Jeff R. Havig

Diversity, Abundance, and Potential Activity of Nitrifying and Nitrate-Reducing Microbial Assemblages in a Subglacial Ecosystem

ABSTRACT Subglacial sediments sampled from beneath Robertson Glacier (RG), Alberta, Canada, were shown to harbor diverse assemblages of potential nitrifiers, nitrate reducers, and diazotrophs, as assessed by amoA, narG, and nifH gene biomarker diversity. Although archaeal amoA genes were detected, they were less abundant and less diverse than bacterial amoA, suggesting that bacteria are the predominant nitrifiers in RG sediments. Maximum nitrification and nitrate reduction rates in microcosms incubated at 4°C were 280 and 18.5 nmol of N per g of dry weight sediment per day, respectively, indicating the potential for these processes to occur in situ. Geochemical analyses of subglacial sediment pore waters and bulk subglacial meltwaters revealed low concentrations of inorganic and organic nitrogen compounds. These data, when coupled with a C/N atomic ratio of dissolved organic matter in subglacial pore waters of ∼210, indicate that the sediment communities are N limited. This may reflect the combined biological activities of organic N mineralization, nitrification, and nitrate reduction. Despite evidence of N limitation and the detection of nifH, we were unable to detect biological nitrogen fixation activity in subglacial sediments. Collectively, the results presented here suggest a role for nitrification and nitrate reduction in sustaining microbial life in subglacial environments. Considering that ice currently covers 11% of the terrestrial landmass and has covered significantly greater portions of Earth at times in the past, the demonstration of nitrification and nitrate reduction in subglacial environments furthers our understanding of the potential for these environments to contribute to global biogeochemical cycles on glacial-interglacial timescales.

Hydrothermal ecotones and streamer biofilm communities in the Lower Geyser Basin, Yellowstone National Park.

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.

Chemolithotrophic Primary Production in a Subglacial Ecosystem

ABSTRACT Glacial comminution of bedrock generates fresh mineral surfaces capable of sustaining chemotrophic microbial communities under the dark conditions that pervade subglacial habitats. Geochemical and isotopic evidence suggests that pyrite oxidation is a dominant weathering process generating protons that drive mineral dissolution in many subglacial systems. Here, we provide evidence correlating pyrite oxidation with chemosynthetic primary productivity and carbonate dissolution in subglacial sediments sampled from Robertson Glacier (RG), Alberta, Canada. Quantification and sequencing of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) transcripts suggest that populations closely affiliated with Sideroxydans lithotrophicus, an iron sulfide-oxidizing autotrophic bacterium, are abundant constituents of microbial communities at RG. Microcosm experiments indicate sulfate production during biological assimilation of radiolabeled bicarbonate. Geochemical analyses of subglacial meltwater indicate that increases in sulfate levels are associated with increased calcite and dolomite dissolution. Collectively, these data suggest a role for biological pyrite oxidation in driving primary productivity and mineral dissolution in a subglacial environment and provide the first rate estimate for bicarbonate assimilation in these ecosystems. Evidence for lithotrophic primary production in this contemporary subglacial environment provides a plausible mechanism to explain how subglacial communities could be sustained in near-isolation from the atmosphere during glacial-interglacial cycles.

Coordinating environmental genomics and geochemistry reveals metabolic transitions in a hot spring ecosystem

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.

Korarchaeota Diversity, Biogeography, and Abundance in Yellowstone and Great Basin Hot Springs and Ecological Niche Modeling Based on Machine Learning

Over 100 hot spring sediment samples were collected from 28 sites in 12 areas/regions, while recording as many coincident geochemical properties as feasible (>60 analytes). PCR was used to screen samples for Korarchaeota 16S rRNA genes. Over 500 Korarchaeota 16S rRNA genes were screened by RFLP analysis and 90 were sequenced, resulting in identification of novel Korarchaeota phylotypes and exclusive geographical variants. Korarchaeota diversity was low, as in other terrestrial geothermal systems, suggesting a marine origin for Korarchaeota with subsequent niche-invasion into terrestrial systems. Korarchaeota endemism is consistent with endemism of other terrestrial thermophiles and supports the existence of dispersal barriers. Korarchaeota were found predominantly in >55°C springs at pH 4.7–8.5 at concentrations up to 6.6×106 16S rRNA gene copies g−1 wet sediment. In Yellowstone National Park (YNP), Korarchaeota were most abundant in springs with a pH range of 5.7 to 7.0. High sulfate concentrations suggest these fluids are influenced by contributions from hydrothermal vapors that may be neutralized to some extent by mixing with water from deep geothermal sources or meteoric water. In the Great Basin (GB), Korarchaeota were most abundant at spring sources of pH<7.2 with high particulate C content and high alkalinity, which are likely to be buffered by the carbonic acid system. It is therefore likely that at least two different geological mechanisms in YNP and GB springs create the neutral to mildly acidic pH that is optimal for Korarchaeota. A classification support vector machine (C-SVM) trained on single analytes, two analyte combinations, or vectors from non-metric multidimensional scaling models was able to predict springs as Korarchaeota-optimal or sub-optimal habitats with accuracies up to 95%. To our knowledge, this is the most extensive analysis of the geochemical habitat of any high-level microbial taxon and the first application of a C-SVM to microbial ecology.

Modeling the habitat range of phototrophs in yellowstone national park: toward the development of a comprehensive fitness landscape.

The extent to which geochemical variation shapes the distribution of phototrophic metabolisms was modeled based on 439 observations in geothermal springs in Yellowstone National Park (YNP), Wyoming. Generalized additive models (GAMs) were developed to predict the distribution of phototrophic metabolism as a function of spring temperature, pH, and total sulfide. GAMs comprised of temperature explained 38.8% of the variation in the distribution of phototrophic metabolism, whereas GAMs comprised of sulfide and pH explained 19.6 and 11.2% of the variation, respectively. These results suggest that of the measured variables, temperature is the primary constraint on the distribution of phototrophs in YNP. GAMs comprised of multiple variables explained a larger percentage of the variation in the distribution of phototrophic metabolism, indicating additive interactions among variables. A GAM that combined temperature and sulfide explained the greatest variation in the dataset (53.4%) while minimizing the introduction of degrees of freedom. In an effort to verify the extent to which phototroph distribution reflects constraints on activity, we examined the influence of sulfide and temperature on dissolved inorganic carbon (DIC) uptake rates under both light and dark conditions. Light-driven DIC uptake decreased systematically with increasing concentrations of sulfide in acidic, algal-dominated systems, but was unaffected in alkaline, cyanobacterial-dominated systems. In both alkaline and acidic systems, light-driven DIC uptake was suppressed in cultures incubated at temperatures 10°C greater than their in situ temperature. Collectively, these quantitative results indicate that apart from light availability, the habitat range of phototrophs in YNP springs is defined largely by constraints imposed firstly by temperature and secondly by sulfide on the activity of these populations that inhabit the edges of the habitat range. These findings are consistent with the predictions from GAMs and provide a quantitative framework from which to translate distributional patterns into fitness landscapes for use in interpreting the environmental constraints that have shaped the evolution of this process through Earth history.

Evidence for high‐temperature in situ nifH transcription in an alkaline hot spring of Lower Geyser Basin, Yellowstone National Park

Genes encoding nitrogenase (nifH) were amplified from sediment and photosynthetic mat samples collected in the outflow channel of Mound Spring, an alkaline thermal feature in Yellowstone National Park. Results indicate the genetic capacity for nitrogen fixation over the entire range of temperatures sampled (57.2°C to 80.2°C). Amplification of environmental nifH transcripts revealed in situ expression of nifH genes at temperatures up to 72.7°C. However, we were unable to amplify transcripts of nifH at the higher-temperature locations (> 72.7°C). These results indicate that microbes at the highest temperature sites contain the genetic capacity to fix nitrogen, yet either do not express nifH or do so only transiently. Field measurements of nitrate and ammonium show fixed nitrogen limitation as temperature decreases along the outflow channel, suggesting nifH expression in response to the downstream decrease in bioavailable nitrogen. Nitrogen stable isotope values of Mound Spring sediment communities further support geochemical and genetic data. DNA and cDNA nifH amplicons form several unique phylogenetic clades, some of which appear to represent novel nifH sequences in both photosynthetic and chemosynthetic microbial communities. This is the first report of in situ nifH expression in strictly chemosynthetic zones of terrestrial (non-marine) hydrothermal systems, and sets a new upper temperature limit (72.7°C) for nitrogen fixation in alkaline, terrestrial hydrothermal environments.

Geobiological feedbacks and the evolution of thermoacidophiles

Oxygen-dependent microbial oxidation of sulfur compounds leads to the acidification of natural waters. How acidophiles and their acidic habitats evolved, however, is largely unknown. Using 16S rRNA gene abundance and composition data from 72 hot springs in Yellowstone National Park, Wyoming, we show that hyperacidic (pH<3.0) hydrothermal ecosystems are dominated by a limited number of archaeal lineages with an inferred ability to respire O2. Phylogenomic analyses of 584 existing archaeal genomes revealed that hyperacidophiles evolved independently multiple times within the Archaea, each coincident with the emergence of the ability to respire O2, and that these events likely occurred in the recent evolutionary past. Comparative genomic analyses indicated that archaeal thermoacidophiles from independent lineages are enriched in similar protein-coding genes, consistent with convergent evolution aided by horizontal gene transfer. Because the generation of acidic environments and their successful habitation characteristically require O2, these results suggest that thermoacidophilic Archaea and the acidity of their habitats co-evolved after the evolution of oxygenic photosynthesis. Moreover, it is likely that dissolved O2 concentrations in thermal waters likely did not reach levels capable of sustaining aerobic thermoacidophiles and their acidifying activity until ~0.8 Ga, when present day atmospheric levels were reached, a time period that is supported by our estimation of divergence times for archaeal thermoacidophilic clades.

Primary productivity of snow algae communities on stratovolcanoes of the Pacific Northwest

Abstract The majority of geomicrobiological research conducted on glacial systems to date has focused on glaciers that override primarily carbonate or granitic bedrock types, with little known of the processes that support microbial life in glacial systems overriding volcanic terrains (e.g., basalt or andesite). To better constrain the role of the supraglacial ecosystems in the carbon and nitrogen cycles, to gain insight into microbiome composition and function in alpine glacial systems overriding volcanic terrains, and to constrain potential elemental sequestration or release through weathering processes associated with snow algae communities, we examined the microbial community structure and primary productivity of snow algae communities on stratovolcanoes in the Cascade Range of the Pacific Northwest. Here, we present the first published values for carbon fixation rates of snow algae communities on glaciers in the Pacific Northwest. We observed varying levels of light‐dependent carbon fixation on supraglacial and periglacial snowfields at Mt. Hood, Mt. Adams, and North Sister. Recovery of abundant 18S rRNA transcripts affiliated with photoautotrophs and 16S rRNA transcripts affiliated with heterotrophic bacteria is consistent with previous studies indicating the majority of primary productivity on snow and ice can be attributed to photoautotrophs. In contrast to previous observations of glacial ecosystems, our geochemical, isotopic, and microcosm data suggest these assemblages are not limited by phosphorus or fixed nitrogen availability. Furthermore, our data indicate these snow algae communities actively sequester Fe, Mn, and P leached from minerals sourced from the local rocks. Our observations of light‐dependent primary productivity on snow are consistent with similar studies in polar ecosystems; however, our data may suggest that DIC may be a limiting nutrient in contrast to phosphorus or fixed nitrogen as has been observed in other glacial ecosystems. Our data underscore the need for similar studies on glacier surfaces and seasonal snowfields to better constrain the role of local bedrock and nutrient delivery on carbon fixation and biogeochemical cycling in these ecosystems.

Hot Spring Microbial Community Composition, Morphology, and Carbon Fixation: Implications for Interpreting the Ancient Rock Record

Microbial communities in hydrothermal systems exist in a range of macroscopic morphologies including stromatolites, mats, and filaments. The architects of these structures are typically autotrophic, serving as primary producers. Structures attributed to microbial life have been documented in the rock record dating back to the Archean including recent reports of microbially-related structures in terrestrial hot springs that date back as far as 3.5 Ga. Microbial structures exhibit a range of complexity from filaments to more complex mats and stromatolites and the complexity impacts preservation potential. As a result, interpretation of these structures in the rock record relies on isotopic signatures in combination with overall morphology and paleoenvironmental setting. However, the relationships between morphology, microbial community composition, and primary productivity remain poorly constrained. To begin to address this gap, we examined community composition and carbon fixation in filaments, mats, and stromatolites from the Greater Obsidian Pool Area (GOPA) of the Mud Volcano Area, Yellowstone National Park, WY. We targeted morphologies dominated by bacterial phototrophs located in close proximity within the same pool which are exposed to similar geochemistry as well as bacterial mat, algal filament and chemotrophic filaments from nearby springs. Our results indicate i) natural abundance δ13C values of biomass from these features (-11.0 to -24.3 ‰) are similar to those found in the rock record; ii) carbon uptake rates of photoautotrophic communities is greater than chemoautotrophic; iii) oxygenic photosynthesis, anoxygenic photosynthesis, and chemoautotrophy often contribute to carbon fixation within the same morphology; and iv) increasing phototrophic biofilm complexity corresponds to a significant decrease in rates of carbon fixation—filaments had the highest uptake rates whereas carbon fixation by stromatolites was significantly lower. Our data highlight important differences in primary productivity between structures despite indistinguishable δ13C values of the biomass. Furthermore, low primary productivity by stromatolites compared to other structures underscores the need to consider a larger role for microbial mats and filaments in carbon fixation and O2 generation during the Archean and Proterozoic.