Single-carbon chemistry of acetogenic and methanogenic bacteria

Author(s): J.G. Zeikus , R. Kerby and J.A. Krzycki
Source: Science. 227 (Mar. 8, 1985): p1167.
Document Type: Article

COPYRIGHT 1985 American Association for the Advancement of Science
http://www.sciencemag.org/

Single-Carbon Chemistry of Acetogenic and Methanogenic Bacteria

There has been considerable chemical research on the technological application of single-carbon (C1) transformations. Synthesis gas (syngas), a mixture of hydrogen and carbon monoxide has long been used as a feedstock for production of industrial chemicals. Although syngas has been derived from methane it is increasingly manufactured from coal and may be derived from nonfossilized forms of biomass. The role of syngas and C1 chemistry in the production of polymeric chemicals and fuels may increase as the supply of petroleum-derived chemicals becomes limiting (1).

The conversion of single-carbon compounds to higher molecular weight products depends on metal-containing catalysts (2). The conversion of syngas to methane and higher hydrocarbons (Fischer-Tropsch reaction) and of methane to ethanol occur in the presence of iron-, cobalt-, or nickel-containing catalysts. The conversion of methanol (itself derived from syngas by copper-zinc chromite-containing catalysts) and CO to acetic acid depends on homogeneous rhodium-containing catalysts (Monsanto process), although cobalt was used initially and efforts to produce suitable nickel-containing catalysts are under way.



There are similarities between many of these C1 transformation reactions and metabolic reactions occurring in at least two groups of anaerobic bacteria (acetogens and methanogens) that consume various single-carbon substrates and acetate as energy sources. The acetogens produce acetic, butyric, or mixtures of both acids, while the methanogens produce methane. These bacteria are notable for (i) their complements of unusual enzymes and coenzymes in which nickel, cobalt, iron, tungsten, molybdenum, selenium, and zinc are present either singly or in various combinations; and (ii) their unusual energy-yielding mechanisms, which do not necessarily rely on the breakdown of carbon substrates. Thus, the usual connection between catabolism and degradation of carbon-carbon bonds is not found in the catabolic processes of acetogens and methanogens when they are grown on C1 substrates.

Early studies on acetogens and methanogens have followed a farily parallel course because they are found in similar environments and because they utilize some of the same substrates (3). Thus, research on cultures enriched for growth of methanogens (3) recorded methane and "vinegar acid' formation from H2 and CO2. Soon representative species were isolated including the acetogen Clostridium aceticum, which consumes saccharides and H2 plus CO2, and the methanol- and acetate-catabolizing methanogen, Methanosarcina barkeri.

We review the C1 chemistry of anaerobes that can consume C1 compounds as their sole electron donors and acceptors including both acetogens and methanogens; however, we exclude those species in which sulfate is used as an electron acceptor (4) and the acetogens that degrade purines and amino acids (5).

Microbial Acetogenic Transformations

Table 1 (top) shows the physiological properties of widely studied acetogenic bacteria. Many other species have also been described, including Clostridium thermoautotrophicum which can grow on methanol at temperatures higher than 60|C (6). Analysis of RNA sequences from diverse acetogens has not revealed a unifying correlation between this catabolic process and phylogeny (7). As in other eubacteria, acetogens have typical ester-linked alkyl lipids and a peptidoglycan wall constructed from polymerized N-acetylmuramic acid and N-acetylglucosamine residues. The cross-linking bridge peptides (8), however, contain unusual sequences (Table 1).

Butyribacterium methylotrophicum is the most metabolically versatile acetogen described to date in that it uses a wide variety of C1 substratges singly or in combination and, depending on the substrate composition, it can form acetate or butyrate as the sole end product, or mixtures of both acids (9). The metabolic processes for various single carbon substrate transformations of B. methylotrophicum with the energetics and growth parmeters are shown in Table 2. For the organism to grow readily at high CO partial pressures (>1 atmosphere) it was necessary to select a CO-tolerant strain. The growth yield appears to be directly related to the free energy available from the various catabolic reactions and, when grown on a medium supplied with CH3OH and CO2, more than 20 percent of the CH3OH is converted into cellular components. This represents a very high efficiency of cellular carbon synthesis for an anaerobic microorganism.

The most oxidized substrates are consumed by B. methylotrophicum either individually or concurrently, resulting predominantly in acetic acid synthesis (9). Formate and carbon monoxide (CO), although of equal oxidation state, are metabolized differently by B. methylotrophicum in that (i) more than twice as much CO as formate is consumed when both are present, and (ii) the consumption of formate gives rise to a large amount of H2, which is consumed in the latter stages of fermentation, whereas H2 is only detected as a trace product of CO consumption at the end of fermentation.

Substantial growth on methanol, a substrate more reduced per carbon atom than either acetate or butyrate, requires an oxidized co-substrate (such as CO2, CO, formate, or acetate). Methanol and the co-substrate are simultaneously consumed. The transformation of methanol and CO during growth is a biochemical curiosity since, unlike typical fermentations, neither substrate needs to be oxidized or reduced when both are directly condensed to acetate. 13C-nuclear magnetic resonance labeling studies show that labeled methanol is incorporated predominantly as the methyl moiety of acetate whereas CO is incorporated predominantly as the carboxyl moiety of acetate.

Research on growth and metabolism based on enzyme activities, as well as fermentation and substrate-product labeling patterns (Tables 1 and 2) have led to a common scheme for single-carbon transformations by acetogenic bacteria (Fig. 1). This model shows entry points for various substrates and three mechanisms of substrate-level adenosine triphosphate (ATP) generation: (i) the conversion of acetyl-coenzyme A (-CoA) to acetate and ATP via acetyl phosphate, (ii) conversion of butyryl-CoA to butyrate and APT, and (iii) during methanol oxidation, the conversion of formyl-tetrahydrofolate (formyl-THF) to formate and THF by formyl-THF) to synthetase. These mechanisms of ATP generation may be involved in the synthesis of ATP by acetogens that metabolize purines (5). The initial reduced product of this catabolic process, acetyl-CoA, also serves as the precursor for cellular carbon (10) and thus efficiently links the catabolic and anabolic pathways. Tetrahydrofolate appears to play a key role in the transformation of C1 units between the methyl and formyl oxidation levels in acetogens (Fig. 2). Acetogens have remarkably higher levels of these enzymes than other organisms (11, 12). Additional information is available concerning the biochemical details of anaerobic synthesis of acetic acid from C1 compounds (12-14).

Hydrogen oxidation by hydrogenase (Fig. 1, reaction 1) is coupled with CO2 reduction to acetate in acetogens. However, less is known about this enzyme from acetogens as compared with those from methanogens. Previous reports indicate that the hydrogenases of Acetobacterium woodii and Clostridium thermoaceticum are not nickel-containing enzymes (15, 16). Recent data support the presence of multiple hydrogenases in C. theremoaceticu, one of which is induced only in the presence of CO.

Formate dehydrogenase (FDH) functions at the oxidized end of the THF sequence (Fig. 1, reaction 3) by coupling the oxidation of formic acid to CO2 with the reduction of an electron acceptor (17). The enzyme functions in the reverse direction under different reaction conditions (18). The oxygen-labile FDH from C. thermoaceticum has been purified (17) and the molar ratio of tungsten, selenium, iron, and sulfur present is approximately 2:2:36:50. The specific activity of the enzyme from different acetogens varies, depending both on the energy source (Table 1) and the concentrations of iron, selenium, tungsten, and molybdenum supplied in the growth medium. For example, the specific activity of FDH in extracts of fructose-grown Clostridium formicoaceticum can vary by a factor of 250, depending on the metals present in the medium (19).

At the opposite end of the THF sequence lies a cobalt-containing corrinoid protein (labeled X in Fig. 2) required in the transfer of the methyl group from methyl-THF to the C-2 position of acetate (20). This protein is oxygen-labile and has only recently been purified. Although biochemical studies of the function of cobalt-corrinoids have been limited to C. thermoaceticum, all acetogens assayed had a higher content of corrinoids (see Table 1) than most other bacteria.

The mechanism of synthesis of the precursor to the acetate carboxyl group requires a carbon monoxide dehydrogenase (CODH) activity whose properties have been reviewed (21). Typically, CODH is assayed by coupling the oxidation of CO with the reduction of an artificial electron acceptor (Table 1). The CODH enzymes (22) from both C. thermoaceticum and A. woodii have been purified to >95 percent purity. The high molecular weight enzymes (440,000 and 460,000, respectively) contain nickel, zinc, iron, and acid-labile sulfur. Iron and sulfur exist as Fe4S4 clustern in these enzymes and are reduced and oxidized in the presence of CO and CO2, respectively. On exposure to CO, both enzymes exhibit electron paramagnetic resonance (EPR) signals consistent with the formation of a nickel-carbon radical species.

The function of CODH in acetogens in vivo appears to be more extensive than the oxidation of CO to CO2 (9, 13, 23) as indicated by (i) in vitro results indicating tht CODH is one component necessary for the conversion of methyl-THF and either CO or the carboxyl group of pyruvate into acetyl-CoA; (ii) higher specific activities of the enzyme found when B. methylotrophicum was grown with methanol rather than CO as the energy substrate and the presence of significant amounts of the enzyme in all acetogens tested, regardless of growth substrate (Table 1); and, (iii) preferential in vivo incorporation of CO into the acetate carboxyl group by A. woodii and B. methylotrophicum. Furthermore, the effects of removal of nickel on the fermentation of fructose by A. woodii are consistent with a role for CODH in the synthesis of the acetate carboxyl group (16). Thus, it has been suggested that the role of CODH involves the synthesis of a carbonyl intermediate ([CO] in Fig. 1) from CO, CO2, and the pyruvate carboxyl group (21). CODH and the cobalt-containing corrinoid protein may exist as a complex (20); however, the synthesis of the methyl-carbonyl bond of acetyl-CoA may be a corrinoid-dependent reaction independent of CODH (24).

Figure 2 is a model that can account for energy conservation during growth of acetogens on carbon monoxide. Similar mechanisms could be devised to explain energy generation on formate or H2-CO2. Electron transport phosphorylation may be linked to the dehydrogenative (CODH) and hydrogenative (THF-linked reductions) reactions because substrate level phosphorylations would consume one ATP by formyl-THF synthetase (Fig. 2, reaction 3) for each ATP produced by acetate kinase (Fig. 2, reaction 9). Numerous soluble (ferredoxin, flavodoxin, and rubredoxin) and membrane bound (cytochrome b and menaquinone) electron carriers (25) have been datected in acetogens, but their function in coupling vectorial electron transfer to ATP synthesis has not been demonstrated.

Microbial Methanogenic Transformations

Methanogens display great diversity in morphology, ultrastructure, wall and membrane chemistry, and uncleic acid homology and more than ten different genera have been described (14, 26-30). Studies of RNA sequences from methanogens suggest that individual genera are phylogenetically separated but all genera have been assigned to the kingdom Archaebacteria and not to the kingdom Eubacteria (31). Methanogen membrane lipids are composed of ether-linked isoprenoid units (32) and their walls lack peptidoglycan but are composed of various polysaccharides, polypetides, or a mixture of N-acetyltalosaminuronic acid, N-acetylglucosamine, and polypeptides (33).

Metabolic studies of the two best-characterized methanogenic species, Methanosarcina barkeri and Methanobacterium thermoautotrophicum (Table 1, bottom) and other methanogens, support the model for their single carbon biochemistry outlined in Fig. 3. Carbon flow from C1 substrates to either a methyl or carbonyl intermediate or a C2 product appears schematically similar to that shown in acetogens; however, the carbon carriers used are distinct from THF derivatives.

Barker first postulated that conversion of CO2, methanol, or acetate to methane took place on carbon carriers (26). The initial steps of CO2 reduction by H2 oxidation are not well characterized, but CO2 is thought to be activated by carbon dioxide reduction factor (CDR factor), an unusual coenzyme now identified as methanofuran (34). After reduction, the C1 unit is transferred to tetrahydrometh-anopterin (THMP), also known as form-aldehyde activation factor (30, 35). This pteridine is a yellow fluorescent coenzyme in methanogenesis (36) and has been characterized as methenyl THMP (31). Further reduction to the methyl level presumably takes place on THMP and the methyl group subsequently is transferred to mercaptoethanesulfonic acid (coenzyme M or CoM), a cofactor present in all methanogens (37). Methylmercaptoethanesulfonic acid (methyl CoM) and methenyl THMP are readily labeled when cells of M. barkeri are incubated with 14CO2 or 14CH3OH (36, 38). Methyl CoM is reduced to methane by the enzyme methylreductase, which is present in Methanosarcina (Table 1, bottom) and can account for up to 12 percent of the soluble protein of M. thermoautotrophicum (39). Methylreductase contains a nickel tetrapyrrole, factor F430, as its prosthetic group with 2 moles of the tetrapyrrole present per mole of enzyme (40). The manner in which factor F430 interacts with methyl CoM during methane production is controversial (41).

Thus, the knowledge of enzyme mechanisms (rather than the bacteria or their enzymes) may be useful in industrial organic acid or methane production, by leading to the use of synthetic or semisynthetic organometallic catalysts, based on their biological counterparts. Such systems would imcorporate the mild conditions and selectivity generally attributed to biological catalysts, and simultaneously allow higher rates as well as higher product yield and concentration in accord with current industrial processes (59).

The biochemical cofactors and components present in these bacteria may also be of industrial interest. Methanol-grown acetogens and methanogens contain levels of B12-like vitamins equivalent to those found in commercially used Propionibacterium species that are grown on more expensive substrates (59).

The biochemical components of these anaerobes may be of importance in understanding the diagenesis of petroleum (62). The ether-linked lipids of methanogens and other Archaebacteria may be microbial precursors of the isoprenoid hydrocarbons present in kerogen. Abelsonite is a nickel tetrapyrrole present in Green River shales (63). Although it was suggested that plant chlorophylls might be the precursor to abelsonite, this component of kerogen is structurally similar to the nickel tetrapyrrole, F430, which appears to occur only in methanogens. Similarly, the high corrinoid contents of acetogens and methanogens could have also contributed as microbial precursors of kerogens which were naturally deposited by bacteria in anaerobic sedimentary environments.

Although the basic carbon flow to products in acetogenic and methanogenic bacteria is predictable, we will need to have a better understanding of the exact biochemistries (enzymes, coenzymes, electron carriers, and their cellular localization) in order to test the various proposed models for carbon and electron flow and energy conservation during growth on C1 compounds. The isolation of metabolic mutants will aid in defining the specific biochemistry and the regulation of carbon and electron flow in these bacteria. Further study of the reaction centers of such metalloenzymes as CO dehydrogenase and their organometallic counterparts should prove mutually beneficial to chemists and biologists.

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71. The literature cited was completed in June 1984. We thank L. Daniels, H. Drake, R. K. Thauer, H. G. Wood, and their co-workers for communicating unpublished results. Supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; Department of Energy Grant AC02-80Er1075; Exxon Research and Development Corporation; Genentech Inc.; Institut Pasteur; National Science Foundation prodoctoral fellowship (R.K.); and a Cellular and Molecular Biology Training Grant from the U.S. Public Health Service 144-T263 (J.K.).

Source Citation (MLA 7th Edition)

Zeikus, J.G., R. Kerby, and J.A. Krzycki. "Single-carbon chemistry of acetogenic and methanogenic bacteria." Science 227 (1985): 1167+. Academic OneFile. Web. 13 June 2013.