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

知识价值：对有机化合物的氢同位素比率（D/H）的化合物特异性分析正在迅速普及。这些分析被广泛应用于生物地球化学领域，包括古气候记录的生成、沉积有机质来源的识别、污染物的生物修复研究、古代代谢途径的识别、石油来源的理解等等。这种快速扩张的一个结果是，有机D/H数据的生成大大超出了我们对如何解释这些数据的基本理解。例如，古气候记录是基于这样的假设，即环境水和植物蜡质之间的大D/H分异是恒定的，但我们既不知道分异的生化基础，也不知道它可能变化的程度。该提案包含了许多相关的研究，其单一目标是了解有机D/H比的基本生物地球化学控制。将致力于四个特别重要的领域。首先，我们将试图了解光合作用D/H分馏的生化基础。主要的问题是，光合作用下的水的分裂，或者生物合成过程中碳骨架的减少，是否是脂质D/H比的主要决定因素。还将研究净分馏的变异性以及生物合成过程中氢交换的潜力。我们将使用蓝细菌和光合紫硫细菌的培养实验研究，以确定关键的生化步骤和相关的分馏。这些结果将对D/H气候代用物的解释具有深远的意义。其次，我们将试图阐明异养微生物中脂质H的来源。特别是，我们将研究脂质的D/H比是否主要反映了它们的食物（有机底物）或水的D/H比。在实验上很难区分两个源的未知比例和与每个源的使用相关的两个未知部分。我们的新方法将是研究利用不含氢的有机基质的生物，如草酸盐。这将使我们能够测量相对于水的净异养分异，然后准确地评估各种其他异养生物和各种基质对有机氢的吸收。第三，我们将开发一个有机氢和水之间的温度依赖平衡D/H分馏的综合数据集。这些数据将立即对广泛的研究有用，例如确定特定样本是否受到氢交换的影响，从而可以作为古气候的代表。我们的方法将实验平衡与从头算分子模型相结合，以产生广泛的有机部分的准确估计。实验将遵循我们最近测试的一种新方法，即间接测量（-羰基）位置的交换。这些实验适用于酮类和羧酸，并将作为后续烃类计算估计的关键校准点。最后，我们将寻求在叶蜡D/H和环境干旱之间建立一个强有力的经验关系。在此过程中，我们将研究各种可能影响净脂/水分离的因素，包括水利用效率和水运输、生化分离的可变性、代谢的季节性变化等。更广泛的影响：这些研究还将为当地高中和大学的推广项目提供一个框架。其目标是让年轻学生就气候变化这一与他们息息相关的问题进行有意义的科学研究。我们将为年轻学生在他们的家庭学校设立独立的研究项目，由他们自己的老师监督。他们将接受介绍讲座，提出假设和研究计划，准备脂质提取物，真空提取叶水，提交样品到我的实验室进行同位素分析，然后分析他们自己的数据。这种方法有很多好处，包括i)他们产生的数据将是科学有用的，并与一个重要的主题相关；ii)它使用最简单的同位素系统之一（D/H蒸发）来教授古气候科学的概念；（三）科学问题适合划分为学生个人可接近的小单元；Iv)方法是安全的，未经训练的学生可以使用，允许他们参与几乎完整的研究过程；v)它提供了一种有效的方式，将加州理工学院的技术专长输出到当地学校，利用学生和教师的时间，同时吸引学生进入科学领域，同时也产生有用的研究。

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.