Methane ($CH_4$) is a potent greenhouse gas (GHG) but, at the same time, is a promising resource as energy source and feedstock for chemical industry. Methanotrophs, the group of bacteria utilizing CH4 as their sole carbon and energy source, have recently gained interest as the biocatalysts for mitigation of GHG emissions and for conversion of $CH_4$ to value-added products; however, their slow growth rates have been the major bottleneck for their industrial applications, barring prompt genetic modifications and biomass generation.
In this study, the chemostat principle was adopted to isolate fast-growing methanotrophs by screening the methanotrophs by their growth rates (i.e., the dilution rate of the chemostat reactor). Sediment from Gapcheon Stream and anaerobic digestion effluent from Daejeon WWTP were collected and enriched in sealed batch culture before inoculation into a continuously stirred tank reactor (CSTR) fed with continuous stream of 20% $CH_4$ (in air) in the headspace and fresh NMS medium in the aqueous phase. After initial fed-batch incubation in the CSTR, the dilution rate was gradually increased from 0.1 $h^{-1}$ with an increment of $0.05^{-1}$ until complete washout was observed. The samples collected at the highest dilution rates were used for isolation. The shift in microbial population and enrichment of fast-growing methanotrophs at each step were monitored with 16S rRNA amplicon sequencing.
Diverse methanotrophs initially constituting minor populations in the environmental samples were enriched in batch incubation, including genera Methylosinus, Methylocaldum, Methylomonas, Methylococcus, Methylomicrobium, Methylocella, and Methylosarcina. After chemostat operation, the OTUs affiliated with Methylomonas sp. LW13 (SS) and Methylosarcina spp. (AD) were exclusively enriched. The isolated strain exhibited exponential growth rates higher than 0.3 $h^{-1}$, the highest growth rate ever reported for any methanotroph grown at 30$\circ C$. The novel isolation method successfully screened for fast-growing methanotrophs with great industrial potential and significantly shortened the time for isolation.
지구온난화가스인 메탄을 에너지원으로 사용하는 메탄산화균은 토양 및 지하수 내 오염물질 처리, 바이오 폴리머, 메탄올 생산 등 공업적인 측면에서도 연구 가치가 높다. 하지만 메탄산화균의 느린 성장속도는 관련 연구를 늦추고 있다. 본 논문에서는 물질환경조절장치를 도입하여 비생장속도가 높은 메탄산화균을 찾는 방법을 소개하였다.
갑천 토양 및 대전하수처리장의 혐기성 소화조에서 나오는 폐수를 비연속 배양 후 연속교반탱크반응기에 주입하였다. 유가 배양 후, 희석속도를 시간당 0.1로 조절하였으며, 이 후 0.05씩 높여나갔다. 미생물이 모두 씻겨나갔을 때, 반응기 운영을 종료하였으며, 가장 높은 희석속도로 운영된 샘플에서 메탄산화균 분리를 시작하였다. 동시에 각 단계별 미생물 군집을 Miseq 기법을 통하며 분석하였다.
환경 시료에는 다양한 메탄산화균이 존재했으나, 전체 미생물 중 차지하는 비율은 낮았다. 비연속 배양을 시작하면서 그 비율은 증가했으며, 반응기 운영 후에는 Methylomoas sp. LW13(갑천)과 Methylosarcina spp.(혐기성 소화조)와 유사한 군집이 우세하였다. 분리된 미생물의 비생장속도는 시간당 0.3 이상으로 현재까지 알려진 메탄산화균에 비해 매우 빠르게 생장했다.