Over the course of this project, Spear and Boyd took several research trips to Yellowstone National Park for field data collection and sample acquisition. In this project, a mineral coupon holding a field deployable, sampling container was developed that could hold several crushed rock types and/or other materials, to establish the growth of biofilms when the coupons were deployed in hot springs for periods of time. We suspended these mineral coupons in several hot springs of varying chemistry, pH and temperature to observe change over time. One objective was to determine basal rates of metabolic transformation of small organic molecules in ecosystems previously found to be supported by heterotrophic metabolism. Co-I Boyd and graduate student Matthew Urschel determined rates of dissolved inorganic carbon (DIC) mineralization / assimilation to evaluate the importance of different carbon substrates that support metabolism. Urschel and Boyd found in 9 of 13 high-temperature hot springs studied (all greater than the upper temperature limit for photosynthesis of 73C), rates of DIC assimilation were greater than those of formate and acetate assimilation, but 2 exhibited rates of formate and acetate assimilation that exceeded those of DIC assimilation. DIC, formate and acetate mineralization and assimilation was positively correlated with spring pH and showed little correlation with spring temperature. Overall, results indicate that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic and are adapted to fluctuating nutrient availability. This likely allows hot-spring microbial communities (and other similar subsurface biosphere organisms) to take advantage of energy-rich organic substrates when they become available. The results substantiate earlier efforts by PI Spear for the importance of chemolithoautotrophs to sustain high-temperature, non-photosynthetic hot spring communities. However, the rate of carbon assimilation by these organisms suggests that members of these non-photosynthetic communities may be reliant upon, or take advantage of, organic carbon to support their metabolism. Results indicate that subsurface microbiota in hot environments are versatile in their ability to adapt to environmental fluctuation.