典型兽用抗生素及其抗性基因在猪粪堆肥过程中消减的微生物分子生态学机制

In recent years, pollution by veterinary antibiotics and its resistance genes gradually aroused the concern of the government and environmental experts. In order to elucidate molecular microbial ecology mechanism for decreases of typical veterinary antibiotics and its resistance genes during livestock and poultry manure composting, three kinds of veterinary antibiotics widely used in the scaled livestock farm with the great amount including tetracyclines, sulfonamides, and quinolones were selected in this study. To characterize the antibiotics degradation and shift in microbial diversity and antibiotics resistance genes during the pig manure composting, the technologies such as HPLC-MS-MS, Fluorescence Real-time Quantitative PCR, and High-throughput Sequencing will be used. The proposed research will mainly focus on the following area: (1) to elucidate the degradation rule for typical veterinary antibiotics during aerobic composting of pig manure and to optimize their composting conditions; (2) to characterize the degradation of typical veterinary antibiotics and to illustrate their molecular microbial ecology mechanism during the pig manure composting under the optimized condition; (3) to elucidate the molecular microbial ecology mechanisms for the dynamic shift or decreasing rule for antibiotics resistance genes (ARGs) during pig manure composting under the optimized condition. The research results will provide a theoretical basis and data supporting for the technologies development for preventing veterinary antibiotics and its resistance genes from entering the environments via application of livestock manure on farm land. These results will also be very useful for reducing the risk of veterinary antibiotics in the environments, and ensuring the quality safety of agricultural products.

近年来，兽用抗生素及其抗性基因环境污染问题逐渐引起了环保专家和政府的关注。大量未经无害化处理的畜禽粪便农用是抗生素及其抗性基因进入环境的主要途径。本项目以规模化养殖场用量较大的四环素类、磺胺类和喹诺酮类三种抗生素为研究对象，以猪抗生素饲喂-猪粪-好氧堆肥为主线，采用实时监测好氧堆肥系统结合荧光实时定量PCR、高通量测序技术和HPLC-MS-MS等技术，重点研究：（1）典型抗生素在猪粪好氧堆肥过程中的降解规律及堆肥条件优化；（2）优化条件下典型抗生素在猪粪好氧堆肥过程中的降解特征及微生物分子生态学机制；（3）优化条件下猪粪堆肥过程中抗生素抗性基因动态变化的微生物分子生态学机制。以期阐明猪堆肥过程中抗生素及其抗性基因消减的分子生态学机制。研究成果可为从猪粪源头上切断抗生素及其抗性基因进入环境技术的建立提供理论依据，进而对于确保农田生态环境和农产品质量安全具有重意义。

本项目以我国畜禽养殖使用量较大的3类兽用抗生素为研究对象，开展了猪粪好氧堆肥研究。揭示猪粪好氧堆肥过程中典型兽用抗生素消减规律及其ARGs行为特征及分子生态学机制。.（1）建立了简便价廉的同时检测畜禽粪便中5大类11种抗生素的固相萃取-高效液相色谱法。抗生素残留加标回收率介于62.65%-99.16%之间，定量限为0.3-5.9 μg•kg-1。本方法成本低，简便实用，可以完成畜禽粪便中抗生素残留的快速定量检测。.（2）构建了预测土霉素（OTC）、环丙沙星（CIP）和磺胺甲基嘧啶（SM1）在猪粪中的Kd的3个定量模型。3个模型的累积交叉验证系数值、标准偏差和外部验证系数变化范围分别为：0.761-0.868、0.027-0.064和0.743-0.827之间。因此，模型具有较强的内部和外部预测性。.（3）经过42 d堆肥，除了SM1单独添加，猪粪堆肥对抗生素均有较高的去除效果（84.9-96.2%）。除了TRGs中的EI和SRGs外，大部分ARGs可被有效去除。3种抗生素和ARGs行为受微生物活性的控制，且在整个堆肥过程中受到C/N、水分和pH显著影响。.（4）研究了抗生素饲喂添加（TD）和直接添加（TS）方法对堆肥中抗生素消减及ARGs行为特征及影响机理。TD处理中OTC和CIP消减率（94.9%和87.8%）明显高于TS处理中两者消减率（83.8%和83.9%）；反之，TD处理中SM1的消减率略低于TS处理中SM1消减率。TD和TS处理能够有效地消减大部分的四环素类抗性基因（TRGs），且TD处理能够增强某些ARGs的抵抗力。抗生素饲喂引起了TD处理细菌群落多样性存在的显著差异。冗余分析表明，堆肥前期TD处理ARGs变化主要受控于抗生素含量，TS处理则为堆体含水量；而堆肥后期二者均受控于pH变化。.（5）从庆大霉素生产过程中所产生的药渣和废水中筛选出了一株能够高效去除庆大霉素的真菌—FZC3。通过ITS鉴定和菌落形态鉴定，该株真菌被鉴定为土曲霉，其在优化条件下对庆大霉素的去除率达到95%以上。.本研究对于确保生态环境和农产品安全具有重意义。