异养硝化-好氧反硝化苯酚降解功能菌株的代谢特性研究

Diaphorobacter sp. PD-7 capable of heterotrophic nitrification - aerobic denitrification as well as phenol degradation was isolated from active sludge previously. This research aims at investgating on the metabolic characteristics of the bacterium during the simultaneous nitrification and denitrification phenol-degrading process. Firstly, the heterotrophic nitrification and aerobic denitrification pathway is determined. Then the kinetic analysis of the enzymes in the heterotrophic nitrification - aerobic denitrification pathway and determination of the rate-limiting reactions and enzymes are conducted. Finally the mathematical model for metabolic flux analysis is constructed. The electron distribution and energy generation between oxidative phosphorylation and heterotrophic nitrification - aerobic denitrification pathway are analyzed to explain the relationship between the two pathways. With the help of genetic engineering methods, the bacterium with high metabolic activity of heterotrophic nitrification - aerobic denitrification is constructed. The metabolic flux analysis is conducted to investigate the effect of genetic manipulation on the generation and consumption of reducing power and energy in the cell, and to study whether cell growth and metabolism is improved. And the metabolic mechanism of heterotrophic nitrification - aerobic denitrification in the view of intracellular metabolism is revealed. This research can make the theory of simultaneous nitrification and denitrification more abundant and make a foundation for the development and design of phenolic wastewater treatment by simultaneous nitrification and denitrification technique.

申请者前期从活性污泥中分离得到一株异养硝化–好氧反硝化苯酚降解功能菌株Diaphorobacter sp. PD-7。本课题旨在深入研究同步硝化反硝化苯酚降解过程中菌株的代谢特性。首先明确异养硝化和好氧反硝化代谢途径；其次对异养硝化-好氧反硝化途径中催化酶进行酶促反应动力学分析，确定代谢途径中的限速反应和限速酶；最后构建代谢通量分析数学模型，考察电子传递在氧化磷酸化途径和异养硝化–好氧反硝化途径的分配比例及两种途径各自的产能水平，阐明两个途径的关系；并利用基因改造手段增强异养硝化–好氧反硝化代谢活性，考察细胞产还原力产能水平变化及其重新分布情况，对细胞生长代谢是否有促进作用，从胞内代谢角度揭示异养硝化–好氧反硝化的代谢作用。本课题研究可丰富同步硝化反硝化理论，为含酚废水同步硝化反硝化处理工艺的开发和设计奠定理论基础。

异养硝化-好氧反硝化是一种新型生物脱氮技术，可以在同一反应器内同时进行硝化和反硝化反应。酚类物质和氨氮是焦化废水中特征污染物，开发同时降酚脱氮新工艺，有利于降低处理成本、提高效率，因此有必要研究降酚脱氮菌株的代谢特征。. 以焦化废水活性污泥中筛选分离得到的异养硝化-好氧反硝化苯酚降解菌Diaphorobacter菌为对象，研究了其最佳降酚脱氮条件，在培养过程中硝态氮和亚硝态氮积累，并在后期降低，且细胞生长动力学符合Haldane模型。. 苯酚可诱导菌株合成邻苯二酚2,3-双加氧酶，推断菌株以间位开环途径降解苯酚。除苯酚外，菌株还可以利用间甲酚、4-氯酚、喹啉和吡啶为碳源进行脱氮。氮平衡分析表明，52.3%的氨氮转化为胞内氮，37.2%转化为氮气，菌株主要通过细胞同化作用和异养硝化-好氧反硝化作用脱氮。在与波兹坦短芽孢杆菌构建的双菌种降解体系中，苯酚降解和脱氮速率均比单菌种时速率提高。. 检测到氨单加氧酶、羟胺氧化酶、亚硝酸盐还原酶和硝酸盐还原酶活性，表明菌株具有完整的异养硝化-好氧反硝化偶联途径。分析了这四种酶活性测定的最佳温度和pH。采用米氏方程进行酶促反应动力学分析，推测亚硝酸盐还原酶是菌株脱氮过程中的限速酶。. 构建了菌株的代谢网络模型，通过代谢通量分析，发现溶氧较低时，反硝化产能占总产能的比例较高，随着溶氧的升高比例降低，而氧化磷酸化产能占总产能的比例随着溶氧升高而升高。说明溶氧较低时，有较多的电子流向反硝化途径产能，弥补氧化磷酸化产能不足。溶氧较高时，更多的电子流向产能效率更高的氧化磷酸化途径产能。. 采用基因工程手段过表达了亚硝酸盐还原酶基因nirS，在以苯酚为碳源的培养基中硝化和反硝化速率没有明显变化，而在以乙酸钠为碳源的培养基中，降解50mg/L氨氮由25h缩短至22h，硝态氮降解时间由25h缩短至23h。代谢通量分析表明，过表达nirS后反硝化途径代谢活力增强，产能增加了16.1%。. 这些研究成果可为同步硝化反硝化降酚新工艺的开发奠定理论基础和设计依据。