Robert A. Mah

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.

The Methanogenic Bacteria

The methanogenic bacteria are unique among pro-karyotes because they produce a hydrocarbon, methane, as a major product of anaerobic metabolism. This physiological property was proposed in 1956 by H. A. Barker as the main taxonomic characteristic of a morphologically diverse group of bacteria which he termed the Methanobacteriaceae. The taxonomy of this physiological family has been obfuscated by the difficulty of obtaining members in pure culture. Consequently, various species were named on the basis of the types of substrates converted to methane by “purified” (i.e., enrichment) cultures containing a predominant morphological type suspected of methanogenesis. Only three species, Methanobacterium formicicum, Methano-coccus vannielii, and Methanosarcina barkeri, were isolated in axenic culture by the time of Barker’s review of these organisms (Barker, 1956).

Isolation and characterization of Methanogenium aggregans sp. nov.

A new mesophilic methanogenic bacterium was isolated from a sewage sludge digestor. The cells were irregular cocci (diameter, 0.5 to 2.0 μm) and occurred singly or in small aggregates. Adjacent cells did not appear to touch, even in aggregates. This bacterium was not motile and did not possess flagella. Colonies appearing after 1 week of incubation were white and 1 mm in diameter. Older colonies were yellow and up to 4 mm in diameter. The organism used H2-CO2 or formate as an energy source. Acetate and either yeast extract or Trypticase peptone were required for growth. The optimum pH for growth was between 6.5 and 7.0. The NaCI requirement for methanogenesis was less than 0.2 g/liter. The deoxyribonucleic acid base composition was 52 mol% guanine plus cytosine. This isolate is named Methanogenium aggregans sp. nov., and the type strain (strain MSt) has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3027.

Emendation of the genus Thermobacteroides: Thermobacteroides proteolyticus sp. nov., a proteolytic acetogen from a methanogenic enrichment

Thermobacteroides proteolyticus sp. nov. was isolated from a methanogenic enrichment culture inoculated from a thermophilic digestor (55°C) that was fermenting tannery wastes and cattle manure. The cells were anaerobic, gram-negative, nonsporeforming, nonmotile rods that were 0.5 μm wide and 1 to 6 μm long. At the end of logarithmic growth, they were pleomorphic, with some filamentous cells. The deoxyribonucleic acid base composition was 45 mol% guanine plus cytosine. The temperature optimum was 63°C (growth range 35, to 75°C); the pH optimum was 7.5 (growth range, pH 5.0 to 8.5). The growth substrates used included yeast extract, peptone, casein, gelatin, and Trypticase peptone. Fructose, glucose, maltose, sucrose, and mannose were weakly used as growth substrates; however, addition of yeast extract and either rumen fluid or Trypticase peptone stimulated utilization of these carbohydrates. Acetate, H2, and CO2 were the major products of growth in medium containing gelatin or glucose. The cells were resistant to kanamycin. The type strain is strain BT (= ATCC 35242).

Transfer of Methanogenium bourgense, Methanogenium marisnigri, Methanogenium olentangyi, and Methanogenium thermophilicum to the Genus Methanoculleus gen. nov., Emendation of Methanoculleus marisnigri and Methanogenium, and Description of New Strains of Methanoculleus bourgense and Methanoculleus marisnigri

Two strains of Methanogenium bourgense, strains MS2T (T = type strain) and LX1, were characterized, and, based in part on previously published DNA hybridization data, this species was transferred to a new genus, Methanoculleus, as Methanoculleus bourgense comb. nov. Methanogenium marisnigri JR1T and a new strain of Methanogenium marisnigri, strain AN8, were also characterized. This species was also transferred to the genus Methanoculleus as Methanoculleus marisnigri comb. nov. et emend., and its description was emended to indicate that the species has a temperature optimum near 40°C, is halotolerant, and is slightly alkaliphilic; in contrast, the previous description of this organism indicates that it has a temperature optimum of 20 to 25°C, is halophilic, and is slightly acidophilic. We also propose the transfer of two other phylogenetically related species, Methanogenium thermophilicum and Methanogenium olentangyi, to the genus Methanoculleus as Methanoculleus thermophilicum and Methanoculleus olentangyi, respectively. Methanogenium cariaci JR1T was also further characterized, and its description is emended.

Methanobacterium alcaliphilum sp. nov., an H2-utilizing methanogen that grows at high pH values

Four strains of alkaliphilic methanogens (strains WeN1, WeN2, WeN3, and WeN4T [T = type strain]) previously enriched and isolated from sediments of four low-salt, high-pH (pH 8.3 to 9.3) lakes in the Wadi el Natrun of Egypt were further characterized. These organisms were H2-oxidizing, CO2-reducing, rod-shaped bacteria which grew best at high pH values (strains WeN1, WeN2, and WeN4T preferred pH 8.4 and grew at pH values up to 9.9; strain WeN3 grew best at pH 7.8 and grew at pH values up to 9.2). Each strain required sulfide as a sulfur source and was resistant to elevated concentrations (growth rates in the presence of 0.5% Na2S · 9H2O were one-third to one-half the maximum observed rates). The guanine-plus-cytosine content of the deoxyribonucleic acid of strain WeN4T was 57 mol%. A new species of methanogen, Methanobacterium alcaliphilum, is described; strain WeN4 (= DSM 3387) is the type strain.

Isolation and characterization ofMethanococcus mazei

Methanococcus mazei strain S-6 was isolated from a laboratory digester; this is the first report of its isolation in pure culture. The present isolate exhibited a life cycle, and the forms produced during its development fit the morphological description ofM. mazei exactly. In young cultures, cell clusters resembleMethanosarcina strain TM-1; in older cultures, these cell clusters become “cysts” which may be physically ruptured to yield myriads of highly irregular coccoid elements.M. mazei used acetate, methanol, methylamine, or trimethylamine for growth and methanogenesis. It used H2−CO2 slowly. It did not use formate, ethanol, butyrate, or propionate. It was Gram negative to Gram variable.

Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium.

An anaerobic, extremely thermophilic, xylanolytic, non-spore-forming bacterium was isolated from a sediment sample taken from Owens Lake, California, and designated strain OLT (T = type strain). Strain OLT had a Gramnegative reaction and occurred as short rods which sometimes formed long chains containing a few coccoid cells. It grew at 50-80 degrees C, with an optimum at 75 degrees C. The pH range for growth was 5.5-9.0 with an optimum at about pH 7.5. When grown on glucose at optimal conditions, its doubling time was 7.3 h. In addition to glucose, the isolate utilized sucrose, xylose, fructose, ribose, xylan, starch, pectin and cellulose. Yeast extract stimulated growth on carbohydrates but was not obligately required. The end products from glucose fermentation were lactate, acetate, ethanol, H2 and CO2. The G + C content of strain OLT was 36.6 mol%. The 16S rDNA sequence analysis indicated that strain OLT was a member of the subdivision containing Gram-positive bacteria with DNA G + C content of less than 55 mol% and clustered with members of the genus Caldicellulosiruptor. Because strain OLT is phylogenetically and phenotypically different from other members of this genus, it is proposed to designate this isolate Caldicellulosiruptor owensensis sp. nov. Strain OLT is the type strain (= ATCC 700167T).

Ecology and microbiology of biogasification

Abstract The biodegradation of organic matter to form methane and carbon dioxide requires the interactions of diverse populations of bacteria. The roles of each of these organisms in the process and how they interact with each other is understood only in a rudimentary way. This paper describes the investigation of the microbial ecology of the anaerobic degradation of biomass feedstocks.

Acetoanaerobium noterae gen. nov., sp. nov.: an Anaerobic Bacterium That Forms Acetate from H2 and CO2

An anaerobic bacterium which produced acetate from H2 and CO2 was isolated. The rod-shaped cells were not lysed by KOH, did not hydrolyze L-alanine-4-nitroanilide, and stained gram negative. However, the cell wall did not resemble a gram-negative wall in structure; it was comprised of two layers. The cells were motile by means of three or four peritrichous flagella. Yeast extract was required for both chemoorganotrophic and chemolithotrophic growth; yeast extract, glucose, maltose, or H2-CO2 could serve as a substrate for growth. Strain NOT-3T(T = type strain) grew best at 37°C and pH 7.6 to 7.8. The deoxyribonucleic acid base composition was 36.8 mol% guanine plus cytosine. Strain NOT-3 (= ATCC 35199) is named Acetoanaerobium noterae gen. nov., sp. nov. and is the type strain of this new species.