Physiological underpinnings of sulfur isotope effects produced by sulfate reducing microbes

硫的生物地球化学循环在调节地球表面氧化还原收支中起着关键作用，并与大气氧的演变密切相关。微生物硫酸盐还原作用是现代海洋沉积物中约一半有机碳再矿化的微生物过程，是地表硫储集层硫同位素分馏的主要过程，但微生物硫酸盐还原过程中硫同位素大小的控制机制尚不清楚。这种不确定性尤其适用于与高分馏（大于50 / ml）相关的生理条件。由于新元古代硫酸盐和硫化物之间的硫同位素分馏超过50 / mil的现象越来越普遍，这一趋势初步归因于大气氧的增加和硫的氧化再循环。本提案通过测试两个主要假设，探讨了在微生物硫酸盐还原过程中导致类似大分馏的生理条件。硫酸盐还原微生物在顽固的有机基质上缓慢生长时，无论是纯培养物还是群体，都能产生较大的硫同位素效应（大于50 / mil）。这些底物通常不用于硫酸盐还原剂的生长，但可能包括葡萄糖、纤维素、木质素和羟基对苯二酚。硫同位素效应的大小是细胞内碳和硫代谢耦合的函数，在非化学计量硫酸盐还原过程中，以及在缺乏一些将还原当量从碳转移到硫的酶的情况下，硫同位素效应会产生很大的影响。这些假设来自pi实验室最近的研究，其中一种新分离的硫酸盐还原细菌（DMSS-1）在葡萄糖上生长的纯培养物中，在电子供体限制的生长条件下产生了广泛的同位素分异（7至66 permil）。值得注意的是，测量到的最大硫同位素效应（66 permil）比以前在纯培养研究中观察到的效应大~20 permil，但完全在观察到的高自然分馏范围内。为了验证第一个假设，将在先前分离的微生物的批量和连续培养中测量多种硫同位素效应，这些微生物生长缓慢，将硫酸盐还原与各种单糖、双糖和其他有机底物的氧化结合起来。第二个假设将通过描述dsss -1在产生大量硫同位素效应过程中对葡萄糖的生长的化学计量学特征，以及通过测量在连续培养中产生的多种硫同位素效应，来测试普通脱硫弧菌野生型和不同突变体，这些突变体缺乏参与从乳酸到硫酸盐还原等量物转移的酶。这项工作的结果将直接测试和约束现在和过去的硫循环、地球的氧化作用和海洋化学演变的模型。它还将有助于解决各种各样的生物地球化学问题，包括修复和监测受污染含水层中的mSR，其中mSR是降解地下水中有机污染物的重要过程。拟议的研究将支持两名初级教员和一名研究生两年。本项目包含多个子项目，将在研究生和pi的指导下提供本科生研究机会。pi在夏季接待了来自波士顿地区的十多名本科生、高中生和K-12科学教师作为实习生。为一名K-12科学教师实习生申请为期6周的暑期津贴。教师、高中生和本科生将参与项目，提供与微生物多样性、地球化学、地球历史以及将基因组信息与地球化学过程联系起来的现代方法相关的各种主题的实践经验。

Biogeochemical cycling of sulfur plays a critical role in regulating the Earth's surface redox budget and is tightly linked to the evolution of atmospheric oxygen. Microbial sulfate reduction, a microbial process that remineralizes about half of organic carbon in modern marine sediments, is the main process that fractionates sulfur isotope ratios of surface sulfur reservoirs but mechanisms that control the magnitude of sulfur isotope during microbial sulfate reduction are still not well understood. This uncertainty particularly applies to physiological conditions associated with high, greater than 50 per mil, fractionations. Because sulfur isotope fractionations between sulfate and sulfide exceeding 50 per mil become increasingly more common in the Neoproterozoic, this trend was tentatively attributed to the increase of atmospheric oxygen and oxidative recycling of sulfur. This proposal explores physiological conditions that lead to similarly large fractionations during microbial sulfate reduction alone by testing two main hypotheses:1. Sulfate reducing microbes can produce large sulfur isotope effects (greater than 50 per mil) when growing slowly on recalcitrant organic substrates, either in pure cultures or in consortia. These substrates are not commonly used to grow sulfate reducers, but may include glucose, cellulose, lignin, and hydroxyhydroquinones.2. The magnitude of sulfur isotope effects is a function of the intracellular coupling of carbon and sulfur metabolisms, with large sulfur isotope effects produced during non-stoichiometric sulfate reduction and in the absence of some enzymes that transfer reducing equivalents from carbon to sulfur.These hypotheses emerge from recent studies in the PIs' laboratories, in which a newly isolated sulfate reducing bacterium (DMSS-1) produced a wide range of isotope fractionation (7 to 66 permil) under electron donor-limited growth conditions in pure culture grown on glucose. Notably, the maximum measured sulfur isotope effect (66 permil) was ~20 permil larger than those observed previously in pure culture studies, but were well within the range of observed high natural fractionations. To test the first hypothesis, multiple sulfur isotope effects will be measured in batch and continuous cultures of previously isolated microbes that grow slowly and couple sulfate reduction with the oxidation of various monosaccharides, disaccharides and other organic substrates. The second hypothesis will be tested by characterizing the stoichiometry of growth of DMSS-1 on glucose during the production of large sulfur isotope effects, and by measuring multiple sulfur isotope effects produced in continuous cultures of Desulfovibrio vulgaris wild type and different mutants that lack enzymes involved in the transfer of reducing equivalents from lactate to sulfate.The results of the proposed work will directly test and constrain models of present and past sulfur cycles, oxygenation of the Earth, and the evolution of ocean chemistry. It will also contribute to a wide variety of biogeochemical problems, including the remediation and monitoring of mSR in contaminated aquifers, where mSR is an important process for degrading organic contaminants in groundwater. The proposed research will support two junior faculty members and one graduate student for two years. The proposed project contains multiple subprojects that will provide undergraduate research opportunities under the guidance of graduate students and PIs. PIs have hosted over ten undergraduate students, high school students, as well as K-12 science teachers from the Boston area as interns during the summer. Funding is requested for a 6-week summer stipend for a K-12 science teacher intern. The teachers, high school students and undergraduates will work on projects that provide a hands-on experience in various topics related to microbial diversity, geochemistry, Earth history and modern methods of linking genomic information to geochemical processes.