Yellowstone Hot Springs Research Articles

Environmental analogs from yellowstone hot springs on geochemical and microbial diversity with implications for the search for life on Mars

From Viking landers to Perseverance rover, Mars has been explored by several in situ missions capable of analyzing organic compounds. Results from the SAM and SHERLOC on Curiosity and Perseverance, respectively, support the detection of lean organic matter (at ppb-ppm levels) in the top surface samples, although the source(s) and preservation mechanisms are still ambiguous. Perseverance is currently exploring a fluvio-lacustrine system at Jezero crater and may explore an ancient volcanic terrain after exiting the crater. As Perseverance would collect samples for potential return to Earth, preparation is needed for sample return efforts through various means including i) the detection of trace organic compounds in various matrices, ii) validation of compounds identified by Martian rovers, and iii) better understanding of mechanisms of their production on Mars. On these returned samples, the community may be able to resolve the timing of organic matter formation and refine hypotheses regarding organic preservation in Martian soils despite the presence of numerous oxidants, salts, and pH-temperature intra and inter-site variations that are less conductive to long-term preservation of organic matter. For instance, acidic conditions promote clay catalyzed isomerization, but seem to benefit for the fatty acid preservation producing organic-salts or favoring salt dissolution in the matrix to protect organic compounds from radiations and water alteration. With a similar aim, we selected samples from Yellowstone National Park hot springs and silica sinters as analogs to locations visited by Curiosity and Perseverance or – in the future – Rosalind Franklin rover. The hot springs in this study developed over hundreds to thousands of years, providing optimal conditions (i.e., matrix composition, temperature, pH) of preservation for organic molecules, extremophilic and mesophilic cells. In our study, the most well preserved organic matter and biosignatures were detected in acidic silica sinters with a surface (water) temperature below 50 °C and a minor crystalline phase. The gas chromatography – mass spectrometry molecular analysis revealed a variety of organic compounds we classified as bioindicators (such as amino acids, nucleobases, and sugars), and biosignatures (such as long-chain branched and/or (poly)unsaturated lipids, secondary metabolites involved in the quorum sensing or communication between individuals). We validated with a SAM/MOMA-like benchtop extracting oven the organic matter extraction protocols performed with the SAM experiment. We identified using the different SAM and MOMA extraction protocols (pyrolysis and wet-chemistry derivatizations) eight microbial classes through a unique untargeted environmental metabolomics’ method embracing space flight technology constraints. Additionally, we identified one (and likely two) agnostic biosignature(s): i) the concomitance of some elements and organic compounds in the analogs (correlation of organic matter elements: C, N, S, P and organic molecules co-located with essential biological elements: Fe, Mg, V, Mn and non-essential biological elements concentrated by microorganisms: As, Cs, Ga), and ii) the negative isotope C and N ratio demonstrating organic molecules rich in 12C and 14N: archaeal, bacterial, and eukaryotic lipids for an efficient low energy-consuming metabolism.

Assessing microbial diversity in Yellowstone National Park hot springs using a field deployable automated nucleic acid extraction system

Microbial diversity estimation involves extracting nucleic acids from intricate sample matrices. Preparing nucleic acid samples is time-consuming, necessitating effective cell lysis and obtaining pure, inhibitor-free nucleic acid purifications before further use. An automated system offers advantages for field deployment due to its ease of use and quick autonomous results. This is especially beneficial for rapid measurement of in situ microbial diversity in remote areas. Our study aimed to assess microbial diversity of Yellowstone hot springs using a field-deployable lab in a resource-limited remote setting and demonstrate on-site nucleic acid sample processing and sequencing. We collected microbial mat and sediment samples from several Yellowstone National Park hot springs, focusing on the Five Sister Springs (FSS), spring LNN010, and Octopus Spring (OS). The samples were processed for DNA extraction on-site and further sequenced in the lab for microbial diversity. In addition, DNA extracted from one sample was sequenced and analyzed on-site as proof-of-concept. Using either Illumina or Oxford Nanopore Technology sequencing, we found similar microbial diversities. Bacteria (over 90%) were predominant at the FSS and OS sites, with archaea accounting for less than 10%. Metagenomic results were taxonomically categorized based on the closest known organism with a sequenced genome. The dominant archaeal community member was Candidatus Caldiarchaeum subterraneum, and among bacteria, Roseiflexus sp. RS-1 was abundant in mat samples. Interestingly, Bacterium HR17 was also frequently found, suggesting the need for more research on this newly recognized bacterial community member. The presence of Bacterium HR17 in these hot springs suggests its potential role in nitrogen cycling, informing both ecological understanding and industrial potential. This pioneering study assessed the microbiome of Yellowstone hot springs in about 8-9 hours using an automated system for nucleic acid extraction. By its deployment, the system’s value in elucidating the microbial diversity of extreme environments without the need to bring samples to the lab for processing had been highlighted. Sample processing and sequencing had been included in the benefits of the field-deployable lab, and the Nanopore platform had been utilized.

Methylotrophic methanogenesis in the Archaeoglobi revealed by cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring.

Over the past decade, environmental metagenomics and polymerase chain reaction-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of thermophilic Euryarchaeota that have long been considered to live non-methanogenic lifestyles. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64-70°C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park, as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems.

Genomic insight and physiological characterization of thermoacidophilic Alicyclobacillus isolated from Yellowstone National Park.

Alicyclobacillus has been isolated from extreme environments such as hot springs, volcanoes, as well as pasteurized acidic beverages, because it can tolerate extreme temperatures and acidity. In our previous study, Alicyclobacillus was isolated during the enrichment of methane oxidizing bacteria from Yellowstone Hot Spring samples. Physiological characterization and genomic exploration of two new Alicyclobacillus isolates, AL01A and AL05G, are the main focus of this study to identify their potential relationships with a thermoacidophilic methanotroph (Methylacidiphilum) isolated from the same hot spring sediments. In the present study, both Alicyclobacillus isolates showed optimal growth at pH 3.5 and 55°C, and contain ω-alicyclic fatty acids as a major lipid (ca. 60%) in the bacterial membrane. Genomic analysis of these strains revealed specific genes and pathways that the methanotroph genome does not have in the intermediary carbon metabolism pathway such as serC (phosphoserine aminotransferase), comA (phosphosulfolactate synthase), and DAK (glycerone kinase). Both Alicyclobacillus strains were also found to contain transporter systems for extracellular sulfate (ABC transporter), suggesting that they could play an important role in sulfur metabolism in this extreme environment. Genomic analysis of vitamin metabolism revealed Alicyclobacillus and Methylacidiphilum are able to complement each other's nutritional deficiencies, resulting in a mutually beneficial relationship, especially in vitamin B1(thiamin), B3 (niacin), and B7 (biotin) metabolism. These findings provide insights into the role of Alicyclobacillus isolates in geothermal environments and their unique metabolic adaptations to these environments.

Hot Is Rich—An Enormous Diversity of Simple Trichal Cyanobacteria from Yellowstone Hot Springs

In 2019, cyanobacterial samples were collected from thermal sites in Yellowstone National Park. In this paper, we discuss the considerable richness of representatives of simple filamentous cyanobacteria. Using a polyphasic approach, one new genus, Copelandiella, with two species (C. yellowstonensis and C. thermalis) and eight additional new species Albertania prattii, K. anagnostidisii, Kovacikia brockii, Leptolyngbya tildenae, L. vaporiphila, Nodosilinea calida, N. igneolacustris, and Oculatella castenholzii were described. At the same time, by analyzing our new molecular data, we concluded that other genera of trichal cyanobacteria can be merged with existing genera: species within the genus Plectolyngbya are herein transferred to Leptolyngbya, and if some nomenclatural issues are resolved, species within the genus Leptothermofonsia will be subsequently transferred to the genus Kovacikia, an earlier synonym.

Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

Relationships between fluid mixing, biodiversity, and chemosynthetic primary productivity in Yellowstone hot springs

Relationships between fluid mixing, biodiversity, and chemosynthetic primary productivity in Yellowstone hot springs

Relationships between fluid mixing, biodiversity, and chemosynthetic primary productivity in Yellowstone hot springs.

The factors that influence biodiversity and productivity of hydrothermal ecosystems are not well understood. Here we investigate the relationship between fluid mixing, biodiversity, and chemosynthetic primary productivity in three co-localized hot springs (RSW, RSN, and RSE) in Yellowstone National Park that have different geochemistry. All three springs are sourced by reduced hydrothermal fluid, but RSE and RSN receive input of vapour phase gas and oxidized groundwaters, with input of both being substantially higher in RSN. Metagenomic sequencing revealed that communities in RSN were more biodiverse than those of RSE and RSW in all dimensions evaluated. Microcosm activity assays indicate that rates of dissolved inorganic carbon (DIC) uptake were also higher in RSN than in RSE and RSW. Together, these results suggest that increased mixing of reduced volcanic fluid with oxidized fluids generates additional niche space capable of supporting increasingly biodiverse communities that are more productive. These results provide insight into the factors that generate and maintain chemosynthetic biodiversity in hydrothermal systems and that influence the distribution, abundance, and diversity of microbial life in communities supported by chemosynthesis. These factors may also extend to other ecosystems not supported by photosynthesis, including the vast subterranean biosphere and biospheres beneath ice sheets and glaciers.

Subsurface Archaea associated with rapid geobiological change in a model Yellowstone hot spring

Despite over a century of study, it is unknown if continental hydrothermal fields support high-temperature subsurface biospheres. Cinder Pool is among the deepest hot springs in Yellowstone and is widely studied due to unique sulfur geochemistry that is attributed to hydrolysis of molten elemental sulfur at ∼18 m depth that promotes several chemical reactions that maintain low sulfide, low oxygen, and a moderate pH of ∼4.0. Following ∼100 years of stability, Cinder Pool underwent extreme visual and chemical change (acidification) in 2018. Here, we show that depth-resolved geochemical and metagenomic-based microbial community analyses pre- (2016) and post-acidification (2020) indicate the changes are likely attributable to feedbacks between geological/geochemical processes, sulfur oxidation by subsurface Sulfolobales Archaea, and the disappearance of molten sulfur at depth. These findings underscore the dynamic and rapid feedback between the geosphere and biosphere in continental hydrothermal fields and suggest subsurface biospheres to be more prevalent in these systems than previously recognized.

Controls on Reactive Oxygen Species Cycles in Yellowstone Hot Springs: Implications for Biosignature Preservation on Mars

Early Earth and Mars had analogous environments. While life developed on our planet, the question of whether it did on Mars remains to be answered. Hot spring deposits are compelling targets for exploration because of their high habitability and potential to retain morphological and chemical biosignatures. As a result in this study, we aim to better understand the potential for biosignature preservation in Fe-bearing hydrothermal systems. Understanding oxidation-reduction reactions involving Fe in hot springs is a key step in elucidating the preservation process. Fe reacts readily with reactive oxygen species (ROS), which are produced in hot spring surface waters through photochemical processes. Furthermore, Fe3+ can bind to cell membranes and preserve complex organic molecules (i.e., biomarkers). ROS formation is typically controlled by photoreactions with dissolved organic matter (DOM). However, Fe redox reactions more likely control ROS formation in these Fe-bearing systems. We deconvolved the relationship of ROS with Fe in hot springs and evaluated the role that DOM and dissolved organic sulfur (DOS) may have in ROS production. To better understand these coupled systems, field and laboratory experiments were conducted in hot springs of Yellowstone National Park. In situ H2O2 concentrations observed in these hot springs were comparable to, or higher than, those of other high-temperature systems. Reaction rates determined by measuring concentrations after specified time intervals varied based on water compositions and the presence of particulate or dissolved matter. Fe speciation (photochemical reactivity), concentration, and solubility further determined ROS cycling rates. Specifically, photochemically active Fe enhanced both ROS formation and decay rates depending on incident UV irradiance, and rates increased along with Fe concentration and solubility (i.e., in acidic conditions). Better understanding how ROS and Fe cycle in predominantly abiotic conditions will eventually aid in distinguishing between biosignatures and abiotic substances in the rock record.

Genome Sequence and Characterization of a Xanthorhodopsin-Containing, Aerobic Anoxygenic Phototrophic Rhodobacter Species, Isolated from Mesophilic Conditions at Yellowstone National Park.

The genus Rhodobacter consists of purple nonsulfur photosynthetic alphaproteobacteria known for their diverse metabolic capabilities. Here, we report the genome sequence and initial characterization of a novel Rhodobacter species, strain M37P, isolated from Mushroom hot spring runoff in Yellowstone National Park at 37 °C. Genome-based analyses and initial growth characteristics helped to define the differentiating characteristics of this species and identified it as an aerobic anoxygenic phototroph (AAP). This is the first AAP identified in the genus Rhodobacter. Strain M37P has a pinkish-red pigmentation that is present under aerobic dark conditions but disappears under light incubation. Whole genome-based analysis and average nucleotide identity (ANI) comparison indicate that strain M37P belongs to a specific clade of recently identified species that are genetically and physiologically unique from other representative Rhodobacter species. The genome encodes a unique xanthorhodopsin, not found in any other Rhodobacter species, which may be responsible for the pinkish-red pigmentation. These analyses indicates that strain M37P is a unique species that is well-adapted to optimized growth in the Yellowstone hot spring runoff, for which we propose the name Rhodobacter calidifons sp. nov.

Meet Me in the Middle: Median Temperatures Impact Cyanobacteria and Photoautotrophy in Eruptive Yellowstone Hot Springs.

ABSTRACTGeographic isolation can be a main driver of microbial evolution in hot springs while temperature plays a role on local scales. For example, cyanobacteria, particularly high-temperature Synechococcus spp., have undergone ecological diversification along temperature gradients in hot spring outflow channels. While water flow, and thus temperature, is largely stable in many hot springs, flow can vary in geysing/eruptive hot springs, resulting in large temperature fluctuations (sometimes more than 40°C). However, the role of large temperature fluctuations in driving diversification of cyanobacteria in eruptive hot springs has not been explored. Here, we examined phototroph community composition and potential photoautotrophic activity in two alkaline eruptive hot springs with similar geochemistry in the Lower Geyser Basin in Yellowstone National Park, WY. We observed distinct cyanobacterial amplicon sequencing variants (ASVs) consistent with allopatry and levels of light-dependent inorganic carbon uptake rates similar to other hot springs, despite large temperature fluctuations. Our data suggest median temperatures may drive phototroph fitness in eruptive hot springs while future studies are necessary to determine the evolutionary consequences of thriving under continuously fluctuating temperatures. We propose that large temperature swings in eruptive hot springs offer unique environments to examine the role of allopatry versus physical and chemical characteristics of ecosystems in driving cyanobacterium evolution and add to the debate regarding the ecology of thermal adaptation and the potential for narrowing niche breadth with increasing temperature.IMPORTANCE Hot spring cyanobacteria have long been model systems for examining ecological diversification as well as characterizing microbial adaptation and evolution to extreme environments. These studies have reported cyanobacterial diversification in hot spring outflow channels that can be defined by distinct temperature ranges. Our study builds on these previous studies by examining cyanobacteria in geysing hot springs. Geysing hot springs result in outflow channels that experience regular and large temperature fluctuations. While community compositions are similar between geysing and nongeysing hot spring outflow channels, our data suggest median, rather than high, temperature drives the fitness of cyanobacteria in geysing hot springs. We propose that large temperature swings may result in patterns of ecological diversification that are distinct from more stable outflows.

Dynamics of the dissolved organic matter in a stream-lake system within an extremely acid to neutral pH range: Agrio-Caviahue watershed.

The Agrio-Caviahue is a natural stream-lake system composed of the Upper Agrio River (UAR) -Lake Caviahue-Lower Agrio River (LAR). The system has a broad pH gradient from extremely acid in the hot spring to almost neutral pH only 60km downstream the source, and varies as a function of the distance. The aim of this study was to analyze the dynamics of the dissolved organic matter (DOM) of this extreme system. For that matter, an absorbance and fluorescence-based characterization was performed on natural water samples and hydrophobic resin DOM extracts during different seasons in nine sampling stations. Between August and December, the hydrological connectivity is maximum due to precipitation and snowmelt, respectively. During these months, the stream that exits the lake governs the chemical characteristics downstream mainly in the period of high connectivity. In contrast, in the dry season when the flow of LAR is minimum, the two major affluents of this river influence its pH and DOM features, and deliver inputs of humified DOM from the wetlands where the tributaries are born. DOM was characterized by having low absorbance. The PARAFAC analysis of the fluorescent DOM (FDOM) validated three fluorescent components in the natural samples (with humic and non-humic features), two of which were also recorded in the extracts, meaning that no additional hydrophobic components were missed under the limit of detection of the fluorometer. The unique features of Agrio-Caviahue DOM resemble those found in DOM from Yellowstone hot springs, both acid and alkaline.

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat.

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (Ci), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of Ci incorporation into different taxa, mat samples were incubated with [13C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13C. Most 13C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci fixation. Other taxa showed much lower [13C]bicarbonate incorporation.IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven Ci fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13C/12C ratios of mat biomass will help geochemists interpret the 13C/12C ratios of organic carbon in the fossil record.

Probing the geological source and biological fate of hydrogen in Yellowstone hot springs.

Hydrogen (H2 ) is enriched in hot springs and can support microbial primary production. Using a series of geochemical proxies, a model to describe variable H2 concentrations in Yellowstone National Park (YNP) hot springs is presented. Interaction between water and crustal iron minerals yields H2 that partition into the vapour phase during decompressional boiling of ascending hydrothermal fluids. Variable vapour input leads to differences in H2 concentration among springs. Analysis of 50 metagenomes from a variety of YNP springs reveals that genes encoding oxidative hydrogenases are enriched in communities inhabiting springs sourced with vapour-phase gas. Three springs in the Smokejumper (SJ) area of YNP that are sourced with vapour-phase gas and with the most H2 in YNP were examined to determine the fate of H2 . SJ3 had the most H2 , the most 16S rRNA gene templates and the greatest abundance of culturable hydrogenotrophic and autotrophic cells of the three springs. Metagenomics and transcriptomics of SJ3 reveal a diverse community comprised of abundant populations expressing genes involved in H2 oxidation and carbon dioxide fixation. These observations suggest a link between geologic processes that generate and source H2 to hot springs and the distribution of organisms that use H2 to generate energy.

Yellowstone Hot Springs are Organic Chemodiversity Hot Spots

Yellowstone National Park hydrothermal springs were investigated according to their organic geochemistry with a special focus on the Yellowstone hot spring dissolved organic matter (YDOM) that was solid-phase extracted. Here we show that YDOM has a unique chemodiversity that has not yet been observed anywhere else in aquatic surface environments and that Yellowstone hot springs are organic chemodiversity hot spots. Four main geochemically classified hot spring types (alkaline-chloride, mixed alkaline-chloride, acid-chloride-sulfate and travertine-precipitating) exhibited distinct organic molecular signatures that correlated remarkably well with the known inorganic geochemistry and manifested themselves in excitation emission matrix fluorescence, nuclear magnetic resonance, and ultrahigh resolution mass spectra. YDOM contained thousands of molecular formulas unique to Yellowstone of which 80% contained sulfur, even in low hydrogen sulfide containing alkaline-chloride springs. This unique YDOM reflects the extreme organic geochemistry present in the hydrothermal features of Yellowstone National Park.

Direct measurement of advective heat flux from several Yellowstone hot springs, Wyoming, USA

Direct measurement of advective heat flux from several Yellowstone hot springs, Wyoming, USA

Ecological differentiation in planktonic and sediment-associated chemotropic microbial populations in Yellowstone hot springs.

National Aeronautics and Space Administration Exobiology and Evolutionary Biology (NNX13AI11G); National Science Foundation (EAR-1529963)

Ecological differentiation in planktonic and sediment-associated chemotrophic microbial populations in Yellowstone hot springs

Chemosynthetic sediment and planktonic community composition and sizes, aqueous geochemistry and sediment mineralogy were determined in 15 non-photosynthetic hot springs in Yellowstone National Park (YNP). These data were used to evaluate the hypothesis that differences in the availability of dissolved or mineral substrates in the bulk fluids or sediments within springs coincides with ecologically differentiated microbial communities and their populations. Planktonic and sediment-associated communities exhibited differing ecological characteristics including community sizes, evenness and richness. pH and temperature influenced microbial community composition among springs, but within-spring partitioning of taxa into sediment or planktonic communities was widespread, statistically supported (P < 0.05) and could be best explained by the inferred metabolic strategies of the partitioned taxa. Microaerophilic genera of the Aquificales predominated in many of the planktonic communities. In contrast, taxa capable of mineral-based metabolism such as S(o) oxidation/reduction or Fe-oxide reduction predominated in sediment communities. These results indicate that ecological differentiation within thermal spring habitats is common across a range of spring geochemistry and is influenced by the availability of dissolved nutrients and minerals that can be used in metabolism.

Stable isotope labeling confirms mixotrophic nature of streamer biofilm communities at alkaline hot springs.

Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75–88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and “Bison Pool,” using various 13C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest 13C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. 13C-glucose showed a similar, but a 10–30 times lower uptake across most fatty acids. 13C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at “Bison Pool” and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of 13C-formate occurred only at very low rates at “Bison Pool” and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. 13C-uptake into archaeal lipids occurred predominantly with 13C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth.