Response of Deep Subsurface Microbial Community to Different Carbon Sources and Electron Acceptors during ∼2 months Incubation in Microcosms.

Acetate plays a key role as electron donor and acceptor and serves as carbon source in oligotrophic deep subsurface environments. It can be produced from inorganic carbon by acetogenic microbes or through breakdown of more complex organic matter. Acetate is an important molecule for sulfate reducers that are substantially present in several deep bedrock environments. Aceticlastic methanogens use acetate as an electron donor and/or a carbon source. The goal of this study was to shed light on carbon cycling and competition in microbial communities in fracture fluids of Finnish crystalline bedrock groundwater system. Fracture fluid was anaerobically collected from a fracture zone at 967 m depth of the Outokumpu Deep Drill Hole and amended with acetate, acetate + sulfate, sulfate only or left unamended as a control and incubated up to 68 days. The headspace atmosphere of microcosms consisted of 80% hydrogen and 20% CO2. We studied the changes in the microbial communities with community fingerprinting technique as well as high-throughput 16S rRNA gene amplicon sequencing. The amended microcosms hosted more diverse bacterial communities compared to the intrinsic fracture zone community and the control treatment without amendments. The majority of the bacterial populations enriched with acetate belonged to clostridial hydrogenotrophic thiosulfate reducers and Alphaproteobacteria affiliating with groups earlier found from subsurface and groundwater environments. We detected a slight increase in the number of sulfate reducers after the 68 days of incubation. The microbial community changed significantly during the experiment, but increase in specifically acetate-cycling microbial groups was not observed.

CO2 and carbonate as substrate for the activation of the microbial community in 180 m deep bedrock fracture fluid of Outokumpu Deep Drill Hole, Finland.

Microbial communities in deep subsurface environments comprise a large portion of Earth's biomass, but the metabolic activities in these habitats are largely unknown. Here the effect of CO2 and carbonate on the microbial community of an isolated groundwater fracture zone at 180 m depth of the Outokumpu Deep Scientific Drill Hole (Finland) was tested. Outokumpu groundwater at 180 m depth contains approximately 0.45 L L-1 dissolved gas of which methane contributes 76%. CO2, on the other hand, is scarce. The number of microbial cells with intracellular activity in the groundwater was low when examined with redox staining. Fluorescence Assisted Cell Sorting (FACS) analyses indicated that only 1% of the microbial community stained active with the redox sensing dye in the untreated groundwater after 4 weeks of starvation. However, carbon substrate and sulfate addition increased the abundance of fluorescent cells up to 7%. CO2 and CO2 + sulfate activated the greatest number of microbes, especially increasing the abundance of Pseudomonas sp., which otherwise was present at only low abundance in Outokumpu. Over longer exposure time (2 months) up to 50% of the bacterial cells in the groundwater were shown to incorporate inorganic carbon from carbonate into biomass. Carbon recapture is an important feature in this ecosystem since it may decrease the rate of carbon loss in form of CO2 released from cellular processes.

Microbiome composition and geochemical characteristics of deep subsurface high-pressure environment, Pyhäsalmi mine Finland.

Pyhäsalmi mine in central Finland provides an excellent opportunity to study microbial and geochemical processes in a deep subsurface crystalline rock environment through near-vertical drill holes that reach to a depth of more than two kilometers below the surface. However, microbial sampling was challenging in this high-pressure environment. Nucleic acid yields obtained were extremely low when compared to the cell counts detected (1.4 × 10(4) cells mL(-1)) in water. The water for nucleic acid analysis went through high decompression (60-130 bar) during sampling, whereas water samples for detection of cell counts by microscopy could be collected with slow decompression. No clear cells could be identified in water that went through high decompression. The high-pressure decompression may have damaged part of the cells and the nucleic acids escaped through the filter. The microbial diversity was analyzed from two drill holes by pyrosequencing amplicons of the bacterial and archaeal 16S rRNA genes and from the fungal ITS regions from both DNA and RNA fractions. The identified prokaryotic diversity was low, dominated by Firmicute, Beta- and Gammaproteobacteria species that are common in deep subsurface environments. The archaeal diversity consisted mainly of Methanobacteriales. Ascomycota dominated the fungal diversity and fungi were discovered to be active and to produce ribosomes in the deep oligotrophic biosphere. The deep fluids from the Pyhäsalmi mine shared several features with other deep Precambrian continental subsurface environments including saline, Ca-dominated water and stable isotope compositions positioning left from the meteoric water line. The dissolved gas phase was dominated by nitrogen but the gas composition clearly differed from that of atmospheric air. Despite carbon-poor conditions indicated by the lack of carbon-rich fracture fillings and only minor amounts of dissolved carbon detected in formation waters, some methane was found in the drill holes. No dramatic differences in gas compositions were observed between different gas sampling methods tested. For simple characterization of gas composition the most convenient way to collect samples is from free flowing fluid. However, compared to a pressurized method a relative decrease in the least soluble gases may appear.

Rapid Reactivation of Deep Subsurface Microbes in the Presence of C-1 Compounds.

Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect the long-term stability of the repository systems, Here, we studied how microorganisms present in fracture fluid from a depth of 500 m in Outokumpu, Finland, respond to simple carbon compounds (C-1 compounds) in the presence or absence of sulphate as an electron acceptor. C-1 compounds such as methane and methanol are important intermediates in the deep subsurface carbon cycle, and electron acceptors such as sulphate are critical components of oxidation processes. Fracture fluid samples were incubated in vitro with either methane or methanol in the presence or absence of sulphate as an electron acceptor. Metabolic response was measured by staining the microbial cells with fluorescent dyes that indicate metabolic activity and transcriptional response with RT-qPCR. Our results show that deep subsurface microbes exist in dormant states but rapidly reactivate their transcription and respiration systems in the presence of C-1 substrates, particularly methane. Microbial activity was further enhanced by the addition of sulphate as an electron acceptor. Sulphate- and nitrate-reducing microbes were particularly responsive to the addition of C-1 compounds and sulphate. These taxa are common in deep biosphere environments and may be affected by conditions disturbed by bedrock intrusion, as from drilling and excavation for long-term storage of hazardous waste.

Heterotrophic communities supplied by ancient organic carbon predominate in deep fennoscandian bedrock fluids.

The deep subsurface hosts diverse life, but the mechanisms that sustain this diversity remain elusive. Here, we studied microbial communities involved in carbon cycling in deep, dark biosphere and identified anaerobic microbial energy production mechanisms from groundwater of Fennoscandian crystalline bedrock sampled from a deep drill hole in Outokumpu, Finland, by using molecular biological analyses. Carbon cycling pathways, such as carbon assimilation, methane production and methane consumption, were studied with cbbM, rbcL, acsB, accC, mcrA and pmoA marker genes, respectively. Energy sources, i.e. the terminal electron accepting processes of sulphate-reducing and nitrate-reducing communities, were assessed with detection of marker genes dsrB and narG, respectively. While organic carbon is scarce in deep subsurface, the main carbon source for microbes has been hypothesized to be inorganic carbon dioxide. However, our results demonstrate that carbon assimilation is performed throughout the Outokumpu deep scientific drill hole water column by mainly heterotrophic microorganisms such as Clostridia. The source of carbon for the heterotrophic microbial metabolism is likely the Outokumpu bedrock, mainly composed of serpentinites and metasediments with black schist interlayers. In addition to organotrophic metabolism, nitrate and sulphate are other possible energy sources. Methanogenic and methanotrophic microorganisms are scarce, but our analyses suggest that the Outokumpu deep biosphere provides niches for these organisms; however, they are not very abundant.

Taxonomically and functionally diverse microbial communities in deep crystalline rocks of the Fennoscandian shield.

Microbial life in the nutrient-limited and low-permeability continental crystalline crust is abundant but remains relatively unexplored. Using high-throughput sequencing to assess the 16S rRNA gene diversity, we found diverse bacterial and archaeal communities along a 2516-m-deep drill hole in continental crystalline crust in Outokumpu, Finland. These communities varied at different sampling depths in response to prevailing lithology and hydrogeochemistry. Further analysis by shotgun metagenomic sequencing revealed variable carbon and nutrient utilization strategies as well as specific functional and physiological adaptations uniquely associated with specific environmental conditions. Altogether, our results show that predominant geological and hydrogeochemical conditions, including the existence and connectivity of fracture systems and the low amounts of available energy, have a key role in controlling microbial ecology and evolution in the nutrient and energy-poor deep crustal biosphere.

Dissecting the deep biosphere: retrieving authentic microbial communities from packer-isolated deep crystalline bedrock fracture zones.

Deep fracture zones in Finnish crystalline bedrock have been isolated for long, the oldest fluids being tens of millions of years old. To accurately measure the native microbial diversity in fracture-zone fluids, water samples were obtained by isolating the borehole fraction spanning a deep subsurface aquifer fracture zone with inflatable packers (500 and 967 m) or by pumping fluids directly from the fracture zone. Sampling frequency was examined to establish the time required for the space between packers to be flushed and replaced by indigenous fracture fluids. Chemical parameters of the fluid were monitored continuously, and samples were taken at three points during the flushing process. Microbial communities were characterized by comparison of 16S ribosomal genes and transcripts and quantification of dsrB (dissimilatory sulfate reduction) gene. Results suggest that fracture-zones host microbial communities with fewer cells and lower diversity than those in the drill hole prior to flushing. In addition, each fracture zone showed a community composition distinct from that inhabiting the drill hole at corresponding depth. The highest diversity was detected from the 967-m fracture zone. We conclude that the applied packer method can successfully isolate and sample authentic microbial fracture-zone communities of deep bedrock environments.

Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu deep borehole, Fennoscandian Shield.

This paper demonstrates the first microbiological sampling of the Outokumpu deep borehole (2516 m deep) aiming at characterizing the bacterial community composition and diversity of sulphate-reducing bacteria (SRB) in Finnish crystalline bedrock aquifers. Sampling was performed using a 1500-m-long pressure-tight tube that provided 15 subsamples, each corresponding to a 100-m section down the borehole. Microbial density measurements, as well as community fingerprinting with 16S rRNA gene-based denaturing gradient gel electrophoresis, demonstrated that microbial communities in the borehole water varied as a function of sampling depth. In the upper part of the borehole, bacteria affiliated to the family Comamonadaceae dominated the bacterial community. Further down the borehole, bacteria affiliated to the class Firmicutes became more prominent and, according to 16S rRNA gene clone libraries, dominated the bacterial community at 1400-1500 m. In addition, the largest number of bacterial classes was observed at 1400-1500 m. The dsrB genes detected in the upper part of the borehole were more similar to the dsrB genes of cultured SRBs, such as the genus Desulfotomaculum, whereas in the deeper parts of the borehole, the dsrB genes were more closely related to the uncultured bacteria that have been detected earlier in deep earth crust aquifers.

Microbial communities in subpermafrost saline fracture water at the Lupin Au mine, Nunavut, Canada.

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). 14C, 3H, and noble gas isotope analyses suggest that the Na-Ca-Cl, suboxic, fracture water represents a mixture of geologically ancient brine, approximately25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were approximately10(3) cells mL(-1). Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH4 isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH4 on Mars.