Geological activity shapes the microbiome in deep-subsurface aquifers by advection.

Subsurface environments host diverse microorganisms in fluid-filled fractures; however, little is known about how geological and hydrological processes shape the subterranean biosphere. Here, we sampled three flowing boreholes weekly for 10 mo in a 1478-m-deep fractured rock aquifer to study the role of fracture activity (defined as seismically or aseismically induced fracture aperture change) and advection on fluid-associated microbial community composition. We found that despite a largely stable deep-subsurface fluid microbiome, drastic community-level shifts occurred after events signifying physical changes in the permeable fracture network. The community-level shifts include the emergence of microbial families from undetected to over 50% relative abundance, as well as the replacement of the community in one borehole by the earlier community from a different borehole. Null-model analysis indicates that the observed spatial and temporal community turnover was primarily driven by stochastic processes (as opposed to deterministic processes). We, therefore, conclude that the observed community-level shifts resulted from the physical transport of distinct microbial communities from other fracture(s) that outpaced environmental selection. Given that geological activity is a major cause of fracture activity and that geological activity is ubiquitous across space and time on Earth, our findings suggest that advection induced by geological activity is a general mechanism shaping the microbial biogeography and diversity in deep-subsurface habitats across the globe.

A global perspective on bacterial diversity in the terrestrial deep subsurface.

While recent efforts to catalogue Earth's microbial diversity have focused upon surface and marine habitats, 12-20 % of Earth's biomass is suggested to exist in the terrestrial deep subsurface, compared to ~1.8 % in the deep subseafloor. Metagenomic studies of the terrestrial deep subsurface have yielded a trove of divergent and functionally important microbiomes from a range of localities. However, a wider perspective of microbial diversity and its relationship to environmental conditions within the terrestrial deep subsurface is still required. Our meta-analysis reveals that terrestrial deep subsurface microbiota are dominated by Betaproteobacteria, Gammaproteobacteria and Firmicutes, probably as a function of the diverse metabolic strategies of these taxa. Evidence was also found for a common small consortium of prevalent Betaproteobacteria and Gammaproteobacteria operational taxonomic units across the localities. This implies a core terrestrial deep subsurface community, irrespective of aquifer lithology, depth and other variables, that may play an important role in colonizing and sustaining microbial habitats in the deep terrestrial subsurface. An in silico contamination-aware approach to analysing this dataset underscores the importance of downstream methods for assuring that robust conclusions can be reached from deep subsurface-derived sequencing data. Understanding the global panorama of microbial diversity and ecological dynamics in the deep terrestrial subsurface provides a first step towards understanding the role of microbes in global subsurface element and nutrient cycling.

Iron-Fueled Life in the Continental Subsurface: Deep Mine Microbial Observatory, South Dakota, USA.

Iron-bearing minerals are key components of the Earth's crust and potentially critical energy sources for subsurface microbial life. The Deep Mine Microbial Observatory (DeMMO) is situated in a range of iron-rich lithologies, and fracture fluids here reach concentrations as high as 8.84 mg/liter. Iron cycling is likely an important process, given the high concentrations of iron in fracture fluids and detection of putative iron-cycling taxa via marker gene surveys. However, a previous metagenomic survey detected no iron cycling potential at two DeMMO localities. Here, we revisited the potential for iron cycling at DeMMO using a new metagenomic data set including all DeMMO sites and FeGenie, a new annotation pipeline that is optimized for the detection of iron cycling genes. We annotated functional genes from whole metagenomic assemblies and metagenome-assembled genomes and characterized putative iron cycling pathways and taxa in the context of local geochemical conditions and available metabolic energy estimated from thermodynamic models. We reannotated previous metagenomic data, revealing iron cycling potential that was previously missed. Across both metagenomic data sets, we found that not only is there genetic potential for iron cycling at DeMMO, but also, iron is likely an important source of energy across the system. In response to the dramatic differences we observed between annotation approaches, we recommend the use of optimized pipelines where the detection of iron cycling genes is a major goal. IMPORTANCE We investigated iron cycling potential among microbial communities inhabiting iron-rich fracture fluids to a depth of 1.5 km in the continental crust. A previous study found no iron cycling potential in the communities despite the iron-rich nature of the system. A new tool for detecting iron cycling genes was recently published, which we used on a new data set. We combined this with a number of other approaches to get a holistic view of metabolic strategies across the communities, revealing iron cycling to be an important process here. In addition, we used the tool on the data from the previous study, revealing previously missed iron cycling potential. Iron is common in continental crust; thus, our findings are likely not unique to our study site. Our new view of important metabolic strategies underscores the importance of choosing optimized tools for detecting the potential for metabolisms like iron cycling that may otherwise be missed.

Kinetics and Identities of Extracellular Peptidases in Subsurface Sediments of the White Oak River Estuary, North Carolina.

Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily low rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River Estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities (Vmax) decreased with increasing sediment depth, although Vmax expressed on a per-cell basis was approximately the same at all depths. Half-saturation constants (Km ) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter (d-phenylalanine and l-ornithine) increased relative to enzymes that act on l-phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded-DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes.IMPORTANCE Burial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, ∼40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO2, are poorly constrained. In this study, we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface.

Tautonia sociabilis gen. nov., sp. nov., a novel thermotolerant planctomycete, isolated from a 4000 m deep subterranean habitat.

A novel aerobic bacterium, designated as strain GM2012T, was isolated from a microbial mat proliferating under the flow of thermal water dissipating from the wall of a 4000 m deep mine in South Africa. The cells were non-motile cocci, capable of budding, occurred in single or gathered in aggregates. The organism is a strictly aerobic chemoorganoheterotroph, preferring simple sugars and polysaccharides as growth substrates. The optimal growth occurred at 42 °C and pH 7.5-7.7. The predominant fatty acids were palmitate, stearate and oleate. The G+C content of the DNA was 70.1 mol%. The 16S rRNA gene sequence analysis placed strain GM2012T within the family Isosphaeraceae of the order Planctomycetales with 88-89 % sequence identity to Isosphaera pallida, Aquisphaeragiovannonii, Singulisphaera acidiphila, Paludisphaera borealis and Tundrisphaera lichenicola type strains. Based on the genotypic and phenotypic distinctive features of the new strain, we propose a novel genus and species Tautonia sociabilis gen. nov., sp. nov. with the type strain GM2012T (=VKM B-2860,=KCTC 72013).

Biofilm formation and potential for iron cycling in serpentinization-influenced groundwater of the Zambales and Coast Range ophiolites.

Terrestrial serpentinizing systems harbor microbial subsurface life. Passive or active microbially mediated iron transformations at alkaline conditions in deep biosphere serpentinizing ecosystems are understudied. We explore these processes in the Zambales (Philippines) and Coast Range (CA, USA) ophiolites, and associated surface ecosystems by probing the relevance of samples acquired at the surface to in situ, subsurface ecosystems, and the nature of microbe-mineral associations in the subsurface. In this pilot study, we use microcosm experiments and batch culturing directed at iron redox transformations to confirm thermodynamically based predictions that iron transformations may be important in subsurface serpentinizing ecosystems. Biofilms formed on rock cores from the Zambales ophiolite on surface and in-pit associations, confirming that organisms from serpentinizing systems can form biofilms in subsurface environments. Analysis by XPS and FTIR confirmed that enrichment culturing utilizing ferric iron growth substrates produced reduced, magnetic solids containing siderite, spinels, and FeO minerals. Microcosms and enrichment cultures supported organisms whose near relatives participate in iron redox transformations. Further, a potential 'principal' microbial community common to solid samples in serpentinizing systems was identified. These results indicate collectively that iron redox transformations should be more thoroughly and universally considered when assessing the function of terrestrial subsurface ecosystems driven by serpentinization.

Petrothermobacter organivorans gen. nov., sp. nov., a thermophilic, strictly anaerobic bacterium of the phylum Deferribacteres isolated from a deep subsurface oil reservoir.

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5-5.0 µm long and 0.6-0.7 µm wide). The isolate grew at 25-60 °C (optimum 55 °C) and pH 6.0-8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae. In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres. Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).

Metalliferous Biosignatures for Deep Subsurface Microbial Activity.

The interaction of microbes and metals is widely assumed to have occurred in surface or very shallow subsurface environments. However new evidence suggests that much microbial activity occurs in the deep subsurface. Fluvial, lacustrine and aeolian 'red beds' contain widespread centimetre-scale reduction spheroids in which a pale reduced spheroid in otherwise red rocks contains a metalliferous core. Most of the reduction of Fe (III) in sediments is caused by Fe (III) reducing bacteria. They have the potential to reduce a range of metals and metalloids, including V, Cu, Mo, U and Se, by substituting them for Fe (III) as electron acceptors, which are all elements common in reduction spheroids. The spheroidal morphology indicates that they were formed at depth, after compaction, which is consistent with a microbial formation. Given that the consequences of Fe (III) reduction have a visual expression, they are potential biosignatures during exploration of the terrestrial and extraterrestrial geological record. There is debate about the energy available from Fe (III) reduction on Mars, but the abundance of iron in Martian soils makes it one of the most valuable prospects for life there. Entrapment of the microbes themselves as fossils is possible, but a more realistic target during the exploration of Mars would be the colour contrasts reflecting selective reduction or oxidation. This can be achieved by analysing quartz grains across a reduction spheroid using Raman spectroscopy, which demonstrates its suitability for life detection in subsurface environments. Microbial action is the most suitable explanation for the formation of reduction spheroids and may act as metalliferous biosignatures for deep subsurface microbial activity.

Draft genome sequence of the deep-subsurface Ciceribacter sp. strain T2.26MG-112.2, a second Rhizobiaceae isolated from the Iberian Pyrite Belt at 492.6 mbs.

T2.26MG-112.2 is a Ciceribacter strain that has been isolated from the deep subsurface of the Iberian Pyrite Belt. We report its draft genome consisting of a chromosome of ≈4.9 Mb and a plasmid of 357 kb. The annotation reveals 4,824 coding sequences, 48 tRNA genes, and 1 rRNA operon.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Microbial ecology of the deep terrestrial subsurface.

The terrestrial subsurface hosts microbial communities that, collectively, are predicted to comprise as many microbial cells as global surface soils. Although initially thought to be associated with deposited organic matter, deep subsurface microbial communities are supported by chemolithoautotrophic primary production, with hydrogen serving as an important source of electrons. Despite recent progress, relatively little is known about the deep terrestrial subsurface compared to more commonly studied environments. Understanding the composition of deep terrestrial subsurface microbial communities and the factors that influence them is of importance because of human-associated activities including long-term storage of used nuclear fuel, carbon capture, and storage of hydrogen for use as an energy vector. In addition to identifying deep subsurface microorganisms, recent research focuses on identifying the roles of microorganisms in subsurface communities, as well as elucidating myriad interactions-syntrophic, episymbiotic, and viral-that occur among community members. In recent years, entirely new groups of microorganisms (i.e. candidate phyla radiation bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoloarchaeota, Nanoarchaeota archaea) have been discovered in deep terrestrial subsurface environments, suggesting that much remains unknown about this biosphere. This review explores the historical context for deep terrestrial subsurface microbial ecology and highlights recent discoveries that shape current ecological understanding of this poorly explored microbial habitat. Additionally, we highlight the need for multifaceted experimental approaches to observe phenomena such as cryptic cycles, complex interactions, and episymbiosis, which may not be apparent when using single approaches in isolation, but are nonetheless critical to advancing our understanding of this deep biosphere.

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Vitamin B12 Auxotrophy in Isolates from the Deep Subsurface of the Iberian Pyrite Belt.

Vitamin B12 is an enzymatic cofactor that is essential for both eukaryotes and prokaryotes. The development of life in extreme environments depends on cofactors such as vitamin B12 as well. The genomes of twelve microorganisms isolated from the deep subsurface of the Iberian Pyrite Belt have been analyzed in search of enzymatic activities that require vitamin B12 or are involved in its synthesis and import. Results have revealed that vitamin B12 is needed by these microorganisms for several essential enzymes such as ribonucleotide reductase, methionine synthase and epoxyqueosine reductase. Isolate Desulfosporosinus sp. DEEP is the only analyzed genome that holds a set core of proteins that could lead to the production of vitamin B12. The rest are dependent on obtaining it from the subsurface oligotrophic environment in which they grow. Sought proteins involved in the import of vitamin B12 are not widespread in the sample. The dependence found in the genomes of these microorganisms is supported by the production of vitamin B12 by microorganisms such as Desulfosporosinus sp. DEEP, showing that the operation of deep subsurface biogeochemical cycles is dependent on cofactors such as vitamin B12.

Subsurface biogeochemical cycling of nitrogen in the actively serpentinizing Samail Ophiolite, Oman.

Nitrogen (N) is an essential element for life. N compounds such as ammonium ( NH 4 + ) may act as electron donors, while nitrate ( NO 3 - ) and nitrite ( NO 2 - ) may serve as electron acceptors to support energy metabolism. However, little is known regarding the availability and forms of N in subsurface ecosystems, particularly in serpentinite-hosted settings where hydrogen (H2) generated through water-rock reactions promotes habitable conditions for microbial life. Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg2+- HCO 3 - type fluids comprised mostly of NO 3 - , and Ca2+-OH- fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as NO 3 - . High concentrations of NO 3 - (>100 µM) with a relict meteoric oxygen isotopic composition (δ18O ~ 22‰, Δ17O ~ 6‰) were observed in shallow aquifer fluids, indicative of NO 3 - sourced from atmospheric deposition (rainwater NO 3 - : δ18O of 53.7‰, Δ17O of 16.8‰) mixed with NO 3 - produced in situ through nitrification (estimated endmember δ18O and Δ17O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH3 (>100 µM) with little NO 3 - detectable. We interpret that NH3 in hyperalkaline fluids is a product of NO 3 - reduction. The proportionality of the O and N isotope fractionation (18ε / 15ε) measured in Samail Ophiolite NO 3 - was close to unity (18ε / 15ε ~ 1), which is consistent with dissimilatory NO 3 - reduction with a membrane-bound reductase (NarG); however, abiotic reduction processes may also be occurring. The presence of genes commonly involved in N reduction processes (narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of NO 3 - . Production of NH 4 + as the end-product of NO 3 - reduction via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.

Shewanella sp. T2.3D-1.1 a Novel Microorganism Sustaining the Iron Cycle in the Deep Subsurface of the Iberian Pyrite Belt.

The Iberian Pyrite Belt (IPB) is one of the largest deposits of sulphidic minerals on Earth. Río Tinto raises from its core, presenting low a pH and high metal concentration. Several drilling cores were extracted from the IPB's subsurface, and strain T2.3D-1.1 was isolated from a core at 121.8 m depth. We aimed to characterize this subterranean microorganism, revealing its phylogenomic affiliation (Average Nucleotide Identity, digital DNA-DNA Hybridization) and inferring its physiology through genome annotation, backed with physiological experiments to explore its relationship with the Fe biogeochemical cycle. Results determined that the isolate belongs to the Shewanella putrefaciens (with ANI 99.25 with S. putrefaciens CN-32). Its genome harbours the necessary genes, including omcA mtrCAB, to perform the Extracellular Electron Transfer (EET) and reduce acceptors such as Fe3+, napAB to reduce NO3- to NO2-, hydAB to produce H2 and genes sirA, phsABC and ttrABC to reduce SO32-, S2O32- and S4O62-, respectively. A full CRISPR-Cas 1F type system was found as well. S. putrefaciens T2.3D-1.1 can reduce Fe3+ and promote the oxidation of Fe2+ in the presence of NO3- under anaerobic conditions. Production of H2 has been observed under anaerobic conditions with lactate or pyruvate as the electron donor and fumarate as the electron acceptor. Besides Fe3+ and NO3-, the isolate also grows with Dimethyl Sulfoxide and Trimethyl N-oxide, S4O62- and S2O32- as electron acceptors. It tolerates different concentrations of heavy metals such as 7.5 mM of Pb, 5 mM of Cr and Cu and 1 mM of Cd, Co, Ni and Zn. This array of traits suggests that S. putrefaciens T2.3D-1.1 could have an important role within the Iberian Pyrite Belt subsurface participating in the iron cycle, through the dissolution of iron minerals and therefore contributing to generate the extreme conditions detected in the Río Tinto basin.

High Abundance of Thaumarchaeota Found in Deep Metamorphic Subsurface in Eastern China.

Members of the Thaumarchaeota phylum play a key role in nitrogen cycling and are prevalent in a variety of environments including soil, sediment, and seawater. However, few studies have shown the presence of Thaumarchaeota in the terrestrial deep subsurface. Using high-throughput 16S rRNA gene sequencing, this study presents evidence for the high relative abundance of Thaumarchaeota in a biofilm sample collected from the well of Chinese Continental Scientific Drilling at a depth of 2000 m. Phylogenetic analysis showed a close relationship of these thaumarchaeotal sequences with known ammonia-oxidizing archaea (AOA) isolates, suggesting the presence of AOA in the deep metamorphic environment of eastern China which is believed to be oxic. Based on fluid geochemistry and FAProTax functional prediction, a pathway of nitrogen cycling is proposed. Firstly, heterotrophic nitrogen fixation is executed by diazotrophic bacteria coupled with methane oxidation. Then, ammonia is oxidized to nitrite by AOA, and nitrite is further oxidized to nitrate by bacteria within the phylum Nitrospirae. Denitrification and anaerobic ammonia oxidation occur slowly, leading to nitrate accumulation in the subsurface. With respect to biogeochemistry, the reaction between downward diffusing O2 and upward diffusing CH4 potentially fuels the ecosystem with a high relative abundance of Thaumarchaeota.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Deep Subsurface Hypersaline Environment as a Source of Novel Species of Halophilic Sulfur-Oxidizing Bacteria.

The sulfur cycle participates significantly in life evolution. Some facultatively autotrophic microorganisms are able to thrive in extreme environments with limited nutrient availability where they specialize in obtaining energy by oxidation of reduced sulfur compounds. In our experiments focused on the characterization of halophilic bacteria from a former salt mine in Solivar (Presov, Slovakia), a high diversity of cultivable bacteria was observed. Based on ARDRA (Amplified Ribosomal DNA Restriction Analysis), at least six groups of strains were identified with four of them showing similarity levels of 16S rRNA gene sequences lower than 98.5% when compared against the GenBank rRNA/ITS database. Heterotrophic sulfur oxidizers represented ~34% of strains and were dominated by Halomonas and Marinobacter genera. Autotrophic sulfur oxidizers represented ~66% and were dominated by Guyparkeria and Hydrogenovibrio genera. Overall, our results indicate that the spatially isolated hypersaline deep subsurface habitat in Solivar harbors novel and diverse extremophilic sulfur-oxidizing bacteria.

Biological, geological and chemical effects of oxygen injection in underground gas storage aquifers in the setting of biomethane deployment.

The last few years have seen the proliferation of anaerobic digestion plants to produce biomethane. Oxygen (O2) traces added to biogas during the desulfurization process are co-injected in the gas network and can be stored in Underground Gas Storage (UGS). However, there are no data available for the undesirable effects of O2 on these anoxic environments, especially on deep aquifers. In addition to mineral alteration, O2 can have an impact on the anaerobic autochthonous microbial life. In our study, the storage conditions of an UGS aquifer were reproduced in a high-pressure reactor and bio-geo-chemical interactions between the aqueous, gas and solid phases were studied. Sulfate was depleted from the liquid phase for three consecutive times during the first 130 days of incubation reproducing the storage conditions (36 °C, 60 bar, methane with 1% CO2). Sulfate-reducers, such as Desulfovibrionaceae, were identified from the high-pressure system. Simulations with PHREEQC were used to determine the thermodynamic equilibrium to confirm any gas consumption. CO2 quantities decreased in the gas phase, suggesting its use as carbon source by microbial life. Benzene and toluene, hydrocarbons found in traces and known to be biodegradable in storages, were monitored and a decrease of toluene was revealed and associated to the Peptococcaceae family. Afterwards, O2 was added as 1% of the gas phase, corresponding to the maximum quantity found in biomethane after desulfurization process. Re-oxidation of sulfide to sulfate was observed along with the end of sulfate reducing activity and toluene biodegradation and the disappearance of most of the community. H2 surprisingly appeared and accumulated as soon as hydrogenotrophic sulfate-reducers decreased. H2 would be produced via the necromass fermentation accomplished by microorganisms able to resist the oxic conditions of 4.42·10-4 mol.Kgw-1 of O2. The solid phase composed essentially of quartz, presented no remarkable changes.

Rock-Hosted Subsurface Biofilms: Mineral Selectivity Drives Hotspots for Intraterrestrial Life.

The continental deep subsurface is likely the largest reservoir of biofilm-based microbial biomass on Earth, but the role of mineral selectivity in regulating its distribution and diversity is unclear. Minerals can produce hotspots for intraterrestrial life by locally enhancing biofilm biomass. Metabolic transformations of minerals by subsurface biofilms may occur widely with the potential to significantly impact subsurface biogeochemical cycles. However, the degree of impact depends upon the amount of biofilm biomass and its relationship to host rock mineralogy, estimates that are currently loosely constrained to non-existent. Here, we use in situ cultivation of biofilms on native rocks and coupled microscopy/spectroscopy to constrain mineral selectivity by biofilms in a deep continental subsurface setting: the Deep Mine Microbial Observatory (DeMMO). Through hotspot analysis and spatial modeling approaches we find that mineral distributions, particularly those putatively metabolized by microbes, indeed drive biofilm distribution at DeMMO, and that bioleaching of pyrite may be a volumetrically important process influencing fluid geochemistry at this site when considered at the kilometer scale. Given the ubiquity of iron-bearing minerals at this site and globally, and the amount of biomass they can support, we posit that rock-hosted biofilms likely contribute significantly to subsurface biogeochemical cycles. As more data becomes available, future efforts to estimate biomass in the continental subsurface should incorporate host rock mineralogy.