Riikka Kietäväinen

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.

The origin, source, and cycling of methane in deep crystalline rock biosphere.

The emerging interest in using stable bedrock formations for industrial purposes, e.g., nuclear waste disposal, has increased the need for understanding microbiological and geochemical processes in deep crystalline rock environments, including the carbon cycle. Considering the origin and evolution of life on Earth, these environments may also serve as windows to the past. Various geological, chemical, and biological processes can influence the deep carbon cycle. Conditions of CH4 formation, available substrates and time scales can be drastically different from surface environments. This paper reviews the origin, source, and cycling of methane in deep terrestrial crystalline bedrock with an emphasis on microbiology. In addition to potential formation pathways of CH4, microbial consumption of CH4 is also discussed. Recent studies on the origin of CH4 in continental bedrock environments have shown that the traditional separation of biotic and abiotic CH4 by the isotopic composition can be misleading in substrate-limited environments, such as the deep crystalline bedrock. Despite of similarities between Precambrian continental sites in Fennoscandia, South Africa and North America, where deep methane cycling has been studied, common physicochemical properties which could explain the variation in the amount of CH4 and presence or absence of CH4 cycling microbes were not found. However, based on their preferred carbon metabolism, methanogenic microbes appeared to have similar spatial distribution among the different sites.

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.

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 × 104 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.

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.

CO 2 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 Earths 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.