The implications of exoenzyme activity on microbial carbon and nitrogen limitation in soil: a theoretical model

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Abstract

Traditional models of soil organic matter (SOM) decomposition are all based on first order kinetics in which the decomposition rate of a particular C pool is proportional to the size of the pool and a simple decomposition constant (dC/dt=kC). In fact, SOM decomposition is catalyzed by extracellular enzymes that are produced by microorganisms. We built a simple theoretical model to explore the behavior of the decomposition–microbial growth system when the fundamental kinetic assumption is changed from first order kinetics to exoenzymes catalyzed decomposition (dC/dt=KC×Enzymes). An analysis of the enzyme kinetics showed that there must be some mechanism to produce a non-linear response of decomposition rates to enzyme concentration—the most likely is competition for enzyme binding on solid substrates as predicted by Langmuir adsorption isotherm theory. This non-linearity also induces C limitation, regardless of the potential supply of C. The linked C and N version of the model showed that actual polymer breakdown and microbial use of the released monomers can be disconnected, and that it requires relatively little N to maintain the maximal rate of decomposition, regardless of the microbial biomass’ ability to use the breakdown products. In this model, adding a pulse of C to an N limited system increases respiration, while adding N actually decreases respiration (as C is redirected from waste respiration to microbial growth). For many years, researchers have argued that the lack of a respiratory response by soil microbes to added N indicates that they are not N limited. This model suggests that conclusion may be wrong. While total C flow may be limited by the functioning of the exoenzyme system, actual microbial growth may be N limited.

Introduction

It has long been dogma in microbiology that soil microbes are most commonly limited by carbon availability, even in organic soils where microbes live in the midst of potentially degradable C. Waksman and Stevens (1929) stated: “The fact that the addition of available nitrogen, phosphorus, and potassium did not bring about any appreciable increase in the evolution of CO2 points definitely to the fact that nitrogen is not a limiting factor in the activities of microörganisms in peat but that the available carbon compounds are”. This logic has never been challenged; rather it has been restated and reinforced over the years (Broadbent and Norman, 1947, Alexander, 1977, Flanagan and Van Cleve, 1983, Fogg, 1988, Vance et al., 2001). These results and conclusions are in contrast to studies on N dynamics that strongly suggest that microbes in natural soils are sometimes N limited. For example, many field and lab incubation studies have shown net immobilization (Nadelhoffer et al., 1984, Giblin et al., 1991, Polglase et al., 1992, Wagener and Schimel, 1998), other studies have shown NO3 assimilation in the presence of a measurable NH4+ pool (Jackson et al., 1989, Hart et al., 1994, Chen and Stark, 2000), and Schimel and Firestone (1989) showed that soil microbes use glutamine synthetase to assimilate NH4+. All these constitute evidence that microbes were N limited. Even in a soil that is immobilizing N (suggesting microbial N limitation); however, adding C enhances respiration (suggesting C limitation by the traditional logic; Vance and Chapin, 2001).

Thus, there are two apparent contradictions that develop from previous studies: first, microbes appear to be C limited in the midst of plenty, and second, microbes can appear to be both C and N limited simultaneously. The normal argument to explain C limitation in organic-rich soils is that the low quality of soil substrates limits their availability to soil microbes, and thus, even if the organisms are not truly C limited, they are energy limited (Waksman and Stevens, 1929, Broadbent and Norman, 1947, Flanagan and Van Cleve, 1983). While this argument has been in circulation for many years, the specific physiological mechanisms involved in causing energy limitation have not been well discussed. The apparent contradiction that C based data suggests C limitation, while N based data suggests N limitation has not been discussed in the literature to our knowledge.

We believe that at least a partial resolution to both these apparent contradictions may lie in the nature of organic matter processing and our conceptual and mathematical models for describing it. Most models of soil organic matter (SOM) breakdown assume first order decomposition kineticsdC/dt=KCMdTd(Partonetal.,1987)In this equation, C is the size of a soil carbon pool, K a first order rate constant, and Md and Td are reducing functions based on temperature and moisture. Each SOM pool has a single K value that defines its quality. This approach to modeling and thinking about SOM dynamics is at the heart of almost all SOM models (Van Veen et al., 1984, Parton et al., 1987, Molina et al., 1990, Chertov et al., 1997, Li, 1996; but see Parnas (1975, 1976) for an exception where decomposition is based on microbial growth), and is so thoroughly integrated in thinking about SOM dynamics that it is found in every soil biology textbook we examined (Alexander, 1977, Killham, 1994, Coleman and Crossley, 1996, Paul and Clark, 1996, Sylvia et al., 1998). The argument that grows out of this approach is that a low enough K limits C supply. Microbial biomass then grows up to the point at which the maintenance demand meets the C supply, inducing C and energy limitation. However, the simple argument that a low K value induces C limitation is flawed in a fundamental mechanistic assumption. The flaw is that, biochemically, SOM decomposition is not simply first order. SOM does not break down spontaneously by itself. Rather, its breakdown is catalyzed by extracellular enzymes that are produced by microorganisms. To accurately describe the kinetics of catalyzed reactions the concentration of the catalyst must be part of the rate equation (Roberts, 1977). The most familiar such rate equation is the Michaelis–Menton equationdC/dt=KEC/(Km+C)(Roberts,1977)where K is the fundamental kinetic constant as defined by the quality of the substrate, E the concentration of catalyst, and Km is the half-saturation constant. The equation is commonly simplified by assuming that E is constant, and thus can be combined with K into a Vmax term (the maximum reaction rate, defined as KE). Under some conditions (e.g. low substrate concentrations), this relationship can be effectively simplified to a pseudo-first order equation, but even in that case, the concentration of the catalyst remains part of the fundamental rate equation (Schimel, 2001). At the microbial scale, considering SOM breakdown kinetics and C supply to microbes requires considering the dynamics of the catalyst to accurately model processes (Parnas, 1975, Parnas, 1976, Vetter et al., 1998, Schimel, 2001). A number of authors have discussed the role of exoenzymes in controlling decomposition rates (Burns, 1982, Sinsabaugh, 1994, Sinsabaugh and Moorhead, 1994, Foreman et al., 1998, Moorhead and Sinsabaugh, 2000). However, we take those ideas further and argue that to understand the basis of microbial C and N limitation in soil, it is necessary to consider the nature of catalysis.

One can argue that if the microbes could increase their investment in exoenzymes, they could accelerate the breakdown of SOM and increase the flow of C back to the microbes, thus alleviating C limitation, regardless of the fundamental K value for organic matter breakdown. This defines the fallacy in the argument that a low K value alone can induce C limitation. As long as catalyst concentration is a term in the reaction rate equation, recalcitrance, in terms of a low K value, cannot by itself induce C limitation.

While the rate at which SOM is processed is strongly controlled by the quality of the material (the fundamental K value), the extent of C limitation to the microorganisms is controlled by the dynamics of exoenzymes. The key control of C limitation becomes the ‘return on investment’ microbes get in producing exoenzymes. If that return is large, i.e. more atoms of C and more useable energy come back than went into producing the enzymes, then microbes should be able to synthesize yet more enzymes and grow rapidly on the substrate, regardless of its recalcitrance. In such a case, microbes would not be C limited. If the return on investment were negative, however, less C and energy would come back than it cost to produce the enzymes and provide basic cellular maintenance needs. If that were the only available substrate, growth would ultimately stop and the organism would starve, although it was in the midst of apparent plenty. In such a case, only if exoenzyme production were subsidized by C and energy from other substrates (e.g. root exudates) would exoenzyme production continue and the substrate be broken down. This is one of the rationales behind the concept of priming (Bingeman et al., 1953, Dalenberg and Jager, 1989); by supplying labile C it should be possible to subsidize and stimulate the breakdown of recalcitrant OM.

In this paper, we develop a simple theoretical model to explore the dynamics of the decomposition–microbial growth system when the fundamental kinetic assumption is changed from simple first order kinetics to decomposition that is catalyzed by an exoenzyme system. We built two versions of the model, one which assumes C limitation and a more complex version that integrates N and allows either C or N to limit microbial growth. We had two primary issues we wanted to examine: (1) how C limitation to soil microbes is induced by the behavior of the exoenzyme system, and (2) how C flow and microbial growth might behave under N limited conditions.

Section snippets

C-only model

The fundamental structure of the C-only model is shown in Fig. 1. The basic concept underlying the model is that the breakdown of SOM is controlled by the activity of exoenzymes, rather than simply the concentration of SOM. This breakdown produces a pool of small dissolved organic molecules that can be taken up and used by soil microbes to provide for their needs (in order of priority):

  • 1.

    exoenzyme synthesis;

  • 2.

    cellular maintenance (respired as CO2);

  • 3.

    biomass production.

The production of exoenzymes and

Kinetic response to enzyme concentration

When the model is run with decomposition as a linear function of enzyme concentration, there is only a knife-edge equilibrium of stable system behavior (Fig. 3a). With a Kd value other than exactly 1.00, the biomass ultimately either grows out of control or crashes. With a Kd>1.00, there is a net positive C supply to the microbes, fueling exoenzyme production and driving a positive feedback cycle among enzyme production, decomposition, and C flow to microbes (Fig. 3a). With other parameter

C-only model

Using this model, we have examined how changing the underlying kinetic assumptions in a litter or SOM decomposition model can produce very different and perhaps counterintuitive results from those generated by traditional first order kinetic models. This alternative construct closely mimics several patterns that are commonly observed in real soils. First, studies have commonly suggested that soil microbes appear to be C limited, even in organic soils where C availability is potentially very

Acknowledgements

We thank Roger Nisbet and Jill Schmidt for valuable discussions on the basic ideas presented here. We thank Patricia Holden for thoughtful comments on the model structure and the manuscript. Support for this research came from the US National Science Foundation Bonanza Creek Long Term Ecological Research, TECO, ATLAS and Microbial Observatories Programs, and from the Andrew W. Mellon Foundation.

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