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 (R2=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 communit

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 sediment

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

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

Deep sub-seafloor prokaryotes stimulated at interfaces over geological time

The sub-seafloor biosphere is the largest prokaryotic habitat on Earth1 but also a habitat with the lowest metabolic rates2. Modelled activity rates are very low, indicating that most prokaryotes may be inactive or have extraordinarily slow metabolism2. 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 90m 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 diatomrich sediments (about 9Myr 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 (∼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

Regulation of anaerobic methane oxidation in sediments of the Black Sea

Anaerobic oxidation of methane (AOM) and sulfate reduction (SRR) were investigated in sediments of the western Black Sea, where upward methane transport is controlled by diffusion. To understand the regulation and dynamics of methane production and oxidation in the Black Sea, rates of methanogenesis, AOM, and SRR were determined using radiotracers in combination with pore water chemistry and stable isotopes. In the Danube Canyon and the Dnjepr palaeo-delta AOM did not consume methane effectively and upwards diffusing methane created an extended sulfate-methane transition zone (SMTZ) that spread over more than 2.5 m and was located in brackish and limnic sediment. Measurable AOM rates occurred mainly in the lower part of the SMTZ, sometimes even at depths where sulfate seemed to be unavailable. The inefficiency of methane oxidation appears to be linked to the paleoceanographic history of the sediment, since in all cores methane was completely oxidized at the transition from the formerly oxic brackish clays to marine anoxic sediments. The upward tailing of methane was less pronounced in a core from the deep sea in the area of the Dnjepr Canyon, the only station with a SMTZ close to the marine deposits. Sub-surface sulfate reduction rates were mostly extremely low, and in the SMTZ were even lower than AOM rates. Rates of bicarbonate-based methanogenesis were below detection limit in two of the cores, but δ13C values of methane indicate a biogenic origin. The most δ13C- depleted isotopic signal of methane was found in the SMTZ of the core from the deep sea, most likely as a result of carbon recycling between AOM and methanogenesis

Bacterial profiles in a sulfide mound (Site 1035) and an area of active fluid venting (Site 1036) in hot hydrothermal sediments from Middle Valley (northwest Pacific)

Sediment samples (1 cm3 each) were obtained from two sites (Ocean Drilling Program [ODP] Sites 1035 and 1036) in the Middle Valley of the northern Juan de Fuca Ridge for direct microscopic determination of bacterial depth distributions in a region influenced by hydrothermal activity. These data were compared to data gathered during Leg 139, Site 858, at the same location. Site 1035 was cored to 170 meters below seafloor (mbsf), and significant numbers of bacterial cells were detected in most samples with 4 x 105 cells/cm3 at the base of the hole. Dividing and divided cells were only found above 64 mbsf. The temperature at the base of the hole was estimated at ~113°C—the current estimated upper temperature limit for bacteria. When the data were divided according to growth-temperature ranges of bacteria (mesophile = 10° -45°C, thermophile = 45° -80°C, and hyperthermophile = >80°C), the bacterial profile clearly displayed three bands of bacterial populations. Only populations in the upper mesophilic section of the hole agreed with a general population profile obtained from many other ODP legs. At higher temperatures bacterial populations were markedly lower than this general profile. Samples from Holes 1036A, 1036B, and 1036C all showed reduced populations when compared to the general profile. The deepest reliable enumeration was at 30 mbsf in Hole 1036B with 5 x 105 cells/cm3. Bacteriological sampling from Hole 1036C stopped before very high temperatures were encountered in the borehole; however, at Holes 1036A and 1034B, samples from temperatures apparently >200°C were obtained. Bacterial populations decreased rapidly from the surface and became nondetectable at ~110°C, but, at ~155° -185°C, intact cells were observed. This was similar to data from Site 858, where intact bacterial cells were also detected in this temperature range. Analysis of geochemical data suggested, however, that the reasons for the presence of these cells may be different. For Site 858, bacterial cells could be explained by a constrained lateral flow of entrained seawater carrying cells from shallower sediments down into the hot sediments containing hydrothermal fluids. This was not the case at Site 1036, where rapid seawater recharge occurred throughout the depths where bacteria were detected, which may have distorted, and significantly reduced, the assumed temperature profile. These populations appear to exist in a thermophilic/hyperthermophilic environment at the edge of hydrothermal sediment layers. There was some chemical evidence of in situ bacterial activity and they may be utilizing the products of hydrothermal alteration (e.g., methane) rising up from deeper sediment layers

Recent studies on bacterial populations and processes in subseafloor sediments: A review

Subsurface bacteria also occur in hydrothermal sediments with large temperature gradients (up to 12 °C/m) and with population numbers similar to non-hydrothermal sites at temperatures from psychrophilic to mesophilic. At greater depths and temperatures, populations decline rapidly, but they are still significant up to hyperthermophilic temperatures and are even stimulated by subsurface seawater flow. These results suggest that temperature alone does not limit bacteria in non-hydrothermal sediments until about 4 km, and evidence exists that bacterial processes may even be sustained by interaction with thermogenic processes as temperatures increase during deep burial. Experiments demonstrate that in the presence of readily degradable organic substrates, actively growing bacteria can move faster than sediment deposition; hence, these bacteria are not necessarily trapped and buried. However, bacterial growth decreases with depth to such an extent that subsurface bacteria would not be able to keep up with sedimentation rate and hence would be buried. In some circumstances, such as in sapropel layers with high organic matter in the Mediterranean, bacteria may be buried within a specific deposition horizon. Subsurface bacteria can utilize old and recalcitrant organic matter, but only very slowly, and they seem to have a strategy of high biomass and low growth rate, commensurate with their geological habitat of generally low energy flux

The geomicrobiology of deep marine sediments from Blake Ridge containing methane hydrate (Sites 994, 995, and 997)

Bacterial populations and activity were quantifid at three sites on the Blake Ridge, Ocean Drilling Program Leg 164, which formed a transect from a point where no bottom-simulating reflector (BSR) was present to an area where a well-developed BSR existed. In near-surface sediments (top ~ 10 mbsf) at Sites 994 and 995, bacterial profiles were similar to previously studied deep-sea sites, with bacterial populations (total and dividing bacteria, viable bacteria, and growth rates [thymidine incorporatio]) highest in surface sedients and decreasing exponentially with depth. The presence of methane hydrate was inferred at depth (~ 190-450 mbsf) within the sediment at all three sites. Associated with these deposits were high concentrations of free methane beneath the inferred base of the hydrate. Bacteria were present in all samples analyzed, to a maximum of 750 mbsf, extending the previous known limit of the deep biosphere in marine sediments by ~ 100 m. Even at this depth, the population was substantial, at 1.8 x 10 (6) cells mL-1. Bacterial populations and numbers of dividing and divided cells were stimulated significantly below the base of the inferred hydrate zone, which may also reflect high concentrations of free gas. Solid methane hydrate, recovered from 331 mbsf at Site 997, contained only 2% of the predicted bacterial population n a sediment from this depth, suggesting reduced bacterial populations in solid hydrate. Bacterial activity in near-surface sediments was dominated by sulfate reduction. Sulfate reduction rates and pore-water sulfate decreased rapidly with depth, concomitant with an accumulation of soild-phase sulfide in the sediment. Once sulfate was depleted (~20-30 mbsf), methane concentrations, methanogenesis, and methane oxidation all increased. Below 100 mbsf, bacterial processes occurred at very low rates. However, bacterial activity increased sharply around 450 mbsf, associated with the base of the inferred hydrate zone and the free-gas zone beneath; anaerobic methane oxidation, methanogenesis from both acetate an H2:CO2, acetate oxidation, sulfate reduction and bacterial productivity were all stimulated (from 1.5 to 15 times), demonstrating that the sediments near and below the BSR form a biogeochemically dynamic zone, with carbon cycling occurring through methane, acetate, and carbon dioxide. At Site 995, pore water acetate was present in surprisingly high concentrations, reaching ~ 15 mM at 691 mbsf, ~ 1000 times higher than "typical" near-surface concentrations (2-20 uM). Potential rates of acetate metabolism were extremely high and could not be sustained without influx of organic carbon into the sediment; hence in situ rates are likely to be lower than these potential rate measurements. However, there is evidence for upward migration of high concentrations of dissolved organic carbon into the sediments at these sites. Rates of acetate methanogenesis below the BSR were 2-3 orders of magnitude higher than H2:CO2 methanogenesis and were associated with extremely high quantities of free gas. Methane oxidation rates at the base of the hydrate zone at Site 995 were 10 times greater than H2:CO2 methanogenesis. However, acetate methanogenesis at Site 995 exceeded methane oxidation through and below the BSR, potentially providing an unexpected source of methane gas for the formation of hydrates, These results confirm and extend previous results from Cascadia Margin, demonstrating that gas hydrate-containing sediments provide a unique deep bacterial habitat in marine sediments