混合菌的构建及其对3,4-二氯苯胺的生物转化

Construction of mixed bacterial strain and its biotransformation of 3,4-dichloroaniline

从染料废水中分离出以3,4-二氯苯胺(3,4-DCA)为唯一碳源和能源生长的革兰氏阴性菌,筛选其中两株形态差异较大的菌株P11-1和L13构建混合菌群,用于3,4-DCA的转化实验以削减其毒性。通过优化两株菌配比构建混合菌群,混合菌群对3,4-DCA转化率达73.33%,明显高于单菌。经菌株形态观察和16S rDNA序列分析,确定两株菌均为铜绿假单胞菌。以单因素实验考察混合菌群在3 d内对100 mg/L 3,4-DCA的转化能力,并通过正交实验进一步优化转化条件,得出最佳条件组合为:接菌量3%(体积分数),温度30℃,pH=8.0,摇床转速190r/min,在该条件下3,4-DCA转化率达89.26%,其中pH对菌群转化能力影响最大。GC-MS分析菌群代谢产物主要为3,4-二氯乙酰苯胺,酶活分析发现N-乙酰基转移酶(NAT)活性较高,即菌群通过乙酰化对3,4-DCA进行解毒。

Gram-negative bacteria growing on 3,4-dichloroaniline(3,4-DCA)as the only carbon source and energy were isolated from dye wastewater.Two strains with great morphological differences,P11-1 and L13,were screened to construct mixed bacterial strain,which was used to the transformation of 3,4-DCA to reduce the toxicity of 3,4-DCA.By optimizing the proportion of the two strains to construct mixed bacterial strain,the conversion rate of 3,4-DCA reached 73.33%,obviously higher than that of single strain.Both strains were identified as Pseudomonas aeruginosa by morphology observation and 16S rDNA sequence analysis.The transformation ability of mixed strain to 100 mg/L 3,4-DCA in 3 d was evaluated by single factor experiment.The transformation conditions were further optimized by orthogonal experiment.The results showed that the best conditions was obtained as follows:inoculum amount 3%(volume fraction),30℃,pH=8.0,shaking velocity 190 r/min.Under these conditions,the transformation rate was up to 89.26%.Especially,pH had the greatest effect on the transformation ability of mixed strain.GC-MS result revealed that the main metabolite of mixed strain was 3,4-dichloroacetanilide.Enzyme activity analysis indicated that the mixed strain had higher N-acetyltransferase(NAT)activity,that is,it detoxified 3,4-DCA by acetylation.