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DOE OSTI2023

Cooperative microbial interactions drive spatial segregation in porous environments

The role of microbial interactions and the underlying mechanisms that shape complex biofilm communities are poorly understood. Here we employ a microfluidic chip to represent porous subsurface environments and show that cooperative microbial interactions between free-living and biofilm-forming bacteria trigger active spatial segregation to promote their respective dominance in segregated microhabitats. During initial colonization, free-living and biofilm-forming microbes are segregated from the mixed planktonic inoculum to occupy the ambient fluid and grain surface. Contrary to spatial exclusion through competition, the active spatial segregation is induced by cooperative interactions which improves the fitness of both biofilm and planktonic populations. We further show that free-living Arthrobacter induces the surface colonization by scavenging the biofilm inhibitor, D-amino acids and receives benefits from the public goods secreted by the biofilm-forming strains. Collectively, our results reveal how cooperative microbial interactions may contribute to microbial coexistence in segregated microhabitats and drive subsurface biofilm community succession.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Methanogenesis and Acetogenesis in Hydrogenotrophy with Carbonate Minerals: Dependence on Mineral Surface Area, Biofilm Growth, and Microbial Community

The production, storage, and use of hydrogen are anticipated to grow substantially to achieve energy and climate goals. Consequently, microbial communities in many terrestrial and subsurface Earth environments could be exposed to elevated hydrogen concentrations. Hydrogen stimulates metabolic processes that reduce aqueous chemical species, such as bicarbonate or sulfate, that can exchange with solid mineral phases, but the controls on microbial hydrogenotrophy with mineral sources of electron acceptors are not fully understood. Herein, we applied laboratory experiments and biogeochemical modeling to study the response of a natural microbial community to an elevated partial pressure of hydrogen in the presence of carbonate minerals of varying composition, solubility, and size. Experimental incubations and simulation results showed that hydrogen consumption by microbial communities was initially dominated by sulfate reduction and, subsequently, transitioned to acetogenesis and methanogenesis. The rates of acetogenesis and methanogenesis were not correlated with the solubility of carbonate minerals. Instead, we observed strong linear correlations between the rates and surface area of carbonate minerals. Methane and acetate production slowed down in all incubations after about 2 weeks of incubation, although biogeochemical modeling predicted that the metabolic processes were not thermodynamically limited. Electron microscopy and infrared spectroscopy showed that biofilms with diverse microorganisms grew on the carbonates during this period. The methane δ13C value significantly increased, consistent with slower growth at elevated pH. This work highlights that microbial communities form biofilm on carbonate mineral surfaces as a response to hydrogen and that biofilm formation could pose a strong kinetic limitation to hydrogenotrophic metabolism utilizing carbonate minerals.

Acetobacterium↗

DOE OSTI2024

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA): Molecular and Computational Technologies for Environmental Microbiology (Final Scientific/Technical Report)

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Predominance of Methanomicrobiales and diverse hydrocarbon–degrading taxa in the Appalachian coalbed biosphere revealed through metagenomics and genome–resolved metabolisms

Coalbed deposits are a unique subsurface environment and represent an underutilized resource for methane generation. Microbial communities extant in coalbed deposits are responsible for key subsurface biogeochemical cycling and could be utilized to enhance methane production in areas where existing gas wells have depleted methane stores, or in coalbeds that are unmined, or conversely be utilized for mitigation of methane release. Here we utilize metagenomics and metagenome-assembled genomes to identify extant microbial lineages and genome-resolved microbial metabolisms of coalbed produced water, which has not yet been explored in the Appalachian Basin. Our analyses resulted in the recovery of over 40 metagenome-assembled genomes (MAGs) from eight coalbed methane wells. The most commonly identified taxa among samples were hydrogenotrophic methanogens from the order Methanomicrobiales and these dominant MAGs were highly similar to one another. Conversely, low-abundance coalbed bacterial populations were taxonomically and functionally diverse, mostly belonging to a variety of Proteobacteria classes, and encoding various hydrocarbon solubilization and degradation pathways. Further, the data presented herein provides novel insights into Appalachian Basin coalbed microbial ecology, and our findings provide new perspectives on underrepresented Methanocalculus species and low-relative abundance bacterial assemblages in coalbed environments, and their potential roles in stimulation or mitigation of methane release.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Deep Unsaturated Zone Contributions to Carbon Cycling in Semiarid Environments

Understanding terrestrial carbon cycling has relied primarily on studies of topsoils that are typically characterized to depths shallower than 0.5 m. At a semiarid site instrumented down to 7 m, here we measured seasonal- and depth-resolved carbon inventories and fluxes and groundwater and unsaturated zone flow rates. Measurements showed that ~30% of the CO 2 efflux to the atmosphere (60% in winter) originates from below 1 m, contrary to predictions of less than 1% by Earth System Model land modules. Respiration from deeper roots and deeper microbial communities is supported by favorable subsurface temperatures, moisture, and oxygen availability. Below 1 m, dissolved organic carbon fluxes from the overlying soil and C from deep roots and exudates are expected to be important in sustaining microbial respiration. Finally, because these conditions are characteristic of semiarid climate regions, we contend that Earth System Model land modules should incorporate such deeper soil processes to improve CO 2 flux predictions.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (Final Scientific/Technical Report)

The ENIGMA science focus area (SFA) is a multi-disciplinary, multi-institutional research effort focused on addressing foundational knowledge gaps in environmental microbial communities by studying groundwater and sediment microbiomes in the shallow subsurface at the contaminated Oak Ridge Field Research Site (FRC). We seek to discover and characterize the reciprocal interactions between the microbial communities and the geochemical and geophysical parameters of the shallow subsurface within the contamination plume. The goal of the Fields lab is to help manage and conduct laboratory experiments informed by field observations using ecological and physiological approaches to study microbial populations in situ as well as ex situ (in the laboratory). In particular, we aim to study and characterize the impact of hydrological constraints on free-living and biofilm biomass and activity with increasing spatial and temporal resolution under static and flow conditions. We also aim to help characterize novel microbial groups that are present and active under relevant field conditions, including the development of molecular techniques for in situ detection as well as metabolic interactions that underlie pertinent physiology and ecology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Microbial Interactions With Dissolved Organic Matter Drive Carbon Dynamics and Community Succession

Knowledge of dynamic interactions between natural organic matter (NOM) and microbial communities is critical not only to delineate the routes of NOM degradation/transformation and carbon (C) fluxes, but also to understand microbial community evolution and succession in ecosystems. Yet, these processes in subsurface environments are usually studied independently, and a comprehensive view has been elusive thus far. In this study, we fed sediment-derived dissolved organic matter (DOM) to groundwater microbes and continually analyzed microbial transformation of DOM over a 50-day incubation. To document fine-scale changes in DOM chemistry, we applied high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and soft X-ray absorption spectroscopy (sXAS). We also monitored the trajectory of microbial biomass, community structure and activity over this time period. Together, these analyses provided an unprecedented comprehensive view of interactions between sediment-derived DOM and indigenous subsurface groundwater microbes. Microbial decomposition of labile C in DOM was immediately evident from biomass increase and total organic carbon (TOC) decrease. The change of microbial composition was closely related to DOM turnover: microbial community in early stages of incubation was influenced by relatively labile tannin- and protein-like compounds; while in later stages the community composition evolved to be most correlated with less labile lipid- and lignin-like compounds. These changes in microbial community structure and function, coupled with the contribution of microbial products to DOM pool affected the further transformation of DOM, culminating in stark changes to DOM composition over time. Our study demonstrates a distinct response of microbial communities to biotransformation of DOM, which improves our understanding of coupled interactions between sediment-derived DOM, microbial processes, and community structure in subsurface groundwater.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

Functional Response of a Near-Surface Soil Microbial Community to a Simulated Underground CO 2 Storage Leak

Understanding the impacts of leaks from geologic carbon sequestration, also known as carbon capture and storage, is key to developing effective strategies for carbon dioxide (CO 2 ) emissions management and mitigation of potential negative effects. Here, we provide the first report on the potential effects of leaks from carbon capture and storage sites on microbial functional groups in surface and near-surface soils. Using a simulated subsurface CO 2 storage leak scenario, we demonstrate how CO 2 flow upward through the soil column altered both the abundance (DNA) and activity (mRNA) of microbial functional groups mediating carbon and nitrogen transformations. These microbial responses were found to be seasonally dependent and correlated to shifts in atmospheric conditions. While both DNA and mRNA levels were affected by elevated CO 2 , they did not react equally, suggesting two separate mechanisms for soil microbial community response to high CO 2 levels. The results did not always agree with previous studies on elevated atmospheric (rather than subsurface) CO 2 using FACE (Free-Air CO 2 Enrichment) systems, suggesting that microbial community response to CO 2 seepage from the subsurface might differ from its response to atmospheric CO 2 increases.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Novel candidate taxa contribute to key metabolic processes in Fennoscandian Shield deep groundwaters

The continental deep biosphere contains a vast reservoir of microorganisms, although a large proportion of its diversity remains both uncultured and undescribed. In this study, the metabolic potential (metagenomes) and activity (metatranscriptomes) of the microbial communities in Fennoscandian Shield deep subsurface groundwaters were characterized with a focus on novel taxa. DNA sequencing generated 1270 de-replicated metagenome-assembled genomes and single-amplified genomes, containing 7 novel classes, 34 orders, and 72 families. The majority of novel taxa were affiliated with Patescibacteria, whereas among novel archaea taxa, Thermoproteota and Nanoarchaeota representatives dominated. Metatranscriptomes revealed that 30 of the 112 novel taxa at the class, order, and family levels were active in at least one investigated groundwater sample, implying that novel taxa represent a partially active but hitherto uncharacterized deep biosphere component. The novel taxa genomes coded for carbon fixation predominantly via the Wood–Ljungdahl pathway, nitrogen fixation, sulfur plus hydrogen oxidation, and fermentative pathways, including acetogenesis. These metabolic processes contributed significantly to the total community’s capacity, with up to 9.9% of fermentation, 6.4% of the Wood–Ljungdahl pathway, 6.8% of sulfur plus 8.6% of hydrogen oxidation, and energy conservation via nitrate (4.4%) and sulfate (6.0%) reduction. Key novel taxa included the UBA9089 phylum, with representatives having a prominent role in carbon fixation, nitrate and sulfate reduction, and organic and inorganic electron donor oxidation. These data provided insights into deep biosphere microbial diversity and their contribution to nutrient and energy cycling in this ecosystem.

Candidatus↗

DOE OSTI2014

Fe-oxide grain coatings support bacterial Fe-reducing metabolisms in 1.7 - 2.0 km-deep subsurface quartz arenite sandstone reservoirs of the Illinois Basin (USA)

The Cambrian-age Mt. Simon Sandstone, deeply buried within the Illinois Basin of the midcontinent of North America, contains quartz sand grains ubiquitously encrusted with iron-oxide cements and dissolved ferrous iron in pore-water. Although microbial iron reduction has previously been documented in the deep terrestrial subsurface, the potential for diagenetic mineral cementation to drive microbial activity has not been well studied. In this study, two subsurface formation water samples were collected at 1.72 and 2.02 km, respectively, from the Mt. Simon Sandstone in Decatur, Illinois. Low-diversity microbial communities were detected from both horizons and were dominated by Halanaerobiales of Phylum Firmicutes. Iron-reducing enrichment cultures fed with ferric citrate were successfully established using the formation water. Phylogenetic classification identified the enriched species to be related to Vulcanibacillus from the 1.72 km depth sample, while Orenia dominated the communities at 2.02 km of burial depth. Species-specific quantitative analyses of the enriched organisms in the microbial communities suggest that they are indigenous to the Mt. Simon Sandstone. Optimal iron reduction by the 1.72 km enrichment culture occurred at a temperature of 40°C (range 20–60°C) and a salinity of 25 parts per thousand (range 25–75 ppt). This culture also mediated fermentation and nitrate reduction. In contrast, the 2.02 km enrichment culture exclusively utilized hydrogen and pyruvate as the electron donors for iron reduction, tolerated a wider range of salinities (25–200 ppt), and exhibited only minimal nitrate- and sulfate-reduction. In addition, the 2.02 km depth community actively reduces the more crystalline ferric iron minerals goethite and hematite. The results suggest evolutionary adaptation of the autochthonous microbial communities to the Mt. Simon Sandstone and carries potentially important implications for future utilization of this reservoir for CO2 injection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

An Evaluation of Subsurface Microbial Activity Conditional to Subsurface Temperature, Porosity, and Permeability at North American Carbon Sequestration Sites

Several nations, including the United States, recognize global climate change as a force transforming the global ecosphere. Carbon dioxide (CO 2 ) is a greenhouse gas that contributes to the evolving climate. Reduction of atmospheric CO 2 levels is a goal for many nations and carbon sequestration which traps CO 2 in the Earth’s subsurface is one method to reduce atmospheric CO 2 levels. Among the variables that must be considered in developing this technology to a national scale is microbial activity. Microbial activity or biomass can change rock permeability, alter artificial seals around boreholes, and play a key role in biogeochemistry and accordingly may determine how CO 2 is sequestered underground. Certain physical parameters of a reservoir found in literature (e.g., temperature, porosity, and permeability) may indicate whether a reservoir can host microbial communities. In order to estimate which subsurface formations may host microbes, this report examines the subsurface temperature, porosity, and permeability of underground rock formations that have high potential to be targeted for CO 2 sequestration. Of the 268 North American wellbore locations from the National Carbon Sequestration Database (NATCARB; National Energy and Technology Laboratory, 2015) and 35 sites from Nelson and Kibler (2003), 96 sequestration sites contain temperature data. Of these 96 sites, 36 sites have temperatures that would be favorable for microbial survival, 48 sites have mixed conditions for supporting microbial populations, and 11 sites would appear to be unfavorable to support microbial populations. Future studies of microbe viability would benefit from a larger database with more formation parameters (e.g. mineralogy, structure, and groundwater chemistry), which would help to increase understanding of where CO 2 sequestration could be most efficiently implemented.

25 ENERGY STORAGE↗

DOE OSTI2014

Metabolic interdependencies between phylogenetically novel fermenters and respiratory organisms in an unconfined aquifer

Fermentation-based metabolism is an important ecosystem function often associated with environments rich in organic carbon, such as wetlands, sewage sludge, and the mammalian gut. The diversity of microorganisms and pathways involved in carbon and hydrogen cycling in sediments and aquifers and the impacts of these processes on other biogeochemical cycles remain poorly understood. Here we used metagenomics and proteomics to characterize microbial communities sampled from an aquifer adjacent to the Colorado River at Rifle, Colorado, USA, and document interlinked microbial roles in geochemical cycling. The organic carbon content in the aquifer was elevated via two acetate-based biostimulation treatments. Samples were collected at three time points, with the objective of extensive genome recovery to enable metabolic reconstruction of the community. Fermentative community members include genomes from a new phylum (ACD20), phylogenetically novel members of the Chloroflexi and Bacteroidetes, as well as candidate phyla genomes (OD1, BD1-5, SR1, WWE3, ACD58, TM6, PER, and OP11). These organisms have the capacity to produce hydrogen, acetate, formate, ethanol, butyrate, and lactate, activities supported by proteomic data. The diversity and expression of hydrogenases suggests the importance of hydrogen currency in the subsurface. Our proteogenomic data further indicate the consumption of fermentation intermediates by Proteobacteria can be coupled to nitrate, sulfate, and iron reduction. Thus, fermentation carried out by previously unstudied members of sediment microbial communities may be an important driver of diverse subsurface biogeochemical cycles.

metabolic interdependencies↗

DOE OSTI2016

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over time revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. As a result, these data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Produced Water DNA Database (PW-DNA): Utilizing KBase to generate an environmental specific curated molecular database

The deep subsurface is estimated to host the majority of Earth’s microbial biomass yet remains one of the most challenging environments to access and study. One common approach to investigate these microbial communities is through the analysis of produced water from subsurface reservoirs, where researchers can assess water and gas chemistry along with molecular (DNA/RNA) sequence data. Advances in high-throughput sequencing have greatly expanded our understanding of these environments and their biotechnological potential. However, further progress requires large-scale, integrative meta-analyses across diverse datasets. To address this need, we developed the Produced Water-DNA (PW-DNA) Database, a curated, publicly available resource that consolidates microbial DNA/RNA sequences, geochemical data, and relevant metadata from in situ hydrocarbon environments such as coal beds, oil reservoirs, and natural gas systems. The PW-DNA database delivers three core benefits to the research community: (1) it improves data sharing by linking environmental microbial datasets with corresponding geochemical parameters, enabling more robust filtering and analysis; (2) it connects with complementary research databases to promote broader dissemination and interoperability; and (3) it supports technological innovation by serving as a resource for identifying microbial trends and exploring genetic potential. While individual studies have highlighted basin-specific microbial communities and functional redundancy in biogeochemical cycling, a comprehensive, system-wide perspective is needed to better understand connectivity and novelty across subsurface ecosystems. By designing the PW-DNA in the KBase platform, we provide a reproducible, visual framework for integrating large-scale genomic and geochemical data, enabling researchers to perform more informed analyses and experimental design. Ultimately, this resource enhances the ability to identify, characterize, and interpret microbial functions across diverse subsurface environments, thereby accelerating discovery in subsurface microbiology and biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Effect of tectonic processes on biosphere–geosphere feedbacks across a convergent margin

The subsurface is among Earth’s largest biomes, but the extent to which microbial communities vary across tectonic plate boundaries or interact with subduction-scale geological processes remains unknown. Here we compare bacterial community composition with deep-subsurface geochemistry from 21 hot springs across the Costa Rican convergent margin. We find that cation and anion compositions of the springs reflect the dip angle and position of the underlying tectonic structure and also correlate with the bacterial community. Co-occurring microbial cliques related to cultured chemolithoautotrophs that use the reverse tricarboxylic acid cycle (rTCA) as well as abundances of metagenomic rTCA genes correlate with concentrations of slab-volatilized carbon. This, combined with carbon isotope evidence, suggests that fixation of slab-derived CO2 into biomass may support a chemolithoautotrophy-based subsurface ecosystem. We calculate that this forearc subsurface biosphere could sequester 1.4 × 10 9 to 1.4 × 10 10 mol of carbon per year, which would decrease estimates of the total carbon delivered to the mantle by 2 to 22%. Furthermore, based on the observed correlations, we suggest that distribution and composition of the subsurface bacterial community are probably affected by deep tectonic processes across the Costa Rican convergent margin and that, by sequestering carbon volatilized during subduction, these chemolithoautotrophic communities could in turn impact the geosphere.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2013

Viral Infection of Subsurface Microorganisms and Metal/Radionuclide Transport (Final Technical Report)

Microbially mediated metabolisms have been identified as a significant factor either directly or indirectly impacting the fate and transport of heavy metal/radionuclide contaminants. To date microorganisms have been isolated from contaminated environments. Examination of annotated finished genome sequences of many of these subsurface isolates from DOE sites, revealed evidence of prior viral infection. To date the role that viruses play influencing microbial mortality and the resulting community structure which directly influences biogeochemical cycling in soils and sedimentary environments remains poorly understood. The objective of this exploratory study was to investigate the role of viral infection of subsurface bacteria and the formation of contaminant-bearing viral particles. This objective was approached by examining the following working hypotheses: (i) subsurface microorganisms are susceptible to viral infections by the indigenous subsurface viral community, and (ii) viral surfaces will adsorb heavy metals and radionuclides. Our results have addressed basic research needed to accomplish the BER Long Term Measure to provide sufficient scientific understanding such that DOE sites would be able to incorporate coupled physical, chemical and biological processes into decision making for environmental remediation or natural attenuation and long-term stewardship by establishing viral-microbial relationships on the subsequent fate and transport of heavy metals and radionuclides. Here we demonstrated that viruses play a significant role in microbial mortality and community structure in terrestrial subsurface sedimentary systems. The production of viral-like particles within subsurface sediments in response to biostimulation with dissolved organic carbon and a terminal electron acceptor resulted in the production of viral-like particles. Organic carbon alone did not result in significant viral production and required the addition of a terminal electron acceptor (nitrate), indicating that nutrients are not limiting viral production, but rather substrates that can be converted into energy for host metabolism. Our results also revealed that cell abundance was not correlated to the mineralization of organic carbon, but rather viruses were positively correlated with carbon mineralization. This is a result of viral-mediated cell lysis and demonstrates that viruses are sensitive indicators of microbial activity. Viruses as an indicator of microbial activity was not unique to batch culture studies as results obtained from an in situ field experiment conducted at the DOE Old Rifle Field site. This study revealed that viral abundance increased in response to the injection of oxygenated groundwater and influx of dissolved organic carbon whereas cell abundance changes were minimal. However, the extent to which viral-mediated cell lysis alters organic matter pools subsequently influencing microbial community structure and biogeochemical function remains a critical question in subsurface biogeochemical cycling.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

R&D Effort of Geologic Hydrogen Production at the National Renewable Energy Lab (NREL)

Geologic hydrogen (geoH2) is an emerging technology with massive current market interest and distinct potential to change the paradigm of hydrogen production. Two major subsurface processes influence the amount of geoH2 that are available for energy extraction: 1) geochemical reactions of H2O and Fe2+-bearing rocks which can produce hydrogen in the subsurface environment, where 2) various active microbial communities consume hydrogen as an energy source before the hydrogen reaches the surface. The net gain of hydrogen from these two competing processes dictates the production rate of geoH2. A recent study (Templeton et al., 2024) suggested that for most natural geoH2 systems, five orders of magnitude of production rate enhancement are needed to make geoH2 production economical in the near term. Effective enhancement of the production rate requires an in-depth understanding of the two geoH2 processes, in order to promote the H2-generating geochemical processes and suppress the H2-consuming microbial processes. However, current significant knowledge gaps in these two processes hinders the efforts to formulate strategies to enhance geoH2 production. The National Renewable Energy Laboratory (NREL) is a U.S. Department of Energy National Laboratory with the core mission of leading research, innovation, and strategic partnership to deliver solutions for a clean energy based economy. NREL's extensive research portfolio in hydrogen, bioenergy, geothermal, industrial decarbonization, and energy analysis makes us well positioned to conduct interdisciplinary research and facilitate technology deployment in the geoH2 space. In this presentation, we will discuss ongoing geoH2 research and engagement efforts at NREL, including: 1) geochemical investigation to understand the reaction mechanisms and production rate and potential of different source minerals and rocks, 2) microbiological investigation to understand methanogenesis and acetogenesis in the subsurface geoH2 environment, and identify effective inhibitors for these microbial processes, and 3) preliminary analysis for geoH2 production in the State of Minnesota, where abundant Fe-rich rocks for stimulated geoH2 production and ample opportunity to utilize geoH2 in transforming iron and steel industries are currently available.

08 HYDROGEN↗

DOE OSTI2019

Microbial Diversity in the Deep Sandstones of a Carbon Sequestration Well

A lack of data on microbes in the deep subsurface hinders our ability to predict how these microbial communities will respond to carbon sequestration. Previous studies predicted how microbial communities may shift due to an influx of carbon dioxide (CO2) by 1) examining waters with naturally-occurring elevated CO2 concentration; and/or 2) via experiments involving exposure of laboratory microbial strains to elevated concentrations of CO2 relative to their native subsurface environment. To identify microbes naturally found in carbon sequestration target reservoirs, water samples were collected from three potential target reservoirs in the Powder River Basin, Wyoming which include the Hulett, Lakota, and Minnelusa sandstones. Water samples were collected at depths of 8,060 ft (Lakota), 8,330 ft (Hulett), 9,380 ft (Minnelusa), 9,463 ft (Minnelusa), and 9,544 ft (Minnelusa) for microbial DNA analysis as well as water chemistry and stable isotope analyses to look for any microbial trends that could be related to water chemistry alone. Identification of microbial communities present in the sampled water was determined via 16S rRNA sequencing; these data were compared to 16S rRNA isolated from drilling mud to distinguish native from any microbes introduced via drilling processes. Enrichment experiments reveal that the collected water samples contain active (e.g., growing) microbes and how the microbial community may adapt upon the introduction of CO2.

Wawrousek, K↗