• Energy Research

Here, we demonstrate that wood-rot fungi produce methane anaerobically without the involvement of methanogenic archaea via a new, halomethane-dependent pathway. These findings of an anaerobic fungal methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Sulfate reduction is the predominant anaerobic microbial process of organic matter mineralization in marine sediments, with recent studies revealing that sulfate reduction not only occurs in sulfate-rich sediments, but even extends to deeper, methanogenic sediments at very low background concentrations of sulfate. Using samples retrieved off the Shimokita Peninsula, Japan, during the Integrated Ocean Drilling Program (IODP) Expedition 337, we measured potential sulfate reduction rates by slurry incubations with 35S-labeled sulfate in deep methanogenic sediments between 1276.75 and 2456.75 meters below the seafloor. Potential sulfate reduction rates were generally extremely low (mostly below 0.1 pmol cm-3 d-1) but showed elevated values (up to 1.8 pmol cm-3 d-1) in a coal-bearing interval (Unit III). A measured increase in hydrogenase activity in the coal-bearing horizons coincided with this local increase in potential sulfate reduction rates. This paired enzymatic response suggests that hydrogen is a potentially important electron donor for sulfate reduction in the deep coalbed biosphere. By contrast, no stimulation of sulfate reduction rates was observed in treatments where methane was added as an electron donor. In the deep coalbeds, small amounts of sulfate might be provided by a cryptic sulfur cycle. The isotopically very heavy pyrites (δ34S = +43‰) found in this horizon is consistent with its formation via microbial sulfate reduction that has been continuously utilizing a small, increasingly 34S-enriched sulfate reservoir over geologic time scales. Although our results do not represent in-situ activity, and the sulfate reducers might only have persisted in a dormant, spore-like state, our findings show that organisms capable of sulfate reduction have survived in deep methanogenic sediments over more than 20 Ma. This highlights the ability of sulfate-reducers to persist over geological timespans even in sulfate-depleted environments. Our study moreover represents the deepest evidence of a potential for sulfate reduction in marine sediments to date.

m CSF = depth of Core below Sea Floor in meters / m CSF-A: Distance from sea floor to sample within recovered core. This scale allows overlap at core and section boundaries. /m CSF-B: Distance from sea floor to sample within recovered core is compressed, if core recovery > 100%.

In subsurface anoxic environments, microbial communities generally produce methane as an end-product to consume organic compounds. This metabolic function is a source of biogenic methane in coastal natural gas aquifers, submarine mud volcanoes, and methane hydrates. Within the methanogenic communities, hydrogenotrophic methanogens, and syntrophic bacteria are converting volatile fatty acids to methane syntrophically via interspecies hydrogen transfer. Recently, direct interspecies electron transfer (DIET) between fermentative/syntrophic bacteria and electrotrophic methanogens has been proposed as an effective interspecies metabolite transfer process to enhance methane production. In this study, in order to stimulate the DIET-associated methanogenic process at deep biosphere-aquifer systems in a natural gas field, we operated a bioelectrochemical system (BES) to apply voltage between an anode and a cathode. Two single-chamber BESs were filled with seawater-based formation water collected from an onshore natural gas well, repeatedly amended with acetate, and operated with 600 mV between electrodes for 21 months, resulting in a successful conversion of acetate to methane via electrical current consumption. One reactor yielded a stable current of ~200 mA/m2 with a coulombic efficiency (CE) of >90%; however, the other reactor, which had been incidentally disconnected for 3 days, showed less electromethanogenic activity with a CE of only ~10%. The 16S rRNA gene-based community analyses showed that two methanogenic archaeal families, Methanocalculaceae and Methanobacteriaceae, were abundant in cathode biofilms that were mainly covered by single-cell-layered biofilm, implicating them as key players in the electromethanogenesis. In contrast, family Methanosaetaceae was abundant at both electrodes and the electrolyte suspension only in the reactor with less electromethanogenesis, suggesting this family was not involved in electromethanogenesis and became abundant only after the no-electron-flow event. The anodes were covered by thick biofilms with filamentous networks, with the family Desulfuromonadaceae dominating in the early stage of the operation. The family Geobacteraceae (mainly genus Geoalkalibacter) became dominant during the longer-term operation, suggesting that these families were correlated with electrode-respiring reactions. These results indicate that the BES reactors with voltage application effectively activated a subsurface DIET-related methanogenic microbiome in the natural gas field, and specific electrogenic bacteria and electromethanogenic archaea were identified within the anode and/or cathode biofilms.

Over the past few decades, the subseafloor biosphere has been explored by scientific ocean drilling to depths of about 2.5km below the seafloor. Although organic-rich anaerobic sedimentary habitats in the ocean margins harbor large numbers of microbial cells, microbial populations in ultraoligotrophic aerobic sedimentary habitats in the open ocean gyres are several orders of magnitude less abundant. Despite advances in cultivation-independent molecular ecological techniques, exploring the low-biomass environment remains technologically challenging, especially in the deep subseafloor biosphere. Reviewing the historical background of deep-biosphere analytical methods, the importance of obtaining clean samples and tracing contamination, as well as methods for detecting microbial life, technological aspects of molecular microbiology, and detecting subseafloor metabolic activity will be discussed.

SummaryAlthough subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal‐associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal‐sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ∼20 million years of depositional history.

Abstract Subseafloor sedimentary environments harbor a remarkable number of microorganisms that constitute anaerobic and aerobic microbial ecosystems beneath the ocean margins and open-ocean gyres, respectively. Microbial biomass and diversity richness generally decrease with increasing sediment depth and burial time. However, there has been a long-standing debate over the contribution and distribution of Archaea in the subseafloor sedimentary biosphere. Here we show the global quantification of archaeal and bacterial 16S rRNA genes in 221 sediment core samples obtained from diverse oceanographic settings through scientific ocean drilling using microfluidic digital PCR. We estimated that archaeal cells constitute 37.3% of the total microbial cells (40.0% and 12.8% in the ocean margin and open-ocean sites, respectively), corresponding to 1.1 × 1029 cells on Earth. In addition, the relative abundance of archaeal 16S rRNA genes generally decreased with the depth of water in the overlying sedimentary habitat, suggesting that Archaea may be more sensitive to nutrient quality and quantity supplied from the overlying ocean.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells (Parkes et al., 1994, doi:10.1038/371410a0; D'Hondt et al., 2004, doi:10.1126/science.1101155). Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (Parkes et al., 1994, doi:10.1038/371410a0) and 303 Pg (Whitman et al., 1998) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem (Schippers et al., doi:10.1038/nature03302; Inagaki et al., doi:10.1073/pnas.0511033103 ; Mauclaire et al., doi:10.1111/j.1472-4677.2004.00035.x; Biddle et al., doi:10.1073/pnas.0600035103). Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells (Biddle et al., doi:10.1073/pnas.0600035103; Sturt et al., 2004, doi:10.1002/rcm.1378), are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

Significance Microbial cells are widespread in diverse deep subseafloor environments; however, the viability, growth, and ecophysiology of these low-abundance organisms are poorly understood. Using single-cell–targeted stable isotope probing incubations combined with nanometer-scale secondary ion mass spectrometry, we measured the metabolic activity and generation times of thermally adapted microorganisms within Miocene-aged coal and shale bed samples collected from 2 km below the seafloor during Integrated Ocean Drilling Program Expedition 337. Microorganisms from the shale and coal were capable of metabolizing methylated substrates, including methylamine and methanol, when incubated at their in situ temperature of 45 °C, but had exceedingly slow growth, with biomass generation times ranging from less than a year to hundreds of years as measured by the passive tracer deuterated water.

SignificanceMarine sediment covers 70% of Earth’s surface and harbors as much biomass as seawater. However, the global taxonomic diversity of marine sedimentary communities, and the spatial distribution of that diversity remain unclear. We investigated microbial composition from 40 globally distributed sampling locations, spanning sediment depths of 0.1 to 678 m. Statistical analysis reveals that oxygen presence or absence and organic carbon concentration are key environmental factors for defining taxonomic composition and diversity of marine sedimentary communities. Global marine sedimentary taxonomic richness predicted by species–area relationship models is 7.85 × 103to 6.10 × 105for Archaea and 3.28 × 104to 2.46 × 106for Bacteria as amplicon sequence variants, which is comparable to the richness in seawater and that in topsoil.