抗生素和重金属复合污染对SBR系统中胞外-胞内活性微生物及抗性基因的影响研究

With more and more pollutants entering the environment, combined pollution becomes a vital environmental pollution problem. As the major source of antibiotics and heavy metals in environment, wastewater treatment plants (WWTPs) have also become one of the reservoirs and sources for antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs). Moreover, the “extracellular free DNA” has been detected in the WWTPs, which is one of the carriers of ARGs and MRGs. However, the ecological toxicity of combined pollution of antibiotics and heavy metals on intracellular and extracellular active microorganisms and behavior of ARGs and MRGs are still unknown. .Normally, the concentrations of antibiotics and heavy metals are gradually increase, however, in some other cases, their concentrations may suddenly increase and then gradually decrease to the environmental concentrations. Therefore, this project is proposed to investigate the long-term effects of antibiotics and heavy metals on the biological wastewater treatment based on these two actual conditions in lab-scale nitrifying sequencing batch reactors (SBRs) relative to control SBRs that received no pollutants. Firstly, the effects of combined pollution on the nitrogen removal and the activity of activated sludge will be investigated. Second, the activity of ammonia-oxidizing bacteria and archaea (AOB and AOA, respectively) and their relative contributions to ammonia oxidation under the influence of antibiotics and heavy metals will be explored using DNA-based stable isotope probing (DNA-SIP) and metagenome sequencing. Thirdly, we aim to determine the response of intracellular and extracellular active microorganisms to combined pollution (sensitive? Resistance? No effect?), and to further reveal the variations of persistent, intermittent and transient active genera. Fourthly, the combined pollution on the occurrence, transfer and removal of ARGs and MRGs, and their coupling effects on wastewater treatment (e.g., nitrogen removal) will be investigated using qPCR, high-throughput qPCR and metagenome sequencing. Finally, the occurrence and variation of ARGs and MRGs will be investigated under different environmental and operational conditions to obtain the reduction strategy of ARGs and MRGs. Overall, this project is important for developing new wastewater treatment processes and improving the reduce performance of ARGs and MRGs under the influence of combined pollution.

污水处理厂是抗生素和重金属共存的典型环境，也是抗生素抗性基因（ARGs）和重金属抗性基因（MRGs）的“汇”与“源”。同时，在WWTPs中检测到“胞外游离DNA”的存在，其可作为ARGs和MRGs的携带者。但是，目前还未开展抗生素和重金属复合污染对胞内-胞外活性微生物及抗性基因影响的研究。因此，本项目拟采用“梯度增加”和“梯度降低”的方式，在SBRs系统中开展抗生素和重金属复合污染的长期影响研究。首先，基于稳定同位素核酸探针技术（DNA-SIP）探究复合污染对胞内-胞外氨氧化微生物活性的影响；其次，探究胞内和胞外持久性、间歇性和瞬时性活性微生物对于复合污染的响应；第三，解析复合污染对ARGs和MRGs赋存特征和传播的影响。最后，研究环境因子和操作参数对ARGs和MRGs的削减作用。该项目能够为开发高效低耗的新型污污水处理工艺，提高对于ARGs和MRGs的消减性能提供微生物依据和技术参考。

抗生素和重金属复合污染对活性污泥系统的生态效应及其对抗生素抗性基因增殖的影响是污水处理领域关注的热点问题。本课题选取典型抗生素（阿奇霉素、环丙沙星、左氧氟沙星、诺氟沙星、恩诺沙星、四环素、克拉霉素、阿莫西林、磺胺甲恶唑、甲氧苄啶和群体感应抑制剂等）和重金属（铜），通过剂量“梯度增加”和“梯度降低”方式探究了环境/低/中/高浓度的单一/复合污染物对系统性能的短期和长期影响及其后效应；基于微生物全尺度分类阐明了稀有菌属和丰富菌属对复合污染的响应及其交互作用，重点对脱氮功能菌群及相应功能基因进行辨识；探究了ARGs的赋存特征、传播机制及潜在细菌/古菌宿主；最后研究不同的高级氧化和消毒技术对ARGs的削减机制。研究明确了不同浓度复合污染对系统硝化作用促进和抑制作用，高浓度复合污染及其后效应对硝化作用的抑制更明显，且在特定高浓度下实现了亚硝酸盐氮的稳定积累与异养硝化-好氧反硝化低碳耗高效脱氮。基于全尺度分类发现复合污染胁迫和后效应改变了细菌和古菌群落的多样性和结构；不同类型稀有和丰富微生物中均存在抗性与恢复特性不同的特定菌属，且存在复杂的交互作用，稀有菌属占据重要的生态位；氨氧化古菌和一步式氨氧化菌对复合污染具有高抗性，同时，系统中出现了抗性好氧反硝化菌的“原位”的自富集；复合污染下检出多种ARGs，高浓度复合污染会引起ARGs的明显增殖，且基因之间多为共现模式；除了丰富细菌属和部分致病菌是ARGs的潜在宿主外，多种稀有菌属、脱氮功能菌和优势古菌菌属是潜在的ARGs宿主且携带多种ARGs；明确定了VUV/PMS去除二级出水ARGs的最优条件及机理，其对颗粒态和自由生长ARGs去除率高于游离态ARGs，不同消毒方式均降低了ARGs的总丰度、减弱了ARGs复杂的交互作用。相关研究成果开发高效低碳耗的新型污水脱氮工艺提供微生物学基础，还可为菌群的优化调控及ARGs的高效削减提供理论依据与技术支撑。