4

Energy Transformations Supporting Growth and Survival of Soil Microbes

Along with the appropriate chemical and physical properties required for microbial survival, the soil habitat must contain the energy resources necessary for growth and sustainability of the microbes. Additionally, the microbial community must possess the genotypic traits required for energy recovery and be capable of expressing them in situ in the microbe's habitat. A simple question that could provide a starting place to examine energy transformations catalyzed by the resident microbes is “What is the ultimate product of microbial existence from the viewpoint of the microbe; that is, why is this energy supply essential?” A consideration of the microbial metabolic processes leads us to a rather simple, or perhaps, simplistic, response. The most important product from the view of life in soil is new microbial cells. Thus, the microbe must be capable of converting the mineral and organic components of its environment into the building blocks of microbial biomass. Some of the cellular components will be synthesized de novo, whereas others will be incorporated into cellular biomass with minimal or no alteration from the organic components of the microbes environment. All will require expenditure of energy. What is the primary energy source supporting the growth and function of the microbial community? It is reasonable to conclude that in the absence of major human‐produced organic matter, the vast majority of the energy recovered by the microbial community is derived from oxidation of plant biomass. That is, the primary source of the energy “pipeline” used by the microbes is the sun.

Were this synthesis of new microbial biomass the sole outcome of microbial energy recovery, life on our planet would most likely be rather tenuous. True, the soil and the microbes contained therein are continually evolving. The biological, chemical, and physical components are continually changing – the microbes therefore must evolve in order to survive in the stressful soil system. This “dance of survival” performed by the growing microbes and their physical environment results in a multifaceted terrestrial ecosystem (Tate 1985, 1987).

Before exploring the basis of microbial energy recovery, one further question requires attention. At this point, the primary energy source exploited by microbes has been elucidated, but the justification of the interaction with their physical environment must be at least a bit more complicated. The revelation that both the microbes and their habitat are constantly changing provides a window to understanding the significance of the complex energy recovery system to be described in this chapter. The microbial community is continually evolving to adjust to a soil environment that is also evolving in response to its every changing environment. But, change is only part of the story; the two components identified here must be interacting in a long‐term, parallel interactive manner.

Are there examples of the physical and chemical changes in the physical properties of the microbial habitat resulting from the metabolic processes of the microbial population? Yes, the microbes (i) synthesize chelators and carbon dioxide that dissolve soil minerals thereby altering the nature and properties of soil minerals, (ii) mineralize and participate in synthesis of soil organic matter, thereby controlling soil aggregate formation and aboveground biomass production, and (iii) synthesize greenhouse gases such as carbon dioxide, methane, and nitrous oxides, thereby affecting terrestrial climate.

Based on these observations, a primary key to understanding microbial processes in soil is to understand the nuances of microbial energy recovery. Therefore, the basic growth and energy recovery mechanisms of the soil microbial community will be examined herein. This will be achieved by (i) evaluating the applicability of principles revealed through laboratory‐based experiments to microbial growth kinetics in the soil ecosystem and (ii) elucidating the diversity of energy‐yielding processes existent in soil microbial communities and how this array of microbial metabolic processes results in decomposition of both biologically and anthropogenically synthesized organic compounds.

4.1 Microbial Growth Kinetics in Soil

Since the primary benefits to microbes in soil are growth and/or maintenance of population densities, consideration of general microbial growth kinetics and specific examples of their replication and survival patterns in native soil ecosystems is a necessary starting point for analysis of soil physiological processes. To achieve this goal, the applicability of laboratory‐derived relationships to field situations will be examined.

Open vs closed biological systems: In the laboratory, microbial growth kinetics are assessed as closed systems (e.g. culture flasks and test tubes, batch soil incubation chambers) or as open systems (e.g. a chemostat or in some primary aspects, soil columns) (Figure 4.1). A closed ecosystem is self‐contained in that external contributions to microbial function are excluded, whereas open systems receive inputs from processes occurring external to the defined boundaries of the site. For example, an open system can be exemplified by the recycling of organic wastes (sewage, biosolids, animal manures) on an agricultural soil. A consequence of the two defined situations is that since no external nutrient sources is provided in a closed system, energy and essential nutrients can become limited and metabolic waste materials or by‐products can accumulate. In open systems, microbial cells can be constantly bathed in externally supplied growth supporting nutrients such as those provided by root exudates in the rhizosphere and metabolic by‐products may not reach toxic levels in the vicinity of the colony or the individual cell. (In reality, all terrestrial ecosystems are open systems due to the input of photosynthetically fixed carbon driven by solar energy).

Figure 4.1 Examples of laboratory‐incubated open and closed systems for study of microbial processes.

Although it is essentially impossible to exclude external influences from any portion of a soil site, conditions may exist locally where microbial population dynamics mimic those of a culture vessel incubated in the laboratory. This situation can be exemplified by microbial community development in soil surrounding a portion of decaying insect biomass or plant debris. Growth of the microbial population is initially controlled locally by the quantities of readily metabolized substrates exuded from the animal or plant tissues. As the microbes grow and replicate, these easily metabolized substituents are depleted from the site or their concentrations may be reduced to growth‐limiting concentrations. In the latter situation, cellular function is controlled by the rate of solubilization or release of the energy supplying nutrients from within the chitinous cell walls of the insect or the cellulosic walls of the plant biomass. In both cases, initially the more easily mineralized organic substrates are protected by the chitin containing structure of the insect or the cellulosic cell walls of the plant tissue. Eventually the energy‐yielding substrates, e.g. sugars and proteins, are released by the action of enzymes such as chitinase or cellulose that catalyze the conversion of chitin or cellulose to easily metabolizable carbohydrates or amino sugars. As the cell wall structures are breached, the easily mineralized material contained within the cell is released. Upon depletion of all energy and nutrient supply pools in the vicinity of the microbial colony (i.e. total catabolism of the fixed carbon contained in the plant or animal debris), microbial growth ceases and the microbial population becomes quiescent. This state could be achieved prematurely, should growth‐limiting cellular waste products accumulate to inhibitory levels prior to nutrient depletion, or if another essential nutrient supply is exhausted (e.g. free oxygen for aerobic microbes).

At this point, continued activity of the microbial population depends on an influx of nutrients from outside the boundaries of its habitats, as described above. A reasonable example of such a situation in field soils is comparison of a fallow field (noncropped) to an adjacent wheat field (Figure 4.2). In the fallow field, the microbial growth is limited by the existing soil organic matter pools with growth rates declining as the quantities of metabolizable organic matter declines whereas in the wheat field microbial metabolic activity and growth is controlled predominantly by the rate of influx of newly fixed carbon resulting from growth of the wheat crop.

The artificiality of this comparison of the native soil condition with closed cultural systems emerges when the totality of soil properties influencing microbial growth are considered. In the site described above, the primary delimiters of microbial replication were energy and nutrients, but imposition of moisture limitations, deficiencies in fixed nitrogen resources, or even difficulties with temperature modulation can easily be envisioned. Even with this small list of soil properties affecting microbial function, it is clear that both internal and external forces are generally involved in sustenance of the soil biological community. In a soil site contiguous with a variety of different soil habitats, stresses and the means of their relief for microbial populations may easily originate in regions beyond the artificial boundaries of an arbitrarily defined closed system. Thus, in soil, closed ecosystem properties may be invoked to simplify data interpretation, but the experimenter must always bear in mind that the microbes of interest are functioning in a system of interconnected and interacting ecosystem types.

Figure 4.2 Idealized bacterial growth curve.

Not only is a closed system an imperfect descriptor of the rates of biologically catalyzed processes in soils, but the soil ecosystem is also poorly described by the kinetics of an open system. (As will be shown below, chemostat systems are generally used to develop mathematical models of an open biological system.) Many delimiters of biological activity in soil are imposed from outside sources (e.g. nutrient and energy sources, moisture). This external control of microbial growth could result from a variety of conditions, such as from fixed carbon contributions of root exudates in rhizosphere soils, leaching of soluble fixed carbon sources from soil surface organic matter accumulations (e.g. forest litter layers, thatch accumulated in grassland soils), or anthropogenically created situations such as situations where industrial or domestic waste systems enter into a soil ecosystem (e.g. sewage or factory effluents). For example, overland flow of water from the cultivation portion of the wheat‐fallow soil system depicted in Figure 4.2 could transport newly fixed carbon from the wheat field into the noncultivated portion of the site. A common occurrence of such transport is exemplified by the erosion of soil from the top or crest of the hill to the surrounding lowlands which can accelerate microbial growth and increase microbial biomass at the base of the hill due to metabolized organic carbon contained in the runoff water. Thus, it is reasonable to consider the impact of nutrient availability in soils to that encountered in laboratory cultures where the growth of a microbial population results in reduction or even elimination of nutrient and energy reserves and imposition of growth‐limiting conditions.

4.2 Microbial Growth Phases: Laboratory‐Observed Microbial Growth Compared to Soil Population Dynamics

The intricacies of microbial growth and the impact of soil properties on growth rate are best exemplified by contrasting microbial growth principles derived from laboratory study to those occurring in the soil ecosystem. A idealized microbial growth curve (Figure 4.3) is commonly divided into four phases: lag (no observable growth), exponential growth (growth rate > death rate), stationary (growth rate approximately unchanging), and decline phase (death rate > growth rate). Each of these portions of the growth curve can be observed in soil ecosystems. It must be noted that major changes in population density generally occur only with an augmented input of easily metabolized organic matter. This pattern of population development is most commonly associated with soil sites where the native microbial growth rates are stimulated by alteration of soil properties, such as introduction of plant debris into a carbon‐ or energy‐limited community, through oxygenation of an anaerobic system where partially decomposed organic matter has accumulated, or through increased availability of native soil organic matter through disruption of a soil structure that precludes its catabolism. For example, conversion of native grasslands to wheat fields disrupts the soil aggregate structure protecting the soil organic matter results in major declines in soil organic matter pools. Once the previously existing impediments to microbial growth are removed, the cells replicate until a new limitation to community development is encountered (e.g. increased fixed nitrogen availability, decreased molecular oxygen levels).

Figure 4.3 Bacterial growth curve resulting from sequential catabolism of two energy substrates (diauxic growth).

Lag phase: A lag phase occurs when conditions are appropriate for microbial growth, yet there is a delay before a measurable change in microbial population density is detectable. This cellular division could be due to site amendment with metabolizable substances that were previously not present (e.g. pesticides, xenobiotic compounds) or alteration of the physical conditions (e.g. imposition of anaerobic conditions, which would stimulate growth of obligate anaerobes, or reduction of soil pH sufficiently to favor acidophilic populations).

In axenic cultures, a sensitive measure of cellular replication is usually employed. Thus, an accurate assessment of the lag time is gained. This assay sensitivity is difficult to achieve in soil. Most methods for quantifying soil microbial population density do not detect small changes in microbial population densities. Thus, several cell division cycles must occur before a measurable change in the microbial populations is detected. Therefore it could be concluded that at least a portion of the delays in response of soil populations due to system modification is the result of limitations in the methods used to quantify microbial population densities in soil.

Several methods are available for assessing increases in microbial biomass and biomass of individual microbial species in soil, including techniques for assessing the synthesis rates of various biomass specific compounds, such as lipids (¹⁴C‐acetate incorporation into microbial lipids (Barnhart and Vestal 1983; Federle et al. 1983; Tate 1985)) and nucleic acids (³H‐thymidine incorporation (Christensen et al. 1989)). Accuracy of the data yielded from these procedures is controlled by such factors as:

· the variability of biomass within soil samples

· the extraction efficiency for recovery of the population or cellular component assayed from the soil sample

· the minimal detection limit of the measurement procedure.

A variety of implications of the occurrence of a lag phase for microbial growth in soils on interpretation of data describing soil process rates can be conceived. A positive aspect of the occurrence of a lag phase in soil microbial growth kinetics is exemplified by considerations associated with use of biodegradable soil amendment, for example, pesticides. Sufficient longevity of the pesticide in soil for it to interact with the target species is requisite. Greatest time is allowed if the population density of the microbes mineralizing (conversion of the organic substrate to mineral components, including carbon dioxide, ammonium, sulfate, and sulfide) of the soil amendment is limited. With populations of microbes capable of mineralizing the soil amendment of a few hundred or even thousands of organisms per g soil, several cell divisions may be required before detectable declines in the pesticide concentration occur. Thus, lag periods of days to weeks could be anticipated with more pristine systems. In such soil systems, the addition of the pesticide to the soil would have an adequate longevity to accomplish the desired ends before its equally desirable biodegradation occurs. Efficacy of repeated uses of the soil amendment in the system would be dependent on the longevity of the microbial populations involved in the decomposition of the pesticide.

A negative environmental implication of extended delays in biological decomposition of soil amendments is the potential for subsoil or groundwater contamination. Again, biodegradable compounds may be added to soil purposely or accidentally through mismanagement or through spills. To minimize the environmental impact in these situations, rapid biodegradation of the contaminant is necessary. The longer the lag in development of requisite biochemical activity, the greater the potential for leaching of the substance to a site where its presence is inappropriate and its decomposition is less likely to occur (for example, leaching of toxicants from the surface soil horizon to subsoil regions where decomposition is limited or groundwater contamination can occur). Similarly, overland flow of contaminated water or erosion of toxicant bound to soil particles becomes more problematic with longer longevity of the contaminant.

A practical example of the potential to exploit the occurrence of the lag phase is the collection of soil samples for estimation of in situ biochemical activities. Disruption of the soil physical structure during soil sampling necessarily causes sufficient changes in soil properties to stimulate microbial growth. For example, oxygen levels may increase as well as metabolizable carbon resources can be increased by the liberation of organic matter sequestered within the soil physical structure. The delay in growth (lag phase) and use of methods such as storage at 4 °C to reduce the microbial growth rate may afford the experimenter several hours to days to complete the laboratory assessments before statistically significant changes in population densities occur. The length of storage allowed before data are compromised by microbial growth depends upon the properties of the species of interest and the sensitivity of the assay method. The more sensitive the assay (i.e. the capacity to detect changes in population density of a few cells per gram soil) or the more prolific the microbes of interest (i.e. growth rates of a few hours under the storage conditions), the greater will be the problems associated with estimation of in situ soil microbial population densities. Less storage stability is anticipated for samples to be assayed using polymerase chain reaction procedures or fluorescent antibody detection of microbial cells, since these methods are sufficiently sensitive to allow detection of increases of population densities of a few cells per g of soil. In contrast, considerable increase in populations quantified by most‐probable‐number procedures is possible before significant changes in data are demonstrable. Confidence limits of ±300% are not unusual with these types of procedures. In either case, a control study must be conducted to determine the acceptability of the chosen storage procedure for the soils and populations in question.

Exponential growth phase: Conceptually, occurrence of a period of exponential growth, a blooming per se, of specific microbial populations in soil is easily envisioned. Amendment of soil with essentially any biodegradable substance at a nontoxic, growth‐supporting concentration results in an increase in the energy supply supporting microbial growth. The genetic diversity of the indigenous community as well as the commonly occurring immigration of propagules from adjacent as well as distant ecosystems (e.g. via wind and water erosion) virtually assure inoculation of the incorporated materials with organisms capable of the oxidizing them for energy and nutrients.

Other less appreciated, but perhaps more commonly occurring processes inducing microbes to enter an exponential growth phase are associated with indigenous ecosystem processes. Intrusion of predators, such as amoebae, nematodes, or earthworms, into a microsite where bacterial growth is controlled by space limitations may reduce the population densities sufficiently to allow several rounds of cell division. Indeed, balanced feeding of the predator could maintain the prey population in an active growth phase for extended time periods. When this occurs, the replicating microbial population could be dividing at a relatively rapid rate, but ingestion of the newly produced cells by the predators would prevent an observable increase in prey population density.

Similarly, a burst of cell division occurs following freeze–thaw cycles in soil. Freezing and thawing of soil results in death of susceptible microbial populations. For example, Biederbeck and Campbell (1971) found that one freeze–thaw cycle in recently cropped soil resulted in death of 92% of the bacterial population, 55% of the fungi, and 33% of the actinomycetes. The organic substances constituting the cell mass of the deceased organisms provided carbon and energy as well as a variety of cofactors for replication of the surviving microbial populations. This nutrient reservoir plus the reduction in space limitations results in a rapid increase in microbial biomass. Practical implications of freeze–thaw cycles interactions with soil biogeochemical cycles and microbial populations, especially relating to the more frequent and higher intensity freeze–thaw cycles anticipated in high‐latitude regions due to anticipated global climate change, have been noted. For example, Haei et al. (2011) examined the impact of freeze–thaw cycles in soils from a Swedish boreal forest on fungal and bacterial populations resulted in higher fungal growth and reduced bacterial growth ultimately resulting in an increased fungi/bacterial ratio. Others have noted significant impacts of these temperature cycles on soil respiration as well as soil fertility and carbon sequestration (for example, see Jenkins and Adams 2011; Nielsen et al. 2001).

Soil disturbance, natural through activity of soil animals or the result of anthropogenic intervention such as cultivation, may also induce at least a temporary increase in microbial biomass. The disturbance may release occluded organic matter (i.e. substances trapped in soil aggregates) and/or relieve space or free oxygen limitations to growth. Reduction in the quantities of organic matter sequestered in a variety of cultivated soils and associated changes in microbial populations has been demonstration in a variety of agricultural situations. This relationship between soil management and the quality of the soil as assessed by measuring properties of the microbial community (including biomass, diversity, etc.) has long been appreciated. The initial disruption of the soil structure commonly increases the bioavailability of the sequestered soil carbon resulting in microbial growth. With time, due to the changing properties of the residual organic matter and the decreasing quantities resulting from disruption of the aggregate structure, the microbial biomass level declines and microbes more capable of degrading the more resistant soil organic matter components dominate. For example, plowing of a meadow soil results in decreases in native organic matter and an increase in microbial biomass carbon in the top 6 cm of soil by 40–50% (Angers et al. 1992). Related recent studies for reference of this topic include Bailey et al. 2012; Blackwood et al. 2006; Motschenbacher et al. 2014; Six et al. 2000; Smith et al. 2014.

Stationary phase: For many microbial species in soil, the stationary phase is their primary mode of existence. Growth is limited to the degree that new cell production is dependent upon accompanying cell death. For example, although most of the surface area in soil is not occupied by microbial colonies, colonial development is controlled by the space available for expansion in the region of available nutrient and energy resources. Organisms lacking the capacity to migrate to new habitats soon become constrained by the accessible volume of the soil pore in which they are developing. Thus, division of the parent cells depends upon production of space by death and lysis of companion cells. Similar situations may result from the immobilization of a growth‐limiting, essential nutrient into microbial biomass. Again, replication is controlled by the release of the limiting nutrient from biomass via cellular death and decomposition.

Decline (death phase): This portion of the bacterial growth curve is essential for the maintenance of soil community homeostasis. Under relatively constant conditions, system survival depends upon maintenance of the status quo. This includes a balance in microbial population size. Population densities may increase in response to ecosystem perturbation, but to maintain community stability, the augmented population must return to its preexisting density. This conclusion is predicated on the assumption that a permanent change or relatively long‐lived modification of system dynamics has not occurred. In that case, all microbial population densities are modulated to the new levels optimal for the newly established system parameters. In community with inputs and outputs in equilibrium, population increases are dampened by such mechanisms as death and decay of the newly synthesized cell mass, predation by protozoa or other predators, and subsequent decay of their populations.

Diauxic growth: In laboratory culture, microbes are generally grown in a medium containing a single carbon and energy source plus a variety of cofactors. The growth curve depicted in Figure 4.4 is characteristics of such a situation. Since soil microbes rarely, if ever, encounter chemically pure growth substrates, this growth model must be modified to reflect the more complex carbon and energy supplies existent in soil and the microbial response to them. If the microorganism lacks the genetic capacity to metabolize more than one of the fixed carbon substrates present in its habitat, the presence of multiple organic compounds is of little consequence. This situation is rare. More commonly microbes with the means of catabolizing a variety of carbon sources predominate in a soil.

In a uniform system with no other limitations, microbial population increases reflect catabolite repression kinetics. Catabolite repression, also called the glucose effect, occurs when the presence of one growth substrate represses synthesis of enzymes necessary for the metabolism of alternate compounds. Thus, utilization of a second substrate for growth cannot occur until the initial substance is exhausted or reduced in concentration below growth supportive levels. This results in diauxic growth kinetics (Figure 4.3). The growth pattern reflects successive exhaustion of the available substrates. Once the first nutrient and energy resource is depleted, a lag in growth is observed before the microbes commence to use the second substrate for energy and carbon. During the lag period, the microbes are synthesizing the enzymes needed for mineralization of the second growth‐supporting substrate. This transition between energy and nutrient sources can be repeated several times should the microbes be existing in a complex mixture of metabolizable organic compounds.

Figure 4.4 Classical Monod equations describing microbial growth, cell mass yield, and growth substrate interactions.

Demonstration of diauxic growth in the heterogeneous world of soil microbes can be difficult. A preferred carbon and energy source for a particular soil population may become exhausted in one microsite while an adjacent site retains a reasonable supply of the substance. Thus, in a cross‐section of a soil community, a variety of microsites can be envisioned to occur wherein many different organic substances are being concurrently metabolized. Studies in pure culture may predict occurrence of diauxic growth kinetics, but the true field data may not reflect its occurrence because of the nonhomogeneity of the field soil. A more specialized growth curve as exemplified by diauxic growth kinetics may be more significant in a soil contaminated with a mixture of organic substance, such as a soil contaminated by an oil spill where sequential mineralization of the substituents could occur.

4.3 Mathematical Representation of Soil Microbial Growth

A variety of complex mathematical models have been developed to describe microbial metabolism in soil ecosystems. These relationships are commonly based on combinations of simple microbial growth relationships derived from assessment of population behavior in laboratory culture. One set of such equations that may be used to elucidate the nuances of microbial growth in soil are the classical Monod equations (Figure 4.5).

Growth rate relationships to microbial biomass: Equation 1 (dM/ dt = μM) depicts the fundamental association of growth rate with total population biomass present. The specific growth rate (u) is not only intrinsic to the microbe itself (i.e. there is a maximal growth rate characteristic of the organism functioning under optimal conditions) but is also controlled by the physical and chemical conditions of the environment in which the organism exists. In soil, the chemical and physical limitations of the habitat result in microbial population replication at a rate far below its maximum capabilities.

Figure 4.5 Glycolytic conversion of glucose to pyruvate. Note the net production of two ATP and two NADH molecules. Under fermentative conditions, organic intermediates such as acetaldehyde or pyruvate serve as the terminal electron acceptors.

Since bacteria divide by binary fission, the growth rate is related to the generation time as follows:

Under the harsh conditions of the soil environment, generation times of several hours to days are common. These extended generation times compare to division cycles of as short a duration as 10–20 minutes under laboratory culture conditions.

Whereas individual microbial populations in a soil community may be actively replicating, changes in cell mass as well as total microbial biomass may remain unchanged. Individual populations frequently increase or decline, but it is not uncommon to detect a relatively constant level of microbial biomass in an undisturbed soil site. This phenomenon is explained by the fact that (i) most microbes in the surface soil are not replicating – they are in some form of resting state, (ii) some of the microbial populations are replicating, and (iii) others are declining due to cell death, predation, and so forth. Thus, a small portion of the microbial community could be actively dividing, but their increase in number results in an insignificant alteration of the total soil biomass due to the quasi buffering effect of the inactive and declining portions of the microbial community. Similarly, microbial growth can be shown to be occurring, yet population densities appear to be unchanging. Microbes that decompose fixed carbon in soil are fed upon by a variety of predators and parasites. Thus, the microbes could be consumed by predators at a rate approximating their growth rate. Thus, quantification of growth rate by a method such as ³H incorporation into DNA would indicate an actively multiplying microbial community whereas cell numbers would remain constant.

Growth substrate effects on microbial growth rate: Although it is reasonable to assume that the microbial growth rate in soil is proportional to the concentration of growth‐limiting carbon and energy substrates as indicated by Monod equation 2 (Figure 4.4), the relationship is not applicable to soil communities. It is essentially impossible to determine the concentration of growth substrate occurring in the soil microsites where microbial growth occurs. Assumptions underlying the use of concentrations of total substrate in a soil sample as a predictor of microbial growth rate are (i) the substrates and microbes are evenly distributed throughout the soil sample and (ii) all of the carbon and energy sources present in the culture (or vicinity) of the microbial cell are equally available for catabolism. These assumptions clearly are inapplicable in a heterogeneous system such as soil.

Along with the variability in microbial oxidation of their energy sources resulting from the admixture of living cells, soil particulate mineral matter, and colloidal organic matter, heterogeneity of fixed carbon distribution within the soil matrix also results from the exhaustion of nutrients in microsites due to the activity of resident microbial populations. Once the local nutrient supply is depleted due to microbial growth, subsequent growth rates are controlled by the nutrient input rate. Growth kinetics then are described by diffusion equations (static systems) or flow dynamics (where energy resources are contained in water flowing through soil pores – dynamic systems).

Similarly, adequate energy or carbon sources may be available to the microbes in soil microsites but may occur in a form that precludes microbial attack. That is, they may be water insoluble or retained within water‐insoluble structures. Decomposition of these materials and hence the growth rate of microbial populations are controlled by the surface area of the particulate material, the rate of breaching of the protective barriers, or even the dissolution rate of the particulate substance.

Surface area‐controlled microbial growth is exemplified by the oxidation rate of elemental sulfur particles. Sulfur is minimally soluble in water, but its oxidation to sulfate provides an excellent energy source to a variety of sulfur‐oxidizing bacteria. Physical attachment of the bacteria to the sulfur particle is not only required for oxidation of this substance but is frequently the rate‐controlling factor. Early studies by Volger and Umbreit (1941) revealed the necessity for this physical interaction. Similarly, in an examination of the kinetics of oxidation of sulfur by Thiobacillus ferrooxidans, Espejo and Romero (1987) noted that only the bacteria attached to the sulfur particles were capable of growth. Therefore, since the elemental sulfur particles are not soluble in water and direct contact of the microbial cells is necessary for its oxidation, it can be concluded that the surface area of the particles controls not only substrate oxidation rate but also resultant microbial growth since sulfur oxidation is providing the microbial energy supply. Thus, in this situation, microbial growth rate is proportional to substrate surface area, not its concentration.

In contrast, Stucki and Alexander (1987) found that dissolution rate, not surface area of the particles, controlled the decomposition of biphenyl by Moraxella sp. Pseudomonas sp., and Flavobacterium sp. Once the water‐soluble substrates were depleted, dissolution rate of the hydrocarbon apparently controlled the microbial growth rates. That is, growth was controlled by the rate of diffusion of the minimally water‐soluble biphenyl into the water phase.

Many biodecomposable xenobiotic compounds are amended to soils at micro‐ and nanogram concentrations per gram soil. This is especially true of a variety of herbicides that are effective at low concentrations. Due in part to the common practice of evaluating biodegradation potential in culture media containing at least a thousand fold these concentrations, it has been concluded that microorganisms could not mineralize the minute quantities of organic substrates (i.e. the conclusion was based on the assumption that no growth indicated no interaction with the pesticide). Several laboratory studies have shown that mineralization of nanogram concentrations of a variety of organic substrates in culture (Simkins and Alexander 1984), in sewage (Simkins and Alexander 1984, 1985), and in soil (Scow et al. 1986). In the latter study, soil was amended with phenol or aniline and incubated in biometer flasks at 70% or −0.3 bar soil moisture. The soil moisture level resembled one that would be commonly encountered in a well‐drained field soil. The phenol and aniline were mineralized, but not at rates resembling Monod kinetics. A two‐compartment model provided the best fit of the data. These data suggest that biological oxidation can occur in soil at concentrations that would not be anticipated to support synthesis of cell mass.

Growth yield as a measure of biomass production in soil: In culture, the quantity of biomass yielded is proportional to the rate of substrate oxidation (Monod equation 3, Figure 4.4). This relationship is applicable to microbial growth under well‐defined cultural conditions where biomass production is determined by oxidation of a known energy source, such as occurs in a chemostat. Because of the simplicity of the relationship and the general ability to quantify microbial biomass and growth substances, application of this relationship to soil ecosystems is tempting. Unfortunately, the idealized conditions of the chemostat are rarely, if ever, replicated in soil. Use of this relationship with soil systems is precluded (i) by the variable contributions of energy resources to biomass production in soil, (ii) by difficulties in attribution of biomass yields with oxidation of a specific growth substance, and (iii) by complexities in linking biomass synthesis and growth substrate oxidation chronologically in complex soil ecosystems. In soil, a variable proportion of the energy yielded from oxidation of the growth substrate by the slowly replicating cells is utilized for cellular maintenance (maintenance energy). Thus, a clear relationship between energy production and biomass yields is not always evident in soil ecosystems. Furthermore, with the number of fixed carbon and other energy sources available in the soil microbe's habitat, at least a portion of the growth assessed in an experiment could result from oxidation of energy sources not quantified or even considered in the experimental design. Over the duration of an experiment, microbes may catalyze a number of different energy sources.

A further complication associated with soil systems relates to the capability to attribute changes in concentration of energy substrates directly with microbial growth yields. The concurrent observation of products of microbial metabolism in soil is not sufficient evidence to assume their linkage in a cause‐and‐effect relationship. For example, nitrate is commonly detected in soil samples. This compound is the product of energy production by nitrifiers that oxidize nitrite to nitrate. It could be easily assumed that the nitrite oxidizers in a soil sample arose from the production of nitrate associated with them in their habitat, but this nitrate could as easily have entered the soil from external sources (carried with percolating water) or have been produced by previous generations of nitrite oxidizers. Direct linkage of the two variables, nitrifier biomass production and nitrite oxidation, is appropriate only if their augmentation occurs concurrently. Since this prerequisite cannot be generally assumed to be met, application of the third Monod equation to analysis of soil systems should be avoided.

4.4 Uncoupling Energy Production from Microbial Biomass Synthesis

Cometabolism: Organic carbon compounds may also be oxidized in soils in a manner in which energy production, substrate oxidation, and microbial biomass yields are uncoupled. That is, a microbe produces enzymes that catalyze the oxidation of a compound, but no metabolic benefit is provided to the microbe. The energy required to maintain the microbial cell results from the oxidation of a second substrate, which may or may not be chemically related to the cometabolized material. Many aromatic ring‐containing compounds are catabolized in soil through this mechanism. For example, catabolism of a variety of polychlorinated biphenyls (PCBs) constituting the mixture Arochlor was stimulated by the addition of biphenyl to soil samples (Focht and Brunner 1985). In the presence of the biphenyl, between 48% and 49% of the PCBs were converted to carbon dioxide, compared to less than 2% in the absence of the biphenyl. Similar cometabolic responses have been noted for aniline, phenol, and their monochlorinated derivatives (Janke and Ihn 1989); catabolism of trichloroethylene in the presence of methane (Henry and Grbic‐Galic 1991); and chloroparaffinic hydrocarbons (Beam and Perry 1973, 1974). Cometabolism is particularly important in the metabolism of halogenated organic compounds (e.g. Boyle 1989; Wackett 1995).

A fortuitous observation from the view of environmental reclamation is the fact that the energy source and the cometabolized substance need not be present in equivalent amounts. Growth‐supporting concentrations of an energy‐producing substrate can facilitate the catabolism of nanogram concentrations of the cometabolized substrate (e.g. see Schmidt and Alexander 1985; Schmidt et al. 1987; Wang et al. 1984; Wiggins and Alexander 1988). Thus, amendment of soil with metabolizable substrates may encourage decomposition of contaminating organic compounds present in trace quantities.

Maintenance energy: A proportion of the energy derived from oxidation of growth substrates is used for cell maintenance. This is termed maintenance energy (the portion of the energy yielded by substrate metabolism that is not used for biosynthetic purposes). Maintenance energy is used to provide for such basic cellular functions as motility and maintenance of internal osmotic pressure. Under energy‐limiting conditions, the growth yield of microbial cells is reduced by the amount of energy expended to meet these needs. The slower the generation time of a microbe, the larger the proportion of energy required to maintain cellular integrity. Because of the long generation times of soil microbial populations and the observation that these organisms are generally energy limited, significant reductions in growth yield results from maintenance energy expenditures by these populations.

An example of the impact of maintenance energy expenditures on growth yield of microbes growing at reduced growth rates was provided by Traore et al. (1982). Reduction of the apparent growth rate in chemostat culture from 0.206 to 0.0125 h⁻¹ reduced the growth yield of Desulfovibrio vulgaris from 6.65 to 2.06 g mol⁻¹. That is, at the lowest growth rate, 68% of the energy was used for cellular maintenance. This phenomenon combined with the low‐free energy efficiencies explain how relatively high respiration rates may be detected in soil with a minimal increase in microbial biomass. (Free‐energy efficiency equals the free energy captured by the microbe divided by the free energy available in the substrate oxidized for energy. See the section “Chemoautotrophic Existence in Soils” later in this chapter for a comparison of free‐energy efficiencies of soil microbes.) The microbes are replicating, but they are doing so rather inefficiently.

4.5 Implications of Microbial Energy and Carbon Transformation Capacities for Soil Biological Processes

Although elucidation of biochemical pathways utilized by soil microbes and the physiological control of the expression of these capacities generally is studied in axenic culture, the real value of microbial metabolic processes resides in their expression by the organisms in their native environment.

The metabolic diversity of the soil microbial community has long been appreciated. In fact, the list of organic substances that can be mineralized by soil microbes is so extensive that it was considered conceptually to be essentially endless. Soil microbes were deemed to be infallible (the microbial infallibility principle) in their capability to rid our soils of unwanted organic compounds. Thus, it was commonly believed in the mid‐twentieth century that microbes that are capable of decomposing essentially any organic compound could be expected to occur in soil. This opinion provided the justification for the optimistic consideration of soil as a vast repository for degradation of organic wastes. With the advent of the chemical era and initiation of what could be termed a time of excessive exploitation of soil resources, this concept of microbial infallibility has frequently been transformed into a feeling of extreme microbial fallibility. Many difficulties have arisen because organic compounds, which contain chemical linkages rarely if ever encountered in biologically synthesized compounds, are produced industrially and added to soil systems on a routine basis. These same compounds may have a negative or positive effect on the function of the soil microbial community, but the primary environmental impact of consideration is their extended persistence. Accordingly, the concept of microbial infallibility has to be modified to include the concept of “biological” synthesis; that is, the soil microbial community is considered to be capable of decomposing all biologically synthesized organic compounds (or synthetic chemicals that mimic the covalent linkages produced biologically), assuming that all other physical and chemical requirements for microbial function are met. It must be noted that this inclusion of a biological synthesis similarity consideration to assumptions regarding environmental longevity of xenobiotic chemicals in soil does not provide insights into their decomposition rate.

4.5.1 Energy Acquisition in Soil Ecosystems

Soil microbes, as do all living entities, derive the energy necessary for cellular function through oxidation reactions. A basic principle of biochemistry is that all oxidation must be balanced by reductions; that is, electrons generated in the oxidation processes must be transported to an electron acceptor. Common terminal electron acceptors include oxygen, nitrate, nitrite, sulfate, and a variety of organic compounds, including acetate and pyruvate.

Figure 4.6 Production of ATP via transport of electrons through an electron transport chain with molecular oxygen as the terminal electron acceptor.

The quantity of energy provided to the growing cell in the form of adenosine triphosphate (ATP) is dependent upon the intermediate steps between the initial oxidative reaction and the final electron acceptor. A direct transfer of the electron from the oxidized substrate to an organic recipient as occurs in glycolysis (Figure 4.6) yields a single ATP molecule per electron transfer, whereas passage of the electron through a series of intermediate reduction and oxidation reactions (Figure 4.7), as occurs in respiratory metabolism of glucose through the citric acid cycle, yields as many as three ATP molecules per electron transferred. Thus, the incomplete oxidation of glucose to organic intermediates by glycolysis produces a net gain of two ATP molecules, whereas a maximum of 38 ATP molecules is produced by the complete conversion of glucose to carbon dioxide (Figure 4.8).

Aerobic versus anoxic processes: Environmental metabolic processes can conveniently be grouped into two rather broad categories: aerobic and anoxic conversions. Maximal biological energy is made available for cellular function when free oxygen is the terminal electron acceptor. Inversely, incomplete oxidation of fixed carbon substrates in the absence of free oxygen results in reduced energy recovery and reduced biomass production. Based on the above discussion of substrate‐level phosphorylation and the recovery of energy through an electron‐transport chain, such conclusions are reasonable, and are applicable to the majority of metabolic processes.

Fortunately, the underlying assumption of this generalization – cytochrome‐based respiration (the basic process when molecular oxygen serves as the terminal electron acceptor) only occurs under aerobic conditions – is not true. Denitrification (e.g. Koike and Hattori 1975; Kristjansson et al. 1978) and sulfate reduction (e.g. see Kim and Akagi 1985; Postgate 1984), two conversions that require anoxic (oxygen free) conditions, yield energy to the growing cell through cytochrome‐based electron‐transfer chains similar to that depicted in Figure 4.5. Thus, for these two anoxic processes, the quantity of cell mass produced approaches that achieved when free oxygen is the terminal acceptor.

Figure 4.7 The tricarboxylic acid cycle (TCA).

A basic principle to be considered in these dissimilatory processes (oxidation of carbon substrates coupled to cytochrome oxidation under anoxic conditions) is that the utilization of the specific electron acceptors is mutually exclusive. That is, the dissimilatory reduction of nitrate or sulfate does not occur until the molecular oxygen supply in the microsite of the microbial cell is exhausted. This results in a reduction in the local reduction–oxidation potential sufficient to allow utilization of the alternate electron acceptors. Each of these substances functions at specific reduction–oxidation potentials (Table 4.1). That is, the redox potential of the environment of the microbial cells is perched at the specific value associated with a given electron acceptor until it is exhausted, then the potential drops to a new level characteristic of the next electron acceptor. For example, in a system containing nitrogen oxides (but no molecular oxygen), the reduction potential remains at the level dictated by the nitrogen oxides until all of them have been reduced through denitrification, then organic substances or other acceptors (e.g. sulfate), if present, become the primary terminal electron acceptors for the active biological community.

Figure 4.8 General organic matter transformations associated with methanogenesis. Processes enclosed in the box are catalyzed by methanogenic bacteria.

A complex example of this phenomenon involves the sequential oxidation and reduction of growth substrates and terminal electron acceptors in a flooded soil containing nitrate, ammonium, manganese (II), and ferrous iron (Patrick and Jugsujinda 1992). No overlap in the oxidation or reduction of nitrate and manganese was detected, and little overlap in the transformation of manganese and ferrous ions occurred.

The environmental implications of the potential for dissimilatory oxidation of fixed carbon with nitrate or sulfate as the final electron acceptor include the fact that greater biomass is supported by the processes than occurs with classical fermentation reactions, and that the organic substances providing the energy are completely oxidized to carbon dioxide and water. The latter trait is particularly significant in bioremediation processes (Tiedje et al. 1984). This is especially important in situations where the oxygen supply is exhausted due to the rapid catabolism of the energy‐supplying substrate. Should nitrogen oxides or sulfate be present, the contaminant could still be reduced rapidly to carbon dioxide and water even under the anoxic conditions.

The potential for exploiting dissimilatory processes under anoxic conditions for decomposition of soil contaminants is underscored by the variety of substrates catabolized by denitrifiers and sulfate reducers. Denitrifiers have been shown to oxidize a variety of aromatic ring‐containing compounds, including phthalic acid (Aftring et al. 1981; Nozawa and Maruyama 1988), toluene (Altenschmidt and Fuchs 1991; Evans et al. 1991a), xylene (Evans et al. 1991b), benzene and a variety of alkyl benzenes (Hutchins 1991), resorcyclic acids and resorcinol (Kludge et al. 1990), plus the more complex polycyclic aromatic hydrocarbons (McNally et al. 1998; Mihelcic and Luthy 1988a, b). An added advantage of oxidation of these substances via denitrification is that a second soil pollutant, nitrate, is also removed from the system through its conversion to nitrous oxide and dinitrogen. Similarly, sulfate can serve as a terminal electron acceptor for oxidation of such compounds as chlorophenol (Haggblom and Young 1990), aniline and dihydroxybenzenes (Schnell et al. 1989), benzoate (Tsaki et al. 1991), catechol (Szewzk and Pfennig 1987), and m‐cresol (Ramanand and Suflita 1991). Microbial decomposition of similar aromatic compounds with sulfate or nitrate as the terminal electron acceptor by microbial consortia in anoxic conditions is exemplified by Li et al. (2012) and So et al. (2003).

Table 4.1 Reduction potentials of some common electron acceptors functioning in soil biological systems

Redox couple	E0 (volts)
O2/H2O	0.82
Fe3+/Fe2+	0.77
NO3−/NO2−	0.42
Cytochrome c oxidized/reduced	0.25
Acetaldehyde/ethanol	0.20
Cytochrome b oxidized/reduced	0.07
SO42−/S2−	−0.22
NAD+/NADH	−0.32
CO2/CH4	−0.35
H+/H2	−0.42

4.5.2 Microbial Contribution to Soil Energy and Carbon Transformation

The diversity of habitats available to soil microbes is reflected in their versatility in carbon and energy metabolic capacities. Essentially all microbial groupings (Table 4.2) are found at least to a limited extent in soil ecosystems. Some are important contributors to total microbial biomass and are versatile in their capacity to oxidize fixed carbon substrates (e.g. the heterotrophs), whereas others, although limited contributors to total soil microbial biomass, are important because of their pivotal role in soil biogeochemical processes (e.g. nitrifiers). Others pique the interest of soil microbiologists not because of the impact of their presence on soil system function, but more due to their unusual or precarious existence in the system (e.g. soil algae or photosynthetic bacteria). Whether they are major or minor components of the total soil microbial biomass, all replicating microbial are important, if not essential, to total soil ecosystem function.

Table 4.2 Some groupings of soil microbes based on their primary energy sources

Microbial group	Energy source	Carbon source	Terminal electron acceptors	Examples
Heterotrophs
Aerobes	Fixed carbon	Fixed carbon	O2, NOx, SO42−	Arthrobacter spp. Pseudomonas spp. Bacillus spp. Denitrifiers
Anaerobes	Fixed carbon	Fixed carbon	Fixed carbon	Clostridium spp.
Fermenters	Fixed carbon	Fixed carbon or CO2	Fixed carbon	Enterics, lactic acid bacteria
Autotrophs
Photoautotrophs	Light	CO2	CO2	Methanogens algae, photosynthetic bacteria

Chemoautotrophic existence in soils: Chemoautotrophs gain their energy through the oxidation of inorganic compounds. Primary electron sources are ammonium, nitrite, hydrogen, and a variety of sulfur‐based anions. A characteristic of these microbes is their specificity for particular energy sources. For example, autotrophs oxidizing nitrogenous compounds use only ammonium or nitrite as electron sources, whereas sulfur autotrophs are restricted to oxidation of sulfur compounds. Furthermore, the specificity generally extends to individual compounds within each grouping – for example, nitrifiers are divided into ammonium and nitrite oxidizers. Chemoautotrophs may assimilate simple organic compounds, but their primary cellular structure is provided through the reduction of carbon dioxide. Many of the autotrophic bacteria are obligate autotrophs; that is, they can use only carbon dioxide as a carbon source. Facultative autotrophs are capable of growing either autotrophically or heterotrophically.

Chemoautotrophic organisms are of pivotal importance to soil biotic function. Although their contribution of fixed carbon to the ecosystem is minor compared to that incorporated by higher plants, oxidation of their inorganic substrates is frequently central to the completion of various soil biogeochemical cycles. For example, ammonium oxidation to nitrate is primarily catalyzed by autotrophic nitrifiers in soil.

Although neither heterotrophs or chemoautotrophs capture all of the energy released from oxidation of their substrate, chemoautotrophic bacteria are especially inefficient in energy recovery (free energy efficiency). The free energy efficiency is generally measured in culture by quantifying the heat of combustion of the cells produced by metabolism of a specific substrate and the heat of combustion of the quantity of substrate oxidized in this process. This is not measured directly in soil because of the difficulty in determining the specific substrate catabolized among the vast array available to the cell. The free energy efficiency can be as low as 5–10% for oxidation of nitrite to nitrate by Nitrobacter species (Alexander 1977) to 50% for the oxidation of elemental sulfur to sulfate by Thiobacillus thiooxidans.

For the obligate autotrophs, all of the cellular substituents must be synthesized from the reduction of carbon dioxide. Considering that the conversion of ammonium to nitrate yields 66 kcal of energy per mole of substrate oxidized, and the oxidation of nitrate to nitrate produces only 20 kcal of energy per mole of substrate, and that the cellular substituents of both the ammonium and nitrite oxidizers are the same, approximately threefold more nitrite must be oxidized to produce the same biomass of nitrite oxidizers as for the ammonium oxidizers. Approximately 35 nitrogen atoms are oxidized per carbon fixed for the ammonium oxidizers as compared to about 100 nitrogen atoms for the nitrite oxidizers. Thus, the microorganisms using these low‐energy substrates must oxidize large quantities of their substrates to produce the same biomass as those growing by more efficient metabolic processes.

In summary, chemoautotrophs are a small portion of the total soil microbial biomass but they are essential participants in the provision of soil ecosystem services. A major role in ecosystem function and sustainability resides in their capacity to oxidize and to reduce soil mineral components. For example, they convert ammonium to plant available nitrate, oxidize a variety of reduced sulfur compounds to sulfate, and oxidize ferrous iron to ferric iron. Conversely, examples of reductive autotrophic processes include conversion of nitrate to dinitrogen, thereby closing the loop of the nitrogen cycle and reduce sulfate to elemental sulfur and sulfide.

Anaerobic heterotrophs and the soil ecosystem: Anaerobes are microorganisms that grow and reproduce in the absence of free oxygen. (Organisms using cytochrome‐based electron‐transport systems, e.g. sulfate reducers and denitrifiers, even when functioning under anoxic conditions, are by definition excluded from this class of organisms.) Although there is in reality a gradient of sensitivity to free oxygen, oxygen is lethal to anaerobes.

Anaerobic microbes may be divided into two classes, strict or obligate anaerobes, and facultative anaerobes. Strict anaerobes do not grow or survive in the vegetative phase for extended periods in the presence of molecular oxygen. Examples of this group of organisms include the clostridia (which are ubiquitous in soil), methanogens, protozoa, and anaerobic fungi. Facultative anaerobes are capable of growth in the presence or absence of free oxygen. Examples of these organisms include the commonly encountered enteric bacteria.

In spite of their sensitivity to molecular oxygen and the abundance of this molecule in our terrestrial system, anaerobic microbes are nearly ubiquitous in soil systems. In fact, in a well‐aggregated soil, anoxic and aerobic processes generally occur concurrently, as the result of the intermixing of aerobic and anoxic sites in and around the individual soil aggregates. For this to occur, aerobic bacteria and fungi consume molecular oxygen on or near the surface of a soil aggregate at a rate sufficient to lower the free oxygen levels sufficiently that essentially no free oxygen remains to diffuse into the aggregate. Thus, the internal portions of the aggregate become anoxic. Since it is not uncommon for the nutrients requisite to support growth of anaerobic microbes to be contained within the aggregate structure, these populations not only survive but contribute significantly to overall soil metabolic activity.

Flooding of the soil, as can result from a heavy rainfall, may result in expansion of the anaerobic microbial biomass. It must be stressed that a flooded soil is not necessarily an anaerobic soil. The oxygen tension of a flooded soil system depends on (i) the oxygen diffusion rate in water, (ii) the rate of consumption of this molecule by the living community plus any depletion due to chemical processes, as well as (iii) the potential for oxygenation of the sites through influxes of oxygen‐bearing water. That is, a flooded system in which the water level is maintained by inputs of oxygenated waters may not become anoxic. Soil sites commonly associated with oxygen‐depleted conditions are swamps or marshes, rice paddies, sediments, and any other sites receiving inputs of easily decomposed organic matter. The latter soils include a variety of environmentally stressed situations such as buried organic matter in landfills or soils affected by spills of biodecomposable societal products.

The products of complete mineralization of an organic substance containing carbon, hydrogen, oxygen, and sulfur in the absence of oxygen are carbon dioxide, methane, ammonium ion, hydrogen sulfide, and water. This list of products contrasts to the carbon dioxide, ammonium ion, water, and hydrogen sulfide yielded by aerobic decomposition of the same compound. Of more general importance to soil systems are the products of incomplete mineralization of organic substances by facultative and obligate anaerobic organisms. In these situations, alcohols and organic acids accumulate. These fermentations leave a major portion of the energy contained in the substrates in the fermentation products. Thus, microbial biomass production is limited compared to what would be produced through complete, aerobic oxidation of the fixed carbon sources.

Methanogenesis: Methane generation provides an excellent example of complete mineralization of organic compounds under anaerobic conditions and the interaction of a variety of anaerobic bacteria to achieve these ends. Complex substrates are decomposed by anaerobic and fermentative bacteria to the simple substrates that can be converted to methane. Methane synthesis is continuous in marshes and bogs where the extremely reducing environment necessary for the process (−200 to −1000 mV) commonly occurs.

Methane is produced by a highly specialized group of obligately anaerobic microbes, the methanogenic bacteria. This unique group of anaerobic bacteria includes the following species: Methanobacterium arbophilicum, Methanobacterium formicum, Methanobacterium rumination, Methanobacterium mobile, Methanobacterium thermoautotrophicum, Methanococcus vanielli, Methanosarcina barkeri, and Methanospirillum hungatii. These organisms reduce carbon dioxide to methane with hydrogen generally serving as the electron donor by the following reaction:

Therefore, hydrogen is serving as a sole source of reducing power for both methanogenesis and cell carbon synthesis. Other substrates that can be converted to methane include methanol, formate, acetate, and methylamines. With the latter substrates, the carbon substrate serves as both the electron donor and the final electron acceptor. For example, acetate is converted to methane and carbon dioxide as follows:

The acetate, carbon dioxide, and hydrogen used by these methanogenic bacteria are generated by the fermentation of complex polymers, including cellulose and other polysaccharides, by obligate anaerobes, and fermentative bacteria (Figure 4.9). When it is considered that most other carbon pools entering soil are products of decomposition of plant based organic carbon compounds (animal biomass, composts, and sludges, as well as petroleum products), the intense dependence of belowground life processes on plant productivity is highlighted.

Transformation of long‐term stable soil organic matter pools: Biomass carbon entering soil can be divided into two primary pools: biodegradation resistant and readily or easily metabolized carbon. Clearly, the biodegradation‐resistant categorization is a relative term in that these substances (e.g. lignified materials) are decomposed in soil, albeit slowly. The readily metabolized fractions are defined as those fixed carbon materials that provide a carbon and energy source for the soil microbial community with minimal energy expenditure by the microbe. Included in these energy expenditures are the activities necessary to synthesize catabolic enzymes as well as those necessary to activate the substrate molecule to enhance its degradation susceptibility – for example, the phosphorylation of hexoses with ATP to form hexose‐monophosphates (Figure 4.5). Lignin and humic acid do contain sufficient chemical energy to support growing microbes, but the energy expended to synthesize the array of enzymes required for the process far exceeds the amount of energy liberated by the total mineralized of the complex aromatic ring structures.

Figure 4.9 Conceptual model of carbon catabolism by the soil heterotrophic microbial community.

Occurrence of necessary but undetectable metabolic activities: It is not unusual to know a priori that a certain metabolic activity must be present in a soil sample, but due to difficulties associated with analysis of a complex mixture of abiotic and biotic components, detection, or quantification of the process is precluded. Fortunately, the properties of comparative biochemistry dictate that metabolic reactions occurring in bacteria or fungi that can be studied in laboratory culture are common with “minor variations” to those occurring in the soil microbial community. Thus, if a fixed carbon substance is amended to a soil sample and its decomposition is documented, it is reasonable to conclude that the decomposition is occurring, with minor variations, by commonly described biochemical pathways – for example, Embden Meyerhoff Parnas, citric acid cycle pathways.

This observation allows construction of a model predictive of the fate of the primary classes of organic carbon compounds entering soil (Figure 4.9). As in other ecosystems, in soil, most fixed carbon is degraded to simple organic acids that can be activated to acetyl‐coA and fed into the tricarboxylic acid cycle. For example, proteins are hydrolyzed by proteases and deaminated to organic acids. Aromatic ring‐containing substances are hydroxylated to facilitate ring cleavage (Figure 4.10). In this process, molecular oxygen is incorporated directly into the aromatic ring. Thus, it is logical to predict that within any soil sample containing viable microbial populations, all of the common enzymes for intermediary metabolism associated with these cycles plus those activities, such as proteases and hydrolases necessary to produce the precursors of these cycles, are present in that soil sample.

Humification: The complexity of the soil system, including the potential for a variety of spontaneous chemical reactions, provides a number of fates for reactive biochemical intermediates that may not be detected in laboratory culture. Essentially any reactive organic compound entering soil could be incorporated into soil humic acids; that is, it can be humified. Since many of the reactions associated with covalent linkage of organic compounds to humic acids are chemically catalyzed, the reaction rate is controlled in large part by the reactivity of the substrates and the probability of interaction of the reactants. In typical soils containing less than 2% organic matter, the probability of such reactions is limited, although significant. Since in most soil systems the factor controlling the rate of humification is the probability of collision of the reactants, the survival time of the molecule in soil and its concentration in soil become major predictor of whether the substance will be decomposed totally or at least partially, or whether it will be humified. The complex humic acid molecules are formed through a combination of biologically catalyzed reactions and spontaneous chemical reactions. The more long‐lasting organic components, such as complex polyaromatics in petroleum or lignin, are more likely to be humified than the more ephemeral proteins and simple sugars.

Figure 4.10 Examples of pathways leading to entry of the carbons of aromatic compounds into the TCA cycle. The specific pathway depicted is the catechol pathway. Other aromatic ring‐degrading processes include the gentisate and protocatechuate pathways.

4.6 Concluding Comments

The soil microbial community is clearly distinguished by its diversity in metabolic capacity. Along with the common variety of fixed carbon substances decomposed by living organisms, soil microbes also oxidize a number of soil minerals (e.g. sulfides), metals (e.g. ferrous ion), and several cations and anions containing atoms existing in numerous oxidation states (e.g. ammonium and thiosulfate). Combined with the capacity to utilize several different terminal electron acceptors (e.g. molecular oxygen, nitrate, nitrite, sulfate, and numerous organic compounds), microbial life is capable of existing in soil under essentially any chemical and physical condition that allows for sustenance of cellular integrity. The societal legacy of these myriad metabolic capabilities is the potential for the soil microbial community to survive severe chemical insults and to return to previously existing conditions. For example, influxes of acidic solutions of sulfate (acid mine drainage) may be ameliorated by anaerobic reduction of the sulfate to sulfide (i.e. the sulfate is used as a terminal electron acceptor). Toxic organic compounds may be mineralized in the presence or absence of molecular oxygen. Heavy metal contaminants can be removed from interstitial waters by precipitation with the sulfide produced by sulfate reduction. Survivability and sustenance of the soil microbial community are built to a large extent on its vast catalog of catabolic capabilities.

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