抗生素残留对湖库沉积物中反硝化作用的影响及作用机制研究

Denitrification plays an important role in nitrogen removal and regulating regional nitrogen cycling in water ecosystem, this process is mainly catalyzed by denitrifiers, but the key factors influencing denitrification rate is not clear. Our previous study found that the denitrification rate of natural lakes were much higher than those of aquaculture lakes. Compared with natural lakes, feed and drugs which contained large amounts of antibiotics were thrown into aquaculture lakes, the redundant and the excreted antibiotics were mainly suspended in water or deposited in sediments. So we put forward the scientific hypothesis that antibiotics change the structure of denitrifying microorganisms in the sediments, reduce the activity of denitrifiers and weaken the denitrification function. To test our hypothesis, field investigation and laboratory simulation are conducted in ten lakes of natural and aquaculture lakes from Wuhan city and Shiyan city, relationship between antibiotic content and sediment denitrification rate were firstly investigated, and then, molecular biology analysis of T-RFLP, high-throughput sequencing and qPCR were used to identify the community structure and gene copy number of different kinds of denitrifiers as well as microorganism, simulation experiments were eventually conductivitied to verify the hypothesis. This project provides theoretical basis of the influence of antibiotics on nitrogen cycle, the work is of great environmental and ecological significance.

反硝化作用在去除水体氮素、调节区域氮循环过程中起关键作用，这一过程主要由反硝化微生物催化完成，但调控反硝化作用强度的因素尚不清晰。前期研究发现营养状况相近的湖泊，天然湖泊反硝化速率显著高于养殖湖泊。与天然湖泊相比，养殖湖泊中人为投放了大量含抗生素的饵料、药物等，未被吸收及随粪便排泄的抗生素大量残留于水体或沉降于沉积物中。由此提出假设，抗生素改变了沉积物中反硝化微生物群落，降低了反硝化微生物活性，从而弱化反硝化功能。项目拟采用野外调查与实验室模拟相结合的方法，以武汉市和十堰市十个养殖及天然湖泊为研究对象，首先根据野外大样本数据求证抗生素含量与反硝化速率之间的关系，再运用分子生物学手段T-RFLP及高通量测序解析微生物群落结构、qPCR技术定量检测抗性基因及不同类型反硝化菌的数量，最后通过模拟实验证明科学假设，为抗生素干扰微生物介导的生物地球化学循环提供理论依据，具有重要的环境和生态学意义。

近年来，湖泊富营养化已经日益成为最严重的环境问题之一，湖泊中的氮素是影响富营养化的关键元素之一，而反硝化作用是将氮素排出水体，实现水体自净的最主要途径。前期调查结果显示天然湖泊反硝化速率显著高于养殖湖泊，水体中残留的新型环境污染物如抗生素对反硝化作用具有潜在调控作用。本项目系统分析了武汉市东湖、汤逊湖、青菱湖、后官湖和天鹅湖水体及沉积物中抗生素残留状况、沉积物反硝化微生物活性、反硝化菌群和耐药菌数量及反硝化菌群落结构。结果显示，浅水湖泊水体及沉积物中抗生素残留较为普遍，检出率高。相关性分析显示反硝化速率与三种抗性基因存在显著正相关关系，nirK及nosZ型反硝化菌基因拷贝数与恩诺沙星呈显著负相关关系，而抗性基因sulI 和 sulII与nirK及nosZ型反硝化菌基因拷贝数呈现出显著正相关关系。逐步分析结果显示抗性基因tetG对反硝化速率存在显著正调控作用、营养状况同样存在显著正调控作用，而抗生素磺胺甲恶唑对反硝化速率具有显著负向影响，模型对于反硝化速率的解释量达89.5%。而NMDS分析表明，nirS型反硝化细菌的群落结构与tetG显著相关，nirS型反硝化细菌群落结构与反硝化速率显著相关。综上所述，环境抗生素残留通过调控反硝化菌群落结构和反硝化菌数量达到影响反硝化速率的结果，而抗性基因的产生能在一定程度上提高脱氮能力。这些结果揭示了抗生素对浅水富营养化湖泊反硝化作用的影响。然而，反硝化菌抗性基因的产生及影响因素、环境污染物之间的联合作用关系值得进一步研究。本项目的实施有助于剖析抗生素对微生物介导的生物地球化学循环的影响，提高公众对新型污染物的重视，为湖泊富营养化的防治提供新的思路，具有一定的理论与实践意义。