Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem.

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 (~103 cells 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 including Candidatus Desulforudis 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-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-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 to Ca. Desulforudis audaxviator.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

Replicated life-history patterns and subsurface origins of the bacterial sister phyla Nitrospirota and Nitrospinota.

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.

Metabolic Potential of Microbial Communities in the Hypersaline Sediments of the Bonneville Salt Flats.

The Bonneville Salt Flats (BSF) appear to be entirely desolate when viewed from above, but they host rich microbial communities just below the surface salt crust. In this study, we investigated the metabolic potential of the BSF microbial ecosystem. The predicted and measured metabolic activities provide new insights into the ecosystem functions of evaporite landscapes and are an important analog for potential subsurface microbial ecosystems on ancient and modern Mars. Hypersaline and evaporite systems have been investigated previously as astrobiological analogs for Mars and other salty celestial bodies, but these studies have generally focused on aquatic systems and cultivation-dependent approaches. Here, we present an ecosystem-level examination of metabolic pathways within the shallow subsurface of evaporites. We detected aerobic and anaerobic respiration as well as methanogenesis in BSF sediments. Metagenome-assembled genomes of diverse bacteria and archaea encode a remarkable diversity of metabolic pathways, including those associated with carbon fixation, carbon monoxide oxidation, acetogenesis, methanogenesis, sulfide oxidation, denitrification, and nitrogen fixation. These results demonstrate the potential for multiple energy sources and metabolic pathways in BSF and highlight the possibility for vibrant microbial ecosystems in the shallow subsurface of evaporites. IMPORTANCE The Bonneville Salt Flats is a unique ecosystem created from 10,000 years of desiccation and serves as an important natural laboratory for the investigation of the habitability of salty, halite, and gypsum-rich environments. Here, we show that gypsum-rich mineral deposits host a surprising diversity of organisms and appear to play a key role in stimulating the microbial cycling of sulfur and nitrogen compounds. This work highlights how diverse microbial communities within the shallow subsurface sediments are capable of maintaining an active and sustainable ecosystem, even though the surface salt crust appears to be completely devoid of life.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

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 molecular 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 Nitrospirota) 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 environments where H2 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 alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE 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 (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Genomic Evidence for Formate Metabolism by Chloroflexi as the Key to Unlocking Deep Carbon in Lost City Microbial Ecosystems.

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 MethanosarcinalesIMPORTANCE 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.

Robust Archaeal and Bacterial Communities Inhabit Shallow Subsurface Sediments of the Bonneville Salt Flats.

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.

Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field.

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.