Robert L. Sinsabaugh

MICROBIAL ENZYME SHIFTS EXPLAIN LITTER DECAY RESPONSES TO SIMULATED NITROGEN DEPOSITION

Some natural ecosystems near industrialized and agricultural areas receive atmospheric nitrogen inputs that are an order of magnitude greater than those presumed for preindustrial times. Because nitrogen (N) often limits microbial growth on dead vegetation, increased N input can be expected to affect the ecosystem process of decomposition. We found that extracellular enzyme responses of a forest-floor microbial community to chronically applied aqueous NH4NO3 can explain both increased and decreased litter decomposition rates caused by added N. Microbes responded to N by increasing cellulase activity in decaying leaf litter of flowering dogwood, red maple, and red oak, but in high-lignin oak litter, the activity of lignin-degrading phenol oxidase declined substantially. We believe this is the first report of reduced ligninolytic enzyme activity caused by chronic N addition in an ecosystem. This result provides evidence that ligninolytic enzyme suppression can be an important mechanism explaining decreased ...

The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil

Anthropogenic N deposition affects litter decomposition and soil organic matter (SOM) storage through multiple mechanisms. Microbial community responses to long-term N deposition were investigated in a sugar maple-dominated forest in northern Michigan during the 1998– 2000 growing seasons. Litter and soil were collected from three fertilized plots (30 kg N ha 21 y 21 ) and three control plots. The activities of 10 extracellular enzymes (EEA) were assayed. ANOVA and meta-analysis techniques were used to compare treatment responses. EEA responses to N amendment were greater in litter than in soil (litter mean effect size [d ] ¼ 0.534 std. dev.; soil d ¼ 0:308). Urease, acid phosphatase and glycosidase (b-glucosidase, a-glucosidase, cellobiohydrolase, b-xylosidase) activities increased in both soil and litter; mean responses ranged from 7 to 56%. N-Acetylglucosaminidase activity increased 14% in soil but decreased 4% in litter. Phenol oxidase activity dropped 40% in soil, but increased 63% in litter. These responses suggest that N deposition has increased litter decomposition rate and depressed SOM decomposition. In previous studies, loss of phenol oxidase activity in response to N deposition has been attributed to suppression of lignin-degrading basidiomycetes. However, the decline of this activity in bacterially-dominated soil suggest that N inhibition of recalcitrant organic matter decomposition may be a more general phenomenon. q 2002 Elsevier Science Ltd. All rights reserved.

Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

Decomposition of plant material is a complex process that requiresinteraction among a diversity of microorganisms whose presence and activity issubject to regulation by a wide range of environmental factors. Analysis ofextracellular enzyme activity (EEA) provides a way to relate the functionalorganization of microdecomposer communities to environmental variables. In thisstudy, we examined EEA in relation to litter composition and nitrogendeposition. Mesh bags containing senescent leaves of Quercusborealis (red oak), Acer rubrum (red maple) andCornus florida (flowering dogwood) were placed on forestfloor plots in southeastern New York. One-third of the plots were sprayedmonthly with distilled water. The other plots were sprayed monthly withNH4NO3 solution at dose rates equivalent to 2 or 8 g N m−2 y−1. Mass loss, litter composition, fungal mass, and the activities ofeight enzymes were measured on 13 dates for each litter type. Dogwood wasfollowed for one year, maple for two, oak for three. For each litter type andtreatment, enzymatic turnover activities were calculated from regressions of LN(%mass remaining) vs. cumulative activity. The decomposition of dogwood litterwas more efficient than that of maple and oak. Maple litter had the lowestfungal mass and required the most enzymatic work to decompose, even though itsmass loss rate was twice that of oak. Across litter types, N amendment reducedapparent enzymatic efficiencies and shifted EEA away from N acquisition andtoward P acquisition, and away from polyphenol oxidation and towardpolysaccharide hydrolysis. The effect of these shifts on decomposition ratevaried with litter composition: dogwood was stimulated, oak was inhibited andmaple showed mixed effects. The results show that relatively small shifts intheactivity of one or two critical enzymes can significantly alter decompositionrates.

Resource allocation to extracellular enzyme production : a model for nitrogen and phosphorus control of litter decomposition

Abstract Most models for plant litter decomposition link degradation rates to measures of climate or litter composition, rather than directly to microbial activity. We developed a model based on the premise that saprotrophic microbial communities maximize their productivity by optimizing their allocation of resources in the production of extracellular carbon, nitrogen and phosphorus-acquiring enzymes. In this model, enzyme activity indicators are used to estimate decomposition rates and to assess relative N and P availability. This approach may facilitate estimation of decomposition rates in the field and improve ecological forecasting.

Wood decomposition: Nitrogen and phosphorus dynamics in relation to extracellular enzyme activity

Because plant litter decomposition is directly mediated by extracellular enzymes (ectoenzymes), analyses of the dynamics of their activity may clarify the mechanisms that link decomposition rates to substrate quality and nutrient availability. We investigated this possibility by placing arrays of white birch sticks at eight upland, riparian, and lotic sites on a forested watershed in northern New York. For 3 yr, samples were analyzed for mass loss, protein, total Kjeldahl nitrogen (TKN), and total phosphorus (TP) accumulation, and the activity of 11 classes of extracellular enzymes involved in C, N, and P cycling. The relationship between lignocellulase activity and mass loss did not differ among sites. TKN and TP immobilization exhibited some spatial variation; rates of accumulation per 1% loss of initial mass, estimated from linear regressions, ranged from 2.2 to 4.4 mg/g OM for TP and from 43 to 139 mg/g OM for TKN, with maximum concentrations reached at °80% mass loss. The relationship between the activities of acid phosphatase (AcPase) and N—acetylglucosaminidase (NAGase), enzymes involved in the acquisition of P and N from organic sources, and mass loss displayed even greater variation among sites; the slopes of linear regressions relating mass loss and temporally integrated activity ranged from 0.019 to 0.135 activity—months per mass loss point and 0.107 to 0.775 activity—months per mass loss point, respectively, suggesting that edaphic rather than substrate quality factors were regulating activity. The extent of N limitation at each site was inferred by plotting TKN accumulation, defined as the slope of the linear regression TKN concentration vs. mass loss, in relation to NAGase activity accumulation, defined as the slope of the linear regression cumulative NAGase activity—months vs. mass loss. P limitation at each site was similarly assessed from an analogous plot of TP accumulation in relation to AcPase activity accumulation. Low N or P accumulation in conjunction with high acquisition activity was taken as an indication of nutrient limitation while the converse indicated surfeit. The diagrams suggested that decomposition at the upland hemlock and lotic sites, which displayed intermediate rates of OM loss (zero order k = 0.29 g/mo and 0.23 g/mo, respectively), was primarily N limited, while the riparian sites, which had the lowest rates of OM loss (k = 0.14 g/mo), appeared to be P limited. Relative to the others, OM loss at the upland deciduous sites (k = 0.38 g/mo) was not limited by either N or P. The concordance of field observations with predictions based on ectoenzyme regulation mechanisms suggest that enzyme activity assays in conjunction with nutrient concentration measurements may be a useful indicator of nutrient limitation. An economic model is proposed that directly links N and P availability to litter decomposition rates on the basis of microbial allocation of resources of extracellular enzyme production.

Nitrogen Deposition Modifies Soil Carbon Storage Through Changes In Microbial Enzymatic Activity

Atmospheric nitrogen (N) deposition derived from fossil-fuel combustion, land clearing, and biomass burning is occurring over large geographical regions on nearly every continent. Greater ecosystem N availability can result in greater aboveground carbon (C) sequestration, but little is understood as to how soil C storage could be altered by N deposition. High concentrations of inorganic N accelerate the degradation of easily decom- posable litter and slow the decomposition of recalcitrant litter containing large amounts of lignin. This pattern has been attributed to stimulation or repression of different sets of microbial extracellular enzymes. We hypothesized that soil C cycling in forest ecosystems with markedly different litter chemistry and decomposition rates would respond to anthro- pogenic N deposition in a manner consistent with the biochemical composition of the dominant vegetation. Specifically, oak-dominated ecosystems with low litter quality should gain soil C, and sugar maple ecosystems with high litter quality should lose soil C in response to high levels of N deposition (80 kg N-ha-1-yr-1). Consistent with this hypothesis, we observed over a three-year period a significant loss of soil C (20%) from a sugar maple- dominated ecosystem and a significant gain (10%) in soil C in an oak-dominated ecosystem, a result that appears to be mediated by the regulation of the microbial extracellular enzyme phenol oxidase. Elevated N deposition resulted in changes in soil carbon that were ecosystem specific and resulted from the divergent regulatory control of microbial extracellular en- zymes by soil N availability.

Microbial production, enzyme activity, and carbon turnover in surface sediments of the Hudson River estuary

The detrital food web is a major nexus of energy flow in nearly all aquatic ecosystems. Energy enters this nexus by microbial assimilation of detrital carbon. To link microbiological variables with ecosystem process, it is necessary to understand the regulatory hierarchy that controls the distribution of microbial biomass and activity. Toward that goal, we investigated variability in microbial abundance and activities within the tidal freshwater estuary of the Hudson River. Surface sediments were collected from four contrasting sites: a mid-channel shoal, two types of wetlands, and a tributary confluence. These samples, collected in June to August 1992, were sorted into two to four size fractions, depending on the particle size distribution at each site. Each fraction was analyzed for bacterial biomass (by acridine orange direct counting), bacterial production (by 3H-thymidine incorporation into DNA), fungal biomass (by ergosterol extraction), fungal production (by biomass accrual), and the potential activities of seven extracellular enzymes involved in the degradation of detrital structural molecules. Decomposition rates for particulate organic carbon (POC) were estimated from a statistical model relating mass loss rates to endocellulase activity. Within samples, bacterial biomass and productivity were negatively correlated with particle size: Standing stocks and rates in the <63-μm class were roughly twofold greater than in the >4-mm class. Conversely, fungal biomass was positively correlated with particle size, with standing stocks in the largest size class more than 1OX greater than in the smallest. Extracellular enzyme activities also differed significantly among size classes, with high carbohydrase activities associated with the largest particles, while oxidative activities predominated in the smallest size classes. Among sites, the mid-channel sediments had the lowest POC standing stock (2% of sediment dry mass) and longest turnover time (approximately 1.7 years), with bacterial productivity approximately equal to fungal (56 vs. 46 μg C per gram POC per day, respectively). In the Typha wetland, POC standing stock was high (10%); turnover time was about 0.3 years; and 90% of the microbial productivity was fungal (670 vs. 84 μg C per gram POC per day). The other two sites, a Trapa wetland and a tributary confluence, showed intermediate values for microbial productivity and POC turnover. Differences among sites were described by regression models that related the distribution of microbial biomass (r2 = 0.98) and productivity (r2 = 0.81) to particle size and carbon quality. These factors also determined POC decomposition rates. Net microbial production efficiency (production rate/decomposition rate) averaged 10.6%, suggesting that the sediments were exporting large quantities of unassimilated dissolved organic carbon into the water column. Our results suggest that studies of carbon processing in large systems, like the Hudson River estuary, can be facilitated by regression models that relate microbial dynamics to more readily measured parameters.

Soil microbial activity in a Liquidambar plantation unresponsive to CO2-driven increases in primary production

The indirect responses of soil microbiota to changes in plant physiology effected by elevated atmospheric carbon dioxide have the potential to alter nutrient availability and soil carbon storage. We measured fine root density, microbial biomass nitrogen, rates of nitrogen mineralization and nitrification, substrate utilization by soil bacteria and extracellular enzyme activities (EEA) associated with bulk soil and fine root rhizoplanes within a 3-year period at the Oak Ridge National Laboratory (ORNL) Free Air Carbon Enrichment (FACE) experiment, situated in a Liquidambar styraciflua plantation. Rhizoplane EEA was similar to that of bulk soil. Prior studies have reported a 21% increase in net primary production (NPP) in the enrichment plots and evidence that additional carbon is reaching the soil system, however we observed no response in any of the variables we measured. These results, which contrast with those from other temperate forest FACE sites, suggest that soil characteristics can influence the magnitude and timing of belowground responses.

Rapid assay for amidohydrolase (urease) activity in environmental samples

The use of microplate technology for enzyme assays has made it economical to measure a wide range of activities in environmental samples. Urease is one of the most widely measured soil enzyme activities, but current methods are cumbersome. We have developed a rapid, safe and sensitive assay that can be performed on microplates.

The enzymic basis of plant litter decomposition: emergence of an ecological process

Abstract Plant litter decomposition is an integral process in the macronutrient cycles of ecosystems. The absence of fine-scale models for this process hampers attempts to simulate ecosystem responses to disturbance. Two recent studies have suggested that mass loss from decomposing plant litter can be directly related to the temporally-integrated activity of extracellular lignocellulose-degrading enzymes. To evaluate the generality and potential application of such relationships, we surveyed the literature and found eight studies that included mass loss data and some enzymic measure of lignocellulose degradation potential. Although the number of suitable studies was small, they encompassed a broad range of ecosystem and litter types. For all studies, there were strong linear relationships between temporally integrated enzyme activity, expressed as cumulative activity-days, and mass loss. No single enzyme gave the best fit in all cases; multiple linear regressions that included all enzymes measured within a particular study generally yielded better goodness of fit statistics than single enzyme models. Where methodological compatibility permitted, direct comparisons of apparent enzymatic efficiency (relative mass loss/activity-day) were made between studies; values for particular enzymes varied by a factor of ten and were strongly correlated with mean exposure temperature ( r 2 = 0.88). Activation energy for enzymatic decomposition was estimated at 58 kJ mol −1 . Principal components analysis (PCA) was used to generate a composite lignocellulase variable from each study, providing a common format for comparison. The results suggested that the three microcosm studies differed from the field investigations: enzymatic efficiency was approximately half that estimated for field studies and the data were more stochastic. We attributed these differences to disruptions in microbial succession caused by the lack of exogenous sources of colonists, nutrients and grazers. PCA also permitted the calculation of a global regression model for mass loss as a function of cumulative lignocellulase activity. For the five field studies, this model had an r 2 value of 0.73 in linear form and 0.76 in logarithmic form. Our analyses suggest that enzymatic decomposition models retain predictive value even when viewed from the ecosystem perspective. This hierarchal penetrance suggests useful applications: as a monitoring tool for the estimation of litter turnover in the field and as a basis for the simulation of decomposition processes at the microbial community level.