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Fluctuations in the production of renewable-based electricity have to be compensated by converting and storing the energy for later use. Underground methanation reactors (UMR) are a promising technology to address this issue. The idea is to create a controlled bio-reactor system in a porous underground formation, where hydrogen obtained from renewable energy sources by electrolysis and carbon dioxide from industrial sources are fed into the reactor and converted into methane. Microorganisms, known as methanogenic archaea, catalyze the chemical reaction by using the two non-organic substrates as nutrients for their growth and for their respiratory metabolism. The generated synthetic methane is renewable and capable to compete with the fossil methane. Mathematical models play an important role in the design and planning of such systems. Usually, a numerical solution of the model is required since complex initial-boundary problems cannot be solved analytically. In this paper, an existing bio-reactive transport model for UMR is simplified to such an extent that an analytical solution of the advection-dispersion-reaction equation can be applied. A second analytical solution is used for the case without dispersion. The analytical solutions are shown for both the educt (hydrogen) and the reaction product (methane). In order to examine the applicability of the analytical models, they are compared with the significantly more complex numerical model for a 1D case and a 3D case. It was shown that there is an acceptable agreement between the two analytical solutions and the numerical solution in different spatial plots of hydrogen and methane concentration and in the methane concentration in the withdrawn gas. The mean absolute error in the mole fraction is well below 0.015 in most cases. The spatial distribution of the hydrogen concentration in the comparison to the 3D case shows a higher deviation with a mean absolute error of approx. 0.023. As expected, the model with dispersion shows a slightly lower error in all cases, as only here the gas mixing resulting in smeared displacement fronts can be represented. It is shown that analytical modeling is a good tool to get a first estimation of the behavior of an UMR. It allows to help in the design of well spacing in combination with the injection rate and injected gas composition. Nevertheless, it is recommended to use more complex models for the later detailed analysis, which require a numerical solution. [ABSTRACT FROM AUTHOR]

The Soudan Underground Mine State Park, found in the Vermilion Iron Range in northern Minnesota, provides access to a ~ 2.7 billion-year-old banded iron formation. Exploratory boreholes drilled between 1958 and 1962 on the 27th level (713 m underground) of the mine intersect calcium and iron-rich brines that have recently been subject to metagenomic analysis and microbial enrichments. Using concentrated brine samples pumped from a borehole depth of up to 55 m, a novel Gram-positive bacterium was enriched under anaerobic, acetate-oxidizing, and Fe(III) citrate-reducing conditions. The isolated bacterium, designated strain MK1, is non-motile, rod-shaped, spore-forming, anaerobic, and mesophilic, with a growth range between 24°C and 30°C. The complete circular MK1 genome was found to be 3,720,236 bp and encodes 25 putative multiheme cytochromes, including homologs to inner membrane cytochromes in the Gram-negative bacterium Geobacter sulfurreducens and cytoplasmic membrane and periplasmic cytochromes in the Gram-positive bacterium Thermincola potens. However, MK1 does not encode homologs of the peptidoglycan (CwcA) and cell surface-associated (OcwA) multiheme cytochromes proposed to be required by T. potens to perform extracellular electron transfer. The 16S rRNA gene sequence of MK1 indicates that its closest related isolate is Desulfitibacter alkalitolerans strain sk.kt5 (91% sequence identity), which places MK1 in a novel genus within the Desulfitibacterac eae family and Moorellales order. Within the Moorellales order, only Calderihabitans maritimus strain KKC1 has been reported to reduce Fe(III), and only D. alkalitolerans can also grow in temperatures below 40°C. Thus, MK1 represents a novel species within a novel genus, for which we propose the name "Metallumcola ferriviriculae" strain MK1, and provides a unique opportunity to study a cytochrome-rich, mesophilic, Gram-positive, spore-forming Fe(III)-reducing bacterium. [ABSTRACT FROM AUTHOR]

This document is a collection of articles that highlight the significant roles that microbes play in caves. The articles cover various topics such as the presence of fungi in caves, the microbial communities in quartz-rich rocks, the variation of microbial communities in cave rocks, the potential for microbial-induced calcite precipitation, the formation of biospeleothems in caves, fungal infections in amphibians, the gut microbiota of cave-dwelling insects, and the effects of electrical lighting on cave-dwelling cyanobacteria. These articles offer insights into the diverse biogeochemical processes that occur in caves and the ways in which microbes adapt to cave environments. [Extracted from the article]

Little is known about how microbial activity affects the hydraulic properties of karst aquifers. To explore the potential impacts of microbial activity on the hydraulic properties of karst aquifers, microbiological analysis, heat tracer, isotope (dissolved inorganic carbon isotope, δ13CDIC) and aqueous geochemical analyses were conducted at six monitoring wells in Northern Guangdong Province, China. Greater hydraulic conductivity corresponded to a low temperature gradient to an extent; the temperature gradient in karst groundwater aquifers can reflect the degree of dissolution. Higher HCO3− concentrations coupled with lower d-excess and pH values at B2 and B6 reflect potential microbial activity (e.g., Sulfuricurvum kujiense) causing carbonate dissolution. Microbial activity or the input of anthropogenic acids, as evidenced by significantly more positive δ13CDIC values, potentially affect carbonate dissolution in deep karst aquifers, which eventually alters hydraulic properties of karst aquifer. However, more direct evidence is needed to quantify the effects of microbial activity on carbonate dissolution in karst aquifers.

The global trend of transiting to more renewable energy sources requires transition fuels, such as coal seam gas, to supplement and secure energy needs. In order to optimise strategies and technologies for enhancing gas production, an understanding of the fundamental microbial processes and interactions would be advantageous. Models have recently begun mapping the microbial roles and interactions in coal seam environments, from direct coal degradation to methanogenesis. This study seeks to expand those models by observing community compositional shifts in the presence of differing organic matter by conducting 16S rRNA microbial surveys using formation water from the Surat and Sydney Basins grown on varying types of organic matter (black and brown coal, oil shale, humic acid, and lignin). A total of 135 microbes were observed to become enriched in the presence of added organic matter in comparison to carbon-free treatments. These surveys allowed detailed analysis of microbial compositions in order to extrapolate which taxa favour growth in the presence of differing organic matter. This study has experimentally demonstrated shifts in the microbial community composition due to differing carbon sources and, for the first time, generated a conceptual model to map putative degradation pathways regarding subsurface microbial consortia. [ABSTRACT FROM AUTHOR]

ABSTRACT Balneotherapeutic water springs, such as those with thermal, saline, sulfur, or any other characteristics, have recently been the subject of phylogenetic studies with a closer focus on the description and/or isolation of phylogenetically novel or biotechnologically interesting microorganisms. Generally, however, most such microorganisms are rarely obtained in pure culture or are even, for now, unculturable under laboratory conditions. In this culture-dependent study of radioactive water springs of Jáchymov (Joachimstahl), Czech Republic, we investigated a combination of classical cultivation approaches with those imitating sampling source conditions. Using these environmentally relevant cultivation approaches, over 1,000 pure cultures were successfully isolated from 4 radioactive springs. Subsequent dereplication yielded 121 unique taxonomic units spanning 44 genera and 9 taxonomic classes, ~10% of which were identified as hitherto undescribed taxa. Genomes of the latter were sequenced and analyzed, with a special focus on endogenous defense systems to withstand oxidative stress and aid in radiotolerance. Due to their origin from radioactive waters, we determined the resistance of the isolates to oxidative stress. Most of the isolates were more resistant to menadione than the model strain Deinococcus radiodurans DSM 20539T. Moreover, isolates of the Deinococcacecae, Micrococcaceae, Bacillaceae, Moraxellaceae, and Pseudomonadaceae families even exhibited higher resistance in the presence of hydrogen peroxide. In summary, our culturomic analysis shows that subsurface water springs contain diverse bacterial populations, including as-yet-undescribed taxa and strains with promising biotechnological potential. Furthermore, this study suggests that environmentally relevant cultivation techniques increase the efficiency of cultivation, thus enhancing the chance of isolating hitherto uncultured microorganisms. IMPORTANCE The mine Svornost in Jáchymov (Joachimstahl), Czech Republic is a former silver-uranium mine and the world’s first and for a long time only radium mine, nowadays the deepest mine devoted to the extraction of water which is saturated with radon and has therapeutic benefits given its chemical properties. This healing water, which is approximately 13 thousand years old, is used under medical supervision for the treatment of patients with neurological and rheumatic disorders. Our culturomic approach using low concentrations of growth substrates or the environmental matrix itself (i.e., water filtrate) in culturing media combined with prolonged cultivation time resulted in the isolation of a broad spectrum of microorganisms from 4 radioactive springs of Jáchymov which are phylogenetically novel and/or bear various adaptive or coping mechanisms to thrive under selective pressure and can thus provide a wide spectrum of capabilities potentially exploitable in diverse scientific, biotechnological, or medical disciplines.

Rocks that react with liquid water are widespread but spatiotemporally limited throughout the solar system, except for Earth. Rock-forming minerals with high iron content and accessory minerals with high amounts of radioactive elements are essential to support rock-hosted microbial life by supplying organics, molecular hydrogen, and/or oxidants. Recent technological advances have broadened our understanding of the rocky biosphere, where microbial inhabitation appears to be difficult without nutrient and energy inputs from minerals. In particular, microbial proliferation in igneous rock basements has been revealed using innovative geomicrobiological techniques. These recent findings have dramatically changed our perspective on the nature and the extent of microbial life in the rocky biosphere, microbial interactions with minerals, and the influence of external factors on habitability. This study aimed to gather information from scientific and/or technological innovations, such as omics-based and single-cell level characterizations, targeting deep rocky habitats of organisms with minimal dependence on photosynthesis. By synthesizing pieces of rock-hosted life, we can explore the evo-phylogeny and ecophysiology of microbial life on Earth and the life's potential on other planetary bodies. [ABSTRACT FROM AUTHOR]

ABSTRACT The deep biosphere hosts uniquely adapted microorganisms overcoming geochemical extremes at significant depths within the crust of the Earth. Attention is required to understand the near subsurface and its continuity with surface systems, where numerous novel microbial members with unique physiological modifications remain to be identified. This surface-subsurface relationship raises key questions about networking of surface hydrology, geochemistry affecting near-subsurface microbial composition, and resiliency of subsurface ecosystems. Here, we apply molecular and geochemical approaches to determine temporal microbial composition and environmental conditions of filtered borehole fluid from the Edgar Experimental Mine (∼150 m below the surface) in Idaho Springs, CO. Samples were collected over a 4-year collection period from expandable packers deployed to accumulate fluid in previously drilled boreholes located centimeters to meters apart, revealing temporal evolution of borehole microbiology. Meteoric groundwater feeding boreholes demonstrated variable recharge rates likely due to a complex and undefined fracture system within the host rock. 16S rRNA gene analysis determined that unique microbial communities occupy the four boreholes examined. Two boreholes yielded sequences revealing the presence of Desulfosporosinus, Candidatus Nitrotoga, and Chelatococcus associated with endemic subsurface communities. Two other boreholes presented sequences related to nonsubsurface-originating microbiota. High concentration of sulfate along with detected sulfur reducing and oxidizing microorganisms suggests that sulfur related metabolic strategies are prominent within these near-subsurface boreholes. Overall, results indicate that microbial community composition in the near-subsurface is highly dynamic at very fine spatial scales (

Rocks that react with liquid water are widespread but spatiotemporally limited throughout the solar system, except for Earth. Rock-forming minerals with high iron content and accessory minerals with high amounts of radioactive elements are essential to support rock-hosted microbial life by supplying organics, molecular hydrogen, and/or oxidants. Recent technological advances have broadened our understanding of the rocky biosphere, where microbial inhabitation appears to be difficult without nutrient and energy inputs from minerals. In particular, microbial proliferation in igneous rock basements has been revealed using innovative geomicrobiological techniques. These recent findings have dramatically changed our perspective on the nature and the extent of microbial life in the rocky biosphere, microbial interactions with minerals, and the influence of external factors on habitability. This study aimed to gather information from scientific and/or technological innovations, such as omics-based and single-cell level characterizations, targeting deep rocky habitats of organisms with minimal dependence on photosynthesis. By synthesizing pieces of rock-hosted life, we can explore the evo-phylogeny and ecophysiology of microbial life on Earth and the life’s potential on other planetary bodies.

Caves are semi-closed ecosystems that experience the absence of sunlight and often, poor organic matter conditions due to physical isolation. Therefore, microbial activity is a crucial factor for sustaining life in caves. However, the heterogeneity of microbial diversity and community structure in relation to different types of habitats in karstic caves (e.g., drip water residue, mineral deposits, rock varnish) remains largely unknown. In this study, cave deposit samples in various morphology were analyzed to reveal its bacterial and archaeal diversity of İnsuyu Cave in Burdur Province that is located in central Turkey. Illumina MiSeq sequencing of seven cave deposit samples (S1–S7) from the zones with a diverse array of environmental conditions revealed a total of four bacterial phyla including (1) Proteobacteria ranging from 80.20% to 98.72%, (2) Actinobacteria ranging from 0.23–13.03%, (3) Firmicutes ranging from 0.18 to 12.61% and (4) Bacteroidetes ranging from 0.00% to 3.80%, while none of the samples exhibits sign for archaeal life form. The heatmap diagram on abundance and distribution of the genera, referring to the results of 16S rRNA gene-based metagenomic analysis of the cave-dwelling microbial communities in İnsuyu Cave, revealed that the structures of the bacterial communities were significantly different across the samples (S1–S7). The high abundance of taxa that is relevant to anthropogenic activity revealed a strong impact of agricultural activity on the bacterial composition of İnsuyu Cave. [ABSTRACT FROM AUTHOR]

The effects of high radiation as a biological extreme have historically been, and continue to be, extensively researched in the fields of radiation biology and astrobiology. However, the absence of radiation as an extreme has received relatively limited attention from the scientific community, with its effects on life remaining unclear. The currently accepted model of the radiation dose-damage relationship for organisms is the linear no-threshold (LNT) model, which predicts a positive linear correlation between dose and damage that intercepts at zero dose corresponding to zero damage. Despite its wide-spread implementation, the LNT model is continuously being challenged by various new models, with the hormesis model as one of its main competitors. This model also postulates damage at high doses but, in contrast to the LNT model, it predicts beneficial stimulation of growth at low doses. Experiments to date have not yet been able to conclusively validate or dismiss either of these models. The aim of the collaborative Subsurface Experiment of Life in Low Radiation (SELLR) project was to test these competing models on prokaryotes in a well-characterised environment and provide a robust experimental set up to investigate low radiation in terrestrial and non-terrestrial environments. Bacterial growth assays using Bacillus subtilis and Escherichia coli were performed under ultra low ionising radiation in the Boulby International Subsurface Astrobiology Laboratory (BISAL) facilities of the Boulby Underground Laboratory at Boulby mine (Redcar & Cleveland, UK) and were used to investigate effects on viability and signs of preconditioning. No significant effect on bacterial growth was observed from exposure to radiation doses ranging from 0.01 times the levels of background radiation typically found in terrestrial surface environments to 100 times that background. Additionally, no preconditioned susceptibility to stress was observed in the bacterial strains grown in sustained low radiation. These data suggest that the extremes of low radiation do not alter growth parameters of these two organisms and that an improved model should be considered for prokaryotes, consisting of a dose-damage response with a threshold at ultra low radiation. We discuss the implications of these data for low radiation as a novel microbiological “extreme.”

Contaminant biodegradation, multi-omics approaches, petroleum hydrocarbons, chlorinated organic compounds, subsurface microbiology Keywords: contaminant biodegradation; multi-omics approaches; petroleum hydrocarbons; chlorinated organic compounds; subsurface microbiology EN contaminant biodegradation multi-omics approaches petroleum hydrocarbons chlorinated organic compounds subsurface microbiology N.PAG N.PAG 3 05/06/21 20210504 NES 210504 Contamination of the subsurface ecosystems with organic compounds originating from the petrochemical, pharmaceutical and agricultural industries is a persistent and growing problem globally. Laczi et al. aimed to review the anaerobic hydrocarbon degradation processes, the most important hydrocarbon degraders, and their diverse metabolic pathways. The acquired genome data revealed small genome size (~1 Mbps) without the capability of hydrocarbon degradation. [Extracted from the article]

The dominant initial phase formed during microbially mediated manganese oxidation is a poorly crystalline birnessite-type phyllomanganate. The occurrence of manganese deposits containing this mineral is of interest for increased understanding of microbial involvement in the manganese cycle. A culture independent molecular approach is used as a first step to investigate the role of microorganisms in forming rare earth element enriched birnessite-type manganese oxides, associated with water bearing rock fractures in a tunnel of the Ytterby mine, Sweden. 16S rRNA gene results show that the chemotrophic bacterial communities are diverse and include a high percentage of uncultured unclassified bacteria while archaeal diversity is low with Thaumarchaeota almost exclusively dominating the population. Ytterby clones are frequently most similar to clones isolated from subsurface environments, low temperature milieus and/or settings rich in metals. Overall, bacteria are dominant compared to archaea. Both bacterial and archaeal abundances are up to four orders of magnitude higher in manganese samples than in fracture water. Potential players in the manganese cycling are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium, and a group of Bacteroidetes sequences that cluster within an uncultured novel genus most closely related to the Terrimonas. This study strongly suggest that the production of the YBS deposit is microbially mediated. [ABSTRACT FROM AUTHOR]

Radioactive 129I, a byproduct of nuclear power generation, can pose risks to human health if released into the environment, where its mobility is highly dependent on speciation. Based on thermodynamic principles, 129I should exist primarily as iodide (I-) in most terrestrial environments; however, organo-129I and 129iodate are also commonly detected in contaminated soils and groundwater. To investigate the capability of biogenic manganese oxides to influence iodide speciation, 17 manganese-oxidizing bacterial strains, representing six genera, were isolated from soils of the Savannah River Site, South Carolina. The isolates produced between 2.6 and 67.1 nmole Mn oxides (ml-1 media after 25 days, pH 6.5). Results from inhibitor assays targeting extracellular enzymes and reactive oxygen species indicated that both play a role in microbe-induced Mn(II) oxidation among the strains examined. Iodide oxidation was not observed in cultures of the most active Mn-oxidizing bacteria, Chryseobacterium sp. strain SRS1 and Chromobacterium sp. strain SRS8, or the fungus, Acremonium strictum strain KR21-2. While substantial amounts of Mn(III/IV) oxides were only generated in cultures at ≥pH 6, iodide oxidation was only observed in the presence of Mn(III/IV) oxides when the pH was ≤5. Iodide oxidation was promoted to a greater extent by synthetic Mn(IV)O2 than biogenic Mn(III/IV) oxides under these low pH conditions (≤pH 5). These results indicate that the influence of biogenic manganese oxides on iodide oxidation and immobilization is primarily limited to low pH environments. [ABSTRACT FROM AUTHOR]

Precambrian Shield rocks host the oldest fracture fluids on Earth, with residence times up to a billion years or more. Water–rock reactions in these fracture systems over geological time have produced highly saline fluids, which can contain millimolar concentrations of H2. Mixing of these ancient Precambrian fluids with meteoric or palaeo-meteoric water can occur through tectonic fracturing, providing microbial inocula and redox couples to fuel blooms of subsurface growth. Here, we present geochemical and microbiological data from a series of borehole fluids of varying ionic strength (0.6–6.4 M) from the Thompson Mine (Manitoba) within the Canadian Precambrian Shield. Thermodynamic calculations demonstrate sufficient energy for H2-based catabolic reactions across the entire range of ionic strengths during mixing of high ionic strength fracture fluids with meteoric water, although microbial H2 consumption and cultivable H2-utilizing microbes were only detected in fluids of ≤1.9 M ionic strength. This pattern of microbial H2 utilization can be explained by the higher potential bioenergetic cost of organic osmolyte synthesis at increasing ionic strengths. We propose that further research into the bioenergetics of osmolyte regulation in halophiles is warranted to better constrain the habitability zones of hydrogenotrophic ecosystems in both terrestrial subsurface, including potential future radioactive waste disposal sites, and other planetary body crustal environments, including Mars. [ABSTRACT FROM AUTHOR]

Microbial communities and their functions in subsurface petroleum reservoirs are crucially important for better understanding of biogeochemical processes and life forms in extreme environment, and developing new strategies and technologies for microbial enhanced energy recovery (MEER) and control of reservoir souring and corrosion in oil production systems as well as bioremediation of contaminated sites. Over the last decades, broad phylogenetic and functional diverse microbial communities of different subsurface oil reservoirs have been described by using the newly available molecular techniques, but the information has been scattered in individual publications. In this review, we present a synthesis on critical analysis of the bacterial and archaeal composition and diversity in oil reservoir systems for a more comprehensive understanding of microbiota and their affiliation to specific oil reservoir conditions, including temperature, salinity and production practices. In particular the frequently reported core and common species of microorganisms with specific characteristics of oil reservoirs including temperature, water flooding, oil and water phases were identified. This review gives an up-to-date information synthesis of the current data available and also a discussion on the challenging questions and the future research needs. [ABSTRACT FROM AUTHOR]

Microbiological studies related to the geological disposal of radioactive waste have been conducted at the Mont Terri rock laboratory in Opalinus Clay, a potential host rock for a deep geologic repository, since 2002. The metabolic potential of microorganisms and their response to excavation-induced effects have been investigated in undisturbed and disturbed claystone cores and in pore- (borehole) water. Results from nearly 15 years of research at the Mont Terri rock laboratory have shown that microorganisms can potentially affect the environment of a repository by influencing redox conditions, metal corrosion and gas production and consumption under favourable conditions. However, the activity of microorganisms in undisturbed Opalinus Clay is limited by the very low porosity, the low water activity, and the largely recalcitrant nature of organic matter in the claystone formation. The presence of microorganisms in numerous experiments at the Mont Terri rock laboratory has suggested that excavation activities and perturbation of the host rock combined with additional contamination during the installation of experiments in boreholes create favourable conditions for microbial activity by providing increased space, water and substrates. Thus effects resulting from microbial activity might be expected in the proximity of a geological repository i.e., in the excavation damaged zone, the engineered barriers, and first containments (the containers). [ABSTRACT FROM AUTHOR]

Dissimilatory microbial reduction of solid-phase Fe(III)-oxides and Fe(III)-bearing phyllosilicates (Fe(III)-phyllosilicates) is an important process in anoxic soils, sediments and subsurface materials. Although various studies have documented the relative extent of microbial reduction of single-phase Fe(III)-oxides and Fe(III)-phyllosilicates, detailed information is not available on interaction between these two processes in situations where both phases are available for microbial reduction. The goal of this research was to use the model dissimilatory iron-reducing bacterium (DIRB)Geobacter sulfurreducensto study Fe(III)-oxide vs. Fe(III)-phyllosilicate reduction in a range of subsurface materials and Fe(III)-oxide stripped versions of the materials. Low-temperature (12 K) Mossbauer spectroscopy was used to infer changes in the relative abundances of Fe(III)-oxide, Fe(III)-phyllosilicate, and phyllosilicate-associated Fe(II) (Fe(II) phyllosilicate). A Fe partitioning model was employed to analyze the fate of Fe(II) and assess the potential for abiotic Fe(II)-catalyzed reduction of Fe(III)-phyllosilicates. The results showed that in most cases Fe(III)-oxide utilization dominated (70–100%) bulk Fe(III) reduction activity, and that electron transfer from oxide-derived Fe(II) played only a minor role (ca. 10–20%) in Fe partitioning. In addition, the extent of Fe(III)-oxide reduction was positively correlated to surface area-normalized cation exchange capacity and the Fe(III)-phyllosilicate/total Fe(III) ratio. This finding suggests that the phyllosilicates in the natural sediments promoted Fe(III)-oxide reduction by binding of oxide-derived Fe(II), thereby enhancing Fe(III)-oxide reduction by reducing or delaying the inhibitory effect that Fe(II) accumulation on oxide and DIRB cell surfaces has on Fe(III)-oxide reduction. In general our results suggest that although Fe(III)-oxide reduction is likely to dominate bulk Fe(III) reduction in most subsurface sediments, Fe(II) binding by phyllosilicates is likely to play a key role in controlling the long-term kinetics of Fe(III) oxide reduction [ABSTRACT FROM PUBLISHER]

Microorganisms found in terrestrial subsurface environments make up a large proportion of the Earth’s biomass. Biogeochemical cycles catalysed by subsurface microbes have the potential to influence the speciation and transport of radionuclides managed in geological repositories. To gain insight on factors that constrain microbial processes within a formation with restricted groundwater flow we performed a meta-community analysis on groundwater collected from multiple discrete fractures underlying the Chalk River Laboratories site (located on Ontario, Canada). Bacterial taxa were numerically dominant in the groundwater. Although these were mainly uncultured, the closest cultivated representatives were from the phenotypically diverse Betaproteobacteria, Deltaproteobacteria, Bacteroidetes, Actinobacteria, Nitrospirae and Firmicutes. Hundreds of taxa were identified but only a few were found in abundance (>1%) across all assemblages. The remainder of the taxa were low abundance. Within an ecological framework of selection, dispersal and drift, the local and regional diversity revealed fewer taxa within each assemblage relative to the meta-community, but the taxa that were present were more related than predicted by chance. The combination of dispersion at one phylogenetic depth and clustering at another phylogenetic depth suggest both niche (dispersion) and filtering (clustering) as drivers of local assembly. Distance decay of similarity reveals apparent biogeography of 1.5 km. Beta diversity revealed greater influence of selection at shallow sampling locations while the influences of dispersal limitation and randomness were greater at deeper sampling locations. Although selection has shaped each assemblage, the spatial scale of groundwater sampling favored detection of neutral processes over selective processes. Dispersal limitation between assemblages combined with local selection means the meta-community is subject to drift, and therefore, likely reflects the differential historical events that have influenced the current bacterial composition. Categorizing the study site into smaller regions of interest of more closely spaced fractures, or of potentially hydraulically connected fractures, might improve the resolution of an analysis to reveal environmental influences that have shaped these bacterial communities.

Subglacial Lake Whillans (SLW), located beneath ~800 m of ice on the Whillans Ice Stream in West Antarctica was sampled in January of 2013, providing the first opportunity to directly examine water and sediments from an Antarctic subglacial lake. To minimize the introduction of surface contaminants to SLW during its exploration, an access borehole was created using a microbiologically clean hot water drill designed to reduce the number and viability of microorganisms in the drilling water. Analysis of 16S rRNA genes (rDNA) amplified from samples of the drilling and borehole water allowed an evaluation of the efficacy of this approach and enabled a confident assessment of the SLW ecosystem inhabitants. Based on an analysis of 16S rDNA and rRNA (i.e., reverse-transcribed rRNA molecules) data, the SLW community was found to be bacterially dominated and compositionally distinct from the assemblages identified in the drill system. The abundance of bacteria (e.g., Candidatus Nitrotoga, Sideroxydans, Thiobacillus, and Albidiferax) and archaea (Candidatus Nitrosoarcheaum) related to chemolithoautotrophs was consistent with the oxidation of reduced iron, sulfur, and nitrogen compounds having important roles as pathways for primary production in this permanently dark ecosystem. Further, the prevalence of Methylobacter in surficial lake sediments combined with the detection of methanogenic taxa in the deepest sediment horizons analyzed (34-36 cm) provided evidence for methane cycling beneath the West Antarctic Ice Sheet. Large ratios of rRNA to rDNA were observed for several OTUs abundant in the water column and sediments (e.g., Albidiferax, Methylobacter, Candidatus Nitrotoga, Sideroxydans, and Smithella), suggesting a potentially active role for these taxa in the SLW ecosystem. Our findings are consistent with chemosynthetic microorganisms serving as the ecological foundation in this dark subsurface environment, providing new organic matter that sustains a microbial ecosystem beneath the West Antarctic Ice Sheet.

Continental subsurface environments can present significant energetic challenges to the resident microorganisms. While these environments are geologically diverse, potentially allowing energy harvesting by microorganisms that catalyze redox reactions, many of the abundant electron donors and acceptors are insoluble and therefore not directly bioavailable. Extracellular electron transfer (EET) is a metabolic strategy that microorganisms can deploy to meet the challenges of interacting with redox-active surfaces. Though mechanistically characterized in a few metal-reducing bacteria, the role, extent, and diversity of EET in subsurface ecosystems remains unclear. Since this process can be mimicked on electrode surfaces, it opens the door to electrochemical techniques to enrich for and quantify the activities of environmental microorganisms in situ. Here, we report the electrochemical enrichment of microorganisms from a deep fractured-rock aquifer in Death Valley, California, USA. In experiments performed in mesocosms containing a synthetic medium based on aquifer chemistry, four working electrodes were poised at different redox potentials (272, 373, 472, 572 mV vs. SHE) to serve as electron acceptors, resulting in anodic currents coupled to the oxidation of acetate during enrichment. The anodes were dominated by Betaproteobacteria from the families Comamonadaceae and Rhodocyclaceae. A representative of each dominant family was subsequently isolated from electrode-associated biomass. The EET abilities of the isolated Delftia strain (designated WE1-13) and Azonexus strain (designated WE2-4) were confirmed in electrochemical reactors using working electrodes poised at 522 mV vs. SHE. The rise in anodic current upon inoculation was correlated with a modest increase in total protein content. Both genera have been previously observed in mixed communities of microbial fuel cell enrichments, but this is the first direct measurement of their electrochemical activity. While alternate metabolisms (e.g. nitrate reduction) by these organisms were previously known, our observations suggest that additional ‘hidden’ interactions with external electron acceptors are also possible. Electrochemical approaches are well positioned to dissect such extracellular interactions that may be prevalent in the subsurface.

Thermococcus is a genus of hyperthermophilic archaea that is ubiquitous in marine hydrothermal environments growing in anaerobic subsurface habitats but able to survive in cold oxygenated seawater. DNA analyses of Thermococcus isolates were applied to determine the relationship between geographic distribution and relatedness focusing primarily on isolates from the Juan de Fuca Ridge and South East Pacific Rise. Amplified fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) were used to resolve genomic differences in 90 isolates of Thermococcus, making biogeographic patterns and evolutionary relationships apparent. Isolates were differentiated into regionally endemic populations however there was also evidence in some lineages of cosmopolitan distribution. The biodiversity identified in Thermococcus isolates and presence of distinct lineages within the same vent site suggests the utilization of varying ecological niches in this genus. In addition to resolving biogeographic patterns in Thermococcus, this study has raised new questions about the closely related Pyrococcus genus. The phylogenetic placement of Pyrococcus type strains shows the close relationship between Thermococcus and Pyrococcus and the unresolved divergence of these two genera.

Subglacial Lake Whillans (SLW) is located beneath ~800 m of ice on the Whillans Ice Stream in West Antarctica and was sampled in January of 2013, providing the first opportunity to directly examine water and sediments from an Antarctic subglacial lake. To minimize the introduction of surface contaminants to SLW during its exploration, an access borehole was created using a microbiologically clean hot water drill designed to reduce the number and viability of microorganisms in the drilling water. Analysis of 16S rRNA genes (rDNA) amplified from samples of the drilling and borehole water allowed an evaluation of the efficacy of this approach and enabled a confident assessment of the SLW ecosystem inhabitants. Based on an analysis of 16S rDNA and rRNA (i.e., reverse-transcribed rRNA molecules) data, the SLW community was found to be bacterially dominated and compositionally distinct from the assemblages identified in the drill system. The abundance of bacteria (e.g., Candidatus Nitrotoga, Sideroxydans, Thiobacillus, and Albidiferax) and archaea (Candidatus Nitrosoarchaeum) related to chemolithoautotrophs was consistent with the oxidation of reduced iron, sulfur, and nitrogen compounds having important roles as pathways for primary production in this permanently dark ecosystem. Further, the prevalence of Methylobacter in surficial lake sediments combined with the detection of methanogenic taxa in the deepest sediment horizons analyzed (34-36 cm) supported the hypothesis that methane cycling occurs beneath the West Antarctic Ice Sheet. Large ratios of rRNA to rDNA were observed for several operational taxonomic units abundant in the water column and sediments (e.g., Albidiferax, Methylobacter, Candidatus Nitrotoga, Sideroxydans, and Smithella), suggesting a potentially active role for these taxa in the SLWecosystem. Our findings are consistent with chemosynthetic microorganisms serving as the ecological foundation in this dark subsurface environment, providing new organic matter that sustains a microbial ecosystem beneath the West Antarctic Ice Sheet. [ABSTRACT FROM AUTHOR]

The high UV radiation and scarcity of liquid water in desert areas make these regions among the most challenging environments for life. Here we studied the multitolerant microbial communities of deserts in Iran. Three bacterial strains were isolated and identified asMicrobacterium phyllosphaeraeMG3,Kocuria flavaMG5 andExiguobacterium acetylicumMG7. UV resistance and desiccation tolerance in MG7 and MG5 strains were comparable to multiresistantDeinococcus radiodurans. Also, resistance to mitomycin C and hydrogen peroxide in all isolated strains were significant. The strain MG3 showed moderate to poor salt tolerance but strains MG5 and MG7 showed high NaCl tolerance. [ABSTRACT FROM AUTHOR]

Continental subsurface environments can present significant energetic challenges to the resident microorganisms. While these environments are geologically diverse, potentially allowing energy harvesting by microorganisms that catalyze redox reactions, many of the abundant electron donors and acceptors are insoluble and therefore not directly bioavailable. Extracellular electron transfer (EET) is a metabolic strategy that microorganisms can deploy to meet the challenges of interacting with redox-active surfaces. Though mechanistically characterized in a few metal-reducing bacteria, the role, extent, and diversity of EET in subsurface ecosystems remains unclear. Since this process can be mimicked on electrode surfaces, it opens the door to electrochemical techniques to enrich for and quantify the activities of environmental microorganisms in situ. Here, we report the electrochemical enrichment of microorganisms from a deep fractured-rock aquifer in Death Valley, CA, USA. In experiments performed in mesocosms containing a synthetic medium based on aquifer chemistry, four working electrodes (WEs) were poised at different redox potentials (272, 373, 472, 572 mV vs. SHE) to serve as electron acceptors, resulting in anodic currents coupled to the oxidation of acetate during enrichment. The anodes were dominated by Betaproteobacteria from the families Comamonadaceae and Rhodocyclaceae. A representative of each dominant family was subsequently isolated from electrodeassociated biomass. The EET abilities of the isolated Delftia strain (designated WE1-13) and Azonexus strain (designated WE2-4) were confirmed in electrochemical reactors using WEs poised at 522 mV vs. SHE. The rise in anodic current upon inoculation was correlated with a modest increase in total protein content. Both genera have been previously observed in mixed communities of microbial fuel cell enrichments, but this is the first direct measurement of their electrochemical activity. While alternate metabolisms (e.g., nitrate reduction) by these organisms were previously known, our observations suggest that additional 'hidden' interactions with external electron acceptors are also possible. Electrochemical approaches are well positioned to dissect such extracellular interactions that may be prevalent in the subsurface. [ABSTRACT FROM AUTHOR]

Researchers at the Center for Frontiers of Subsurface Energy Security (CFSES) have conducted laboratory and modeling studies to better understand the interplay between microorganisms and geochemistry for geological carbon storage (GCS). We provide evidence of microorganisms adapting to high pressure CO 2 conditions and identify factors that may influence survival of cells to CO 2 stress. Factors that influenced the ability of cells to survive exposure to high-pressure CO 2 in our experiments include mineralogy, the permeability of cell walls and/or membranes, intracellular buffering capacity, and whether cells live planktonically or within biofilm. Column experiments show that, following exposure to acidic water, biomass can remain intact in porous media and continue to alter hydraulic conductivity. Our research also shows that geochemical changes triggered by CO 2 injection can alter energy available to populations of subsurface anaerobes and that microbial feedbacks on this effect can influence carbon storage. Our research documents the impact of CO 2 on microorganisms and in turn, how subsurface microorganisms can influence GCS. We conclude that microbial presence and activities can have important implications for carbon storage and that microorganisms should not be overlooked in further GCS research. [ABSTRACT FROM AUTHOR]

Microbially mediated reduction of Fe(III) in chlorite and biotite byShewanella oneidensisMR-1 leads to a significant reduction in sorption of both Cs and Sr compared to the abiotic systems. As seen in previous studies, biotite is a more efficient sorbent than chlorite. Reduction of the mineral-associated Fe(III) causes increased dissolution of both minerals and reduces the sites available for Cs and Sr sorption. As this dissolution progresses it causes desorption of Cs and Sr from chlorite, but not biotite. Subsequent exposure to air increases sorption due to precipitation of secondary Fe(III) oxyhydroxide minerals derived from the Fe(II) released by bio-reduction. In contrast to successful bioremediation of redox active elements, this study suggests that microbial Fe(III) reduction could enhance the migration of Cs and Sr through phyllosilicate-dominated strata. [ABSTRACT FROM AUTHOR]

The prokaryotic community at the “La Cira-Infantas” oil field, located in Colombia's Middle Magdalena Valley Basin, was characterized using 16S rRNA gene sequence analysis. This characterization is a first-step in assessing the dynamics of microbial degradation and defining strategies that may increase oil recovery and quality at the site. Two 16S rRNA gene libraries were generated from the total community DNA extracted from production water using both Eubacterial and Archaea universal primers. Sequence analysis of the libraries indicated that a large percentage of Eubacteria clones were affiliated with class α-, β- and δ-Proteobacteria, ClostridiaandBacteroidetes. Archaea clones were dominated byMethanobacteriaandMethanococci. Annotations at these generic levels indicate that the prokaryotic community has the following metabolic capacities: i) reduction of sulfur-compounds and fermentation, ii) nitrate reduction and sulfide oxidation, iii) decomposition of aromatic compounds, and iv) methane production. These results are discussed in the context of the importance of the characterized metabolic capacities for oil biodegradation in the mesothermic and water-flooded environment of this reservoir. [ABSTRACT FROM AUTHOR]

Little is known about how microbial activity affects the hydraulic properties of karst aquifers. To explore the potential impacts of microbial activity on the hydraulic properties of karst aquifers, microbiological analysis, heat tracer, isotope (dissolved inorganic carbon isotope, δ 13 C DIC ) and aqueous geochemical analyses were conducted at six monitoring wells in Northern Guangdong Province, China. Greater hydraulic conductivity corresponded to a low temperature gradient to an extent; the temperature gradient in karst groundwater aquifers can reflect the degree of dissolution. Higher HCO 3 - concentrations coupled with lower d-excess and pH values at B2 and B6 reflect potential microbial activity (e.g., Sulfuricurvum kujiense ) causing carbonate dissolution. Microbial activity or the input of anthropogenic acids, as evidenced by significantly more positive δ 13 C DIC values, potentially affect carbonate dissolution in deep karst aquifers, which eventually alters hydraulic properties of karst aquifer. However, more direct evidence is needed to quantify the effects of microbial activity on carbonate dissolution in karst aquifers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liang, Li, Wang, Li, Yang, Chen, Gao and Sun.)

The moonmilk deposits within the alpine Hundsalm cave in Austria offered the opportunity to investigate anthropogenically uninfluenced microbiomes. Via cultivation experiments we were able to show that the communities were cold-adapted and oligotrophic. Combined qPCR, DGGE, cloning and sequencing data further highlighted that the archaeal community basically comprises a low number of species, though highly abundant. These organisms are assumed to form new lineages within theEuryarchaeota, while the detectedThaumarchaeota, closely related to ammonium oxidizers, form a second, but minor, abundant group within the moonmilk deposits. Moreover, in terms of abundance the archaeal community clearly outnumbers bacteria (e.g., generaPseudomonas, FlavobacteriumandRhodococcus) and fungi within the investigated microbiomes. In contrast to the highly complex bacterial and fungal communities, only a low number of archaeal species form a constant and essential element within the moonmilk speleothems and other cave-internal habitats. [ABSTRACT FROM AUTHOR]

Thermococcus is a genus of hyperthermophilic archaea that is ubiquitous in marine hydrothermal environments growing in anaerobic subsurface habitats but able to survive in cold oxygenated seawater. DNA analyses of Thermococcus isolates were applied to determine the relationship between geographic distribution and relatedness focusing primarily on isolates from the Juan de Fuca Ridge and South East Pacific Rise. Amplified fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) were used to resolve genomic differences in 90 isolates of Thermococcus, making biogeographic patterns and evolutionary relationships apparent. Isolates were differentiated into regionally endemic populations however there was also evidence in some lineages of cosmopolitan distribution. The biodiversity identified in Thermococcus isolates and presence of distinct lineages within the same vent site suggests the utilization of varying ecological niches in this genus. In addition to resolving biogeographic patterns in Thermococcus, this study has raised new questions about the closely related Pyrococcus genus. The phylogenetic placement of Pyrococcus type strains shows the close relationship between Thermococcus and Pyrococcus and the unresolved divergence of these two genera. [ABSTRACT FROM AUTHOR]

Fe oxidation is often the first chemical reaction that initiates weathering and disaggregation of intact bedrock into regolith. Here we explore the use of pyrosequencing tools to test for evidence that bacteria participate in these reactions in deep regolith. We analyze regolith developed on volcaniclastic rocks of the Fajardo formation in a ridgetop within the rainforest of the Luquillo Mountains of Puerto Rico. In the 9-m-deep regolith profile, the primary minerals chlorite, feldspar, and pyroxene are detected near 8.3 m but weather to kaolinite and Fe oxides found at shallower depths. Over the regolith profile, both total and heterotrophic bacterial cell counts generally increase from the bedrock to the surface. Like other soil microbial studies, the dominant phyla detected are Proteobacteria, Acidobacteria, Planctomycetes, and Actinobacteria. Proteobacteria (α,β,γandδ) were the most abundant at depth (6.8–9 m, 41–44%), while Acidobacteria were the most abundant at the surface (1.4–4.4 m, 37–43%). Despite the fact that Acidobacteria dominated surficial communities while Proteobacteria dominated near bedrock, the near-surface and near-bedrock communities were not statistically different in structure but were statistically different from mid-depth communities. Approximately 21% of all sequences analyzed did not match known sequences: the highest fraction of unmatched sequences was greatest at mid-depth (45% at 4.4 m). At the regolith-bedrock interface where weathering begins, several lines of evidence are consistent with biotic Fe oxidation. At that interface, iron-related bacterial activity tests and culturing indicate the presence of iron-related bacteria, and phylogenetic analyses identified sub-phyla containing known iron-oxidizing microorganisms. Cell densities of iron-oxidizers in the deep saprolite were estimated to be on the order of 105cells g−1. Overall Fe loss was also observed at the regolith-bedrock interface, consistent with bacterial production of organic acids and leaching of Fe-organic complexes. Fe-organic species were also detected to be enriched near the bedrock-regolith interface. In this and other deep weathering profiles, chemolithoautotrophic bacteria that use Fe for energy and nitrate or oxygen as an electron acceptor may play an important role in initiating disaggregation of bedrock. [ABSTRACT FROM PUBLISHER]

Subseafloor sediments represent a large reservoir of organic matter and are inhabited by microbial groups of the three domains of life. Besides impacting the planetary geochemical cycles, the subsurface biosphere remains poorly understood, notably questions related to possible metabolic pathways and selective advantages that may be deployed by buried microorganisms (sporulation, response to stress, dormancy). In order to better understand physiological potentials and possible lifestyles of subseafloor microbial communities, we analyzed two metagenomes from subseafloor sediments collected at 31 mbsf (meters below the sea floor) and 136 mbsf in the Canterbury Basin. Metagenomic phylogenetic and functional diversities were very similar. Phylogenetic diversity was mostly represented by Chloroflexi, Firmicutes and Proteobacteria for Bacteria and by Thaumarchaeota and Euryarchaeota for Archaea. Predicted anaerobic metabolisms encompassed fermentation, methanogenesis and utilization of fatty acids, aromatic and halogenated substrates. Potential processes that may confer selective advantages for subsurface microorganisms included sporulation, detoxication equipment or osmolyte accumulation. Annotation of genomic fragments described the metabolic versatility of Chloroflexi, Miscellaneous Crenarchaeotic Group and Euryarchaeota and showed frequent recombination events within subsurface taxa. This study confirmed that the subseafloor habitat is unique compared to other habitats at the (meta)-genomic level and described physiological potential of still uncultured groups. [ABSTRACT FROM AUTHOR]

Conditioning films and biofilms forming on surfaces of solid materials exposed to aqueous media play a key role in in the interaction between the geo- and biospheres. In this study, time-of-flight secondary ion mass spectrometry and scanning electron microscopy were used to investigate the time scale, mode of formation, and chemistry of conditioning films and biofilms that formed on Si substrates exposed to aquifer water in the subsurface Äspö Hard Rock Laboratory, SE-Sweden. The detection of fragment ions of amino acids, carbohydrates, and carboxylic acids revealed that different types of organic compounds had adhered to the Si surface already after 10 min of exposure to the aquifer fluids, whereas the attachment of microbial cells was first observed after 1000 min. The organic compounds first formed isolated μm-sized accumulations and subsequently started to distribute on the wafer surface more homogenously. Simultaneously further microorganisms attached to the surface and formed biofilm-like cell accumulations after 3 months of exposure to aquifer water. [ABSTRACT FROM PUBLISHER]

Processes of iron mineralization are of great significance to the understanding of Early-Earth geochemistry. Of specific interest are processes at circumneutral pH, where chemical oxidation of Fe can outcompete biological oxidation. To better understand microbially-induced mineral formation and the composition of the involved microbial communities, we set up a series of flow-reactors in the Äspö Hard Rock Laboratory, a 3.6 km tunnel that runs under the Baltic Sea. Various aquifers of Fe2+-rich brackish to saline waters penetrate the tunnel through a series of fractions. The reactors were set up with different combinations of light and aeration conditions, and were connected to three aquifers of differing chemical composition and age. Using a combination of 454 pyrosequencing and CAtalyzed Reporter Deposition Fluorescent In Situ Hybridization we analyzed the bacterial community from these reactors in two consecutive seasons half a year apart. A general decrease in diversity was observed towards the deep part of the tunnel. Multivariate modeling of the community composition and environmental parameters shows that the overall diversity of the microbial community is controlled by salinity as well as carbon and nitrogen sources. However, the composition of iron oxidizing bacteria is driven by pH, O2 and the availability of Fe2+. The latter is mostly supplied by Fe3+ reduction in the reactors. Thus the reactors form a self-sustained ecosystem. Several genera of known aerobic and anaerobic iron oxidizing bacteria were found. Mariprofundus sp. was found to be dominant in many of the samples. This is the first detection of this marine species in groundwater. The microbial community in the reactors is unique in each site, while that in the exposed tunnel is more homogenous. Therefore we suggest that the flow reactors are a good model system to study the nonaccessible microbial communities that are likely present in cracks and crevices of the surrounding bedrock. [ABSTRACT FROM AUTHOR]