The role of intermediate sulfur species (ISS) for isotopic fractionation processes during abiotic and chemolithoautotrophic sulfide oxidation in a natural environment

硫同位素分馏法（34S/32S）自20世纪40年代末以来一直用于研究化学和生物硫循环。虽然细菌硫酸盐还原过程中的大同位素分馏被成功地用于解释，例如古代海洋中硫酸盐的积累或早期生命的演化，但对硫化物氧化过程中的分馏所知甚少。两个端元硫化物和硫酸盐之间的分馏通常要小得多，无论底物还是产物富集都存在不一致。这些不一致是由于缺乏对氧化途径和速率以及中间硫物质的了解，如单质硫、多硫化物、硫代硫酸盐、亚硫酸盐或金属-硫化物复合物（如硫代砷酸盐），这些物质可能作为34S汇。在本项目中，我们将开发一种基于制备色谱分离的硫种选择性同位素分析方法。Sn2-和S0的分离将在C18柱上与三氟酸甲酯衍生化后实现，其他硫种在碱性洗脱液中在AS16柱上分离。所收集馏分中的硫将直接用活性铜片（Sn2-, S0）提取，或沉淀为ZnS （S2-）或BaSO4，并通过常规方法作为SO2分析。这种物种选择方法的结果将与以前的端元池测定和顺序沉淀技术进行比较。该方法将应用于美国黄石国家公园中性到碱性温泉的硫化物氧化剖面，在那里我们检测到中间硫种是重要的物种。我们之前的研究仅在硫化物和硫酸盐中测定了34S/32S，从地球化学的角度来看，我们发现了两种具有硫化物氧化剖面的温泉排水的不同分馏模式。同位素变化趋势不同的原因尚不清楚。在目前的项目中，我们将区分物种选择的非生物与生物分离，使用现场孵化实验，以趋化石养硫氧化细菌Thermocrinis橡胶为模式生物。对于选定的样品，我们将测试33S和36S是否进一步阐明物种选择性硫化物氧化模式。我们预计较低的硫化物源浓度会增加元素硫歧化，从而增加氧化还原循环和同位素分馏。我们还预计，中间硫种（包括硫代砷酸盐）的浓度越大，同位素分馏越大。在物种选择池中进行分选后，我们将能够澄清先前报道的底物或产物中34S富集的不一致性，阐明硫化物氧化途径和速率，并揭示硫代谢的细节。我们的新方法和基于现场的数据将成为未来硫化物氧化更一致研究的基础。

Sulfur isotope fractionation (34S/32S) has been used since the late 1940s to study the chemical and biological sulfur cycle. While large isotope fractionations during bacterial sulfate reduction were used successfully to interpret, e.g., accumulation of sulfate in ancient oceans or the evolution of early life, much less is known about fractionation during sulfide oxidation. The fractionation between the two end-members sulfide and sulfate is commonly much smaller and inconsistencies exist whether substrate or product are enriched. These inconsistencies are explained by a lack of knowledge on oxidation pathways and rates as well as intermediate sulfur species, such as elemental sulfur, polysulfides, thiosulfate, sulfite, or metalloid-sulfide complexes (e.g. thioarsenates), potentially acting as 34S sinks.In the proposed project, we will develop a method for sulfur species-selective isotope analysis based on separation by preparative chromatography. Separation of Sn2- and S0 will be achieved after derivatization with methyl triflate on a C18 column, separation of the other sulfur species in an alkaline eluent on an AS16 column. Sulfur in the collected fractions will be extracted directly with activated copper chips (Sn2-, S0), or precipitated as ZnS (S2-) or BaSO4 and analyzed by routine methods as SO2. Results of this species-selective approach will be compared to those from previous techniques of end-member pool determinations and sequential precipitations.The method will be applied to sulfide oxidation profiles at neutral to alkaline hot springs at Yellowstone National Park, USA, where we detected intermediate sulfur species as important species. Determining 34S/32S only in sulfide and sulfate, our previous study has shown different fractionation patterns for two hot spring drainages with sulfide oxidation profiles that seemed similar from a geochemical perspective. The reasons for the different isotopic trends are unclear. In the present project, we will differentiate species-selective abiotic versus biotic fractionation using on-site incubation experiments with the chemolithotrophic sulfur-oxidizing bacteria Thermocrinis ruber as model organism. For selected samples, we will test whether 33S and 36S further elucidate species-selective sulfide oxidation patterns. We expect that lower source sulfide concentrations increase elemental sulfur disproportionation, thus increase redox cycling and isotope fractionation. We also expect that the larger the concentration of intermediate sulfur species, including thioarsenates, the larger the isotope fractionation. Following fractionation in species-selective pools, we will be able to clarify previously reported inconsistencies of 34S enrichment in substrate or product, elucidate sulfide oxidation pathways and rates, and reveal details about sulfur metabolism. Our new methodology and field-based data will be a basis for more consistent studies on sulfide oxidation in the future.