Evidence for Microbial Carbon and Sulfur Cycling in
Deeply Buried Ridge Flank Basalt
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Citation
Lever, M. A., O. Rouxel, J. C. Alt, N. Shimizu, S. Ono, R. M.
Coggon, W. C. Shanks, et al. “Evidence for Microbial Carbon and
Sulfur Cycling in Deeply Buried Ridge Flank Basalt.” Science
339, no. 6125 (March 15, 2013): 1305–1308.
As Published
http://doi.org/10.1126/science.1229240
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American Association for the Advancement of Science (AAAS)
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Original manuscript
Accessed
Wed May 25 23:38:39 EDT 2016
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http://hdl.handle.net/1721.1/87684
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Evidence for Microbial Carbon and Sulfur Cycling in Deeply
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Buried Ridge Flank Basalt
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Mark A. Lever1,2*, Olivier Rouxel3,4, Jeffrey C. Alt5, Nobumichi Shimizu3, Shuhei
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Ono6, Rosalind M. Coggon7, Wayne C. Shanks, III8, Laura Lapham2,9, Marcus
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Elvert10, Xavier Prieto-Mollar10, Kai-Uwe Hinrichs10, Fumio Inagaki11, and Andreas
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Teske1*
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1
Department of Marine Sciences, University of North Carolina, Chapel Hill, NC
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27516, USA.
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8000 Aarhus C, Denmark.
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Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
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IFREMER, Centre de Brest, 29280 Plouzané, France.
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5
Department of Earth and Environmental Sciences, University of Michigan, Ann
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Arbor, MI 48109, USA.
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of Technology, Cambridge, MA 02139, USA.
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Kensington Campus, London SW7 2AZ, UK.
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U.S. Geological Survey, Denver, CO 80225, USA.
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Present address: University of Maryland Center for Environmental Science,
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Solomons, MD 20688, USA.
Center for Geomicrobiology, Department of BioScience, Aarhus University, DK-
Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute
Department of Earth Science and Engineering, Imperial College London, South
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10
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Marine Environmental Sciences, University of Bremen, D-28334 Bremen, Germany.
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for Marine-Earth Science and Technology, Nankoku, Kochi 783-8502, Japan.
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*Corresponding authors, email: mark.lever@biology.au.dk, teske@email.unc.edu.
Organic Geochemistry Group, Department of Geosciences & MARUM Center for
Geomicrobiology Group, Kochi Institute for Core Sample Research, Japan Agency
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One sentence summary:
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Functional gene, geochemical, and incubation evidence reveal active, methane-
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and sulfur-cycling microbial communities in deep basaltic ocean crust.
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Sediment-covered basalt on ridge flanks constitutes most of Earth’s oceanic
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crust, but the composition and metabolic function of its microbial ecosystem is
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largely unknown. Here, we demonstrate the presence of methane- and sulfur (S)-
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cycling microbes in subseafloor basalt of the eastern flank of the Juan de Fuca
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Ridge. Depth horizons with functional genes indicative of methane-cycling and
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sulfate-reducing microorganisms are enriched in solid-phase S and total organic
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carbon (TOC), host δ 13C- and δ 34S-isotopic values with a biological imprint, and
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show clear signs of microbial activity when incubated in the laboratory.
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Downcore changes in C and S cycling show discrete geochemical intervals with
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chemoautotrophic δ 13C-TOC signatures potentially attenuated by heterotrophic
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metabolism.
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Subseafloor basaltic crust represents the largest habitable zone by volume on
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Earth (1). Chemical reactions of basalt with seawater flowing through fractures
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(veins) release energy that may support chemosynthetic communities. Microbes
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exploiting these reactions are known from basalt exposed at the seafloor, where the
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oxidation of reduced S and iron (Fe) from basalt with dissolved oxygen and nitrate
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from seawater supports high microbial biomass and diversity (2, 3). Multiple lines of
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evidence that include textural alterations (4), depletions in δ34S-pyrite (FeS2) (5) and
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δ13C-dissolved inorganic carbon (DIC) (6), and DNA sequences from borehole
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observatories (7, 8) suggest active microbial communities in subseafloor basalt. Yet,
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direct evidence of these communities is still missing. In this study we combine
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sequencing of genes diagnostic of microbial methane- and S-cycling with
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geochemical and isotopic analyses of C- and S-pools and laboratory-based
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incubations to identify microbial ecosystem components in deep subseafloor basalt.
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The 3.5 million-year-old basement at Hole U1301B was sampled during
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Integrated Ocean Drilling Program (IODP) Expedition 301 in 2004 (Fig. S1) (9). Site
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U1301 is covered by a 265-m thick sediment layer and lies ~2 km south of ODP Site
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1026, which it resembles in temperature profile, lithology, and sediment chemistry
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(9). Given anticipated poor recovery due to brecciation of the upper basement (265-
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350 meters below seafloor; mbsf), coring was restricted to an interval of pillow
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basalts and massive lavas (351-583 mbsf). Sulfate concentrations (~16mM) and vein
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carbonates indicate basalt fluids are derived from seawater, which enters ~80 km
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south at Grizzly Bare outcrop and discharges near U1301B, at Baby Bare and Mama
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Bare outcrops (9, 10; Fig. S1B). Yet, the basement at U1301 differs from seafloor-
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exposed basalt in its uniformly high temperature (~64°C) (9) and lack of fresh
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photosynthesis-derived organic matter, dissolved oxygen and nitrate (7, 11). These
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conditions preclude chemosynthetic S- and Fe-oxidation reactions that require oxygen
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or nitrate as electron acceptors (12), but may enable growth of strict anaerobes, such
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as many methanogens, methane-oxidizing archaea and sulfate reducers.
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By sequencing genes encoding the α subunit of methyl coenzyme M reductase
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(mcrA), a gene unique to methanogens and anaerobic methane-oxidizers (13), and the
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β subunit of dissimilatory sulfite reductase (dsrB), a gene found in sulfate- and
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sulfite-reducing microbes (14), we demonstrate the presence of key genes of methane-
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cycling and sulfate-reducing microbes (Methods in Supporting Online Material). We
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detect mcrA in 5 of the 10 samples and dsrB in 4 of the 6 samples tested (Table S1).
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The phylogenetic diversity of mcrA genes is restricted to two groups: the Juan
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de Fuca Methanogen Group (JdFMG), which falls into an uncultivated cluster within
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the Methanosarcinales, and anaerobic methane-oxidizing archaea (ANME-1; Fig.
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1A). Close relatives of the JdFMG were first reported as ‘Unidentified Rice Field Soil
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mcrA’ from paddy soil (15), and have since also been found in marine habitats,
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including JdF Ridge hydrothermal vent chimneys and seafloor-exposed basalt ~100
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km west of U1301B (Fig. S2) (16-17). Recently, a close relative of JdFMG, which
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uses hydrogen (H2), acetate, methanol and trimethylamine as energy substrates for
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methanogenesis, was enriched in wetland soil (18). Given that substrate use follows
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phylogenetic divisions in mcrA (19), the JdFMG are likely to be methanogens with
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similar substrate requirements. ANME-1 occur widely in marine sediments and
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methane seeps, and are believed to gain energy from the anaerobic oxidation of
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methane (AOM) (20). Two distinct ANME phylotypes occur at U1301, one closely
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related to ANME-1 from methane seeps, and another clustering with only one other
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sequence, from subseafloor sediment (Fig. S3). We detect JdFMG in four, and
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ANME-1 in three out of ten basalt samples. Two samples contain both groups (Table
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S1).
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The phylogenetic diversity of dsrB is limited to one group, the Juan de Fuca
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Sulfate Reducing Group (JdFSRG), which falls into Cluster IV, a deeply-branching
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dsrB cluster without cultured members, first reported from hydrothermal sediment
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(Fig. 1B and S4, Table S1) (21). Remarkably, the only other dsr sequences reported
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so far from the subseafloor, i.e. sediment of the Peru Margin (22), fall into this
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cluster, which is widespread in shallow marine sediment and terrestrial aquifers.
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The study of solid-phase S-pools through analyses of acid-volatile sulfide
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(AVS), chromium-reducible S (CRS), and sulfate-S (SO4-S) provides insights to
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redox processes (5, 23), and shows a relationship with dsrB distributions at U1301:
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dsrB was only found in a relatively reduced ‘intermediate depth interval’ (~430-520
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mbsf, 14R-26R), in samples with AVS as the main S pool in alteration halos (14R-1-
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11) or host rock (17R-170, 20R-1-57, 23R-2-21; Fig. S5, Table S1). Samples from
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this interval have higher AVS, CRS, and total S (Fig. S5, Table S2), contain large
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pyrite fronts (14R-1-65P, 15R-4-142P; Fig. S5), and have lower δ34S-AVS, -CRS, and
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-SO4-S, compared to the more oxidized upper (1R-12R) and lower coring intervals
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(30R-36R; Fig. S6, Table S1). Consistent with higher Fe3+/FeTotal ratios, which
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indicate halos to be more oxidized than host rock (Table S1), pyrite is generally
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absent from halos or veins. Outside the intermediate depth interval, the near absence
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of pyrite from host rock, and mixed clay-Fe-oxyhydroxide-dominated halos and veins
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are further evidence of pervasive oxidative alteration.
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To examine micro- and macroscale variations in microbial S-cycling, the δ34S
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of pyrite grains were analyzed (Tables S1 and S3, Fig. 2). Though variable, the δ34S-
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pyrite grains (-72.4 to 1.2‰; Table S3) are typically lower than those of AVS (-9.3 to
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-0.2‰), CRS (-13.7 to 0‰), SO4-S (-6.5 to 0‰), mantle S (0‰) (5), dissolved sulfate
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in bottom sediments at ODP Site 1026 (+30‰) (24), or seawater (+21‰; Fig. 2).
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Locally, the δ34S of pyrite grains reach very negative values (-72‰), consistent with
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the addition of highly 34S-depleted secondary sulfide to basement rock (23). These
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low δ34S-pyrite values indicate single-step sulfate reduction (25) or repeated cycles of
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sulfate reduction and S oxidation (26). The co-occurrence of low δ34S-pyrite, dsrB,
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and mcrA of ANME-1 in two samples (14R-1-11, 17R-1-70) suggests local coupling
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between methane and S-cycling by sulfate-dependent AOM.
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Depth profiles of TOC content, δ13C-TOC, and δ13C-carbonate at U1301B are
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consistent with functional gene- and 34S-data (Fig. 3). The TOC content is highest in
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the intermediate depth interval in cores with mcrA, dsrB, and low δ34S-pyrite (Fig.
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3A, Table S4). The δ13C-TOC is in the range of dissolved organic C (DOC) in fluids
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from nearby 1026B and Baby Bare Springs (BBS; Fig. 3B, Table S4) and lower than
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seawater DOC (-21.1‰; 6). The δ13C-carbonate is higher than δ13C-DIC at 1026B or
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BBS (Fig. 3C, Table S5) and overlaps with δ13C-DIC of bottom seawater (-1.4‰; 10).
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δ13C-TOC values in the upper coring interval (4R-5R) and near the bottom
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(23R-26R; -34.6 to -32.0‰) are close to δ13C-DOC from nearby BBS (-34.6‰; Fig.
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3B). The absence of O2 and the high 13C-TOC depletion relative to carbonate (~ -30
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to -35‰) suggest C fixation by the reductive acetyl CoA pathway – an anaerobic
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pathway found in all methanogens and acetogens, and certain sulfate and iron
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reducers (Fig. S7, Tables S6 and S7; 27). The presence of dsrB but not mcrA in these
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samples suggests that sulfate reducers or other groups, but not methanogens, produce
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this low δ 13C-TOC.
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δ 13C-TOC at the top (2R) and in the intermediate depth interval (-28.4 to -
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21.6‰) are close to δ13C-DOC from borehole 1026B (-26.1‰, Fig. 3B; 6). The 13C-
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depletion relative to inorganic C is lower than in the other layers (~ -20 to -26‰), but
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also falls in the range of the reductive acetyl CoA pathway (Table S7), and, consistent
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with mcrA detection, could be impacted by autotrophic methanogenesis. In addition,
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elevated heterotrophic activity is possible, since degradation of chemoautotrophy-
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derived OC, e.g. by AOM, methanogenesis or fermentation, would lower the δ13C-
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carbonate and potentially raise the δ13C-TOC. The alternative explanation, enhanced
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breakdown of photosynthesis-derived OC in the intermediate depth interval, is
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unlikely given that sediment inclusions are absent (9). Similarly, influx of labile DOC
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or unaltered DIC from seawater is incompatible with the 7-11 kyr greater DOC age
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compared to bottom seawater and the 4-8‰ decrease in δ13C-DIC along the flowpath
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from Grizzly Bare outcrop to 1026B and BBS, respectively (6, 10).
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To rule out a fossil origin of functional genes, C and S compounds in subseafloor
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basalt, we incubated pieces from the interior of three rocks used for functional gene
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analyses (1R-1-79, 14R-1-11, 23R-2-21) at 65°C in anoxic, sulfate-rich media
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containing H2, acetate, methanol, and dimethyl sulfide as energy substrates (Table
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S8). After two years, aliquots were transferred to fresh media, and incubated for
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another five years using triple-autoclaved basalt pieces as substrata. By then, low
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concentrations of 13C-depleted methane (-54 to -65‰) had formed indicating the
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presence of active methanogenic microorganisms (Table S9).
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The variability in δ34S-pyrite, δ13C-TOC and δ13C-carbonate indicates that
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micro- and macro-scale geochemical changes related to mineralogy, fracturing and/or
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fluid flow strongly influence microbial activity. These chemical microniches may
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explain the coexistence of sulfate reducers and methanogens at U1301 and in other
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igneous habitats, despite higher energy yields of sulfate reduction compared to
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methanogenesis (28). In addition, some methanogens can survive in the presence of
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sulfate reducers by consuming non-competitive methyl substrates (19); this
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explanation is consistent with the ability of a close relative of JdFMG to use methanol
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(18), and the production of biogenic methane in basalt incubations containing sulfate
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and methanol (Table S9, Fig. S8). The sources of energy substrates to sulfate reducers
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and methanogens at U1301B remain unknown. A potential source of H2 is the
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oxidative alteration of Fe or S minerals, while short-chain organic acids and alcohols
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might be produced by breakdown of TOC (19, 28-29) or Fischer-Tropsch-type
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synthesis (30).
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Samples from the eastern flank of the Juan de Fuca Ridge provide the first
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integrated view of microbial community structure and activity in subseafloor basalt.
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Our analyses of functional gene, geochemical, and isotopic data reveal strong micro-
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and macroscale heterogeneity of microbial C- and S-cycling, and indicate a key role
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of chemoautotrophy in supporting Earth’s oceanic crustal biosphere.
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Acknowledgments. We thank B. B. Jørgensen, M. L. Sogin and the IODP Expedition
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301 Scientists. Funding was obtained from a Schlanger Ocean Drilling Fellowship, a
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University of North Carolina Dissertation Completion Fellowship, a Marie-Curie
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Intra-European Fellowship (#255135; all to M. A. Lever), the Danish National
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Research Foundation, the Max Planck Society (both to B. B. Jørgensen), Europole
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Mer (to O. Rouxel), the European Research Council Advanced Grant DARCLIFE (to
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K.-U. Hinrichs), the NASA Astrobiology Institute Subsurface Biospheres (to A.
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Teske), the Japan Society for the Promotion of Science (JSPS) Funding Program for
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Next Generation World-Leading Researchers (NEXT Program; to F. Inagaki), and the
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National Science Foundation (NSF-OCE 0622949 and OCE 1129631 to J. Alt, OCE-
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0753126 to S. Ono and O. Rouxel, and NSF-ODP 0727175 and NSF-STC for Dark
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Energy Biosphere Investigations to A. Teske). The geochemical data are fully
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documented and available in the Supplementary Tables. The functional gene sequence
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data are available from Genbank database.
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FIGURES
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Figure 1. Phylogenetic trees of functional genes. (A) McrA sequences from U1301B
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are in bold magenta type face. Close relatives based on microarray analyses of JdF
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Ridge hydrothermal vent chimneys and seafloor basalt are in green (16) and cyan
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(17), respectively. (B) DsrB sequences from U1301B are in bold magenta type face,
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and sequences from subseafloor sediment off Peru in cyan (22). Bootstrap support (in
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%, 1,000 replications) is indicated at each branching point.
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Figure 2. Macro-and micro-scale distribution of S-isotopic data. On the left, δ34S-
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depth profile of pyrite granules, analyzed by laser ablation and secondary ion mass
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spectrometry (SIMS), and bulk S pools (AVS, CRS). On the right, thin section
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micrograph showing individual pyrite granules and their δ34S. The dashed magenta
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line indicates the sampling depth of the thin section. The dashed black lines mark the
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intermediate depth interval. Pyrite grains with a sufficient diameter for δ34S-
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determination (10 µm) were limited to this interval. The scale bar is 200 µm.
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Figure 3. Depth-related trends in (A) TOC content, (B) δ13C-TOC, and (C) δ13C-
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carbonate. Cores with functional gene detection are indicated in A and B. Dashed
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vertical lines indicate δ13C-DOC (B) and –DIC (C) values from 1026B and BBS.
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Because the carbonate content of rock samples used in (A) and (B) was too low for
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analyses, δ13C from carbonate veins are shown in (C). The reduced intermediate depth
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interval falls between the dashed horizontal lines. All δ13C are in ‰ vs. VPDB.