温度对污泥厌氧消化系统中抗生素抗性基因水平传播及整合子重组的影响机制研究

The high abundance of various antibiotic resistance genes (ARGs) in activated sludge makes the disposal of excess sludge at a high risk of ARGs dissemination. The removal of ARGs in sludge by anaerobic digestion is not effective enough in previous studies. The different types of ARGs show different changes at different digestion temperatures, and the internal mechanism has not been studied deeply. In this study, we are going to construct anaerobic digestion systems with different digestion temperature, and quantitatively analyze the abundances of plasmid, chromosome, cell free DNA, and ARGs in corresponding DNA. It will reveal the dynamics of different types of DNA and ARGs abundances at different digestion temperatures. The effect of temperature on the dissemination efficiency of ARGs in cell free DNA and intracellular plasmids in sludge anaerobic digestion system will be evaluated by tracing the plasmid and bacteria tracers. The effect of temperature on the recombination frequency and the diversity of the variable region of integrons in the sludge anaerobic digestion system will be investigated by dynamic analysis of the transcription activity of integrase and the sequences of integron variable regions. The results of the study will systematically clarify the mechanism of temperature on the dissemination of antibiotic resistance genes and the recombination of integron in sludge anaerobic digestion system, which is of great theoretical and practical significance to the effective removal of ARGs in sludge anaerobic digestion system.

活性污泥丰富的抗生素抗性基因（ARGs）使得剩余污泥的处置具有极高的ARGs传播风险。已有报道中厌氧消化技术对污泥ARGs去除效果还不理想，不同消化温度下不同种类ARGs表现出巨大变化差异，其内在机制还没有深入研究。本研究将构建不同消化温度污泥厌氧消化系统，对质粒、染色体、游离DNA含量和相应DNA上ARGs丰度进行定量跟踪分析，揭示不同类型DNA及其所赋存的ARGs在不同消化温度污泥厌氧消化系统中的消长动力学；通过示踪质粒和示踪菌的构建、投加与跟踪，阐明温度对污泥厌氧消化系统中游离ARGs和胞内质粒ARGs传播效率的影响；通过对整合酶转录活性和整合子可变区的动态分析，解析温度对污泥厌氧消化系统中整合子重组频率及可变区多样性的影响。研究结果将系统阐明温度对污泥厌氧消化系统中抗生素抗性基因水平传播及整合子重组的影响机制，对指导污泥厌氧消化系统有效控制ARGs传播具有重要的理论价值和现实意义。

剩余污泥丰富的抗生素抗性基因（ARGs）使污泥处理处置具有极高的ARGs传播风险。污泥厌氧消化工艺存在ARGs去除效果不理想，温度对污泥厌氧消化系统中ARGs赋存特征影响机制不明的问题。本项目针对性地考察了不同预处理技术对污泥ARGs的去除特性，阐明了预处理污泥中ARGs的分布特征，揭示了有利于污泥ARGs去除的预处理方法；构建了25℃、35℃、55℃三组不同温度半连续污泥厌氧消化系统，探明了不同温度污泥厌氧消化系统中污泥总DNA、质粒DNA和游离DNA携带ARGs丰度的动态变化，揭示了55℃高温污泥厌氧消化系统比25℃和35℃中温污泥厌氧消化具有更低的的污泥总ARGs和质粒ARGs以及丰度相近的游离ARGs，阐明了高温厌氧消化较中温厌氧消化更有利于削减污泥ARGs，而独特的微生物群落结构是高温污泥厌氧消化系统中ARGs丰度较低的主要原因，并发现了Firmicutes是污泥厌氧消化系统中ARGs重要的潜在宿主；解析了不同温度污泥厌氧消化系统中I型整合子丰度与多样性的动态变化，发现了55℃高温污泥厌氧消化系统较25℃和35℃中温厌氧消化系统更低的I型整合子丰度，阐明了不同温度污泥厌氧消化系统中I型整合子序列的差异化变化，识别到了中温污泥厌氧消化系统中I型整合子携带的多ARGs基因盒阵列，揭示了高温厌氧消化有利于削减污泥长片段多基因盒I型整合子；解析了不同温度污泥厌氧消化系统中I型整合子ARGs的赋存特征，发现了55℃高温污泥厌氧消化系统I型整合子携带ARGs种类高于25℃和35℃厌氧消化系统，但其整合子ARGs丰度低于25℃和35℃厌氧消化系统。研究系统阐明了温度对污泥厌氧消化系统中不同类型DNA携带ARGs丰度特征、I型整合子丰度与多样性动态变化、以及整合子ARGs种类与丰度的影响机制，为指导厌氧消化系统有效控制污泥ARGs传播提供理论基础。