4 Microbial Transformations in Autotrophs and Chemolithotrophs

This article throws light upon the five microbial transformations that take place in autotrophs and chemolithotrophs. The transformations are: 1. Nitrogen Fixation 2. Oxidation of H₂S with Reduction of CO₂ 3. Oxidation of N with Reduction of CO₂ 4. Oxidation of Reduced C

Transformation # 1. Nitrogen Fixation:

After C fixation through photosynthesis, N₂ fixation is the second fundamental reaction needed to introduce essential elements from the atmosphere to the pedosphere (Table 18.12). Representatives of both the photoaquatrophs (Eq. [4] in Table 18.12) and the photolithotrophs (Eq. [5]) carry out N₂ fixation, but it is not restricted to phototrophs.

Consequently, both the oxygenic and the anoxygenic forms of photosynthesis are associated with the ability to fix N₂ as well as to create reduced C for release to subsequent organisms along the food chain.

The amount of N₂ fixed per mole of e^– generated varies because nitrogenase also coreduced H⁺ to H₂ in varying amounts. The values for number of ATPs produced in Table 18.12 are based on 1 mol of H₂ generated per mole of N₂ fixed and an ATP/2e^– ratio of 4.

Aerobic Chemolithotrophic Examples:

The aerobic chemolithtrophic group contains organisms with the ability to reduce CO₂ to CH₂O using the Calvin cycle and energy derived from oxidation of reduced minerals. It cannot operate independent of oxygenic photosynthesis, however because of its strict requirement for O₂.

Transformation # 2. Oxidation of H₂S with Reduction of CO₂:

Oxidation of H₂S yields SO₄^2-, electrons, and hydrogen ions (protons) as in the half-cell reactions depicted in Table 18.13. Water contributes O during formation of SO₄^2-. Electrons are passed down the respiratory chain from S to reduce O₂ to water and generate proton motive force for synthesis of ATP.

Consequently water is both used and formed in such oxidations and there may be no change in total water. Oxidation of S⁰ to SO₄^2- yields only 6e^– per mole of S (Table 18.14, Eq. [8]) and consequently has a lower energy yield (Eq. [9]) than does oxidation of H₂S to SO₄^2- (Table 18.13, Eq. [7]). The overall reaction shows the correct stoichiometry between S, O and H together with generation of acidity.

In such autotrophic (obtain C from CO₂) chemolithotrophs (energy from a chemical source and electrons from a mineral source), CO₂ must be reduced to form biomass, represented here as CH₂O. Electrons for reduction of CO₂ are provided by the oxidation of S (Table 18.14. using S⁰ as an example). Consequently additional S must be oxidized to provide electrons to reduce CO₂[11].

Reverse e^– transport reduces NAD⁺ to NADH for subsequent reduction of NADP⁺, probably by trans-hydrogenation, to NADPH, which is used in the Calvin cycle to reduce C. These reactions generate acidity in soil environments.

For example, the combined set of reactions (Eq. [11]) releases 2 mol of H⁺ per mole of S° oxidized during the processes of energy generation and of biomass formation. Such acidity influences soil pH and mineral weathering.

Large quantities of S may be oxidized to generate ATP for growth and reducing equivalents for the Calvin cycle. A way to relate S oxidation to ATP formation would add precision to understanding about soil microbial ecosystems. ATP yield can be estimated roughly from free energy change.

Because of the non-standard condition of the cytoplasm, free energy change in organisms is less than the “standard” free energy change. ATP synthesis under “cytoplasmic” conditions captures only about 40% of the “standard” free energy change.

Consequently, although ATP hydrolysis yields a standard free energy change of 30 kJ mol^-1, a free energy change of about 30/0.4 = 75 kJ mol^-1 will be expected to yield only 1 mol of ATP.

Accepting this estimate, calculating the expected ATP yield using Eq. [10] yields 584.9/75 = 7.8 mol. The Calvin cycle requires 3 mol of ATP per mole of C fixed.

Rounding to 8 mol of ATP per mole S⁰ oxidized (ATP/2e^– = 2.7) and 3 mol ATP used per mole of C fixed, one can write an overall summary of the amount of S oxidized both to generate ATP and to provide the reducing equivalents needed to reduce CO₂ to CH₂O (Table 18,15 Eq. [12]).

Transformation # 3. Oxidation of N with Reduction of CO₂:

Nitrogen is an important agronomic, ecological, and environmental element that is oxidized by autotrophic chemolithotrophs during nitrification. The N atoms in NH₄⁺ are oxidized to release protons and electrons; and water contributes O for formation of NO₂^– (Table 18.16). The O₂ is the terminal electron acceptor and reacts with electrons and H⁺ to form water [13].

Transport of the electrons and H⁺ down the respiratory chain for final reaction with O² to form water generates the proton motive force used to synthesize ATP. The H⁺ in excess of that used in reacting with O₂ and electrons to form water is released to the environment, causing acidification [14]. Reverse e^– transport is used to transfer e^– transport from NH₄⁺ to NADH as in S⁰ oxidation.

Large amounts of NH₄⁺ must be oxidized, and large amount of H⁺ are released into the environment during N oxidation from NH₄⁺ to NO₃^– (nitrification) to reduce CO₂ for CH₂O production [15]. In contrast, H⁺ is not released during oxidation of NO₂^– to NO₃^– (Table 18.17 Eq. [16]). Consequently one should not conclude that all chemolithotrophic oxidation reactions release H⁺ into the soil environment.

As with S oxidation, it is possible to estimate how much N must be oxidized to provide energy to synthesize microbial biomass of nitrifies. To this end the amount of N that would be oxidized for combined energy and C reduction by Nitrosomonas spp. are estimated in Table 18.18. and for Nitrobacter spp. in Table 18.19.

From Eq. [14] there could 270.7/75 = 3.6 mol ATP mol^-1 of NH₄⁺ oxidized (ATP/2e^– = 1.2). The net estimate is that 12 mol of H⁺ are released, and 4 mol of C are reduced for every 6 mol of NH₄⁺ oxidized to NO₂⁺ (Eq. [18]). This NO₂^– rarely accumulates in soil; it is further oxidized to NO₃^– (Eq. [19]). Less energy is obtained by oxidizing a mole of NO₂^– (Eq. [16]) than is obtained by oxidizing a mole of NH₄⁺ (Eq. [14]).

This partly reflects the smaller number of electrons released in Eq. [16] (2 mol^-1 of N) than in Eq. [14] (16 mol^-1 of N). Because NO₂^– oxidation yield little energy, the ratio of NO₂^– oxidized/C fixed is 5: 1 (Eq. [19]), whereas it is only 1.5: 1 for NH₄⁺ oxidation (Eq. [18]) and the corresponding ratio for S⁰ oxidation is 1.04: 1 (Eq. [12]).

Comparing S oxidation with N oxidation, it is also seen that more energy is released during oxidation of S⁰ to SO₄^2- than in oxidizing NH₄⁺ to NO₂^‑.

This is not due to the different number of electrons involved, because each oxidation removes 6e^– from the element donating them. The difference here lies in the free energy change and associated potential developed for each redox couple and the corresponding difference in location at which the e^– and H⁺ enter the respiratory chain.

Transformation # 4. Oxidation of Reduced C:

Aerobic oxidation of CH₂O to CO₂ is an important source of energy for chemo-organotrophs. It is simplest form this process is the reverse of the overall oxygen is photosynthesis reaction (Table 18.20, Eq. [20]). This group of transformations also includes oxidation of N- or S-containing organic molecules as a source of energy.

Oxidation of L-glutamic acid is one example. Such oxidations to yield energy are responsible for mineralization reactions that release plant nutrients such as NH₄⁺ (Table 18.21). In Table 18.20, oxidation of 1 mol of C as CH₂O generates 4e^–, whereas in Table 18.22, oxidation of 1 mol of C as L-glutamic acid generates only 3.4e^–.

Consequently the amount of ATP synthesized from the oxidation of 1 mol of C from L-glutamate would be expected to be less than during the oxidation of 1 mol of C from glucose. Such N-containing energy sources are less energetically favourable than pure carbohydrates or lipids.

In turn amino acids are best reserved for protein biosynthesis. The search for energy at the expense of N-containing organic molecules (Eq. [21]) in the absence of more energetically favourable alternatives is the basis for much of the N supply to crops from decomposition of humus, plant residues and manures.

Use of oxidized minerals as alternate external electron acceptors leads to reduction of oxidized elements. O₂ prevents reduction of NO₃^–, SO₄^2-, and N₂ however; it is a major product from oxygenic photosynthesis. Consequently, anaerobic-aerobic environments must function in sequence forming a syntrophic system.

A syntrophic system may be defined as one in which two or more species of organisms with contrasting characteristics require each other in order to function or survive.

For example, one species may produce a product or environmental condition that is essential for a second species, which in turn produces a product or condition essential for the first. In the case of the above soil situation, the anaerobic portion of the environment and the organisms in it generates reduced minerals from oxidized forms; the aerobic portion generates O₂ and oxidized minerals from reduced forms.

The sequence of aerobic-anaerobic environments may be organized either temporally or spatially. Oxidized products are typical of the oxygenic cycle. Without a way to reduce such materials, the system would stop. The anaerobic phase, which shares elements with the anoxygenic cycle, reduces oxidized minerals to allow completion of the cycle (Table 18.22).

Reduction of oxidized forms of elements serves diverse purposes designated by specific terms. For example NO₃^– may be reduced for two distinctly different reasons. The first is for incorporation into monomers such as amino acids and assimilation into polymers such as protein, which is called assimilatory NO₃^– reduction.

The second is to use NO₃^– as a terminal electron acceptor in electron transport phosphorylation, which is termed dissimilatory NO₃^– reduction because the N is not assimilated. Alternatively it is called nitrate respiration because of its parallel to oxygen respiration. When dissimilatory NO^–₃ reduction leads to N₂ production it is called de-nitrification.

When it leads to NH₃, it is called dissimilatory NO₃^– reduction to NH₃. Similar distinctions apply to reduction of oxidized forms of S, such that assimilatory S reduction leads to incorporation of S into amino acids or other monomeric building blocks and eventually into biomass. On the other hand, S may be reduced as a terminal electron acceptor during oxidation of C, in which case it is called dissimilatory S reduction.

Two important distinctions exist between organisms responsible for oxidizing reduced C using NO₃^– as a terminal electron acceptor (Table 18.22 Eq. [22]) and those that oxidize C using SO₄^2- as an electron acceptor (termed dissimilatory SO₄^2- reduction or sometimes SO₄^2- respiration; Eq. [23]).

First, NO₃^– respiration is mediated by facultative anaerobes (e.g. Pseudomonas dentrificans), whereas SO₄^2- respiration is mediated by strict anaerobes (e.g. Desulfavibrio desulfuricans). Second, the free energy change and ATP/2e^– ratio is greater for NO₃^– respiration than for SO₄^2- respiration.

Because of these differences some people prefer to restrict the term anaerobic respiration to de-nitrification. This metabolic distinction and the associated greater energy yield from NO₃^– respiration (Eq. [22]) compared to dissimilatory SO₄^2- reduction (Eq. [23]) suggest that one would not expect to find dissimilatory SO₄^2- reduction in a soil well supplied with NO₃^–.

The elemental cycling implications should be noted. For example, production of 1 mol of NO₃^– from NH₄⁺ consumes 2 mol of O₂. Subsequent reduction of that mole of NO₃^– back of NH₄⁺ will support as much oxidation of C as would the original 2 mol of O₂. This is because O accepts 8 mol of electrons from a mole of N and in turn a mole of N gains 8 mol of electrons from C.

Similarly using O₂ to oxidize S^2- to SO₄^2- and then using SO₄^2- to oxidize C yields the same amount of CO₂ as if the C had been oxidized directly with O₂. So where is the distinction between anaerobic oxidation by anaerobic chemoorganotrophs using NO₃^– or SO₄^2- and aerobic chemoorganotrophs using O₂?

The difference is in the yield of ATP and hence potential for growth of biomass, which subsequently controls the rate of the process. The ATP yield is much lower under NO₃^– respiration or SO₄^2- respiration (for those who accept that term for S reduction during C oxidation) than when using O₂.

What about the NO₃^– or SO₄^2-generated during reduction of CO₂ to CH₂O for growth? The treatment above considers only the production of NO₃^– or SO₄^2- associated with O₂ reduction. But more biogenic NO₃^– and SO₄^2- are present in ecosystems than are generated by O₂ reduction.

For example, from Eq. [12] about 1.8 times as much SO₄^2- is generated in reducing C with S⁰ as is used to generate energy by reducing O₂.

Similarly, from Tables 18.14 and 18.15, about 42% of the NO₃^– generated from NH₄⁺ originates through production of reducing equivalents to reduce CO₂ to CH₂O. Consequently the potential for oxidation of C using NO₃^– or SO₄^2- in ecosystems is greater than the consumption of O₂ for their production would indicate.

From a practical environmental perspective, dissimilatory NO₃^– reduction, or the organisms involved in it, may be helpful in facilitating oxidation of persistent organic pollutants under anaerobic conditions.

Is it possible to generate ATP in the absence of external electron acceptors? To do so would require an internal electron acceptor and a net H balance of 0. Further, without an external electron acceptor, there would be no need to transport electrons through a respiratory chain and hence no opportunity for electron transports phosphorylation.

Consequently ATP generation would be by substrate-level phosphorylation. Fermentation is the mechanisms by which ATP is generated in organisms without access to external electron acceptors.

A wide range of substrates is fermented, including carbohydrates, organic acids, amino acids, and purine and pyrimidine bases. Chemically fermentation can be treated as a disproportionation reaction (sometimes called dis-mutation; an oxidation-reduction reaction in which a reactant or element is both oxidized and reduced leading to two different products) in which both the source of e^– and the e^– acceptor is an organic molecule.

Ethanol fermentation is shown in detail in Table 18.23 and several fermentation systems are summarized below:

Ethanol fermentation by yeast

Glucose ←→ 2 enthanol + 2CO₂ + 2ATP

Lactic acid homo-fermentation by bacteria

Glucose ←→ 2 lactate + 2ATP

Lactic acid homo-fermentation by bacteria

Glucose ←→ enthanol + lactate + CO₂ + 1ATP

H₂ production, e.g. butyrate fermentation by bacteria

Glucose ←→ butyrate + 2CO₂ + 2H₂ + 3ATP

Taking ethanol fermentation as an example, the average oxidation state of the C atoms in glucose is 0 (C₆H₁₂O₆). Four electrons are removed from each of 2C atoms to generate 2CO₂ (oxidation state of C + 4), which are distributed by transferring 2 electrons to each of 4 atoms of C to form 2 ethanol molecules (2C₂H₆O; oxidation state of C-2).

The more reduced and the more oxidized moieties can be seen in each of the above examples except for lactic acid fermentation, in which subsequent reduction of one product may yield two identical molecules.

Why bother with all this if energy as ATP is the goal? The reason is that formation of phosphorylated intermediates needed for substrate level phosphorylation requires oxidation of the organic substrate. Such oxidation removes e^– + H⁺, which must go somewhere.

With no external electron acceptor, a vast array of internal mechanisms is used to relocate the e^– + H⁺; hence the great diversity of fermentation systems and of fermentation intermediates within anaerobic ecosystems. Extent of oxidation, energy yield, and hence, growth under such conditions is several folds lower than under aerobic conditions.

Methanogens are the strictest anaerobes normally found in nature and use a limited array of substrates: H₂ + CO₂, formate, methanol, methylamines, and acetate. These substrates are formed during fermentation or converted from fermentation products in anaerobic systems. Two groups of organisms produce methane.

The first is strictly chemolithotrophic organisms that grow on H₂ and CO₂. They are fascinating in their ability to produce all their needs for energy and C from H₂ and CO₂ alone. Clearly some of the reducing equivalents from the H₂ are used for CO₂ reduction as well as for energy generation. The reaction is CO₂ + H₂→ CH₄ + HOH.

Hydrogen can be produced by reactions such as fermentation of butyric acid to produce acetate according to C₄HgO₂ + 2HOH → 2C₂H₄O₂ + 2H₂. Such thermodynamically unfavourable free energy change would suggest that H₂ could not be released. Thermodynamically the actual free energy change is related to the difference between equilibrium concentration and the current concentration.

Rapid utilization of H₂ by methanogens (among others) in close proximity to H₂ producers keeps the H₂ concentration exceedingly low compared to the equilibrium concentration thereby driving the above reaction to the right. The net free energy change for the combined reactions is negative and hence thermodynamically favourable. Hence, syntrophic associations of organisms are important in soils.

The second group of organisms that produces methane is chemo-organotrophic; they produce CH₄ from substrates such as methanol, acetate, or methylamines, which contain methyl groups. For methanol fermentation to CH₄ the overall reaction is 4CH₃OH → 3CH₄ + CO₂ + 2HOH. Acetate is the most common and important, and its conversion to methane is written simply as C₂H₄O₂ → CH₄ + CO₂.

In the presence of SO₄^2- acetate is oxidized to CO₂ rather than being split to CH₄ and CO₂. The preference of SO4^2- is to act as a terminal electron acceptor over methane fermentation. The arrangement of anaerobic and aerobic microsites close to each other in soils facilitates communalistic associations that favour the use of fermentation products either in methanogenesis or by aerobic or anaerobic chemoorganotrophs.

Microbial Contributions:

Two principles are fundamental to this simplified concept.

They are:

1. The mechanisms by which soil organisms achieve their major functions are centered on the supply and inter conversions of diverse forms of energy.

2. These mechanisms are also responsible for most other biological transformations observed in soils.

Transformations mediated by soil organisms result from their search for energy. Energy for soil organisms is obtained by passing electrons from e^– donors to e^– acceptors to produce ATP. Flow of e^– between donors and receptors changes the oxidation states of elements.

These donors and acceptors form multiple interconnected oxidation-reduction couples, which lead to cycles through which electrons flow. Because of the central role of oxidation-reduction reactions, O₂ availability would be expected to be, and is, a major control on how these interconnected oxidation-reduction couples operate.

Electron donors and acceptors in each couple are often different elements. Consequently these flows of e^– unite cycles of elements, alter mobility and functions of elements, and regulate soil biological transformations.

Flow of electrons among these cycles unites activities of extremely diverse groups of soil organisms. Therefore, one can understand most soil biological transformations as a simple framework of interconnected cycle of e^–.

Such a conceptualization is a simple and robust way to unite the myriad details about transformations mediated by soil organisms. An electron cycle model accommodates the full spectrum of understanding from detailed presentations of reactions to global biogeochemical cycles and earth history.