污水中抗生素抗性基因的微观界面扩散特征与调控机理

Antibiotic resistance genes (ARGs) detected ubiquitously in water environment and spread by sophisticated pathway, were expected to threaten the safety of human health and ecological system. WWTPs obviously reduced the ARG from influent, but on the contrary, their higher ARG transferring intensity than natural environment made them the risk sources of ARG to environment, due to the abundant species in microbiome and favor nutrient conditions. Above findings not only clear understanding of ARG spreading and control in water environment in macro scale, but also distilled into more deep scientific questions about dynamic kinetics of ARG spreading, multiphase transfer in micro-environment, which requests more explore on the scale of single cell and micrometer scale. In this study, the microfluidics methodology will be grafted to study the micro-scale, in-situ and dynamic behavior of ARG multiphase transfer in water treatment process. Firstly, we plant to establish the microfluidic platform to observe the dynamic features of ARG transfer, by realizing the time-lapse recording, in-situ spatial distribution, and simulated microcosm control for target bacteria and community. Secondly, to study the key factors for gene transfer in micro-environment, by quantifying the effects from bacterial characteristics, aggregation states and environmental conditions. Thirdly, to analyze the gene transfer properties on the micro-interphase, by simulating micro-bubble aeration, cell lysis and vapour deposition process in microfluidic chip and inducing the transfer mechanisms. Lastly, to abstract ARG transfer model and identify parameters, in order to propose the principles and ideas to control the ARG in WWTPs.

抗生素抗性基因（ARG）在水环境中分布广泛、转移途径复杂，对人体健康和生态安全造成威胁。城市污水处理工艺能够有效削减ARG，同时又因出水残留多种ARG成为扩散风险源。污水处理设施内ARG转移率明显高于自然环境，且影响因素复杂。上述研究加深了对水环境中ARG扩散与控制的理论认识，同时也促成提出ARG转移动态特征、多相微界面行为等更深入的科学问题，有必要在单细胞微米尺度上进一步探索。.本研究借鉴微流控芯片技术开展ARG多相扩散过程的微观、原位和动态研究。首先创新实验手段观测ARG转移的动态特征，实现时间连续、空间原位、微环境可控。其次研究微环境中ARG在细胞间转移的影响因素，包括细菌生理特征、环境条件和聚集状态。第三观察多相微界面上ARG的转移行为，解析微孔曝气、细胞裂解、气相沉积等过程关联的ARG扩散机制。最后建立ARG扩散模型，提出工艺调控的原理和思路。

抗生素抗性基因（antibiotic resistance gene, ARG）导致抗生素失效，对人体健康构成了较大风险。城市污水处理过程是多种ARG的转移热点和储存库。因此，了解ARG在其中的行为特征，研究其去除和控制方法，具有重要的意义。本项目从微观、反应器和工艺等三个层面开展研究。. 在微观层面上，创新了实验手段观测ARG转移动态特征，探究了微环境中ARG转移的影响因素和动力学。（1）构建微流控芯片实验体系，选取携ARG质粒的供体菌，通过激光共聚焦显微镜获取ARG转移图像，通过细胞流式分析观测供体菌和结合子数量。（2）获取ARG质粒在纯菌和混合菌中的转移特征，分析了供受体菌类型和接种比例的影响。（3）使用单细胞跟踪芯片识别ARG水平转移事件，首次使用流行病传染模型建立ARG水平转移动力学。. 在反应器层面上，获取了ARG多相转移特征，探究了影响ARG转移的过程和条件。（1）使用流式分选和高通量测序获取了活性污泥中易接受ARG质粒的菌属，确定了垂直转移是活性污泥体系ARG扩散的主要途径。（2）在SBR反应器中观测ARG在气液固三相的分布，解析了曝气强度、水温等条件对ARG扩散的影响。（3）研究次氯酸、二氧化氯、紫外辐射等消毒处理后存活细菌接受ARG质粒的能力，发现氯类消毒剂促进了ARG转移。. 在工艺层面上，研究污水处理工程系统的ARG分布和去除规律，提出了优化设计和运行的建议。（1）研究典型城市和农村污水处理系统中ARGs的分布特征，确定了深度处理工艺对ARG去除的贡献显著。（2）研究了MBR和对照传统工艺的ARG去除效果及设计运行参数的影响。（3）除抗生素外，一些非抗生素药物也与出水ARG显著相关。（4）建议选择低污泥产率、少生物膜的工艺，运行时降低曝气量、稳定化污泥等，以控制ARG扩散风险。. 本项目为控制水环境中ARG传播提供一定的理论依据。