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DOE OSTI2022

Variation in Root Exudate Composition Influences Soil Microbiome Membership and Function

Root exudation is one of the primary processes that mediate interactions between plant roots, microorganisms, and the soil matrix, yet the mechanisms by which exudation alters microbial metabolism in soils have been challenging to unravel. Here, utilizing distinct sorghum genotypes, we characterized the chemical heterogeneity between root exudates and the effects of that variability on soil microbial membership and metabolism. Distinct exudate chemical profiles were quantified and used to formulate synthetic root exudate treatments: a high-organic-acid treatment (HOT) and a high-sugar treatment (HST). To parse the response of the soil microbiome to different exudate regimens, laboratory soil reactors were amended with these root exudate treatments as well as a nonexudate control. Amplicon sequencing of the 16S rRNA gene illustrated distinct microbial diversity patterns and membership in response to HST, HOT, or control amendments. Exometabolite changes reflected these microbial community changes, and we observed enrichment of organic and amino acids, as well as possible phytohormones in the HST relative to the HOT and control. Linking the metabolic capacity of metagenome-assembled genomes in the HST to the exometabolite patterns, we identified microorganisms that could produce these phytohormones. Our findings emphasize the tractability of high-resolution multiomics tools to investigate soil microbiomes, opening the possibility of manipulating native microbial communities to improve specific soil microbial functions and enhance crop production.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Meta-analysis identifies pleiotropic loci controlling phenotypic trade-offs in sorghum

Abstract Community association populations are composed of phenotypically and genetically diverse accessions. Once these populations are genotyped, the resulting marker data can be reused by different groups investigating the genetic basis of different traits. Because the same genotypes are observed and scored for a wide range of traits in different environments, these populations represent a unique resource to investigate pleiotropy. Here, we assembled a set of 234 separate trait datasets for the Sorghum Association Panel, a group of 406 sorghum genotypes widely employed by the sorghum genetics community. Comparison of genome-wide association studies (GWAS) conducted with two independently generated marker sets for this population demonstrate that existing genetic marker sets do not saturate the genome and likely capture only 35–43% of potentially detectable loci controlling variation for traits scored in this population. While limited evidence for pleiotropy was apparent in cross-GWAS comparisons, a multivariate adaptive shrinkage approach recovered both known pleiotropic effects of existing loci and new pleiotropic effects, particularly significant impacts of known dwarfing genes on root architecture. In addition, we identified new loci with pleiotropic effects consistent with known trade-offs in sorghum development. These results demonstrate the potential for mining existing trait datasets from widely used community association populations to enable new discoveries from existing trait datasets as new, denser genetic marker datasets are generated for existing community association populations.

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 OSTI2021

The NIH Somatic Cell Genome Editing program

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Reference-free structural variant detection in microbiomes via long-read co-assembly graphs

Motivation: The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. Results: We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays. Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant-microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Yadav, Archana↗

NASA NTRSDate not supplied

Analytical Needs in a Sample Receiving Facility: Input from the MSR Operation Definition Team

The return of scientifically selected samples from Mars would provide a rare opportunity forinvestigation with the full range of the latest technology available, but to take full advantageof this opportunity, it is important to plan ahead to ensure the pristine nature of the samplesupon arrival within the Earth environment until scientific investigations can begin.The NASA/ESA science community-driven MSR Science Planning Group – Phase 2 (MSPG2)delivered recommendations and guidance regarding curation (1) and science (2, 3) activities tobe performed on the samples under containment. High-level requirements for the infrastruc-ture were also developed by MSPG2 (4). In order to prepare infrastructure-targeted input forthe ESA and NASA facility studies planned in the 2022-2023 timeframe, the agency-led MSROperational Scenarios Definition Team (MOSDT) was assembled to conceptualize the sampleoperations that will inform future architecture teams. Emphasis was placed on the respon-sibility of MOSDT to use community-defined requirements and to represent the view of the international scientific community.All necessary and sufficient instruments and analytical needs described in MSPG2 were inte-grated in MOSDT main deliverable, the operational workflow (see Hays et al, this conference).In MSPG2, notional instruments were split between curation analytical needs, and objective-driven (time-sensitive and sterilization-sensitive) science analytical needs. In MOSDT, whilethe first phases of curation, “pre-Basic Characterization” and “Basic Characterization” wererather streamlined and separate from other analytical needs, “Preliminary Examination” and“Science” instruments were not always physically segregated. In addition to the necessary andsufficient instruments described by MSPG2, the MOSDT recommended additional supportequipment for sterilization, cleanliness and contamination monitoring.It was sometimes necessary for the MOSDT to rely on assumptions to integrate instruments inthe activity workflow. In general, the assumptions were very conservative to limit contaminationand cross-contamination risks. It is expected that future work to refine limits of contaminationwill enable optimization of instrumentation.The community was consulted during the course of the MOSDT work. This abstract’s aimis two-fold: on one hand, inform the scientific community and overall MSR stakeholders, tobring their attention on the analytical needs currently considered as necessary and sufficient;on the other hand, to solicit feedback from a larger community audience to optimize and refineanalytical needs during the next phases of MSR ground-segment preparation.Disclaimer: The decision to implement Mars Sample Return will not be finalized until NASA’scompletion of the program’s National Environmental Policy Act (NEPA) process. This docu-ment is being made available for informational purposes only.[1] Tait et al. (2021) Preliminary planning for Mars Sample Return (MSR) curation activities ina Sample Receiving Facility (SRF). Astrobiology in press, doi:10.1089/ast.2021.0105. [2] Toscaet al. (2021) Time-sensitive aspects of Mars Sample Return (MSR) science. Astrobiologyin press, doi:10.1089/ast.2021.0115. [3] Velbel et al. (2021) Planning implications relatedto sterilization-sensitive science investigations associated with Mars Sample Return (MSR).Astrobiology in press, doi:10.1089/ast.2021.0113. [4] Carrier et al. (2021) Science and curationconsiderations for the design of a Mars Sample Return (MSR) Sample Receiving Facility (SRF).Astrobiology in press, doi:10.1089/ast.2021.0110.

Mars Sample Return↗

NASA NTRS1986

Point of view of the U.S. science community

A special task force was formed by the National Academy of Sciences to examine the applications of Manned Space Station (MSS) platforms, communications and information systems, MSS operations and satellite assembly and repair capabilities on seven areas of space research. The task force reached several conclusions, including comprehensive space science planning by NASA with the MSS being one element of the plan. The STS is to provide a test bed for space research technologies and its basic design should be studied for conversion to an extended duration flight vehicle. The MSS could be categorized as a national facility, and payloads should be developed which specifically take advantage of its capabilities. The repair and maintenance capabilities which the MSS will furnish are concluded to be a significant asset to any future space science activities, and participation in those activities by the international science community is to be encouraged.

Black, D. C.↗

NASA NTRSDate not supplied

Autonomous Robotic Manipulator Software

Autonomous robotic manipulation requires a deep and wide stack of supporting software. This paper presents Autonomous Robotic Manipulator Software (ARMS), a software suite designed at NASA Langley Research Center to support research and development of different algorithms for In-space Servicing, Assembly and Manufacturing (ISAM). ARMS solves common challenges along the autonomous manipulation software stack. Various challenges, such as integration with commercial hardware, simulation, and path planning, are solved through the use of Robot Operating System 2 and its community-developed packages. Other challenges, such as configuration management and task definition, and execution are solved in software built on those tools. The result is a modular approach to robotic system definition, agent actions, and assembly task definitions. ARMS has been used in two ISAM projects at NASA Langley Research Center, the Precision Assembled Space Structures project and the Built On-orbit Robotically assembled Gigatruss project.

Collin J Cresta↗

NASA NTRSDate not supplied

Autonomous Robotic Manipulator Software (ARMS)

Collin J Cresta↗

DOE OSTI2021

Evolution of standardization and dissemination of cryo-EM structures and data jointly by the community, PDB, and EMDB

Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduces new data archival challenges in terms of data size and complexity in the years to come.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

The relationship between gene traits and transcription in soil microbial communities varies by environmental stimulus

Codon and nucleotide frequencies are known to relate to the rate of gene transcription, yet how these traits shape transcriptional profiles of soil microbial communities remains unclear. Here we test the prediction that functional genes with high codon optimization and energetically lower cost nucleotides (i.e., nucleotides requiring less adenosine triphosphate (ATP) for synthesis) have higher transcriptional expression in a soil microbial community. In laboratory incubations, we subjected an agricultural soil to two separate short-term environmental changes: labile carbon (glucose) addition or a sudden 30-min increase in temperature from 20 °C to 60 °C. Using the total genomic codon frequencies to predict preferred codon usage for each taxon, we then estimated codon optimization for each transcript. On the community level, we found a higher average level of codon optimization after the addition of glucose. Synonymous nucleotide composition in the transcript pool also shifted towards energetically cheaper nucleotides, favoring uracil (U) over adenine (A) and cytosine (C) over guanine (G). Similarly, we found that encoded amino acid usage shifted towards energetically cheaper amino acids in response to labile carbon. In contrast, in communities responding to heat shock, there were no significant differences in the averaged gene traits of expressed transcripts. We used metagenome-assembled-genomes to further examine the ability of gene traits to predict transcriptional responses within and between taxa. We found that traits of individual genes could not reliably predict the level of transcription of a gene within or between taxa—highlighting the limits of this approach. However, we did find that when traits were averaged across several related genes, codon optimization was able to predict levels of transcription in metabolic pathways associated with growth and nutrient uptake in response to glucose. Similar relationships were not observed in response to heat, or for functions associated with stress—such as genes associated with sporulation or heat shock. These results demonstrate that gene traits, such as codon usage, nucleotide selection, and amino acid selection, relate to the transcriptional expression of genes in soil microbial communities and suggests that these relationships may be dependent on both gene function and the specific type of environmental stimuli.

Biological and medical sciences↗

DOE OSTI2022

Metagenomes from 25 Low-Abundance Microbes in a Partial Nitritation Anammox Microbiome

Microbial communities using anammox bacteria to remove nitrogen are increasingly important in wastewater treatment. We report on 25 metagenome-assembled genomes of low-abundance microbes from a partial nitritation anammox bioreactor system that have not been described previously. These data add to the body of information about this important wastewater treatment system.

Beach, Natalie K.↗

NASA NTRS1989

Computer-Aided Engineering Of Cabling

Program generates data sheets, drawings, and other information on electrical connections. DFACS program, centered around single data base, has built-in menus providing easy input of, and access to, data for all personnel involved in system, subsystem, and cabling. Enables parallel design of circuit-data sheets and drawings of harnesses. Also recombines raw information to generate automatically various project documents and drawings, including index of circuit-data sheets, list of electrical-interface circuits, lists of assemblies and equipment, cabling trees, and drawings of cabling electrical interfaces and harnesses. Purpose of program to provide engineering community with centralized data base for putting in, and gaining access to, functional definition of system as specified in terms of details of pin connections of end circuits of subsystems and instruments and data on harnessing. Primary objective to provide instantaneous single point of interchange of information, thus avoiding

Billitti, Joseph W.↗

NASA NTRS2001

Hollow Retroreflectors Offer Solid Benefits

A technician who lead a successful team of scientists, engineers, and other technicians in the design, fabrication, and characterization of cryogenic retroreflectors for the NASA Cassini/Composite Infrared Spectrometer (CIRS) mission to Saturn, developed a hollow retroreflector technology while working at NASA Goddard Space Flight Center. With 16 years of NASA experience, the technician teamed up with another NASA colleague and formed PROSystems, Inc., of Sharpsburg, Maryland, to provide the optics community with an alternative source for precision hollow retroreflectors. The company's hollow retroreflectors are front surface glass substrates assembled to provide many advantages over existing hollow retroreflectors and solid glass retroreflectors. Previous to this new technology, some companies chose not to use hollow retroreflectors due to large seam widths and loss of signal. The "tongue and groove" facet design of PROSystems's retroreflector allows for an extremely small seam width of .001 inches. Feedback from users is very positive regarding this characteristic. Most of PROSystems's primary customers mount the hollow retroreflectors in chrome steel balls for laser tracker targets in applications such as automobile manufacturing and spacecraft assembly.

Source record↗

DOE OSTI2023

Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides.

09 BIOMASS FUELS↗

DOE OSTI2021

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) (Final Scientific/Technical Report, Subcontract Award No. 6953691)

The overarching objective the Baliga lab is to develop a mechanistic understanding of the field relevant metabolic processes underlying community partitioning of respiration pathways and physiological state shifts. In order to dissect how environmental perturbations influence microbial regulatory networks, we have constructed data-driven models that capture the dynamic interplay between abiotic and biotic factors during laboratory simulations. Specifically, we have developed multiple computational models (i.e., cMonkey2, EGRIN 2.0, and multiple genome-scale metabolic models) connecting environmental influence to genome-encoded regulatory programs. In addition, we have generated new tools such as Live Anaerobic Cell Sorting (LAnCS) and Fluidized Bed Reactors (FBRs) to investigate the dynamics of community partitioning of metabolic processes supporting sulfate or nitrate respiration, which are two critical activities observed from the field site at Oak Ridge National Labs (ORNL). This framework of connecting field phenomena with laboratory simulations and computational modeling has created the Environmental Simulations and Modelling (EnvSim) campaign within ENIGMA. The campaign, which spans multiple laboratories across ENIGMA and is led by the Baliga group, has established synthetic communities (SynComs) to discover, characterize, and dissect key microbial processes relevant to field observations, such as emissions of the greenhouse gas nitrous oxide (N 2 O). For example, the EnvSim campaign so far has deduced four potential mechanisms that may account for the N 2 O emissions at the FRC and are currently being investigated by labs across ENIGMA. Denitrification may be driven by complete denitrifiers, however, their NosZ enzymes, which catalyze the final step in denitrification by converting N 2 O to N 2 , may be sensitive at a lower pH. Additionally, the denitrification process could be partitioned among organisms and some may have pH-sensitive NosZ genes. Another factor contributing to variable N 2 O emissions relates to the metal co-factors involved in the denitrification pathway. For instance, excess Cu, Al, Mn, U, Ni, Co, Cu, and/or Cd may have inhibitory effects on multiple enzymatic steps during denitrification, while the enzymatic production of nitrite, the precursor of N 2 O, can be limited by the essential metal Mo. Lastly, abiotic production of N 2 O may occur as a result of chemodenitrification, in which metals like Fe, Mn, and some organic compounds can drive redox reactions that convert nitrogen cycle intermediates to N 2 O under the right conditions. Current investigations and analyses have generated multiple transcriptomic profiles aiding the refinement of new metabolic and gene regulatory network model for our denitrifying SynCom. In sum, the previous funding cycle has generated 33 peer-reviewed publications spanning predictive network biology to co-operativity of mutualistic interspecies interactions of evolved communities. Our integration of multiple omics datasets and the development of new network modelling tools has revealed important insights that can explain field observed phenomenon and generated new hypotheses to be investigated in the field.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

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

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

Candidatus↗