CAREER: The Biogeochemical Controls on Hydrogen-Isotope (D/H) Fractionations in Lipids

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Intellectual Merit: Compound-specific analyses of the hydrogen isotope ratio (D/H) of organic compounds are enjoying a rapid boom in popularity. These analyses are being applied to widespread topics of biogeochemical interest, including generation of paleoclimate records, identifying sedimentary organic matter sources, studying bioremediation of pollutants, identifying ancient metabolic pathways, understanding petroleum sources, and many others. One result of this rapid expansion is that the generation of organic D/H data has greatly surpassed our fundamental understanding of how to interpret such data. For example, paleoclimate records are based on the assumption that the large D/H fractionation between environmental water and plant-wax lipids is constant, yet we do not know either the biochemical basis for that fractionation or the extent to which it may vary. This proposal encompasses a number of related studies with the single goal of understanding basic biogeochemical controls on organic D/H ratios.Four areas of particular importance will be pursued. First, we will seek to understand the biochemical basis for photosynthetic D/H fractionations. The main question is whether the splitting of H2O by photosynthesis, or the reduction of carbon skeletons during biosynthesis, is the primary determinant of lipid D/H ratios. Variability in the net fractionation, and the potential for H exchange during biosynthesis, will also be investigated. We will use experimental studies of cyanobacteria and photosynthetic purple sulfur bacteria in culture to identify key biochemical steps and associated fractionations. The results will have profound importance for the interpretation of D/H climate proxies. Second, we will attempt to elucidate sources of lipid H in heterotrophic microbes. In particular, we will investigate whether the D/H ratio of lipids primarily reflects that of their food (organic substrates) or water. It is experimentally difficult to distinguish between the unknown proportion of the two sources and the two unknown fractionations associated with use of each source. Our novel approach will be to study organisms that utilize organic substrates with no hydrogen, such as oxalate. This will allow us to measure the net heterotrophic fractionation with respect to water, and then to accurately assess the uptake of organic H by a variety of other heterotrophs, and for a variety of substrates.Third, we will develop a comprehensive dataset of temperature-dependent equilibrium D/H fractionations between organic H and water. This data will immediately be useful to a wide range of studies, for example in ascertaining whether or not particular samples have been affected by H exchange and thus could serve as a proxy for paleoclimate. Our approach will combine experimental equilibration with ab initio molecular modeling to produce accurate estimates for a wide range of organic moieties. Experiments will follow a new approach we have recently tested, in which exchange of (-carbonyl positions is measured indirectly. These experiments are amenable to ketones and carboxylic acids, and will serve as key calibration points for subsequent computational estimates of hydrocarbons.Finally, we will seek to develop a robust empirical relationship between leaf-wax D/H and environmental aridity. In doing so, we will examine a variety of factors that potentially influence the net lipid/water fractionation, including water-use efficiency and water transport, variability in biochemical fractionations, seasonal changes in metabolism, and others.Broader Impacts: These studies will also provide a framework for an outreach program at local high schools and colleges. The goal is to engage young students with meaningful scientific research on a problem that matters to them: climate change. We will set up independent research projects for young students at their home schools, supervised by their own teachers. They will be provided with introductory lectures, develop hypotheses and research plans, prepare lipid extracts, vacuum extract leaf waters, submit samples for isotopic analysis in my lab, and then analyze their own data. This approach has numerous benefits, including i) the data they produce will be scientifically useful and pertinent to an important topic; ii) it uses one of the simplest possible isotopic systems (D/H evaporation) to teach concepts of paleoclimate science; iii) the scientific questions are suitable for division into small units approachable by individual students; iv) methods are safe and accessible to untrained students, allowing them to participate in nearly the complete research process; v) it provides an efficient way to export the technical expertise of Caltech to local schools, leveraging the time of students and teachers to simultaneously draw students into science while also producing useful research.