猪场废水中耐药病原菌及携带的抗性基因在厌氧消化-农田利用过程中的消减机制研究

Livestock industry constitutes an important position in domestic development, and the amount of pollutants discharged from livestock operations was at the first place among other industries. Animal wastewater is typically applied to farmland as fertilizer. However, large amount of antibiotic resistant pathogenic bacteria contained in the wastewater poses great potential risk to public health, and the study on the occurrence and removal mechanism of antibiotic resistant pathogenic bacteria was deficient. The prevalence of typical pathogenic bacteria from animal production source, including Enterobacteriaceae and Enterococcus, and the characteristics of their antibiotic resistance and the carried antibiotic resistance genes (ARGs) will be investigated in the study. With modern molecular biological analysis and conventional microbial analysis methods, the fate and distribution of antibiotic resistant pathogenic bacteria and the carried ARGs during anaerobic digestion and land application of swine wastewater processes will be investigated by field survey. To improve removal of the antibiotic resistant pathogenic bacteria and the ARGs, anaerobic membrane bioreactor will be used to treat swine wastewater, as well as optimizing operational parameters of anaerobic membrane bioreactor, thus the removal mechanism of antibiotic resistant pathogenic bacteria and the ARGs will be investigated. To analyze the profile of antibiotic resistance pathogenic bacteria and the ARGs during the application of swine wastewater to farmland, pot experiment will be carried out with different pretreatment processes of swine wastewater and characteristics of farmland soils. Overall, the antibiotic resistant pathogenic bacteria and ARGs removal mechanism during the whole process of anaerobic digestion and land application of swine wastewater will be explored, which will be beneficial for the pollution control of livestock industry.

我国是畜禽养殖大国，畜禽养殖污染居全国污染物排放首位，畜禽废水通常作为肥水还田利用。然而畜禽废水中含有大量的耐药病原菌，在废水还田利用过程存在巨大的潜在威胁，其转归特征、消减机制研究较为缺乏。本研究以猪场废水中典型耐药病原菌（肠杆菌科细菌和肠球菌）为研究对象，采用现代分子生物学和常规微生物分析方法，通过现场调研，摸清我国典型规模化猪场废水厌氧消化-农田利用过程耐药病原菌及携带的抗性基因的时空分布特征。在此基础上，为了有效消减耐药病原菌及其抗性基因，重点通过厌氧膜生物反应器工艺及操作参数优化提高厌氧工艺对猪场废水耐药病原菌及携带的抗性基因的去除并对其机制进行解析。通过不同工艺处理的猪场废水和不同特性土壤的废水农田利用实验，研究猪场废水和土壤特性对耐药病原菌及抗性基因转归的影响。揭示猪场废水-农田利用过程耐药病原菌及携带的抗性基因的消减工艺与控制机制，为我国畜禽养殖污染防治提供科技支持。

抗生素抗性污染是二十一世纪公共卫生面临的巨大的威胁，畜禽养殖废水中赋存的耐药病原菌、抗生素抗性基因（Antibiotic resistance genes, ARGs）在生物处理与还田利用过程向环境传播，需要深入研究。本研究以猪场废水中典型的耐药病原菌、ARGs、耐药菌（Antibiotic resistant bacteria, ARB）为研究对象，以现场调研和实验室模拟为研究手段，针对猪场废水的主要处理工艺和利用过程开展研究，探讨猪场废水病原菌、ARGs的时空分布特征、生物处理过程抗性基因去除及其参数优化、以及猪场废水农田利用过程抗性基因的归趋。以期为畜禽养殖废水ARGs、ARB消减提供科学与技术支持。. 通过对我国南北方典型规模化猪场冬、夏季废水处理与农田利用过程ARGs的调研，结果表明tetM、tetX、ermB、ermF、mefA、sul1和sul2是猪场废水中的优势ARGs，冬季ARGs的拷贝数大于夏季。猪场废水处理过程对ARGs拷贝数具有较好去除。在猪场废水农田利用过程，北方冬季土地休耕停止施用沼液可有效降低土壤中ARGs的含量。而南方猪场在全年施用沼液的情况下，冬、夏季土壤中ARGs的拷贝数总数相似（p>0.05）。在猪粪水厌氧消化与土地利用过程中持久性ARGs（sul1和sul2）和逐渐升高的ARGs（tetG和ereA）需要特别关注。. 序批式膜生物反应器（Sequencing batch membrane bioreactor, SMBR）小试装置处理猪场废水的结果表明，SRT是影响ARGs转归最大的单一因子。SMBR对ARGs的拷贝数去除效率最高为2.91 log。提高SRT，生物处理（进水与上清液比较）ARGs拷贝数和丰度的去除率均呈现降低的趋势，反应器中可能发生了较高频率的ARGs增殖以及水平转移。膜分离对出水ARGs削减具有较大贡献。. 猪粪水、沼液土地利用的盆栽试验结果表明，猪粪水还田比沼液还田对土壤的菌群结构、ARGs和ARB影响大；经过猪粪水、沼液还田处理，红土受影响最大，其次是黄土，黑土受粪源细菌、ARGs和ARB污染最小。tetG、tetM和ermF是猪粪水土地利用土壤中的优势ARGs，在土壤中衰减符合一级反应动力学反应，它们的消减速率黑土>黄土>红土。