title: Isolation and characterization of newMethanosarcina mazeistrains KOR-3, -4, -5, and -6 from an anaerobic digester using pig slurry.
abstract: 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 andmcrAgene-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 H2+CO2, formate, or methanol as a growth substrate KOR-5 and KOR-6 strains utilized acetate, methanol, and trimethylamine for methanogenesis but did not use H2+CO2or 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 andmcrAgenes demonstrated that strains KOR-3, -4, -5, and -6 are related toMethanosarcina mazei(M. mazei, 99% sequence similarity).
  
  
  CONCLUSIONS
  On the basis of physiological and phylogenetic characteristics, strains KOR-3, -4, -5, and -6 are proposed to be new strains within the genusMethanosarcina, namedM. mazeiKOR-3, -4, -5, and -6.
authors.affiliation: Graduate School of Future Convergence, Hankyong National University, Anseong 17579, Korea.|School of Animal Science and Biotechnology, Mongolian University of Life Sciences, Ulaanbaatar 17024, Mongolia.
authors.forename: Urantulkhuur
authors.initials: U
authors.lastname: Battumur
authors.affiliation: Biogas Research Center, Hankyong National University, Anseong 17579, Korea.
authors.forename: Youngman
authors.initials: Y
authors.lastname: Yoon
authors.affiliation: Department of Animal Science and Technology, Chung-Ang University, Anseong 17546, Korea.
authors.forename: Gui Sek
authors.initials: GS
authors.lastname: Bae
authors.affiliation: Biogas Research Center, Hankyong National University, Anseong 17579, Korea.|Department of Animal Life and Environment Science, Hankyong National University, Anseong 17579, Korea.
authors.forename: Chang-Hyun
authors.initials: CH
authors.lastname: Kim
keywords: 16S rRNA; Anaerobic Digester; Methanogen; Methanosarcina mazei; mcrA