Lipid D/H ratios as a proxy for microbial metabolism

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Variations in the relative abundance of the two stable isotopes of hydrogen, protium (H) and deuterium (D), are one of the useful natural tracers available to Earth Scientists. For example, changes in the D/H ratio of polar ice cores are arguably the best record of past glacial/interglacial cycles. But current understanding of their behavior in the biosphere is rather limited. It is known that plants preferentially take up H relative D, and so organic matter is strongly depleted in D relative to environmental water. However, it is not known how these isotopes behave in consumers, either animals or microbes, and how isotopic signals propagate through ecosystems. In turn, it is not well known how to interpret ancient records of organic matter D/H, of primary importance to the Earth Science community. Do ancient organic records only record the hydrologic cycle (i.e. climate), or do they also record changes in ecosystem? This proposal tackles that fundamental problem from the standpoint of microbial consumers: bacteria, archaea, and microscopic eukaryotes that live by eating organic materials. The proposed research seeks to answer the questions of how they alter D/H ratios in organic matter, and whether such changes are correlated with their type of metabolism. The problem will be addressed through the use of cultures under well-defined conditions and then directly measuring the D/H ratios of lipid molecules that would be representative of the sedimentary organic record. A number of supporting biochemical measurements will be used to help us understand the mechanistic basis for why such variations exist. The broader impacts consist of developing a web-accessible database for archiving and searching biologic D/H data, involving graduate and undergraduate students, and incorporating research results into an International Geobiology Course as well as standard journal publications and conference presentations. Methods for measuring the D/H ratios of individual lipids have existed for more than a decade, yet almost all such efforts have been directed exclusively towards plants and phytoplankton, with the goal of paleoclimate reconstruction. Recent data suggest, however, that there is a much richer complexity of isotopic fractionations in the world around us, and that it could also be useful for other types of investigations. Variations in the äD values of heterotrophic microbial lipids of up to 500? have been observed, and these signals appear to be controlled by host metabolism, rather than the D/H ratio of growth substrates. If so, H isotopes in naturally occurring lipids could potentially be used to trace microbial metabolism, both in modern and ancient environments, an important scientific goal. The objective of this proposal is to begin to understand the scope and mechanistic basis of such variations in organisms other than plants, and to ultimately move the methodology toward being a useful tool for geobiology. To do this, the PI will undertake three related lines of research: i) The PI will undertake a broad, culture-based survey of microbial D/H fractionations, including bacteria, archaea, and eukaryotes using a range of aerobic and anaerobic metabolisms. The anticipates studying ~10-15 different organisms, each grown under a range of conditions and on different substrates. ii) To obtain a deeper mechanistic understanding of such fractionations, the PI will employ more targeted biochemical studies using abundance measurements of NAD(P)H and transhydrogenase enzyme activity to understand key biochemical processes affecting the isotopic composition of cellular reducing equivalents. iii) The PI will develop compound-specific measurements for amino acids, which will allow us to examine a class of compounds more representative of bulk biomass, as well as specific proteins that are more diagnostic for biotic sources.