Biogeochemical Indicators to a Drawdown and Subsequent Reflood
J. Environ. Qual., November 1, 2004; 33(6): 2357 - 2366.
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 Soil Sci.Home page
J. P. Prenger and K. R. Reddy
Microbial Enzyme Activities in a Freshwater Marsh after Cessation of Nutrient Loading
Soil Sci. Soc. Am. J., September 1, 2004; 68(5): 1796 - 1804.
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A. Chauhan, A. Ogram, and K. R. Reddy
Syntrophic-Methanogenic Associations along a Nutrient Gradient in the Florida Everglades
Appl. Envir. Microbiol., June 1, 2004; 70(6): 3475 - 3484.
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J. R. White and K. R. Reddy
Nitrification and Denitrification Rates of Everglades Wetland Soils along a Phosphorus-Impacted Gradient
J. Environ. Qual., November 1, 2003; 32(6): 2436 - 2443.
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 Soil Sci.Home page
A. L. Wright and K. R. Reddy
Heterotrophic Microbial Activity in Northern Everglades Wetland Soils
Soil Sci. Soc. Am. J., November 1, 2001; 65(6): 1856 - 1864.
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