Archaeal Metagenome-assembled Genomes Research Articles

Microbiology of Big Soda Lake, a multi-extreme meromictic volcanic crater lake in the Nevada desert.

Big Soda Lake, Nevada, is a multi-extreme meromictic lake, whose hypersaline hyperalkaline bottom waters feature permanent anoxia and high concentrations of arsenic, sulphide and ammonia. These properties make Big Soda Lake-and the adjacent Little Soda Lake-a fascinating system for exploring life's boundaries, discovering novel microbial taxa and identifying biotechnologically useful strains. To date, the taxonomic diversity and metabolic capabilities of microorganisms in this system remain largely unknown. Here, we fill this gap using microbiome surveys across the Big and Little Soda Lake water columns, including 16S rRNA sequencing, fungal ITS2 sequencing and gene- and genome-resolved metagenomics. We accompany these surveys with measurements of salinity, pH, temperature, oxygen, ammonium and ammonia concentrations. Our analyses reveal rich bacterial communities, taxonomically and functionally differentiated along Big Soda Lake's oxycline and, to lesser extent, between lakes. Fungal communities were dominated by a small number of families, while nearly no archaea were detected. Pathways related to perchlorate reduction, anoxygenic phototrophy, fermentation, dissimilatory metabolism of arsenite/arsenate, sulphur compounds, nitrogen compounds and hydrogen, were particularly prevalent. A total of 129 high-quality bacterial and archaeal metagenome-assembled genomes (completeness ≥ 80%, contamination ≤ 5%) were recovered, yielding insight into the taxonomic distribution of microbial metabolic pathways.

Metagenome-assembled genomes from mineral tundra soils in Rásttigáisá, northern Norway.

Microbial communities in tundra soils remain largely unknown despite their important roles in the cycling of greenhouse gases. Here, we report 59 non-redundant metagenome-assembled genomes (MAGs) recovered from mineral tundra soils in Rásttigáisá, northern Norway. The MAGs were obtained by clustering contigs according to tetranucleotide frequency and differential coverage and were manually curated to remove contigs with outlying GC content and/or mean coverage. Most MAGs were assigned to the bacterial phyla Candidatus Dormibacterota (n=12), Verrucomicrobiota (n=10), and Acidobacteriota (n=9). All archaeal MAGs (n=4) belong to the genus Candidatus Nitrosopolaris (phylum Thermoproteota). The 59 Rásttigáisá MAGs expand our knowledge of the diversity and ecological roles of tundra microbiomes.

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton.

Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are neither designed to consider eukaryotes nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome-assembled genomes (MAGs) from large-scale metagenomic sequence data sets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8-2,000 µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life.IMPORTANCESingle-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers' efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

Novel complete methanogenic pathways in longitudinal genomic study of monogastric age-associated archaea

BackgroundArchaea perform critical roles in the microbiome system, including utilizing hydrogen to allow for enhanced microbiome member growth and influencing overall host health. With the majority of microbiome research focusing on bacteria, the functions of archaea are largely still under investigation. Understanding methanogenic functions during the host lifetime will add to the limited knowledge on archaeal influence on gut and host health. In our study, we determined lifelong archaea dynamics, including detection and methanogenic functions, while assessing global, temporal and host distribution of our novel archaeal metagenome-assembled genomes (MAGs). We followed 7 monogastric swine throughout their life, from birth to adult (1–156 days of age), and collected feces at 22 time points. The samples underwent gDNA extraction, Illumina sequencing, bioinformatic quality and assembly processes, MAG taxonomic assignment and functional annotation. MAGs were utilized in downstream phylogenetic analysis for global, temporal and host distribution in addition to methanogenic functional potential determination.ResultsWe generated 1130 non-redundant MAGs, representing 588 unique taxa at the species level, with 8 classified as methanogenic archaea. The taxonomic classifications were as follows: orders Methanomassiliicoccales (5) and Methanobacteriales (3); genera UBA71 (3), Methanomethylophilus (1), MX-02 (1), and Methanobrevibacter (3). We recovered the first US swine Methanobrevibacter UBA71 sp006954425 and Methanobrevibacter gottschalkii MAGs. The Methanobacteriales MAGs were identified primarily during the young, preweaned host whereas Methanomassiliicoccales primarily in the adult host. Moreover, we identified our methanogens in metagenomic sequences from Chinese swine, US adult humans, Mexican adult humans, Swedish adult humans, and paleontological humans, indicating that methanogens span different hosts, geography and time. We determined complete metabolic pathways for all three methanogenic pathways: hydrogenotrophic, methylotrophic, and acetoclastic. This study provided the first evidence of acetoclastic methanogenesis in archaea of monogastric hosts which indicated a previously unknown capability for acetate utilization in methanogenesis for monogastric methanogens. Overall, we hypothesized that the age-associated detection patterns were due to differential substrate availability via the host diet and microbial metabolism, and that these methanogenic functions are likely crucial to methanogens across hosts. This study provided a comprehensive, genome-centric investigation of monogastric-associated methanogens which will further improve our understanding of microbiome development and functions.

Genome-centric metagenomics reveals the host-driven dynamics and ecological role of CPR bacteria in an activated sludge system

BackgroundCandidate phyla radiation (CPR) constitutes highly diverse bacteria with small cell sizes and are likely obligate intracellular symbionts. Given their distribution and complex associations with bacterial hosts, genetic and biological features of CPR bacteria in low-nutrient environments have received increasing attention. However, CPR bacteria in wastewater treatment systems remain poorly understood. We utilized genome-centric metagenomics to answer how CPR communities shift over 11 years and what kind of ecological roles they act in an activated sludge system.ResultsWe found that approximately 9% (135) of the 1,526 non-redundant bacterial and archaeal metagenome-assembled genomes were affiliated with CPR. CPR bacteria were consistently abundant with a relative abundance of up to 7.5% in the studied activated sludge system. The observed striking fluctuations in CPR community compositions and the limited metabolic and biosynthetic capabilities in CPR bacteria collectively revealed the nature that CPR dynamics may be directly determined by the available hosts. Similarity-based network analysis further confirmed the broad bacterial hosts of CPR lineages. The proteome contents of activated sludge-associated CPR had a higher similarity to those of environmental-associated CPR than to those of human-associated ones. Comparative genomic analysis observed significant enrichment of genes for oxygen stress resistance in activated sludge-associated CPR bacteria. Furthermore, genes for carbon cycling and horizontal gene transfer were extensively identified in activated sludge-associated CPR genomes.ConclusionsThese findings highlight the presence of specific host interactions among CPR lineages in activated sludge systems. Despite the lack of key metabolic pathways, these small, yet abundant bacteria may have significant involvements in biogeochemical cycling and bacterial evolution in activated sludge systems.A1iT8gFKWRifc6GWDf3ih6Video

Metagenome-Assembled Genomes of Four Southern Ocean Archaea Harbor Multiple Genes Linked to Polyethylene Terephthalate and Polyhydroxybutyrate Plastic Degradation.

Here, we present four archaeal metagenome-assembled genomes (MAGs) (three Thaumarchaeota MAGs and one Thermoplasmatota MAG) from a polar upwelling zone in the Southern Ocean. These archaea harbor putative genes encoding enzymes such as polyethylene terephthalate (PET) hydrolases (PETases) and polyhydroxybutyrate (PHB) depolymerases, which are associated with microbial degradation of PET and PHB plastics.

Diverse secondary metabolites are expressed in particle-associated and free-living microorganisms of the permanently anoxic Cariaco Basin

Secondary metabolites play essential roles in ecological interactions and nutrient acquisition, and are of interest for their potential uses in medicine and biotechnology. Genome mining for biosynthetic gene clusters (BGCs) can be used for the discovery of new compounds. Here, we use metagenomics and metatranscriptomics to analyze BGCs in free-living and particle-associated microbial communities through the stratified water column of the Cariaco Basin, Venezuela. We recovered 565 bacterial and archaeal metagenome-assembled genomes (MAGs) and identified 1154 diverse BGCs. We show that differences in water redox potential and microbial lifestyle (particle-associated vs. free-living) are associated with variations in the predicted composition and production of secondary metabolites. Our results indicate that microbes, including understudied clades such as Planctomycetota, potentially produce a wide range of secondary metabolites in these anoxic/euxinic waters.

Genomic view of the diversity and functional role of archaea and bacteria in the skeleton of the reef-building corals Porites lutea and Isopora palifera.

At present, our knowledge on the compartmentalization of coral holobiont microbiomes is highly skewed toward the millimeter-thin coral tissue, leaving the diverse coral skeleton microbiome underexplored. Here, we present a genome-centric view of the skeleton of the reef-building corals Porites lutea and Isopora palifera, through a compendium of ∼400 high-quality bacterial and archaeal metagenome-assembled genomes (MAGs), spanning 34 phyla and 57 classes. Skeletal microbiomes harbored a diverse array of stress response genes, including dimethylsulfoniopropionate synthesis (dsyB) and metabolism (DMSP lyase). Furthermore, skeletal MAGs encoded an average of 22 ± 15 genes in P. lutea and 28 ± 23 in I. palifera with eukaryotic-like motifs thought to be involved in maintaining host association. We provide comprehensive insights into the putative functional role of the skeletal microbiome on key metabolic processes such as nitrogen fixation, dissimilatory and assimilatory nitrate, and sulfate reduction. Our study provides critical genomic resources for a better understanding of the coral skeletal microbiome and its role in holobiont functioning.

Deciphering the community structure and the functional potential of a hypersaline marsh microbial mat community.

Microbial mats are vertically stratified communities of microorganisms characterized by pronounced physiochemical gradients allowing for high species diversity and a wide range of metabolic capabilities. High Throughput Sequencing has the potential to reveal the biodiversity and function of such ecosystems in the cycling of elements. The present study combines 16S rRNA amplicon sequencing and shotgun metagenomics on a hypersaline marsh in Tristomo bay (Karpathos, Greece). Samples were collected in July 2018 and November 2019 from microbial mats, deeper sediment, aggregates observed in the water overlying the sediment, as well as sediment samples with no apparent layering. Metagenomic samples' coassembly and binning revealed 250 bacterial and 39 archaeal metagenome-assembled genomes, with completeness estimates higher than 70% and contamination less than 5%. All MAGs had KEGG Orthology terms related to osmoadaptation, with the 'salt in' strategy ones being prominent. Halobacteria and Bacteroidetes were the most abundant taxa in the mats. Photosynthesis was most likely performed by purple sulphur and nonsulphur bacteria. All samples had the capacity for sulphate reduction, dissimilatory arsenic reduction, and conversion of pyruvate to oxaloacetate. Overall, both sequencing methodologies resulted in similar taxonomic compositions and revealed that the formation of the microbial mat in this marsh exhibits seasonal variation.

Discovery of integrons in Archaea: Platforms for cross-domain gene transfer.

Horizontal gene transfer between different domains of life is increasingly being recognized as an important evolutionary driver, with the potential to increase the pace of biochemical innovation and environmental adaptation. However, the mechanisms underlying the recruitment of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria. However, they have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here, we discover that integrons are also present in 75 archaeal metagenome-assembled genomes from nine phyla, and are particularly enriched among Asgard archaea. Furthermore, we provide experimental evidence that integrons can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a previously unknown mechanism of cross-domain gene transfer whereby bacteria can incorporate archaeal genes from their surrounding environment via integron activity. These findings have important implications for prokaryotic ecology and evolution.

Genome- and community-level interaction insights into the ecological role of archaea in rare earth element mine drainage in South China

Microbial communities play crucial roles in mine drainage generation and remediation. Despite the wide distribution of archaea in the mine ecosystem, their diversity and ecological roles remain less understood than bacteria. Here, we retrieved 56 archaeal metagenome-assembled genomes from a river impacted by rare earth element (REE) mining activities in South China. Genomic analysis showed that archaea represented four distinct lineages, including phyla of Thaumarchaeota, Micrarchaeota, Nanoarchaeota and Thermoplasmata. These archaea represented a considerable fraction (up to 40%) of the total prokaryote community, which might contribute to nitrogen and sulfur cycling in the REE mine drainage. Reconstructed metabolic potential among diverse archaea taxa revealed that archaea were involved in the network of ammonia oxidation, denitrification, sulfate redox reaction, and required substrates supplied by other community members. As the dominant driver of ammonia oxidation, Thaumarchaeota might provide substrates to support the survival of two nano-sized archaea belonging to Micrarchaeota and Nanoarchaeota. Despite the absence of biosynthesis pathways for amino acids and nucleotides, the potential capacity for nitrite reduction (nirD) was observed in Micrarchaeota, indicating that these nano-sized archaea encompassed diverse metabolisms. Moreover, Thermoplasmata, as keystone taxa in community, might be the main genetic donor for the other three archaeal phyla, transferring many environmental resistance related genes (e.g., V/A-type ATPase and Vitamin B12-transporting ATPase). The genetic interactions within archaeal community through horizontal gene transfer might be the key to the formation of archaeal resistance and functional partitioning. This study provides putative metabolic and genetic insights into the diverse archaea taxa from community-level perspectives, and highlights the ecological roles of archaea in REE contaminated aquatic environment.

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, ‘Brockarchaeota’, named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Metagenomic insights into the metabolism and evolution of a new Thermoplasmata order (Candidatus Gimiplasmatales).

Thermoplasmata is a widely distributed and ecologically important archaeal class in the phylum Euryarchaeota. Because few cultures and genomes are available, uncharacterized Thermoplasmata metabolisms remain unexplored. In this study, we obtained four medium- to high-quality archaeal metagenome-assembled genomes (MAGs) from the filamentous fragments of black-odorous aquatic sediments (Foshan, Guangdong, China). Based on their 16S rRNA gene and ribosomal protein phylogenies, the four MAGs belong to the previously unnamed Thermoplasmata UBA10834 clade. We propose that this clade (five reference genomes from the Genome Taxonomy Database (GTDB) and four MAGs from this study) be considered a new order, Candidatus Gimiplasmatales. Metabolic pathway reconstructions indicated that the Ca. Gimiplasmatales MAGs can biosynthesize isoprenoids and nucleotides de novo. Additionally, some taxa have genes for formaldehyde and acetate assimilation, and the Wood-Ljungdahl CO2 -fixation pathway, indicating a mixotrophic lifestyle. Sulfur reduction, hydrogen metabolism, and arsenic detoxification pathways were predicted, indicating sulfur-, hydrogen-, and arsenic-transformation potentials. Comparative genomics indicated that the H4 F Wood-Ljungdahl pathway of both Ca. Gimiplasmatales and Methanomassiliicoccales was likely obtained by the interdomain lateral gene transfer from the Firmicutes. Collectively, this study elucidates the taxonomic and potential metabolic diversity of the new order Ca. Gimiplasmatales and the evolution of this subgroup and its sister lineage Methanomassiliicoccales.

Impact of process temperature and organic loading rate on cellulolytic / hydrolytic biofilm microbiomes during biomethanation of ryegrass silage revealed by genome-centered metagenomics and metatranscriptomics

BackgroundAnaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage two-phase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To follow the adaptive response of the biomass-attached cellulolytic/hydrolytic biofilms at increasing ammonium/ammonia contents, genome-centered metagenomics and transcriptional profiling based on metagenome assembled genomes (MAGs) were conducted.ResultsIn total, 78 bacterial and archaeal MAGs representing the most abundant members of the communities, and featuring defined quality criteria were selected and characterized in detail. Determination of MAG abundances under the tested conditions by mapping of the obtained metagenome sequence reads to the MAGs revealed that MAG abundance profiles were mainly shaped by the temperature but also by the OLR. However, the OLR effect was more pronounced for the mesophilic systems as compared to the thermophilic ones. In contrast, metatranscriptome mapping to MAGs subsequently normalized to MAG abundances showed that under thermophilic conditions, MAGs respond to increased OLRs by shifting their transcriptional activities mainly without adjusting their proliferation rates. This is a clear difference compared to the behavior of the microbiome under mesophilic conditions. Here, the response to increased OLRs involved adjusting of proliferation rates and corresponding transcriptional activities. The analysis led to the identification of MAGs positively responding to increased OLRs. The most outstanding MAGs in this regard, obviously well adapted to higher OLRs and/or associated conditions, were assigned to the order Clostridiales (Acetivibrio sp.) for the mesophilic biofilm and the orders Bacteroidales (Prevotella sp. and an unknown species), Lachnospirales (Herbinix sp. and Kineothrix sp.) and Clostridiales (Clostridium sp.) for the thermophilic biofilm. Genome-based metabolic reconstruction and transcriptional profiling revealed that positively responding MAGs mainly are involved in hydrolysis of grass silage, acidogenesis and / or acetogenesis.ConclusionsAn integrated -omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs. Genome-based knowledge on the metabolic potential and transcriptional activity of responsive microbiome members will contribute to the development of improved microbiological AD management strategies for biomethanation of renewable biomass.

Anaerobic reactor applied to laundry wastewater treatment: Unveiling the microbial community by gene and genome-centric approaches

Gene and genome-centric approaches were applied to investigate the microbial taxonomic and metabolic diversity profiles from a lab-scale anaerobic reactor applied to laundry wastewater treatment with increasing concentrations of linear alkylbenzene sulfonate (LAS). Therefore, metagenomic analysis was carried out with reactor samples collected under three conditions, (i) Stage I, the reactor was fed with synthetic medium, (ii) Stage II, synthetic medium was replaced by diluted laundry wastewater with specific LAS loading rates (SLLRs) of 1.0 ± 0.3 mgLASgTVS−1d−1 and (iii) Stage III, SLLRs increased to 2.7 ± 0.7 mgLASgTVS−1d−1. Shotgun metagenomic sequencing was performed on an Illumina HiSeq 2 × 150 bp platform. Gene-centric analysis of each step of the metabolic pathway (fumarate addition, β-oxidation, ring cleavage and desulfonation) for anaerobic aromatic degradation showed eight over-representative genera (Achromobacter, Pelodictyon, Pseudomonas, Psychrobacter, Rhodococcus, Stenotrophomonas, Sulfurovum and Syntrophobacter), suggesting a microbial core with an important role in LAS biodegradation. Some of these genera were also recovered through a differential binning method, representing fifteen bacterial and one archaeal metagenome-assembled genomes (MAGs). Biodegradation pathway reconstruction of LAS using six MAGs unveiled the syntrophism for complete degradation of the LAS molecule. Only the MAGs with taxonomic annotation for Syntrophobacter showed genetic potential for fumarate addition, whereas in the ring cleavage, there was a predominance of genes in MAGs with taxonomic annotation for Pseudomonas fragi and Rhodococcus. This work represents the first report of genome-centric approach to study biological reactors applied in anionic surfactant degradation, contributing with detailed metabolic information of the key microbial actors in LAS degradation and opening perspectives for future biotechnological strategies aiming at bioaugmentation and/or biostimulation of indigenous microbial populations.

A Collection of 13 Archaeal and 46 Bacterial Genomes Reconstructed from Marine Metagenomes Derived from the North Sea

Marine bacteria are key drivers of ocean biogeochemistry. Despite the increasing number of studies, the complex interaction of marine bacterioplankton communities with their environment is still not fully understood. Additionally, our knowledge about prominent marine lineages is mostly based on genomic information retrieved from single isolates, which do not necessarily represent these groups. Consequently, deciphering the ecological contributions of single bacterioplankton community members is one major challenge in marine microbiology. In the present study, we reconstructed 13 archaeal and 46 bacterial metagenome-assembled genomes (MAGs) from four metagenomic data sets derived from the North Sea. Archaeal MAGs were affiliated to Marine Group II within the Euryarchaeota. Bacterial MAGs mainly belonged to marine groups within the Bacteroidetes as well as alpha- and gammaproteobacteria. In addition, two bacterial MAGs were classified as members of the Actinobacteria and Verrucomicrobiota, respectively. The reconstructed genomes contribute to our understanding of important marine lineages and may serve as a basis for further research on functional traits of these groups.

GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database.

SummaryThe Genome Taxonomy Database Toolkit (GTDB-Tk) provides objective taxonomic assignments for bacterial and archaeal genomes based on the GTDB. GTDB-Tk is computationally efficient and able to classify thousands of draft genomes in parallel. Here we demonstrate the accuracy of the GTDB-Tk taxonomic assignments by evaluating its performance on a phylogenetically diverse set of 10 156 bacterial and archaeal metagenome-assembled genomes.Availability and implementationGTDB-Tk is implemented in Python and licenced under the GNU General Public Licence v3.0. Source code and documentation are available at: https://github.com/ecogenomics/gtdbtk.Supplementary information Supplementary data are available at Bioinformatics online.

Carbon fixation and energy metabolisms of a subseafloor olivine biofilm.

Earth's largest aquifer ecosystem resides in igneous oceanic crust, where chemosynthesis and water-rock reactions provide the carbon and energy that support an active deep biosphere. The Calvin Cycle is the predominant carbon fixation pathway in cool, oxic, crust; however, the energy and carbon metabolisms in the deep thermal basaltic aquifer are poorly understood. Anaerobic carbon fixation pathways such as the Wood-Ljungdahl pathway, which uses hydrogen (H2) and CO2, may be common in thermal aquifers since water-rock reactions can produce H2 in hydrothermal environments and bicarbonate is abundant in seawater. To test this, we reconstructed the metabolisms of eleven bacterial and archaeal metagenome-assembled genomes from an olivine biofilm obtained from a Juan de Fuca Ridge basaltic aquifer. We found that the dominant carbon fixation pathway was the Wood-Ljungdahl pathway, which was present in seven of the eight bacterial genomes. Anaerobic respiration appears to be driven by sulfate reduction, and one bacterial genome contained a complete nitrogen fixation pathway. This study reveals the potential pathways for carbon and energy flux in the deep anoxic thermal aquifer ecosystem, and suggests that ancient H2-based chemolithoautotrophy, which once dominated Earth's early biosphere, may thus remain one of the dominant metabolisms in the suboceanic aquifer today.