嗜热微好氧偶氮染料高效降解复合菌群的降解机制解析

Azo dyes are widely used in the textile, paper, leather and pharmaceutical industries and their consumption scales can reach very high levels. Meanwhile, these processes are always accompanied with the generation of huge amounts of dyestuff wastewater, which will cause great pollution on ecological environment because of the lack of cost-effective, eco-friendly and effective management measures. In this project, the highly efficient biotreatment of azo dye wastewater is focused as the research guidance. On the research basis of a previously constructed thermophilic and micro-aerobic microbiome for effective azo dye degradation, direct black 19 is used as the research object, and a dilution-to-extinction approach is then employed to find out the dilution critical point of the microbiome for effective azo dye degradation. Subsequently, the metagenomic sequencing and iTRAQ (isobaric tags for relative and absolute quantitation) techniques will be combined to dissect the differential genes, proteins and microbial structures before and after the loss of azo dye degradation ability of the microbiome. The key enzymatic proteins and their corresponding function genes for effective azo dye degradation of the microbiome are then clarified through association analysis of the differential genes and proteins. And the metabolic and signal transduction pathways that involved in azo dye degradation of these proteins are further elucidated. In addition, the vital importance of the key enzymatic proteins during azo dye degradation process is verified through a degradation test with the addition of the prokaryotic expressed products. Synthetically analysis will be further proceeded based on the combination of the changes of the corresponding metabolites and metabolic parameters, and the highly efficient azo dye degradation mechanism of this microbiome will be ultimately elucidated from the angles of functional microbes, genes and proteins respectively, which will further provide a theoretical basis for the highly efficient biotreatment of azo dye wastewater.

偶氮染料在纺织、造纸、皮革、医药等行业使用广泛、用量巨大，与此同时伴随着巨量染料废水的产生，因缺乏经济环保有效的治理措施，对环境造成了巨大的污染。本项目以偶氮染料废水的高效生物处置为研究导向，在前期构建的一组嗜热微好氧高效偶氮染料降解复合菌群的研究基础上，以直接黑19为研究对象，采用减绝稀释法确定染料有效降解的稀释临界点，再结合宏基因组测序和iTRAQ技术，解析菌群失去染料降解功能前后的差异基因、蛋白及微生物组成。随后通过差异基因和蛋白的关联分析确定菌群实现染料高效降解的关键酶蛋白及对应的功能基因，并阐明其参与染料降解的代谢和信号转导途径，通过原核表达产物的添加降解研究验证关键酶蛋白在染料降解中的至关重要性。进一步结合相应的代谢产物和代谢参数的变化情况进行综合分析，最终从功能微生物、基因以及蛋白的层面上阐明菌群的高效偶氮染料降解机制，进而为偶氮染料废水的高效生物处置提供一定的理论研究依据。

偶氮染料在纺织、造纸、皮革、医药等行业使用广泛、用量巨大，与此同时伴随着巨量染料废水的产生，因缺乏经济环保有效的治理措施，对环境造成了巨大的污染。本项目以偶氮染料废水的高效生物处置为研究导向，在前期构建的一组嗜热的微好氧高效偶氮染料降解复合菌群的研究基础上，以直接黑G为研究对象，首先采用梯度稀释，PCR-DGGE及HTST相结合的技术确定了复合菌群有效降解偶氮染料的稀释临界点为10-7，确定了菌株Tepidiphilus sp.对偶氮染料的降解起着极其关键的作用，并在Anoxybacillus sp.、Caloramator sp.、Clostridium sp.、Bacillus sp.等菌株的协同作用下实现偶氮染料的高效降解；其次通过宏基组学技术分析出有无偶氮染料胁迫下复合菌群差异基因富集最显著的代谢通路包括氯环己烷和氯苯的降解、甲苯降解、苯甲酸降解、芳香族化合物的降解、二甲苯降解及二噁英降解，且多涉及含苯环类物质的降解，推测上述代谢通路可能与偶氮染料的脱色降解有重要关系，将这些代谢通路关联到的基因通过荧光定量PCR验证后发现共有16个基因具有显著差异性，可能参与了偶氮染料的降解；随后通过iTRAQ技术进一步确定NADH-ubiquinone氧化还原酶、NADH-醌氧化还原酶亚基(A、L和G)、琥珀酸脱氢酶和FAD/NAD(P)结合蛋白在偶氮染料降解过程中起着至关重要的作用，并采用MRM和原核表达技术进行了验证。此外，经LC-MS技术鉴定得到偶氮染料直接黑G的代谢产物，包括2, 7, 8-triaminonaphthalen-1-ol、苯胺、邻苯二胺、邻苯二甲酸及4-hydroxy-2-oxovaleric acid等物质，并根据代谢产物推测了其可能的降解途径；通过对直接黑G及其降解产物进行植物毒理性研究，发现脱色降解后产物的毒性明显降低，脱色降解液中的物质毒性微弱。最终本研究结合宏基因组、宏蛋白组结果及降解途径进一步揭示了直接黑G可能的共代谢降解机制，上述研究结果可为偶氮染料废水的高效生物处置提供一定的理论研究依据。