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Tofua Arc의 열수구환경으로부터 호열성 혐기성 고세균(Thermococcus)의 농화배양 및 동정

Identification of Anaerobic Thermophilic Thermococcus Dominant in Enrichment Cultures from a Hydrothermal Vent Sediment of Tofua Arc

  • 차인태 (충북대학교 미생물학과) ;
  • 김소정 (충북대학교 미생물학과) ;
  • 김종걸 (충북대학교 미생물학과) ;
  • 박수제 (충북대학교 미생물학과) ;
  • 정만영 (충북대학교 미생물학과) ;
  • 주세종 (한국해양연구원 심해해저자원연구부) ;
  • 권개경 (한국해양연구원 해양바이오연구센터) ;
  • 이성근 (충북대학교 미생물학과)
  • Cha, In-Tae (Department of Microbiology, Chungbuk National University) ;
  • Kim, So-Jeong (Department of Microbiology, Chungbuk National University) ;
  • Kim, Jong-Geol (Department of Microbiology, Chungbuk National University) ;
  • Park, Soo-Je (Department of Microbiology, Chungbuk National University) ;
  • Jung, Man-Young (Department of Microbiology, Chungbuk National University) ;
  • Ju, Se-Jong (Marine Biotechnology Research Center, KORDI) ;
  • Kwon, Kae-Kyoung (Deep-sea and Marine Georesources Research Department, KORDI) ;
  • Rhee, Sung-Keun (Department of Microbiology, Chungbuk National University)
  • 투고 : 2011.11.07
  • 심사 : 2012.02.29
  • 발행 : 2012.03.31

초록

열수구(Hydrothermal vent)는 빛이 없는 환경에서 생명체의 진화가 일어나는 독특한 환경을 유지하고 있다. 남태평양 Tonga의 Tofua arc의 열수구로부터 퇴적물을 채취하여 산화철[iron(III)], 황(elemental sulfur, $S^0$) 그리고 질산염을 전자수용체로 사용하고, 수소($H_2$), yeast extract를 전자공여체로 사용하여 배양에 의한 미생물의 다양성을 연구하였다. 배양 온도는 각각 $65^{\circ}C$$80^{\circ}C$였으며, 연속희석배양법과 16S rRNA 유전자의 PCR-Denaturing Gradient Gel Electrophoresis를 분석하고, 검출된 염기서열의 정보분석을 통하여 고세균을 동정하였다. 16S rRNA 유전자의 계통분류학적 분석 결과 배양된 대부분의 고세균은 Thermococcus 속(T. alcaliphilius, T. litoralis, T. celer, T. barossii, T. thoreducens, T. coalescens)에 속하며 그들과 98-99%의 상동성을 가지고 있었다. Thermococcus 속의 미생물들이 일반적으로 이용할 수 없는 질산염과 산화철을 전자수용체로 첨가한 배양에서 관찰되었으나, 이는 환원제로 첨가한 $Na_2S$의 산화물을 이용하여 성장한 것으로 추정된다. Thermococcus 속에 속하는 고세균 외의 다양한 고세균의 배양을 위해서는 $Na_2S$ 대신 다른 환원제를 사용하는 배양조건의 이용이 요구된다.

Hydrothermal vents (HTV) provide special environments for evolution of lives independent on solar energy. HTV samples were gained from Tofua arc trench in Tonga, South Pacific. We investigated archaeal diversity enriched using combinations of various electron donors (yeast extract and $H_2$) and electron acceptors [Iron (III), elemental sulfur ($S^0$) and nitrate. PCR amplification was performed to detect archaeal 16S rRNA genes after the cultures were incubated $65^{\circ}C$ and $80^{\circ}C$ for 2 weeks. The cultures showing archaeal growth were transferred using the dilution-to-extinction method. 16S rRNA gene PCR-Denaturing Gradient Gel Electrophoresis was used to identify the enriched archaea in the highest dilutions where archaeal growth was observed. Most of cultured archaea belonged to genus of Thermococcus (T. alcaliphilius, T. litoralis, T. celer, T. barossii, T. thoreducens, T. coalescens) with 98-99% 16S rRNA gene similarities. Interestingly, archaeal growth was observed in the cultures with Iron (III) and nitrate as an electron acceptor. It was supposed that archaea might use the elemental sulfur generated from oxidation of the reducing agent, sulfide. To cultivate diverse archaea excluding Thermococcus, it would be required to use other reducing agents instead of sulfide.

키워드

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