Hydrogen produced during water-rock serpentinization reactions can drive the synthesis of organic compounds both biotically and abiotically. We investigated abiotic carbon production and microbial metabolic pathways at the high energy but low diversity serpentinite-hosted Lost City hydrothermal field. Compound-specific 14C data demonstrates that formate is mantle-derived and abiotic in some locations and has an additional, seawater-derived component in others. Lipids produced by the dominant member of the archaeal community, the Lost City Methanosarcinales, largely lack 14C, but metagenomic evidence suggests they cannot use formate for methanogenesis. Instead, sulfate-reducing bacteria may be the primary consumers of formate in Lost City chimneys. Paradoxically, the archaeal phylotype that numerically dominates the chimney microbial communities appears ill suited to live in pure hydrothermal fluids without the co-occurrence of organisms that can liberate CO2. Considering the lack of dissolved inorganic carbon in such systems, the ability to utilize formate may be a key trait for survival in pristine serpentinite-hosted environments.

The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth’s mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H 2 ) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon.

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.

The Lost City hydrothermal field on the Mid-Atlantic Ridge supports dense microbial life on the lofty calcium carbonate chimney structures. The vent field is fueled by chemical reactions between the ultramafic rock under the chimneys and ambient seawater. These serpentinization reactions provide reducing power (as hydrogen gas) and organic compounds that can serve as microbial food; the most abundant of these are methane and formate. Previous studies have characterized the interior of the chimneys as a single-species biofilm inhabited by the Lost City Methanosarcinales, but they also indicated that this methanogen is unable to metabolize formate. The new metagenomic results presented here indicate that carbon cycling in these Lost City chimney biofilms could depend on the metabolism of formate by Chloroflexi populations. Additionally, we present evidence for metabolically diverse, formate-utilizing Sulfurovum populations and new genomic and phylogenetic insights into the unique Lost City Methanosarcinales. IMPORTANCE Primitive forms of life may have originated around hydrothermal vents at the bottom of the ancient ocean. The Lost City hydrothermal vent field, fueled by just rock and water, provides an analog for not only primitive ecosystems but also potential extraterrestrial rock-powered ecosystems. The microscopic life covering the towering chimney structures at the Lost City has been previously documented, yet little is known about the carbon cycling in this ecosystem. These results provide a better understanding of how carbon from the deep subsurface can fuel rich microbial ecosystems on the seafloor.

We report the first census of natural microbial communities of the Bonneville Salt Flats (BSF), a perennial salt pan at the Utah-Nevada border. Environmental DNA sequencing of archaeal and bacterial 16S rRNA genes was conducted on samples from multiple evaporite sediment layers collected from the upper 30 cm of the surface salt crust. Our results show that at the time of sampling (September 2016), BSF hosted a robust microbial community dominated by diverse halobacteria and Salinibacter species. Sequences identical to Geitlerinema sp. strain PCC 9228, an anoxygenic cyanobacterium that uses sulfide as the electron donor for photosynthesis, are also abundant in many samples. We identified taxonomic groups enriched in each layer of the salt crust sediment and revealed that the upper gypsum sediment layer found immediately under the uppermost surface halite contains a robust microbial community. In these sediments, we found an increased presence of Thermoplasmatales, Hadesarchaeota, Nanoarchaeaeota, Acetothermia, Desulfovermiculus, Halanaerobiales, Bacteroidetes, and Rhodovibrio. This study provides insight into the diversity, spatial heterogeneity, and geologic context of a surprisingly complex microbial ecosystem within this macroscopically sterile landscape. IMPORTANCE Pleistocene Lake Bonneville, which covered a third of Utah, desiccated approximately 13,000 years ago, leaving behind the Bonneville Salt Flats (BSF) in the Utah West Desert. The potash salts that saturate BSF basin are extracted and sold as an additive for agricultural fertilizers. The salt crust is a well-known recreational and economic commodity, but the biological interactions with the salt crust have not been studied. This study is the first geospatial analysis of microbially diverse populations at this site using cultivation-independent environmental DNA sequencing methods. Identification of the microbes present within this unique, dynamic, and valued sedimentary evaporite environment is an important step toward understanding the potential consequences of perturbations to the microbial ecology on the surrounding landscape and ecosystem.

25We report the first census of natural microbial communities of the Bonneville Salt Flats 26 (BSF), a perennial salt pan at the Utah-Nevada border. Environmental DNA sequencing of 27 archaeal and bacterial 16S rRNA genes was conducted on samples from multiple evaporite 28 sediment layers of the surface salt crust. Our results show that at the time of sampling 29 (September 2016), BSF hosted a robust microbial community dominated by diverse 30 Halobacteriaceae and Salinibacter species. Desulfuromonadales from GR-WP33-58 are also 31 abundant in all samples. We identified taxonomic groups enriched in each layer of the salt crust 32 sediment and revealed that the upper gypsum sediment layer found immediately under the 33 uppermost surface halite contains a robust microbial community. We found an increased 34 presence of Thermoplasmatales, Nanohaloarchaeota, Woesearchaeota, Acetothermia, 35 Halanaerobium, Parcubacteria, Planctomycetes, Clostridia, Gemmatimonadetes, Marinilabiaceae 36 and other Bacteroidetes in this upper gypsum layer. This study provides insight into the diversity, 37 spatial heterogeneity, and geologic context of a surprisingly complex microbial ecosystem within 38 this macroscopically-sterile landscape. 39 40 IMPORTANCE 41Over the last ~13,000 years the Pleistocene Lake Bonneville, which covered a large 42 portion of Utah, drained and desiccated leaving behind the Bonneville Salt Flats (BSF). Today 43 BSF is famous for its use as a speedway, which has hosted many land-speed records and a 44 community that greatly values this salty landscape. Additionally, the salts that saturate BSF basin 45 are extracted and sold as an additive for agricultural fertilizers. The salt crust is a well-known 46 recreational and economic commodity, but the roles of microbes in the formation and 47 3 maintenance of the salt crust are generally unknown. This study is the first geospatial analysis of 48 microbial diversity at this site using cultivation-independent environmental DNA sequencing 49 methods. Identification of the microbes present within this unique, dynamic, and valued 50 sedimentary evaporite environment is an important step toward understanding the potential 51 consequences of perturbations to the microbial ecology on the surrounding landscape and 52 ecosystem.53 54 65Human land use has altered many aspects of the hydrology and morphology of the environment 66 that facilitated deposition of the ~2m thick evaporite salt crust that caps BSF, and the amount 67 and extent of salt present at the site have been observed to change through time (Bowen, B.B., 68 Bernau, J., Kipnis, E.L., Lerback, J., Wetterlin, L. and Kleba, 2018; Bowen et al., 2018).69 4Examination of modern and ancient salt pan deposits demonstrates that these 70 environments undergo repeated cycles of desiccation (dry saline pan), flooding (brackish lake), 71 and evaporative concentration (drying to dry saline pan) (Bowen and Benison, 2009; 72 Lowenstein, T.K. and Hardie, 1985). While the desiccation stage is most common, it is 73 repeat...

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirae) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal and subsurface alkaline environments where hydrogen and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface environments where energy and carbon are provided by geochemical reactions.

24The Lost City hydrothermal field on the Mid-Atlantic Ridge supports dense microbial life on the 25 lofty calcium carbonate chimney structures. The vent field is fueled by chemical reactions 26 between the ultramafic rock under the chimneys and ambient seawater. These serpentinization 27 reactions provide reducing power (as hydrogen gas) and organic compounds that can serve as 28 microbial food; the most abundant of these are methane and formate. Previous studies have 29 characterized the interior of the chimneys as a single-species biofilm inhabited by the Lost City 30 Methanosarcinales, but also indicated that this methanogen is unable to metabolize formate. The 31 new metagenomic results presented here indicate that carbon cycling in these Lost City chimney 32 biofilms could depend on the metabolism of formate by low-abundance Chloroflexi species. 33Additionally, we present evidence that metabolically diverse, formate-utilizing Sulfurovum 34 species are living in the transition zone between the interior and exterior of the chimneys. 35 36 IMPORTANCE 37 Primitive forms of life may have originated around hydrothermal vents at the bottom of the 38 ancient ocean. The Lost City hydrothermal vent field, fueled by just rock and water, provides an 39 analog for not only primitive ecosystems but also extraterrestrial ecosystems that might support 40 life. The microscopic life covering towering chimney structures at the Lost City has been well 41 characterized, yet little is known about the carbon cycling in this ecosystem. These results 42 provide a better understanding of how carbon from the deep subsurface can fuel rich microbial 43 ecosystems on the seafloor.44 45 INTRODUCTION 46 3 The towering carbonate chimneys of the Lost City hydrothermal vent field protrude from 47 the Atlantis Massif, a dome of ultramafic rock uplifted from the mantle. These chimneys differ 48 from other deep-sea hydrothermal systems because they are driven primarily by rock-water 49 reactions known as serpentinization, rather than magmatic activity. The serpentinization 50 reactions create high pH fluids that mix with surrounding cold seawater to form the calcium 51 carbonate structures. Serpentinite-hosted ecosystems are of astrobiological interest because they 52 provide a source of fuel and energy for life that does not require sunlight or an active planetary 53 body (i.e. geothermal energy). These systems are thought to be present on inactive planetary 54 bodies such as Jupiter's moon Europa (1, 2). 55 The dense microbial biofilms of Lost City chimneys are fueled by the carbon and energy 56 released by serpentinization of the underlying ultramafic rock (3-7). The serpentinization 57 reactions provide high concentrations of hydrogen gas, methane, and other simple organic 58 compounds that serve as food and energy sources for microbes. The more extreme interiors of 59 chimneys are anoxic and continually bathed in the warm serpentinizing fluids. Temperatures of 60 venting fluids can reach >95°C, and pH of the fluids can be as high as 11 ...