Abstract
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.
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Acknowledgements
We thank Yoshiaki Takahashi (Alt Associates, Ltd, Nagaoka, Japan) for constructing of the DHS reactor; Ai Miyashita, Yuto Yashiro, Masyuki Ehara and Dr Sanae Sakai for assistance with the DHS reactor operation; Takuya Kazawa and Taketoshi Nakamura for help in measuring organic substances; Takeshi Terada for help with Q-PCR; Drs Haruhiko Sumino and Masanobu Takahashi for providing valuable information on the DHS reactor; and Mandy Cumpston, Drs Takuro Nunoura, Satoshi Nakagawa and Hisako Hirayama for helpful discussions and comments. We greatly appreciate Professors Hideki Harada and Akiyoshi Ohashi for their continuous encouragement and support. We also thank the shipboard scientists and crews of the Chikyu Shakedown Expedition CK06-06 for helping us to collect the sediment core sample. This study was partly supported by grants from the Institute of Fermentation, Osaka, Japan, the Japan Society for the Promotion of Science, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Imachi, H., Aoi, K., Tasumi, E. et al. Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor. ISME J 5, 1913–1925 (2011). https://doi.org/10.1038/ismej.2011.64
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DOI: https://doi.org/10.1038/ismej.2011.64
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