9713967 Reeburgh Hydrogen plays a central role in microbial metabolism and is kinetically and thermodynamically maintained at low concentrations. Hydrogen partial pressure is a major control on anoxic systems, and can be supplied biologically and abiologically. Hydrogen can be detected and measured with great sensitivity, but reliable measurements on natural systems are difficult, as disruption of microenvironments by sampling may lead to production and consumption hydrogen. Most studies on hydrogen to date have involved the use of co-cultures, where two organisms are used to set and control the hydrogen partial pressure. This project includes the development and application of an apparatus that will permit setting and controlling the hydrogen partial pressure of a culture system. Rate measurements and gas composition changes occurring in the culture vessel detected by upstream-downstream gas measurements will be used to study methanogenesis, anaerobic methane oxidation (reverse methanogenesis) and isotope fractionation associated with both under a range of hydrogen partial pressures. This project has three goals: 1) To build and operate a culture system in which the hydrogen partial pressure can be set and maintained at varying levels. The approach will be to set the system's hydrogen partial pressure and study microbial behavior under a range of partial pressures. 2) To use this apparatus to test the hypothesis that anaerobic methane oxidation is conducted by methanogens operating in reverse at low hydrogen partial pressures. Studies on pure cultures of methanogens, mixed cultures of methanogens and sulfate reducers, and samples from natural environments where anaerobic oxidation is known to occur will be carried out. 3) To determine C and H stable isotope fractionation factors under a range of growth rate and culture conditions. Fractionation factors are available, but are difficult to apply generally because of the range of conditions under which they were obtained. The apparatus developed in this study will permit upstream-downstream gas phase sampling and determination of isotope fractionation factors under a range of growth rate and substrate conditions. In addition to settling the remaining question of the organisms and mechanisms responsible for anaerobic methane oxidation, this approach will permit determination of isotope (C and H) fractionation factors for both methanogenesis and reverse methanogenesis on pure and mixed cultures under a range of conditions. The techniques for controlling hydrogen concentration developed in this study may be useful in studies of hydrogen metabolism and interspecies hydrogen transfer by other groups of organisms in extreme anoxic environments. Funding is being provided by the Directorate for Biological Sciences, the Directorate for Geosciences and the Office of Multidisciplinary Activities of the Directorate for Mathematical and Physical Sciences.
