短程反硝化产N2O菌群定向富集技术及其过程机制

Nitrous oxide (N2O) produced from wastewater biological denitrification is a potent powerful greenhouse gas, and consequently has been a subject of interest in the field of environmental engineering. However, N2O can be beneficial from an energy perspective since it can be used as high-efficiency combustion aid. This beneficial aspect of N2O has not been fully studied. Recovering energy in the form of N2O is a novel wastewater treatment which combines the advantages of nitrogen bio-removal, energy recovery and greenhouse gas emission mitigation and is thus worthy of investigation. High efficiency production of N2O would require a truncated denitrifier which can reduce nitrate/nitrite to N2O as a final product. Even though these types of denitrifiers are not uncommon, no effective method for their enrichment is available. This project will develop such a method by beginning with an enrichment for complete denitrifiers, then, by investigating bacterial growth phenotypes, N2O production efficiency, nosZ gene (the gene encoding the protein that reduces N2O) frequency and transcriptional activity as a collected criterion, a method to optimize inoculum, nutrients and environmental conditions for enrichment of truncated denitrifiers will be developed. Detailed investigation of the bacterial community will be used to identify the selection pressures and allow a directional enrichment methodology. During these efforts the characteristics of enrichment/microbial aggregate/microflora/functional genes during the enrichment process will be actively monitored and connected to a suite of characteristics: 'selective pressure – community composition – functional genes (genotype) – N2O producing performance' to reveal the microbial adaptation strategies underlying the enrichment. This study will promote the development of a novel nitrogen pollution control and energy recovery technology from wastewater treatments and greatly extend the insight of the biological denitrification process, and is therefore of both practical and scientific value.

污水生物脱氮过程产生的氧化亚氮（N2O）是一种强温室气体，为环境工程领域所重视，而其作为能源物质（高效助燃剂）的另一面，尚未得到充分研究。以N2O形式回收污水氮素，兼具废水脱氮、能源回收和温室气体减排三大功能，极具研发价值。短程反硝化产N2O菌群是指将硝氮/亚硝氮还原至终产物N2O的反硝化菌群，有望成为高效回收污水氮素的驱动者。本项目针对短程反硝化产N2O菌群富集技术缺乏、富集过程机制不清等问题，从全程反硝化菌群出发，以菌体生长、产N2O效能、nosZ基因频率及活性为综合判据，优配菌源、营养及环境条件，识别定向选择压，建立富集技术；从混培物/菌胶团/功能菌群/功能基因（型）入手，对富集过程进行阶段性表征，揭示过程特性；探明选择压作用下反硝化群落定向遗传进化机制，通过功能基因（型）频率与活性特征关联产N2O性能，揭示富集过程机制。本项目可推动氮素污染治理与能源化发展，深化生物反硝化过程认识。

污水生物脱氮过程产生的氧化亚氮（N2O）是一种强温室气体，为环境工程领域所重视，而其作为能源物质（高效助燃剂）的另一面，尚未得到充分研究。以N2O形式回收污水氮素具有巨大的环境和经济效益，符合国家碳中和战略需求。针对短程反硝化产N2O菌群在废水生物脱氮过程中生理生态特性及其对产N2O的影响机制尚不明晰的问题，本项目通过比选接种菌源、乙炔浓度、pH等环境条件，以产N2O效能、菌体生长、nosZ基因频率与活性等为综合判据，结果发现接种菌源及反应器流态的差异可导致短程反硝化N2O产量的显著差异，接种活性污泥的序批式反应器是短程反硝化高效产N2O的一种有效形式。利用宏基因组耦合TEM电镜观察解释了以盛宴-饥荒循环为特征的序批式操作过程，可通过积累PHB等聚合物驱动内源性短程反硝化高效产N2O，但该方法的产N2O上限仅为2.3%。乙炔是常用的N2O还原过程抑制物。通过高浓度乙炔（40%的顶空体积）的长期驯化培养能富集高性能的短程反硝化产N2O菌群，产N2O上限可达98.6%。但该方法得到的培养物产N2O效能严格依赖于环境中乙炔浓度，在无乙炔状态经过约5个批次后，产N2O能力显著下降。酸性环境的反硝化过程N2O积累屡有报道。本研究发现低pH（pH为4.5~6.5）环境协同阶段性惰性气体吹扫可富集获得高效而稳定地产生N2O的活性污泥，产N2O可维持在97.5%。通过宏基因组耦合宏转录组技术分析发现，低pH时由非生物还原亚硝酸盐产生的大量NO可使得反硝化菌群体内NosR蛋白的铁硫中心失活，从而抑制了nosZI型反硝化菌（系统内的优势菌群）的N2O还原，从而导致了N2O的大量积累。最后，根据本项目创建的短程反硝化产N2O菌群定向富集技术，提出了未来应用的可能的污水脱氮耦合N2O回收的流程图。本项目的研究成果可推动氮素污染治理与能源化发展，同时深化生物反硝化过程认识，促进污水处理过程N2O的精准控制。