Yellowstone National Park Hot Spring Research Articles

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.

The Geochemical Habitats that Favored the Origin of Thermophilic Lineages

Evidence in the fossil and isotopic records suggests that life inhabited hot springs by ~3.5 Ga. Further, phylogenetic evidence places Bacteria and Archaea from high temperature environments as among the earliest evolving lineages. Moreover, contemporary hot spring communities host an extensive level of biodiversity coinciding with extensive geochemical variation due to spatial and temporal heterogeneity in available oxidants and reductants generated by variable mixing of reduced volcanic and oxidized meteoric fluids. Thus, thermophilic archaeal and bacterial lineages have been co-evolving with their hydrothermal environments since early in the history of life and through dramatic changes in Earth’s geologic history. Yet, little is known of the environmental characteristics that enabled the extensive diversification of microbial life and their metabolic functionalities in these environments. To begin developing a framework to understand the environmental characteristics that enabled expansive microbial taxonomic and functional innovation in thermophilic lineages - coordinated geochemical, metagenomic, and phylogenetic analyses were conducted on 37 high-temperature Yellowstone National Park (YNP) hot spring ecosystems that spanned the range of geochemistry (pH ~1.5-10) in YNP springs. Considerable variation in dissolved solutes and gases were identified, consistent with spatial and temporal variation in the geological, geochemical, and hydrologic processes that influence the YNP hydrothermal system. Shotgun sequencing and bioinformatics analyses yielded 1,154 archaeal and bacterial metagenome-assembled-genomes (MAGs) from the 37 springs. Genomic diversity and metabolic functions encoded by the MAGs were not uniformly distributed among spring types based on geochemistry, with moderately acidic springs (pH 5-7) harboring the greatest overall diversity, despite these spring types being relatively rare among continental hydrothermal systems. Phylogenomic analyses of MAGs indicated that their divergence times (estimated by distance to phylogenetic roots) were variable, but highly associated with spring geochemistry. Specifically, spring types hosting the highest genomic, taxonomic, and functional diversity also predominantly harbored microbial lineages with the oldest inferred divergence times. In addition, MAG-encoded metabolic functions related to carbon fixation, methane, sulfur, iron, arsenic, hydrogen, and nitrogen metabolism were discretely distributed across spring geochemical types. The distribution of metabolic functions coincided with variation in spring geochemical parameters related to those metabolisms. For example, iron metabolism was most prevalent in acidic springs exhibiting the highest iron concentrations, gas metabolism was most prominent in moderately acidic springs that exhibited the highest dissolved gas concentrations, and arsenic metabolism was prevalent among alkaline pH springs where arsenic concentrations were highest. Lastly, preliminary comparisons of microbial communities from YNP and other global continental hydrothermal systems suggest that geologic setting (e.g., bedrock type) significantly influences hot spring geochemistry and ultimately, microbial compositional and functional profiles that coincide with distinct evolutionary trajectories of hot spring taxa and functional genes. Results will be discussed in the context of the early evolution of life and the co-evolution of microbial lineages and their geologic settings in hydrothermal systems.

Whole-Genome Sequence of a Unique Elioraea Species Strain Isolated from a Yellowstone National Park Hot Spring.

The genus Elioraea has only one species characterized microbiologically and two genomes sequenced. We have sequenced the genome of a unique Elioraea strain isolated from Yellowstone National Park and found it to be a distinct new species. Elioraea is suggested to be a member of the aerobic anoxygenic photosynthetic bacteria.

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.

Mechanisms of Mineral Substrate Acquisition in a Thermoacidophile.

The thermoacidophile Acidianus is widely distributed in Yellowstone National Park hot springs that span large gradients in pH (1.60 to 4.84), temperature (42 to 90°C), and mineralogical composition. To characterize the potential role of flexibility in mineral-dependent energy metabolism in contributing to the widespread ecological distribution of this organism, we characterized the spectrum of minerals capable of supporting metabolism and the mechanisms that it uses to access these minerals. The energy metabolism of Acidianus strain DS80 was supported by elemental sulfur (S0), a variety of iron (hydr)oxides, and arsenic sulfide. Strain DS80 reduced, oxidized, and disproportionated S0 Cells growing via S0 reduction and disproportionation did not require direct access to the mineral to reduce it, whereas cells growing via S0 oxidation did require direct access, observations that are attributable to the role of H2S produced by S0 reduction/disproportionation in solubilizing and increasing the bioavailability of S0 Cells growing via iron (hydr)oxide reduction did not require access to the mineral, suggesting that the cells reduce Fe(III) that is being leached by the acidic growth medium. Cells growing via oxidation of arsenic sulfide with Fe(III) did not require access to the mineral to grow. The stoichiometry of reactants to products indicates that cells oxidize soluble As(III) released from oxidation of arsenic sulfide by aqueous Fe(III). Taken together, these observations underscore the importance of feedbacks between abiotic and biotic reactions in influencing the bioavailability of mineral substrates and defining ecological niches capable of supporting microbial metabolism.IMPORTANCE Mineral sources of electron donor and acceptor that support microbial metabolism are abundant in the natural environment. However, the spectrum of minerals capable of supporting a given microbial strain and the mechanisms that are used to access these minerals in support of microbial energy metabolism are often unknown, in particular among thermoacidophiles. Here, we show that the thermoacidophile Acidianus strain DS80 is adapted to use a variety of iron (hydro)oxide minerals, elemental sulfur, and arsenic sulfide to support growth. Cells rely on a complex interplay of abiologically and biologically catalyzed reactions that increase the solubility or bioavailability of minerals, thereby enabling their use in microbial metabolism.

A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments.

The application of viral and cellular metagenomics to natural environments has expanded our understanding of the structure, functioning, and diversity of microbial and viral communities. The high diversity of many communities, e.g., soils, surface ocean waters, and animal-associated microbiomes, make it difficult to establish virus-host associations at the single cell (rather than population) level, assign cellular hosts, or determine the extent of viral host range from metagenomics studies alone. Here, we combine single-cell sequencing with environmental metagenomics to characterize the structure of virus-host associations in a Yellowstone National Park (YNP) hot spring microbial community. Leveraging the relatively low diversity of the YNP environment, we are able to overlay evidence at the single-cell level with contextualized viral and cellular community structure. Combining evidence from hexanucelotide analysis, single cell read mapping, network-based analytics, and CRISPR-based inference, we conservatively estimate that >60% of cells contain at least one virus type and a majority of these cells contain two or more virus types. Of the detected virus types, nearly 50% were found in more than 2 cellular clades, indicative of a broad host range. The new lens provided by the combination of metaviromics and single-cell genomics reveals a network of virus-host interactions in extreme environments, provides evidence that extensive virus-host associations are common, and further expands the unseen impact of viruses on cellular life.

Genomic inference of the metabolism of cosmopolitan subsurface Archaea, Hadesarchaea.

The subsurface biosphere is largely unexplored and contains a broad diversity of uncultured microbes(1). Despite being one of the few prokaryotic lineages that is cosmopolitan in both the terrestrial and marine subsurface(2-4), the physiological and ecological roles of SAGMEG (South-African Gold Mine Miscellaneous Euryarchaeal Group) Archaea are unknown. Here, we report the metabolic capabilities of this enigmatic group as inferred from genomic reconstructions. Four high-quality (63-90% complete) genomes were obtained from White Oak River estuary and Yellowstone National Park hot spring sediment metagenomes. Phylogenomic analyses place SAGMEG Archaea as a deeply rooting sister clade of the Thermococci, leading us to propose the name Hadesarchaea for this new Archaeal class. With an estimated genome size of around 1.5 Mbp, the genomes of Hadesarchaea are distinctly streamlined, yet metabolically versatile. They share several physiological mechanisms with strict anaerobic Euryarchaeota. Several metabolic characteristics make them successful in the subsurface, including genes involved in CO and H2 oxidation (or H2 production), with potential coupling to nitrite reduction to ammonia (DNRA). This first glimpse into the metabolic capabilities of these cosmopolitan Archaea suggests they are mediating key geochemical processes and are specialized for survival in the subsurface biosphere.

Prokaryotic cells separated from sediments are suitable for elemental composition analysis

Cell‐sediment separation methods can potentially enable determination of the elemental composition of microbial communities by removing the sediment elemental contribution from bulk samples. We demonstrate that a separation method can be applied to determine the composition of prokaryotic cells. The method uses chemical and physical means to extract cells from benthic sediments and mats. Recovery yields were between 5% and 40%, as determined from cell counts. The method conserves cellular element contents to within 30% or better, as assessed by comparing C, N, P, Mg, Al, Ca, Ti, Mn, Fe, Ni, Cu, Zn, and Mo contents in Escherichia coli. Contamination by C, N, and P from chemicals used during the procedure was negligible. Na and K were not conserved, being likely exchanged through the cell membrane as cations during separation. V, Cr, and Co abundances could not be determined due to large (>100%) measurement uncertainties. We applied this method to measure elemental contents in extremophilic communities of Yellowstone National Park hot springs. The method was generally successful at separating cells from sediment, but does not discriminate between cells and detrital biological or noncellular material of similar density. This resulted in Al, Ti, Mn, and Fe contamination, which can be tracked using proxies such as metal:Al ratios. With these caveats, we present the first measurements, to our knowledge, of the elemental abundances of a chemosynthetic community. The communities have C:N ratios typical of aquatic microorganisms, are low in P, and their metal abundances vary between hot springs by orders of magnitude.

Division-Specific Differences in Bacterial Community Assembly of Two Alkaline Hot Spring Ecosystems from Yellowstone National Park

A fundamental issue in ecology is whether communities are random assemblages or, alternatively, whether there are rules that determine which combinations of taxa can co-occur. For microbial systems, in particular, the question of whether taxonomic groups exhibit differences in community organization remains unresolved but is critical for our understanding of community structure and function. Here, we used presence-absence matrices derived from bar-coded pyrosequencing data to evaluate the assembly patterns of eight bacterial divisions distributed along two Yellowstone National Park hot spring outflow channels. Four divisions (Cyanobacteria, Chloroflexi, Acidobacteria, and Cytophaga-Flavobacterium-Bacteroides) exhibited less co-occurrence than expected by chance, with phototrophic taxa showing the strongest evidence for nonrandom community structure. We propose that both differences in environmental tolerance and competitive interactions within divisions contribute to these nonrandom assembly patterns. The higher degree of nonrandom structure observed for phototrophic taxa compared with the other divisions may be due in part to greater overlap in resource usage, as has been previously proposed for plant communities.

INFLUENCE OF GAS PRODUCTION AND FILAMENT ORIENTATION ON STROMATOLITE MICROFABRIC

Alternating light-dark laminae within stromatolites have been attributed to a phototactic response of the constituent microbial communities, whereby filaments orient vertically during the day and recline at night. This study examines the orientation of cyanobacterial filaments within a laminated siliceous stromatolite from a Yellowstone National Park hot spring to identify the controls on microfabric development and whether phototaxis plays a role. Results indicate that filament orientation is predominantly perpendicular or parallel to lamination, even when laminae are steeply inclined. Thus, phototaxis is not a significant control of microfabric development in these stromatolites. Vertical aspects of the fabric are dominated by hourglass-shaped filament bundles (hourglass structures) adjacent to rounded pores, rather than being defined by individual filaments. The rounded pores likely represent oxygen-rich bubbles generated during photosynthesis. Upon stabilization by filaments, upward buoyancy of the bubbles rotated the bundles toward a vertical orientation. Thus, vertical aspects of the fabric in this stromatolite result from buoyancy forces rather than phototaxis. Examples from the Neoproterozoic Beck Spring Dolomite reveal that similar subvertical hourglass structures are present in the rock record and may be better preserved than individual filaments. The presence of rounded pores (fenestrae) and hourglass structures in ancient microbialites, here termed the hourglass-associated fenestral fabric, can serve as an indication of biogenic influence in stromatolites, especially in the absence of preserved filaments, and may be an indication of oxygenic photosynthesis.

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Hydrogen-isotopic variability in fatty acids from Yellowstone National Park hot spring microbial communities

We report the abundances and hydrogen-isotopic compositions (D/H ratios) of fatty acids extracted from hot-spring microbial mats in Yellowstone National Park. The terrestrial hydrothermal environment provides a useful system for studying D/H fractionations because the numerous microbial communities in and around the springs are visually distinct, separable, and less complex than those in many other aquatic environments. D/H fractionations between lipids and water ranged from −374‰ to +41‰ and showed systematic variations between different types of microbial communities. Lipids produced by chemoautotrophic hyperthermophilic bacteria, such as icosenoic acid (20:1), generally exhibited the largest and most variable fractionations from water (−374‰ to −165‰). This was in contrast to lipids characteristic of heterotrophs, such as branched, odd chain-length fatty acids, which had the smallest fractionations (−163‰ to +41‰). Mats dominated by photoautotrophs exhibited intermediate fractionations similar in magnitude to those expressed by higher plants. These data support the hypothesis that variations in lipid D/H are strongly influenced by central metabolic pathways. Shifts in the isotopic compositions of individual fatty acids across known ecological boundaries show that the isotopic signature of specific metabolisms can be recognized in modern environmental samples, and potentially recorded in ancient ones. Considering all sampled springs, the total range in D/H ratios is similar to that observed in marine sediments, suggesting that the trends observed here are not exclusive to the hydrothermal environment.

First Cultivation and Ecological Investigation of a Bacterium Affiliated with the Candidate Phylum OP5 from Hot Springs

The phylogenetic group termed OP5 was originally discovered in the Yellowstone National Park hot spring and proposed as an uncultured phylum; the group was afterwards analyzed by applying culture-independent approaches. Recently, a novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in Japan that was enriched through various isolation procedures. Phylogenetic analyses of the isolate have revealed that it is closely related to the OP5 phylum that has mainly been constructed with the environmental clones retrieved from thermophilic and mesophilic anaerobic environments. It appears that the lineage is independent at the phylum level in the domain Bacteria. Therefore, we designed a primer set for the 16S rRNA gene to specifically target the OP5 phylum and performed quantitative field analysis by using the real-time PCR method. Thus, the 16S rRNA gene of the OP5 phylum was detected in some hot-spring samples with the relative abundance ranging from 0.2% to 1.4% of the prokaryotic organisms detected. The physiology of the above-mentioned isolate and the related environmental clones indicated that they are scavengers contributing to the sulfur cycle in nature.

Voltammetric (Micro)Electrodes for the In Situ Study of Fe2+ Oxidation Kinetics in Hot Springs and S2O$\rm{ _3^{2 - } }$ Production at Hydrothermal Vents

AbstractWe have used solid‐state Au/Hg voltammetric electrodes to understand redox and biogeochemical processes in hot spring and deep sea hydrothermal environments. These electrodes are non‐specific and have the capability of measuring simultaneously a suite of chemical species including several of the principal redox species involved in early diagenesis (O2, Mn2+, Fe2+, H2S/HS−, and I−) as well as some Fe species (FeS and Fe3+) and sulfur species (Sx2− and S2O$\rm{ _3^{2 - } )}$. Here we demonstrate how in situ data obtained in complex environments can be used to study specific iron and sulfur reactions and processes at (sub)millimeter to centimeter resolution and over short time scales. Examples include the oxidation of Fe2+ by O2 produced by cyanobacterial mats in Yellowstone National Park hot springs and the formation of S2O$\rm{ _3^{2 - } }$ in diffuse flow waters from the hydrothermal vents at Lau Basin. In one example, profiles of redox species in cyanobacterial mats from Yellowstone National Park hot springs show that in the light dissolved Fe2+ is completely removed from the source waters as cyanobacterial mats produce O2 and oxidize the Fe2+. Performing kinetic experiments in the dark and light at the depth of maximum O2 production indicates that the decay of Fe2+ follows a zero order rate law consistent with photosynthesis as the source of O2. These dynamic environments show how kinetic data can be obtained in situ and be used to understand the interactions between biology and chemistry. We know of no other analytical technique that can provide this information in both clear and turbid waters on the time scales (seconds) observed.

Travertine

The term ‘travertine’ encompasses a wide variety of continental carbonate rocks deposited from groundwaters and surface waters. These include, but are not limited to, mineral spring deposits, stream and lake deposits, and cave formations and are recognized by interesting morphologies such as spring mounds, fissure ridges, cascades, step pools, and speleothems. These beautiful deposits are often popular tourist destinations, such as Havasu Falls, Ariz., and Yellowstone National Park hot springs. Larger economic deposits are mined for building stone such as that from Bagni di Tivoli, Italy, which was used for the construction of many famous buildings in ancient Rome. Humankind has used travertine systems literally for millennia for art, building stone, and water sources.

16S rRNA Phylogenetic Investigation of the Candidate Division “ Korarchaeota ”

The environmental distribution and phylogeny of "Korarchaeota," a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9 degrees N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.

Molecular Diversity of Cyanobacteria Inhabiting Coniform Structures and Surrounding Mat in a Yellowstone Hot Spring

Lithified coniform structures are common within cyanobacterial mats in Yellowstone National Park hot springs. It is unknown whether these structures and the mats from which they develop are inhabited by the same cyanobacterial populations. Denaturing gradient gel electrophoresis and sequencing and phylogenetic analysis of 16S rDNA was used to determine whether (1) three different morphological types of lithified coniform structures are inhabited by different cyanobacterial species, (2) these species are partitioned along a vertical gradient of these structures, and (3) lithified and non-lithified sections of mat are inhabited by different cyanobacterial species. Our results, based on multiple samplings, indicate that the cyanobacterial community compositions in the three lithified morphological types were identical and lacked any vertical differentiation. However, lithified and non-lithified portions of the same mat were inhabited by distinct and different populations of cyanobacteria. Cyanobacteria inhabiting lithified structures included at least one undefined Oscillatorialean taxon, which may represent the dominant cyanobacteria genus in lithified coniform stromatolites, Phormidium, three Synechococcus-like species, and two unknown cyanobacterial taxa. In contrast, the surrounding mats contained four closely related Synechococcus-like species. Our results indicate that the distribution of lithified coniform stromatolites may be dependent on the presence of one or more microorganisms, which are phylogenetically different from those inhabiting surrounding non-lithified mats.

Effects of visible light and UV radiation on photosynthesis in a population of a hot spring cyanobacterium, a synechococcus sp., subjected to high-temperature stress

Assays of photosynthesis were conducted with a biofilm population of a cyanobacterium, a Synechococcus sp., growing at approximately 70 degreesC in a Yellowstone National Park hot spring to test whether cells growing near the upper temperature limit of photosynthetic life are optimally adapted to their mean environmental temperature. Cell suspensions were assayed at 70, 65, and 55 degreesC while being simultaneously exposed to modified solar environments, including reduction of total irradiance and exclusion of UV radiation. Carbon fixation was greatest at 65 degreesC, while 70 and 55 degreesC were always supraoptimal and suboptimal for photosynthesis, respectively. The degree of temperature stress was dependent upon light intensity, and this light-dependent temperature effect may involve both reduced quantum efficiency at subsaturating irradiances and a lower saturating irradiance at both supraoptimal and suboptimal temperatures. The Synechococcus sp. was also more susceptible to UV inhibition of photosynthesis at nonoptimal temperatures. These results suggest that this population is persisting at a nearly lethal temperature and is consequently subject to greater damage by both visible and UV radiation, but it is speculated that these cells may be avoiding competition with other photoautotrophs under these nonoptimal conditions. In separate experiments monitoring diurnal patterns of photosynthesis, cells exhibited peak productivity during the morning, followed by an afternoon decline. No recovery of photosynthesis was observed during the remaining daytime, and carbon fixation was always UV inhibited under conditions of photosynthetically saturating light.

Novel division level bacterial diversity in a Yellowstone hot spring.

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Modification of organosulfur compounds and water-soluble coal-derived material by anaerobic microorganisms

The transformation of organosulfur compounds by anaerobic microorganisms may be of interest for coal desulfurization. An anaerobic microbial community indigenous to cyanobacterial mats of a Yellowstone National Park hot spring was assessed for the ability to evolve volatile sulfur compounds from benzyl methyl sulfide, benzyl methyl disulfide, methionine and water-soluble lignite-derived material. The addition of methionine or benzyl methyl disulfide stimulated the production of methanethiol by slurries made with cyanobacterial mat material. Transformation of methionine appeared to be the direct result of microbial activity. However, the evolution of methanethiol from benzyl methyl disulfide appeared to be the indirect result of microbial activity, that is, the cleavage of the disulfide linkage may have resulted from the reduction by microbially produced hydrogen sulfide. Results indicated a transient interaction of hydrogen sulfide and methanethiol with the coal material. In separate studies, sulfate-reducing bacteria were examined for the modification of water-soluble lignite-derived material. As expected, the sulfur content of the soluble material increased under conditions of sulfate respiration. However, when grown fermentatively without the production of sulfide, two of the nine cultures examined reduced the sulfur content of the soluble material. Most of the cells in all the cultures immediately lysed upon the addition of the soluble material. By 2 weeks, cell densities were greater in cultures amended with soluble coal material than in control cultures.