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DOE OSTI2021

Biogeochemical dynamics and microbial community development under sulfate- and iron-reducing conditions based on electron shuttle amendment

Iron reduction and sulfate reduction are two of the major biogeochemical processes that occur in anoxic sediments. Microbes that catalyze these reactions are therefore some of the most abundant organisms in the subsurface, and some of the most important. Due to the variety of mechanisms that microbes employ to derive energy from these reactions, including the use of soluble electron shuttles, the dynamics between iron- and sulfate-reducing populations under changing biogeochemical conditions still elude complete characterization. Here, we amended experimental bioreactors comprised of freshwater aquifer sediment with ferric iron, sulfate, acetate, and the model electron shuttle AQDS (9,10-anthraquinone-2,6-disulfonate) and monitored both the changing redox conditions as well as changes in the microbial community over time. The addition of the electron shuttle AQDS did increase the initial rate of Fe III reduction; however, it had little effect on the composition of the microbial community. Our results show that in both AQDS- and AQDS+ systems there was an initial dominance of organisms classified as Geobacter (a genus of dissimilatory Fe III -reducing bacteria), after which sequences classified as Desulfosporosinus (a genus of dissimilatory sulfate-reducing bacteria) came to dominate both experimental systems. Furthermore, most of the ferric iron reduction occurred under this later, ostensibly “sulfate-reducing” phase of the experiment. This calls into question the usefulness of classifying subsurface sediments by the dominant microbial process alone because of their interrelated biogeochemical consequences. To better inform models of microbially-catalyzed subsurface processes, such interactions must be more thoroughly understood under a broad range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Geochemical and Microbial Community Attributes in Relation to Hyporheic Zone Geological Facies

The hyporheic zone (HZ) is the active ecotone between the surface stream and groundwater, where exchanges of water, nutrients, and organic matter occur in response to variations in discharge and riverbed properties. Within this region, a confluence of surface-derived organic carbon and subsurface nitrogen (in the form of nitrate) has been shown to stimulate microbial activity and transformations of carbon and nitrogen species. For example, production of gases such as CO 2 , N 2 and N 2 O indicate hyporheic zones might have a significant effect on energy and nutrient flows between the atmosphere and the subsurface. Managed and seasonal river stage changes and geomorphology-controlled sediment texture drive water flow within the HZ of the Columbia River. To examine the relationship between sediment texture, biogeochemistry, and biological activity in the HZ, the grain size distributions for sediment samples taken across 320 m of shoreline were characterized to define geological facies, and the relationships among physical properties of the facies, physicochemical attributes of the local environment, and the structure and activity of associated microbial communities were examined. Mud and sand content and the presence of carbon and nitrogen oxidizers were found to explain the variability in many biogeochemical attributes. Microbial community analysis revealed a high relative abundance of putative ammonia-oxidizing Thaumarchaeota and nitrite-oxidizing Nitrospirae, together comprising ~20% of the total community across all samples, but scant ammonia-oxidizing Bacteria. Network analysis of operational taxonomic units and the measured geophysical, chemical, and functional parameters showed negative relationships between abundance-based modules of organisms and sand and mud contents, and positive relationships with total organic carbon. The relationships identified in this work indicate grain size distribution is a good predictor of biogeochemical properties, and that subsets of the overall microbial community respond to different sediment texture. Some member populations of these sub-communities appear to respond directly to environmental conditions, while others may be dependent on the function of the first group. For example, nitrification is a strong primary response to the observed conditions, and this activity appears to support a larger heterotrophic community. Relationships between facies and hydrobiogeochemical properties enables facies-based conditional simulation/mapping of these properties to inform multiscale modeling of hyporheic exchange and biogeochemical processes.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2017

Developing methanogenic microbial consortia from diverse coal sources and environments

Biogenic gas production is a promising alternative or supplement to conventional methane extraction from coalbeds. Adsorbed and free gas, generated over geologic time, can be supplemented with biogenic gas during short-term engineering operations. There are two generic protocols for doing this. The first is to contact the coal with nutrients to support native bacterial development. The second approach is to inject appropriately cultured ex-situ consortia into subsurface coal accumulations. Research has mainly focused on the former: in-situ stimulation of native microbial communities with added nutrients. Relatively few studies have been conducted on the strategies for enriching ex-situ microbial populations under initial atmospheric exposure for subsequent injection into coal seams to stimulate biodegradation, and methanogenesis. In order to evaluate the feasibility of ex-situ cultivation, natural microbial populations were collected from various hydrocarbon-rich environments and locations characterized by natural methanogenesis. Different rank coals (i.e., lignite, sub-bituminous, bituminous), complex hydrocarbon sources (i.e., oil shale, waxy crude), hydrocarbon seeps, and natural biogenic environments were incorporated in the sampling. Three levels of screening (down-selection to high grade the most productive consortia) allowed selection of microbial populations, favorable nutrient amendments, sources of the microbial community, and quantification of methane produced from various coal types. Incubation periods of up to twenty-four weeks were evaluated at 23 °C. Headspace concentrations of CH 4 and CO 2 were analyzed by gas chromatography. After a two-week incubation period of the most promising microbes, generated headspace gas concentrations reached 873,400 ppm (154 sft 3 /ton or 4.8 scm 3 /g) for methane and 176,370 ppm (31 sft 3 /ton or 0.9 scm 3 /g) for carbon dioxide. Rudimentary statistical assessments – variance analysis (ANOVA) of a single factor - were used to identify trends and levels of significance or impact of the consortia enrichment. We then demonstrated that microbial communities from coal and lake sediments can be enriched and adapted to effectively generate methane under initial atmospheric exposure. The development and enrichment of these methanogenic consortia is described.

01 COAL, LIGNITE, AND PEAT↗

DOE OSTI2025

Distinct Fracture Mineralogy That is Out of Equilibrium With Modern Groundwaters Provides Important Context for Subsurface Life

Rock fracture surfaces in the crust are essential habitat for microorganisms. Fracture‐groundwater interfaces provide physical substrates for biofilm growth and are sources of carbon, nutrients, and electron donors and acceptors. To better understand geochemical processes impacting fracture surfaces and the subsurface microbiome, we identified fractures in archived rock cores from the Soudan formation, which is known to host saline groundwaters and isolated microbial communities dependent on rock‐water interactions. Cores with open fractures were thin sectioned and studied via electron microprobe and synchrotron X‐ray fluorescence microprobe. Most fracture surfaces had mineralogy distinct from that of the bulk rock. Chlorite minerals were abundant on fracture surfaces and had elemental compositions suggesting deposition during late‐stage hydrothermal alteration. Fracture‐lining chlorites likely limit access to iron oxide and sulfide minerals that are active in subsurface biogeochemical cycles. Calcium‐rich rinds were also observed along fracture edges. These rinds were too thin and poorly ordered to be identified via light microscopy or X‐ray diffraction; however, Ca K‐edge micro‐X‐ray absorption near‐edge structure spectroscopy identified them as carbonates, minerals not observed in the bulk rock. Thermodynamic modeling shows that carbonate precipitation is largely unfavorable in Soudan groundwaters, indicating that fracture edge conditions differed from those in modern water samples. Because of the low carbon concentrations in Soudan groundwaters, carbonate rinds likely play an important role in subsurface carbon cycling and may mark fracture surfaces that once hosted biofilms. Overall, this study suggests that fracture alteration can both play an active role in and suppress rock‐water interactions essential to subsurface life.

36 MATERIALS SCIENCE↗

DOE OSTI2022

Cave Thiovulum ( Candidatus Thiovulum stygium) differs metabolically and genomically from marine species

Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O 2 (~7%). The cave’s surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O 2 and NO 3 - as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks

Background Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling. Results We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, or single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric “MW-score” (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut. Conclusion METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 at https://github.com/AnantharamanLab/METABOLIC.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Influence of Microbial Membranes on Biofilm Formation, Scaling Reactions, and Production Chemistry in Engineered Energy Systems (Final Report)

Humans are engineering the deep subsurface for the purposes of energy-capture, energy-storage, and environmental mitigation. Despite the potential for microbial communities to significantly alter the effectiveness of these technologies, we have historically lacked a clear understanding of how microbial cells interact in a fractured shale system. Existing knowledge has been heavily based on genomic information, lacking studies on in situ physiologies, kinetic rates, and attachment mechanisms governing biogeochemical reactions in the system. The overarching goal of this research was to advance understanding of engineered hydrocarbon extraction systems as they evolve from newly developed environments to mature use. Plasma membranes protect microbial cells, mediate many critical functions, and respond to intracellular cues and ecological perturbations through physicochemical modifications. The project objectives were to 1) characterize variables influencing growth and membrane features of shale taxa, 2) characterize the interactions between shale matrices and microorganisms and 3) elucidate engineered and environmental processes driving biogeochemical signatures at the field scale. Objectives were realized through laboratory experiments involving growth of pure cultures and enrichments in continuous culture and biofilms; access to samples from two DOE-NETL funded energy-capture sites: Marcellus Shale Energy and Environmental Laboratory (MSEEL and MSEEL II); and through collaborations with scientists and engineers at the Environmental Molecular Science Laboratory (EMSL) and National Energy Technology Laboratory (NETL). Key findings showed (a) Halanaerobium congolense WG10 makes its membrane more fluid and thicker through increasing its polyunsaturated fatty acid (PUFA) composition under non-optimal salinities (7% and 20%) compared to the optimum (13% NaCl); (b) anionic phospholipids within the plasma membrane are important for high salinity tolerance in H. congolense WG10 while zwitterionic species modulate biofilm physiologies in both isolate and mixed cultures, and (c) tyrosine accumulation with overexpression of Na+/H+ antiporters are essential for osmoprotection in H. congolense WG10 under high salinity while upregulation of aromatic amino acid synthesis and cell envelope maintenance underpin biofilm formation. It is clear from these results that microbial persistence in fractured shale reservoirs under environmental and engineered perturbations requires strategic plasma membrane lipid chemistry adjustments and metabolic restructuring, with implications for improved shale microbiology management and more efficient hydrocarbon extraction approaches targeting microorganisms.

03 NATURAL GAS↗

DOE OSTI2015

Genomic expansion of domain archaea highlights roles for organisms from new phyla in anaerobic carbon cycling.

Archaea represent a significant fraction of Earth's biodiversity, yet they remain much less well understood than Bacteria. Gene surveys, a few metagenomic studies, and some single-cell sequencing projects have revealed numerous little-studied archaeal phyla. Certain lineages appear to branch deeply and may be part of a major phylum radiation. The structure of this radiation and the physiology of the organisms remain almost unknown.We used genome-resolved metagenomic analyses to investigate the diversity, genomes sizes, metabolic capacities, and potential roles of Archaea in terrestrial subsurface biogeochemical cycles. We sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River (USA) and reconstructed the first complete genomes for Archaea using cultivation-independent methods. To provide taxonomic context, we analyzed an additional 151 newly sampled archaeal sequences. We resolved two new phyla within a major, apparently deep-branching group of phyla (a superphylum). The organisms have small genomes, and metabolic predictions indicate that their primary contributions to Earth's biogeochemical cycles involve carbon and hydrogen metabolism, probably associated with symbiotic and/or fermentation-based lifestyles.The results dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum. This study, in combination with recently published work on bacterial phyla lacking cultivated representatives, reveals a fascinating phenomenon of major radiations of organisms with small genomes, novel proteome composition, and strong interdependence in both domains.

Castelle, Cindy J↗

NASA NTRS1994

Subsurface Intertidal Microbes: A Cryptic Source Of Organic Carbon For Beach Ecosystems

Some freshwater, marine or hotspring beaches have no visible source of primary production, yet beneath the surface is an interstitial photosynthetic microbial community. To assess the significance of this source of organic carbon, we measured in situ carbon fixation rates in an intertidal marine beach through a diurnal cycle. Gross fixation for a transect (99 x 1 m) perpendicular to the shore was approx. 4041 mg C fixed/ day, or approx. 41 mg C fixed/ sq m day. In contrast, an adjacent well-established cyanobacterial (Lyngbya) mat was approx. 12 x as productive (approx. 490 mg C fixed/sq m day). Thus, subsurface sand mats may be an overlooked, yet important, endogenous source of organic carbon for intertidal ecosystems, as well as a sink in the global carbon cycle.

Rothschild, Lynn J.↗

DOE OSTI2016

Modern water/rock reactions in Oman hyperalkaline peridotite aquifers and implications for microbial habitability

The Samail ophiolite in Oman is undergoing modern hydration and carbonation of peridotite and may host a deep subsurface biosphere. Previous investigations of hyperalkaline fluids in Oman have focused on fluids released at surface seeps, which quickly lose their reducing character and precipitate carbonates upon contact with the O 2 /CO 2 -rich atmosphere. In this study, geochemical analysis of rocks and fluids from the subsurface provides new insights into the operative reactions in serpentinizing aquifers. Serpentinite rock and hyperalkaline fluids (pH > 10), which exhibit millimolar concentrations of Ca 2+ , H 2 and CH 4 , as well as variable sulfate and nitrate, were accessed from wells situated in mantle peridotite near Ibra and studied to investigate their aqueous geochemistry, gas concentrations, isotopic signatures, mineralogy, Fe speciation and microbial community composition.The bulk mineralogy of drill cuttings is dominated by olivine, pyroxene, brucite, serpentine and magnetite. At depth, Fe-bearing brucite is commonly intermixed with serpentine, whereas near the surface, olivine and brucite are lost and increased magnetite and serpentine is detected. Micro-Raman spectroscopy reveals at least two distinct generations of serpentine present in drill cuttings recovered from several depths from two wells. Fe K-edge X-ray absorption near-edge spectroscopy (XANES) analysis of the lizardite shows a strong tetrahedral Fe coordination, suggesting a mixture of both Fe(II) and Fe(III) in the serpentine. Magnetite veins are also closely associated with this second generation serpentine, and 2-10 μm magnetite grains overprint all minerals in the drill cuttings. Thus we propose that the dissolved H 2 that accumulates in the subsurface hyperalkaline fluids was evolved through low temperature oxidation and hydration of relict olivine, as well as destabilization of pre-existing brucite present in the partially serpentinized dunites and harzburgites. In particular, we hypothesize that Fe-bearing brucite is currently reacting with dissolved silica in the aquifer fluids to generate late-stage magnetite, additional serpentine and dissolved H 2 . Dissolved CH 4 in the fluids exhibits the most isotopically heavy carbon in CH 4 reported in the literature thus far. The CH 4 may have formed through abiotic reduction of dissolved CO 2 or through biogenic pathways under extreme carbon limitation. The methane isotopic composition may have also been modified by significant methane oxidation. 16S rRNA sequencing of DNA recovered from filtered hyperalkaline well fluids reveals an abundance of Meiothermus, Thermodesulfovibrionaceae (sulfate-reducers) and Clostridia (fermenters). The fluids also contain candidate phyla OP1 and OD1, as well as Methanobacterium (methanogen) and Methylococcus sp. (methanotroph). Finally, the composition of these microbial communities suggests that low-temperature hydrogen and methane generation, coupled with the presence of electron acceptors such as nitrate and sulfate, sustains subsurface microbial life within the Oman ophiolite.

58 GEOSCIENCES↗

DOE OSTI2016

Stygofauna enhance prokaryotic transport in groundwater ecosystems

More than 97% of the world’s freshwater reserves are found in aquifers, making groundwater one of the most important resources on the planet. Prokaryotic communities in groundwater underpin the turnover of energy and matter while also maintaining groundwater purity. Thus, knowledge of microbial transport in the subsurface is crucial for maintaining groundwater health. Here, we describe for the first time the importance of stygofauna as vectors for prokaryotes. The “hitch-hiking” prokaryotes associated with stygofauna may be up to 5 orders of magnitude higher in abundance and transported up to 34× faster than bulk groundwater flow. We also demonstrate that prokaryotic diversity associated with stygofauna may be higher than that of the surrounding groundwater. Stygofauna are a newly recognized prokaryotic niche in groundwater ecosystems that have the potential to transport remediating, water purifying and pathogenic prokaryotes. Furthermore, stygofauna may influence ecosystem dynamics and health at a microbial level, and at a larger scale could be a new source of prokaryotic diversity in groundwater ecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2016

Measurement of bacterial replication rates in microbial communities

Culture-independent microbiome studies have increased our understanding of the complexity and metabolic potential of microbial communities. However, to understand the contribution of individual microbiome members to community functions, it is important to determine which bacteria are actively replicating. We developed an algorithm, iRep, that uses draft-quality genome sequences and single time-point metagenome sequencing to infer microbial population replication rates. The algorithm calculates an index of replication (iRep) based on the sequencing coverage trend that results from bi-directional genome replication from a single origin of replication. We apply this method to show that microbial replication rates increase after antibiotic administration in human infants. We also show that uncultivated, groundwater-associated, Candidate Phyla Radiation bacteria only rarely replicate quickly in subsurface communities undergoing substantial changes in geochemistry. Our method can be applied to any genome-resolved microbiome study to track organism responses to varying conditions, identify actively growing populations and measure replication rates for use in modeling studies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Culturing of “Unculturable” Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria From Groundwater

Recovery and cultivation of diverse environmentally-relevant microorganisms from the terrestrial subsurface remain a challenge despite recent advances in modern molecular technology. Here, we applied complex carbon (C) sources, i.e., sediment dissolved organic matter (DOM) and bacterial cell lysate, to enrich groundwater microbial communities for 30 days. As comparisons, we also included enrichments amended with simple C sources including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex C is far more effective in enriching diverse and distinct microorganisms from groundwater than simple C. Simple C enrichments yield significantly lower biodiversity, and are dominated by few phyla (e.g., Proteobacteria and Bacteroidetes ), while microcosms enriched with complex C demonstrate significantly higher biodiversity including phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia , Planctomycetes , and Armatimonadetes ). Subsequent isolation from complex C enrichments yielded 228 bacterial isolates representing five phyla, 17 orders, and 56 distinct species, including candidate novel, rarely cultivated, and undescribed organisms. Results from this study will substantially advance cultivation and isolation strategies for recovering diverse and novel subsurface microorganisms. Obtaining axenic representatives of “once-unculturable” microorganisms will enhance our understanding of microbial physiology and function in different biogeochemical niches of terrestrial subsurface ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Reactive transport benchmarks for subsurface environmental simulation

Over the last 20 years, we have seen firsthand the evolution of multicomponent reactive transport modeling and the expanding range and increasing complexity of subsurface applications it is being used to address. There is a growing reliance on reactive transport modeling (RTM) to address some of the most compelling issues facing our planet: climate change, nuclear waste management, contaminant remediation, and pollution prevention. While these issues are motivating the development of new and improved capabilities for subsurface environmental modeling using RTM (e.g., biogeochemistry from cell-scale physiology to continental-scale terrestrial ecosystems, nonisothermal multiphase conditions, coupled geomechanics), there remain longstanding challenges in characterizing the natural variability of hydrological, biological, and geochemical properties in subsurface environments and limited success in transferring models between sites and across scales. An equally important trend over the last 20 years is the evolution of modeling from a service sought out after data has been collected to a multifaceted research approach that provides (1) an organizing principle for characterization and monitoring activities; (2) a systematic framework for identifying knowledge gaps, developing and integrating new knowledge; and (3) a mechanistic understanding that represents the collective wisdom of the participating scientists and engineers. There are now large multidisciplinary projects where the research approach is model-driven, and the principal product is a holistic predictive simulation capability that can be used as a test bed for alternative conceptualizations of processes, properties, and conditions. Much of the future growth and expanded role for RTM will depend on its continued ability to exploit technological advancements in the earth and environmental sciences. Advances in measurement technology, particularly in molecular biology (genomics), isotope fractionation, and high-resolution X-ray spectroscopy, have created new lines of research that can be used to inform the conceptualization of reactions and rate laws and validate mechanistic models. For example, spectroscopy has identified the oxidation states of key components and elemental distributions at increasingly smaller scales and lower concentrations; molecular biology has progressed from identifying the presence of microbes to characterization of which microbial communities are active and what they are doing (i.e., microbial function), which has led in turn to the identification of active processes under conditions beyond what analytical chemistry can discern; isotope ratios in pore water and solid phases that can be used to distinguish between biotic from abiotic processes, sorption from precipitation, and origin and age of groundwater. The other noteworthy development that is expanding the role of RTM in subsurface environmental modeling is he advance in computational technology that is enabling the simulation of more coupled processes with increasing mechanistic detail. In some cases, this involves the inclusion of more reactive species and/or microbial populations in the simulations; in other cases, the impact is through the ability to achieve high resolution of property distributions over longer simulated times. To achieve these ambitious objectives for subsurface reactive transport simulation, the subsurface science and engineering community is being driven to provide accurate assessments of engineering performance and risk for important issues with far-reaching consequences. As a result, the complexity and detail of subsurface processes, properties, and conditions that can be simulated have significantly expanded. This expansion was enabled, in part, by advances in measurement technology, computing technology, and numerical techniques.

benchmarking↗

DOE OSTI2017

Methane-Oxidizing Bacteria Shunt Carbon to Microbial Mats at a Marine Hydrocarbon Seep

The marine subsurface is a reservoir of the greenhouse gas methane. While microorganisms living in water column and seafloor ecosystems are known to be a major sink limiting net methane transport from the marine subsurface to the atmosphere, few studies have assessed the flow of methane-derived carbon through the benthic mat communities that line the seafloor on the continental shelf where methane is emitted. We analyzed the abundance and isotope composition of fatty acids in microbial mats grown in the shallow Coal Oil Point seep field off Santa Barbara, CA, USA, where seep gas is a mixture of methane and CO 2 . We further used stable isotope probing (SIP) to track methane incorporation into mat biomass. We found evidence that multiple allochthonous substrates supported the rich growth of these mats, with notable contributions from bacterial methanotrophs and sulfur-oxidizers as well as eukaryotic phototrophs. Fatty acids characteristic of methanotrophs were shown to be abundant and 13 C-enriched in SIP samples, and DNA-SIP identified members of the methanotrophic family Methylococcaceae as major 13 CH 4 consumers. Members of Sulfuricurvaceae, Sulfurospirillaceae, and Sulfurovumaceae are implicated in fixation of seep CO 2 . The mats’ autotrophs support a diverse assemblage of co-occurring bacteria and protozoa, with Methylophaga as key consumers of methane-derived organic matter. This study identifies the taxa contributing to the flow of seep-derived carbon through microbial mat biomass, revealing the bacterial and eukaryotic diversity of these remarkable ecosystems.

16S rRNA gene↗

DOE OSTI2019

In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing

In the last decade, extensive application of hydraulic fracturing technologies to unconventional low-permeability hydrocarbon-rich formations has significantly increased natural gas production in the United States and abroad. The injection of surface-sourced fluids to generate fractures in the deep subsurface introduces microbial cells and substrates to low-permeability rock. A subset of injected organic additives has been investigated for their ability to support biological growth in shale microbial community members; however, to date, little is known on how complex xenobiotic organic compounds undergo biotransformations in this deep rock ecosystem. Here, high-resolution chemical, metagenomic, and proteomic analyses reveal that widely-used surfactants are degraded by the shale-associated taxa Halanaerobium, both in situ and under laboratory conditions. These halotolerant bacteria exhibit surfactant substrate specificities, preferring polymeric propoxylated glycols (PPGs) and longer alkyl polyethoxylates (AEOs) over polyethylene glycols (PEGs) and shorter AEOs. Enzymatic transformation occurs through repeated terminal-end polyglycol chain shortening during co-metabolic growth through the methylglyoxal bypass. Furthermore, this work provides the first evidence that shale microorganisms can transform xenobiotic surfactants in fracture fluid formulations, likely affecting the efficiency of hydrocarbon recovery, and demonstrating an important association between injected substrates and microbial growth in an engineered subsurface ecosystem.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2019

WHONDRS 48 Hour Diel Cycling Study at the Columbia River in Washington, USA

This dataset is from a sampling campaign completed over the course of 48 hours to investigate the effects of the upstream Priest Rapids Dam-induced water level variation on aqueous chemistry, dissolved organic matter chemistry, and metabolic potential of microbial communities at the Columbia River, WA, USA. Filtered surface water (SW) and pore water (PP) samples were collected every three hours in August 2018. This dataset is one of seven companion datasets that were part of the WHONDRS 48 Hour Diel Cycling Study of freshwater rivers. All seven datasets are available on ESS-DIVE; they can be found by searching for WHONDRS 48 Hour.This package contains a variety of chemical and hydrologic variables. All data types are provided in a standard format. Basic geochemistry analyses (e.g., non-purgeable organic carbon concentration, cations, anions, SpC, pH) were conducted, standardized photos were taken, surface water hydrographs were collated from existing instrumentation, and extensive metadata were captured. Aqueous chemistry variables include USGS codes in the first portion of the variable name (e.g., 00940_Cl_mg_per_L). These codes allow for interoperability with USGS water quality data. In addition, samples were delivered to the Environmental Molecular Sciences Laboratory (EMSL; https://www.pnnl.gov/environmental-molecular-sciences-laboratory) for high resolution characterization of dissolved organic matter via 12 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The data package contains instructions to process the FTICR data based on the user’s specific needs, and sample-level summary indices derived from the FTICR data will be added to the data package. Beyond the data in this data package, metagenomic data are being generated on associated microbial communities. When these data are available, the data package will be updated with information on accessing them. Please contact WHONDRS@pnnl.gov for more information. Please use the data package’s DOI to cite the data package. We ask that you email us at WHONDRS@pnnl.gov to let us know that you're using the data and acknowledge WHONDRS and the U.S. Department of Energy's Subsurface Biogeochemical Research program—which generously provides funding to WHONDRS—in your publications, proposals, presentations, etc. All data are free to be used for any purpose, such as for manuscripts, presentations, and grant proposals. There is no obligation to include WHONDRS members as co-authors.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2019

WHONDRS 48 Hour Diel Cycling Study at the Jordan River, Israel

This dataset is from a sampling campaign completed over the course of 48 hours to investigate the effects of groundwater pumping induced water level variation on aqueous chemistry, dissolved organic matter chemistry, and metabolic potential of microbial communities at the Jordan River, Israel. Filtered surface water (SW) and pore water (PP) samples were collected every three hours in August 2018. This dataset is one of seven companion datasets that were part of the WHONDRS 48 Hour Diel Cycling Study of freshwater rivers. All seven datasets are available on ESS-DIVE; they can be found by searching for WHONDRS 48 Hour.This package contains a variety of chemical and hydrologic variables. All data types are provided in a standard format. Basic geochemistry analyses (e.g., non-purgeable organic carbon concentration, cations, anions, SpC, pH) were conducted, standardized photos were taken, surface water hydrographs were collated from existing instrumentation, and extensive metadata were captured. Aqueous chemistry variables include USGS codes in the first portion of the variable name (e.g., 00940_Cl_mg_per_L). These codes allow for interoperability with USGS water quality data. In addition, samples were delivered to the Environmental Molecular Sciences Laboratory (EMSL; https://www.pnnl.gov/environmental-molecular-sciences-laboratory) for high resolution characterization of dissolved organic matter via 12 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The data package contains instructions to process the FTICR data based on the user’s specific needs, and sample-level summary indices derived from the FTICR data will be added to the data package. Beyond the data in this data package, metagenomic data are being generated on associated microbial communities. When these data are available, the data package will be updated with information on accessing them. Please contact WHONDRS@pnnl.gov for more information. Please use the data package’s DOI to cite the data package. We ask that you email us at WHONDRS@pnnl.gov to let us know that you're using the data and acknowledge WHONDRS and the U.S. Department of Energy's Subsurface Biogeochemical Research program—which generously provides funding to WHONDRS—in your publications, proposals, presentations, etc. All data are free to be used for any purpose, such as for manuscripts, presentations, and grant proposals. There is no obligation to include WHONDRS members as co-authors.

54 ENVIRONMENTAL SCIENCES↗