Global diversity of microbial communities in marine sediment Tatsuhiko Hoshinoa,1, Hideyuki Doib,1, Go-Ichiro Uramotoa,2, Lars Wörmerc,d, Rishi R. Adhikaric,d, Nan Xiaoa,e, Yuki Moronoa, Steven D’Hondtf, Kai-Uwe Hinrichsc,d, and Fumio Inagakia,e,1 aKochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Nankoku, 783-8502 Kochi, Japan; bGraduate School of Simulation Studies, University of Hyogo, Kobe, 650-0047 Hyogo, Japan; cMARUM-Center for Marine Environmental Sciences and Faculty of Geosciences, University of Bremen, 28359 Bremen, Germany; dDepartment of Geosciences, University of Bremen, 28359 Bremen, Germany; eMantle Drilling Promotion Office, Institute for Marine-Earth Exploration and Engineering, JAMSTEC, Yokohama, Kanagawa 236-0001, Japan; and fGraduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882 Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved September 4, 2020 (received for review November 17, 2019) Microbial life in marine sediment contributes substantially to biomass, even from 2-km-deep anoxic Miocene sediment (9–12) global biomass and is a crucial component of the Earth system. and 101.5-Ma oxic sediment. Subseafloor sediment includes both aerobic and anaerobic micro- Factors that may limit the deep sedimentary biosphere are not bial ecosystems, which persist on very low fluxes of bioavailable restricted to scarcity of nutrients and scarcity of energy-yielding energy over geologic time. However, the taxonomic diversity of substrates. Limiting factors may also include temperature, pres- the marine sedimentary microbial biome and the spatial distribu- sure, pH, salinity, water availability, sediment porosity, or per- tion of that diversity have been poorly constrained on a global meability. For example, without fluid transport in sediment, scale. We investigated 299 globally distributed sediment core dispersal of subseafloor microbial cells may be limited to diffu- samples from 40 different sites at depths of 0.1 to 678 m below sive transport since it is unlikely that proton pump-driven fla- the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gellar motility can occur under such low energy flux (13). In this gene sequences using consistent clean subsampling and experi- case, transport may only reach 6 m over 1 million years, even in mental procedures, which enabled accurate and unbiased compar- very porous layers (8, 14, 15). Recent studies of community ison of all samples. Statistical analysis reveals significant correlations compositional relationships between shallow sediment and sea- between taxonomic composition, sedimentary organic carbon con- water have demonstrated that deep-subseafloor sediment is MICROBIOLOGY centration, and presence or absence of dissolved oxygen. Extrapo- populated by descendants of seafloor-sediment communities, – lation with two fitted species area relationship models indicates which become predominant through preferential survival as the taxonomic richness in marine sediment to be 7.85 × 103 to 6.10 × 5 × 4 × 6 communities are buried over thousands to hundreds of thou- 10 and 3.28 10 to 2.46 10 amplicon sequence variants for sands of years (16–18). Some deeply buried cells may later be Archaea and Bacteria, respectively. This richness is comparable introduced to the ocean through fluid transport (flow through to the richness in topsoil and the richness in seawater, indicat- faults, mud volcanism, and hydrocarbon seepage) at plate- ing that Bacteria are more diverse than Archaea in Earth’s global convergent margins, being dispersed as “deep-biosphere seeds” biosphere. subseafloor life | microbial diversity | marine sediment Significance ’ ver the last two decades, scientists have explored the nature Marine sediment covers 70% of Earth s surface and harbors as Oand extent of subseafloor life through scientific ocean much biomass as seawater. However, the global taxonomic drilling in various oceanographic settings. The total number of diversity of marine sedimentary communities, and the spatial microbial cells in marine sediment is presently estimated as 2.9 × distribution of that diversity remain unclear. We investigated microbial composition from 40 globally distributed sampling 1029 to 5.4 × 1029 cells, accounting for 0.18 to 3.6% of Earth’s locations, spanning sediment depths of 0.1 to 678 m. Statistical total living biomass (1, 2). The abundance of microbes in marine analysis reveals that oxygen presence or absence and organic sediment generally decreases with increasing depth and in- carbon concentration are key environmental factors for defin- creasing sediment age (1, 3). Cell concentrations are usually ing taxonomic composition and diversity of marine sedimen- orders of magnitude higher in the organic-rich anoxic sediment tary communities. Global marine sedimentary taxonomic of continental margins than in the organic-poor oxic sediment of richness predicted by species–area relationship models is 7.85 × the open ocean (4). A recent study used microfluidic digital PCR 103 to 6.10 × 105 for Archaea and 3.28 × 104 to 2.46 × 106 for to estimate that archaeal cells constitute 37.3% of all marine Bacteria as amplicon sequence variants, which is comparable to sedimentary cells, with notably higher percentages of archaeal the richness in seawater and that in topsoil. cells in ocean-margin sediment than in open-ocean sediment (40.0% and 12.8%, respectively; ref. 5). Another recent study Author contributions: T.H., K.-U.H., and F.I. designed research; T.H., H.D., G.-I.U., L.W., estimates that 2.5 × 1028 to 1.9 × 1029 bacterial endospores (4.6 R.R.A., N.X., Y.M., and F.I. performed research; T.H. and H.D. analyzed data; and T.H., to 35 Pg of biomass carbon) exist in the uppermost kilometer of H.D., S.D., and F.I. wrote the paper. marine sediment (6). The authors declare no competing interest. Profiles of cell counts and porewater chemistry indicate that This article is a PNAS Direct Submission. microbial activity in subseafloor sediment is usually extraordi- This open access article is distributed under Creative Commons Attribution-NonCommercial- × −18 NoDerivatives License 4.0 (CC BY-NC-ND). narily low, with mean respiration rates ranging from 2.8 10 1 × −14 To whom correspondence may be addressed. Email: [email protected], hideyuki. to 1.1 10 moles of electrons per cell per year, depending on [email protected], or [email protected]. the availability of electron donors and acceptors (4, 7, 8). Single 2Present address: Center for Advanced Marine Core Research, Kochi University, Nankoku, cell-targeted, stable isotope-probing incubation and nanometer- 783-8502 Kochi, Japan. scale secondary ion mass spectrometry have shown that most This article contains supporting information online at https://www.pnas.org/lookup/suppl/ microbial cells in samples of subseafloor sediment can assimilate doi:10.1073/pnas.1919139117/-/DCSupplemental. a diverse range of carbon and nitrogen compounds into cellular First published October 19, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1919139117 PNAS | November 3, 2020 | vol. 117 | no. 44 | 27587–27597 Downloaded by guest on September 25, 2021 (19–22). Based on mathematical models of energy availability understand microbial taxonomic richness and diversity, the same and microbial processes, the energy required to simply survive, primer sets and sequencing technology were used throughout the rather than to grow, comprises a substantial component of the study, including negative experimental controls. total power consumed by subseafloor-sedimentary communities Archaea-specific, Bacteria-specific, and universal primers (23–25). were used to amplify 16S rRNA gene sequences from 236, 299, Despite the ecological and evolutionary significance of marine and 287 sediment samples, respectively (Materials and Methods). sedimentary life, the spatial distribution and environmental Total samples amplified with the Archaea-specific primer and constraints of microbial diversity in marine sediment are poorly with the universal primer are fewer than 299 because those delineated, in part due to limited microbiological sampling of primer sets did not yield amplicons from several samples. After subseafloor sediment and partly due to use of different quality filtering the raw data, totals of 12.5, 16.0, and 18.6 million se- controls and different analytical protocols by different studies quence reads were obtained using the Archaea-specific, (26–28). Despite these limitations, studies of 16S ribosomal Bacteria-specific, and universal primers, respectively. The data- RNA (rRNA) gene sequences have demonstrated that 1) diverse sets obtained with these primer sets are referred to in the fol- bacterial and archaeal taxa are ubiquitous in organic-rich anoxic lowing sections as “Archaea,”“Bacteria,” and “Universal.” sediment, 2) microbial communities are stratified by sediment depth, and 3) geochemical and sedimentological properties in- Taxonomic Composition of Archaeal Communities. The taxonomic fluence microbial community composition (10, 28–36). In addi- composition of the archaeal community in anoxic subseafloor tion, single-cell genomic, metagenomic, and functional gene sediment differs markedly from that in oxic subseafloor sediment analyses have demonstrated that predominant bacterial and ar- (Fig. 2 A and C and SI Appendix, Fig. S1). Fig. 2A shows the chaeal taxa (e.g., members of the Atribacteria and Bathy- results of the Archaea-sequencing library.