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Published online 1 May 2008
Published in J Environ Qual 37:972-976 (2008)
DOI: 10.2134/jeq2007.0248
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TECHNICAL REPORTS

Wetlands and Aquatic Processes

Enzyme-Based Resource Allocated Decomposition and Landscape Heterogeneity in the Florida Everglades

C. Ryan Pentona,b,* and Susan Newmanc

a Soil and Water Science Dep., Univ. of Florida, IFAS, 106 Newell Hall, Gainesville, FL 32611
b current address, Crop and Soil Science Dep., The Center for Microbial Ecology, Michigan State Univ., 540 Plant and Soil Sciences Bldg., East Lansing, MI 48824-1325
c Everglades Div., South Florida Water Management District, P.O. Box 24680, West Palm Beach, FL 33416-4680

* Corresponding author (pentonch{at}msu.edu).

Received for publication May 15, 2007.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
Enzyme catalyzed reactions are generally considered the rate-limiting step in organic matter degradation and may be significantly influenced by the structure and composition of plant communities. Changes in these rates have the potential to effect long-term peat accumulation and influence the topography of a wetland ecosystem. To determine habitat influences on enzyme activities, we examined slough and sawgrass plots within enriched and reference phosphorus (P) sites in the Everglades. Assays were performed for the enzymes involved in carbon (C), nitrogen (N), and P cycling and lignin depolymerization. Enzyme activities were normalized and analyzed in terms of a resource allocation strategy. Plant composition was found to significantly alter the allocation of enzymatic resources due to varying substrate complexities. Potential decomposition in the slough was less influenced by lignin than in the sawgrass habitats. Additionally, an index relating hydrolytic and oxidative enzymes was significantly greater in the slough habitats, whereas C/N ratios were significantly lower. These indices suggest more favorable decomposition conditions and thus slower peat accretion within the slough communities, which may contribute to the development of elevation differences within the sawgrass ridge and slough topography of the Everglades.

Abbreviations: BGL, β-glucosidase • CBH, cellobiohydrolase • EICQ, Enzyme Index of Carbon Quality • leucine aminopeptidase • MUF, methylumbelliferyl • PHE, phenol oxidase • PHO, phosphatase • TOC, total organic carbon


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
THE chemical and physical properties of available substrates such as lignin, nitrogen (N), and phosphorus (P) (DeBusk and Reddy, 1998; Kourtev et al., 2002) are key factors controlling plant decomposition. As a consequence, plant litter degradation has been shown to be most influenced by the enzymes involved in lignocellulose degradation, P cycling, and N cycling (Sinsabaugh and Moorhead, 1994). These factors are often considered the rate-limiting steps in degradation (Chróst and Rai, 1993). Litter breakdown rates and enzyme activities may vary among plant species due to differences in their intrinsic structure (Kourtev et al., 2002) and chemical composition (Carreiro et al., 2000) and with site quality (Hobbie and Vitousek, 2000; Rejmánková and Houdková, 2006). A current strategy for predicting microbial degradation rates involves the use of a resource allocation rationale for exposing linkages between individual enzymes (Sinsabaugh and Moorhead, 1994). This approach is based on the premise that enzyme-regulated decomposition of complex molecules is the rate-limiting step. Enzyme expression is tied to environmental nutrient availability, so the distribution of enzyme activities can be interpreted as energetic relationships (Sinsabaugh et al., 2002).

In the Everglades, organic matter decomposition is involved in the development, accumulation, and maintenance of the peat profile. This landscape consisted of dense sawgrass ridges with soil surfaces 2 to 3 ft higher than the adjacent, deeper sloughs (Baldwin and Hawker, 1915). However, after almost 100 yr of altered surface water depth, hydroperiod, and flow, the area covered by the historically deeper sloughs is diminishing and is being replaced by shallower, monotypic sawgrass (Cladium jamaicense Crantz) stands (Sklar et al., 2002). The geomorphological contribution to the development of the Everglades topography is unknown, but differential decomposition is hypothesized to be one of the primary drivers (SCT, 2003). Anthropogenic P enrichment has also contributed to a substantial loss of ridge and slough habitat. In enriched areas, monotypic stands of Typha domingensis (Pers.) have replaced sawgrass and slough species (McCormick et al., 2002).

The objective of this study was to determine the effects of plant habitat types and nutrient loading on enzyme activity. By using a resource allocation strategy that is based on the premises proposed by Sinsabaugh and colleagues (2002), we focused on the apparent P, N, and lignin influences on C mineralization. The differences in potential decomposition were then qualitatively applied to the observed elevation of sawgrass and slough habitats. Our hypothesis was that the allocation among enzymes would be different between habitats, influenced by the composition and lability of the plants occupying each area.


   Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Methods
Results and Discussion
 Conclusions
 REFERENCES
 
Study Sites
Field study sites were located within Water Conservation Area 3A, Broward County, Florida, a 2339-km2 freshwater marsh incompletely impounded by levees and canals. The area is a mosaic of sawgrass stands (ridges) and sloughs interspersed with tree islands. Over 30 yr of agricultural runoff into the Everglades has resulted in the establishment of a P gradient originating in the northern regions of the Everglades, downstream from discharges.

Four sites were selected for sampling to represent P-enriched and slightly P-enriched (designated reference) sawgrass and slough sites. Global positioning satellite coordinates were 26°04.90'N, W80°49.52'S for the enriched site and 26°02.41'N, 80°48.87'W for the reference site, with the habitat subplots located within approximately 10 m of each other at each site. Three samples were taken from within each habitat subplot. Dense cattail, sparse sawgrass, and slough areas were present at the P-enriched sites located approximately 1.22 km from the L-28 intercept canal inflow. The slough areas at the enriched sites were characterized by open water areas surrounded by dense cattail stands and were thus not characteristic sloughs. Sparse cattail, stands of sawgrass, and classic Everglades slough communities consisting of water lily (Nymphaea sp.), spikerush (Eleocharis sp.), and periphyton constituted the reference site, which is located approximately 5.83 km from the canal inflow.

Soil Sampling and Enzyme Analysis
Soil cores were obtained in triplicate to a depth of approximately 30 cm at each habitat–site combination using a 5-cm stainless steel piston type corer with butyrate inserts on 9 Sept. 2001. The sampled benthic matter, defined as the unconsolidated or pourable core fraction, and the soil layer (0 to –10 cm) were stored and analyzed separately. Large objects were discarded, and the samples were homogenized with a Biospec Biohomogenizer (Biospec, Bartlesville, OK). Ten grams of the slurry was added to 100 mL deionized H2O and homogenized for an additional 5 min. An aliquot (10 mL) of the suspension was brought to 100 mL with deionized H2O and refrigerated. Relative site comparisons have been shown to be stable with cool storage (Verchot, 1999), although a reduction in metabolic activity and biomass does occur.

Hydrolytic enzyme activity was determined 24 h after field collection using methylumbelliferyl (MUF) and aminomethylcoumarin substrates. Substrate concentrations were optimized at saturating conditions. The activities of the hydrolytic enzymes β-glucosidase (BGL), cellobiohydrolase (CBH), phosphatase (PHO), and leucine aminopeptidase (LEU) were assayed in quadruplicate using MUF–β-D-glucoside (Sigma M3633; Sigma, St. Louis, MO), MUF-cellobioside (Sigma M6018), MUF-phosphate (Sigma M8168), and L-Leucine amidomethylcoumarin (Sigma L2145). The oxidative enzymes phenol oxidase (PHE) and peroxidase were assayed using L-3,4 dihydroxyphenylalanine (DOPA) and DOPA + H2O2, respectively.

Methylumbelliferyl and aminomethylcoumarin substrate conversion were measured using a Cytofluor 600 automated spectrofluorimeter (PerSeptive Biosystems, Inc., Framingham, MA) with Kineticalc software at 360 nm excitation and 460 nm emission at 20°C. Hydrolytic assay conditions were as described in Penton and Newman (2007), with the addition of a 60 µM in-well concentration of MUF-cellobioside. Phenol oxidase activities and peroxidase activities were determined per Sinsabaugh and Linkins (1990), with quench corrections established for each sample. Peroxidase was not included in the final analysis due to the lack of detectable activities. Final enzyme activities were calculated as µmoles substrate released g–1 ash-free dry mass h–1.

Sample nutrient analysis was performed by DB Labs (Rockledge, FL). Total P (EPA 365.2), total N (method validation package), total organic carbon (TOC) (method validation package), calcium (Ca) (SW7140), and magnesium (Mg) (SW7450) analyses were performed using standard methods on homogenized samples (USEPA 1983, 1986). Nutrient ratios, such as N/P, C/N, and C/P were expressed in molar units and are reported in Table 1 .


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  		Table 1. Mean chemical properties of the soil and benthic layers. Significant differences are indicated between habitats at each site.

 
Models
Extracellular enzymes can be grouped into four categories: Ec (BGL and CBH), En (LEU), Ep (PHO), and Eox (PHE); these categories separate the enzyme into C, N, and P mineralization groups and lignin degradation, respectively. This method has been used as part of the MARCIE (Microbial Allocation of Resources among Community Indicator Enzymes) model (Sinsabaugh et al., 2002). To compare different enzymes on the same scale, data for the model and ratios were normalized by dividing each enzyme activity by the highest activity in that category such that activities ranged from 0 to 1 (Sinsabaugh et al., 1997). Ec/En reflects apparent N control over C mineralization, Ec/Ep is a relative measure indicating P control over C mineralization, and Ec/Eox reflects potential lignin control over C mineralization. The Enzyme Index of Carbon Quality (EICQ), a relative measure of hydrolytic to oxidative enzymes, was calculated as the mean normalized hydrolytic enzyme activities (BGL, CBH, LEU, PHO) divided by the mean normalized oxidative enzyme activities (PHE) for each field rep (Sinsabaugh and Linkins, 1990; Sinsabaugh and Findlay, 1995). The EICQ and Ec/Eox calculations were modified for statistical analysis due to numerous nonresolvable oxidative activities leading to zeros in the denominator. Minimum activity was treated as the limit of detection for all enzymes, and that value was added to each measured activity, which permitted statistical analysis of all replicates. Actual values are reported in Table 2 .


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  		Table 2. Mean enzyme activities and enzyme ratios of the soil and benthic layers. Data reflect means with corresponding SEs. Significant differences are indicated between habitats at each site.

 
Statistical Analysis
Data were log transformed before performing statistical analysis to resolve normality and heteroscedescity using SAS version 8 statistical software (SAS Institute, 1999). A split-plot model (sawgrass-enriched, slough-enriched, sawgrass-reference, slough-reference) for the data was adopted for subsequent ANOVA analysis using SAS PROC GLM (SAS Institute, 1999). The whole plot corresponded to the sites in reference to the nutrient gradient, whereas the subplots referred to the specific plant communities within the whole plots. Specific contrasts were made between habitat–site combinations, and error terms were specified using LSMEANS. Analyses were performed on benthic and soil layers independently. Stated significant differences are based on habitat x site interaction terms at the P < 0.05 level.


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Methods
 Results and Discussion
Conclusions
 REFERENCES
 
The use of enzyme ratios, such as Ec/Ep and Ec/En, is based on the premise that the energy requirement for the production of certain extracellular enzymes reduces the net available energy for the expression of other enzymes. With generally higher enzyme activities, the benthic layer seemed to be the most responsive to environmental conditions. The discussion is, therefore, primarily focused on the benthic layer.

Sawgrass and Slough Comparisons
The potential for peat accumulation in each habitat is directly tied to local littoral inputs, the chemical and structural properties of the substrates, and the activities of the resident heterotrophic microbial decomposers. Above ground productivity in Everglades sawgrass marsh averaged 2991 ± 891 g m–2 yr–1, compared with 409 ± 160 g m–2 yr–1 in an Eleocharis spp. wet prairie (Daoust and Childers, 1998), which indicates an eightfold greater C input that ultimately enters the microbial food web. This is reflected in the higher benthic TOC values found in the sawgrass habitat at the reference site, which more closely represents the historical Everglades.

By combining Ec/Ep and Ec/En for each habitat, the influences of P and N on decomposition were constructed for the benthic and soil layers (Fig. 1 ). Relative benthic N and P limitation (Ec/En and Ec/Ep) indicated more favorable decomposition conditions in the sawgrass sites, whereas Ec/Eox and EICQ predicted lower decomposition rates than the slough. Specifically, EICQ has been positively correlated with productivity and microbial biomass and is negatively related to POC turnover time (Sinsabaugh and Findlay, 1995). This is supported in our data by lower slough C/N ratios, which are often used as predictors of litter quality (Carreiro et al., 2000) and indicate faster decomposition potential. Although the differences between the enriched and reference sites seem to be largely mediated by changes in N and P availability, which is expected due to the nutrient gradient, the sawgrass–slough comparisons are marked by larger changes in Ec/Eox, which also heavily influences the EICQ calculation. It seems that phenol oxidase, which has been described as an "enzymic latch" for global C stores (Freeman et al., 2001), is a primary controller of decomposition between habitats.


Figure 1
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  		Fig. 1. Benthic and soil microbial N and P resource allocation in terms of C mineralization at sawgrass and slough habitats. The y axis represents apparent P limitation, and the x axis represents apparent N limitation on C mineralization. Error bars represent SEs on each axis. Values are log-transformed for comparison. Numerical values reflect Ec/Eox values with associated SEs. Nitrogen and phosphorus conditions reflect microbially perceived concentrations. Site designations: BR, benthic reference; BE, benthic enriched; SR, soil reference; SE, soil enriched.

 
Nutrient Loading Comparisons
In slough habitats, total nitrogen, total phosphorus, and TOC concentrations were higher (P < 0.05) at the enriched sites in both layers. Significantly higher C/N ratios and lower LEU and PHO (P < 0.05) levels were found in the soil layer of enriched slough habitats. As expected, PHO was highly variable at the enriched site. In some cases, benthic PHO activities were 50 times higher than those found in the soil layer.

Enrichment differences are not denoted in Tables 1 or 2; however, Ec/Ep and Ec/Eox values were significantly greater at the enriched sites, except within the benthic layer of the sawgrass. Benthic and soil Ec/En values were similar, although only soil Ec/En was greater (P < 0.05) at the enriched site compared with the reference site. Values for EICQ were higher (P < 0.05) in the benthic layer at the enriched slough site and in the soil layer at the enriched sawgrass site. Because low oxidative activities increase the magnitude of each of these parameters, these values are conservative.

The effect of nutrient loading on decomposition within our different habitats is reflected by apparent decreases in N, P, and lignin influences on decomposition and by the markedly higher EICQ values. The higher EICQ values indicate an enzyme-based potential for faster decomposition from a more robust microbial community. As such, significantly higher microbial biomass C and microbial biomass P has been documented with P loading in the Everglades (Wright and Reddy, 2001), along with faster decomposition (Newman et al., 2003; Penton and Newman, 2007). Increased bacterial, periphyton, and plant productivity with decreased nutrient limitations have also been documented in the Everglades (McCormick et al., 2002) and other systems (DeBruyn et al., 2004). The higher PHO activity in the benthic layer has been observed in other studies (Newman and Reddy, 1993; Wright and Reddy, 2001, Sirová et al., 2006) and is attributed to the higher microbial productivity in this layer. In some cases, Ec/Ep values in this study are fourfold lower than values reported in an aquatic study (Sinsabaugh et al., 1997), which reflects the high P limitation in this system and spans an almost 20-fold greater range than Ec/En.

Although both habitats exhibit identical trends, nutrient loading at the enriched site resulted in larger decreases in apparent N (Ec/En), P (Ec/Ep), and lignin (Ec/Eox) control in the slough. Similarly, EICQ showed a larger increase in the slough, suggesting that the slough microbial community may be more highly influenced by nutrient loading than the sawgrass habitat. The faster comparative changes in decomposition rates in the slough may be a result of increased organic matter export from the surrounding sawgrass. The abundance of TOC in the reference slough is about 65% that of the sawgrass, although the amounts of TOC of the slough and sawgrass within the enriched site are virtually identical. These data suggest that within the enriched sites there may be more substantial carbon flux from the sawgrass into the slough as a result of faster decomposition.


   Conclusions
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Methods
 Results and Discussion
 Conclusions
REFERENCES
 
Nutrient loading has the potential to increase enzyme-based decomposition rates by relieving the effects of N and P limitation on microbial energetics. With a higher proportion of available energy being used in the production of enzymes involved in C mineralization, a faster organic matter turnover would be expected. Among the habitats, the combination of higher EICQ, lower apparent lignin influence on C mineralization, and lower C/N ratios supports faster decomposition and the long-term potential for the development of lower elevation sloughs. Increased decomposition would, in particular, lower the slough elevation relative to the sawgrass habitat. This is even more likely when increased decomposition is coupled with decreased C input due to the lower primary productivity occurring in the slough habitats. Conversely, lower decomposition and greater C input at sawgrass habitats would have the potential to exceed the metabolic capability of the resident microbial community, resulting in the accumulation of organic matter regardless of more favorable P and N availability.

Even with the use of corresponding in situ decomposition studies, the heterogeneity of substrates and complex microbial interactions may limit accurate relationships (Sinsabaugh et al., 2002). However, this study points to the potential role differential decomposition may play in the development and maintenance of the Everglade landscape. Subsequent investigations of differential decomposition processes, POC transport, hydrologic effects, and algal–bacterial relationships should more thoroughly explain the complex interactions involved in the development and maintenance of the Everglades topography.


   ACKNOWLEDGMENTS
 
This project was funded by the South Florida Water Management District and part of a larger Everglades research project conducted by the Marsh Ecology Research Group. The authors thank Megan Jacoby for sampling and laboratory assistance. This manuscript was improved by comments from Scot Hagerthey and Fred Sklar.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 Methods
 Results and Discussion
 Conclusions
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 Methods
 Results and Discussion
 Conclusions
 REFERENCES
 





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