난배양성 토양세균의 배양법 평가 및 신 분류군의 순수분리 = Evaluation of Various Oligotrophic Media for Cultivation of Previously Uncultured Soil Bacteria

국문 초록 (Abstract)

난배양성 세균의 배양효율을 증진시킬 수 있다고 보고된 배지 첨가물들이 포함된 다양한 종류의 빈영양 배지들을 대상으로 배양효율을 비교평가하고 최적의 배양조건을 모색하였으며, 평가된 배지를 사용하여 토양시료로부터 순수 분리된 난배양성 세균들의 계통분류학적 위치를 분석하였다. 배지 첨가물로는 토양의 화학적 조성을 반영하기 위한 토양추출액(soil extract), 부식질산의 유사체(humic acid analogue)인 anthraquinone disulfonate, 정족수인식 신호물질(quorum-signaling compounds)인 acyl homoserine lactones, 과산화물(exogenous peroxide)로부터 세균을 보호하기 위한 catalase가 사용되었다. $CO_2$ 과분압(5%, v/v) 조건에서 60일간 배양하였을 때, catalase가 첨가된 배지가 가장 높은 세균집락수(CFU)를 보였다. 이 배지로부터 147개의 균주를 무작위적으로 선택하여 순수분리하고 16S rRNA 유전자의 염기서열을 이용한 계통학적 분석을 실시한 결과, 순수분리된 균주의 약30%가 이전에 배양 또는 발견된 적이 없는 새로운 종(species)에 속하며, 이 중 약 25%는 새로운 과(family)에 속하는 세균일 가능성이 있는 것으로 나타났다. 또한 난배양성 토양세균으로 알려진 phylum Acidobacteria에 속하는 세균들이 성공적으로 배양되었다는 결과를 고려하면, 본 연구에서 사용된 배지 및 배양조건은 난배양성 토양세균의 배양은 물론 신 분류군의 발굴에도 큰 기여를 할 것으로 기대된다.

다국어 초록 (Multilingual Abstract)

We evaluated cultivation methods to obtain pure cultures of previously uncultivated bacteria from soil. Soil bacteria (suspensions) were inoculated onto various oligotrophic media with one of the following additives: 1) soil extract; 2) anthraquinone disulfonate (humic acid analogue); 3) acyl homoserine lactones (quorum-signaling compounds); 4) catalase (for the protection of bacteria from exogenous peroxides). After the relatively long period (60 days) of incubation with elevated concentrations of $CO_2$ (5%, v/v), the media containing catalase showed the highest colony count. We purified 147 randomly selected colonies from the media and the isolates were subjected to the phylogenetic analyses of their 16S rRNA gene sequences. Phylogenetic analysis revealed that approximately 30% of the isolates might belong to novel species or novel family, suggesting that the media and incubation conditions used could be useful for the cultivation of as-yet-uncultured bacteria. Especially, bacteria belonging to the phylum Acidobacteria, ubiquitous bacterial taxon known as an uncultured bacterial group (at least difficult to culture from environmental samples), were successfully cultured in this study.

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