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中活跃而多样的一部分。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强。锰是一种营养物质，分布在整个低氧海洋系统中，在主要元素循环中发挥着重要作用，包括由微生物进行的循环。因此，锰与海洋微生物的健康、新陈代谢和功能有着千丝万缕的联系。尽管低氧和微咸水生态系统中的活性真菌可能对锰和其他营养循环做出重大贡献，但人们对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不同的海洋环境中被认为是重要的营养和碳循环生物，但对化学跃层环境(氧气浓度变化的水柱)中的真菌的研究很少。已知真菌分离株在低氧/缺氧(零氧)海洋环境中将锰(Mn(II)氧化)的化学转化与有机碳的降解和活性氧物种(ROS)的产生联系起来，而真菌ROS的产生可能在低氧/缺氧(零氧)海洋环境中金属以及碳和氮的循环和生物有效性中发挥核心作用。由于人类活动的影响，富含锰的波罗的海是研究未来沿海海洋及其盐度梯度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锰、氮和硫循环中的作用，以及在低氧和微咸水海洋生态系统中产生活性氧物种。这些生境中的真菌活动可能会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。通过这项研究产生的培养物集合有望回收许多新的真菌菌株，其生态、新的性质和潜在的与医学相关的生物活性化合物可以被感兴趣的研究人员探索。这项研究包括每年6名本科生、1名研究生和2名高中生，并与当地一名高中美术教师建立合作，教授一个科学中的艺术单位，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，以及对职业生涯早期和代表性不足的少数族裔科学家的培训。这项提议利用2024年初预定巡航期间的采样机会，在波罗的海的两个站沿趋化层的3个深度收集水样，这些水样的O2浓度以及N和Mn物种的分布情况截然不同。该项目的总体目标是描述这些样本中存在的原核生物(细菌和古菌)和真菌分类群的多样性，以及那些表达与锰、碘、氧和氮转化有关的生物地球化学循环的基因的多样性，并特别强调真菌的作用。该工作计划结合了对真菌和微型真核生物以及原核生物的分类标记基因的分析，以及催化报告沉积荧光原位杂交(CARD-FISH)，以估计主要真菌组的原位丰度。水样的Poly-A和Non Polya转录组学提供了表达的代谢基因的总体概述，并专门识别了与锰氧化有关的基因。结合激光散射比浊法的高通量培养工作被用来收集这些趋化层生境中最广泛的可培养海洋真菌的代表，并识别那些携带与Mn(II)氧化和有机碳降解有关的感兴趣基因的海洋真菌。结合真菌和原核生物抑制剂的船上培养研究被用来确定真菌对锰转化过程的贡献程度。结合RT-qPCR和培养研究的偏转录组分析被用来阐明与锰转化(例如，锰过氧化物酶)、氮循环(例如，关键的真菌反硝化基因p450nor，nirK)和ROS产生/衰退(SOD1，NOXA)相关的真菌基因的表达。这一奖项反映了NSF的法定使命，并通过使用基金会的智力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.