鞘氨醇单胞菌SW-2降解H酸的机理研究

1-Amino-8-hydroxynaphthalene-3,6-disulfonic acid (H-acid) is an important intermediate of naphthalene series dyestuff industry. It is a kind of organic naphthalene sulfonic acid compound which is very resistant to be biodegradation. To date, there are few studies about the biodegradation of H-acid, and the H-acid biodegradation mechanism remains unknown. Our group previously isolated Sphingomonas yabuuchiae SW-2, which can degrade H-acid. In this study, a BAC library will be generated from the strain SW-2 genomic DNA to investigate genes involved in H-acid degradation, and the H-acid will be selected as the only carbon source to screen the clone which can degrade the H-acid. Then, upstream and downstream genes will be mutated to accurately define the boundary of the H-acid degradation synthesis gene cluster and the minimum required genes for H-acid degradation. In addition, the metabolic intermediates of degradation will be purified and identified by HPLC, UV-Vis, MS and NMR. Then, the possible degradation pathway of H-acid will be proposed accoring to the known degradation pathway for naphthalene and above analyses in this work. Finally, the possible degradation pathway will be veried by knocking out the key or special gene. This study will reveal the H-acid degradation mechanism of Sphingomonas yabuuchiae SW-2, which provided the theory basis for the treatment of H-acid industrial wastewater by microorganisms.

H-酸属于有机萘磺酸类化合物，是萘系染料工业的重要中间体，是一种典型的难生物降解有机物，目前有关微生物降解H-酸的报道较少，H-酸生物降解的机理更是不清楚。本课题组在前期的研究中已经筛选出了能降解H-酸的鞘氨醇单胞菌SW-2，本项目拟通过构建菌株SW-2的全基因组BAC文库，筛选以H-酸为唯一碳源的克隆子，发掘H-酸降解基因簇；突变基因簇上下游边界的基因，确定SW-2菌株H-酸降解的最少必需基因；通过HPLC分离纯化SW-2降解H-酸的中间产物，利用紫外-可见光谱、质谱、核磁共振氢谱等手段鉴定其分子结构，根据目前已知的萘的降解途径，结合SW-2菌株降解H-酸的最少必需基因，推测出可能的H-酸降解代谢途径；通过基因敲除验证关键酶及特殊酶基因的功能，对推测的H-酸降解途径进行验证。本课题将阐明SW-2菌株降解H-酸的代谢途径，为微生物处理H-酸工业废水提供理论依据。

H-酸属于有机萘磺酸类化合物，是萘系染料工业的重要中间体，主要用于生产酸性染料、直接染料、活性染料和医药。H-酸工业废水CODCr浓度高、酸性强、颜色深，对微生物具有很大的毒性。目前，对于H-酸废水的处理主要包括两方面，一是添加无机或有机絮凝剂沉淀废水中的悬浮物，二是用萃取法、微波法、光催化法、膜分离法、化学氧化法等物理化学方法清除废水中的H-酸，这些絮凝和H-酸处理方法具有成本高、易造成二次污染等缺点。本研究以染料工业废水中筛选到的能以H-酸为唯一碳源的絮凝剂产生菌株鞘氨醇单胞菌SW-2为研究对象，研究该菌的生长特点，优化菌株SW-2降解H-酸的发酵条件；发掘H-酸降解的基因簇，分析SW-2菌株降解H-酸的中间产物，初步推测SW-2 菌株降解H-酸的途径；检测菌株SW-2对高岭土及重金属离子的絮凝活性，优化最佳絮凝条件，鉴定SW-2产生的生物絮凝剂的组成成分及分子结构。菌株SW-2具有良好的pH值、盐度和H-酸的耐受性，降解H-酸的同时能絮凝水中的固体悬浮物及重金属离子，本研究将为开发安全无毒、无二次污染的H-酸废水处理工艺提供相应的理论依据和治理措施。