Alexander B. Michaud

A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the worlds oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.

Cyanobacterial diversity across landscape units in a polar desert: Taylor Valley, Antarctica

Life in the Taylor Valley, Antarctica, is dominated by microorganisms, with cyanobacteria being key primary producers in the region. Despite their abundance and ecological importance, the factors controlling biogeography, diversity, dispersal of cyanobacteria in Taylor Valley and other polar environments are poorly understood. Owing to persistent high winds, we hypothesize that the cyanobacterial diversity across this polar landscape is influenced by aeolian processes. Using molecular and pigment analysis, we describe the cyanobacterial diversity present in several prominent habitats across the Taylor Valley. Our data show that the diversity of cyanobacteria increases from the upper portion of the valley towards the McMurdo Sound. This trend is likely due to the net transport of organisms in a down-valley direction, consistent with the prevailing orientation of high-energy, episodic föhn winds. Genomic analysis of cyanobacteria present in aeolian material also suggests that wind mixes the cyanobacterial phylotypes among the landscape units. Our 16S rRNA gene sequence data revealed that (1) many of the cyanobacterial phylotypes present in our study site are common in polar or alpine environments, (2) many operational taxonomic units (OTUs) (22) were endemic to Antarctica and (3) four OTUs were potentially endemic to the McMurdo Dry Valleys.

A microbiologically clean strategy for access to the Whillans Ice Stream subglacial environment

Abstract The Whillans Ice Stream Subglacial Access Research Drilling (WISSARD) project will test the overarching hypothesis that an active hydrological system exists beneath a West Antarctic ice stream that exerts a major control on ice dynamics, and the metabolic and phylogenetic diversity of the microbial community in subglacial water and sediment. WISSARD will explore Subglacial Lake Whillans (SLW, unofficial name) and its outflow toward the grounding line where it is thought to enter the Ross Ice Shelf seawater cavity. Introducing microbial contamination to the subglacial environment during drilling operations could compromise environmental stewardship and the science objectives of the project, consequently we developed a set of tools and procedures to directly address these issues. WISSARD hot water drilling efforts will include a custom water treatment system designed to remove micron and sub-micron sized particles (biotic and abiotic), irradiate the drilling water with germicidal ultraviolet (UV) radiation, and pasteurize the water to reduce the viability of persisting microbial contamination. Our clean access protocols also include methods to reduce microbial contamination on the surfaces of cables/hoses and down-borehole equipment using germicidal UV exposure and chemical disinfection. This paper presents experimental data showing that our protocols will meet expectations established by international agreement between participating Antarctic nations.

Microbial sulfur transformations in sediments from Subglacial Lake Whillans

Diverse microbial assemblages inhabit subglacial aquatic environments. While few of these environments have been sampled, data reveal that subglacial organisms gain energy for growth from reduced minerals containing nitrogen, iron, and sulfur. Here we investigate the role of microbially mediated sulfur transformations in sediments from Subglacial Lake Whillans (SLW), Antarctica, by examining key genes involved in dissimilatory sulfur oxidation and reduction. The presence of sulfur transformation genes throughout the top 34 cm of SLW sediments changes with depth. SLW surficial sediments were dominated by genes related to known sulfur-oxidizing chemoautotrophs. Sequences encoding the adenosine-5′-phosphosulfate (APS) reductase gene, involved in both dissimilatory sulfate reduction and sulfur oxidation, were present in all samples and clustered into 16 distinct operational taxonomic units. The majority of APS reductase sequences (74%) clustered with known sulfur oxidizers including those within the “Sideroxydans” and Thiobacillus genera. Reverse-acting dissimilatory sulfite reductase (rDSR) and 16S rRNA gene sequences further support dominance of “Sideroxydans” and Thiobacillus phylotypes in the top 2 cm of SLW sediments. The SLW microbial community has the genetic potential for sulfate reduction which is supported by experimentally measured low rates (1.4 pmol cm-3d-1) of biologically mediated sulfate reduction and the presence of APS reductase and DSR gene sequences related to Desulfobacteraceae and Desulfotomaculum. Our results also infer the presence of sulfur oxidation, which can be a significant energetic pathway for chemosynthetic biosynthesis in SLW sediments. The water in SLW ultimately flows into the Ross Sea where intermediates from subglacial sulfur transformations can influence the flux of solutes to the Southern Ocean.

Methane sources in arctic thermokarst lake sediments on the North Slope of Alaska

The permafrost on the North Slope of Alaska is densely populated by shallow lakes that result from thermokarst erosion. These lakes release methane (CH4 ) derived from a combination of ancient thermogenic pools and contemporary biogenic production. Despite the potential importance of CH4 as a greenhouse gas, the contribution of biogenic CH4 production in arctic thermokarst lakes in Alaska is not currently well understood. To further advance our knowledge of CH4 dynamics in these lakes, we focused our study on (i) the potential for microbial CH4 production in lake sediments, (ii) the role of sediment geochemistry in controlling biogenic CH4 production, and (iii) the temperature dependence of this process. Sediment cores were collected from one site in Siqlukaq Lake and two sites in Sukok Lake in late October to early November. Analyses of pore water geochemistry, sedimentary organic matter and lipid biomarkers, stable carbon isotopes, results from CH4 production experiments, and copy number of a methanogenic pathway-specific gene (mcrA) indicated the existence of different sources of CH4 in each of the lakes chosen for the study. Analysis of this integrated data set revealed that there is biological CH4 production in Siqlukaq at moderate levels, while the very low levels of CH4 detected in Sukok had a mixed origin, with little to no biological CH4 production. Furthermore, methanogenic archaea exhibited temperature-dependent use of in situ substrates for methanogenesis, and the amount of CH4 produced was directly related to the amount of labile organic matter in the sediments. This study constitutes an important first step in better understanding the actual contribution of biogenic CH4 from thermokarst lakes on the coastal plain of Alaska to the current CH4 budgets.

Solute sources and geochemical processes in Subglacial Lake Whillans, West Antarctica

National Science Foundation (NSF, grants OPP-0838933, 1346250, and 1439774); Italian National Antarctic Program (Barbante); graduate fellowships from the NSF Integrative Graduate Education and Research Traineeship program (0654336) and NSF Center for Dark Energy Biosphere Investigations; dissertation grant from the American Association of University Women

Microbial Community Structure of Subglacial Lake Whillans, West Antarctica

Subglacial Lake Whillans (SLW) is located beneath ∼800 m of ice on the Whillans Ice Stream in West Antarctica and was sampled in January of 2013, providing the first opportunity to directly examine water and sediments from an Antarctic subglacial lake. To minimize the introduction of surface contaminants to SLW during its exploration, an access borehole was created using a microbiologically clean hot water drill designed to reduce the number and viability of microorganisms in the drilling water. Analysis of 16S rRNA genes (rDNA) amplified from samples of the drilling and borehole water allowed an evaluation of the efficacy of this approach and enabled a confident assessment of the SLW ecosystem inhabitants. Based on an analysis of 16S rDNA and rRNA (i.e., reverse-transcribed rRNA molecules) data, the SLW community was found to be bacterially dominated and compositionally distinct from the assemblages identified in the drill system. The abundance of bacteria (e.g., Candidatus Nitrotoga, Sideroxydans, Thiobacillus, and Albidiferax) and archaea (Candidatus Nitrosoarchaeum) related to chemolithoautotrophs was consistent with the oxidation of reduced iron, sulfur, and nitrogen compounds having important roles as pathways for primary production in this permanently dark ecosystem. Further, the prevalence of Methylobacter in surficial lake sediments combined with the detection of methanogenic taxa in the deepest sediment horizons analyzed (34–36 cm) supported the hypothesis that methane cycling occurs beneath the West Antarctic Ice Sheet. Large ratios of rRNA to rDNA were observed for several operational taxonomic units abundant in the water column and sediments (e.g., Albidiferax, Methylobacter, Candidatus Nitrotoga, Sideroxydans, and Smithella), suggesting a potentially active role for these taxa in the SLW ecosystem. Our findings are consistent with chemosynthetic microorganisms serving as the ecological foundation in this dark subsurface environment, providing new organic matter that sustains a microbial ecosystem beneath the West Antarctic Ice Sheet.

Biological ice nucleation initiates hailstone formation

Cloud condensation and ice nuclei in the troposphere are required precursors to cloud and precipitation formation, both of which influence the radiative balance of Earth. The initial stage of hailstone formation (i.e., the embryo) and the subsequent layered growth allow hail to be used as a model for the study of nucleation processes in precipitation. By virtue of the preserved particle and isotopic record captured by hailstones, they represent a unique form of precipitation that allows direct characterization of the particles present during atmospheric ice nucleation. Despite the ecological and economic consequences of hail storms, the dynamics of hailstone nucleation, and thus their formation, are not well understood. Our experiments show that hailstone embryos from three Rocky Mountain storms contained biological ice nuclei capable of freezing water at warm, subzero (°C) temperatures, indicating that biological particles can act as nucleation sites for hailstone formation. These results are corroborated by analysis of δD and δ18O from melted hailstone embryos, which show that the hailstones formed at similarly warm temperatures in situ. Low densities of ice nucleation active abiotic particles were also present in hailstone embryos, but their low concentration indicates they were not likely to have catalyzed ice formation at the warm temperatures determined from water stable isotope analysis. Our study provides new data on ice nucleation occurring at the bottom of clouds, an atmospheric region whose processes are critical to global climate models but which has challenged instrument-based measurements.

Bacterial responses to environmental change on the Tibetan Plateau over the past half century

Climate change and anthropogenic factors can alter biodiversity and can lead to changes in community structure and function. Despite the potential impacts, no long-term records of climatic influences on microbial communities exist. The Tibetan Plateau is a highly sensitive region that is currently undergoing significant alteration resulting from both climate change and increased human activity. Ice cores from glaciers in this region serve as unique natural archives of bacterial abundance and community composition, and contain concomitant records of climate and environmental change. We report high-resolution profiles of bacterial density and community composition over the past half century in ice cores from three glaciers on the Tibetan Plateau. Statistical analysis showed that the bacterial community composition in the three ice cores converged starting in the 1990s. Changes in bacterial community composition were related to changing precipitation, increasing air temperature and anthropogenic activities in the vicinity of the plateau. Collectively, our ice core data on bacteria in concert with environmental and anthropogenic proxies indicate that the convergence of bacterial communities deposited on glaciers across a wide geographical area and situated in diverse habitat types was likely induced by climatic and anthropogenic drivers.

Physiological Ecology of Microorganisms in Subglacial Lake Whillans

Subglacial microbial habitats are widespread in glaciated regions of our planet. Some of these environments have been isolated from the atmosphere and from sunlight for many thousands of years. Consequently, ecosystem processes must rely on energy gained from the oxidation of inorganic substrates or detrital organic matter. Subglacial Lake Whillans (SLW) is one of more than 400 subglacial lakes known to exist under the Antarctic ice sheet; however, little is known about microbial physiology and energetics in these systems. When it was sampled through its 800 m thick ice cover in 2013, the SLW water column was shallow (~2 m deep), oxygenated, and possessed sufficient concentrations of C, N, and P substrates to support microbial growth. Here, we use a combination of physiological assays and models to assess the energetics of microbial life in SLW. In general, SLW microorganisms grew slowly in this energy-limited environment. Heterotrophic cellular carbon turnover times, calculated from 3H-thymidine and 3H-leucine incorporation rates, were long (60 to 500 days) while cellular doubling times averaged 196 days. Inferred growth rates (average ~0.006 d−1) obtained from the same incubations were at least an order of magnitude lower than those measured in Antarctic surface lakes and oligotrophic areas of the ocean. Low growth efficiency (8%) indicated that heterotrophic populations in SLW partition a majority of their carbon demand to cellular maintenance rather than growth. Chemoautotrophic CO2-fixation exceeded heterotrophic organic C-demand by a factor of ~1.5. Aerobic respiratory activity associated with heterotrophic and chemoautotrophic metabolism surpassed the estimated supply of oxygen to SLW, implying that microbial activity could deplete the oxygenated waters, resulting in anoxia. We used thermodynamic calculations to examine the biogeochemical and energetic consequences of environmentally imposed switching between aerobic and anaerobic metabolisms in the SLW water column. Heterotrophic metabolisms utilizing acetate and formate as electron donors yielded less energy than chemolithotrophic metabolisms when calculated in terms of energy density, which supports experimental results that showed chemoautotrophic activity in excess of heterotrophic activity. The microbial communities of subglacial lake ecosystems provide important natural laboratories to study the physiological and biogeochemical behavior of microorganisms inhabiting cold, dark environments.