Methylotrophy

 Methylotrophy refers to the ability of microorganisms to utilize one-carbon compounds more reduced than CO2 as sole energy sources and to assimilate carbon into cell biomass at the oxidation level of formaldehyde. Methylotrophic organisms must synthesize all cellular constituents from methylotrophic compounds, such as methane, methanol, methylated amines, halogenated methanes, and methylated sulfur species. A diverse range of both aerobic and anaerobic prokaryotes and eukaryotes can utilize methanotrophic substrates for growth. 

I. HISTORICAL PERSPECTIVE Methylotrophs were first discovered in 1892 by Leow, who described a pink bacterium growing on methanol, methylamine, formaldehyde, and also on a variety of multi-carbon compounds. This organism was called Bacillus methylicus and was almost certainly what is now know as the pink-pigmented facultative methylotroph (PPFM) Methylobacterium extorquens. Bacillus methanicus was the first methaneoxidizing bacterium, reported by Söhngen in 1906. This methanotroph was isolated in pure culture from aquatic plants. This isolate was subsequently lost but was reisolated in 1956 by Dworkin and Foster and renamed Pseudomonas methanica. At around that time, the PPFM Pseudomonas AM1 was isolated on methanol by Quayle and colleagues. This was to become the “workhorse” organism for many of the biochemical and molecular biological studies on the metabolism of methanol and has now been renamed as Methylobacterium extorquens AM1. In 1970, Whittenbury and colleagues isolated over 100 new strains of methane-oxidizing bacteria. The characterization of these organisms was carried out and the scheme proposed still remains the basis for current classification schemes for methanotrophs. 

II. SIGNIFICANCE A. Global carbon cycle Methane is the most abundant organic gas in the atmosphere. It is a very potent greenhouse gas and it absorbs infrared radiation considerably more efficiently than CO2 and, therefore, makes a significant contribution to global warming. Current understanding of the global methane budget suggests that methane-oxidizing bacteria play an important role in oxidizing a large proportion of the methane produced by methanogenic bacteria in environments such as wetlands, ricefields, tundra, and the marine environment. Therefore, these bacteria are a significant sink for methane in the environment in modulating net emissions of methane and may provide an important negative feedback on future methane increases in wetland and soil environments. It is, therefore, important to learn more about the role of methanotrophs in the global carbon cycle. B. Biotechnology Both methane and methanol are relatively cheap feedstocks for fermentation processes and methylotrophs have received considerable attention for a number of biotechnological applications. Initial work on the production of single-cell protein (SCP) was carried out with methanotrophs. Growth yields of these organisms on methane were high but two drawbacks included the high oxygen demand in the fermentation process and the explosive nature of their substrates methane and oxygen. Methanol-utilizing bacteria, e.g., Methylophilus methylotrophus, have been successfully used for SCP production in very large-scale fermentation processes (ICI Pruteen Process). However, due to the fall in price of agricultural protein products, such as soya protein, over recent years, SCP from methanol has not been particularly competitive on a commercial scale. Methanol-utilizing bacteria have also been exploited for the production of vitamins, polymers, and amino acids and these processes may be more economically viable. For example, auxotrophs of thermophilic gram-positive methanol utilizers can excrete relatively large amounts of lysine and other amino acids, which can then be used for animal feedstock supplements. The possibilities of genetically engineering methylotrophs for the overproduction of amino acids is also being explored. Methanotrophs have also been investigated for the production of bulk chemicals, such as propylene oxide. Methane monooxygenase is able to insert oxygen into a number of aliphatic and aromatic compounds other than methane. 

These co-oxidation properties of methanotrophs, particularly those that contain soluble methane monooxygenase (sMMO), are unique and also unusual, since these organisms appear to derive no benefit from this process. sMMO is able to cooxidize propylene to propylene oxide, a valuable compound in organic synthesis. However, due to the toxic nature of propylene oxide, alternatives to the use of whole cells, such as immobilized enzymes, together with the problem of regenerating reductant for sMMO may need to be considered. C. Bioremediation Methanotrophs have received considerable attention for their potential use in bioremediation processes. The enzyme soluble methane monooxygenase not only oxidizes methane but will co-oxidize a wide variety of aliphatic, substituted aliphatic, and aromatic compounds. sMMO is able to degrade several pollutants, including vinyl chloride, trichloroethylene, and other halogenated hydrocarbons that contaminate soil and groundwater. Challenges facing the use of methanotrophs in situ in bioremediation processes include ensuring supply of the substrates methane and oxygen and/or reductant and also overcoming problems associated with the negative regulation of sMMO by copper ions. Methylotrophs containing specific dehalogenases may also be useful in clean-up of industrial solvent-contaminated sites. For example, dichloromethane is metabolized by some Methylobacterium species using a dehalogenase. More recently, methylotrophs with the ability to degrade methyl chloride and methyl bromide have been isolated. Degradation of methyl bromide, a potent ozone-depleting gas currently used as a pesticide in agriculture, is a particularly interesting trait for methylotrophs and may be useful in mitigating methyl bromide loss to the atmosphere during soil fumigation processes. Methylotrophic bacteria can also grow on some methylated sulfur compounds found in toxic wastes, such as paper mill effluents. Others can degrade aliphatic sulfonates and, therefore, may be important in degradation of detergents and related compounds in the environment. Research is under way to investigate the metabolism of halogenated methanes and one-carbon compounds containing sulfur, in order to be able to explore the bioremediation potential and to exploit the properties of these novel methylotrophs. D. Expression systems The methylotrophic yeast Pichia pastoris is now becoming one of the best hosts for the production of foreign proteins because of the presence of the strong methanol-inducible promoter AOX1. This allows high level expression of a large number of biotechnologically and pharmaceutically important proteins in a controlled fashion during growth of a yeast on a relatively cheap substrate.   

III. HABITATS AND ECOLOGY Methane is produced by methanogenic bacteria in a number of diverse environments in the biosphere. Methane-oxidizing bacteria, which require both methane and oxygen for growth, are generally found on the fringes of anaerobic environments and are probably responsible for oxidizing much of the methane derived from methanogens, before it escapes to the atmosphere. They appear to be ubiquitous in nature and have been isolated from many different environments, including freshwater and lake sediments, rivers, groundwater aquifers, seawater, marine sediments, rice paddies, sewage sludge, decaying plant material, acidic peat bogs, and alkaline lakes. Psychrophilic representatives may also be isolated from Arctic and Antarctic tundra and thermophilic methanotrophs growing at temperatures as high as 70 _C have recently been obtained from hot springs. They can also be isolated from polluted environments. Methanotrophs also appear to exist as symbionts, for example, in the gill tissue of marine mussels or tube worms. The carbon assimilated by these methanotrophic endosymbionts probably supplies much of the organic carbon necessary for growth of these marine organisms. Methanotrophs from certain environments, for example, the putative symbionts already described, do not respond well to conventional enrichment and isolation techniques and molecular ecology experiments employing phylogenetic and functional gene probes suggest the presence of many new, and as yet uncultivated, methanotrophs in the environment. Methanol is also a relatively abundant substrate for the growth of methylotrophs in the environment. Methanol is released during the decomposition of plant lignins and pectins and other compounds that contain methoxy groups. Bacteria that utilize methanol are frequently found in association with methaneoxidizing bacteria, presumably growing on the methanol excreted by methanotrophs. 

Methanol utilizers such as Methylobacterium are frequently found on the leaves of aquatic and terrestrial plants and on decaying plant material. Methanol-utilizers have also been isolated from the marine environment. Bacteria that utilize methyamine, many of which also grow on methanol, are also widespread in nature. Methylated amines are the products of degradation of some pesticides, lecithin and carnitine derivatives, and of trimethylamine oxide. Methylamine-utilizing bacteria are common in both terrestrial and marine environments. Methylated sulfur compounds, such as dimethylsulfoxide (DMS), dimethylsulfide, and dimethyldisulfide, are capable of supporting the growth of certain methylotrophs, such as Hyphomicrobium and Thiobacillus. DMS is the most abundant organic sulfur gas in the environment, produced in the marine environment from the cleavage of dimethylsulfoniopropionate, an algal osmoregulator. DMS is oxidized in the upper atmosphere to sulfur dioxide and the C1 sulfur compound methanesulfonic acid (MSA). This MSA falls to earth by wet and dry deposition and, recently, it has been demonstrated that terrestrial and marine bacteria can grow methylotrophically on this C1 substrate. Halogenated methanes are widely used as industrial solvents in the chemical industry and some, e.g., dichloromethane, have been shown to be methylotrophic substrates for some strains of Methylobacterium in polluted soils. Methyl bromide and methyl chloride are natural products released into the biosphere in large amounts from marine phytoplankton, algae, and wood-decaying fungi and these are also methylotrophic substrates for newly isolated bacteria. 

IV. METHANOTROPHS (METHANE UTILIZERS) A. Physiology and biochemistry Methanotrophs grow by oxidizing methane to methanol using the pathway shown in Fig. 58.1. Methanol is further metabolized to formaldehyde by the pyrolloquinoline quinone- (PQQ-)linked enzyme methanol dehydrogenase (MDH), an enzyme found in all gramnegative methylotrophic bacteria. Approximately half of the formaldehyde produced is further oxidized to yield carbon dioxide, resulting in generation of reducing power for biosynthesis and the initial oxidation of methane. The carbon dioxide is not fixed into cell carbon in significant amounts but is lost to the atmosphere. The remainder of the formaldehyde is assimilated into cell carbon by one of two pathways. In Type I methanotrophs, formaldehyde is condensed with ribulose phosphate into hexulose-6-phosphate in the ribulose monophosphate pathway. Type II methanotrophs utilize the serine pathway for the incorporation of formaldehyde into the cell. The initial reaction in the RuMP pathway involves the addition of 3 mol formaldehyde to 3 mol ribulose 5-phosphate to produce 3 mol hexulose 6-phosphate. Rearrangement reactions similar to those in the Calvin cycle for carbon dioxide fixation result in the production of glyceraldehyde 3-phosphate and the regeneration of 3mol of ribulose 5-phosphate. Glyceraldehyde 3-phosphate is used for the synthesis of cell material. The serine pathway is very different from other formaldehyde assimilation pathways. There are no enzymatic reactions in common with the RuMP or methylotrophic yeast assimilatory pathways. In the serine pathway, 2mol formaldehyde are condensed with 2 mol glycine to form 2 mol serine. The serine is converted to 2 mol of 2-phosphoglycerate. One phosphoglycerate is assimilated into cell material while the other is converted to phosphoenolpyruvate (PEP). The carboxylation of PEP yields oxaloacetate that is subsequently converted to 2 mol glyoxylate. Transamination of glyoxylate with serine as the amino donor regenerates the two glycine acceptor molecules. The phosphoglycerate, which is assimilated, undergoes transformations in central metabolic routes to provide carbon backbones for the synthesis of all cell materials. Methane oxidation is carried out by the enzyme methane monooxygenase (MMO). A membranebound, particulate methane monooxygenase (pMMO) appears to be present in all methanotrophs grown in the presence of relatively high concentrations of copper ions. pMMO appears to consist of at least 3 polypeptides of approximately 46, 23, and 20 kDa, and contains a number of copper clusters. Further characterization of pMMO at the biochemical level is ongoing in a number of research groups. 

In some methanotrophs, a second form of MMO, a cytoplasmic, soluble form (sMMO) is synthesized in growth conditions when the copper-to-biomass ratio is low. This sMMO is structurally and catalytically distinct from the pMMO and has a broad substrate specificity, oxidizing a wide range of aliphatic and aromatic compounds. The sMMO enzymes of M. capsulatus (Bath) and M. trichosporium OB3b both consist of three components—A, B, and C. Protein Ais the hydroxylase component of the enzyme complex and contains a binuclear iron–oxo center, believed to be the reactive center for catalysis. Protein B, is a single polypeptide, contains no metal ions or cofactors, and functions as a regulatory, or coupling, protein. Protein C is a reductase containing 1 mol each of FAD and a 2Fe2S cluster which accepts electrons from NADH and transfers them to the diiron site of the hydroxylase component. The x ray crystal structure of the hydroxylase component of sMMO is now known, a fact which has further stimulated research on the mechanism of oxidation of methane by this unique enzyme. B. Molecular biology The genes encoding sMMO complex have been cloned from several methanotrophs. The gene cluster contains genes encoding the _, _, and _ subunits of Protein A (mmoX, Y, and Z), Protein B (mmoB), and Protein C (mmoC). Derived polypeptide sequences of sMMO components from three methanotrophs showed a high degree of identity, highlighting the conserved nature of this enzyme complex. Amino acid sequences within the _ subunit of Protein Aalign well with the four helix iron coordination bundle of the R2 protein of ribonucleotide reductase and is characteristic of a family of proteins that contains a catalytic carboxylate-bridged diiron center. Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copperbiomass ratios. The transcriptional regulation of the sMMO gene cluster appears to be under the control of a copper-regulated promoter. Transcription of the sMMO gene cluster is negatively regulated by copper ions. Activation of pmo transcription by copper ions is concomitant with repression of sMMO gene transcription in both methanotrophs, suggesting that a common regulatory pathway may be involved in the transcriptional regulation of sMMO and pMMO. C. Molecular ecology Difficulties in using traditional culture-based techniques to study the ecology of methanotrophs (e.g., slow growth of methanotrophs and scavenging of nonmethanotrophs on agar plates) has hampered studies. 

Application of molecular biology techniques to methanotrophs has been aided considerably by the sequencing of a number of 16S rRNA genes of methanotrophs and methylotrophs and the cloning of several methanotroph-specific genes. Hanson and colleagues (1996) have used 16S rRNA sequence data to examine phylogenetic relationships within genera of methanotrophs and methylotrophs. 16S rRNA data coupled with PCR technology have also been used successfully in analyzing methanotrophs in the marine environment. Seawater samples were enriched for methanotrophs by adding essential nutrients and methane. Changes in composition of the bacterial population was then monitored by analysis of 16S rDNA libraries. The dominant 16S rRNA sequence that was present in samples after enrichment on methane was found to show a close phylogenetic relationship to Methylomonas, indicating that novel methanotrophs related to extant Methylomonas spp. were present in enrichment cultures. Genes unique to methanotrophs include those encoding sMMO and pMMO polypeptides. The high degree of identity between sMMO genes has enabled the design of PCR primers which specifically amplify sMMO genes directly from a variety of different freshwater, marine, soil, and peat samples. Results suggest that there is considerable diversity of methanotrophs in these environments. Another functional gene probe for methanotrophs is one based on the pMMO, present in all extant methanotrophs. Sequence data on pmo and amo (ammonia monooxygenase—a related enzyme found in nitrifying bacteria) genes has allowed the design of degenerate PCR primers which will specifically amplify DNA genes encoding pmoA or amoA from many different methanotrophs and nitrifiers. Analysis of the predicted amino acid sequences of these genes from representatives of each of the phylogenetic groups of methanotrophs (_ and _ Proteobacteria) and ammonia oxidizing nitrifiers (_ and _ Proteobacteria) suggests that the particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related enzymes. Another potentially useful marker is mxaF, encoding the large subunit of methanol dehydrogenase, which is present in virtually all gram-negative methylotrophs. mxaF is highly conserved and is, therefore, a good indicator of the presence of these organisms in the natural environment. 

D. Phylogeny and taxonomy Methanotrophs are all gram-negative bacteria and can be classified into two groups. Type I methanotrophs of the genera Methylomonas, Methylobacter, Methylomicrobium, “Methylothermus,” Methylococcus, Methylosphaera, and “Methylocaldum” utilize the ribulose monophosphate (RuMP) pathway for the assimilation of formaldehyde into cell carbon, possess bundles of intracytoplasmic membranes, and are members of the _-subdivision of the purple bacteria (class Proteobacteria). Type II methanotrophs, such as Methylosinus and Methylocystis, utilize the serine pathway for formaldehyde fixation, possess intracytoplasmic membranes arranged around the periphery of the cell, and fall within the _-subdivision of the Proteobacteria. Classification schemes, based on phenoand chemo-taxonomic studies have been strengthened as a result of the nucleotide sequencing of both 5S and 16S ribosomal RNA (rRNA) from a large number of methanotrophs and methylotrophs (Fig. 58.2). The key features of representative genera of methanotrophs are summarized in Table 58.1. Properties such as mol% G_C content of DNA, membrane fatty acid composition, nitrogen fixation, and some morphological features, can be used to discriminate between Type I and Type II methanotrophs.  

V. AEROBIC METHYLOTROPHS A. Methanol utilizers The bacteria capable of growth on methanol are more diverse than those capable of growing on methane. They include a variety of gram-negative and gram-positive strains and include both facultative and obligate methylotrophs (Table 58.2). The methanol-utilizing bacteria can be divided according to their carbon assimilation pathway. The methanolutilizers that contain the serine cycle for formaldehyde assimilation include Methylobacterium and Hyphomicrobium strains. Most of the gram-negative methanol utilizers that contain the RuMP cycle are obligate methylotrophs, with the exception of the facultative Acidomonas strains. The gram-positive methanol utilizers, which all contain the RuMP cycle, are facultative methylotrophs. All of the known gramnegative methanol- and methane-utilizing bacteria contain an enzyme for oxidizing methanol called methanol dehydrogenase. This enzyme, which oxidizes primary alcohols, contains the cofactor pyrroloquinoline quinone (PQQ). Methanol dehydrogenase is one of a family of PQQ-linked enzymes known as quinoproteins. The electrons from the oxidation of methanol are transferred from the PQQ cofactor to a specific soluble cytochrome c and, from there, through to other carriers to the terminal oxidase. Methanol dehydrogenases are highly conserved throughout the gram-negative methylotrophic bacteria. Studies of the molecular genetics of this system have revealed that the synthesis of a fully active methanol oxidizing pathway requires a total of at least 32 genes, among which are the pqq genes involved in cofactor biosynthesis and a comprehensive set of mxa, mxb, mxc, and mxd genes, some of which encode the structural enzymes and others which encode proteins involved in regulation of gene expression and protein activity. Different types of methanol dehydrogenases occur in the gram-positive methylotrophs. A methylotrophic Amycolatopsis species contains an unusual quino alcohol dehydrogenase, and the methylotrophic Bacillus species contain a methanol dehydrogenase enzyme that is not PQQ linked, but instead is linked to NAD. Recently, an aerobic methylotrophic bacterium, Methylobacterium extorquens, was found to contain a cluster of genes that are predicted to encode some of the enzymes from methanogenic and sulfate-reducing Archaea involved in C1 transfer, thought to be unique to this group of strictly anaerobic microorganisms. Enzyme activities were also detected in M. extorquens and mutants defective in some of these genes were unable to grow on C1 compounds, suggesting that the archaeal enzymes also function in aerobic C1 metabolism. Thus, methylotrophy and methanogenesis involve common genes that cross the bacteria/ archaeal boundaries. Some bacteria, such as Paracoccus, 

Thiosphaera, and Xanthobacter, can grow on methanol by oxidizing this methylotrophic substrate to carbon dioxide but then fixing this carbon into cell biomass using the enzyme ribulose bisphosphate carboxylase/ oxygenase (RuBISCO). These types of organisms are, therefore, considered as autotrophic methylotrophs. B. Utilization of methylated amines Many of the bacteria that grow on methanol are also capable of utilizing methylated amines. Some bacteria have been isolated that are capable of growth on methylated amines but do not grow on methanol or methane, such as Pseudomonas aminovorans and Arthrobacter P1. Several species of methylotrophic bacteria are able to utilize methylamine as a sole source of carbon and energy. Three different systems for the oxidation of primary amines are known. These are methylamine dehydrogenase, found in some gram-negative methylotrophs; amine oxidase, found in gram-positive methylotrophs; and indirect methylamine oxidation via N-methyl-glutamate dehydrogenase, found in the remaining gram-negative methylotrophs. The methylamine dehydrogenases (MADH) are periplasmic proteins, consisting of two small and two large subunits. Each small subunit has a covalently bound prosthetic group called tryptophan tryptophylquinone (TQQ). MADHs can be divided into two groups, based on the electron acceptors that they use. The MADHs from restricted facultative methylotrophic bacteria belonging to the genus Methylophilus, use a c-type cytochrome as a electron acceptor, whereas all other MADHs use blue copper proteins called amicyanins. A group of genes called the mau genes are responsible for the synthesis of MADH. 

The mau gene cluster of Paracoccus denitrificans consists of 11 genes, 10 of which encode the structural proteins or proteins involved in cofactor biosynthesis and which are transcribed in one direction, whereas the 11th regulatory gene (mauR) is located upstream and is divergently transcribed. C. Utilization of halomethanes Certain Methylobacterium and Methylophilus species can grow on dichloromethane as sole carbon and energy source (Fig. 58.2). The key enzyme in the aerobic degradation of CH2Cl2 is dichloromethane dehalogenase, which catalyzes the glutathione (GSH)- dependent dehalogenation of CH2Cl2 to formaldehyde and chloride ions. The formaldehyde is subsequently assimilated via the serine pathway. Some Hyphomicrobium and Methylobacterium species also grow on methyl chloride, although the exact mechanism for utilization of this C1 compound is not yet known. Methyl bromide also appears to be a C1 substrate for certain bacteria and is currently receiving considerable attention as a potential methylotrophic substrate. Again, the bacteria responsible have not been fully characterized but it is likely that these novel bacteria are widespread in the terrestrial and marine environment and play an important role in cycling of halogenated methanes. D. Utilization of methylated sulfur species There are some methylotrophic bacteria which are capable of utilizing methylated sulfur compounds, such as dimethylsulfoxide (DMSO), dimethylsulfide (DMS), and dimethyldisulfide (DMDS). Most of these are of the genus Hyphomicrobium but certain Thiobacillus strains have been reported. DMSO and DMS metabolism has not been studied in great detail in methylotrophic bacteria but it is believed that in Hyphomicrobium DMSO is reduced to DMS, which is, in turn, converted to formaldehyde and methanethiol. The methanethiol may then be converted by an oxidase to formaldehyde and H2S with the concomitant production of hydrogen peroxide. The formaldehyde produced would then be assimilated into cell carbon by the serine pathway in Hyphomicrobium. Methanesulfonic acid (MSA) is also a C1 source for certain methylotrophs, which appear to be ubiquitous in the environment. The terrestrial strain Methylosulfonomonas and the marine strain Marinosulfonomonas oxidize MSA to formaldehyde and sulfite, using a methanesulfonic acid monooxgenase. The formaldehyde is subsequently assimilated into the cell via the serine pathway. Some strains of Methylobacterium and Hyphomicrobium can also utilize MSA. Most of the MSA-utilizers also grow well on other C1 compounds such as methanol, methylamine, and formate, but not methane. Certain Hyphomicrobium species can grow on monomethylsulfate. 

VI. ANAEROBIC METHYLOTROPHS All extant methanotrophs are obligate aerobes. However, there is now good biogeochemical and biological evidence that methane oxidation occurs in marine environments, such as sulfate-rich sediments, alkaline soda lakes, and some freshwater lakes. However, to date, no anaerobic bacteria that will grow on or oxidize methane have been isolated from these environments and cultivated in the laboratory. It is not known if such bacteria are true methanotrophs or if a consortium of bacteria is involved in these processes. One hypothesis is that sulfate is the terminal electron acceptor for anaerobic methane oxidation but further experimental work is required here. Some studies indicate that, although methane is oxidized to carbon dioxide, the methane carbon is not assimilated into cell biomass. Methanol can also be oxidized by facultatively anaerobic bacteria of the genus Hyphomicrobium, using nitrate as a terminal electron acceptor. Some acetogenic and methanogenic bacteria, which are strict anaerobes, are capable of growth on C1 compounds, such as methanol and methylamines. During anaerobic growth, these bacteria convert such C1 compounds to methane, acetate, or butyrate, rather than carbon dioxide. Carbon from the original C1 substrate is not assimilated via formaldehyde but their methyl groups are incorporated into acetyl CoA, a precursor for cellular constituents. Therefore, following the original definition of methylotrophy, these obligate anaerobes that utilize C1 compounds, such as methanol and methylamine, are not normally considered as methylotrophs. 

VII. METHYLOTROPHIC YEASTS The ability of some yeasts to grow on methanol as a source of carbon and energy has been discovered only relatively recently. They can be isolated from soil, rotting fruits, and vegetables, or plant material, again suggesting that methanol derived from methoxy groups in wood lignin or pectin is an important factor in the ecology of these yeasts. Methylotrophic yeasts belong to the fungi perfecti, form ascospores that are hat-shaped and homothallic. They are members of the genera Hansenula, Pichia, and Candida and they metabolize methanol via alcohol oxidases in peroxisomes. Assimilation of formaldehyde is accomplished by the xylulose monophosphate cycle. Yeast cultures that use methane as a sole carbon and energy source have also been described. These strains were slow growing and have received very little attention over the past 20 years.