基于抗药基因高效控制的畜禽粪便好氧堆肥工艺原理研究

The excessive use of antibiotics in the livestock farming makes the livestock manure become one of major pollution sources of antibiotic resistance genes (ARGs). As the main disposal and recycling mode of livestock manure in China, aerobic composting becomes the key link of preventing ARGs from propagation to the environment matrices. However, the behavior mechanisms of ARGs in the composting process are still unclear. The efficiency for controlling ARGs by composting has not been identified. Taking typical ARGs as the research project, this study will mainly explore the following issues: 1) to clarify the dynamic mechanisms of typical ARGs in the composting process by simulating experiments, and screen out the bacterial communities and horizontal transfer elements which highly correlate with the dissemination of ARGs; 2) to investigate the influence of composting process parameters on the behaviors of ARGs and identify the key factors for controlling ARGs; 3) to seek the optimal regulation of process parameters for the coupling of compost maturity and reduction of risk from ARGs, and establish the technical principle for efficient control of ARGs during aerobic composting of livestock manure. The output of this work will provide strong technical support for preventing the dissemination of ARGs from the livestock farming to the environment and providing green organic fertilizer for agriculture.

抗生素在畜禽养殖业中的过量使用，使畜禽粪便成为环境中抗药基因的重要污染源之一。好氧堆肥作为我国畜禽粪便处置及资源化的主要方式，是阻断粪便中抗药基因向环境介质中传播的关键环节，然而目前堆肥过程中抗药基因的行为机制尚不明确，对于堆肥工艺是否可有效控制抗药基因的传播亟待深入研究。本项目以典型抗药基因为研究对象，通过模拟堆肥试验解析堆肥过程中抗药基因的动态变化机制，甄别与抗药基因传播行为密切相关的菌群及水平转移元件；深入探讨堆肥工艺参数对抗药基因行为的影响机制，明确抗药基因高效控制的关键要素。在此基础上，通过堆肥工艺参数的优化调控，达到堆肥腐熟及抗药基因风险削减的双重目标，从而构建基于抗药基因高效控制的畜禽粪便好氧堆肥工艺原理，既能从源头阻断抗药基因向环境中的传播，又能为农作物提供绿色有机肥料。

本项目通过实验室模拟堆肥试验，改变辅料、调控高温期、添加复合微生物菌剂等不同工艺条件，探讨了堆肥进程中抗生素抗性基因（ARGs）动态变化的普适规律，研究堆肥过程中主导水平转移元件（MGEs）变化、微生物群落演替及堆料性质改变等因素与ARGs行为的关联，并提出了有效可行的猪粪堆肥工艺中ARGs的抑制策略，构建了基于ARGs高效控制的畜禽粪便好氧堆肥工艺原理。研究结果显示；1）市售有机肥中普遍存在ARGs，充分腐熟和未腐熟猪粪肥中的ARGs分布特征差异显著；2）采用小麦秸秆、玉米秸秆、杨木木屑和蘑菇渣作为猪粪堆肥的辅料，发现堆肥结束时总ARGs绝对和相对丰度分别增高0.43‒2.73 log值和0.19‒1.61 log值，堆肥过程中ARGs、MGEs分布的变化和微生物群落的演替过程明显分为升温-高温和降温-腐熟两阶段；3）采用提高高温期温度和延长高温期时长等措施，发现堆肥结束时总ARGs绝对丰度增高0.71–0.99 log值，相对丰度降低49.7–66.2%，可见高温期调控不能达到抑制ARGs的效果，堆肥过程中有机质等营养元素通过影响微生物群落（如ARGs宿主）进而影响ARGs的分布；4）添加复合微生物菌剂的堆肥组中总ARGs绝对和相对丰度分别降低29.3–77.2%和27.3–64.5%，高剂量添加和过渡期二次添加菌剂可有效抑制ARGs，同时可加快堆肥腐熟进程，是控制堆肥中ARGs的可行策略；5）堆肥过程中不同种类ARGs的动态变化规律呈现多样性，其中磺胺和甲氧苄啶类抗性基因、氨基糖苷类抗性基因难以通过堆肥去除；6）堆肥过程中存在较活跃的ARGs水平转移过程、多样化的ARGs宿主种类及广泛的多重耐药菌，值得注意的是，整合子intI1介导的水平转移过程增强，且与数种增多的ARGs正相关；多数潜在病原微生物得到有效去除，但Mycobacterium、Bordetell和Bacillus等病原微生物与增多的ARGs正相关；整合子intI1和这些潜在病原微生物可能提高猪粪堆肥中抗生素抗性传播的风险。