Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

Methanogen activity and microbial diversity in Gulf of Cádiz mud volcano sediments.

The Gulf of Cádiz is a tectonically active continental margin with over sixty mud volcanoes (MV) documented, some associated with active methane (CH4) seepage. However, the role of prokaryotes in influencing this CH4 release is largely unknown. In two expeditions (MSM1-3 and JC10) seven Gulf of Cádiz MVs (Porto, Bonjardim, Carlos Ribeiro, Captain Arutyunov, Darwin, Meknes, and Mercator) were analyzed for microbial diversity, geochemistry, and methanogenic activity, plus substrate amended slurries also measured potential methanogenesis and anaerobic oxidation of methane (AOM). Prokaryotic populations and activities were variable in these MV sediments reflecting the geochemical heterogeneity within and between them. There were also marked differences between many MV and their reference sites. Overall direct cell numbers below the SMTZ (0.2-0.5 mbsf) were much lower than the general global depth distribution and equivalent to cell numbers from below 100 mbsf. Methanogenesis from methyl compounds, especially methylamine, were much higher than the usually dominant substrates H2/CO2 or acetate. Also, CH4 production occurred in 50% of methylated substrate slurries and only methylotrophic CH4 production occurred at all seven MV sites. These slurries were dominated by Methanococcoides methanogens (resulting in pure cultures), and prokaryotes found in other MV sediments. AOM occurred in some slurries, particularly, those from Captain Arutyunov, Mercator and Carlos Ribeiro MVs. Archaeal diversity at MV sites showed the presence of both methanogens and ANME (Methanosarcinales, Methanococcoides, and ANME-1) related sequences, and bacterial diversity was higher than archaeal diversity, dominated by members of the Atribacterota, Chloroflexota, Pseudomonadota, Planctomycetota, Bacillota, and Ca. "Aminicenantes." Further work is essential to determine the full contribution of Gulf of Cádiz mud volcanoes to the global methane and carbon cycles.

Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano, Eastern Mediterranean Sea.

Submarine mud volcanoes are a significant source of methane to the atmosphere. The Napoli mud volcano, situated in the brine-impacted Olimpi Area of the Eastern Mediterranean Sea, emits mainly biogenic methane particularly at the centre of the mud volcano. Temperature gradients support the suggestion that Napoli is a cold mud volcano with moderate fluid flow rates. Biogeochemical and molecular genetic analyses were carried out to assess the methanogenic activity rates, pathways and diversity in the hypersaline sediments of the centre of the Napoli mud volcano. Methylotrophic methanogenesis was the only significant methanogenic pathway in the shallow sediments (0-40 cm) but was also measured throughout the sediment core, confirming that methylotrophic methanogens could be well adapted to hypersaline environments. Hydrogenotrophic methanogenesis was the dominant pathway below 50 cm; however, low rates of acetoclastic methanogenesis were also present, even in sediment layers with the highest salinity, showing that these methanogens can thrive in this extreme environment. PCR-DGGE and methyl coenzyme M reductase gene libraries detected sequences affiliated with anaerobic methanotrophs (mainly ANME-1) as well as Methanococcoides methanogens. Results show that the hypersaline conditions in the centre of the Napoli mud volcano influence active biogenic methane fluxes and methanogenic/methylotrophic diversity.

Prokaryotic cells of the deep sub-seafloor biosphere identified as living bacteria.

Chemical analyses of the pore waters from hundreds of deep ocean sediment cores have over decades provided evidence for ongoing processes that require biological catalysis by prokaryotes. This sub-seafloor activity of microorganisms may influence the surface Earth by changing the chemistry of the ocean and by triggering the emission of methane, with consequences for the marine carbon cycle and even the global climate. Despite the fact that only about 1% of the total marine primary production of organic carbon is available for deep-sea microorganisms, sub-seafloor sediments harbour over half of all prokaryotic cells on Earth. This estimation has been calculated from numerous microscopic cell counts in sediment cores of the Ocean Drilling Program. Because these counts cannot differentiate between dead and alive cells, the population size of living microorganisms is unknown. Here, using ribosomal RNA as a target for the technique known as catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH), we provide direct quantification of live cells as defined by the presence of ribosomes. We show that a large fraction of the sub-seafloor prokaryotes is alive, even in very old (16 million yr) and deep (> 400 m) sediments. All detectable living cells belong to the Bacteria and have turnover times of 0.25-22 yr, comparable to surface sediments.

Deep sub-seafloor prokaryotes stimulated at interfaces over geological time.

The sub-seafloor biosphere is the largest prokaryotic habitat on Earth but also a habitat with the lowest metabolic rates. Modelled activity rates are very low, indicating that most prokaryotes may be inactive or have extraordinarily slow metabolism. Here we present results from two Pacific Ocean sites, margin and open ocean, both of which have deep, subsurface stimulation of prokaryotic processes associated with geochemical and/or sedimentary interfaces. At 90 m depth in the margin site, stimulation was such that prokaryote numbers were higher (about 13-fold) and activity rates higher than or similar to near-surface values. Analysis of high-molecular-mass DNA confirmed the presence of viable prokaryotes and showed changes in biodiversity with depth that were coupled to geochemistry, including a marked community change at the 90-m interface. At the open ocean site, increases in numbers of prokaryotes at depth were more restricted but also corresponded to increased activity; however, this time they were associated with repeating layers of diatom-rich sediments (about 9 Myr old). These results show that deep sedimentary prokaryotes can have high activity, have changing diversity associated with interfaces and are active over geological timescales.

Subsurface microbiology and biogeochemistry of a deep, cold-water carbonate mound from the Porcupine Seabight (IODP Expedition 307).

The Porcupine Seabight Challenger Mound is the first carbonate mound to be drilled (approximately 270 m) and analyzed in detail microbiologically and biogeochemically. Two mound sites and a non-mound Reference site were analyzed with a range of molecular techniques [catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), quantitative PCR (16S rRNA and functional genes, dsrA and mcrA), and 16S rRNA gene PCR-DGGE] to assess prokaryotic diversity, and this was compared with the distribution of total and culturable cell counts, radiotracer activity measurements and geochemistry. There was a significant and active prokaryotic community both within and beneath the carbonate mound. Although total cell numbers at certain depths were lower than the global average for other subseafloor sediments and prokaryotic activities were relatively low (iron and sulfate reduction, acetate oxidation, methanogenesis) they were significantly enhanced compared with the Reference site. In addition, there was some stimulation of prokaryotic activity in the deepest sediments (Miocene, > 10 Ma) including potential for anaerobic oxidation of methane activity below the mound base. Both Bacteria and Archaea were present, with neither dominant, and these were related to sequences commonly found in other subseafloor sediments. With an estimate of some 1600 mounds in the Porcupine Basin alone, carbonate mounds may represent a significant prokaryotic subseafloor habitat.

Active and diverse viruses persist in the deep sub-seafloor sediments over thousands of years.

Viruses are ubiquitous and cause significant mortality in marine bacterial and archaeal communities. Little is known about the role of viruses in the sub-seafloor biosphere, which hosts a large fraction of all microbes on Earth. We quantified and characterized viruses in sediments from the Baltic Sea. The results show that the Baltic Sea sub-seafloor biosphere harbors highly abundant viruses with densities up to 1.8 × 1010 viruses cm-3. High potential viral production down to 37 meters below seafloor in ca. 6000-years-old sediments and infected prokaryotic cells visible by transmission electron microscopy demonstrate active viral infection. Morphological and molecular data indicate that the highly diverse community of viruses includes both allochthonous input from the overlying seawater and autochthonous production. The detection of cyanophage-like sequences showed that viruses of phototrophic hosts may persist in marine sediments for thousands of years. Our results imply that viruses influence sub-seafloor microbial community dynamics and thereby affect biogeochemical processes in the sub-seafloor biosphere.

Prokaryotic biodiversity and activity in the deep subseafloor biosphere.

The deep subseafloor biosphere supports a diverse population of prokaryotes belonging to the Bacteria and Archaea. Most of the taxonomic groups identified by molecular methods contain mainly uncultured phylotypes. Despite this several cultured strains have been isolated from this habitat, but they probably do not represent the majority of the population. Evidence is starting to suggest that some of the activities measured, such as sulphate reduction and methanogenesis, reflected in geochemical profiles, are carried out by a small subset of the community detected by molecular methods. It is further possible that heterotrophy may be the most important mode of metabolism in subsurface sediments and heterotrophic microorganisms could dominate the uncultured prokaryotic population. Although, heterotrophy is limited by the increasing recalcitrance of organic matter with depth, this may be counteracted by thermal activation of buried organic matter providing additional substrates at depth.

Prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin.

The community compositions of Bacteria and Archaea were investigated in deep, sub-seafloor sediments from the highly productive Peru Margin (ODP Leg 201, sites 1228 and 1229, c. 25 km apart) down to nearly 200 m below the seafloor using taxonomic (16S rRNA) and functional (mcrA and dsrA) gene markers. Bacterial and archaeal groups identified from clone libraries of 16S rRNA gene sequences at site 1229 agreed well with sequences amplified from bands excised from denaturing gradient gel electrophoresis (DGGE) depth profiles, with the exception of the Miscellaneous Crenarchaeotic Group (MCG). This suggested that the prokaryotic community at site 1228, obtained from DGGE profiling alone, was reliable. Sites were dominated by Bacteria in the Gammaproteobacteria, Chloroflexi (green non-sulphur bacteria) and Archaea in the MCG and South African Gold Mine Euryarchaeotic Group, although community composition changed with depth. The candidate division JS1 was present throughout both sites but was not dominant. The populations identified in the Peru Margin sediments consisted mainly of prokaryotes found in other deep subsurface sediments, and were more similar to communities from the Sea of Okhotsk (pelagic clays) than to those from the low organic carbon Nankai Trough sediments. Despite broad similarities in the prokaryotic community at the two sites, there were some differences, as well as differences in activity and geochemistry. Methanogens (mcrA) within the Methanosarcinales and Methanobacteriales were only found at site 1229 (4 depths analysed), whereas sulphate-reducing prokaryotes (dsrA) were only found at site 1228 (one depth), and these terminal-oxidizing prokaryotes may represent an active community component present at low abundance. This study clearly demonstrates that the deep subsurface sediments of the Peru Margin have a large diverse and metabolically active prokaryotic population.

Analysis of DGGE profiles to explore the relationship between prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin.

The aim of this work was to relate depth profiles of prokaryotic community composition with geochemical processes in the deep subseafloor biosphere at two shallow-water sites on the Peru Margin in the Pacific Ocean (ODP Leg 201, sites 1228 and 1229). Principal component analysis of denaturing gradient gel electrophoresis banding patterns of deep-sediment Bacteria, Archaea, Euryarchaeota and the novel candidate division JS1, followed by multiple regression, showed strong relationships with prokaryotic activity and geochemistry (R(2)=55-100%). Further correlation analysis, at one site, between the principal components from the community composition profiles for Bacteria and 12 other variables quantitatively confirmed their relationship with activity and geochemistry, which had previously only been implied. Comparison with previously published cell counts enumerated by fluorescent in situ hybridization with rRNA-targeted probes confirmed that these denaturing gradient gel electrophoresis profiles described an active prokaryotic community.

Complex coupled metabolic and prokaryotic community responses to increasing temperatures in anaerobic marine sediments: critical temperatures and substrate changes.

The impact of temperature (0-80°C) on anaerobic biogeochemical processes and prokaryotic communities in marine sediments (tidal flat) was investigated in slurries for up to 100 days. Temperature had a non-linear effect on biogeochemistry and prokaryotes with rapid changes over small temperature intervals. Some activities (e.g. methanogenesis) had multiple 'windows' within a large temperature range (∼10 to 80°C). Others, including acetate oxidation, had maximum activities within a temperature zone, which varied with electron acceptor [metal oxide (up to ∼34°C) and sulphate (up to ∼50°C)]. Substrates for sulphate reduction changed from predominantly acetate below, and H2 above, a 43°C critical temperature, along with changes in activation energies and types of sulphate-reducing Bacteria. Above ∼43°C, methylamine metabolism ceased with changes in methanogen types and increased acetate concentrations (>1 mM). Abundances of uncultured Archaea, characteristic of deep marine sediments (e.g. MBGD Euryarchaeota, 'Bathyarchaeota') changed, indicating their possible metabolic activity and temperature range. Bacterial cell numbers were consistently higher than archaeal cells and both decreased above ∼15°C. Substrate addition stimulated activities, widened some activity temperature ranges (methanogenesis) and increased bacterial (×10) more than archaeal cell numbers. Hence, additional organic matter input from climate-related eutrophication may amplify the impact of temperature increases on sedimentary biogeochemistry.

Bacterial and Archaeal direct counts: a faster method of enumeration, for enrichment cultures and aqueous environmental samples.

A new presence/absence method has been developed to count fluorochrome-stained bacterial and archaeal cells on membrane filters using epifluorescence microscopy. This approach was derived from the random distribution of cells on membranes that allowed the use of the Poisson distribution to estimate total cell densities. Comparison with the standard Acridine Orange Direct Count (AODC) technique shows no significant difference in the estimation of total cell populations, or any reduction in the precision of these estimations. The new method offers advantages over the standard AODC in considerably faster counting, as there is no need to discriminate between every potential cell visible on a field and fluorescent detritus, it is only necessary to confirm the presence of one cell. Additionally, the new method requires less skill, so has less reliance on expert counters, and that should reduce inter-counter variability. Although this work used the fluorochrome Acridine Orange, clearly the results are applicable to any fluorochrome used to count bacterial and archaeal cells. This method was developed using enrichment cultures for use with enrichment cultures and aqueous environmental samples where interfering detrital and mineral particles are minimal e.g., freshwater/seawater, therefore, it is not suitable for estimating total cells from sediment samples. This method has the potential for use in any situation where counts of randomly distributed items are made using a grid or quadrat system.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea.

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.

Enrichment and cultivation of prokaryotes associated with the sulphate-methane transition zone of diffusion-controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions.

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10 mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (~2 mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives.

Extending the sub-sea-floor biosphere.

Sub-sea-floor sediments may contain two-thirds of Earth's total prokaryotic biomass. However, this has its basis in data extrapolation from ~500-meter to 4-kilometer depths, whereas the deepest documented prokaryotes are from only 842 meters. Here, we provide evidence for low concentrations of living prokaryotic cells in the deepest (1626 meters below the sea floor), oldest (111 million years old), and potentially hottest (~100 degrees C) marine sediments investigated. These Newfoundland margin sediments also have DNA sequences related to thermophilic and/or hyperthermophilic Archaea. These form two unique clusters within Pyrococcus and Thermococcus genera, suggesting unknown, uncultured groups are present in deep, hot, marine sediments (~54 degrees to 100 degrees C). Sequences of anaerobic methane-oxidizing Archaea were also present, suggesting a deep biosphere partly supported by methane. These findings demonstrate that the sub-sea-floor biosphere extends to at least 1600 meters below the sea floor and probably deeper, given an upper temperature limit for prokaryotic life of at least 113 degrees C and increasing thermogenic energy supply with depth.

Biogeochemistry and biodiversity of methane cycling in subsurface marine sediments (Skagerrak, Denmark).

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190.

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.

Distributions of microbial activities in deep subseafloor sediments.

Diverse microbial communities and numerous energy-yielding activities occur in deeply buried sediments of the eastern Pacific Ocean. Distributions of metabolic activities often deviate from the standard model. Rates of activities, cell concentrations, and populations of cultured bacteria vary consistently from one subseafloor environment to another. Net rates of major activities principally rely on electron acceptors and electron donors from the photosynthetic surface world. At open-ocean sites, nitrate and oxygen are supplied to the deepest sedimentary communities through the underlying basaltic aquifer. In turn, these sedimentary communities may supply dissolved electron donors and nutrients to the underlying crustal biosphere.