Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

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 1C. 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.

IODP Expedition 337: Deep Coalbed Biosphere off Shimokita – Microbial processes and hydrocarbon system associated with deeply buried coalbed in the ocean

“…Using a continuous-flow bioreactor system called down-flow hanging sponge reactor, phylogenetically diverse, strictly anaerobic microbes were also successfully cultivated, including methanogens such as the genera Methanobacterium, Methanoccoides, and Methanosarcina (Imachi et al, 2011). Several attempts of traditional batch-type cultivations also successfully led to the pure culture isolation of several subseafloor anaerobic microbes, including new Bacterioidetes species of Geofilum rubicundum and Sunxiuqinia faeciviva , new Spirochaetes species of Spirochaeta psychrophila and Sphaerochaeta multiformis (Miyazaki et al, 2014a, b), and a new species of a new genus of Pelolinea submarina within the phylum Chloroflexi .…”

Spirochaeta psychrophila sp. nov., a psychrophilic spirochaete isolated from subseafloor sediment, and emended description of the genus Spirochaeta

“…Inagaki et al, 2006;Inagaki & Nakagawa, 2008;Beal et al, 2009;Mills et al, 2012). However, the first report on the isolation of a spirochaete from a deep-sea sedimentary environment was quite recent (Imachi et al, 2011).…”

“…We successfully isolated a spirochaete, strain MO-SPC1 T , from subseafloor sediment samples collected off the shore of the Shimokita Peninsula of Japan, in the north-western Pacific Ocean (site C9001, 1180 m water depth) (Imachi et al, 2011). Enrichment and isolation of the strain were achieved by a continuous-flow bioreactor approach.…”

“…Enrichment and isolation of the strain were achieved by a continuous-flow bioreactor approach. In a previous publication, we described the isolation of the micro-organism, but we did not characterize the strain completely (Imachi et al, 2011). In this study, we describe details of the isolation procedure, as well as the morphological, physiological and genetic properties of strain MO-SPC1 T .…”

“…Procedures for DNA extraction, PCR amplification, cloning and sequencing were as described previously (Imachi et al, 2011). The nearly full-length 16S rRNA gene sequence of the isolate was amplified with bacterial universal primer pair 8f/ UN1490R.…”