Collaborative Research: IODP-enabled Insights into Fungi and Their Metabolic Interactions with Other Microorganisms in Deep Subsurface Hydrothermal Sediments

海洋地下是地球上探索最少的栖息地之一。国际海洋发现计划（IODP） 385探险队在墨西哥瓜伊马斯盆地的海底进行了钻探，并首次直接钻探了活跃热液盆地的地下沉积物和沉积物为主体的玄武岩基侵入体。这次考察提供了一个直接的微生物窗口，通过一个活跃的板块扩张中心进入深海热液生物圈，在那里，复杂的碳氢化合物是通过在高温高压下加热埋藏的有机物而产生的。越来越多的证据表明，真菌构成了地下生物圈群落的一个活跃和生态重要的部分。在富含有机物的大陆边缘沉积物中尤其如此，这些沉积物是好氧和厌氧真菌定植的理想场所，真菌活动可能对养分循环做出重大贡献。我们对地下微生物群知识的主要知识缺口使我们无法估计它们的全部影响：活性真菌如何沿着温度和深度梯度分布，活性细胞利用的底物范围，以及真菌如何与细菌合作降解复杂的有机物，包括碳氢化合物。众所周知，真菌参与难降解有机物和金属循环的降解，并产生具有有趣性质的新型代谢物。这个项目告诉我们地球上微生物生命的不同谱系的起源，海洋地下碳循环的程度，生命的极限，生命如何适应环境变化，以及真菌加速复杂碳氢化合物生物降解的潜力。考虑到潜在的地下生物圈的范围，真菌可能在全球营养循环中发挥重要作用。该项目创建的真菌分离物培养集将为其他感兴趣的研究人员探索其生态学和新特性提供帮助，也可能对基础真菌谱系产生见解。这些生物地球化学和潜在的进化结果引起了其他研究学科、教育工作者和学生的极大兴趣。该项目的K-16教育计划利用旨在增加代表性不足的本科生群体参与研究的计划。参与研究的有高中生、本科生、一名研究生和博士后。与当地一所高中合作的科学艺术项目正在社区图书馆展出，同时展出的还有有关海洋真菌及其生态作用的教育资料。该项目研究了真菌、共生细菌和古细菌的丰度、多样性和分布在地下沉积物样品中的变化，这些样品显示了广泛的原位温度和压力，沿着这些梯度的活性细胞部分，以及真菌是否/如何通过与细菌的代谢相互作用来分解碳氢化合物底物来影响生物圈中的碳循环。该项目正在评估活跃热液地下生物圈中原位微生物的活动，采用分子方法和基于培养的富集和微生物分离研究的尖端组合，应用于瓜伊马斯盆地8个地点的大量样本收集，这些地点的温度剖面、古老的埋藏岩浆断层的存在和地球化学条件都不同。研究人员正在使用新的生物正交非规范氨基酸标记（BONCAT）方法检查1)分类活性细胞的标记基因和宏基因组，2)细菌，古细菌和真菌细胞及其标记基因沿深度和地球化学梯度的分布，使用显微镜，“元组学”和脂质生物标志物分析，3)真菌分离物的底物使用，4)代谢物池，营养物和碳氢化合物。5)真菌对复杂有机物（真菌和细菌之间的同质物）的代谢，利用基于培养的RNA的时程稳定同位素探测，结合对表达基因和代谢物池的分析。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The marine subsurface is one of the least explored habitats on Earth. International Ocean Discovery Program (IODP) Expedition 385 drilled into the seafloor in Guaymas Basin, Mexico, and was the first to drill directly into subsurface sediments and sediment-hosted basalt sill intrusions of an active hydrothermal basin. This expedition provides a direct microbiological window into a deep hydrothermal biosphere across an active plate spreading center where complex hydrocarbons are generated by heating of buried organic matter under high temperature and pressure. Mounting evidence suggests that Fungi constitute an active and ecologically important fraction of the subsurface biosphere community. This is especially true in organic-rich continental margin sediments that are ideal for colonization by aerobic and anaerobic Fungi, where fungal activities may contribute significantly to nutrient cycling. Major knowledge gaps in our knowledge of subsurface microbiota preclude our ability to estimate their full impact: how active Fungi are distributed along temperature and depth gradients, the range of substrates utilized by active cells, and how Fungi may cooperate with bacteria in degradation of complex organic matter, including hydrocarbons. Fungi are known to participate in degradation of refractory organics and cycling of metals and to produce novel metabolites with interesting properties. This project informs us on origins of different lineages of microbial life on Earth, the extent of marine subsurface carbon cycling, limits of life, how life adapts to environmental change, and the potential for Fungi to accelerate the biodegradation of complex hydrocarbons. Given the extent of the potential subsurface biosphere, Fungi likely play an important role in global nutrient cycling. The culture collection of fungal isolates created by this project will be available for exploration of their ecology and novel properties by other interested researchers, and may also yield insights into basal fungal lineages. These biogeochemical and potential evolutionary outcomes are of great interest to other research disciplines, educators, and students alike. The project’s K-16 education program capitalizes on programs aimed at increasing involvement of under-represented undergraduate populations in research. High school students, undergraduates, a graduate student, and postdoc are involved in the research. An art-in-science project with a local high school is being displayed at the community library along with education materials on marine Fungi and their ecological roles. This project examines how abundance, diversity and distribution of Fungi and co-inhabiting bacteria and archaea changes in subsurface sediment samples exhibiting a wide range of in situ temperatures and pressure, what the active fraction of cells is along these gradients, and whether/how Fungi impact carbon cycling in this biosphere by interacting metabolically with bacteria to break down hydrocarbon substrates. The project is assessing the activities of in situ microorganisms in this active hydrothermal subsurface biosphere using a cutting-edge combination of molecular approaches and culture-based studies of enrichments and microbial isolates applied to an extensive collection of samples from 8 sites in Guaymas Basin varying in temperature profile, presence of old, buried magmatic sills, and geochemical conditions. The investigators are examining 1) marker genes and metagenomes of sorted active cells using new bioorthogonal non-canonical amino acid tagging (BONCAT) approaches, 2) the distribution of bacterial, archaeal, and fungal cells and their marker genes along depth and geochemical gradients using microscopy, ‘meta-omics’ and lipid biomarker analyses, 3) substrate usage by fungal isolates, 4) metabolite pools, nutrients, and hydrocarbons with depth, and 5) fungal metabolism of complex organics (and syntrophies between Fungi and Bacteria) using time-course stable isotope probing of RNA from culture-based studies coupled with analyses of expressed genes and pools of metabolites.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.