畜禽粪便中兽用抗生素污染驱动下菜田土壤微生物响应特征研究

Land use of manure wastes has been considered as a main source of antibiotic pollution in agricultural soils. Natural manure or manure products often contain more than two antibiotics. However, referred to previous experiments, only effects of individual antibiotic on soil microbial systems were studied under single action by the application of antibiotic reagents onto soils rather than the natural antibiotic contaminated manure, in most cases. Thus, there is still a certain gap between actual results of the course and the current expectations. In this project, individual antibiotic and multiple antibiotics contaminated manure were collected from pigs receiving veterinary antibiotics through feed and used for fertilization. Investigations would be carried out under three conditions such as the long-term fertilization field, the artificial soil microcosm and the pot culture condition. This project can be divided into following sections: (1) The distribution and fates of antibiotic will be determined in vegetable-soil systems using two different soils following manure application; (2) Effects of antibiotics, particularly multiple antibiotics, on soil microbial community structure and function, soil nutrients transformation and antibiotic resistance expansion will be studied on the basis of DNA-SIP, High-throughput sequencing, DNA microarray, MicroRESPTM analysis as well as traditional soil enzyme activity determination. The microorganisms significantly affected by antibiotics will further be characterized to explore their distribution profile, phylogenetic information and ecological function in soil; (3) This work aims to elaborate the microbial mechanism of the environmental behavior of antibiotics in soil by building the relationship among soil metabolism, antibiotic resistance and soil microorganism. This research will provide new insights into a more deep understand of the ecological risk of the land use of manure with residual antibiotics. Meanwhile, the result would be reference for the safe use of manure based fertilizer.

农田抗生素污染主要源于养殖废弃物农用且以复合抗生素污染为主，但现有研究主要集中于单一抗生素的单独作用，与实际有一定差距，且鲜少深入探讨微生物在响应抗生素污染时所呈现的特征。本项目基于生产实践，采用“单一及复合兽用抗生素喂养-猪粪收集-施肥”的方式，在长期定位试验的基础上，利用微宇宙土壤培养和蔬菜盆栽试验：研究粪便有机肥中抗生素在不同类型菜田土壤中的分布、迁移和转化；结合DNA-SIP、高通量测序、基因芯片等分子生态技术和MicroRESP、土壤酶测定等代谢分析手段明确粪便有机肥中抗生素，尤其是复合抗生素，对土壤微生物群落、养分转化和耐药性扩散的影响，着重探讨主要响应微生物种群分布特征、进化关系及生态功能；最后整合代谢功能、耐药性和微生物特征数据，勾勒典型兽用抗生素土壤环境效应的微生物机制。研究将从一个更加深入且微观的角度阐述有机肥中抗生素的土壤环境效应，有助于养殖源有机肥的安全合理施用。

农田抗生素污染主要源于养殖废弃物农用且以复合抗生素污染为主，但现有研究主要应用土壤培养试验且集中于单一抗生素的作用，鲜少深入探讨微生物在响应抗生素污染时所呈现的特征。抗生素的土壤微生物生态学效应对于抗生素生态环境风险评估以及预测抗生素在土壤中的分解命运均至关重要。本项目以2种典型南方菜田土壤（青紫泥和小粉土）为对象，结合田间定位调研、原位土柱、盆栽和微宇宙土壤培养等试验，系统研究主要兽用抗生素在菜田的迁移、转化特征，探讨抗生素对土壤微生物群落结构、养分转化代谢和耐药性传播的影响，侧重对比单一和复合污染间的差异，探明土壤类型和蔬菜栽培的作用。研究发现：（1）长期施用有机肥加剧表层土壤中抗生素残留累积，特别是氟喹诺酮类抗生素，其中恩诺沙星在菜田具有明显向下迁移趋势。（2）小粉土中磺胺二甲嘧啶和强力霉素降解总体高于青紫泥，两种抗生素共存抑制二者在土壤中的降解，增加土壤微生物群落代谢多样性，复合污染对土壤微生物代谢的影响总体源于强力霉素的作用。（3）土壤微生物群落代谢性能和多样性影响不同菜田系统抗生素降解转化，特别是强力霉素降解转化与土壤微生物群落对酸类碳源和碳水化合物的利用能力紧密相关。（4）植物对土壤的修饰改变抗生素对土壤微生物氮循环的影响，恩诺沙星胁迫上调非种植土壤中hao、nxrA、narG、nirK、nirS和norB，增强氮循环转化，下调nosZ，抑制N2O向N2转变，增加土壤N2O释放量，但蔬菜栽培可在一定程度上缓解这种效应，降低N2O释放，萝卜优于青菜；相比对照，青菜和萝卜土壤细菌群落对恩诺沙星胁迫更强的缓冲能力。（5）磺胺甲恶唑增加菜田土壤磺胺抗性基因和I型整合子丰度，青菜土壤增加幅度高于萝卜土壤，根际土壤增加幅度高于非根际土壤；证实可通过土壤中添加抗生素降解菌促进抗生素降解，降低相应的抗生素抗性细菌的数量。研究为养殖源有机肥使用的科学性、安全性提供参考。