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Proteins potentially involved in stress response and associated EPS formation in H. congolense WG8. Black outlined boxes represent a significant difference (P < 0.05, Student’s t test) in protein abundances between low- and high-pressure conditions. Boxes without outlines represent changes in protein concentration that were not statistically significant (P > 0.05, Student’s t test).
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The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demo...
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Citations
... The common mechanisms of ameliorating this "souring," such as NO 3 injection, represent intentional modulation of the subsurface biosphere at industrial scales. Another is the introduction of Halanaerobium in deep hydraulically fractured shale gas reservoirs, which were previously sterile or near sterile (Booker et al., 2019). In some cases, oil and gas companies have intentionally stimulated existing microbial populations to degrade hydrocarbons and produce methane by injecting amendments, such as yeast or algal extracts and nutrients (Barnhart et al., 2022;Ritter et al., 2015). ...
The Anthropocene has been framed around humanity's impact on atmospheric, biologic, and near‐surface processes, such as land use and vegetation change, greenhouse gas emissions, and the above‐ground hydrologic cycle. Groundwater extraction has lowered water tables in many key aquifers but comparatively little attention has been given to the impacts in the deeper subsurface. Here, we show that fluid fluxes from the extraction and injection of fluids associated with oil and gas production and inflow of water into mines likely exceed background flow rates in deep (>500 m) groundwater systems at a global scale. Projected carbon capture and sequestration (CCS), geothermal energy production, and lithium extraction to facilitate the energy transition will require fluid production rates exceeding current oil and co‐produced water extraction. Natural analogs and geochemical modeling indicate that subsurface fluid manipulation in the Anthropocene will likely appear in the rock record. The magnitude and importance of these changes are unclear, due to a lack of understanding of how deep subsurface hydrologic and geochemical cycles and associated microbial life interact with the rest of the Earth system.
... The common mechanisms of ameliorating this "souring," such as NO3 injection, represent intentional modulation of the subsurface biosphere at industrial scales. Another is the introduction of Halanaerobium in deep hydraulically fractured shale gas reservoirs, which were previously sterile or near sterile (Booker et al., 2019). ...
... How the microbial community develops over time when a new cavern is leached, needs to be studied in more detail. It is known from fracking operations in the US that fresh water injected into shale becomes highly saline over time, which gives rise to the activity of halophiles (especially Halanaerobium) [34,35]. ...
Hydrogen will be one of the key components for renewable energy storage in the future energy systems as it can be stored in significant volumes to overcome daily to seasonal energy fluctuations. Subsurface storage in salt caverns will be a first step. These caverns are created by solution mining in underground salt formations. Despite the high salinity in this environment, salt caverns harbor microbial life. These microorganisms can not only survive in these caverns by using unique adaptation mechanisms, but they actually cause several risks to hydrogen storage. Different metabolisms can use hydrogen as electron donor, leading to hydrogen loss and in the worst case also to H2S formation. The knowledge on salt cavern microbiology and subsequent possible effects of hydrogen is still in its infancy and only a limited number of salt caverns have been investigated so far. This review summarizes the current knowledge and key questions about halophilic (salt-loving) microbes, their adaptation strategies, their origin and potential consequences of their metabolisms. It also discusses the major factors influencing microbial activities and potential risks. This review emphasizes that more research and field trials with extensive microbial monitoring are needed before hydrogen storage in a biologically active system can be safely achieved at a global scale.
... However, since thiosulfate (S 2 O 3 − ) or elemental sulphur (S 0 ) were not added and therefore not measured in the ISW:PW incubations, it would not be possible to quantify the use of these as electron acceptors by the enriched Halanaerobium sp., which leads the way to the only other possibility of fermentative metabolism of carbohydrates being the most plausible explanation for the observed enrichments of Halanaerobium sp. in this ISW:PW experiment, and consistent with earlier literature (Abdeljabbar, et al., 2013;Booker, et al., 2017Booker, et al., , 2019Kögler, et al., 2021;Lipus, et al., 2017). ...
... Glycine betaine, an osmoprotectant found across all three domains of life (60,61), has been previously observed in elevated concentrations in saline environments (62). Its expression under high-pressure culture conditions may persist in deeper habitats (63,64). Methylamines derived from glycine betaine serve as noncompetitive substrates for methanogenesis; however, most methanogens are incapable of efficiently converting glycine betaine to methane (45). ...
Deep-sea and subseafloor sedimentary environments host heterotrophic microbial communities that contribute to Earth’s carbon cycling. However, the potential metabolic functions of individual microorganisms and their biogeographical distributions in hadal ocean sediments remain largely unexplored. In this study, we conducted single-cell genome sequencing on sediment samples collected from six sites (7,445–8,023 m water depth) along an approximately 500 km transect of the Japan Trench during the International Ocean Discovery Program Expedition 386. A total of 1,886 single-cell amplified genomes (SAGs) were obtained, offering comprehensive genetic insights into sedimentary microbial communities in surface sediments (<1 m depth) above the sulfate-methane transition zone along the Japan Trench. Our genome data set included 269 SAGs from Atribacterota JS1, the predominant bacterial clade in these hadal environments. Phylogenetic analysis classified SAGs into nine distinct phylotypes, whereas metagenome-assembled genomes were categorized into only two phylotypes, advancing JS1 diversity coverage through a single cell-based approach. Comparative genomic analysis of JS1 lineages from different habitats revealed frequent detection of genes related to organic carbon utilization, such as extracellular enzymes like clostripain and α-amylase, and ABC transporters of oligopeptide from Japan Trench members. Furthermore, specific JS1 phylotypes exhibited a strong correlation with in situ methane concentrations and contained genes involved in glycine betaine metabolism. These findings suggest that the phylogenomically diverse and novel Atribacterota JS1 is widely distributed in Japan Trench sediment, playing crucial roles in carbon cycling within the hadal sedimentary biosphere.
IMPORTANCE
The Japan Trench represents tectonically active hadal environments associated with Pacific plate subduction beneath the northeastern Japan arc. This study, for the first time, documented a large-scale single-cell and metagenomic survey along an approximately 500 km transect of the Japan Trench, obtaining high-quality genomic information on hadal sedimentary microbial communities. Single-cell genomics revealed the predominance of diverse JS1 lineages not recoverable through conventional metagenomic binning. Their metabolic potential includes genes related to the degradation of organic matter, which contributes to methanogenesis in the deeper layers. Our findings enhance understanding of sedimentary microbial communities at water depths exceeding 7,000 m and provide new insights into the ecological role of biogeochemical carbon cycling in the hadal sedimentary biosphere.
... KEYWORDS membrane adaptation, Halanaerobium, fractured shale, intact polar lipids, lipidomics, salinity, hydraulic retention time S ome microorganisms that are inadvertently introduced into the deep biosphere during hydraulic fracturing to extract natural gas and oil from shale formations survive numerous stressors and persist for long periods of time (1)(2)(3)(4). The persisting microbial communities in engineered shale reservoirs are mostly comprised of anaerobic halophilic and halotolerant taxa (5,6). Halanaerobium has been found to be ubiquitous and dominate many geologically distinct formations (3,6). ...
... Fractured shale is an extreme environment for microbial growth and, by virtue of drilling/hydraulic fracturing, a highly disturbed ecosystem. It is characterized by elevated temperatures, nutrient limitation, anoxia, elevated pressures, and brine-level salinities (1,5,16). For instance, the salinity of flowback and produced water, which is co-collected with natural gas, could increase more than fourfold up to 6,000 ppm in just about 30 days post-fracturing (17,18). ...
Microorganisms that persist in fractured shale reservoirs cause several problems including secreting foul gases and forming biofilms. Current biocontrol measures often fail due to limited knowledge of their in situ activities. The plasma membrane protects the cell, mediates many of its critical functions, and responds to intracellular cues and ecological perturbations through physicochemical modifications. As such, it provides valuable insight into the physiological adaptation of microorganisms in disturbed environmental systems. Here, we (i) demonstrate how changes in salinity and hydraulic retention time (HRT) influence the plasma membrane intact polar lipid (IPL) chemistry of model bacterium, Halanaerobium congolense WG10, and mixed microbial consortia enriched from shale-produced fluids and (ii) elucidate adjustments in membrane IPL chemistry during biofilm growth relative to planktonic cells. We incubated H. congolense WG10 in chemostats under three salinities (7%, 13%, and 20% NaCl), operated under three HRTs (19.2, 24, and 48 h), and in drip flow biofilm reactors under the same salinity gradients. Also, mixed microbial consortia in produced fluids were enriched in triplicate chemostat vessels under three HRTs (19.2, 24, and 72 h) and biofilm reactors. Lipids were analyzed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that phosphatidylglycerols, cardiolipins, and phosphatidylethanolamines were predominantly enriched in planktonic H. congolense WG10 cells grown at hypersalinity (20%) compared to optimum (13%). In addition, several zwitterionic phosphatidylcholines and phosphatidylethanolamines were higher in abundance during biofilm growth. These observations suggest that microbial adaptation and biofilm formation in fractured shale are enabled by strategic plasma membrane IPL chemistry adjustments.
IMPORTANCE
Microorganisms inadvertently introduced into the shale reservoir during fracturing face multiple stressors including brine-level salinities and starvation. However, some anaerobic halotolerant bacteria adapt and persist for long periods of time. They produce hydrogen sulfide, which sours the reservoir and corrodes engineering infrastructure. In addition, they form biofilms on rock matrices, which decrease shale permeability and clog fracture networks. These reduce well productivity and increase extraction costs. Under stress, microbes remodel their plasma membrane to optimize its roles in protection and mediating cellular processes such as signaling, transport, and energy metabolism. Hence, by observing changes in the membrane lipidome of model shale bacteria, Halanaerobium congolense WG10, and mixed consortia enriched from produced fluids under varying subsurface conditions and growth modes, we provide insight that advances our knowledge of the fractured shale biosystem. We also offer data-driven recommendations for improving biocontrol efficacy and the efficiency of energy recovery from unconventional formations.
... Hence, we draw the general conclusion that some of the injected species such as Halanaerobium, Arhodomonas, Desulfohalobium, and Methanohalophilus are dominant due to their capacity to thrive in the surface facility and under reservoir's conditions. This is supported by [13,41,63] who have found similar patterns in shale gas and high salinity oil reservoirs. Through this study, we observed the potential resistance against the applied biocide in the injection water sample and thus propose the injection of nitrate or molybdate [64] as alternative biocides since the field is already encompassing an active intrinsic nitrate-reducing community such as ...
... Metabolic plasticity is the capacity to alter a physiological response to environmental conditions. It is generally described based on single microorganisms grown in pure cultures at different environmental conditions to determine whether they can adapt to changes at their physiological limits [46,47]. Using the metabolic niche framework provides the possibility to not only study metabolic plasticity of individual phylotypes that are not yet cultured, but also investigate individual genes or whole changes in pathways for hundreds of phylotypes at a time. ...
The environmental niche concept describes the distribution of a taxon in the environment and can be used to understand community dynamics, biological invasions, and the impact of environmental changes. The uses and applications are still restricted in microbial ecology, largely due to the complexity of microbial systems and associated methodological limitations. The development of shotgun metagenomics and metatranscriptomics opens new ways to investigate the microbial niche by focusing on the metabolic niche within the environmental space. Here, we propose the metabolic niche framework, which, by defining the fundamental and realised metabolic niche of microorganisms, has the potential to not only provide novel insights into habitat preferences and the metabolism associated, but also to inform on metabolic plasticity, niche shifts, and microbial invasions.
... Halanaerobium species have received attention as the dominant microbial taxon in flowback fluids recovered from numerous shale gas wells. Halanaerobium isolates have the metabolic potential to produce corrosive sulfide (via thiosulfate reduction), acids and form biofilms [61,62]. Other studies have linked their presence with the potential for sulfide generation [42,63] The family Shewanellaceae was detected in 55% of the samples, exhibiting greater abundance in the Bowland and Sichuan formations (Fig. 4). ...
Background:
Hydraulically fractured shales offer a window into the deep biosphere, where hydraulic fracturing creates new microbial ecosystems kilometers beneath the surface of the Earth. Studying the microbial communities from flowback fluids that are assumed to inhabit these environments provides insights into their ecophysiology, and in particular their ability to survive in these extreme environments as well as their influence on site operation e.g. via problematic biofouling processes and/or biocorrosion. Over the past decade, research on fractured shale microbiology has focused on wells in North America, with a few additional reported studies conducted in China. To extend the knowledge in this area, we characterized the geochemistry and microbial ecology of two exploratory shale gas wells in the Bowland Shale, UK. We then employed a meta-analysis approach to compare geochemical and 16S rRNA gene sequencing data from our study site with previously published research from geographically distinct formations spanning China, Canada and the USA.
Results:
Our findings revealed that fluids recovered from exploratory wells in the Bowland are characterized by moderate salinity and high microbial diversity. The microbial community was dominated by lineages known to degrade hydrocarbons, including members of Shewanellaceae, Marinobacteraceae, Halomonadaceae and Pseudomonadaceae. Moreover, UK fractured shale communities lacked the usually dominant Halanaerobium lineages. From our meta-analysis, we infer that chloride concentrations play a dominant role in controlling microbial community composition. Spatio-temporal trends were also apparent, with different shale formations giving rise to communities of distinct diversity and composition.
Conclusions:
These findings highlight an unexpected level of compositional heterogeneity across fractured shale formations, which is not only relevant to inform management practices but also provides insight into the ability of diverse microbial consortia to tolerate the extreme conditions characteristic of the engineered deep subsurface.
... To address this knowledge gap, we used a temporally-resolved dataset from six subsurface fractured shale wells to interrogate host-virus dynamics and CRISPR arrays in a natural ecosystem. Subsurface fractured shales, which are relatively closed ecosystems with limited immigration, elevated temperatures, lower microbial diversity and likely dominated by biofilms, present an opportunity to address these questions through strong CRISPR-based host-viral linkages (21,23,(46)(47)(48)(49). We hypothesized and found that CRISPR viral defense systems were widely encoded across hosts within shale microbial communities. ...
... Taxa unable to tolerate high temperatures and elevated salinity are likely outcompeted, while biofilms and spatially distinct niches likely emerge and expand (55,56). Thus, we expect that microbial communities within the established wells are more spatially heterogeneous and partitioned into more stabilized niches, while microbial communities in the new wells are initially well mixed, more spatially homogenous, and lack established biofilms (49,57). ...
Viruses are the most ubiquitous biological entities on earth. Even so, elucidating the impact of viruses on microbial communities and associated ecosystem processes often requires identification of strong host-virus linkages – an undeniable challenge in many ecosystems. Subsurface fractured shales present a unique opportunity to first make these strong linkages and subsequently reveal complex long-term host-virus dynamics and trends in CRISPR array size and frequency. Here, we sampled two replicated sets of fractured shale wells for nearly 800 days (Denver-Julesburg Basin, Colorado, USA). We identified a relatively diverse microbial community with widely encoded CRISPR viral defense systems, which facilitated 2,110 CRISPR-based viral linkages across 90 host MAGs representing 25 phyla. Leveraging these linkages with timeseries data across differing well ages, we observed how patterns of host-virus co-existence develop and converge in this closed ecosystem through time. We observed a transition to smaller CRISPR arrays in older, more established wells, potentially reflecting a natural progression where CRISPR arrays harbor fewer, yet more effective spacers that target viral genes with fewer mutations. Together, our findings shed light on the complexities of host-virus temporal dynamics as well as possible drivers of spacer loss and acquisition within CRISPR arrays of diverse microbial populations through time.
