微生物-电极耦合系统强化底泥中氯代抗菌剂降解及分子机制

The extensive use and abuse of antibiotics and antimicrobials have led to their release into various environmental settings, especially in urban rivers and sediments. Their persistence could evolve multi-drug resistant bacteria, which constitute great risk for human health. In order to remediate the contaminated aquatic and sediment ecosystems, a sustainable and controllable biologically enhanced in situ remediation technology is urgently needed. In this project, the applicants propose an innovative microbe-electrode coupled system, where antimicrobials are decomposed via a combination processes of biodegradation by introduced bacteria and respiration of electrophilic microbes on electrode placed in the sediment phase. In this project, we select triclosan and triclocarban as the targeted chlorinated antimicrobials due to their wide spread and persistence features, we aim to investigate the bioremediation efficiency and roles of the introduced electrode and chlorinated antimicrobials degrading bacteria and understand the regulation strategies in the microbe-electrode coupled system. The ultimate goal is to elucidate the metabolic pathways and the key genes that are responsible for the chlorinated antimicrobials biodegradation and clarify the biological niche and abundance of the chlorinated antimicrobials degrading bacteria and the electrochemically active bacteria during the bioremediation process. The symbiotic molecular ecologic network within electrode and sediment microbial communities was also reconstructed. This project would lay technological foundation toward a better bioremediation of representative antibiotics and antimicrobials in contaminated sediment environment.

人类过度使用和滥用抗生素/抗菌剂导致城市河道水体和底泥环境严重污染，污染环境中残存抗生素/抗菌剂可以诱发多重耐药细菌，对人类健康危害极大。因此亟待开发一种绿色可持续与可控性强的生物强化修复技术。本研究创新性地将降解菌殖入底泥与嗜电极微生物厌氧降解过程耦合，提出一种微生物-电极耦合原位修复抗菌剂污染底泥的新型工艺。以地表水和底泥中频繁检出的氯代抗菌剂三氯生和三氯卡班为研究对象，重点探讨引入电极和降解菌强化修复氯代抗菌剂的效率，掌握微生物-电极耦合系统修复污染底泥的调控策略。同时，揭示微生物降解氯代抗菌剂的代谢途径和关键基因，明确氯代抗菌剂降解菌和嗜电极活性微生物在修复过程中的功能地位与丰度，阐明微生物群落协作分子生态网络，为强化生物修复底泥环境中典型抗生素/抗菌剂的污染提供理论与技术基础。

三氯卡班是一种在个人护理品中使用量巨大的卤代芳烃抗菌剂，是全球十大新兴有机污染物之一，在环境介质如污水处理厂的剩余污泥和河道底质中残留浓度高达mg/kg水平。针对三氯卡班微生物脱毒关键机制不清、强化生物修复手段有限，该项目以三氯卡班为研究对象，分离筛选高效降解菌株资源；研究三氯卡班生物降解特征及降解途径；克隆新颖的脱毒关键酶基因，研究其催化特征；构建污染底泥微生物-电极耦合强化修复系统等。通过项目的实施，分离获得了2株三氯卡班降解菌（Ochrobactrum sp. TCC-1和TCC-2）及1株氯苯胺产物降解菌（Diaphorobacter sp. LD72），三氯卡班降解菌在好氧与厌氧脱氮条件下均可以水解三氯卡班至氯苯胺，氯苯胺降解菌可以矿化多类卤代苯胺，两降解菌能实现协同矿化三氯卡班；克隆获得了三氯卡班双酰胺键新水解酶TccA，单体组成型表达；酶学特性研究表明该酶可耐受≤60oC、pH=5-10和多数金属离子和化学抑制剂。同源性比对分析表明TccA与生化鉴定的酰胺酶氨基酸同源性仅为27-38%，具有酰胺酶超家族关键催化的Lys-Ser-Ser位点，除了对三氯卡班及脱卤同系物有很高的水解活性，对一些含酰胺键的除草剂、杀虫剂、化学合成中间体等均具有水解能力；构建了微生物-电极耦合的生物强化修复工艺，耦合系统不仅对于底泥中传统有机污染物如多环芳烃的去除起到了显著的强化作用（去除率提高7-30%），而且对三氯卡班的降解效率大幅增加，是未添加降解菌耦合系统的4.4倍。微生物群落测序结果表明降解菌在与土著微生物竞争生存空间和营养物质时可能处于劣势地位，使其在耦合系统中的相对丰度随时间不断变小。克隆获得的TCC双酰胺键水解酶关键基因tccA，为表征其在生物修复过程中（如污水处理厂、黑臭河道底质）的催化活性和在实际环境中微生物介导的归趋、转化活性和抗性风险提供了重要的分子生态标记，为强化微生物处理环境中三氯卡班提供理论依据与技术基础。微生物-电极耦合系统对于河道底泥中有机与常规污染物的强化去除效果表明其具有深度净化污染底泥的工程价值。