Collaborative Research: Examination of Diverse Anaerobic Methane Oxidizing Archaea and Associated Syntrophic Relationships Using High Resolution Molecular and Isotopic Methods

在微观尺度上研究甲烷消耗微生物的特征及其活动的控制因素，对于我们理解全球尺度上甲烷的生物地球化学循环具有重要意义。世界上的海洋是甲烷最大的来源之一，然而这种温室气体只有一小部分被释放到大气中，因为它被缺氧的海洋沉积物中的微生物消耗了。最近的跨学科研究表明，甲烷厌氧氧化（AOM）的过程是由新型未培养的古细菌ANME-1和ANME-2介导的，并与硫酸盐还原菌共生。本项目采用细尺度分子rna和同位素分析相结合的一系列实验，研究了甲烷渗透环境中的甲烷营养古菌（包括ANME-1和ANME-2）和纯培养的methanoarcina acetivorans和Methanococcoides methylutens （ANME-2的近亲）。目标是通过三个广泛的研究领域来确定负责和理解AOM过程的微生物。本研究的目标是：(1)通过扩展和完善目前富甲烷沉积物中细胞的天然碳同位素组成数据库，表征AOM微生物的多样性和代谢多样性；(2)通过确定天然ANME-2/Desulfosarcina菌群中活性细胞的位置，建立AOM的约束热力学模型；(3)通过限制该过程的中间体和识别可能涉及的维生素和辅助因子来阐明AOM的代谢方面。这些跨学科研究是假设驱动的研究，旨在通过结合细胞特异性分子和同位素分析来了解甲烷氧化微生物细胞和联合体的潜在机制和共生关系。这项研究将产生一个独特的高分辨率同位素数据集，这将作为模拟AOM热力学的基础，并有助于定义参与甲烷循环的微生物之间复杂的相互作用。这种方法是对地球化学家和微生物学家正在进行的厌氧甲烷氧化研究的高度补充，并提供了一个独特的视角来帮助阐明这一全球重要但特征不明确的过程。教育和推广计划将在小学、本科和研究生阶段扩大对地球微生物学的兴趣。该项目将在微生物地理生物学方面提供团队导向的研究生教育，通过旨在增加科学多样性的现有项目吸引本科生，并在现有基础设施的基础上进行K-12教师教育。具体来说，两位pi将在PSU的年度教师研讨会上讲授微生物在调节地球气候方面发挥的重要作用。通过与北卡罗莱纳州格林斯伯勒的贝内特学院（Bennett College）的贝尼塔·贝尔（Benita Bell）博士的联系，pi们在贝内特空间科学周期间举办了一个研讨会，向未被充分代表的学生介绍地球生物学领域。在研究生阶段，该项目将为宾夕法尼亚州立大学和加州理工学院的研究生提供教育机会。

The characterization of methane-consuming microorganisms and the factors that control their activity on the micro scale is important in our understanding the biogeochemical cycling of methane on a global scale. The world's oceans are one of the largest sources of methane, yet only a fraction of this greenhouse gas is released into the atmosphere because it is consumed by microorganisms in anoxic marine sediments. Cross-disciplinary investigations have recently shown that the process of anaerobic oxidation of methane (AOM) is mediated by novel uncultured Archaea, the ANME-1 and ANME-2, in syntrophic association with sulfate-reducing bacteria. This project is studying methanotrophic Archaea (including ANME-1 and ANME-2) from methane seep environments and pure cultures of Methanosarcina acetivorans and Methanococcoides methylutens (close cultured relatives of the ANME-2) using a series of experiments combining fine scale molecular RNA-based and isotopic analyses. The objectives are aimed at identifying the microbes responsible for and understanding the process of AOM through three broad areas of research. The goals of this research are: (1) to characterize the diversity and metabolic versatility of microorganisms responsible for AOM by expanding and refining the current database on the natural carbon isotopic composition of cells in methane-rich sediments, (2) to develop constraining thermodynamic models of AOM by identifying where active cells reside in natural ANME-2/Desulfosarcina consortia, (3) to elucidate metabolic aspects of AOM by constraining intermediates of the process and by identifying possible vitamins and co-factors involved. These cross-disciplinary studies are hypothesis-driven investigations aimed at understanding the underlying mechanisms and syntrophic relationships of methane-oxidizing microbial cells and consortia through combined cell-specific molecular and isotopic analyses. This research will generate a unique high-resolution isotopic data set that will serve as a basis for modeling the thermodynamics of AOM as well as assist in defining the complex interactions between microorganisms involved in the cycling of methane. This approach is highly complementary to ongoing studies of anaerobic methane oxidation by geochemists and microbiologists and provides a unique perspective to assist in elucidating this globally important, but poorly characterized process.The education and outreach program will expand interest in geomicrobiology at the grade school, undergraduate, and graduate level. This project will provide team-oriented graduate education in microbial geobiology, engaging undergraduates through existing programs aimed at increasing diversity in the sciences, and building on existing infrastructure aimed at K-12 teacher education. Specifically, the PIs are contributing to the annual PSU teacher's workshop by teaching a session on the important role microbes play in regulating climate on Earth. Through connections with Dr. Benita Bell from Bennett College, a historically Black women's college in Greensborough, North Carolina, the PIs are introducing underrepresented students to the field of geobiology through a workshop during Space Science Week at Bennett. At the graduate level, the project will provide educational opportunities to graduate students at Penn State and Caltech.