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.

Implications of a short carbon pulse on biofilm formation on mica schist in microcosms with deep crystalline bedrock groundwater.

Microbial life in the deep subsurface occupies rock surfaces as attached communities and biofilms. Previously, epilithic Fennoscandian deep subsurface bacterial communities were shown to host genetic potential, especially for heterotrophy and sulfur cycling. Acetate, methane, and methanol link multiple biogeochemical pathways and thus represent an important carbon and energy source for microorganisms in the deep subsurface. In this study, we examined further how a short pulse of low-molecular-weight carbon compounds impacts the formation and structure of sessile microbial communities on mica schist surfaces over an incubation period of ∼3.5 years in microcosms containing deep subsurface groundwater from the depth of 500 m, from Outokumpu, Finland. The marker gene copy counts in the water and rock phases were estimated with qPCR, which showed that bacteria dominated the mica schist communities with a relatively high proportion of epilithic sulfate-reducing bacteria in all microcosms. The dominant bacterial phyla in the microcosms were Proteobacteria, Firmicutes, and Actinobacteria, whereas most fungal genera belonged to Ascomycota and Basidiomycota. Dissimilarities between planktic and sessile rock surface microbial communities were observed, and the supplied carbon substrates led to variations in the bacterial community composition.

Epilithic Microbial Community Functionality in Deep Oligotrophic Continental Bedrock.

The deep terrestrial biosphere hosts vast sessile rock surface communities and biofilms, but thus far, mostly planktic communities have been studied. We enriched deep subsurface microbial communities on mica schist in microcosms containing bedrock groundwater from the depth of 500 m from Outokumpu, Finland. The biofilms were visualized using scanning electron microscopy, revealing numerous different microbial cell morphologies and attachment strategies on the mica schist surface, e.g., bacteria with outer membrane vesicle-like structures, hair-like extracellular extensions, and long tubular cell structures expanding over hundreds of micrometers over mica schist surfaces. Bacterial communities were analyzed with amplicon sequencing showing that Pseudomonas, Desulfosporosinus, Hydrogenophaga, and Brevundimonas genera dominated communities after 8-40 months of incubation. A total of 21 metagenome assembled genomes from sessile rock surface metagenomes identified genes involved in biofilm formation, as well as a wide variety of metabolic traits indicating a high degree of environmental adaptivity to oligotrophic environment and potential for shifting between multiple energy or carbon sources. In addition, we detected ubiquitous organic carbon oxidation and capacity for arsenate and selenate reduction within our rocky MAGs. Our results agree with the previously suggested interaction between the deep subsurface microbial communities and the rock surfaces, and that this interaction could be crucial for sustaining life in the harsh anoxic and oligotrophic deep subsurface of crystalline bedrock environment.

Climatic Factors Influencing the Anthrax Outbreak of 2016 in Siberia, Russia.

In 2016, an outbreak of anthrax killing thousands of reindeer and affecting dozens of humans occurred on the Yamal peninsula, Northwest Siberia, after 70 years of epidemiological situation without outbreaks. The trigger of the outbreak has been ascribed to the activation of spores due to permafrost thaw that was accelerated during the summer heat wave. The focus of our study is on the dynamics of local environmental factors in connection with the observed anthrax revival. We show that permafrost was thawing rapidly for already 6 years before the outbreak. During 2011-2016, relatively warm years were followed by cold years with a thick snow cover, preventing freezing of the soil. Furthermore, the spread of anthrax was likely intensified by an extremely dry summer of 2016. Concurrent with the long-term decreasing trend in the regional annual precipitation, the rainfall in July 2016 was less than 10% of its 30-year mean value. We conclude that epidemiological situation of anthrax in the previously contaminated Arctic regions requires monitoring of climatic factors such as warming and precipitation extremes.

Rock Surface Fungi in Deep Continental Biosphere-Exploration of Microbial Community Formation with Subsurface In Situ Biofilm Trap.

Fungi have an important role in nutrient cycling in most ecosystems on Earth, yet their ecology and functionality in deep continental subsurface remain unknown. Here, we report the first observations of active fungal colonization of mica schist in the deep continental biosphere and the ability of deep subsurface fungi to attach to rock surfaces under in situ conditions in groundwater at 500 and 967 m depth in Precambrian bedrock. We present an in situ subsurface biofilm trap, designed to reveal sessile microbial communities on rock surface in deep continental groundwater, using Outokumpu Deep Drill Hole, in eastern Finland, as a test site. The observed fungal phyla in Outokumpu subsurface were Basidiomycota, Ascomycota, and Mortierellomycota. In addition, significant proportion of the community represented unclassified Fungi. Sessile fungal communities on mica schist surfaces differed from the planktic fungal communities. The main bacterial phyla were Firmicutes, Proteobacteria, and Actinobacteriota. Biofilm formation on rock surfaces is a slow process and our results indicate that fungal and bacterial communities dominate the early surface attachment process, when pristine mineral surfaces are exposed to deep subsurface ecosystems. Various fungi showed statistically significant cross-kingdom correlation with both thiosulfate and sulfate reducing bacteria, e.g., SRB2 with fungi Debaryomyces hansenii.

Controlling fluid-induced seismicity during a 6.1-km-deep geothermal stimulation in Finland.

We show that near-real-time seismic monitoring of fluid injection allowed control of induced earthquakes during the stimulation of a 6.1-km-deep geothermal well near Helsinki, Finland. A total of 18,160 m3 of fresh water was pumped into crystalline rocks over 49 days in June to July 2018. Seismic monitoring was performed with a 24-station borehole seismometer network. Using near-real-time information on induced-earthquake rates, locations, magnitudes, and evolution of seismic and hydraulic energy, pumping was either stopped or varied-in the latter case, between well-head pressures of 60 and 90 MPa and flow rates of 400 and 800 liters/min. This procedure avoided the nucleation of a project-stopping magnitude M W 2.0 induced earthquake, a limit set by local authorities. Our results suggest a possible physics-based approach to controlling stimulation-induced seismicity in geothermal projects.

Diversity and functionality of archaeal, bacterial and fungal communities in deep Archaean bedrock groundwater.

The diversity and metabolic functions of deep subsurface ecosystems remain relatively unexplored. Microbial communities in previously studied deep subsurface sites of the Fennoscandian Shield are distinctive to each site. Thus, we hypothesized that the microbial communities of the deep Archaean bedrock fracture aquifer in Romuvaara, northern Finland, differ both in community composition and metabolic functionality from the other sites in the Fennoscandian Shield. We characterized the composition, functionality and substrate preferences of the microbial communities at different depths in a 600 m deep borehole. In contrast to other Fennoscandian deep biosphere communities studied to date, iron-oxidizing Gallionella dominated the bacterial communities, while methanogenic and ammonia-oxidizing archaea were the most prominent archaea, and a diverse fungal community was also detected. Potential for methane cycling and sulfate and nitrate reduction was confirmed by detection of the functional genes of these metabolic pathways. Organotrophs were less abundant, although carbohydrates were the most preferred of the tested substrates. The microbial communities shared features with those detected from other deep groundwaters with similar geochemistry, but the majority of taxa distinctive to Romuvaara are different from the taxa previously detected in saline deep groundwater in the Fennoscandian Shield, most likely because of the differences in water chemistry.

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.

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.

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.