David R. Boone

Diversity and Taxonomy of Methanogens

The production of methane is a ubiquitous, defining characteristic of methanogens. The production of methane or any other hydrocarbon as a major catabolic product is unique to this group of microbes, which share many other characteristics that are not common among other microbes. Phylogenetically, methanogens are Archaeobacteria (Woese et al., 1978), a group of microbes that are distinguished from true bacteria by a number of characteristics, including the possession of membrane lipids composed of isoprenoids ether-linked to glycerol or other carbohydrates (De Rosa and Gambacorta, 1988; Jones et al., 1987; Langworthy, 1985), a lack of peptidoglycan containing muramic acid (Kandier and Hippe, 1977), and distinctive ribosomal RNA sequences (Balch et al., 1979; Woese, 1987). This group also includes some extreme halophiles and some extremely thermophilic, sulfur-dependent microbes (Woese, 1987) and is phylogenetically distinct from eukaryotes and true bacteria (Figure 1.1).

Bacillus infernus sp. nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61 degrees C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16S rRNA indicated that B. infernus should be classified as a new species of the genus Bacillus. B. infernus is the only strictly anaerobic species in the genus Bacillus.

Characterization of the anaerobic propionate-degrading syntrophs Smithella propionica gen. nov., sp. nov. and Syntrophobacter wolinii.

A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen. nov., sp. nov. The strain was enriched from an anaerobic digestor and isolated. Initial isolation was as a monoxenic propionate-degrading co-culture containing Methanospirillum hungateii JF-1T as an H2- and formate-using partner. Later, an axenic culture was obtained by using crotonate as the catabolic substrate. The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO2 and methane from propionate. However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 16S rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter.

Methanogenium frigidum sp. nov., a psychrophilic, H2-Using methanogen from Ace Lake, Antarctica

Methanogenium frigidum sp. nov. was isolated from the perennially cold, anoxic hypolimnion of Ace Lake in the Vesfold Hills of Antarctica. The cells were psychrophilic, exhibiting most rapid growth at 15 degrees C and no growth at temperatures above 18 to 20 degrees C. The cells were irregular, nonmotile coccoids (diameter, 1.2 to 2.5 microns) that occurred singly and grew by CO2 reduction by using H2 as a reductant. Formate could replace H2, but growth was slower. Acetate, methanol, and trimethylamine were not catabolized. Cells grew with acetate as the only organic compounds in the culture medium, but growth was much faster in medium also supplemented with peptones and yeast extract. The cells were slightly halophilic; good growth occurred in medium supplemented with 350 to 600 mM Na+, but no growth occurred with 100 or 850 mM Na+. The pH range for growth was 6.5 to 7.9; no growth occurred at pH 6.0 or 8.5. Growth was slow (maximum specific growth rate, 0.24 day-1; doubling time, 2.9 days). This is the first report of a psychrophilic methanogen growing by CO2 reduction.

Proposal of Minimal Standards for Describing New Taxa of Methanogenic Bacteria

The Subcommittee for Taxonomy of Methanogenic Bacteria has agreed in principle on minimum requirements for the description of new taxa of methanogenic bacteria. These requirements, as well as methods for determining specified characteristics, are indicated here and are proposed as minimal standards for the taxonomic description of new taxa of methanogens. The specified phenotypic characteristics are often not sufficient to distinguish among taxa or to determine the phylogenetic placement of a taxon, and, in these cases, additional chemotaxonomic, molecular, or genetic data may be required. The placement of a new taxon should be consistent with phylogeny, usually based on tests such as nucleic acid sequencing or cataloging studies or on protein fingerprinting. Suggestions are welcome for the improvement of these requirements, which are tentative until given final approval by the International Committee on Systematic Bacteriology.

Isolation of a Methanogen from Deep Marine Sediments That Contain Methane Hydrates, and Description of Methanoculleus submarinus sp. nov.

ABSTRACT We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 μm) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45°C (no growth at ≤10°C or ≥55°C). Cells grew with a maximum specific growth rate of 2.43 day−1 at 45°C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na+. The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

Methanohalophilus zhilinae sp. nov., an Alkaliphilic, Halophilic, Methylotrophic Methanogen

Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described. Strain WeN5T (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized. This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added. Sulfide (21 mM) inhibited cultures growing on trimethylamine. The antibiotic susceptibility pattern of strain WeN5T was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol%. Characterization of the 16S ribosomal ribonucleic acid sequence indicated that strain WeN5T is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic methanogens into their own genus.

Isolation and Characterization of a Dimethyl Sulfide-Degrading Methanogen, Methanolobus siciliae HI350, from an Oil Well, Characterization of M. siciliae T4/MT, and Emendation of M. siciliae

We isolated strain HI350 from a gas and oil well in the Gulf of Mexico, characterized it, and found that it is closely related to Methanolobus siciliae T4/MT (T = type strain), which we also characterized. The previously published characterization of the type strain of M. siciliae was limited to the optimum temperature for growth, and our characterization suggested the species description given below. Cells are irregular, nonmotile, coccoid, and 1.5 to 3 microns in diameter. The catabolic substrates used include methanol, trimethylamine, and dimethyl sulfide, but not H2-CO2, formate, or acetate. Growth is fastest in the presence of 0.4 to 0.6 M Na+, in the presence of 60 to 200 mM Mg2+, at pH 6.5 to 6.8, and at 40 degrees C. Growth on trimethylamine is stimulated by yeast extract. An electrophoretic analysis confirmed that strain HI350 is closely related to strain T4/MT and indicated that major changes in the intracellular proteins of M. siciliae HI350 occur when the growth substrate is switched between dimethyl sulfide and trimethylamine.

Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T

Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H2 plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCl. The catabolic substrates which supported most rapid growth were H2 plus methanol; this combination sometimes allowed growth of a strain under extreme environmental conditions which prevented growth on other substrates. The cell morphology of all strains was affected by growth conditions.

Methanohalophilus oregonense sp. nov., a methylotrophic methanogen from an alkaline, saline aquifer

Methanohalophilus oregonense WAL1T (= OGI 99T = DSM 5435T) (T = type strain) was isolated from an anoxic aquifer (pH 10, with 100 g of dissolved solids per liter of pore water) 3 m deep near Alkali Lake, an alkaline, desert lake in south central Oregon. An examination of the subsurface sediments revealed no methanogens that were capable of growth on methanogenic substrates, such as H2-CO2, formate, or acetate, which is consistent with the results of other studies of hypersaline, sulfate-containing anoxic environments. Strain WAL1T grew on trimethylamine and grew slowly on methanol or dimethylsulfide, but did not catabolize H2-CO2, formate, or acetate. The cells were irregular coccoids (diameter, 1 to 1.5 μm), and cells growing in liquid media also formed clumps of 2 to 15 or more cells. The cells were mesophilic and required one or more vitamins present in yeast extract. Like the only previously described strain of alkaliphilic, methylotrophic methanogen (Methanohalophilus zhilinae WeN5T), strain WAL1T grew most rapidly in medium of moderate salinity; strain WAL1T grew well in the presence of 0.1 to 1.4 M Na+ and grew most rapidly at an Na+ concentration of 0.35 M (specific growth rate, 0.1 h-1). Best growth occurred with about 50 mM Mg2+ and at a pH of 8.4 to 9.0. K+ appeared to be required, with 13 to 130 mM K+ supporting most rapid growth. The guanine-plus-cytosine content of the DNA was 40.9 ± 0.1 mol%.