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Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~10³cells mL⁻¹) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers includingCandidatusDesulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell⁻¹h⁻¹. Scaled to volume, this equates to a bulk environmental rate of ~10³pmol sulfate L⁻¹d⁻¹, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable toCa.Desulforudis audaxviator. 

Abstract The phyla Nitrospirota and Nitrospinota have received significant research attention due to their unique nitrogen metabolisms important to biogeochemical and industrial processes. These phyla are common inhabitants of marine and terrestrial subsurface environments and contain members capable of diverse physiologies in addition to nitrite oxidation and complete ammonia oxidation. Here, we use phylogenomics and gene-based analysis with ancestral state reconstruction and gene-tree–species-tree reconciliation methods to investigate the life histories of these two phyla. We find that basal clades of both phyla primarily inhabit marine and terrestrial subsurface environments. The genomes of basal clades in both phyla appear smaller and more densely coded than the later-branching clades. The extant basal clades of both phyla share many traits inferred to be present in their respective common ancestors, including hydrogen, one-carbon, and sulfur-based metabolisms. Later-branching groups, namely the more frequently studied classes Nitrospiria and Nitrospinia, are both characterized by genome expansions driven by either de novo origination or laterally transferred genes that encode functions expanding their metabolic repertoire. These expansions include gene clusters that perform the unique nitrogen metabolisms that both phyla are most well known for. Our analyses support replicated evolutionary histories of these two bacterial phyla, with modern subsurface environments representing a genomic repository for the coding potential of ancestral metabolic traits. 

Leading deep-sea research expeditions requires a breadth of training and experience, and the opportunities for Early Career Researchers (ECRs) to obtain focused mentorship on expedition leadership are scarce. To address the need for leadership training in deep-sea expeditionary science, the Crustal Ocean Biosphere Research Accelerator (COBRA) launched a 14-week virtual Master Class with both synchronous and asynchronous components to empower students with the skills and tools to successfully design, propose, and execute deep-sea oceanographic field research. The Master Class offered customized and distributed training approaches and created an open-access syllabus with resources, including reading material, lectures, and on-line resources freely-available on the Master Class website (cobra.pubpub.org). All students were Early Career Researchers (ECRs, defined here as advanced graduate students, postdoctoral scientists, early career faculty, or individuals with substantial industry, government, or NGO experience) and designated throughout as COBRA Fellows. Fellows engaged in topics related to choosing the appropriate deep-sea research asset for their Capstone “dream cruise” project, learning about funding sources and how to tailor proposals to meet those source requirements, and working through an essential checklist of pre-expedition planning and operations. The Master Class covered leading an expedition at sea, at-sea operations, and ship-board etiquette, and the strengths and challenges of telepresence. It also included post-expedition training on data management strategies and report preparation and outputs. Throughout the Master Class, Fellows also discussed education and outreach, international ocean law and policy, and the importance and challenges of team science. Fellows further learned about how to develop concepts respectfully with regard to geographic and cultural considerations of their intended study sites. An assessment of initial outcomes from the first iteration of the COBRA Master Class reinforces the need for such training and shows great promise with one-quarter of the Fellows having submitted a research proposal to national funding agencies within six months of the end of the class. As deep-sea research continues to accelerate in scope and speed, providing equitable access to expedition training is a top priority to enable the next generation of deep-sea science leadership. 

Abstract The ocean–atmosphere exchange of CO 2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton) 1–3 , their respiration usually is measured in bulk and treated as a ‘black box’ in global biogeochemical models 4 ; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy 3,5–7 probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO 2 for its energy demands and growth may be lower than commonly assumed and variable among lineages. 

Abstract The deep seafloor covers two-thirds of Earth's surface area, but our understanding of the ecosystems and resources found in the deep ocean, as well as the ability of deep-sea ecosystems to withstand human perturbation, is limited. These deep-sea habitats demand urgent study as there are emergent human uses in the form of deep-sea mining and carbon sequestration that will fundamentally alter physical, chemical, and biological conditions that took millions of years to establish. We propose the international network COBRA, a research accelerator for the crustal ocean biosphere. COBRA will bring together diverse stakeholders and experts, including interdisciplinary academic and government scientists, private institutions, policy makers, data systems engineers, industry experts, and others to coordinate efforts that generate new knowledge and inform decision making about activities that could affect the deep ocean and, by extension, all of society. We will also train the next generation of leaders in ocean exploration, science, and policy through an innovative virtual program to carry this effort into future generations of ocean and earth science research. COBRA will inform policies relating to emergent industrial uses of the deep ocean, decrease the likelihood of serious harm to the environment, and maintain ecosystem services for the benefit of society. 

Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 10 3 cells ml –1 ) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis. 

Permafrost sediments contain one of the largest reservoirs of organic carbon on Earth that is relatively stable when it remains frozen. As air temperatures increase, the shallow permafrost thaws which allows this organic matter to be converted into potent greenhouse gases such as methane (CH4) and carbon dioxide (CO2) through microbial processes. Along the Beaufort Sea coast in the vicinity of the Tuktoyaktuk Peninsula, Northwest Territories, Canada, warming air temperatures are causing the active layer above permafrost to deepen, and a number of active periglacial processes are causing rapid erosion of previously frozen permafrost. In this paper, we consider the biogeochemical consequences of these processes on the permafrost sediments found at Tuktoyaktuk Island. Our goals were to document the in situ carbon characteristics which can support microbial activity, and then consider rates of such activity if the permafrost material were to warm even further. Samples were collected from a 12mpermafrost core positioned on the top of the island adjacent to an eroding coastal bluff. Downcore CH4, total organic carbon and dissolved organic carbon (DOC) concentrations and stable carbon isotopes revealed variable in situ CH4 concentrations down core with a sub-surface peak just below the current active layer. The highest DOC concentrations were observed in the active layer. Controlled incubations of sediment from various depths were carried out from several depths anaerobically under thawed (5°C and 15°C) and under frozen (−20°C and −5°C) conditions. These incubations resulted in gross production rates of CH4 and CO2 that increased upon thawing, as expected, but also showed appreciable production rates under frozen conditions. This dataset presents the potential for sediments below the active layer to produce potent greenhouse gases, even under frozen conditions, which could be an important atmospheric source in the actively eroding coastal zone even prior to thawing.