Subsurface Geobiology Advanced Research (SUGAR) Project, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.
ISME J. 2011 Dec;5(12):1913-25. doi: 10.1038/ismej.2011.64. Epub 2011 Jun 9.
Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.
地下沉积物中的微生物产甲烷作用是地球碳循环中的一个关键过程。然而，基于培养的证据还很少得到证实。在这里，我们报告了使用连续流动型生物反应器和聚氨酯海绵作为微生物栖息地，从海底沉积物中培养产甲烷微生物群落的方法，称为下流式悬挂海绵（DHS）反应器。我们在 10°C 的反应器中，无氧孵育了从日本下北半岛采集的富含甲烷的岩芯沉积物，持续 826 天。补充有葡萄糖、酵母提取物、乙酸盐和丙酸盐作为潜在能源的合成海水被提供到反应器中。经过 289 天的运行，微生物甲烷生产变得明显。荧光原位杂交分析显示，在反应器中存在具有各种形态的代谢活跃的微生物细胞。针对 16S rRNA 的 DNA 和 RNA 基于系统发育的分析表明，在反应器中培养过程中成功地生长了具有多种系统发育的微生物成分。一旦反应器开始产生甲烷，其中的大多数类群与培养序列比与地下环境序列更密切相关。与甲烷杆菌属、甲烷球菌属和产甲烷菌属相关的潜在产甲烷类群与甲烷生成同时被检测到，同时也检测到未培养的古菌类群。使用产甲烷群落的富集作为后续接种物，传统的分批培养导致了几种厌氧微生物的成功分离，包括那些产甲烷菌。我们的结果证实，DHS 生物反应器是一种从海底沉积物中富集多种难培养微生物的有用系统，将能够对以前未培养的海底微生物生命的纯培养物进行生理和生态特征分析。
