FeEDTA强化传质效应下BioDeNOx体系的微生物及还原酶作用机制

NOx is one of the major hazardous atmospheric compounds from industrial flue gases, therefore, the research and development of new technology of the flue gas denitration has been imminent. BioDeNOx is employed to achieve the purpose of the removal of nitric oxide (NO) from the simulated flue gas with the advantages of low cost, complete reduction of NO and high removal efficiency. Focusing on the microbial activity and the reductase mechanism of BioDeNOx, the investigation aims to be conducted in the following aspects to enhance the NO bio-removal efficiency: The effect of Fe(II)EDTA on the microbial activity, biological denitrification capacity and microbial community will be analyzed; The effect of Fe(II)EDTA as electron donor on the microbial denitrification and the suitability of microbes to the different substrates will be elucidated. The dominant strains of Fe(II)EDTA-NO biodegradation will be isolated and the gene mapping of it will be sequenced. The key reductase (Nor, Nos) genes will be cloned and expressed; The degradation pathways and products of dominant strains will be analyzed at the genetic level. The optimization of the regulation of key reductase (Nor, Nos) activity will be investigated by constructing of engineering bacteria. The results of this investigation will provide the insight on the system design, configuration, and operation of the waste gas purification by using biological method.

NOx是主要大气污染物之一，BioDeNOx是新发展起来的一项高效、低耗的脱硝技术，但受限微生物活性和稳定性，阻碍工业化应用。本研究围绕该体系中的微生物及还原酶作用机制与净化效率：分析Fe(II)EDTA对微生物活性及反硝化能力的影响，解析Fe(II)EDTA强化NO气液传质效应下BioDeNOx的微生物群落结构变化规律，阐明体系中强化微生物反应的优先电子供体，探明微生物对不同底物的适配性和还原反应的协同调控机制；分离Fe(II)EDTA-NO高效还原菌株，获得高效菌株的基因图谱，克隆表达关键还原酶（Nor、Nos）基因，获得BioDeNOx降解酶系统，在基因水平上解析高效菌株的降解途径和产物；研究并优化调控关键还原酶活性，通过生物工程的方法构建工程菌，从而提高BioDeNOx体系运行的稳定性和高效性。本项目的研究，有望突破现有BioDeNOx的技术瓶颈，为生物法净化废气研究提供技术支撑。

在BioDeNOx体系中，Fe(II)EDTA-NO的加入强化气液传质并且充当电子供体参与微生物的还原过程，但缺少对于传质过程中的优势菌株深入分析，因此研究脱氮还原过程下的微生物作用机制及对关键还原酶基因的调控，对于推动BioDeNOx技术的应用具有重要意义。.本研究考察了强化过程中高效还原菌株的脱氮机制和还原过程中的脱氮性能，同时测定优势还原菌株的基因图谱，克隆表达高效菌株的关键还原酶，成功构建重组工程菌，并且优化后可高效表达，取得了以下研究结论：.1）Fe(II)EDTA-NO强化传质过程中筛选出的菌株TB可实现同步硝化反硝化。.2）研究碳源对脱氮过程影响发现菌株TB以丁二酸钠为碳源时，氨氮与总氮的去除率分别可达92.36％；pH在7.0～8.0范围时，脱氮效果最佳；不同温度研究比较发现，菌株TB为最适生长温度为20～30℃；控制转速在150~200rpm时，利于TB菌同步脱氮。.3）De Novo测序表明Achromobacter sp. TB中含有两类硝酸盐还原酶的编码基因，且均成簇存在；含铜型亚硝酸盐还原酶的结构基因仅发现nirK调控基因；一氧化氮还原酶由单个norB基因编码；氧化亚氮还原酶编码基因成簇存在。.4）norB、norZ基因被成功克隆表达，分别构建成 E. coli BL21(DE3)-pET28a-norB、E.coli BL21(DE3)-pET28b-nosZ工程菌，在OD600为0.6左右时加入终浓度为1.0 mM的IPTG，30℃下诱导5 h后norB基因表达效果最佳；使用IPTG终浓度为0.5 mM，温度为28°C，诱导6 h时，可成功诱导优化E.coli BL21(DE3)-pET28b-nosZ，其对N2O的还原率约是原菌株的1.92倍。