Isolation and characterization of new Methanosarcina mazei strains KOR-3, -4, -5, and -6 from an anaerobic digester using pig slurry

OBJECTIVE: An experiment was conducted to isolate and identify new methanogens in Korea from an anaerobic digester that uses pig slurry. METHODS: An anaerobic digestate sample was collected from an anaerobic digester using pig slurry. Pre-reduced media were used for the growth and isolation of methanogens. Growth temperature range, pH range, NaCl concentration range, substrate utilization, and antibiotic tolerance were investigated to determine the physiological characteristics of isolated methanogens. The isolates were also examined microscopically for their morphology and Gram-stained. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification purpose. RESULTS: Four strains, designated KOR-3, -4, -5, and -6, were isolated and were non-motile, irregular coccoid, and 0.5 to 1.5 μm in diameter. Moreover, the cell walls of isolated strains were Gram-negative. KOR-3 and KOR-4 strains used acetate for methane production but did not use H(2)+CO(2), formate, or methanol as a growth substrate KOR-5 and KOR-6 strains utilized acetate, methanol, and trimethylamine for methanogenesis but did not use H(2)+CO(2) or formate as a growth substrate. The optimum temperature and pH for growth of four strains were 39°C and 6.8 to 7.2, respectively. The optimum concentration of NaCl for growth of KOR-3, KOR-5, and KOR-6 were 1.0% (w/v). The optimum NaCl concentration for KOR-4 was 0.5% (w/v). All of the strains tolerated ampicillin, penicillin G, kanamycin, streptomycin, and tetracycline; however, chloramphenicol inhibited cell growth. Phylogenetic analysis of 16S rRNA and mcrA genes demonstrated that strains KOR-3, -4, -5, and -6 are related to Methanosarcina mazei (M. mazei, 99% sequence similarity). CONCLUSION: On the basis of physiological and phylogenetic characteristics, strains KOR-3, -4, -5, and -6 are proposed to be new strains within the genus Methanosarcina, named M. mazei KOR-3, -4, -5, and -6.