2015 Annual Science Report

University of Colorado, Boulder Reporting  |  JAN 2015 – DEC 2015

Physiology of Microbial Populations From W/R Hosted Ecosystems

Project Summary

Microbial communities supported by chemical energy (chemotrophic communties) released through water / rock interactions are widespread in contemporary Earth environments, including the subsurface where light is excluded and in surface environments where physical or chemical conditions preclude photosynthetic metabolisms. Chemotrophic microorganisms are key targets of astrobiological investigation due to the strong likelihood that they predate photosynthetic metabolisms and because they can be physiologically tested to define the habitable limits for life on Earth, including those associated with extremes of temperature, pH, salinity, and energy availability. Research by RPL scientists is focused on identifying and characterizing the physiological strategies or mechanisms that allow life to persist under extreme conditions at the habitable limits. By combining this information with phylogenetic approaches, we aim to determine how and when these mechanisms evolved and what role they played in the diversification of early life. As such, this research effort is highly interdisciplinary and employs both traditional (e.g., activity assays, cultivation) and contemporary (genomics, transcriptomics, metabolomics) microbiological approaches in combination with geochemical approaches. In addition, RPL investigators are studying the evolution of these communities to hone in on the nature of key physiological processes (e.g., central carbon metabolism, nitrogen metabolism, and iron-sulfur metabolism) in chemotrophs prior to the onset of photosynthetic metabolisms. Field-based RPL investigations of microbial physiology in water/rock ecosystems to date have focused on populations inhabiting subglacial environments (cold-adaptation), hot springs (adaptation to acidity, high temperature), and subsurface peridotite environments (adapation to energy stress, nutrient stress, alkalinity).

4 Institutions
3 Teams
2 Publications
4 Field Sites
Field Sites

Project Progress

Physiological Adaptations to Cold Temperature. The comminution or grinding of bedrock by glaciers promotes subglacial weathering processes by exposing fresh minerals. Water chemistry profiles from glacial meltwater streams, field- and laboratory-based microcosm experiments, and molecular analyses together indicate the presence of active and diverse subglacial microbiomes founded on chemical energy that function to enhance rates of mineral weathering. In glacial catchments with pyritic carbonate bedrock, DNA-based molecular data show the presence of a number of taxa closely related to organisms capable of Fe and S oxidation, consistent with their role in pyrite (FeS2) oxidation. Additional evidence for the role of FeS2 in supporting subglacial microbial metabolism comes from the recovery of 16S rRNA transcripts from pyritic subglacial sediments, which also exhibit close affiliation with known Fe and S oxidizing taxa.

Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O32-), which represents an electron donor capable of supporting microbial metabolism. However, recent work at Robertson Glacier (RG) indicates concentrations of S2O32- in subglacial meltwater streams that are typically below limits of detection (<0.3 µM) despite the presence of available pyrite and several orders of magnitude higher concentrations of the FeS2 oxidation product sulfate (SO42-). We recently isolated a chemolithoautotrophic facultative anaerobe, denoted as ‘strain RG5’, from the subglacial environment at RG and subjected it to a battery of physiological and genomic characterizations. The RG5 genome encoded pathways for the complete oxidation of S2O32-, CO2 fixation, and aerobic and anaerobic respiration. The energy required to synthesize a cell under oxygen or nitrate reducing conditions with S2O32- as electron donor was lower at 5.1 °C than 14.4 °C, indicating that this organism is cold-adapted. In support of this notion, the generation times of cultures grown under oxygen or nitrate reducing conditions with S2O32- as electron donor were shorter at 5.1 °C than 14.4 °C. RG sediment-associated soxB transcripts, which encode a component of the S2O32--oxidizing complex, were closely affiliated to soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. Moreover, these results suggest that the organisms identified in subglacial W / R hosted environments are adapted to the cold, potentially nutrient poor conditions in those environments.

Physiological Adaptations to High Temperature. Key questions remain as to the preferred mode of metabolism of chemosynthetic organisms inhabiting high temperature environments. These questions are of central interest for our understanding of the metabolisms most likely to have supported early forms of life. The relevancy of such studies comes from phylogenetic evidence indicating that life most likely evolved in a high temperature environment and was dependent on chemical forms of energy. Organic compounds, including formate, have been reported in the thermal fluids of marine and terrestrial hydrothermal systems; these are environments that are often assumed to be supported by autotrophic modes of metabolism.

Genetic characterization and cultivation-based studies suggest facultative autotrophs, or organisms that can switch between autotrophic and heterotrophic metabolisms, are abundant components of communities inhabiting high temperature geothermal springs. However, the influence of uptake kinetics and energetics on preference for inorganic or organic carbon substrates is not well understood in these organisms. Using a novel cultivation based strategy, we isolated a facultatively autotrophic crenarchaeote, strain CP80, from Cinder Pool (CP, 88.7°C, pH 4.0), Yellowstone National Park. The 16S rRNA gene sequence from CP80 is 99% identical to the most abundant sequence identified in CP sediments. Strain CP80 reduces elemental sulfur (S8°) and demonstrates hydrogen (H2)-dependent autotrophic growth. H2-dependent autotrophic activity is suppressed by amendment with formate at low concentration. Synthesis of a cell during growth with low concentrations of formate required less energy when compared to autotrophic growth with H2. These results, coupled to data indicating an order of magnitude greater C assimilation efficiency when grown with formate when compared to autotrophic growth, are consistent with formate being the preferred carbon source for energetic reasons. Collectively, these results provide new insights into the kinetic and energetic factors that influence the physiology and ecology of organisms that inhabit W/R hosted, high temperature environments. These observations also allude to the importance of energetic considerations (e.g., limitation) in constraining the distribution of microbial life and the metabolic strategies that sustain it.

Physiology of methane evolving and consuming life at high pH. The origin of methane detected in hyperalkaline fluids hosted by peridotite rocks is highly enigmatic, and it is not yet known whether methane is commonly produced in low-temperature serpentinizing systems through biological or abiotic pathways. Often, methane-producing organisms are in low abundance or absent from 16S rRNA gene surveys, and there are few known reaction pathways to generate methane at near-surface temperatures. However, we have recently detected the 16S rRNA sequences of the methanogen “Methanobacterium” in CH4-rich deep subsurface hyperalkaline fluids in Oman, and we have used targeted culturing strategies to successfully isolate Methanobacterium spp. in defined media. Thus we can now conduct controlled growth experiments in order to determine their physiology under extremes of high pH and C limitation. We are also conducting isotopic studies of the methane produced under varying environmental conditions and carbon sources to compare to unique, highly enriched δ13C CH4 values observed in the field system (Miller et al., in press).

The Oman Methanobacterium isolates can grow using H2, CO2 and/or formate in medium with a pH ranging from 7.0 to 10.3, producing an average δ13C CH4 isotopic signature of -60‰. Additionally, when Methanobacterium is grown in unfiltered fluids containing a diversity of oxidants such as sulfate and Fe(III)-oxides, we can enrich for and sustain a complex consortia or organisms that produces CH4 and then consumes it, producing an extremely positive δ13C CH4 isotopic signature up to +12.2‰, similar to in-situ observations. Concomitant reduction of sulfate and Fe(III) also occurs. From this work, we have initiated several laboratory experiments designed to identify the coupled metabolic interactions between methanogens and sulfate and iron reducers within serpentinite-hosted ecosystems.

Physiological Adaptations to Alkalinity/Energy Limitation. High pH (>11) and reduced fluids produced by serpentinization present a number of physiological challenges to microbial communities inhabiting these habitats. The CROMO field site provides a convenient platform for performing high resolution studies of the physiology of microorganisms in groundwaters in serpentinizing systems. Studies at the CROMO are investigating the preference of microbial populations for different oxidants in these systems. Earlier work showed that the addition of acetate and intermediate sulfur compounds stimulated the growth of taxa found in the highest pH, most reducing environments. New work is investigating the role of both aqueous and solid iron and sulfur compounds of varying oxidation states (e.g. thiosulfate, polysulfide, sulfite, etc.) in supporting microbial metabolism. This data, when coupled with a systematic record of aqueous geochemistry at the site, will provide new insights into the oxidants supporting growth of microbial populations in situ. Moreover, growth, activity, and community diversity profiles are being monitored and compared to measurements from the natural environment. This new work will provide greater details about the mechanisms of microbe-mineral interaction, including how they are reflected in genetic signatures and how the geochemical record.

Another microbial physiology project associated with CROMO is aimed at investigating the diversity of small (<600 Da) organic compounds dissolved in the alkaline groundwater to help delineate biotic versus abiotic contribution to carbon cycling in serpentinites. Laboratory experiments using microbial isolates from CROMO are being used to generate global metabolomics profiles. Metabolomics profiles are being compared to genomic data to help identify shifts in cytoplasmic and extracellular metabolites in response to environmental stimuli. These studies will be expanded as new isolates are obtained.

Evolutionary Studies of Chemotrophic Physiology. Determining how key processes evolved in response to the onset and ecological expansion of oxygenic photosynthetic provides key insights into how these processes operated early in earth history (>2.8 Ga). One key microbial process of key relevance for RPL studies is biological nitrogen fixation catalyzed by molybdenum nitrogenase. Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth history. Phylogenetic evidence indicates that oxygen (O2) sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. In this study, RPL investigators show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss resulting in a substantial increase in the number of nif-encoded genes. While the observed increase in the number of nif-encoded genes and their phylogenetic distribution is strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to be in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth’s oxygen, nitrogen, and carbon biogeochemical cycles.

Figure 1. Outflow channel of Grand Prismatic hot spring, Midway Geyser Basin, Yellowstone National Park, Wyoming, USA. Photo credit: Eric Boyd.
Figure 2. Silica terraces line one of many outflow channels of Grand Prismatic hot spring, Midway Geyser Basin, Yellowstone National Park, Wyoming, USA. Photo credit: Eric Boyd.