Collaborative Research: Stable isotope-based differentiation of vinyl chloride assimilators from cometabolizers in contaminated groundwater

氯乙烯（VC）是一种已知的人类致癌物，也是一种常见的地下水污染物。本研究项目的重点是鉴定和表征VC生物修复中涉及的关键微生物。VC羽流是在厌氧条件下通过氯化溶剂的还原性脱氯产生的，但经常逃逸到下梯度的好氧地下水中。需氧、生长偶联的VC氧化（即VC同化）细菌很容易从VC污染的地方分离出来，这支持了它们在降解中起作用的观点。然而，直接证据表明vc同化细菌存在并在原位活跃仍然难以捉摸。此外，尚不清楚同化vc的分离株是否是地下的优势微生物，或者它们的培养是否是富集偏差的结果。此外，VC同化剂不能与较大的相关乙烯氧化剂群区分开来，乙烯氧化剂是偶然的VC氧化剂。为了解决这些知识空白，本研究将使用稳定同位素探测（SIP）来区分VC同化细菌和偶然氧化VC的细菌（即共代谢菌）。SIP涉及微生物在生长偶联生物降解过程中将13C标记的底物同化为核酸。随后，分析13C标记的核酸，以确定负责污染物降解的生物体。中心假设是，SIP技术在与现有分子工具结合使用时，将在实验室和实地应用中区分异养生物和vc同化物。该研究涉及以下具体目标：i)使用来自vc污染地点的土壤和/或地下水样本，并应用DNA-SIP来揭示vc同化微生物的存在、身份和活性；ii)使用vc同化和com代谢实验室培养物来开发基于sip的分析，以区分这些密切相关的微生物群；iii)提供直接证据，证明vc同化细菌存在并在原位活跃。地下水被有毒污染物污染是威胁人类健康和环境的一个重大社会问题。原位生物修复实践有望使地下水恢复到污染前的水平。旨在更好地了解生物降解微生物群落的存在、丰度和活性的研究将为基础科学和生物修复实践架起桥梁。本研究的长期目标是通过开发分子工具来表征环境中污染物降解微生物，从而推动生物修复领域的发展。这项研究将包括训练环境工程专业的本科生和研究生应用分子生物学工具来解决环境问题。这项工作将增加代表性不足的少数民族学生的数量，还将涉及K-12外展活动的发展。这项研究将通过解决地下水污染物造成的人为水质恶化问题而造福社会。

1233087/1233154Mattes/CupplesVinyl chloride (VC) is a known human carcinogen and a common groundwater contaminant. This research project focuses on identifying and characterizing the key microorganisms involved in VC bioremediation at field sites. VC plumes are generated under anaerobic conditions via reductive dechlorination of chlorinated solvents, but often escape into downgradient aerobic groundwater zones. Aerobic, growth-coupled VC-oxidizing (i.e. VC assimilating) bacteria are readily isolated from VC-contaminated sites, supporting the idea that they play a role in the degradation. However, direct evidence that VC-assimilating bacteria are present and active in situ remains elusive. Further, it is unclear if VC-assimilating isolates are the dominant microbes in the subsurface or if their cultivation is a result of enrichment bias. Also, VC-assimilators cannot be distinguished from the larger related group of ethene-oxidizers, which are fortuitous VC oxidizers. To address these knowledge gaps, this research will use stable isotope probing (SIP) to differentiate VC-assimilating bacteria from bacteria that fortuitously oxidize VC (i.e. the cometabolizers). SIP involves microbial assimilation of 13C labeled substrates into nucleic acids during growth-coupled biodegradation. Following this, 13C labeled nucleic acids are analyzed to identify the organisms responsible for contaminant degradation. The central hypothesis is that SIP techniques, when used in conjunction with existing molecular tools, will differentiate between etheneotrophs and VC-assimilators in both laboratory and field-based applications. The research involves the following specific aims: i) to use soil and/or groundwater samples from VC-contaminated sites andapply DNA-SIP to reveal the presence, identity, and activity of VC-assimilating microorganisms, ii) to use both VC-assimilating and cometabolizing laboratory cultures to develop a SIP-based assay that differentiates between these closely related microbial groups and iii) to provide direct evidence that VC-assimilating bacteria are present and active in situ. Groundwater contamination by toxic pollutants is a major societal problem that threatens human health and the environment. In situ bioremediation practice hold promise for restoring groundwater to pre-contamination levels. Studies aimed at better understanding the presence, abundance and activity of biodegrading microbial communities will serve to bridge fundamental science and the practice of bioremediation. The long-term goal of this research is to advance the bioremediation field by developing molecular tools to characterize pollutant-degrading microorganisms in the environment. The research will involve the training of undergraduate and graduate environmental engineering students to apply molecular biology tools to environmental problems. The work will increase the number of underrepresented minority students and will also involve the development of K-12 outreach activities. The research will benefit society by addressing anthropogenic deterioration of water quality by groundwater pollutants.