Sediment-covered basalt on the flanks of mid-ocean ridges constitutes most of Earth's oceanic crust, but the composition and metabolic function of its microbial ecosystem are largely unknown. By drilling into 3.5-million-year-old subseafloor basalt, we demonstrated the presence of methane-and sulfurcycling microbes on the eastern flank of the Juan de Fuca Ridge. Depth horizons with functional genes indicative of methane-cycling and sulfate-reducing microorganisms are enriched in solid-phase sulfur and total organic carbon, host delta C-13-and delta S-34-isotopic values with a biological imprint, and show clear signs of microbial activity when incubated in the laboratory. Downcore changes in carbon and sulfur cycling show discrete geochemical intervals with chemoautotrophic delta C-13 signatures locally attenuated by heterotrophic metabolism. Main text Subseafloor basaltic crust represents the largest habitable zone by volume on Earth (1). Chemical reactions of basalt with seawater flowing through fractures release energy that may support chemosynthetic communities. Microbes exploiting these reactions are known from basalt exposed at the seafloor, where the oxidation of reduced sulfur (S) and iron (Fe) from basalt with dissolved oxygen and nitrate from seawater supports high microbial biomass and diversity (2, 3). Multiple lines of indirect evidence that include textural alterations (4), depletions in δ 34 S-pyrite (FeS 2) (5) and δ 13 C-dissolved inorganic carbon (DIC) (6), and DNA sequences from

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 M coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.

Dissolved methane concentrations in the ocean are close to equilibrium with the atmosphere. Because methane is only sparingly soluble in seawater, measuring it without contamination is challenging for samples collected and processed in the presence of air. Several methods for analyzing dissolved methane are described in the literature, yet none has conducted a thorough assessment of the method yield, contamination issues during collection, transport and storage, and the effect of temperature changes and preservative. Previous extraction methods transfer methane from water to gas by either a "sparge and trap" or a "headspace equilibration" technique. The gas is then analyzed for methane by gas chromatography. Here, we revisit the headspace equilibration technique and describe a simple, inexpensive, and reliable method to measure methane in fresh and seawater, regardless of concentration. Within the range of concentrations typically found in surface seawaters (2-1000 nmol L -1 ), the yield of the method nears 100% of what is expected from solubility calculation following the addition of known amount of methane. In addition to being sensitive (detection limit of 0.1 ppmv, or 0.74 nmol L -1 ), this method requires less than 10 min per sample, and does not use highly toxic chemicals. It can be conducted with minimum materials and does not require the use of a gas chromatograph at the collection site. It can therefore be used in various remote working environments and conditions.

[1] Down core concentration gradients of dissolved methane and sulfate; isotope gradients of methane, dissolved inorganic carbon, and authigenic carbonate; and organic matter elemental ratios are incorporated into a vent evolution model to describe spatial and temporal variability of sedimentary microbial activity overlying acoustic wipeout zones at Mississippi Canyon (MC) 118, Gulf of Mexico. We tested the hypothesis that these zones indicate areas where sediments are exposed to elevated fluid flux and therefore should contain saturated methane concentrations and enhanced microbial activity from sulfate reduction (SR), anaerobic oxidation of methane (AOM), and methanogenesis (MP). Thirty surficial cores (between 22 and 460 cm deep) were collected from sediments overlying and outside the wipeout zones and analyzed for pore water and solid phase constituents. Outside the wipeout zones, sulfate and methane concentrations were similar to overlying-water values and did not vary with depth; indicating low microbial activity. Above the wipeouts, nine cores showed moderate activity with gently sloping sulfate and methane concentration gradients, methane concentrations <20 mM, and isotope depth gradients indicative of organic matter oxidation. In stark contrast to this moderate activity, four cores showed high microbial activity where sulfate concentrations were depleted by $50 cm below seafloor, maximum methane concentrations in the decompressed cores were above 4 mM, and down core profiles of d 13 C-CH 4 and d 13 C-dissolved inorganic carbon (DIC) indicated distinct depth zones of SR, AOM, and MP. Bulk organic matter analysis suggested that the high activity was supported by an organic source that was enriched in carbon (C:N $15) and depleted in d 15 N and d 13 C compared to other activity groups, possibly due to the influx of petroleum or chemosynthetically fixed carbon. Within high activity cores, the d 13 C-DIC values were similar to the d 13 C-CaCO 3 values, a result expected for authigenic carbonate recently precipitated. However, these values were dissimilar in moderate activity cores, suggesting that microbial activity was higher in the past. This study provides evidence that the fluid flux at MC 118 varies over time and that the microbial activity responds to such variability. It also suggests that sediments overlying wipeout zones are not always saturated with respect to methane, which has implications for the formation and detection of gas hydrate.

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists—all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earth's global biogeochemical cycles, and for understanding how microorganisms in these “extreme” environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) “theme team” on microbial activity (www.darkenergybiosphere.org).

The Chesapeake Bay is a eutrophic estuary that undergoes seasonal bottom water hypoxia. Methane (CH 4 ) gas bubbles are found within the upper sediment layers, but whether this greenhouse gas escapes to the water column or the atmosphere is not well known. Here, we hypothesize that when bottom waters become anoxic, CH 4 is released from the sediments, builds up under the pycnocline, and escapes to the atmosphere at the end of hypoxia. Osmotically powered pumps (OsmoSamplers) were used for the first time to test this hypothesis.

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