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Asian-Australas J Anim Sci > Accepted
doi: https://doi.org/10.5713/ajas.16.0830    [Accepted]
Isolation and Characterization of New Methanosarcina mazei Strains KOR-3, -4, -5, and -6 from an Anaerobic Digester Using Pig Slurry
Urantulkhuur Battumur1,5, Youngman Yoon2, Gui Sek Bae3, Chang-Hyun Kim4,4,*
1Graduate School of Future Convergence, Hankyong National University, Anseong, Korea
2Biogas Research Center, Hankyong National University, Anseong, Korea
3Department of Animal Science and Technology, Chung-Ang Unversity, Anseong, Korea
4Department of Animal Life and Environment Science, Hankyong National University, Anseong, Korea
5School of Animal Science and Biotechnology, Mogolian University of Life Sciences, Ulaanbaatar, Mongolia
* Corresponding Author: Chang-Hyun Kim ,Tel: 031-670-5095, Fax: 031-670-5099, Email: kimch@hknu.ac.kr
Received: 26 October 2016;  Revised: 28 November 2016.  Accepted: 29 December 2016.  Published online 2 January 2017.

ABSTRACT
Objective:
An experiment was conducted to isolate and identify new methanogens in Korea from an anaerobic digester that uses pig slurry.
Method:
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. PCR 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 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+CO2 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 (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.
Keywords: Methanogen; Anaerobic digester; 16S rRNA; mcrA; Methanosarcina mazei
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