养殖废水中抗生素对人工湿地关键脱氮微生物的作用机理及其反馈响应研究

In China, fast development of livestock industry induced to severe environmental pollution problems, and the antibiotic residue pollution generated by animal feeding operations has also aroused widespread concern. As an ecological treatment technology for treating livestock and poultry wastewater, antibiotic effects on nitrogen biogeochemical cycle are still unknown. In this project, a three-stage free water surface wetlands would be constructed to study nitrogen and antibiotics removal from swine wastewater through the field long-term testing method with periodic sampling and indoor incubation experiment. The abundance, diversity and microbial community structure of nitrification and denitrification based on functional genes analysis by using real-time quantitative PCR detecting system (qPCR), high-throughput genetic sequencing technique were investigated, and antibiotics concentrations in plant and soil of the constructed wetlands were determined to evaluate antibiotic environmental behavior with a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The main aims of this project were to: (1) discover the microbial drive mechanisms and critical pathways of nitrogen removal from swine wastewater in combination with the characteristics of spatial distribution and dynamic change regularity of functional microorganisms in wetlands system; (2) understand the effects of antibiotics on structure and activity of key microbial community with nitrogen conversion; (3) reveal how the dynamics of microbial population response to antibiotic stress in constructed wetlands. The results are useful to reveal the coupling mechanisms of antibiotics removal and key microbial nitrogen removal processes in the constructed wetlands for swine wastewater treatment. This project will provide a scientific basis for the development of constructed wetland technology to efficiently remove nitrogen and antibiotics from livestock and poultry wastewater.

当前畜禽养殖业所带来的环境污染问题日益突出，养殖中抗生素污染也是社会关注的热点。作为一种有效的养殖废水生态处理技术，人工湿地中抗生素对氮的生物地球化学过程影响还不十分清楚。本项目通过野外定位试验，以3级表面流人工湿地为研究对象，通过定期采样、长期监测和室内培养试验，利用qPCR、高通量测序等分子生物学和液相色谱-质谱/质谱联用技术等研究手段，研究人工湿地系统中硝化和反硝化微生物种群基因丰度、群落结构和功能活性，以及植物吸收、土壤吸附和降解等抗生素环境行为，结合人工湿地系统中环境因子、抗生素和含氮污染物的空间分布与动态变化的关联性，明确人工湿地对高氨氮养殖废水脱氮的微生物驱动机制，阐明脱氮功能微生物种群对抗生素胁迫的响应规律，揭示人工湿地处理养殖废水中抗生素和高氨氮复合污染的关键微生物驱动机制。该研究将为研发养殖废水高效人工湿地处理工艺提供科学依据。

当前畜禽养殖业所带来的环境污染问题日益突出，养殖中抗生素污染也是社会关注的热点。作为一种有效的养殖废水生态处理技术，人工湿地中抗生素对氮的生物地球化学过程影响还不十分清楚。本项目以表面流人工湿地为研究对象，利用分子生物学和液相色谱-质谱/质谱联用技术等研究手段，探明了养猪废水人工湿地处理系统水质变化特征，解析了养猪废水人工湿地处理系统关键氮转化过程及去除途径，分析了抗生素在人工湿地消减行为特征，明确了抗生素对湿地氮组分转化影响，揭示了硝化、反硝化微生物种群对抗生素胁迫的响应特征。结果表明，人工湿地系统对不同强度养殖废水均有较好的处理效果，TN去除率高达84%~97%。微生物脱氮作用和植物吸收是人工湿地主要脱氮途径，分别占总氮去除的33.0~67.5%和15.2~57.3%。当抗生素添加到人工湿地，人工湿地对不同种类抗生素均有降解能力，四环素降解率高达95%以上，磺胺嘧啶降解率为56.4~91.6%。抗生素在人工湿地降解过程影响湿地氮转化，与无抗生素处理相比，抗生素（四环素和磺胺嘧啶）添加可不同程度抑制水体氨氮去除，且浓度越高抑制效果越显著；相反，抗生素降低了水体中硝态氮积累。上述结果表明抗生素可能抑制了人工湿地微生物硝化过程，进而导致底泥氨氮含量显著高于无抗生素处理，抗生素处理底泥总氮累积量也提高了11.2~29.8%。抗生素显著降低了人工湿地硝化反硝化微生物种群数量。与无抗生素相比，抗生素处理降低了16.8~32.3%的AOB基因丰度，同时显著降低了反硝化微生物narG、nirK、nirS和nosZ基因丰度。Pearson相关性分析表明，在抗生素影响下，除AOB外，各硝化反硝化基因丰度均与水体总氮显著性正相关，与底泥总氮呈负相关。该结果表明抗生素是通过降低底泥硝化/反硝化微生物数量，导致底泥总氮累积，进而影响水体氮去除。开展本研究有助于深入认识抗生素对微生物脱氮过程的影响机理，进而为研发养殖废水中氮和抗生素污染高效去除的人工湿地工艺提供科学依据。