Methanogenesis

Biological Methanogenesis has a significant role in the global carbon cycle. This process is one of several anaerobic degradative processes that complement aerobic degradation by utilizing alternative electron acceptors in habitats where O2 is not available. The hierarchy of electron acceptors is O2_NO3 _ _Fe_3_Mn_4_SO_2 4 _CO2, where reduction of CO2 to form CH4 is the ultimate terminal process catalyzed by degradative microbial consortia in the absence of all other electron acceptors. Methanogenesis is catalyzed exclusively by prokaryotic single-cell microorganisms, classified as methanogenic Archaea. Although these microorganisms require highly reduced, anaerobic conditions for growth, methanogenesis is ubiquitous in the environment and occurs in many habitats, including sewage digestors, waste landfills, marine and freshwater sediments, algal mats, wet wood of living trees, mammalian gastrointestinal tracts, ruminants, termite guts, geothermal vents, and deep subsurface rock (Fig. 57.1). CH4 generated by methanogens is ultimately converted to CO2 by CH4-oxidizing bacteria or released into the atmosphere where it accumulates as a greenhouse gas. The combined processes of aerobic and anaerobic degradation, which include methanogenesis, ensures that CO2 “fixed” as cell carbon by photosynthetic organisms, and consumed by higher organisms in the food chain, such as herbivores an carnivores, is eventually restored to the atmosphere as CO2, thus completing the global carbon cycle. I. HISTORICAL OVERVIEW The generation of combustible gas, presumably CH4, has been reported by Pliny as early as during the Roman Empire. Legendary manifestations of methanogenesis include the will-o-the-wisp, hypothesized to have resulted from the spontaneous combustion of marsh gas, and fire-breathing dragons, conjectured to have resulted from the accidental ignition of gas from CH4-belching ruminants. The close association between decaying plant material and the generation of “combustible air” was first described by the Italian physicist Alessandro Volta in 1776, when he reported that gas released after disturbing marsh and lake sediments produced a blue flame when ignited by a candle. Bechamp, a student of Pasteur, was the first to establish that methanogenesis was a microbial process, which was corroborated by others throughout the remainder of the nineteenth century and early twentieth centuries. Because of the methanogens’ requirement for strict anaerobic conditions, the first isolates were not reported until the 1940s. The approach used for isolation, the shake culture, involved adding microorganisms to molten agar growth medium containing a reductant, such as pyrogallol-carbonate, to prevent O2 from diffusing into the agar. However, this approach was not suitable for isolating and maintaining methanogens in pure culture for long periods of time, as the medium was not sufficiently anaerobic. It was not until 1950 that a simple, effective technique was developed that provided the rigorous conditions required for routine isolation and culturing of methanogenic Archaea. The technique, referred to as the “Hungate Technique,” employs gassing cannula, O2-free gases, and cysteinesulfide reducing buffers to prepare a highly reduced, O2-free medium. Boiling initially deoxygenates medium and a cannula connected to an anaerobic gas line, such as N2 or CO2, is inserted into the vessel to displace air as the medium cools. The medium is dispensed into culture tubes or serum vials while purging with anaerobic gas and then the medium is sealed with a rubber stopper or septum. The vessel containing reduced medium and anaerobic gas effectively becomes an anaerobic chamber for culture growth. The development of the anaerobic glove box has further simplified culturing of methanogens by providing a means for colony isolation in petri plates containing anaerobic medium (Fig. 57.2). Inoculated plates are then transferred to an anaerobe jar that is purged with anaerobic gas and hydrogen sulfide to create conditions necessary for growth. II. DIVERSITY AND PHYLOGENY The methanogens are members of the Archaea, one of three domains of life proposed by C. Woese on the basis of 16S rRNA sequence, which also include the Bacteria and Eukarya (Fig. 57.3). Archaea have morphological features that resemble the Bacteria; they are unicellular microorganisms that lack a nuclear membrane and intracellular compartmentalization. In contrast, several molecular features of the Archaea have similarity to the Eukarya; these feature include histone-like DNA proteins, a large multicomponent RNA polymerase, and eukaryal-like transcription initiation. Despite the similarities to the other domains, Archaea also have unique characteristics that distinguish them from the Bacteria and Eukarya. These distinguishing features include membranes composed of isoprenoids ether-linked to glycerol or carbohydrates, cell walls that lack peptidoglycan, synthesis of unique enzymes, and enzyme cofactor molecules. An additional unifying characteristic among the Archaea is their requirement for extreme growth conditions, such as high temperatures, extreme salinity, and, in the case of the methanogens, highly reduced, O2-free anoxic environments. Although the methanogens are a phylogenetically coherent group and have a limited substrate range, they are morphologically and physiologically diverse. They include psychrophilic species from Antarctica that grow at 1.7 _C to extremely thermophilic species from deep submarine vents that grow at 110 _C; acidophiles from marine vents that grow at pH 5.0 to alkaliphiles from alkaline lake sediments that grow at pH 10.3; species from freshwater lake sediments that grow at saline concentrations below 0.1 M to extreme halophiles from solar salterns that grow at nearly saturated NaCl concentrations; autotrophs that use only CO2 for cell carbon and methylotrophs that utilize methylated carbon compounds. Despite the range and diversity of growth habitats where methanogens are found, methanogens have one common attribute: they all generate CH4 during growth. There are currently over 60 described species of methanogens in five orders within the archaeal kingdom Euryarchaeota (Fig. 57.3). Characteristics of methanogenic Archaea are described in Table 57.1. The order Methanococcales includes marine autotrophs that grow exclusively by CO2 reduction with H2. Morphologically, these species form irregularly shaped cocci. Instead of a rigid cell wall, typical of most Bacteria, these species form an S-layer, composed of an array of protein subunits, and are subject to osmotic lysis at NaCl concentrations below seawater. This order includes mesophilic Methanococcus spp., the moderate thermophile Methanothermococcus thermolithotrophicus, and the extreme thermophilic Methanocaldococcus spp. The order Methanobacteriales is composed predominantly of rod-shaped cells that grow by CO2 reduction with H2. The exception is the genus Methanosphaera, which grows as cocci and uses H2 to reduce methanol instead of CO2. Cells have a rigid cell wall approximately 15–20 nm thick and, when stained for thinsection electron microscopy, resemble the electron dense monolayer cell wall of gram-positive bacteria. These archaeal cell walls are composed of pseudomurein, which is chemically distinguishable from bacterial murein by the substitution of N-talosaminuronic acid for N-acetylmuramic acid and substitution of _(1,3) for _(1,4) linkage in the glycan strands, and substitution of D-amino acids for L-amino acids in the peptide cross-linkage. Methanothermus species also have an additional cell-wall layer, composed of glycoprotein S-layer that surrounds the pseudomurein. Methanobrevibacter species are all mesophilic, Methanobacterium includes mesophiles and moderate thermophiles with optimal growth temperatures are high as 75_C, Methanothermobacter species are exclusively moderate thermophiles, and Methanothermus species are extreme thermophiles, with maximum growth temperatures as high as 97 _C. The order Methanomicrobiales contains genera that are diverse in morphology and physiology. Most species grow as cocci and rods. In addition, Methanoplanus forms flat plate-like cells with characteristically angular ends. Another species, Methanospirillum hungateii, forms a helical spiral. The cell walls in this order are composed of a protein S-layer and are sensitive to osmotic shock or detergents. In addition to the S-layer, M. hungateii also has an external sheath that is composed of concentric rings stacked together. Species are generally slightly halophilic and include mesophiles, moderate, and extreme thermophiles. Most species grow by CO2 reduction with H2, but some species also use formate or secondary alcohols as electron donors for CO2 reduction. The order Methanosarcinales is the most catabolically diverse species of methanogens. In addition to growth and methanogenesis by CO2 reduction with H2, some species grow by the dismutation, or “splitting,” of acetate and by methylotrophic catabolism of methanol, methylated amines, pyruvate, and dimethylsulfide. While some species of Methanosarcina can grow by all three catabolic pathways, Methanosaeta species are obligate acetotrophs and all other genera are obligate methylotrophs. All species have a protein S-layer cell wall and most species grow as irregularly shaped cocci. However, several species of Methanosarcina also synthesize a heteropolysaccharide matrix external to the S-layer. This external layer can be up to 200nm thick and is composed primarily of a nonsulfonated polymer of N-acetylgalactosamine and D-glucuronic or D-galacturonic acids. The matrix is called methanochondroitin because of its chemical similarity to a mammalian connective tissue component, known as chondroitin. At freshwater NaCl concentrations, Methanosarcina spp. that synthesize methanochondroitin grow in multicellular aggregates rather than as single cells, but when grown at marine salt concentrations or with high concentrations of divalent cations, such as Mg_2, they no longer synthesize methanochondroitin and grow as single cells. Methnaosaeta species have an external sheath that appears similar in structure to that previously described for M. hungateii. The order Methanopyrales is the most deeply branching methanogenic archaeon and presently includes only one species, Methanopyrus kandleri. This species is an obligate hydrogenotroph and grows as a rod with a pseudomurein cell wall surrounded by a protein S-layer, similar to that described for Methanothermus. III. HABITATS A. Interspecies H2 transfer As has been described, methanogens utilize a limited number of simple substrates. In most habitats, they depend on other anaerobes to convert complex organic matter into substrates that they can catabolize. Therefore, unlike aerobic habitats, where a single microorganism can catalyze the mineralization of a polymer by oxidation to CO2, degradation in anaerobic habitats requires consortia of interacting microorganisms to convert polymers to CH4. These interactions are dynamic with the methanogens affecting the pathway of electron flow, and, consequently, carbon flow, by a process called interspecies H2 transfer. In this association, the H2-utilizing methanogens maintain a low H2 partial pressure that allows certain reactions to be thermodynamically favorable. One physiological group of microorganisms affected by this process is the H2-producing acetogens. The reactions carried out by these microorganisms for growth are not thermodynamically favorable (i.e. __G0  under physiological growth conditions), as the H2 they generate accumulates and growth is subsequently inhibited. However, in association with H2-consuming microorganisms, such as the methanogens, the H2 partial pressure is maintained at levels low enough to make the reaction thermodynamically favorable (i.e. __G0  under physiological growth conditions). Because of their dependence on the H2-consuming microorganisms, the H2-producing acetogens are often referred to as obligate syntrophs. Another physiological group of microorganisms affected by interspecies H2 transfer is the fermentative anaerobes that synthesize a hydrogenase. In many of these microorganisms, substrate oxidation is linked to the electron carrier nicotinamide adenine dinucleotide (NAD), which has higher redox potential (_320mV) than H2 (_414 mV) under standard conditions. However, if the H2 partial pressure is maintained at a low level (10 Pa) by H2-utilizing microorganisms, then H2 production from NAD becomes thermodynamically favorable. This enables the fermentative microorganisms to reoxidize NADH by reducing protons to form H2 rather than reducing pyruvate to form dicarboxylic acids and alcohols. This synergistic process enables the fermentor to conserve ATP by synthesizing more acetate and less reduced products. In addition, the net products, acetate and H2, serve as substrates for growth by the methanogen. The result is that carbon and electron flow is directed towards more efficient degradation by the consortium to the gases CH4 and CO2. CO2 reenters the carbon cycle directly and CH4 is either oxidized by methylotrophs to CO2 as it diffuses into the aerobic zone or it enters the atmosphere. Synergistic interspecies substrate transfer has also been reported to occur with formate and acetate. Removal of formate by formate-utilizing methanogens and the removal of acetate by acetotrophic methanogens have been shown to create a thermodynamic shift that favors butyrate and propionate degradation in syntrophic co-cultures of acetogenic Bacteria and methanogenic Archaea. B. Freshwater sediments As organic matter accumulates on lake and river basins, the sediment becomes anaerobic, because oxygen is depleted by the activities of aerobic microorganisms. In eutrophic environments with high organic loading, the anaerobic region occurs immediately below the sediment surface and even into the water column if the activities of the aerobic microorganisms exceed the rate of oxygen diffusion. Once oxygen and alternative electron acceptors such as NO3_, Fe_3, Mn_4, and SO_2 4 are depleted, methanogenesis becomes the ultimate degradative process. The flow of carbon in anaerobic freshwater sediments is shown in Fig. 57.4. Fermentative bacteria that synthesize hydrolytic enzymes, such as cellulases, proteases, amylases, and lipases, catalyze degradation of complex polymers to soluble monomers. The fermentative bacteria then ferment the soluble products to H2, CO2, simple alcohols, and fatty acids, including significant generation of acetate, due to interspecies H2 transfer. H2-producing acetogenic bacteria then catalyze the oxidation of alcohols and fatty acids to H2, CO2, and acetate. The third consortium member, the methanogenic Archaea, utilizes the simple substrates H2, CO2, and acetate generated by the fermentative and acetogenic bacteria, to generate CH4. The net result of this consortium is that carbon and electrons are directed toward the synthesis of CH4 and CO2, which then reenter the global carbon cycle. CH4 is oxidized by aerobic methanotrophic bacteria as it diffuses through the aerobic regions of the water column and reenters the atmosphere as CO2. However, some CH4 escapes from shallow water bodies, such as rice fields, and through the vascular systems of aquatic plants and enters the atmosphere. C. Anaerobic bioreactors Methanogenic bioreactors are used for the conversion of organic wastes to CH4 and CO2. These anaerobic digestors are found in nearly all sewage treatment plants where wastes in the form of sewage sludge, polymeric particulate material generated by settling of raw sewage, are converted to CH4 and CO2 by a consortium of microorganisms in freshwater sediments similar to that previously described. In contrast to most sediment environments, microbial metabolism is higher in a bioreactor because of greater rates of organic loading and higher temperatures (35–40 _C) generated by heating the reactor, often by combusting the CH4 generated in the degradative process. As a result, the rate-limiting factor in a bioreactor is the slow growth rates of the acid-consuming H2-producing acetogens and the acetate-utilizing methanogens, which require retention times of 14 days or greater, to compensate for their slow metabolic activities. Another critical factor is the requirement for H2-utilizing methanogens to maintain H2 partial pressures below 10 Pa. Perturbations in the process that result in inhibition and subsequent “washout” of either of these metabolic groups will result in a drop in pH, resulting in acid accumulation and subsequent inhibition of the entire reactor process. Perturbations can include sudden overloading with a readily fermented organic substrate that results in rapid accumulation of H2 and fatty acids or introduction of a toxic compound that disrupts the microbial balance. Anaerobic bioreactors have also been tested as a low-cost method for treatment of other types of particulate organic wastes, including animal manure and crop wastes. Nonparticulate industrial wastes, including many food processing by-products, and organic solvents, such as chlorinated aliphatic and aromatic compounds, are often too dilute to be economically treated by standard bioreactor configurations, which would require long retention times. In order to decrease the hydraulic retention time of the waste without washing out the biomass, high-rate anaerobic bioreactor configurations have been developed. Examples of these reactors include fixed-film reactors, in which biofilms of microbial consortia are retained in the vessel on solid supports, such as plastic or ceramic matrixes, glass beads, or sand grains. Other designs exist, such as the anaerobic upflow sludge blanket process, in which biomass is immobilized by the aggregation of microbial consortia into distinct granules (Fig. 57.5). The granules usually consist of three discrete layers of microorganisms that include acetate-utilizing methanogens in the interior, H2- producing acetogens and H2-utilizing methanogens in the middle layer, and fermentative microorganisms in the outermost layer. Settler screens separate the granules from the treated water and gas is collected at the top of the reactor. In contrast to particulate waste reactors, in which microbial biomass is generated at the same rate of hydraulic washout, requiring retention times as long as several days, growth of biomass in these high-rate reactors is uncoupled from retention time of the waste and can have retention times as short as 1h. Hydraulic retention times have also been reduced in anaerobic fermentors that operate at temperatures of up to 60 _C, since the metabolic rates of thermophilic microorganisms, including acetate-utilizing methanogens, are greater than those of mesophiles. D. Marine habitats In marine habitats where substrates are limited, sulfate-reducing bacteria outcompete methanogens as the terminal members of the anaerobic consortium (Fig. 57.6). Their predominance is a result of the availability of the electron acceptor sulfate in seawater (ca. 30 mM). Their lower Ks for substrate utilization enables the sulfate-reducing bacteria to use low concentrations of H2 and acetate at rates that are greater than those of methanogens. Methanogenesis is predominant in environments where SO_2 4 has been depleted. These environments include the lower depths of sediments and elevated coastal marshes, where the rate of SO_2 4 reduction is greater than the rate of diffusion from seawater, and also sediments that receive large amounts of organic matter, such as eutrophic coastal regions and submarine trenches. In these regions, the three-member methanogenic consortium is similar to that in freshwater environments, but it is composed of halophilic and halotolerant species. Although the acetotrophic methanogens Methanosarcina and Methanosaeta have been isolated from marine methanogenic enrichments, isotope studies performed in sediment suggest that most of the acetate is oxidized by a H2-producing syntroph rather than by splitting to CH4. Methanogens also generate CH4 from methylated amines and thiols, which are readily available in the marine environment as metabolic osmolytes. Since methylated amines are not used by SO_2 4 -reducing bacteria, this class of compound is “noncompetitive” and can be used by methanogens in habitats that contain high SO_2 4 concentrations. CH4 generated in sediments is often consumed as it diffuses through the SO_2 4 -reducing region of the sediments, before reaching the aerobic regions. However, the microbes that catalyze this anaerobic oxidation have not been described. Although much of the CH4 generated in sediments is consumed in the SO_2 4 -reducing regions, the water column in the open ocean is supersaturated with CH4, compared with the atmospheric concentration. This may result from a combination of unoxidized CH4 that escapes from sediments, and methanogenic activity in the gastrointestinal tracts and fecal material of marine animals. Biologically generated CH4 in some organic-rich buried sediments can accumulate as gas deposits. Since natural gas deposits generated abiotically are used as indicators of petroleum, these biologically generated CH4 deposits can act as false indicators of petroleum deposits during oil exploration. Under high hydrostatic pressures generated in deep ocean sediments, biogenic CH4 can also accumulate as solidified CH4 hydrates. E. Ruminant animals Ruminant animals include both domestic (cows, sheep, camels) and wild (deer, bison, giraffes) animals. These animals have a large chamber, called the rumen, before the stomach, in which polymers, such as cellulose, are fermented by bacteria to shortchain fatty acids, H2, CO2, and CH4. The rumen is similar to an anaerobic bioreactor, except that the short retention time created by swallowing saliva is less than the generation time of H2-producing fatty-acid oxidizers and acetate-utilizing methanogens (Fig. 57.7). As a result, acetate, propionate, and butyrate are not degraded by the consortium, but are absorbed into the bloodstream of the host animal as carbon and energy sources. The volatile gases (e.g., CH4, CO2) are removed from the animal by belching. Acidification of the system by the acids is prevented by bicarbonate in the saliva of the animal. Carbon diverted to CH4 and belched into the atmosphere represents a loss of energy to the animal. Ruminant nutritionists have been attempting to increase feed efficiency by adding methanogenic inhibitors, such as monensin, to feed, thereby, diverting carbon flow to metabolites that can be utilized by the animal. F. Xylophagous termites All known termites harbor a dense microbial community of anaerobic bacteria, and, in the case of lower termites, they also contain cellulolytic protozoa that catalyze the digestion of lignocellulose from wood. As in the rumen, these microorganisms have a synergistic relationship with the termites by converting polymers to short-chain fatty acids that are used as carbon and energy sources for their host. The carbon flow in the hindgut of soil-feeding and fungus-cultivating termites is similar to that in rumen, but in wood- and grass-eating termites, most H2–CO2 is converted to acetate instead of CH4. Generally, methanogenesis outcompetes H2–CO2 acetogenesis and the factors that cause the predominance of acetogenesis in some termites in not known. Two species of H2-utilizing Methanobrevibacter have been isolated from the hindgut of the subterranean termite that exhibit catalase activity and are particularly tolerant to oxygen exposure. Oxygen tolerance may be important for recolonization by bacterial consortia after expulsion of the hindgut contents during molting. Reinoculation is achieved by transfer of hindgut contents from other colony members, which are exposed to air during the process. G. Human gastrointestinal tract The human colon serves as a form of hindgut where undigested polymers and sloughed off intestinal epithelium and mucin are dewatered, fermented by bacterial consortia, treated with bile acids, and held until defecation. Fatty acids generated by fermentation are absorbed into the bloodstream and can provide approximately 10% of human nutritional needs. Methanogenesis occurs in 30–40% of the human population, with the remaining population producing H2 and CO2 instead. Acetogenesis and SO_2 4 reduction from H2–CO2 also occur in the human colon, but studies with colonic bacterial communities suggest that these activities are only prevalent in the absence of methanogenic activity. The level of CH4 produced by an individual corresponds to the population levels of Methanobrevibacter, but factors controlling the occurrence of methanogens in the human population are not known. Diet does not appear to have a significant role in determining whether an individual harbors an active methanogenic population, but hereditary and individual physiological factors may have a role. For example, methanogens may be absent from individuals that excrete higher levels of bile acids, which are inhibitory to methanogens. H. Protozoan endosymbionts Methanogens are present as endosymbionts in many free-living marine and freshwater anaerobic protozoa, where they are often closely associated with hydrogenosomes, organelles that produce H2, CO2, and acetate from the fermentation of polymeric substrates. The products of the hydrogenosomes are substrates for methanogenesis. It is conceivable that the methanogens have a synergistic role by lowering the H2 partial pressure to create a favorable thermodynamic shift in the protozoan’s fermentation reaction. Also, evidence suggests that excretion of undefined organic compounds by the methanogen provides an advantage to the protist host. Endosymbionts are also found in flagellates and ciliates that occur in the hindgut of insects, such as termites, cockroaches, and tropical millipeds. Although rumen ciliates do not harbor endosymbionic methanogens, many have ectosymbionic methanogens that may have an analogous function. I. Other habitats Habitats that have a source of organic carbon and a high water content can become anaerobic as a result of respiratory depletion of oxygen, and, subsequently, support methanogenic communities. Examples include soils waterlogged by heavy rainfall, marshes, rice paddies, rotting heartwood of trees, and landfills. Most of the CH4 generated in these habitats is released into the atmosphere. However, many landfills are now vented and collected to prevent a buildup of potentially combustible CH4 underground or in nearby dwellings, and some communities harvest the vented biogas for heat and energy. Methanogenic habitats are also found in geohydrothermal outsources, such as terrestrial hot springs and deep-sea hydrothermal vents. Methanogens from these environments use geothermally generated H2_CO2 for methanogenesis and most are hyperthermophilic, requiring growth temperatures as high as 110_C. In terrestrial sites, methanogens are usually associated with microbial mats composed of photosynthetic and heterotrophic consortia. However, in deep sea hydrothermal vents, where there is no light available for photosynthetic production of organic carbon, the methanogens and other autotrophic bacteria serve as primary producers of cell carbon for a complex community of heterotrophic microbes and animals that accumulates in the vicinity of a hydrothermal vent. Methanogenesis also occurs in high saline environments, such as Great Salt Lake, Utah, Mono Lake, California, and solar salt ponds. Methanogens from these environments generate CH4 from methylated amines and dimethylsulfide, which are synthesized by animals and plants as osmolytes. Methanogenesis has been detected in subsurface aquifers, where, it has been proposed, that H2 generated by an abiotic reaction between iron-rich minerals in basalt and ground water is used as a substrate for methanogenesis. Methanogenesis has also been detected in deep subsurface sandstone, where it is hypothesized that methanogenic consortia use organic compounds that diffuse from adjacent organic-rich shale layers.   IV. PHYSIOLOGY AND BIOCHEMISTRY A. Catabolic pathways All methanogens generate CH4 during growth by three basic catabolic pathways: autotrophic CO2 reduction with H2, formate, or secondary alcohols; acetotrophic cleavage of acetate; or methylotrophic dismutation of methanol, methylated amines, or methylthiols. The common reactions for methanogenesis are shown in Table 57.2. Six new coenzymes were discovered that serve as carbon carriers in the methanogenic pathway (Fig. 57.8). Methanofuran (MFR) is an analog of molybdopterins, which occur in enzymes that catalyze similar reactions in the Bacteria and Eukarya. Tetramethanopterin (H4MPT) is an analog of tetrahydrofolate (H4THF), which is also a one-carbon carrier in bacterial and eukaryal systems. Although H4MPT was initially found to be unique to methanogens, it has since been detected in other Archaea and, more recently, enzymes catalyzing the methyl transfer for MFR and H4MPT have been found to coexist with the H4THF pathway in the CH4-utilizing methanotrophs. Methyl coenzyme M (CoM-SH), 7-mercaptoheptanoylthreonine phosphate (HS-HTP) and cofactor F430 are currently unique to the methanogens. The methanogenic sequence is initiated by a two-electron reduction of CO2 and methanofuran by formyl-MFR dehydrogenase (a) to form formyl- MFR (Fig. 57.9). The formyl group is then transferred to H4MPT by formyl-MFR:H4MPT formyltransferase (b) yielding formyl-H4MPT. A homolog of H4MPT, tetrahydrosarcinapterin (H4SPT), found in Methanosarcina spp., differs by an additional glutamyl moiety in the substituted R group. Formyl-H4MPT cyclization to methenyl-H4MPT is catalyzed by N5,N10-methenyl- H4MPT cyclohydrolase (c) N5,N10-methylene-H4MPT dehydrogenase (d) and N5,N10-methylene-H4MPT reductase (e) catalyze the sequential reduction of methenyl-H4MPT by the electron carrier coenzyme F420 to methylene-H4MPT and methyl-H4MPT. The methyl group is then transferred to CoM-SH by N5-methyl- H4MPT:CoM-SH methyltransferase (f) forming methyl- S-CoM. Methyl CoM reductase (g) catalyzes the terminal reduction of methyl-S-CoM by two electrons from HS-HTP to CH4. CoM-SS-HTP is the product of the terminal reaction, which is subsequently reduced by heterodisulfide reductase to regenerate the reduced forms CoM-SH and HTP-SH. Methylotrophic catabolism of methanol and methylated amines requires three polypeptides. Methanol is catabolized by transfer of its methyl group to a corrinoid-binding protein, which is methylated by a substrate-specific methyltransferase, methanol:5-hydroxybenzinidazolyl (MT1). The methyl group is then transferred from the corrinoid protein to coenzyme HS-CoM by methylcobamide: CoM methyltransferase (MT2). Trimethylamine, dimethylamine, and monomethylamine each require a distinct corrinoid-binding protein, which is methylated by a substrate-specific methyltransferase. The methyl group is then transferred from the corrinoid protein to coenzyme HS-CoM by a common MT2 homolog. In contrast, catabolism of the methylthiols dimethylsulfide and methylmercaptopropionate is catalyzed by only two polypeptides: a corrinoid-binding protein tightly bound to a methylcobamide:CoM methyltransferase homolog of MT2. Methyl-S-CoM generated from methanol, methylated amines, and methylthiols is reduced to CH4 in the methanogenic pathway, as has been described. A portion of the methyl groups generated from methylotrophic catabolism is oxidized in reverse sequence in a pathway identical to the CO2 reduction pathway, after what appears to be a direct transfer of the methyl groups to H4MPT. However, the mechanism of this transfer is not yet known. This oxidative sequence generates electrons for the reduction of CoM-S-S-HTP in the methyl-S-CoM reductase system. The acetotrophic pathway for acetate catabolism proceeds by initial “activation” of acetate by formation of acetyl CoA. Methanosarcina spp. synthesize acetyl CoA by sequential activities of phosphotransacetylase and acetyl kinase. In contrast, activation of acetate to acetyl CoA by Methanosaeta spp. is catalyzed in a single step by acetyl-CoA synthase. In both genera, cleavage of the C–C and C–S bonds of acetyl-CoA is then catalyzed by the acetyl-CoA decarbonylase/ synthase complex, yielding enzyme-bound methyl and carbonyl groups. The complex contains CO:acceptor oxidoreductase, Co-_-methylcobamide: tetrahydropterine methyltransferase, and acetyl-CoA synthase activities. The five subunit complex consists of a two polypeptide CO-oxidizing nickel/iron-sulfur component, a two-polypeptide corrinoid/iron-sulfur component, and a single polypeptide of unknown function. The nickel/iron-sulfur component catalyzes the cleavage of acetyl-CoA, the oxidation of the bound carbonyl to CO2, and methyl transfer to the corrinoid/ iron-sulfur component. The methyl group is sequentially transferred to H4SPT by a currently unknown process and to HS-CoM by H4MST:CoM-SH methyltransferase. Methyl-S-CoM is reductively demethylated to CH4 by methylreducase as previously described. The enzyme-bound carbonyl group is oxidized to CO2. Methanosarcina spp. and autotrophic methanogens growing on H2/CO2 synthesize acetate, presumably by reversing the direction of the acetyl-CoA decarbonylase/synthase complex in a reaction analogous to acetyl CoAsynthase in acetate-utilizing Clostridia. B. Bioenergetics The methanogenic Archaea derive their metabolic energy from autotrophic CO2 reduction with H2, formate or secondary alcohols, cleavage of acetate, or methylotrophic dismutation of methanol, methylated amines, or methylthiols (Table 57.2). Currently, there is little evidence to convincingly support substrate level phosphorylation. Evidence that redox reactions in the catabolic pathways are catalyzed, in part, by membrane-bound enzyme systems and are dependent upon electrochemical sodium ion or proton gradients indicates that electron transport phosphorylation is responsible for ATP synthesis. Both gradients generate ATP for metabolic energy via membrane ATP synthases. Several reactions in the methanogenic pathway are sufficiently exergonic to be coupled with energy conversion, including reduction of methyl CoM (_29 kJ/mol) and methyl transfer from H4MPT/ H4SPT to HS-CoM (_85 kJ/mol). In addition, the oxidation of formyl-MF (_16 kJ/mol) during methyl oxidation in the methylotrophic pathway and CO oxidation (_20 kJ/mol) in the acetotrophic pathway are also exergonic. During growth on H2–CO2 or H2-methanol, the H2:heterodisulfide oxidoreductase system catalyzes the H2-dependent reduction of CoM-S-S-HTP to HS-CoM (Fig. 57.10). Electron translocation across the membrane generates a proton gradient for ATP synthesis via an A1A0 ATPase. During methylotrophic growth of methanol or methylamines, heterodisulfide dehydrogenase is linked to F420 dehydrogenase instead of hydrogenase for CoM-S-S-HTP reduction and generation of a proton gradient. Reduced F420 is generated by methylene-H4MPT dehydrogenase and methylene-H4MPT reductase. There is evidence for a third type of heterodisulfide oxidoreductase system in the acetotrophic pathway that is linked to acetyl CoA decarbonylase via ferridoxin. In addition to proton gradients, methanogens also use sodium ion gradients to drive endogonic reactions and generate ATP. During growth on H2–CO2 or acetate, vectoral Na_ translocation is coupled to methyl-H4MPT:CoM-SH methyltransferase. The Na_ gradient generates ATP via a F1F0 ATPase. During growth on methanol and methylamines, a sodium ion gradient is formed by a Na_/H_ antiporter and the methyl-H4MPT:CoM-SH methyltransferase sodium pump is used in reverse to drive the endergonic methyl transfer from methyl CoM to methyl-H4MPT, for subsequent oxidation of the methyl groups.   Formyl-MFR dehydrogenase is involved in the bioenergetics of autotrophic growth on H2–CO2 or methylotrophic growth on methanol. During hydrogenotrophic growth, the exergonic CO2 reduction to formyl-MF is driven by a hydrogenasegenerated H_ or Na_ gradient. The reaction is likely to be reversible during CO2 generation by methylotrophs, resulting in a net H_ or Na_ gradient. C. Biosynthetic pathways Most methanogens assimilate carbon by CO2 fixation or acetate uptake and current evidence indicates that assimilation occurs via acetyl-CoA synthesis in a pathway analogous to the Ljungdahl–Wood pathway in acetotrophic clostridia. This conclusion is supported: (a) by labeling studies; (b) by the presence of acetyl CoA synthase in autotrophic methanogens; and (c) by the absence or partial absence of enzymes required for other routes of CO2 fixation, including the Calvin cycle, reverse tricarboxylic acid cycle, the serine pathway, and ribulose monophosphate cycle of the methylotrophs and the hydroxypropionate pathway of Chloroflexus. Acetotrophic methanogens likely utilize acetyl CoA directly synthesized by acetate kinase and phosphotransacetylase in the catabolic acetate pathway. In the autotrophic methanogens, current evidence suggests that methyl-H4MPT, formed by CO2 fixation in the H4MPT reductive pathway, likely donates a methyl group to a Fe/S corrinoid protein where it is subsequently transferred to HS-CoA by acetyl CoA synthase. This pathway is a reversal of the acetotrophic pathway for acetate catabolism. During growth on methanol or methylated amines, methyl-H4MPT, formed by direct methyl transfer to H4MPT, is also the likely precursor for acetyl CoA synthesis in methylotrophs. Acetyl CoA is reductively carboxylated by pyruvate oxidoreductase and enters the predominant biosynthetic pathways as pyruvate via an incomplete tricarboxylic acid cycle (TCA). Both reductive and oxidative partial TCA cycles are detected in methanogens and their distribution among species is based on the bioenergetics of _-ketoglutarate synthesis. The oxidative incomplete TCA pathway requires a second acetyl CoA and is restricted to acetotrophic methanogens, which readily synthesize acetyl CoA from acetate via acetate kinase and phosphotransacetylase. In contrast, the reductive incomplete TCA pathway requires two additional reductive reactions and a reductive carboxylation to synthesis of _-ketoglutarate. This pathway is commonly found in autotrophic methanogens, which have a steady reducing potential from H2. Studies based on labeling and identification of specific enzymatic activities in selected species, thus far, indicate that biosynthesis of the aromatic aspartate, glutamate, histidine, pyruvate, and serine families of amino acids occurs by pathways similar to those described in the Bacteria and Eukarya. Biosynthesis of hexoses, required as precursors for cells walls and reserve polysaccharides, such as glycogen and trehalose, proceeds via gluconeogenesis from phosphoenolpyruvate. Pentoses are formed from fructose 1,6-phosphate and glyceride 3-phosphate via transketolase and transaldolase in Methanobacterium and Methanococcus. In contrast, Methanospirillum forms pentoses by oxidation of glucose-6-phosphate. Labeling studies in Methanospirillum and Methanobacterium indicate that pyrimidine and purines are synthesized by the expected pathways and they also have a purine salvage pathway. The polar core lipids, which include diphytanyl glycerol ethers and diphytanyl diglycerol tetraethers, are synthesized via the mevalonate pathway for isoprenoids. V. MOLECULAR GENETICS A. Genome structure The methanogens a have single circular chromosome that ranges from 1.6_106 to 5.7_106 base pairs in size. The percentage guanosine_cytosine (%G_C) ranges from 23 to 62% and high %G_C content is not always associated with high growth temperatures. Like the Bacteria replication starts at a single origin. The relatively small genome sizes and short generation times suggest that the mechanism to control timing of DNA replication may be similar to that found in the Bacteria. Adenosine–thymidine intergenic spacer regions have been detected in species of Methanococcus, but the role of these regions is unknown. Insertion elements have been detected in chromosomal DNA from methanogens. Methanobrevibacter smithii harbors a 1381 basepair (bp) element that is flanked by 29bp terminal repeat sequences and a 1501bp sequence flanked by 6bp repeats has been detected in strains of Methanobacter thermoautotrophicus. Both elements have an open reading frame hypothesized to be a transposase. Extrachromosomal DNA has been detected in methanogens in the form of plasmids, viruses, and virus like particles. At this time, eleven plasmids, ranging in size from 4.4 to 20 kbp, have been detected in four genera of methanogens. Plasmids from M. thermoautotrophicus encode a type II DNA restriction–modification system, and open reading frames in a plasmid from Methanosarcina acetivorans show homology to site-specific recombinases and RepA-like replication initiation proteins. The functions of the remaining plasmids are cryptic at this time. The mechanism of plasmid replication in methanogens is not known, but RepA proteins are associated with plasmids and phage that replicate by a rolling circle mechanism. Lytic viruses have been detected in three strains of Methanothermobacter and one strain of Methanobrevibacter smithii. These viruses show a varying range of host specificities. A temperate virus-like particle has been isolated that can integrate into the chromosome of Methanococcus voltae. In contrast to the similarities of DNA structure in methanogenic Archaea and Bacteria, DNA-modifying proteins in the methanogens share features common to both the Bacteria and Eukarya (Table 57.3). All cells must package their genomic DNA within the limited space of the cells. Although the Bacteria do not appear to have a conserved mechanism for DNA packaging, Archaea and Eukarya chromosomal DNAs are compacted by histones into defined structures called nucleosomes, which are further assembled to form chromatin. The small, basic histone-like proteins from Methanothermus fervidus have homology to eukaryal histones and appear to have conserved the minimal structure for creating positive superturns, required to build a nucleosome. Archaeal histone-like proteins increase the melting temperature of linear DNA in vitro by as much as 25_C and likely protect DNA from heat denaturation in vivo. Reverse gyrase, also detected in M. fervidus, may contribute to heat resistance of DNA by creating stable positive supercoils in inter-histone regions. Histone-like proteins isolated from mesophilic Methanosarcina species cause concentration-dependent inhibition or stimulation of gene transcriptions in vitro, which suggests that they may also have a role in gene regulation. B. DNA replication, repair, modification, and metabolism Although our understanding of DNA replication and repair in the Bacteria and Eukarya is well advanced, comparatively little is known about these processes in the methanogenic Archaea. Studies on DNA replication show that aphidicolin, a specific inhibitor of eukaryal DNA replication, inhibits DNA polymerases from Methanococcus vannielii and Methanococcus voltae, but does not inhibit DNA polymerase from M. thermoautotrophicus. Sequences of genes encoding both the aphidicolin-sensitive and -insensitive methanogen DNA polymerases reveal they are homologous to family B and X DNA polymerases from the Bacteria and Eukarya. Methanocaldococcus jannaschii contains a single gene with two inteins that encode a B-type DNA polymerase. In contrast, M. thermoautotrophicus has two polymerases: a B-type DNA polymerase composed of two polypeptides and an X-type DNA polymerase. DNA repair mechanisms have also been identified in methanogens. A photoreactivation system in M. thermoautotrophicus is mediated by class II photolyase with homology to metazoan photolyases. Genes encoding putative eukaryal DNA repair proteins RAD2, RAD25, and RAD51, and bacterial DNA repair proteins uvrABC, mutL and mutS have been identified in genomic sequences of methanogenic Archaea. Several type II restriction endonuclease-methyltransferase systems have been identified and four methanogen endonucleases are available commercially. In addition, putative type I restriction-modification enzymes have been identified by sequence annotation of the M. thermoautotrophicus genome. Despite their sensitivity to oxygen, a Fe-superoxide dismutase has been characterized from M. thermoautotrophicus and catalase activity has been detected in species of Methanobrevibacter. Putative genes encoding for related DNA replication and repair proteins, such as helicases, ligases, topoisimerases, endonucleases, recombinases, and replication factors, have also been identified in genomic sequences of methanogens, but their function has not yet been confirmed. C. Gene structure and transcription The organization of methanogenic genes in tightly linked clusters is similar to the operon configuration found in the Bacteria. As in the Bacteria, the archaeal operons are transcribed from an upstream promoter into polycistronic RNAs. However, archaeal genes are transcribed by a multicomponent RNA polymerase that is structurally homologous to eukaryal RNA polymerase and recognizes a TATA promoter with high sequence homology to the consensus motif for the Eukarya. Unlike the variable upstream distance of eukaryal promoters, the archaeal promoter element is located at a consistent distance, approximately 20–30 bp upstream from the transcription initiation site, a range similar to the conserved _35 bp region observed in the Bacteria. Site-directed deletion studies conducted with methanogenic Archaea by in vitro techniques indicate that efficient transcription and startsite selection is dependent upon a TATA promoter. This arrangement closely resembles the core structure of RNA polymerase II promoters in the Eukarya. Purified archaeal RNAPs from M. thermoautotrophicus and M. voltae fail to initiate site-specific transcription without the addition of TATA binding protein (TBP) and transcription factor TFIIB. Yeast and human TATA-binding proteins can substitute for TBP in a Methanococcus-derived archaeal cell-free transcription system, indicating that they are functionally homologous. Additional genes that have sequence similarity to the eukaryal-like transcription factors TFIIIC, TFIIE, and TFIIS have been putatively identified in the genomic sequences of methanogenic Archaea. Although the spatial and temporal nature of these transcription mechanisms is not yet known, the results suggest that a DNA-protein recognition site, analogous to that in the Eukarya, is required for sitespecific RNAP recognition in the Archaea. This mechanism would involve sequence recognition by TFIIBTBP and recruitment of other transcription factors to form a recognition complex that would be recognized by polymerase and initiate transcription. The mechanisms of transcriptional regulation in highly regulated genes has not yet been determined, but in vitro studies on Methanococcus maripaludis reveals that point mutations in a palindrome located downstream of the nifH transcription start site results in derepression of expression. This suggests that a bacterial-type repressor may mediate gene expression in some highly regulated methanogen genes. Genes encoding three distinct TBPs have been detected in the M. acetivorans genome, which raises the possibility that gene regulation is mediated by the formation of alternative TBP-TFB pairing. In addtion to multiple TBPs, multiple TFBs have also been detected in the genome of the extreme halophile Halobacterium, but there is no evidence for multiple TFBs in M. acetivorans. Generally, transcription is terminated following an inverted repeat sequence located downstream of methanogen genes. Transcription termination sites are similar to the _-independent terminators in the Bacteria, and likely form a stem-loop secondary structure to mediate termination. A second type of transcription terminator, which consists of a single or several tandemly arranged oligo-T sequences, is found in hyperthermophilic methanogens. The occurrence of this terminator in hyperthermophiles suggests that the stem-loop structures characteristic of -independent promoters may be unstable at higher growth temperatures. D. RNA structure and translation The stable RNAs transcribed by methanogen genes have been investigated in some detail. Methanogen ribosomes resemble bacterial ribosomes. They are composed of two protein subunits of 30S and 50S and three rRNA components of 23S, 16S, and 5S, which, assembled, yield a ribosome of 70S. Archaeal and bacterial ribosomal proteins are functionally homologous and can be interchanged to create an active ribosome in vitro. Genes encoding rRNA are arranged in the order 16S–23S–5S and the number of operon copies varies in number from one to four. The organization and order of genes encoding methanogen ribosomal proteins also resembles that found in the Bacteria. Methanogen tRNAs contain the sequence 1-methyl_ CG substituted for the sequence T_CG, typically found in the arm of bacterial and eukaryal tRNAs. Introns have been detected in genes encoding tRNA. A unique feature of the archaeal genomes of M. jannaschii and M. thermoautotrophicus is the absence of a gene encoding cyteine-tRNA synthetase. This function is carried out by a dual-specificity prolyl-tRNA synthetase that recognizes and aminoacylates both tRNAPro and tRNACys. Some methanogen mRNAs have poly-A_ tails, but, as in the Bacteria, they average only 12 bases in length. Protein-encoding genes employ the same genetic code as Bacteria and Eukarya, and codon preferences reflect the overall base composition of the genome and the level of gene expression. The codon ATG is used frequently as an initiation codon, as well as GTG and TGG. A ribosomal-binding site located upstream of structural genes, when transcribed, is complementary to the 3  terminal sequence of methanogen 16S rRNA. There is little information on the mechanisms of translation based on biochemical experimentation. However, genome analysis reveals that methanogens possess protein initiation factors that share homology with both bacterial and eukaryal IF proteins. Inteins have been identified by genomic sequencing of M. jannaschii, which suggests that protein splicing occurs in methanogens. In addition, evidence has been found for phosphorylation of proteins at a tyrosine residue, which is a mechanism of post-translational control in the Bacteria and Eukarya. E. Genomics and gene function analysis The genomes of the hyperthermophiles M. jannaschii (1.66 megabases) and Methanopyrus kandleri (1.7 megabases), the thermophile M. thermotrophicus (formerly M. thermoautotrophicum, 1.8 megabases), and the mesophiles Methanosarcina acetivorans (5.7 megabases) and Methanosarcina mazei (4.1 mega bases) have been completely sequenced and annotated. In general similar genes are found for the CO2 reducing catabolic pathways in all four genomes. Genes encoding multiple methyltransferases and acetyl-CoA decarbonylase/synthases are only found in the genome of M. acetivorans, which is consistent with the ability of this species to also grow by methylotrophic and aceticlastic pathways. The large genome size of M. acetivorans likely reflects this species’ ability to use a greater range of substrates, adapt to a broader range of environments and form complex multicellular structures compared with the more limited capabilities of the hydrogen-utilizing species. In contrast the hyperthermophiles are more “minimalist” exhibiting a paucity of genes encoding proteins for signaling and gene regulation, grpE-dnaJ-dnaK heat shock operon, proteasome-chaperonin, several DNA repair proteins, DNA helicases, nitrogenase subunits, ribonucleotide reductase and proteases. Overall, the majority of archaeal open reading frames with similarity to bacterial sequences include genes for small molecule biosynthesis, intermediary metabolism, transport, nitrogen fixation, and regulatory functions. Archaeal open reading frames with similarity to eukaryal sequences include genes for DNA metabolism, transcription, and translation. The presence of Cdc6 homologs and histones suggests that DNA replication initiation and chromosome packaging is eukaryal, but detection of ftsZ suggests that bacterialtype cell division occurs. Additional unique features include an archaeal B-type DNA polymerase with two subunits, putative RNAP A  subunits that suggest that possibility of additional mechanisms for gene selection, and two introns in the same tRNAPro (CCC) gene, which establishes a new precedent. The availability of genome sequences of methanogens and current activities to sequence others make the development of archaeal gene-transfer systems essential for confirmation of gene function. However, advances in the genetics of methanogens have been limited to studies in vitro or using heterologous systems, such as E. coli, because of the lack of tractable gene transfer systems for these microorganisms. Two gene-transfer systems have recently been developed for species of Methanococcus and Methanosarcina. Both systems utilize hybrid shuttle vectors derived from native archaeal plasmids. Since plasmids occur in low copy number in the methanogens, the DNA to be transferred is ligated into the vector, which is then amplified in E. coli with ampicillin as a selectable marker. The modified plasmid is then transferred into the methanogen by polyethylene glycol-mediated (Methanococcus sp.) or liposome-mediated (Methanosarcina spp.) transformation. Asecond selectable marker in the plasmid, such as pac (puromycin acetyl transferase) controlled by a highly expressed methanogen promoter mcr (methyl CoM reductase), provides selection in the methanogen. Both autonomously replicating plasmids for introducing specific phenotypes and integration plasmids for disrupting genes by homologous recombination have been designed. These systems are highly efficient, yielding 107–109 transformants per _g DNA. Methods for transposon-mediated mutagenesis have also been developed. Atransducing phage has been isolated from M. thermoautotrophicus, but it is not currently useful for gene transfer because of its limited burst size (~6 per cell). Conjugation has not been observed in methanogens. Although gene-transfer systems are somewhat limited at this time, development of new and more sophisticated systems is ongoing. Impediments that need to be overcome include a lack of understanding of mechanisms for vector replication, retention and segregation, and the limited number of selectable and phenotypic markers that are currently available for the methanogens. VI. SUMMARY The methanogenic Archaea have a pivotal role in the global carbon cycle by complementing aerobic processes that ultimately lead to the oxidation of organic carbon to CO2. However, a steady increase in the levels of atmospheric CH4 that has coincided with the increase in the human population is a cause for concern, since CH4 is a greenhouse gas. Methane’s contribution to global warming results from its high infrared absorbance and its role in complex chemical reactions in the stratosphere that affect the levels of ozone. Increased waste disposal activities, such as landfills, are a significant source of atmospheric CH4. Another significant source results from agricultural activities, such as the increased use of domesticated ruminants for production of meat and dairy products and the increased development of rice paddies. Understanding the properties of methanogens and the roles they have in the global carbon cycle will have important implications in addressing the issue of global warming as the human population increases. The application of methanogens in biotechnology has been largely limited to waste management, which is often coupled to limited biogas production. Although the petroleum crisis of the 1970s led to an interest in methanogenic biogas production by fermentation of sources ranging from agricultural products to marine kelp, cost-effective technologies were never fully developed and interest has since waned with the drop in petroleum prices. Other potential applications for methanogens include the production of novel pharmaceuticals, corrinoids, and thermo-stable enzymes. To date, methanogens have yielded only a few restriction endonucleases as commercial products. However, significant advances in our understanding of the physiology and biochemistry of methanogens over the past two decades, combined with the recent developments in gene transfer systems and advances in genome sequencing, make the application of methanogens for biotechnology more plausible in the near future.