An overlooked source of N-nitrosamine precursors: Examining the role of biofilm in chloraminated drinking water distribution systems

消毒副产物是在饮用水处理过程中形成的化学物质，通常与消毒过程有关。消毒副产品是过去40年来美国和世界各地研究的主要焦点。这项研究项目意义重大，因为它将证明覆盖在配水管上的生物膜是一类特殊的消毒产品N-亚硝胺的重要来源。这一系列消毒副产物，至少其中一些是已知的人类致癌物。一种新的N-亚硝胺的形成途径将被调查涉及一个以前未确定的中间体在水处理系统中使用氯胺消毒。此外，控制生物膜生长和促进生物膜分离的策略将被系统地评估，以确定实现细菌稳定的水所需的条件，同时控制N-亚硝胺的形成。本提案的研究目的是检验加氯饮用水配水系统中生物膜胞外聚合物（生物膜）是重要的N-亚硝胺前体的假设。N-亚硝胺是一种高毒性、非卤化的消毒副产物，主要在使用氯胺的饮用水分配系统中形成，氯胺是一种常见的二级消毒剂，用于抑制受管制的消毒副产物的形成。最近的一项研究使用EPA方法521，它可以定量测定七种不同的N-亚硝胺，被证明只包括约5%的总N-亚硝胺形成的饮用水分配系统。这一结果意味着其他重要的N-亚硝胺可能被忽略了。生物膜细胞外聚合物物质含有诸如仲胺的官能团，已知仲胺与氯胺反应以形成N-亚硝基二甲胺（NDMA），这是EPA方法521种类中最普遍的。本研究将利用总N-亚硝胺测定来评估生物膜衍生材料对总体前体池的贡献。初步数据表明，外泌多糖分离物的氯胺化产生总N-亚硝胺，具有直接的剂量-反应关系。纯和混合文化生物膜，相关的饮用水分配系统，将使用传统的方法和下一代测序方法相结合的特点。 然后将这些系统进行氯胺化，以阐明细胞外聚合物在N-亚硝胺家族形成产物的扩展列表中的作用。拟议的研究将评估以下三个目标：1.）描述模拟饮用水分配系统中生物膜的动态特性。纯培养物和混合培养物生物膜将分别在间歇式和环形反应器中生长。生物膜EPS组合物（例如，胞外多糖和细胞外蛋白质）和细菌物种将被表征。2.）测量生物膜衍生材料和前体分离物的氯胺化产生的N-亚硝胺产率。将对附着和分离的纯培养物和混合培养物生物膜进行氯胺化，以评估EPA方法521物种和总N-亚硝胺的形成。生物膜分离株和已知的NDMA前体将进行类似的评估，并将完成一系列非生物实验，以帮助揭示潜在的反应机制。3.）第三章评估游离氯对生物膜特性和N-亚硝胺形成的影响。在不同消毒剂浓度、持续时间和温度下，在生物膜环形反应器中模拟游离氯烧伤。生物膜特性和N-亚硝胺前体将在加入游离氯之前、期间和之后进行评估。这项研究标志着第一次调查生物膜的作用，无处不在的所有饮用水分配系统，作为N-亚硝胺前体。本研究的结果将形成一个科学依据，以证明在全国范围内发生的调查，总N-亚硝胺氯化饮用水分配系统。教育和推广计划的目标是让研究生和来自代表性不足群体的高绩效工程本科生参与研究活动，为阐明的反应机制制作科学准确的动画，并向广泛的利益攸关方传播研究成果。

Disinfection by-products are chemicals which are formed during drinking water treatment and are usually associated with the process of disinfection. Disinfection by-products have been a major focus of research in the US and around the world for the past 40 years. This research project is significant because it will demonstrate that biofilms which coat the water distribution pipes are an important source of a particular class of disinfection by products known as N-nitrosamines. This family of disinfection by products, at least some of them, are known human carcinogens. A novel N-nitrosamine formation pathway will be investigated involving a previously unidentified intermediates in water treatment systems which use chloramine disinfection. Further, strategies to control biofilm growth and facilitate biofilm detachment will be systematically evaluated to determine the conditions required to achieve bacterial stable water, while controlling formation of N-nitrosamines. The research objective of this proposal is to test the hypothesis that biofilm extracellular polymeric substances (biofilms) in chloraminated drinking water distribution systems are important N-nitrosamine precursors. N-nitrosamines are a highly toxic, non-halogenated group of disinfection by-products formed primarily in drinking water distribution systems which use chloramine, a common secondary disinfectant used to curb formation of regulated disinfection by products. A recent study using EPA Method 521, which can quantitatively determine seven different N-nitrosamines, were shown to comprise only approximately 5% of the total N-nitrosamines formed in drinking water distribution systems. This result implies that other important N-nitrosamines may have been overlooked. Biofilm extracellular polymeric substances contains functional groups such as secondary amines, known to react with chloramines to form N-nitrosodimethylamine (NDMA), the most prevalent of the EPA Method 521 species. This study will utilize a total N-nitrosamines assay to assess the contribution of biofilm-derived materials to the overall precursor pool. Preliminary data demonstrate that chloramination of exopolysaccharide isolates yielded total N-nitrosamines with a direct dose-response relationship. Pure and mixed culture biofilms, relevant to drinking water distribution systems, will be characterized using a combination of traditional methodologies and next generation sequencing methodology. These systems will then be chloraminated to elucidate the role of extracellular polymeric substances in the formation of an expanded list of formation products in the N-nitrosamine family. The proposed research will evaluate the following three objectives: 1.) Characterize biofilm dynamics in simulated drinking water distribution systems. Pure and mixed culture biofilm will be grown in batch and annular reactors, respectively. The biofilm EPS composition (e.g., exopolysaccharides and extra-cellular proteins) and bacteria species will be characterized. 2.) Measure N-nitrosamine yields from chloramination of biofilm-derived materials and precursor isolates. Attached and detached pure and mixed culture biofilm will be chloraminated to assess the formation of the EPA Method 521 species and total N-nitrosamines. Biofilm isolates and known NDMA precursors will be assessed similarly and a series of abiotic experiments will be completed to help reveal the underlying reaction mechanism. 3.) Assess the impact of free chlorine on biofilm characteristics and formation of N-nitrosamines. Free chlorine burns will be simulated in biofilm annular reactors at different disinfectant concentrations, durations, and temperatures. Biofilm characteristics and N-nitrosamine precursors will be assessed before, during, and after the free chlorine addition. This research marks the first investigation into the role of biofilm, ubiquitous in all drinking water distribution systems, as N-nitrosamine precursors. Results from this study will form a scientific basis to justify a nationwide occurrence survey of total N-nitrosamines in chloraminated drinking water distribution systems. The objective of the education and outreach plan is to engage graduate students and high-performing engineering undergraduates from underrepresented groups in the research activities, create scientifically accurate animations of the elucidated reaction mechanisms, and disseminate the research findings to a wide range of stakeholders.