Deep Subsurface Ecosystems Research Articles

Dynamics of Methane-Consuming Biomes from Wieliczka Formation: Environmental and Enrichment Studies.

The rocks surrounding Wieliczka salt deposits are an extreme, deep subsurface ecosystem that as we studied previously harbors many microorganisms, including methanotrophs. In the presented research bacterial community structure of the Wieliczka Salt Mine was determined as well as the methanotrophic activity of the natural microbiome. Finally, an enrichment culture of methane-consuming methanotrophs was obtained. The research material used in this study consisted of rocks surrounding salt deposits in the Wieliczka Salt Mine. DNA was extracted directly from the pristine rock material, as well as from rocks incubated in an atmosphere containing methane and mineral medium, and from a methanotrophic enrichment culture from this ecosystem. As a result, the study describes the composition of the microbiome in the rocks surrounding the salt deposits, while also explaining how biodiversity changes during the enrichment culture of the methanotrophic bacterial community. The contribution of methanotrophic bacteria ranged from 2.614% in the environmental sample to 64.696% in the bacterial culture. The methanotrophic enrichment culture was predominantly composed of methanotrophs from the genera Methylomonas (48.848%) and Methylomicrobium (15.636%) with methane oxidation rates from 3.353 ± 0.105 to 4.200 ± 0.505 µmol CH4 mL-1 day-1.

Integrative Genomics Sheds Light on Global Deep Terrestrial Biosphere Communities

Active microbial lineages inhabit the deep terrestrial subsurface (Fry et al. 1997, Lopez-Fernandez et al. 2018) with estimates suggesting deep subsurface ecosystems to host ca. 90% of the total bacterial and archaeal biomass on Earth and about 10-20% of all terrestrial biomass (McMahon and Parnell 2014, Bar-On et al. 2018, Magnabosco et al. 2018, Drake and Reiners 2021). This highlights deep subsurface ecosystems as a vast reservoir of genetic innovation that remains poorly characterized. The biological diversity of deep subsurface ecosystems seem to be influenced by parameters such as temperature, pressure, water residence times, and geochemistry (Drake and Reiners 2021). The deep subsurface environment remains underrepresented in metagenomic databases, and yet the publicly available datasets are not synoptically analyzed in an integrated way. In this project, we collected a large set of publicly available and published metagenomes, published metagenome assembled genomes (MAGs) as well as single cell amplified genomes (SAGs) originating from global deep oligotrophic terrestrial subsurface samples (>70 m). Reconstructed MAGs/SAGs from this survey were integrated in to our own existing Fennoscandian Shield Genomic Database (FSGD) (Holmfeldt et al. 2021, Mehrshad et al. 2021). This expanded global database of the deep oligotrophic terrestrial subsurface contains >4000 MAGs/SAGs. In this dataset, representatives of phyla Firmicutes, Desulfobacterota, Patescibacteria, and Chlroflexota were the most prevalent taxa. The presence of Candidatus Desulforudis audaxviator across different continents and samples highlighted its adaptation to different deep biosphere environments. In addition, novel bacterial lineages at the class level were identified in several locations, pointing to the importance and necessity of further studies to characterize deep biosphere microbial diversity. This extensive study of the global deep oligotrophic terrestrial subsurface will reveal whether deep terrestrial populations are ubiquitous and how their distribution patterns are affected by different physicochemical variables such as water types and geology. By Identifying key populations present across multiple global groundwaters, our study will advance our understanding of the global diversity hosted in deep oligotrophic terrestrial subsurface ecosystems.

Microbial colonization and persistence in deep fractured shales is guided by metabolic exchanges and viral predation

BackgroundMicrobial colonization of subsurface shales following hydraulic fracturing offers the opportunity to study coupled biotic and abiotic factors that impact microbial persistence in engineered deep subsurface ecosystems. Shale formations underly much of the continental USA and display geographically distinct gradients in temperature and salinity. Complementing studies performed in eastern USA shales that contain brine-like fluids, here we coupled metagenomic and metabolomic approaches to develop the first genome-level insights into ecosystem colonization and microbial community interactions in a lower-salinity, but high-temperature western USA shale formation.ResultsWe collected materials used during the hydraulic fracturing process (i.e., chemicals, drill muds) paired with temporal sampling of water produced from three different hydraulically fractured wells in the STACK (Sooner Trend Anadarko Basin, Canadian and Kingfisher) shale play in OK, USA. Relative to other shale formations, our metagenomic and metabolomic analyses revealed an expanded taxonomic and metabolic diversity of microorganisms that colonize and persist in fractured shales. Importantly, temporal sampling across all three hydraulic fracturing wells traced the degradation of complex polymers from the hydraulic fracturing process to the production and consumption of organic acids that support sulfate- and thiosulfate-reducing bacteria. Furthermore, we identified 5587 viral genomes and linked many of these to the dominant, colonizing microorganisms, demonstrating the key role that viral predation plays in community dynamics within this closed, engineered system. Lastly, top-side audit sampling of different source materials enabled genome-resolved source tracking, revealing the likely sources of many key colonizing and persisting taxa in these ecosystems.ConclusionsThese findings highlight the importance of resource utilization and resistance to viral predation as key traits that enable specific microbial taxa to persist across fractured shale ecosystems. We also demonstrate the importance of materials used in the hydraulic fracturing process as both a source of persisting shale microorganisms and organic substrates that likely aid in sustaining the microbial community. Moreover, we showed that different physicochemical conditions (i.e., salinity, temperature) can influence the composition and functional potential of persisting microbial communities in shale ecosystems. Together, these results expand our knowledge of microbial life in deep subsurface shales and have important ramifications for management and treatment of microbial biomass in hydraulically fractured wells.FLC2hWdvCQdk4dv7tGhc3oVideo

Microbial Involvement in Carbon Transformation via CH4 and CO2 in Saline Sedimentary Pool.

Simple SummaryMethane and carbon dioxide are commonly found in the environment and are considered the most important greenhouse gases. Transformation of these gases is in large carried by microorganisms, which occur even in extreme environments. This study presents methane-related biological processes in saline sediments of the Miocene Wieliczka Formation, Poland. Biological activity (carbon dioxide and methane production or methane oxidation), confirmed by stable isotope indices, occurred in all of the studied Wieliczka rocks. CH4-utilizing microbes constituted 0.7–3.6% while methanogens (represented by Methanobacterium) only 0.01–0.5% of taxa present in the Wieliczka Salt Mine rocks. Water activity was the key factor regulating microbial activity in saline subsurface sediments. Generally, CO2 respiration was higher in anaerobic conditions while methanogenic and methanotrophic activities were dependent on the type of rock.Methane and carbon dioxide are one of the most important greenhouse gases and significant components of the carbon cycle. Biogeochemical methane transformation may occur even in the extreme conditions of deep subsurface ecosystems. This study presents methane-related biological processes in saline sediments of the Miocene Wieliczka Formation, Poland. Rock samples (W2, W3, and W4) differed in lithology (clayey salt with veins of fibrous salt and lenses of gypsum and anhydrite; siltstone and sandstone; siltstone with veins of fibrous salt and lenses of anhydrite) and the accompanying salt type (spiza salts or green salt). Microbial communities present in the Miocene strata were studied using activity measurements and high throughput sequencing. Biological activity (i.e., carbon dioxide and methane production or methane oxidation) occurred in all of the studied clayey salt and siltstone samples but mainly under water-saturated conditions. Microcosm studies performed at elevated moisture created more convenient conditions for the activity of both methanogenic and methanotrophic microorganisms than the intact sediments. This points to the fact that water activity is an important factor regulating microbial activity in saline subsurface sediments. Generally, respiration was higher in anaerobic conditions and ranged from 36 ± 2 (W2200%t.w.c) to 48 ± 4 (W3200%t.w.c) nmol CO2 gdw−1 day−1. Methanogenic activity was the highest in siltstone and sandstone (W3, 0.025 ± 0.018 nmol CH4 gdw−1 day−1), while aerobic methanotrophic activity was the highest in siltstone with salt and anhydrite (W4, 220 ± 66 nmol CH4 gdw−1 day−1). The relative abundance of CH4-utilizing microorganisms (Methylomicrobium, Methylomonas, Methylocystis) constituted 0.7–3.6% of all taxa. Methanogens were represented by Methanobacterium (0.01–0.5%). The methane-related microbes were accompanied by a significant number of unclassified microorganisms (3–64%) and those of the Bacillus genus (4.5–91%). The stable isotope composition of the CO2 and CH4 trapped in the sediments suggests that methane oxidation could have influenced δ13CCH4, especially in W3 and W4.

Procaryotic Diversity and Hydrogenotrophic Methanogenesis in an Alkaline Spring (La Crouen, New Caledonia).

(1) Background: The geothermal spring of La Crouen (New Caledonia) discharges warm (42 °C) alkaline water (pH~9) enriched in dissolved nitrogen with traces of methane, but its microbial diversity has not yet been studied. (2) Methods: Cultivation-dependent and -independent methods (e.g., Illumina sequencing and quantitative PCR based on 16S rRNA gene) were used to describe the prokaryotic diversity of this spring. (3) Results: Prokaryotes were mainly represented by Proteobacteria (57% on average), followed by Cyanobacteria, Chlorofexi, and Candidatus Gracilibacteria (GN02/BD1-5) (each > 5%). Both potential aerobes and anaerobes, as well as mesophilic and thermophilic microorganisms, were identified. Some of them had previously been detected in continental hyperalkaline springs found in serpentinizing environments (The Cedars, Samail, Voltri, and Zambales ophiolites). Gammaproteobacteria, Ca. Gracilibacteria and Thermotogae were significantly more abundant in spring water than in sediments. Potential chemolithotrophs mainly included beta- and gammaproteobacterial genera of sulfate-reducers (Ca. Desulfobacillus), methylotrophs (Methyloversatilis), sulfur-oxidizers (Thiofaba, Thiovirga), or hydrogen-oxidizers (Hydrogenophaga). Methanogens (Methanobacteriales and Methanosarcinales) were the dominant Archaea, as found in serpentinization-driven and deep subsurface ecosystems. A novel alkaliphilic hydrogenotrophic methanogen (strain CAN) belonging to the genus Methanobacterium was isolated, suggesting that hydrogenotrophic methanogenesis occurs at La Crouen.

Disentangling the distinct mechanisms shaping the subsurface oil reservoir bacterial and archaeal communities across northern China

Microbes in surface ecosystem exhibit strong biogeographic patterns, and are less apparent after human management. However, in contrast with the considerable knowledge on the surface ecosystem, the microbial biogeographic patterns in deep subsurface ecosystem under artificial disturbance is poorly understood. Here, we explored the spatial scale-dependence patterns of bacterial and archaeal communities in oil reservoirs under different artificial flooding duration and environmental conditions across northern China. Bacterial and archaeal communities of oil reservoirs exhibited distinct assembly patterns with a stronger distance-decay relationship in archaeal communities than bacterial communities, as different environmental factors linked to the diversity of bacteria and archaea. Specifically, bacterial and archaeal network properties revealed a significant correlation with spatial reservoir isolation by distinct co-occurrence patterns. The co-occurrences of bacterial communities were more complex in high temperature and alkaline pH, while archaeal co-occurrences were more frequent in low temperature and neutral pH. Potential functions in bacterial communities were more connected with chemoheterotrophy, whereas methanogenesis was abundant in archaeal communities, as confirmed by both keystone taxa and main ecological clusters in networks. This revealed that different mechanisms underlain geography and co-occurrence patterns of bacteria and archaea in oil reservoirs, providing a new insight for understanding biogeography and coexistence theory in deep subsurface ecosystem.

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.

Ecological Assembly Processes Are Coordinated between Bacterial and Viral Communities in Fractured Shale Ecosystems.

The ecological drivers that concurrently act upon both a virus and its host and that drive community assembly are poorly understood despite known interactions between viral populations and their microbial hosts. Hydraulically fractured shale environments provide access to a closed ecosystem in the deep subsurface where constrained microbial and viral community assembly processes can be examined. Here, we used metagenomic analyses of time-resolved-produced fluid samples from two wells in the Appalachian Basin to track viral and host dynamics and to investigate community assembly processes. Hypersaline conditions within these ecosystems should drive microbial community structure to a similar configuration through time in response to common osmotic stress. However, viral predation appears to counterbalance this potentially strong homogeneous selection and pushes the microbial community toward undominated assembly. In comparison, while the viral community was also influenced by substantial undominated processes, it assembled, in part, due to homogeneous selection. When the overall assembly processes acting upon both these communities were directly compared with each other, a significant relationship was revealed, suggesting an association between microbial and viral community development despite differing selective pressures. These results reveal a potentially important balance of ecological dynamics that must be in maintained within this deep subsurface ecosystem in order for the microbial community to persist over extended time periods. More broadly, this relationship begins to provide knowledge underlying metacommunity development across trophic levels.IMPORTANCE Interactions between viral communities and their microbial hosts have been the subject of many recent studies in a wide range of ecosystems. The degree of coordination between ecological assembly processes influencing viral and microbial communities, however, has been explored to a much lesser degree. By using a combined null modeling approach, this study investigated the ecological assembly processes influencing both viral and microbial community structure within hydraulically fractured shale environments. Among other results, significant relationships between the structuring processes affecting both the viral and microbial community were observed, indicating that ecological assembly might be coordinated between these communities despite differing selective pressures. Within this deep subsurface ecosystem, these results reveal a potentially important balance of ecological dynamics that must be maintained to enable long-term microbial community persistence. More broadly, this relationship begins to provide insight into the development of communities across trophic levels.

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.

Technological challenges to understanding the microbial ecology of deep subsurface ecosystems.

Terrestrial subsurface geomicrobiology is a new frontier in environmental microbiology. It seeks to determine whether life can be sustained in the absence of solar radiation. Subsurface ecosystems are also intriguing astrobiological models useful in the re-creation of life in early Earth scenarios or ascertaining its possible exist- ence on other planetary bodies. Although Darwin pre- Scanning electron microscopy allows the presence of diverse mineral substrates to be correlated with identified biological structures through elemental analysis (EDAX) and morphology, but the metabolic status and the type of functional metabolism of these biological structures cannot be determined using these techniques. The adap- tation of rRN-targeted FISH (rRNA-FISH) to the study the microorganisms associated to semisolid substrates (cata- lysed reporter deposition-FISH (CARD-FISH)) was an important breakthrough in the microbial ecology study of sediments. Even though this technique has not been used intensively in the identification and quantification of micro- organisms in continental drilling projects, it is obvious that in the near future, it will play a significant role in clarifying the ambiguous results generated by more conventional techniques such as comparative sequence analysis, which, as mentioned earlier, require sample sizes and preparation methodologies that average or destroy compartmentalization. Fluorescent oligonucleotide probes were originally designed to target rRNA for identification of microorgan- isms in environmental studies. Recently, a wide range of FISH procedures have been developed targeting not only rRNA, but also mRNA or single genes (Moraru et al., 2010). It is easy to foresee that these procedures will be

Evaluating reaction pathways of hydrothermal abiotic organic synthesis at elevated temperatures and pressures using carbon isotopes

Experiments were performed to better understand the role of environmental factors on reaction pathways and corresponding carbon isotope fractionations during abiotic hydrothermal synthesis of organic compounds using piston cylinder apparatus at 750°C and 5.5kbars. Chemical compositions of experimental products and corresponding carbon isotopic values were obtained by a Pyrolysis–GC–MS–IRMS system. Alkanes (methane and ethane), straight-chain saturated alcohols (ethanol and n-butanol) and monocarboxylic acids (formic and acetic acids) were generated with ethanol being the only organic compound with higher δ13C than CO2. CO was not detected in experimental products owing to the favorable water–gas shift reaction under high water pressure conditions. The pattern of δ13C values of CO2, carboxylic acids and alkanes are consistent with their equilibrium isotope relationships: CO2>carboxylic acids>alkanes, but the magnitude of the fractionation among them is higher than predicted isotope equilibrium values. In particular, the isotopic fractionation between CO2 and CH4 remained constant at ∼31‰, indicating a kinetic effect during CO2 reduction processes. No “isotope reversal” of δ13C values for alkanes or carboxylic acids was observed, which indicates a different reaction pathway than what is typically observed during Fischer–Tropsch synthesis under gas phase conditions. Under constraints imposed in experiments, the anomalous 13C isotope enrichment in ethanol suggests that hydroxymethylene is the organic intermediate, and that the generation of other organic compounds enriched in 12C were facilitated by subsequent Rayleigh fractionation of hydroxymethylene reacting with H2 and/or H2O. Carbon isotope fractionation data obtained in this study are instrumental in assessing the controlling factors on abiotic formation of organic compounds in hydrothermal systems. Knowledge on how environmental conditions affect reaction pathways of abiotic synthesis of organic compounds is critical for understanding deep subsurface ecosystems and the origin of organic compounds on Mars and other planets.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Mechanism Analysis of Indigenous Microbial Enhancement for Residue Oil Recovery

Biotechnological nutrient flooding was applied to the North block of the Kongdian Oilfield during 2001-2005. The biotechnology involved the injection of a water-air mixture made up of mineral nitrogen and phosphorous salts with the intent of stimulating the growth of indigenous microorganisms. During monitoring of the physico-chemical, microbiological and production characteristics of the North block of the Kongdian bed, it was revealed significant changes took place in the ecosystem as a result of the technological treatment. The microbial oil transformation was accompanied by an accumulation of carbonates, lower fatty acids and biosurfactants in water formations, which is of value to enhanced oil recovery. The microbial metabolites changed the composition of the water formation, favored the diversion of the injected fluid from closed, high permeability zones to upswept zones and improved the sweep efficiency. The results of the studies demonstrated strong hydrodynamic links between the injection wells and production wells. Microbiological monitoring of the deep subsurface ecosystems and the filtration properties of the fluids are well modified, producing 40000 additional tons of oil in the test areas.

The state, potential distribution, and biological implications of methane in the Martian crust

The search for life on Mars has recently focused on its potential survival in deep (>2 km) subpermafrost aquifers where anaerobic bacteria, similar to those found in deep subsurface ecosystems on Earth, may have survived in an environment that has remained stable for billions of years. An anticipated by‐product of this biological activity is methane. The detection of large deposits of methane gas and hydrate in the Martian cryosphere, or as emissions from deep fracture zones, would provide persuasive evidence of indigenous life and confirm the presence of a valuable in situ resource for use by future human explorers.