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Cultivable microbial community
in 2-km-deep, 20-million-year-
old subseafloor coalbeds through
Received: 15 October 2018
Accepted: 9 January 2019 ~1000 days anaerobic bioreactor
Published: xx xx xxxx
cultivation
Hiroyuki Imachi 1,2, Eiji Tasumi1, Yoshihiro Takaki 1,3, Tatsuhiko Hoshino2,4,
Florence Schubotz5, Shuchai Gan5, Tzu-Hsuan tu1,6, Yumi saito1, Yuko Yamanaka1,
Akira Ijiri 2,3, Yohei Matsui 2,3, Masayuki Miyazaki 1, Yuki Morono 2,4, Ken takai1,2,
Kai-Uwe Hinrichs5 & Fumio Inagaki 2,4,7
Recent explorations of scientific ocean drilling have revealed the presence of microbial communities
persisting in sediments down to ~ km below the ocean floor. However, our knowledge of these
microbial populations in the deep subseafloor sedimentary biosphere remains limited. Here, we
present a cultivation experiment of 2-km-deep subseafloor microbial communities in 20-million-
year-old lignite coalbeds using a continuous-flow bioreactor operating at 40 °C for 1029 days with
lignite particles as the major energy source. Chemical monitoring of effluent samples via fluorescence
emission-excitation matrices spectroscopy and stable isotope analyses traced the transformation
of coalbed-derived organic matter in the dissolved phase. Hereby, the production of acetate and
13C-depleted methane together with the increase and transformation of high molecular weight humics
point to an active lignite-degrading methanogenic community present within the bioreactor. Electron
microscopy revealed abundant microbial cells growing on the surface of lignite particles. Small subunit
rRNA gene sequence analysis revealed that diverse microorganisms grew in the bioreactor (., phyla
Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Spirochaetes, Tenericutes,
Ignavibacteriae, and SBR1093). These results indicate that activation and adaptive growth of 2-km-deep
microbes was successfully accomplished using a continuous-flow bioreactor, which lays the groundwork
to explore networks of microbial communities of the deep biosphere and their physiologies.
Over the past two decades, scientific ocean drilling has demonstrated that numerous microbes exist in the
global deep subseafloor sediment, from the continental margins to open ocean gyres, comprising approximately
1029 microbial cells and 4 Pg of biomass carbon on our planet1,2. Porewater geochemistry suggests that organic
matter-fueled microbial energy respiratory activity is extraordinary low, ranging from × 10−18 to × 10−14
moles/e−/cell/year between the anoxic eastern equatorial Pacific and the oxic South Pacific Gyre sediments,
respectively3–5. Culture-independent molecular ecological studies (., PCR-mediated 16S rRNA and functional
gene analysis, or metagenomics) of the above-mentioned subseafloor settings showed that they harbor diverse
1Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth
Science and Technology (JAMSTEC), Yokosuka, Kanagawa, 237-0061, Japan. 2Research and Development center
for Submarine Resources, JAMSTEC, Yokosuka, Kanagawa, 237-0061, Japan. 3Project team for Development of
New-generation Research Protocol for Submarine Resources, JAMSTEC, Yokosuka, Kanagawa, 237-0061, Japan.
4Kochi Institute for Core Sample Research, JAMSTEC, Nankoku, Kochi, 783-8502, Japan. 5MARUM Center for Marine
Environmental Sciences and Department of Geosciences, University of Bremen, D-28359, Bremen, Germany.
6Institute of Oceanography, National Taiwan University, Taipei, 106, Taiwan. 7Research and Development center
for Ocean Drilling Science, JAMSTEC, Yokohama, Kanagawa, 236-0001, Japan. Correspondence and requests for
materials should be addressed to . (email: ******@)
Scientific RepoRts | (2019) 9:2305 | /s41598-019-38754-w 1 : .
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microbial communities, most of which are phylogenetically distinct from those living in the Earth’s surface envi-
ronments6–9; hence, their physiology and metabolic functions still remain largely unknown10,11.
To gain insight into deep subseafloor microbial life, cultivation is crucial. Previous cultivation efforts on sedi-
ment core samples, however, indicated a high resistance of deeply buried microbial communities to conventional
batch-type cultivation techniques. Consequently, only a small fraction of indigenous deep microbes could be iso-
lated thus far from ≥10 m below seafloor (mbsf) sediment samples, whose members are primarily affiliated with
the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes or Euryarchaeota genera Methanoculleus,
Methanococcus, and Methanosarcina8,12–14. Nevertheless, stable isotope tracer incubation experiments combined
with nanometer-scale secondary ion mass spectrometry (NanoSIMS) analysis confirmed that more than 70%
of the total microbial cells are viable, despite having very slow biomass turnover rates15,16. Thus, cultivation of
deep subseafloor microbes through batch-type techniques may be impeded by their extraordinarily low meta-
bolic activity under energy-limited conditions5 and/or the “substrate-accelerated death” phenomenon, wherein
microbial cells are damaged when suddenly exposed to high substrate concentrations in rich laboratory media17.
Given the limited success of previous efforts to cultivate deep subseafloor microbes, new cultivation
approaches are needed. Parkes et applied a high-pressure anaerobic enrichment system (., DeepIsoBUG)
for gas hydrate-bearing sediments and successfully obtained some anaerobic bacteria (., genera Acetobacterium
and Clostridium). Imachi and co-workers (2011, 2014, 2017)19–21 applied a continuous-flow bioreactor cultiva-
tion technique to overcome the limitation of batch-type cultivation and successfully enriched previously uncul-
tured lineages from deep subseafloor sediments. These studies employed a down-flow hanging sponge (DHS)
reactor system, which was originally developed for treating municipal sewage in developing countries at a low
cost22. Specifically, a polyurethane sponge used in the DHS reactor ensures medium pore space to provide a
larger surface area for microbial colonization and extended cell residence time. Such continuous-flow bioreactor
cultivation can maintain the low concentrations of substrates found in the natural environments and outflow
the accumulated metabolic products that may inhibit microbial growth. These continuous-flow reactors thereby
might increase the culturability of subseafloor microorganisms in a controlled manner and serve as better sources
(incubators) for the isolation of microorganisms than the original samples.
Recently, using a DHS reactor, Inagaki et al .23 established a methanogenic enrichment culture from
~2-km-deep subseafloor coalbed samples obtained using the riser-drilling technology of the deep-sea drilling
vessel Chikyu during the Integrated Ocean Drilling Program (IODP) Expedition 337. In this study, we report the
extensive microbiological and biogeochemical investigations over 1000 days of DHS reactor operation, including
the detailed cultivation procedure, microbial community structure, and microbial metabolism during the course
of the bioreactor operation. We observed that phylogenetically diverse indigenous microbial populations were
cultivated in the bioreactor. The cultivars seemingly grow on and transform coalbed-derived organic matter.
Additionally, three anaerobic microorganisms, including a methanogenic archaeon, were obtained in pure culture
from the bioreactor enrichment culture.
Results
Microbial metabolism during enrichment in the DHS bioreactor. DHS reactor (Fig. 1) operation at
a near in situ temperature of 40 °C over the course of 1029 days yielded effluent at the mean oxidation-reduction
potential (ORP) value of −430 ± 47 mV (n = 478), indicating strict maintenance of anaerobic conditions. The
mean pH of the effluent was ± (Supplementary Fig. S1).
As an indicator of microbial metabolic activity, methane