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

甲烷消耗微生物的特征及其在微观尺度上控制其活动的因素对于我们理解全球范围内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的最大来源之一，然而这种温室气体中只有一小部分被释放到大气中，因为它被缺氧海洋沉积物中的微生物消耗。最近的跨学科研究表明，甲烷厌氧氧化(AOM)过程是由新的未培养的古生菌ANME-1和ANME-2介导的，它们与硫酸盐还原细菌共养。本项目通过一系列实验，结合精细分子RNA分析和同位素分析，研究了甲烷渗漏环境中的甲烷营养古生菌(包括ANME-1和ANME-2)，以及与ANME-2近缘培养的乙酸甲烷八叠球菌和甲基甲烷球菌的纯培养。这些目标旨在通过三个广泛的研究领域确定负责AOM的微生物并了解AOM的过程。这项研究的目标是：(1)通过扩展和完善现有的富甲烷沉积物中细胞的天然碳同位素组成的数据库，来表征AOM的微生物的多样性和代谢的多样性；(2)通过确定AOM的活性细胞在天然ANME-2/Desulfosarcina联合体中的位置来开发AOM的约束性热力学模型；(3)通过限制AOM过程的中间产物和确定可能涉及的维生素和辅助因素来阐明AOM的代谢方面。这些跨学科研究是以假设为导向的研究，旨在通过结合特定细胞的分子和同位素分析，了解甲烷氧化微生物细胞和联合体的潜在机制和同养关系。这项研究将产生一个独特的高分辨率同位素数据集，作为模拟AOM热力学的基础，并有助于确定参与甲烷循环的微生物之间的复杂相互作用。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佳的过程。教育和推广计划将扩大小学、本科生和研究生对地质微生物学的兴趣。该项目将提供面向团队的微生物地球生物学研究生教育，通过旨在增加科学多样性的现有课程吸引本科生，并建立旨在K-12教师教育的现有基础设施。具体地说，PIS正在为一年一度的PSU教师讲习班做出贡献，教授一节关于微生物在调节地球气候中的重要作用的课程。通过与北卡罗来纳州格林斯伯勒的班尼特学院(Bennett College)的贝尼塔·贝尔(Benita Bell)博士建立联系，PI正在通过班尼特空间科学周期间的研讨会，将代表人数不足的学生介绍给地球生物学领域。班尼特学院是北卡罗来纳州格林斯伯勒的一所黑人女子学院。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.