title: A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor.
abstract: Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan; Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Aoki
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ehara
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Institute for Geo-resources and Environment, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Yoshioka
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Miyazaki
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan; Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Miyashita
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan; Department of Construction Systems Engineering, Anan National College of Technology, Anan, Tokushima, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Kawakami
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Social and Environmental Engineering, Hiroshima University, Higashihiroshima, Hiroshima, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Ohashi
authors.affiliation: Research and Development Center for Marine Biosciences, JAMSTEC, Yokosuka, Kanagawa, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Nunoura
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Takai
authors.affiliation: Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imachi
mesh_terms: D001105:Archaea / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D019149:Bioreactors / Q000382:microbiology*; D020563:Gammaproteobacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D008697:Methane / Q000378:metabolism*; D064307:Microbiota; D008969:Molecular Sequence Data; D012623:Seawater / Q000382:microbiology*
chemical_list: D008697:Methane