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Project Progress

Extended Redfield Ratios: During the 2009 and 2010 field seasons, we collected 200+ sediment and microbial mat samples for determination of the Extended Redfield Ratio (ERR). We measured C, N, P, and trace metal content of bulk sediments and microbial mat samples collected in 2009. We have now isolated cells from this bulk material for ~30 samples using density gradient centrifugation. We have recovered 10% to 40% of the microbial cells from samples and established that their composition is not affected by contamination from the solutions used in the separation procedure (Neveu et al., in preparation). Analysis of these cells for C, N, P, and trace metal content yielded C:N:P ratios typical of microbes, even for chemosynthetic communities. Comparison with bulk material elemental compositions showed systematic enrichments in C, N, P, Fe, and ~10 other bio-essential metals in the cells. For some elements (e.g., Fe, Cr), cell content was independent of bulk material content; for others (e.g., Cu, Zn), cell content varied linearly with bulk material content. For elements such as P or Mn, the enrichment patterns were different depending on whether the cells belonged to primarily photosynthetic or chemosynthetic communities. We are investigating explanations for these patterns. We are also using X-ray microanalysis to measure C, N, and P of single cells. Using scanning transmission electron microscopy equipped with an EDX (energy dispersive X-ray spectroscopy) detector (STEM-EDX), measurements on cultured microbes show good accordance between the ratio of C:N obtained via EA-IRMS and by STEM-EDX.

Nutrient-addition experiments: We completed T-RFLP analysis and quantitative PCR analysis of ¹⁶S rRNA genes in DNA and cDNA (derived from RNA) from an alkaline photosynthetic hot spring (Bison Pool). The ¹⁶S rRNA copy number in DNA and cDNA did not appear to vary significantly between the control and nutrient amended samples; however, the cDNA-derived T-RFLP patterns between the control and nutrient amended samples differed. This suggests nutrient amendment resulted in a shift in the active microbial community. We are currently processing samples from the remaining experiments. We probed for expression of genes involved in N, P, and Fe metabolism. Of these genes, we found that N and Fe addition increased the expression of an Fe acquisition gene (M. Knowlton Thorne, MS thesis).

¹⁵N Tracer incubations: We continue to analyze δ¹⁵N samples from the incubation experiments conducted in previous field seasons and are beginning to develop manuscripts for publication based on this work. In collaboration with M. Altabet (UMass) we have developed a method to analyze δ¹⁵N[NO3~] samples from the in situ experiments conducted in 2010. These analyses are in progress and once completed will allow us to complete our interpretation of the N-cycle process studies. An abstract was submitted for the 2012 AGU meeting based on the NanoSIMS analysis of ¹⁵N in single cells (A. Poret-Peterson). We used the NanoSIMS 50L to image individual microbial cells from the ¹⁵N-tracer incubation experiments conducted at acidic and alkaline hot springs during the 2010 field season. Results show that only a portion of the microbial community may be responsible for most of the N assimilation in both systems based on the number of ¹⁵N enriched cells (<2/3 of cells enriched). We also observed that the atom percent enrichment of ¹⁵N varied between cells (0.39 to 14%), which suggests there was differential N uptake during the incubations.

One-carbon experiments: In July of 2012 we conducted field experiments in Yellowstone National Park to further study one-carbon (C1) metabolism in hot springs. Analysis of singles cells via NANOSims to examine ¹³C incorporation are ongoing. A. Poret-Peterson has a manuscript in preparation for E_nv. Microbiology_ based on the C1 experiments from Summer 2011. These recent experiments to further explore methane cycling in Yellowstone hot springs used ¹³C-labeled methane to trace the uptake and aerobic oxidation of CH₄ and ¹³C-labeled acetate as a substrate for aceticlastic methanogenesis. We are in the process of analyzing sediment and CO₂ for ¹³C-enrichment.

MEMS sensor development. We deployed previously developed MEMS sensors to collect data at Yellowstone National park and continued efforts to develop new sensor technologies (liquid conductivity sensors) also based on MEMS (Micro-Electrical Mechanical Systems) technology. In the spring of 2012, we set up a long-term temperature monitoring station – with hourly measurements reported via satellite communication from 15 different MEMS temperature sensors in a privately owned hot spring in Gerlach, NV with current continuous measurements spanning 9 months. In the summer of 2012, we deployed arrays of temperature sensors at specifically targeted sites at Yellowstone National Park with transition zones between different microbial communities. In addition we developed arrays of conductivity sensors that were used at YNP in concert with the temperature arrays. This work was submitted in an abstract (Oiler) for the 2013 IEEE Transducers conference and will be submitted in an abstract for the 2013 Goldschmidt conference.