The Role of Fungi in Biogeochemical Transformations of Mn and Other Micro- and Macro-nutrients Along Chemoclines of the Baltic Sea

越来越多的证据表明，真菌构成了生活在海洋环境中的微生物群落的一个活跃和多样化的部分。其中，全球分布的低氧、低盐度公海和沿海水域预计将随着气候变化而扩大和加剧。锰（Mn）是一种分布在低氧海洋系统中的营养物质，在主要元素循环中起着至关重要的作用，包括由微生物进行的循环。因此，锰与海洋微生物群的健康、新陈代谢和功能有着错综复杂的联系。尽管在低氧和微咸生态系统中，活性真菌可能对锰和其他养分循环做出重大贡献，但真菌在这些过程中的作用和影响知之甚少。子囊菌门和担子菌门真菌在各种海洋环境中被认为是重要的营养和碳循环者，然而在趋化环境（氧浓度变化的水柱）中真菌的研究很少。已知真菌分离物将Mn的化学转化（Mn（II）氧化）与有机碳降解和活性氧（ROS）的产生联系起来，真菌产生活性氧（ROS）可能在低氧/缺氧（零氧）海洋环境中Mn等金属以及碳和氮的循环和生物利用度中发挥核心作用。由于人类活动的影响，富锰的波罗的海及其盐度梯度是研究未来沿海海洋的理想模式生态系统。该项目旨在帮助了解真菌多样性及其在低氧和半咸淡海洋生态系统中锰、氮和硫循环中的作用，以及活性氧的产生。真菌在这些栖息地的活动可能影响重要的全球海洋生物地球化学循环，了解它们的作用和影响可以更准确地预测未来海洋和气候的生物地球化学。本研究产生的培养收集有望恢复许多新的真菌菌株，其生态学，新特性和潜在的医学相关生物活性化合物可以由感兴趣的研究人员进行探索。每年有六名本科生、一名研究生和两名高中生参与这项研究，并与当地一名高中美术教师合作，教授科学中的艺术单元，并在社区图书馆展示其产品，以及关于海洋真菌及其生态作用的教育材料。该项目涉及国际合作，以及培训早期职业和代表性不足的少数民族科学家。该提案利用2024年初预定巡航期间的采样机会，在波罗的海的两个站点沿着趋化斜区的三个深度收集水样，具有不同的O2浓度，N和Mn种类。该项目的总体目标是表征这些样品中存在的原核生物（细菌和古细菌）和真菌分类群的多样性，以及与锰、碘、氧和氮转化相关的生物地球化学循环表达基因的多样性，并特别强调真菌的作用。该工作计划包括真菌、微真核生物和原核生物的分类标记基因分析，以及催化报告沉积荧光原位杂交（CARD-FISH）来估计主要真菌类群的原位丰度。水样的多聚a和非多聚a转录组学提供了表达代谢基因的总体概述，并专门鉴定参与锰氧化的基因。采用激光浊度测定法的高通量培养工作用于收集这些趋化环境中可培养海洋真菌的最广泛代表，并确定那些携带与Mn（II）氧化耦合有机碳降解相关的基因。结合真菌和原核抑制剂的船上孵育研究用于确定真菌对锰转化过程的贡献程度。结合RT-qPCR和培养研究的元转录组分析来阐明与Mn转化（例如Mn过氧化物酶）、氮循环（例如关键的真菌反硝化基因p450nor、nirK）和ROS产生/腐烂（SOD1、NOXA）相关的真菌基因的表达。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。

Mounting evidence suggests that fungi constitute an active and diverse fraction of the microbial community inhabiting marine environments. Among these, globally-distributed low-oxygen, and low salinity open-ocean and coastal waters are expected to expand and intensify with climate change. Manganese (Mn) is a nutrient that is distributed throughout low-oxygen marine systems that plays an essential role in major elemental cycles, including those performed by microorganisms. Manganese is thus intricately linked to the health, metabolism, and function of the ocean microbiome. Despite the potential for active fungi in low-oxygen and brackish ecosystems to make significant contributions to Mn and other nutrient cycling, little is known about the roles and impacts of fungi on those processes. Ascomycota and Basidiomycota fungal species are identified as important nutrient and carbon recyclers in various marine settings, however there are few studies of fungi in chemocline settings (water columns with transitions in oxygen concentration). Fungal isolates are known that link chemical transformation of Mn (Mn(II) oxidation) to organic carbon degradation and production of reactive oxygen species (ROS), and fungal production of ROS may play a central role in the cycling and bioavailability of metals like Mn, as well as carbon and nitrogen in low-oxygen/anoxic (zero oxygen) marine environments. The Mn-rich Baltic Sea is an ideal model ecosystem for studying the future coastal ocean due to anthropogenic impacts experiences, and its salinity gradients. This project aims to contribute to understanding of fungal diversity and roles in manganese, nitrogen and sulfur cycling, as well as production of reactive oxygen species in low oxygen and brackish marine ecosystems. Fungal activities in these habitats may influence important global marine biogeochemical cycles, and knowledge of their role(s) and impacts allows more accurate predictions of the biogeochemistry of a future ocean and climate. The culture collection generated by this study is anticipated to recover many new fungal strains, whose ecology, novel properties, and potential medically-relevant bioactive compounds can be explored by interested researchers. Six undergraduate students, one graduate student, and 2 high school students per year are included in this research, and a collaboration is established with a local high school art teacher to teach an art-in-science unit, and to displayed its product at the community library, along with education materials on marine fungi and their ecological roles. The project involves international collaboration, as well as training of early career and under-represented minority scientists. This proposal leverages a sampling opportunity during a scheduled cruise in early 2024 to collect water samples from 3 depths along the chemocline at two stations in the Baltic Sea with distinct profiles of O2 concentration, and N and Mn species. The overall goal of this project is to characterize the diversity of prokaryotic (bacteria and archaea) and fungal taxa present in these samples, and those expressing genes involved in biogeochemical cycles related to transformations of manganese, iodine, oxygen, and nitrogen, with a specific emphasis on the role of fungi. The workplan incorporates analysis of taxonomic marker genes for fungi and micro-eukaryotes, and prokaryotes, as well as catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) to estimate the in situ abundance of major fungal groups. Poly-A and non polyA transcriptomics of water samples provide a general overview of expressed metabolic genes, and specifically identify genes involved in Mn oxidation. High-throughput culturing efforts incorporating laser nephelometry are used to gather the broadest possible representation of culturable marine fungi in these chemocline habitats, and to identify those that carry genes of interest involved in coupling Mn(II) oxidation to organic carbon degradation. Shipboard incubation studies incorporating fungal and prokaryotic inhibitors are used to determine the extent to which fungi contribute to Mn transformation processes. Coupled RT-qPCR and metatranscriptome analyses of incubation studies are used to elucidate expression of fungal genes related to Mn transformations (e.g., Mn peroxidases), nitrogen cycling (e.g., key fungal denitrification genes p450nor, nirK), and ROS production/decay (SOD1, NOXA).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.