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The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil

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The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil 2001-2006 Mission Kearney Foundation of Soil Science: Soil Carbon and California's Terrestrial Ecosystems Final Report: 2003318, 1/1/2004-12/31/2006 The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil Joshua P. Schimel1, Shinichi Asao2, Allen Doyle3, Keri Holland4, Eric Seabloom5, Elizabeth Borer5 Summary It has become increasingly clear that extracellular enzymes regulate decomposition and C flow in the environment. However, the specific kinetics and mechanisms regulating enzyme activity and function have been poorly understood. This project took a novel theoretical framework and tested key predictions of that theory that described how C and N availability regulate enzymes and overall C flow. Objectives Exoenzymes mediate breakdown of soil organic matter, and therefore must be included in models of SOM breakdown. Exoenzyme production must be mediated by microbial community composition, and should not ensue unless the returns on production exceed the metabolic costs. High rates of diffusion (Ekschmitt et al. 2005) or high concentrations of cheaters (microbes not producing enzymes but taking up product) can limit rates of production because of low returns on investment (Allison 2005). In contrast, saturation of substrate on enzymes also inhibits production by lowering the return on investment (Schimel and Weintraub 2003). High returns on investment should favor producer community and, therefore, the functionality of enzymes in soils may in part structure microbial communities in soils. A significant portion of enzymes become stabilized in soils, and maintain some activity although kinetic parameters can change as enzymatic conformation is altered by sorption and clay-binding; the large degree of stabilized enzymes in soils may drive enzyme evolution for functionality in the stabilized state (Leprince and Quiquampoix 1996). Consequently, the key control of C limitation in soil is the return on investment microbes received from exoenzyme production. That return is dependent on the amount of resource allocation to enzyme production, the functional kinetics of active enzymes in soil and stabilized enzymes in soil, the functional lifetime of the enzymes in active and stabilized phases, and competition for and diffusion of enzyme products. In continuation of our previous Kearney research, we proposed to focus more specifically on developing an improved quantitative understanding of the linkages between nutrient limitations, exoenzyme activity, C flow, microbial growth, and the fate of processed C. To this end, we proposed to test four hypotheses: • Carbon flow to microbes becomes C limited regardless of the potential availability of polymeric C through non-linear exoenzyme kinetics. 1Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara 2Graduate Student 3Lab Manager 4Postdoctoral Researcher 5Collaborators, Oregon State University: field fertilization The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil—Schimel • Polymer breakdown and microbial monomer use become disconnected following stress events. • Adding C will always increase microbial respiration, but when microbes are N limited it will not increase microbial biomass. • Adding N will not increase soil respiration, rather when microbes are N limited it may reduce respiration by diverting C flow from overflow respiration to microbial growth. To these specific hypotheses, we have added research objectives of 1) evaluating methods to separate out the two-step decomposition process-enzyme driven breakdown of soil organic matter (SOM), and 2) developing more sophisticated SOM models that incorporate enzyme activity. Approach Breaking apart the two-step decomposition process. We evaluated the enzymatic generation of microbial substrate in tundra soils, in which we have seen that decomposition is limited by exoenzyme activity (constrained by N availability). We also used the tundra soils as a powerful model system for developing a method for evaluating enzymatic substrate generation. One challenge in evaluating the role of enzymes in decomposition is that the intermediate products are consumed rapidly. We developed a system to trap the intermediates before they could be consumed by microbial activity. This system involves regular episodic leaching to remove intermediates and constant CHCl3 fumigation to reduce microbial activity and substrate consumption to the minimum possible without damaging enzyme activity. Soil (1 g dry weight equivalent) was placed in a 50 mL syringe and 40 mL DI water was added. It was allowed to sit for 12 hours, at which point, the extractant was drained off. This procedure was repeated for 20 extraction cycles (every 12 hours over 10 days). A parallel set of extractions was done on soil that had been fumigated with CHCl3 (48 h) and was extracted with CHCl3 saturated water. We used CHCl3 fumigation to reduce microbial activity because standard sterilization treatments are likely to denature extracellular enzymes as well. For example the intense heating involved in autoclaving is almost certain to damage enzymes, while heavy metals can bind to them and inactivate them. Modeling We expanded the modeling work started on this project through two approaches. First, by collaborating with Dr. Jason Neff at the University of Colorado; and second,through expanding an international collaboration with Dr. Andreas Richter and a Ph.D. student of his, Tina Kaiser, who took the basic ideas of the exoenzyme model and developed them into a whole-soil C and N model. As a result, Schimel became a member of an advisory group for a large project of Dr. Richter’s that was funded by the Austrian Science Foundation. 2 The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil—Schimel Results Generation of microbial substrates by exo-enzyme activity. Here we report on an additional experiment that addresses a somewhat different aspect of the role of enzymes in mediating decomposition and that is evaluating the intermediate products released in soil by exoenzymes. We found that the CHCl3 treatment dramatically reduced respiration potential, but had relatively limited effect on key enzyme activities (fig. 1), as was consistent with our previous measurements. During the extraction experiment, we found that initial levels of extracted DOC and DON were very high, but dropped off rapidly with multiple extraction cycles. By the end of five cycles, DOC concentrations had declined to a stable baseline value. Thus, there are two notable measurements from this experiment: the initial levels of different compounds and the baseline amount of C released by Figure 1. Effect of CHCl3 fumigation on respiration, protease, and cellulose activity extractions after cycle 5. This baseline is in upslope (black) and footslope (gray) presumably the C that is released in an tundra soils. Activity is measured relative to ongoing way by the action of extracellular an unfumigated control. enzymes. As interesting as the amounts of material, is the composition of the material. The initial material was largely carbohydrate, with only small amounts of amino compounds and phenolics. Since the material that is initially present is likely the material that is “hanging around” because microbes haven’t consumed it, it is surprising that so much of it should be carbohydrate, which we normally assume should be labile. Equally surprising, the material coming off in the later extraction cycles is chemically undefined with no more than 10% being characterized as carbohydrates, or phenolics. The composition of the DOC generated in later extraction cycles remains unclear, but we hope to do follow up chemical analyses using NMR or IR to understand the nature of the material. Adding CHCl3 dramatically altered the amounts and chemical nature of DOC generated. Initial levels of DOC were greatly increased, as would be expected, since this is the basis of the CHCl3 fumigation microbial biomass technique. However, baseline levels of DOC were also increased substantially. The amounts of DOC generated under CHCl3 was on the order of 1 mg C -1 -1 g d , a value quite similar to the amount of CO2 respired by these soils in long-term static incubations. This reinforces the conclusion that the material we were extracting was, in fact, the material that would have been consumed by microbial activity. 3 The Implications of Exoenzyme Activity on C Flow and Microbial Carbon and Nitrogen Limitation in Soil—Schimel We assessed biodegradability by incubating the DOC extracted from the soils after addition of nutrients and inoculation (with an inoculum prepared from a soil composite). Biodegradability was measured by the decrease in DOC over a 20-day incubation. In all soils, the biodegradability of extracted materials increased with extraction cycles. Under control conditions, roughly 10% of the DOC from the initial extraction was Figure 2. Extraction time courses for DOC in consumed under those incubation footslope soils. Upslope soils show similar patterns. conditions, while 25-35% of DOC was consumed from the materials released during the last incubation cycle. This finding reinforces the conclusion that the DOC present in the soil at the beginning of the experiment was relatively recalcitrant, despite the large concentration of carbohydrates. CHCl3 fumigation increased the biodegradability of the material initially extracted, which is consistent with those
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