基于生物强化的异养硝化－好氧反硝化复合菌剂脱氮特性及微生态响应机制研究

The bioaugmentation of the heterotrophic nitrification - aerobic denitrification microbial consortium could improve the denitrification effect effectively. Therefore, it has great application potential in rural sewage treatment engineering. However, the heterotrophic nitrification - aerobic denitrification microbial consortium compositional formulation is imperfect, meanwhile, denitrification characteristics and micro ecological response mechanism is not clear. Thus, it is difficult to used in industrial and scale application. In this study, for solving these problems, on the basis of the heterotrophic nitrification - aerobic denitrification strains in our laboratory, the biochemical test combing with the mathematical statistics, molecular biology methods were used to: (1) optimize the microbial consortium compositional formulation by the boundary point test and cubic spline interpolation; (2) explore the mechanism of heterotrophic nitrification - aerobic denitrification strain and microbial consortium by intermediate product analysis and functional genes and enzyme detection methods; (3) use droplet digital PCR and MiSeq high-throughput sequencing combined with sensitivity analysis means to evaluate micro-ecological response mechanism of microbial consortium affected by the indigenous microbial communities and environmental factors change, furthermore, feedback evaluation the compositional formulation and dosing solution. The results will not only provide key parameters for the bioaugmentation of heterotrophic nitrification - aerobic denitrification microbial consortium, and also will offer an important technical support to upgrade the rural sewage treatment plant denitrification process.

异养硝化-好氧反硝化复合菌剂生物强化作用能有效提高脱氮效率，在农村污水处理工程中应用潜力巨大，但异养硝化－好氧反硝化复合菌剂复配方案不完善、脱氮特性及微生态响应机制不明确，成为该方法规模化和工业化应用的技术瓶颈。本研究从解决上述问题出发，在实验室已有的异养硝化-好氧反硝化菌株基础上，采用生化试验结合数理统计学、分子生物学等方法，开展如下研究：（1）采用边界点组合试验和三次样条插值法优化菌剂复配方案；（2）采用中间产物分析与功能基因和酶学检测方法，探索异养硝化-好氧反硝化菌株及复合菌剂脱氮机理；（3）以ddPCR、MiSeq高通量测序结合敏感性分析等手段评价复合菌剂生物强化过程受土著微生物群落及环境因子变化的微生态响应机制，反馈评价复合菌剂配制与投加技术方案。本研究不仅为异养硝化-好氧反硝化复合菌剂生物强化应用提供关键参数，而且为农村污水处理厂脱氮工艺提升改造提供重要的技术支撑。

异养硝化-好氧反硝化复合菌剂生物强化作用能有效提高脱氮效率，在农村污水处理工程中应用潜力巨大，但异养硝化－好氧反硝化复合菌剂复配方案不完善、脱氮特性及微生态响应机制不明确，成为该方法规模化和工业化应用的技术瓶颈。本研究从解决上述问题出发，采用生化试验结合数理统计学、分子生物学等方法，开展如下研究：（1）HN-AD菌株筛选、脱氮性能及机理测定。通过富集驯化、筛选、分离、检测等手段从污水厂活性污泥中分离筛选8株具有异养硝化-好氧反硝化功能的菌株并进行观察、鉴定以及保存。通过一系列脱氮性能测定 (HNM, DM和NDM) 以及脱氮基因（napA, nirS, nirK和nosZ）扩增综合分析脱氮机理，为复合菌剂的脱氮机理研究提供基础。（2）菌剂复配方案、机制及环境条件优化。优选出三株协同作用Pseudomonas sp. strain ID2、Pseudomonas sp. strain ID5和Pseudomonas sp. strain ID8进行菌剂复配，通过三次函数拟合得出最佳复配比例2:1:3，其实现95%的降解率的培养时间最短为19.59 h；复合菌剂适宜的碳源类型为柠檬酸三钠，C/N值为15；采用曲面响应法优化脱氮的环境条件得出脱氮率最高：pH 7.88、温度30.19℃、摇床转速 101 r/min。（3）高效脱氮复合菌剂投加曝气生物滤池进行生物强化应用，水质检测结果显示，滤池中的氨氮和总氮去除效果明显提高。采用MiSeq高通量测序和荧光定量PCR手段检测脱氮功能菌与脱氮基因的变化过程，探究了复合菌剂与土著微生态系统结构、环境因子产生响应机制。