Microbiological and geochemical surveys were conducted at three hot springs (Obsidian Pool, Sylvan Spring, and ‘Bison Pool’) in Yellowstone National Park (Wyoming, USA). Microbial community structure was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA gene sequences from DNA extracted from sediments of each hot spring, followed by molecular cloning. Both bacterial and archaeal DNA was retrieved from all samples. No Euryarchaea were found, but diverse Crenarchaea exist in all three pools, particularly affiliating with deep‐branching, but uncultivated organisms. In addition, cloned DNA affiliating with the Desulphurococcales and Thermoproteales was identified, but the distribution of taxa differs in each hot spring. The bacterial community at all three locations is dominated by members of the Aquificales and Thermodesulfobacteriales, indicating that the ‘knallgas’ reaction (aerobic hydrogen oxidation) may be a central metabolism in these ecosystems. To provide geochemical context for the microbial community structures, energy‐yields for a number of chemolithoautotrophic reactions are provided for >80 sampling sites in Yellowstone, including Obsidian Pool, Sylvan Spring, and ‘Bison Pool’. This energy profile shows that the knallgas reaction is just one of many exergonic reactions in the Yellowstone hot springs, that energy‐yields for certain reactions can vary substantially from one site to the next, and that few of the demonstrated exergonic reactions are known to support microbial metabolism.

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We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the “Bison Pool” (BP) Environmental Genome and a complementary contextual geochemical dataset of ∼75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92°C chemotrophic streamer biofilm community in the BP source pool to a 56°C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic “transition” community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability.

[1] Thermophilic microbes in hydrothermal ecosystems have multiple metabolic strategies for taking up carbon and nitrogen, which may result in distinct stable isotopic compositions of C and N in living biomass, as well as other biosignatures that accumulate in the geologic record. Biofilms from "Bison Pool" at Yellowstone National Park display large variations in carbon and nitrogen isotopic values as a function of downstream sampling and illustrate the presence of large shifts in ecological functions as temperature decreases. This is the first study to couple isotopic data with community genomic analysis to predict dominant carbon fixation pathways that create hydrothermal biofilm signatures. The results also suggest nitrogen limitation through the chemotrophic zone and nitrogen fixation in the lower-temperature phototrophic zone.

In Yellowstone National Park, a small percentage of thermal features support streamer biofilm communities (SBCs), but their growth criteria are poorly understood. This study investigates biofilms in two SBC hosting, and two non-SBC springs. Sequencing of 16S rRNA clones indicates changing community structure as a function of downstream geochemistry, with many novel representatives particularly among the Crenarchaeota. While some taxonomic groups show little genetic variation, others show specialization by sample location. The transition fringe environment between the hotter chemosynthetic and cooler photosynthetic zones hosts a larger diversity of organisms in SBC bearing springs. This transition is proposed to represent an ecotone; this is the first description of an ecotone in a hydrothermal environment. The Aquificales are ubiquitous and dominate among the Bacteria in the hottest environments. However, there is no difference in species of Aquificales from SBC and non-SBC locations, suggesting they are not responsible for the formation of SBCs, or that their role in SBC formation is competitively suppressed in non-SBC sites. In addition, only SBC locations support Thermotogales-like organisms, highlighting the potential importance these organisms may have in SBC formation. Here, we present a novel view of SBC formation and variability in hydrothermal ecosystems.