IV. Residue degradation kinetics Top

• Microbial degradation of a plant resdiue typically results in an exponential decrease in the amount of substrate over time (FIG. 6). This process is described as following first order kinetics. The "first order" part refers to the fact that the degradation rate is directly proportional to the amount of one of the reactants-the substrate.

• Mathematically, this is represented as: dS/dt = -kS [Equation 1]
  
  which is read as "the change in amount of substrate (dS) per change in a unit of time (dt) is equal to a constant (k) times the amount of substrate present." In this equation "k" is termed the "first order rate constant" and is the only parameter that remains constant (at least in theory).

• As shown in FIG 6 and FIG 7A below, an arithmetic plot of residue degradation following first order kinetics gives the exponential curve. However, Eqn. 1 can be integrated and log transformed to give:
  

ln(S_t/S_o) = -kt

S_o = initial amount of substrate

S_t = substrate amount at time t

     FIG 7A:          arithematic plot                    FIG. 7B:     log plot

• FIGs. 6-7A,B show that with time the rate of residue degradation decreases as the amount of substrate present decreases. Again, k is the only parameter that theoretically remains constant (by definition).

• k is often used to characterize residues or residue components; the greater the value of k the faster the rate of decomposition (FIG. 8).

• k can be also be used to derive a half-life (t_1/2) for degradation, which as the name implies is the time it takes for half of the residue to degrade under a given set of environmental conditions. The half-life is calculated as:
  

t_1/2 = 0.693/k

Thus, higher k values correspond to shorter half-lives.
• Half-lives are useful benchmarks for comparison of natural compounds as well as xenobiotic chemicals such as pesticides; the latter are required to be characterized for soil half-lives before approved for use.

• Its important to note that k is influenced by two factors, the chemical nature of the residue (e.g., lignin content) and the environmental conditions to which the residue is exposed. You can imagine that a change in any factor that affects microbial activity (pH, temperature, moisture/oxygen, etc.) will be reflected in the apparent value for k (or t_1/2).

• A. Definitions
  
• SOM (synonomous with "humus") includes all organic material in soil exclusive of non decomposed lant/animal/insect/microbial residues and live microbial biomass.

• The organic materials comprising SOM are termed "humic substances" (HS). Parts of plant, animal, insect, and microbial biomolecules are the building blocks for HS (FIG. 9).

  Because of the conglomeration of materials that go into HS, similarties exist between HS and known biomolecules; lignin components are beleived to be particularly important constituent of HS. Yet, overall HS are chemically distinct from any known material, and as such SOM is considered a unique entity.
  
  

B. Chemical analysis and characteristics

• The unique chemical nature of SOM has required development of analytical approaches different from those used for other biological materials. The most widely used approach (called "proximal analysis"; FIG. 10) is a series of base/acid extractions, which fractionates SOM into "humin", "humic acid" (HA), and "fulvic acid" (FA).

• Most attention has focused on the HA and FA fractions. Generally, the average MW of HAs is greater that of FAs, while FAs tend to be more acidic (Table 1).

• The relative proportions and chemical composition of HAs and FAs from different soils or environments (i.e., soil vs. lake sediment) would be expected to vary, and refelect the difference in biological inputs and physical-chemical conditions underwhich HS formation occured (FIG. 11).
  

C. Roles and importance of SOM

• SOM is a key component affecting the natural physical-chemical and biological properties of soil.

• Phys.-chem aspects: SOM is responsible for structure (i.e., aggregrate formation), which in turn affects, bulk density, aeration, and water-holding characteristics.

• Biological aspects: SOM largest pool of nutrients for heterotrophic microbes and contains the bulk of the soil organic carbon and most (50-90%) of the total nitrogen, phosphorus, and sulfur.
  
  The large nutrient pool is distrubed (conceptually) across three SOM phases: active (degraded in days-weeks), slowly available (months-years), and inactive (tens-hundreds of years). The majority of the organic nutrient pool appears to reside in the latter two categories, thus SOM acts as a type of slow release nutrient source.
  
  The physical basis for the existence of these phases is ill-defined. Humic materials, because of their heterogeneous chemical composition (and "lignin-like" composition), are intrinsically resistent to microbial degradation (FIG. 12). However, sorption to clays at least partly contributes to "protecting" SOM from degradation.
  
  
• SOM also plays a key role in controlling the behavior and fate of organic xenobiotic compounds (e.g., pesticides) in soil. The SOM provides a organic phase into which the xenobiotics migrate or "partition"; this behavior reflects the tendency of organics to dissolve more in organic solvents than in water.

• Generally, sorption of a xenobiotic into SOM results in immobilization of the xenobiotic, which is reflected in 1.) decreased potential for movement downward with percolating water and 2.) decreased biodegradation rates, which is considered to reflect a decrease in "bioavailability" of the xenobiotic. Conceptually, bioavailability is the existence of a substance in a state accessible to organisms; the physical basis is ill-defined.

  
  

A. Comparative overview of processes in aerobic and anaerobic environments

• As mentioned above, the majority of organic material entering soils and other terrestrial systems is polymeric. Regardless of whether the enviornment is aerobic or anaerobic, the high MW character of polymers neccesitates that these be broken down to smaller units before they can be transported across cell walls/membranes (FIG. 13).

• While this initial phase of aerobic and anaerobic biodegradation is mechanistically similar, subsequent degradation processes differ significantly in that aerobic process tend to result in essentially complete degradation (products = CO₂, H₂O, biomass) while one or more intermediates may accumulate during anaerobic degradation.
  
  
• The intermediates produced by one organism during anaerobic metabolism often serve as a growth substrate for another microbe. Thus, many if not most anaerobic biodegradation processes involve microbial "consortia" wherein one organism feeds off the activity of another (FIG. 14). Consortia might also mediate many aerobic processes, but are not as well-documented.
  

1. Hydrolases

• There are many types of hydrolases that function in a wide variety of biodegradation reactions and which are produced by bacteria, fungi and other organisms. The common feature of all of these enzymes is that they use water to break chemical bonds. We'll see later on that anaerobic organisms make extensive use of hydrolases as well.

• Hydrolases are operative at many stages of residue decomposition, but one of the key functions is to initiate degradation of large polymers outside microbial cells. Thus, hydrolases form one the largest groups of enzymes that are truely "extracellular", i.e., which are produced by an organism with the intention of operating outside the cell.
  
  Examples of extracellular hydrolases and substrates are given in FIG 15.

• It should be noted that the chemical diversity (e.g., different bonds, physical arrangements, etc.) in even relatively simple polymers like cellulose prevents efficient biodegradation by a single enzyme. Thus, enzymes such as cellulase are actually "enzyme complexes" composed of two or three seperate enzymes that differ somewhat in their activities (FIG 16).

• Hydrolases also serve key functions as intercellular enezymes breaking down sugar oligomers to single units, and releasing groups containing N, P, or S nutrients (FIG. 15). Extracellular hydrolases catalyzing these functions can also be demonstrated in soil. For these, it is often unclear whether the soil is the targeted site of activity or whether the enzymes have simply persisted after leaking out of dead or dying cells.

• In addition to decomposing natural substances, hydrolases are also important in mediating degradation of xenobiotic compounds: Pesticides are particularly notable since there are many kinds that contain bonds readliy broken by hydrolases (FIG. 17).

• This is the first example of several that we'll encounter where the world's of natural and xenobiotic chemicals overlap. In all of these cases, a basic question (often unanswered) will be whether degradation is essentially accidental or if it represents a "directed effort" on the part of the microbial population.

• Accidental degradation might occur when a hydrolase does not efficiently discriminate between natural compounds that it was produced to degrade and xenobiotic compounds that also happen to be in the surrounding environment. In this case, the xenobiotic is not considered the "true" substrate; the presence of the xenobiotic may not affect hydrolase expression, the organism might not benefit, and after the hydolysis reaction there may or may not be further decomposition.

• In directed degradation, the hydrolase contributes to a broader degradation "pathway" from which the organism derives a nutritional and/or energetic benefit. Hydrolases used in these pathways may have been borrowed or "recruited" from a natural function and thus represent an adaptation that allows the organism to utilize the xenobiotic as a growth substrate.

  An example of enzyme recruitment is the utilization of "beta oxidation" patways. Beta oxidation is a basic mechanism used for metabolism of fatty acids (FIG. 18). Enzymes used in this pathway have been adapted to function in metabolism of pesticides (FIG. 19).
  

2. Oxygen-dependent transformations

• The use of molecular oxygen (O₂) to assist in degradation of organic compounds is a hallmark of aerobic organisms, and confers on these microbes a much broader degradation spectrum than that of their anaerobic counter parts.

• Two general catagories of oxygen-dependent enzymes are peroxidases and oxygenases.
  
  

a) Peroxidases

• Peroxidases are multifunctional enzymes that first form hydrogen peroxide (H₂O₂) from molecular oxygen and water, and then uses the hydrogen peroxide to effect single electron oxidations of organic compounds (FIG. 20). The "free radicals" generated by this process may react further with molecular oxygen or other organic compounds.

• There are different types of peroxidases but all share the characteristic of being relatively non-specific. This means that a variety of natural as well as xenobiotic compounds may serve as substrates and their transformation may result in a spectrum of degradation products.

• Peroxidases are believed to be the primary enzyme catalyzing degradation of lignin (FIG. 21A), and are another example of an enzyme that by nature is extracellular. Both bacteria and fungi produce peroxidases that degrade lignin, but it's yet to be established whether one or the other is more important for mediating this process in soil.

• Because peroxidases may non-specifically attack a variety of xenobiotic chemicals, organisms producing these enzymes have been explored as potentially useful for bioremediation of contaminated soils (FIG. 21B). Wood-degrading fungi of the "white rot" group have attracted the greatest attention; this subject will be discussed indepth later in the bioremediation section.

b.) Oxygenases

• Oxygenases use molecular oxygen to directly oxidize organic compounds, and are categorized as mono- or di-oxygenases depending on whether they incorporate one or both molecular oxygen atoms into the substrate (FIG. 22).

• Monooxygenases act on both aromatic compounds (benzene ring-based structures) and aliphatic compounds (chemicals lacking benzene ring structures), and mediate a multitude of reactions. Often, these transformations are part of a pathway that allows an organism to grow on the compound. For example, with aliphatic substrates, the alcohols produced by monooxygenases are further oxidized to carboxylic acids, the starting point for ß oxidation (FIG. 23). Hydroxylation is a common reaction for many aromatic compounds and results in the formation of central intermediates (i.e., protocatechate, catechols) that are funneled into basic energy-generating pathways (FIG. 24-22).

• Methane monooxygenase (MMO) mediates the oxidation of methane to methanol. This is a key enzymatic transformation allowing microbial growth on methane, and is particularly noteworthy on two accounts.

  First, methane is a "greenhouse gas" atmospheric enrichments of which are implicated in global warming. "Methanotrophs" (literally "methane-eaters") are estimated to consume up to 90% of methane produced in wetlands and thus greatly reduce the amount of methane released to the atmosphere.

  Second, MMO is a relatively broad-spectrum enzyme and, like the hydrolases discussed above, can also accidentally oxidize some xenobiotic compounds. "Methanotrophs" (literally "methane-eaters") have thus been explored for their potential utility in bioremediation.

• Dioxygenases primarily attack aromatic compounds and mediate two general types of reactions (FIG. 22): adding two oxygen atoms to the benzene ring ("hydroxylation") and breaking the ring ("fission" or "cleavage").

  Collectively, the variety of transformations mediated by hydrolases, oxygenases and other enzymes allows organisms to funnel a wide variety of compounds into central metabolic pathways that support growth (FIG. 24-26).

A. Overview

• As described above, aerobes use oxygen to directly oxidize substrates and to serve as a terminal electron acceptor in respiration. Anaerobic organisms obviously must find alternatives for oxygen in both roles, and may encounter difficulties in developing substitute oxidation mechanisms for some types of organics.

• Examples comparing cellulose and lignin
  Cellulose is degraded relatively easily by anaerobes. The polymer is attacked by hydrolases in a manner similar to that described above for aerobes and the glucose monomers that are produced are then fermented to give alcohols, fatty acids. Oxidation of frementation products may then be linked to a variety of electron acceptors by a variety of organisms and ultimately releasing CO₂ and CH₄ as products. (FIG. 14)

  In contrast, lignin biodegradation by anaerobes is hindered by the apparent lack of an efiificent biodegradation system. Lignin degradation is thus very slow under anaerobic conditions, and the mechanisms by which it does occur (albeit slowly) are ill-defined.

B. Anaerobic degradation of aromatics

• While break-down of the lignin polymer is more difficult for anaerobes than it is for aerobes, anaerobes are able to grow on aromatic compounds that compose this material.

  
  

• Benzoate has been widely used as a model in studies of anaerobic biodegradation of aromatics (FIG. 27). Three phases identified during degradation of this compound are: 1.) Reductive phase: ring staturation (hydrogenases), 2.) beta-oxidation phase, 3.) Ring cleavage.

  The beta-oxidation phase is another example of how microbes have adapted a basic function for other uses.

• Similar pathways have been determined for other aromatics (FIG. 28). A common feature of all is that cyclohexanones are intermediates preceding ring cleavage. Cyclohexanones can be considered central intermediates similar to catechol in the aerobic pathway.

C. Aliphatic hydrocarbons

• Anaerobic organisms appear to be unable to degrade aliphatic hydrocarbons that lack double bonds (saturated hydrocarbons). This can be compared to aerobic organisms, which use oxygenases to activate aliphatics for degradation (FIG. 23).

• Unsaturated hydrocarbons are susceptible to degradation.

D. terminal electron acceptor process (TEAP)


• The foregoing discussion of anaerobic degradation focused on mechanisms used to oxidize the substrate. The other aspect is that anaerobes need to couple oxidation to an appropriate electron acceptor to complete the energy generation process.

• As described above there are a wide variety of alternate electron acceptors that organisms could utilize. Populations will "choose" the electron acceptor that has the greatest potential energetic benefit (Table 2).

• Generally, if a redox couple is calculated to give a favorable energy yield than chances are good that the process is mediated by some microorganism somewhere. However, exceptions to this rule occur. For example, the energy yield of benzene oxidation coupled to nitrate reduction is very similar to that with oxygen. Yet, whether the former process occurs is at best uncertain as denitrifying organisms appear to lack approipriate mechanims for oxidizing benzene (aerobes use oxygenases).

• Most of the electron acceptors that organisms might couple to organic matter degradation will be discussed later in connection with appropriate elemental cycle (e.g., NO₃ reduction in the N cycle). In this section, we'll examine the use of CO₂ as an electron acceptor to complete the C cycle.

• One of the main reactions in which anaerobes use CO₂ as an electron acceptor is:

  H₂ + 0.25 CO₂ ---> 0.25 CH₄ + 0.5H₂O

  The reaction is mediated by methanogens (literally "methane creators"), which are strict anaerobes (cannot tolerate exposure to molecular oxygen) and are classified as Archaea. All organisms within this group have names beginning with the prefix "Methano" (e.g., Methanobacterium, Methanococcus). In the above reaction, methanogens are growing as chemolithotrophs. Some may also ferment acetate and thus grow as chemoorganotrophs:
  
  CH₃CO^2- + H⁺ ---> CH₄ + CO₂ DG'o = -8.9 kcal

• The reactions mediated by methanogens are important in being the terminal transformation processes of carbon in anaerobic environments. Methanogens are also important as the primary biological source of CH₄.

  Another function of methanogens is their role in "interspecies H₂ transfer". This is a process inwhich methanogens are physiologically linked with a different group of organisms, the "proton-reducers". An example reaction mediated by the latter organisms is:
  
  acetate + H₂O ---> CO₂ + H₂ DG'o = +111 kJ

• Obviously, the high positive DG'o makes this reaction unfavorable for supporting growth. However, if the H₂ produced from the reaction is rapidly consumed at kept at a very low level the energetics become favorable (FIG. 29).

• Methanogens consume H₂ and making the reaction energetically favorable. This is an example of "syntrophism"