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Project Progress

In the 2010-2011 reporting period we focused on laboratory analysis and data interpretation based on the field studies from 2009 and 2010. We anticipate a third field season to complete these efforts in Summer 2012. We highlight results and progress for specific sub-tasks below.

2a-1, ERR: During the 2009 and 2010 field seasons, we collected 200+ sediment and microbial mat samples for determination of the Extended Redfield Ratio (ERR). We have measured the C, N, P, and trace metal content of bulk sediment and microbial mat samples collected in 2009. We are currently isolating cells from this bulk material using density gradient centrifugation. Results so far indicate that we recover about 35% of the microbial cells from samples. We plan to analyze these cells for C, N, P, and trace metal content for comparison to bulk material. Analysis of 2010 ERR samples has not yet begun.
Personnel: Marcia Kyle, Marc Neveu, Zarraz Lee, Laura Steger, and Amisha Poret-Peterson, James Elser, Ariel Anbar, Everett Shock,

2a-2, ¹⁵N Tracer incubations: ¹⁵N tracer incubation studies were conducted during the 2009 and 2010 field seasons. Isotopic analyses (EA-IRMS) of bulk sediment or microbial mat show nitrate assimilation, ammonium uptake, and N2 fixation. We are working with the Colorado Plateau Stable Isotope Lab and Northern Arizona University to complete analyses of the ¹⁵NO₃ and ¹⁵NH₄ samples collected during the incubation studies. Nanoscale secondary ion mass spectrometry (nanoSIMS) enables high resolution imaging (<50 μm; i.e., cellular scale) of the elemental and isotopic composition of biological and non-biological materials. ASU recently acquired a NanoSIMS and we have examined samples from 15N tracer incubation studies conducted during the 2010 field season. Using the nanoSIMS, we have also detected ¹⁵N enrichment of structures that resemble filamentous cyanobacteria in microbial mat microcosms amended with ¹⁵N-nitrate. We plan to pair nanoSIMS with transmission electron microscopy (TEM) to look at the distribution of ¹⁵N into cellular structures in these and other samples. We also plan to use fluorescence in situ hybridization (FISH)-nanoSIMS to determine which groups of assimilated ¹⁵N-labeled compounds with probes based on metagenomic analysis (Swingley et al., 2011 submitted) and on previous ¹⁶S rRNA sequencing efforts (Meyer-Dombard et al. 2011).

Personnel: Hilairy Hartnett, Steve Romaniello, Katie Alexander, Amisha Poret-Peterson, and Marie Nahlik (ASU Space Grant intern); nanoSIMS staff: Jitao Zhang, and Maitrayee Bose (Postdoc)

2a-3, Nutrient-addition experiments: During the summer 2010, we performed microcosm nutrient enrichment experiments (addition of N, P, and Fe in a full factorial design) at four hot springs above and below the boundary of photosynthesis. We have optimized protocols for simultaneous extraction of RNA and DNA from these samples and are currently performing the extractions. We have also generated terminal restriction fragment length polymorphism (TRFLP) patterns for these samples that show community composition changes in response to nutrient addition. We are also planning to enumerate 16S rRNA gene copy number in DNA and cDNA (derived from RNA) and to examine expression of genes for N, P, and Fe metabolism in these samples.
Personnel: Marcia Kyle, Jessica Corman, Zureyma Martinez, Christie Sabin, Amisha Poret-Peterson, Michele Knowlton, James Elser

2a-4, Metal storage genes: Molybdenum (Mo) is an important metal co-factor in enzymes involved in nitrogen cycling, most notably, the assimilatory and dissimilatory nitrate reductases and nitrogenase. Mo concentrations in hot springs are high (250 to 300 nM) compared to those in marine (~100 nM) and freshwater (<20 nM) environments, although whether that Mo is in a biologically accessible form is not known. An undergraduate researcher, Zureyma Martinez is evaluating the distribution and expression of genes for Mo storage (mop) in hot springs of Yellowstone National Park. Results thus far indicate that mop is present and expressed in alkaline microbial mats. Future work will involve examining mop expression in sediment or microbial mat samples used for the nutrient amendment experiments.
Personnel: Zureyma Martinez, Jennifer Glass, Amisha Poret-Peterson, Ariel Anbar

2a-5, One-carbon experiments: We conducted ¹³C tracer experiments at four hot springs in the summer 2010 to investigate methylotrophy or the use of one-carbon compounds (e.g., methanol and methane) by microbial communities. Methylotrophs, including methanotrophs, assimilate methanol into biomass or oxidize it to carbon dioxide. We have analyzed all samples for (1) C and N content and isotopic composition via EA-IRMS, (2) ¹³C enrichment of headspace gas samples, and (3) ¹³C in the dissolved organic and inorganic carbon pools. These results show that methylotrophs were present and active in a slightly acidic hot spring. We have also detected expression of genes for methanol and methane oxidation in this hot spring. We are currently drafting a manuscript for publication (Stable isotope and genetic evidence for active methylotrophy in a Yellowstone National Park hot spring, lead author: Poret-Peterson) based on these results. Attempts to identify the microbes responsible for methanol assimilation via separation of ¹³C-DNA from unlabeled DNA have been made and this protocol will be optimized in the near future. Furthermore, we plan to use TEM-nanoSIMS and FISH-nanoSIMS to show cellular level assimilation of the added methanol.
Personnel: Amisha Poret-Peterson, Hilairy Hartnett, Steve Romaniello, Zuri Martinez, Ariel Anbar, James Elser, Everett Shock

2a-6, Thermodynamics and genomics of methanogenesis and methanotrophy: Everett Shock (ASU) and Eric Boyd (MSU NAI group) have initiated a collaboration to correlate geochemical processes with functions of microbial communities This work is now funded in part through an NSF-Geobiology grant: “Combining Methods from Geochemistry and Molecular Biology to Predict the Functions of Microbial Communities.” The focus of this project is to use the contrasting microbial processes of methane production (e.g., methanogenesis) and methane consumption (e.g., methanotrophy) as a framework for evaluating the linkages between geochemical predictions and the distribution, diversity, and activity of organisms that catalyze these processes. The overarching rationale for targeting these biological processes is that the combined activities of methanogenesis and methanotrophy largely control the flux of the potent greenhouse gas methane to our atmosphere, the extent of which may significantly impact global climate. Defining the constraints on the distribution of microbial populations catalyzing these two processes in nature can significantly advance our understanding of the impact that a perturbation to their environment would have on their respective activities and the consequence that this may have on the global carbon cycle. Existing geochemical predictions from hydrothermal ecosystems in Yellowstone National Park indicate that the occurrence of populations catalyzing methane production should be highly proscribed, but that aerobic and anaerobic methanotrophy should be widespread and that populations engaged in these activities should display significant genetic diversity as a function of hot spring fluid composition. The thermodynamic predictions will be used to guide experiments aimed to interpret data on the distribution of methanogens and methanotrophs and their respective activities. The integration of geochemical data and biological data will be achieved using newly developed ecological modeling tools. These models will provide a more comprehensive understanding of the extent to which the distribution, diversity, and activity of functional groups of microorganisms reflect the physical and chemical characteristics of their environment. Defining the extent to which such relationships exist using this framework has critical implications for our understanding of the constraints which led to extant biodiversity and will enable predictions of how changes in environmental conditions will affect the functioning of those microbial ecosystems.

2a-7, MEMS sensor development: We continued efforts to develop new sensor technologies based on MEMS (Micro-Electrical Mechanical Systems) technology for exploring hydrothermal ecosystems. In the summer of 2011, we successfully deployed an array of temperature sensors in hotsprings to obtain a 2-D map of vertically resolved (2 cm) profiles through a microbial mat as well as at depths of up to one meter in a hot spring. This work resulted in the submission of an abstract (Oiler) for the 2011 Fall AGU meeting, and a special session on micro- and nano-sensors for extreme environemnts at the 2011 Fall AGU meeting (Yu, Hartnett, Shock).
Personnel: Jon Oiler, Hongyu Yu, Hilairy Hartnett, Everett Shock.