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

消毒副产物是指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。在过去的40年里，消毒副产品一直是美国和世界各地研究的主要焦点。这一研究项目意义重大，因为它将证明，覆盖在供水管道上的生物膜是一种被称为N-亚硝胺的特定类别消毒产品的重要来源。这一家族的消毒副产品，至少其中一些是已知的人类致癌物质。在使用氯胺消毒的水处理系统中，将研究一种新的N-亚硝胺生成途径，涉及一种以前未知的中间体。此外，将对控制生物膜生长和促进生物膜脱离的策略进行系统评估，以确定实现细菌稳定水所需的条件，同时控制N-亚硝胺的形成。这项建议的研究目的是验证氯胺饮用水供水系统中的生物膜胞外聚合物(生物膜)是重要的N-亚硝胺前体的假设。N-亚硝胺是一组剧毒、非卤化的消毒副产物，主要形成于使用氯胺的饮用水分配系统中，氯胺是一种常见的辅助消毒剂，用于遏制受管制消毒副产物的形成。最近使用EPA方法521进行的一项研究表明，它可以定量地测定七种不同的N-亚硝胺，仅占饮用水分配系统中形成的N-亚硝胺总量的大约5%。这一结果意味着其他重要的N-亚硝胺可能被忽视了。生物膜胞外聚合物物质含有功能基团，如仲胺，已知与氯胺反应生成N-亚硝基二甲胺(NDMA)，这是EPA方法中最普遍的521种。这项研究将利用总N-亚硝胺分析来评估生物膜衍生材料对整个前体池的贡献。初步数据表明，胞外多糖分离的氯胺化产生总N-亚硝胺，并具有直接的剂量-反应关系。与饮用水分配系统相关的纯和混合培养生物膜将使用传统方法和下一代测序方法相结合的方法进行表征。然后将对这些体系进行氯胺化，以阐明胞外聚合物在形成N-亚硝胺家族形成产物的扩展清单中的作用。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的生物膜动力学。纯培养生物膜和混合培养生物膜将分别在间歇和环形反应器中生长。将描述生物膜EPS的组成(例如胞外多糖和胞外蛋白质)和细菌种类。2.)测量来自生物膜衍生材料和前体分离株的氯胺化的N-亚硝胺产量。将附着和分离的纯和混合培养生物膜进行氯胺化，以评估EPA方法521种和总N-亚硝胺的形成。将对生物膜分离株和已知的NDMA前体进行类似的评估，并将完成一系列非生物实验，以帮助揭示潜在的反应机理。3.)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，将在生物膜环形反应器中模拟游离氯烧伤。在加入游离氯之前、期间和之后，将对生物膜特性和N-亚硝胺前体进行评估。这项研究标志着对生物膜作为N-亚硝胺前体的作用的首次调查，生物膜普遍存在于所有饮用水分配系统中。这项研究的结果将为在全国范围内对氯胺饮用水供水系统中总N-亚硝胺的存在情况进行调查提供科学依据。教育和推广计划的目标是让来自代表性不足群体的研究生和表现优异的工程学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，并将研究结果传播给广泛的利益攸关方。

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.