Groundwater shapes sediment biogeochemistry and microbial diversity in a submerged Great Lake sinkhole

For a large part of earth's history, cyanobacterial mats thrived in low‐oxygen conditions, yet our understanding of their ecological functioning is limited. Extant cyanobacterial mats provide windows into the putative functioning of ancient ecosystems, and they continue to mediate biogeochemical transformations and nutrient transport across the sediment–water interface in modern ecosystems. The structure and function of benthic mats are shaped by biogeochemical processes in underlying sediments. A modern cyanobacterial mat system in a submerged sinkhole of Lake Huron (LH) provides a unique opportunity to explore such sediment–mat interactions. In the Middle Island Sinkhole (MIS), seeping groundwater establishes a low‐oxygen, sulfidic environment in which a microbial mat dominated by Phormidium and Planktothrix that is capable of both anoxygenic and oxygenic photosynthesis, as well as chemosynthesis, thrives. We explored the coupled microbial community composition and biogeochemical functioning of organic‐rich, sulfidic sediments underlying the surface mat. Microbial communities were diverse and vertically stratified to 12 cm sediment depth. In contrast to previous studies, which used low‐throughput or shotgun metagenomic approaches, our high‐throughput 16S rRNA gene sequencing approach revealed extensive diversity. This diversity was present within microbial groups, including putative sulfate‐reducing taxa of Deltaproteobacteria, some of which exhibited differential abundance patterns in the mats and with depth in the underlying sediments. The biological and geochemical conditions in the MIS were distinctly different from those in typical LH sediments of comparable depth. We found evidence for active cycling of sulfur, methane, and nutrients leading to high concentrations of sulfide, ammonium, and phosphorus in sediments underlying cyanobacterial mats. Indicators of nutrient availability were significantly related to MIS microbial community composition, while LH communities were also shaped by indicators of subsurface groundwater influence. These results show that interactions between the mats and sediments are crucial for sustaining this hot spot of biological diversity and biogeochemical cycling.     

Holocene paleodepositional changes reflected in the sedimentary microbiome of the Black Sea

Subsurface microbial communities are generally thought to be structured through in situ environmental conditions such as the availability of electron acceptors and donors and porosity, but recent studies suggest that the vertical distribution of a subset of subseafloor microbial taxa, which were present at the time of deposition, were selected by the paleodepositional environment. However, additional highly resolved temporal records of subsurface microbiomes and paired paleoenvironmental reconstructions are needed to justify this claim. Here, we performed a highly resolved shotgun metagenomics survey to study the taxonomic and functional diversity of the subsurface microbiome in Holocene sediments underlying the permanently stratified and anoxic Black Sea. Obligate aerobic bacteria made the largest contribution to the observed shifts in microbial communities associated with known Holocene climate stages and transitions. This suggests that the aerobic fraction of the subseafloor microbiome was seeded from the water column and did not undergo post‐depositional selection. In contrast, obligate and facultative anaerobic bacteria showed the most significant response to the establishment of modern‐day environmental conditions 5.2 ka ago that led to a major shift in planktonic communities and in the type of sequestered organic matter available for microbial degradation. No significant shift in the subseafloor microbiome was observed as a result of environmental changes that occurred shortly after the marine reconnection, 9 ka ago. This supports the general view that the marine reconnection was a gradual process. We conclude that a high‐resolution analysis of downcore changes in the subseafloor microbiome can provide detailed insights into paleoenvironmental conditions and biogeochemical processes that occurred at the time of deposition.     

Potentially Active Iron,Sulfur, and Sulfate Reducing Bacteria in Skagerrak and Bothnian Bay Sediments

In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscured by sulfate reduction, which is regarded as the predominant anaerobic microbial respiration process. However, many dissimilatory sulfate and sulfur reducing microorganisms are known to utilize alternative electron acceptors such as metal oxides. In this study, we tested whether sulfate and sulfur reducing bacteria are linked to metal oxide reduction based on biogeochemical modeling of porewater concentration profiles of Mn²⁺ and Fe²⁺ in Bothnian Bay (BB) and Skagerrak (SK) sediments. Steady-state modeling of Fe²⁺ and Mn²⁺ porewater profiles revealed zones of net Fe (0–9 cm BB; ～10 and 20 cm SK) and Mn (0–5 cm BB; 2–8 cm SK) species transformations. 16S rRNA pyrosequencing analysis of the in-situ community showed that Desulfobacteraceae, Desulfuromonadaceae and Desulfobulbaceae were present in the zone of Fe-reduction of both sediments. Rhodobacteraceae were also detected at high relative abundance in both sediments. BB sediments appeared to harbor a greater diversity of potential Fe-reducers compared to SK. Additionally, when the upper 10 cm of sediment from the SK was incubated with lepidocrocite and acetate, Desulfuromonas was the dominant bacteria. Real-time quantitative polymerase chain reaction (qPCR) results showed decreasing dsrA gene copy numbers with depth coincided with decreased Fe-reduction activity. Our results support the idea that sulfur and sulfate reducing bacteria contribute to Fe-reduction in the upper centimeters of both sediments.     

Effect of biostimulation on the microbial community in PCB-contaminated sediments through periodic amendment of sediment with iron

Reductive dehalogenation of polychlorinated biphenyls (PCBs) by indigenous dehalorespiring microorganisms in contaminated sediments may be enhanced via biostimulation by supplying hydrogen generated through the anaerobic corrosion of elemental iron added to the sediment. In this study, the effect of periodic amendment of sediment with various dosages of iron on the microbial community present in sediment was investigated using phospholipid fatty acid analysis (PLFA) over a period of 18 months. Three PCB-contaminated sediments (two freshwater lake sediments and one marine sediment) were used. Signature biomarker analysis of the microbial community present in all three sediments revealed the enrichment of Dehalococcoides species, the population of which was sustained for a longer period of time when the sediment microcosms were amended with the lower dosage of iron (0.01 g iron per g dry sediment) every 6 months as compared to the blank system (without iron). Lower microbial stress levels were reported for the system periodically amended with 0.01 g of iron per g dry sediment every 6 months, thus reducing the competition from other hydrogen-utilizing microorganisms like methanogens, iron reducers, and sulfate reducers. The concentration of hydrogen in the system was found to be an important factor influencing the shift in microbial communities in all sediments with time. Periodic amendment of sediment with larger dosages of iron every 3 months resulted in the early prevalence of Geobacteraceae and sulfate-reducing bacteria followed by methanogens. An average pH of 8.4 (range of 8.2–8.6) and an average hydrogen concentration of 0.75% (range of 0.3–1.2%) observed between 6 and 15 months of the study were found to be conducive to sustaining the population of Dehalococcoides species in the three sediments amended with 0.01 g iron per g dry sediment. Biostimulation of indigenous PCB dechlorinators by the periodic amendment of contaminated sediments with low dosages of iron metal may therefore be an effective technology for remediation of PCB-contaminated sediments.     

Microbial communities in low permeability,high pH uranium mine tailings: characterization and potential effects

Aims

To describe the diversity and metabolic potential of microbial communities in uranium mine tailings characterized by high pH, high metal concentration and low permeability.

Methods and Results

To assess microbial diversity and their potential to influence the geochemistry of uranium mine tailings using aerobic and anaerobic culture‐based methods, in conjunction with next generation sequencing and clone library sequencing targeting two universal bacterial markers (the 16S rRNA and cpn60 genes). Growth assays revealed that 69% of the 59 distinct culturable isolates evaluated were multiple‐metal resistant, with 15% exhibiting dual‐metal hypertolerance. There was a moderately positive correlation coefficient (R = 0·43, P < 0·05) between multiple‐metal resistance of the isolates and their enzyme expression profile. Of the isolates tested, 17 reduced amorphous iron, 22 reduced molybdate and seven oxidized arsenite. Based on next generation sequencing, tailings depth was shown to influence bacterial community composition, with the difference in the microbial diversity of the upper (0–20 m) and middle (20–40 m) tailings zones being highly significant (P < 0·01) from the lower zone (40–60 m) and the difference in diversity of the upper and middle tailings zone being significant (P < 0·05). Phylotypes closely related to well‐known sulfate‐reducing and iron‐reducing bacteria were identified with low abundance, yet relatively high diversity.

Conclusions

The presence of a population of metabolically‐diverse, metal‐resistant micro‐organisms within the tailings environment, along with their demonstrated capacity for transforming metal elements, suggests that these organisms have the potential to influence the long‐term geochemistry of the tailings.

Significance and Impact of the study

This study is the first investigation of the diversity and functional potential of micro‐organisms present in low permeability, high pH uranium mine tailings.     

The distribution of active iron‐cycling bacteria in marine and freshwater sediments is decoupled from geochemical gradients

Microaerophilic, phototrophic and nitrate‐reducing Fe(II)‐oxidizers co‐exist in coastal marine and littoral freshwater sediments. However, the in situ abundance, distribution and diversity of metabolically active Fe(II)‐oxidizers remained largely unexplored. Here, we characterized the microbial community composition at the oxic‐anoxic interface of littoral freshwater (Lake Constance, Germany) and coastal marine sediments (Kalø Vig and Norsminde Fjord, Denmark) using DNA‐/RNA‐based next‐generation 16S rRNA (gene) amplicon sequencing. All three physiological groups of neutrophilic Fe(II)‐oxidizing bacteria were found to be active in marine and freshwater sediments, revealing up to 0.2% anoxygenic photoferrotrophs (e.g., Rhodopseudomonas, Rhodobacter, Chlorobium), 0.1% microaerophilic Fe(II)‐oxidizers (e.g., Mariprofundus, Hyphomonas, Gallionella) and 0.3% nitrate‐reducing Fe(II)‐oxidizers (e.g., Thiobacillus, Pseudomonas, Denitromonas, Hoeflea). Active Fe(III)‐reducing bacteria (e.g., Shewanella, Geobacter) were most abundant (up to 2.8%) in marine sediments and co‐occurred with cable bacteria (up to 4.5%). Geochemical profiles of Fe(III), Fe(II), O₂, light, nitrate and total organic carbon revealed a redox stratification of the sediments and explained 75%–85% of the vertical distribution of microbial taxa, while active Fe‐cycling bacteria were found to be decoupled from geochemical gradients. We suggest that metabolic flexibility, microniches in the sediments, or interrelationships with cable bacteria might explain the distribution patterns of active Fe‐cycling bacteria.     

Linking sedimentary sulfur and iron biogeochemistry to growth patterns of a cold‐water coral mound in the Porcupine Basin,S.W. Ireland (IODP Expedition 307)

Challenger Mound, a 150‐m‐high cold‐water coral mound on the eastern flank of the Porcupine Seabight off SW Ireland, was drilled during Expedition 307 of the Integrated Ocean Drilling Program (IODP). Retrieved cores offer unique insight into an archive of Quaternary paleo‐environmental change, long‐term coral mound development, and the diagenetic alteration of these carbonate fabrics over time. To characterize biogeochemical carbon–iron–sulfur transformations in the mound sediments, the contents of dithionite‐ and HCl‐extractable iron phases, iron monosulfide and pyrite, and acid‐extractable calcium, magnesium, manganese, and strontium were determined. Additionally, the stable isotopic compositions of pore‐water sulfate and solid‐phase reduced sulfur compounds were analyzed. Sulfate penetrated through the mound sequence and into the underlying Miocene sediments, where a sulfate–methane transition zone was identified. Small sulfate concentration decreases (<7 mm ) within the top 40 m of the mound suggested slow net rates of present‐day organoclastic sulfate reduction. Increasing δ³⁴S‐sulfate values due to microbial sulfate reduction mirrored the decrease in sulfate concentrations. This process was accompanied by oxygen isotope exchange with water that was indicated by increasing δ¹⁸O‐sulfate values, reaching equilibrium with pore‐water at depth. Below 50 mbsf, sediment intervals with strong ³⁴S‐enriched imprints on chromium‐reducible sulfur (pyrite S), high degree‐of‐pyritization values, and semi‐lithified diagenetic carbonate‐rich layers characterized by poor coral preservation, were observed. These layers provided evidence for the occurrence of enhanced microbial sulfate‐reducing activity in the mound in the past during periods of rapid mound aggradation and subsequent intervals of non‐deposition or erosion when geochemical fronts remained stationary. During these periods, especially during the Early Pleistocene, elevated sulfate reduction rates facilitated the consumption of reducible iron oxide phases, coral dissolution, and the subsequent formation of carbonate cements.     

Barite in hydrothermal environments as a recorder of subseafloor processes: a multiple‐isotope study from the Loki's Castle vent field

Barite chimneys are known to form in hydrothermal systems where barium‐enriched fluids generated by leaching of the oceanic basement are discharged and react with seawater sulfate. They also form at cold seeps along continental margins, where marine (or pelagic) barite in the sediments is remobilized because of subseafloor microbial sulfate reduction. We test the possibility of using multiple sulfur isotopes (δ³⁴S, Δ³³S, ?³⁶S) of barite to identify microbial sulfate reduction in a hydrothermal system. In addition to multiple sulfur isotopes, we present oxygen (δ¹⁸O) and strontium (⁸⁷Sr/⁸⁶Sr) isotopes for one of numerous barite chimneys in a low‐temperature (~20 °C) venting area of the Loki's Castle black smoker field at the ultraslow‐spreading Arctic Mid‐Ocean Ridge (AMOR). The chemistry of the venting fluids in the barite field identifies a contribution of at least 10% of high‐temperature black smoker fluid, which is corroborated by ⁸⁷Sr/⁸⁶Sr ratios in the barite chimney that are less radiogenic than in seawater. In contrast, oxygen and multiple sulfur isotopes indicate that the fluid from which the barite precipitated contained residual sulfate that was affected by microbial sulfate reduction. A sulfate reduction zone at this site is further supported by the multiple sulfur isotopic composition of framboidal pyrite in the flow channel of the barite chimney and in the hydrothermal sediments in the barite field, as well as by low SO₄ and elevated H₂S concentrations in the venting fluids compared with conservative mixing values. We suggest that the mixing of ascending H₂‐ and CH₄‐rich high‐temperature fluids with percolating seawater fuels microbial sulfate reduction, which is subsequently recorded by barite formed at the seafloor in areas where the flow rate is sufficient. Thus, low‐temperature precipitates in hydrothermal systems are promising sites to explore the interactions between the geosphere and biosphere in order to evaluate the microbial impact on these systems.     

Fluids from the Oceanic Crust Support Microbial Activities within the Deep Biosphere

The importance of crustal fluid chemical composition in driving the marine deep subseafloor biosphere was examined in northeast Pacific ridge-flank sediments. At IODP Site U1301, sulfate from crustal fluids diffuses into overlying sediments, forming a transition zone where sulfate meets in situ-produced methane. Enhanced cell counts and metabolic activity suggest that sulfate stimulates microbial respiration, specifically anaerobic methane oxidation coupled to sulfate reduction. Cell counts and activity are also elevated in basement-near layers. Owing to the worldwide expansion of the crustal aquifer, we postulate that crustal fluids may fuel the marine deep subseafloor biosphere on a global scale.     

Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.     

Bacterial dominance in subseafloor sediments characterized by methane hydrates

The degradation of organic carbon in subseafloor sediments on continental margins contributes to the largest reservoir of methane on Earth. Sediments in the Andaman Sea are composed of ~ 1% marine-derived organic carbon and biogenic methane is present. Our objective was to determine microbial abundance and diversity in sediments that transition the gas hydrate occurrence zone (GHOZ) in the Andaman Sea. Microscopic cell enumeration revealed that most sediment layers harbored relatively low microbial abundance (10(3)-10(5) cells cm(-3)). Archaea were never detected despite the use of both DNA- and lipid-based methods. Statistical analysis of terminal restriction fragment length polymorphisms revealed distinct microbial communities from above, within, and below the GHOZ, and GHOZ samples were correlated with a decrease in organic carbon. Primer-tagged pyrosequences of bacterial 16S rRNA genes showed that members of the phylum Firmicutes are predominant in all zones. Compared with other seafloor settings that contain biogenic methane, this deep subseafloor habitat has a unique microbial community and the low cell abundance detected can help to refine global subseafloor microbial abundance.     

Diversity decoupled from sulfur isotope fractionation in a sulfate‐reducing microbial community

The extent of fractionation of sulfur isotopes by sulfate‐reducing microbes is dictated by genomic and environmental factors. A greater understanding of species‐specific fractionations may better inform interpretation of sulfur isotopes preserved in the rock record. To examine whether gene diversity influences net isotopic fractionation in situ, we assessed environmental chemistry, sulfate reduction rates, diversity of putative sulfur‐metabolizing organisms by 16S rRNA and dissimilatory sulfite reductase (dsrB) gene amplicon sequencing, and net fractionation of sulfur isotopes along a sediment transect of a hypersaline Arctic spring. In situ sulfate reduction rates yielded minimum cell‐specific sulfate reduction rates < 0.3 × 10^?15 moles cell^?1 day^?1. Neither 16S rRNA nor dsrB diversity indices correlated with relatively constant (38‰–45‰) net isotope fractionation (ε³⁴S_{sulfide‐sulfate}). Measured ε³⁴S values could be reproduced in a mechanistic fractionation model if 1%–2% of the microbial community (10%–60% of Deltaproteobacteria) were engaged in sulfate respiration, indicating heterogeneous respiratory activity within sulfate‐reducing populations. This model indicated enzymatic kinetic diversity of Apr was more likely to correlate with sulfur fractionation than DsrB. We propose that, above a threshold Shannon diversity value of 0.8 for dsrB, the influence of the specific composition of the microbial community responsible for generating an isotope signal is overprinted by the control exerted by environmental variables on microbial physiology.     

Abrupt burial imparts persistent changes to the bacterial diversity of turbidite‐associated sediment profiles

The emplacement of subaqueous gravity‐driven sediment flows imposes a significant physical and geochemical impact on underlying sediment and microbial communities. Although previous studies have established lasting mineralogical and biological signatures of turbidite deposition, the response of bacteria and archaea within and beneath debris flows remains poorly constrained. Both bacterial cells associated with the underlying sediment and those attached to allochthonous material must respond to substantially altered environmental conditions and selective pressures. As a consequence, turbidites and underlying sediments provide an exceptional opportunity to examine (i) the microbial community response to rapid sedimentation and (ii) the preservation and identification of displaced micro‐organisms. We collected Illumina MiSeq sequence libraries across turbidite boundaries at ~26 cm sediment depth in La Jolla Canyon off the coast of California, and at ~50 cm depth in meromictic Twin Lake, Hennepin County, MN. 16S rRNA gene signatures of relict and active bacterial populations exhibit persistent differences attributable to turbidite deposition. In particular, both the marine and lacustrine turbidite boundaries are sharply demarcated by the abundance and diversity of Chloroflexi, suggesting a characteristic sensitivity to sediment disturbance history or to differences in organic substrates across turbidite profiles. Variations in the abundance of putative dissimilatory sulfate‐reducing Deltaproteobacteria across the buried La Jolla Canyon sediment–water interface reflect turbidite‐induced changes to the geochemical environment. Species‐level distinctions within the Deltaproteobacteria clearly conform to the sedimentological boundary, suggesting a continuing impact of genetic inheritance distinguishable from broader trends attributable to selective pressure. Abrupt, <1‐cm scale changes in bacterial diversity across the Twin Lake turbidite contact are consistent with previous studies showing that relict DNA signatures attributable to sediment transport may be more easily preserved in low‐energy, anoxic environments. This work raises the possibility that deep subsurface microbial communities may inherit variations in microbial diversity from sediment flow and deformation events.     

Harnessing a methane‐fueled,sediment‐free mixed microbial community for utilization of distributed sources of natural gas

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy‐rich, yet the most efficient methane‐activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by‐products at a comparable rate and in near‐stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost‐effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep‐sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep‐sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full‐scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane‐oxidizing, sulfide‐generating mesocosm incubations. Metabolic activity required >～40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane‐dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth‐based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane‐activating microbial communities using a low‐mass and sediment‐free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Isolation of sulfate‐reducing bacteria from deep sediment layers of the pacific ocean

Bacterial populations exist at great depths in marine sediments, but little is known about the type and characteristics of organisms in this unique bacterial environment. Cascadia Margin sediments from the Pacific Ocean have deep bacterial activity and bacterial populations, which are stimulated around a gas hydrate zone (215–225 m below sea floor [mbsf]). Bacterial sulfate reduction is the dominant anaerobic process within these sediments, and the depth distribution of sulfate‐reducing activity corresponds with distributions of viable sulfate‐reducing bacteria (SRB). Anaerobically stored sediments from this site were used to isolate sulfate‐reducing bacteria using a temperature‐gradient system, elevated pressure and temperatures, different media, and a range of growth substrates. A variety of enrichments on lactate were obtained from 0.5 and 222 mbsf, with surprisingly more rapid growth from the deeper sediments. The temperature range of enrichments producing strong growth from 222 mbsf was markedly wider than those from the near surface sediment (15–45°C and 9–19°C, respectively). This presumably reflects a temperature increase in deeper sediments. Only a few of these enrichments were successfully isolated due to very slow or no growth on subculture, despite the use of a wide range of different media and growth conditions. Psychrophilic and mesophilic sulfate‐reducing isolates were obtained from 0.5 m depth. As the minimum growth temperature of the mesophile (probably a Desulfotomaculum sp.) was above the in situ temperature of 3°C, it must have been present in the sediment as spores. A larger number of isolates (23) was obtained from 222 mbsf, and these barophilic SRB were closely related (based on 16S rRNA gene analysis), but not identical to, Desulfovibrio profundus, recently isolated from deep sediments from the Japan Sea. Bacteria related to D. profundus may be widespread in deep marine sediments.     

Microbiological Assessment of Circulation Mud Fluids During the First Operation of Riser Drilling by the Deep-Earth Research Vessel Chikyu

Quality assurance and control (QA/QC) is significant for the scientific drilling in order to accurately characterize physical, geochemical, and biological properties in the cored deep subseafloor materials. To explore the deep subseafloor life and its biosphere, identification and control of microbial contamination in drilling cores is critical for highly sensitive molecular analyses as well as cultivations, especially for the evaluation of low biomass and/or extremely harsh deep environments. Here we report some microbiological characteristics of circulation mud fluids before and after the first riser drilling operation by the newly constructed deep-earth research vessel Chikyu. During the Chikyu shakedown expedition CK06-06 in 2006, we used the riser system for drilling 547 to 647 meter below the seafloor into the sediments offshore the Shimokita Peninsula of Japan. Cultivation experiments showed that no microbial growth was observed in the precirculation mud fluid, while 4 × 10⁵ colonies per 1 ml were observed in the postcirculation mud fluid; all cultured bacterial isolates were found to be Halomonas. Using culture-independent molecular analysis, 16S rRNA gene sequences of Xanthomonas, which is used for industrial production of the mud fluid viscosifier “xanthan gum”, were predominantly detected in the precirculation mud fluid, while Halomonas sequences consistently dominated the clone library constructed from the postcirculation mud fluid. Archaeal 16S rRNA genes were amplified only from the postcirculation mud fluid; these archaeal clone sequences were affiliated to the Marine Crenarchaeota Group I (MGI), Marine Euryarchaeota Group II (MGII), Miscellaneous Crenarchaeotic Group (MCG), South African Gold Mine Euryarchaeotic Group (SAGMEG), Soil Group, and Methanococcus aeolicus. These results suggest that Halomonas contaminated and grew in the tank of circulation mud fluids, and other indigenous deep subseafloor microbial components, especially deep subsurface archaea, were also mixed into the post-circulation mud fluid.     

Diversity of dimethylsulfide-degrading methanogens and sulfate-reducing bacteria in anoxic sediments along the Medway Estuary,UK

Methane is a powerful greenhouse gas but the microbial diversity mediating methylotrophic methanogenesis is not well-characterized. One overlooked route to methane is via the degradation of dimethylsulfide (DMS), an abundant organosulfur compound in the environment. Methanogens and sulfate-reducing bacteria (SRB) can degrade DMS in anoxic sediments depending on sulfate availability. However, we know little about the underlying microbial community and how sulfate availability affects DMS degradation in anoxic sediments. We studied DMS-dependent methane production along the salinity gradient of the Medway Estuary (UK) and characterized, for the first time, the DMS-degrading methanogens and SRB using cultivation-independent tools. DMS metabolism resulted in high methane yield (39%–42% of the theoretical methane yield) in anoxic sediments regardless of their sulfate content. Methanomethylovorans, Methanolobus and Methanococcoides were dominant methanogens in freshwater, brackish and marine incubations respectively, suggesting niche-partitioning of the methanogens likely driven by DMS amendment and sulfate concentrations. Adding DMS also led to significant changes in SRB composition and abundance in the sediments. Increases in the abundance of Sulfurimonas and SRB suggest cryptic sulfur cycling coupled to DMS degradation. Our study highlights a potentially important pathway to methane production in sediments with contrasting sulfate content and sheds light on the diversity of DMS degraders.     

Evaluation of enrichments of sulfate reducing bacteria from pristine hydrothermal vents sediments as potential inoculum for reducing trichloroethylene

The evaluation of enrichments from pristine hydrothermal vents sediments on its capability of reducing trichloroethylene (TCE) under sulfate reducing conditions with lactate and volatile fatty acids (VFAs) as substrates was performed. Effect of the possible TCE biodegradation intermediates cis and trans 1,2 dichloroethenes on sulfate reduction (SR) was also evaluated. The influence of cyanocobalamin (CNB₁₂) and riboflavin (RF) on the SR and biodegradation of TCE was also determined. Sediments from the vents were incubated at 37°C and supplemented with 4 g l⁻¹ SO₄ ²⁻, lactate or VFAs and amended in the corresponding treatments with either CNB₁₂ or RF in separated experiments. A percentage of TCE removal of 86 (150 μmol l⁻¹ initial concentration) was attained coupled to 48% sulfate depletion with lactate as substrate. Up to 93% removal of TCE (300 μmol l⁻¹ initial concentration) and 40% of sulfate was reached for VFAs as electron donor. A combination of lactate and CNB₁₂ yielded the best SR. The overall results suggest a syntrophic association in this microbial community in which sulfate reducers, dehalogenating, and probably halorespiring bacteria may be interacting and taking advantage of the fermentation of substrates differently, but without interruption of SR in spite of the fact that TCE was always present. It was also clear that sulfate reduction must be established in the cultures before any degradation can occur. The microbial community present in these hydrothermal vents sediments could be a new source of inoculum for bioreactors designed for dechlorination purposes.