三苯甲烷类染料高效降解微生物新资源的发掘及其生态学功能研究

Triphenylmethane dyes, as high-yield dyes of which the majority is teratogenic, carcinogenic and mutagenic, are still widely used to date. The long-term and extensive use of triphenylmethane dyes poses great threats to human health and ecological environment. Microbial degradation technology is considered as one of the most effective ways to eliminate the pollution of dye residues. This proposal proposed to study the biodegradation of triphenylmethane dyes by microbial communities MGD5 enriched with triphenylmethane dyes as the sole carbon resources. The microbial population in the triphenylmethane dyes biodegradation communities will be characterized for their species diversity with culture-dependent and culture-independent techniques. Metagenomic sequencing techniqueis will be used to identify the functional species that may play a vital role in degradation. The not-yet-cultured or low relative abundance microbial species in the communities will be cultivated by culturomics and microfluidic streak plate techniques and studied for their unique physiological functions. Metabolites and intermediates formed during the biodegradationof triphenylmethane dyes will be identified and analyzed for the metabolic pathway deduction. The aims of the project are providing (a) a systematic investigation of biodegradation communities; (b) the ecological function of not-yet cultured or low relative abundance species; and (c) new ideas for isolation of rare or not-yet-cultured microbial species.

三苯甲烷类染料生产量大、应用范围广，长期大量使用对生态环境和人类健康构成严重的安全威胁。微生物降解技术被公认为消除该类染料残留污染的有效途径之一。本项目以前期获得的三苯甲烷类染料降解富集培养物MGD5为研究对象，通过高通量测序发现与降解作用积极响应的关键菌群，重点关注之前经常被忽略的低丰度或未培养等特殊微生物类群。运用元基因组测序技术解析可能对降解发挥核心作用的功能微生物类群。综合运用微生物培养组学、微流控平板等技术手段实现菌群中降解微生物资源的高通量筛选，利用MALDI-TOF鉴定和16S rRNA基因测序技术进行快速鉴定。研究新分离微生物对三苯甲烷类染料的降解能力并推定代谢途径，明确这些微生物类群在降解菌群中的生态学功能和存在意义。最终，为揭示三苯甲烷类染料微生物完全降解机制奠定理论基础，也为今后分离环境中稀有或未培养特殊微生物资源提供新的思路。

三苯甲烷类染料生产量大、应用范围广，长期大量使用对生态环境和人类健康构成严重的安全威胁。微生物降解技术被公认为消除该类染料残留污染的有效途径之一。依据项目的实验安排，项目组发掘了三苯甲烷类染料的高效降解微生物新资源，解析了高效降解富集物中不同微生物的群落组成和功能，明确了降解相关核心微生物，进一步分离到了13株孔雀石绿降解微生物新资源，对其中降解效果较好的Pseudomonas veronii JW3-6进行了深入研究。结果表明Pseudomonas veronii JW3-6最适降解条件温度为32.4℃，pH为7.1，接种浓度为2.5×107 cfu/mL。在该条件下，JW3-6对50 mg/L孔雀石绿7天内降解率93.5%，依据降解产物鉴定结果，首次报道了Pseudomonas veronii JW3-6降解孔雀石绿的代谢途径，推断该菌降解孔雀石绿的中间产物可进一步通过TCA循环实现完全矿化。结合前期的研究成果，深入研究了与三苯甲烷类染料降解过程高度相关的漆酶基因LaclK，根据降解菌株全基因组的生物信息学结果，分析并推导出了与降解过程高度相关的LaclK基因，成功的异源重组表达了LaclK，分析验证了LaclK酶学特性，研究了LaclK对三苯甲烷类染料降解能力。综上所述，本研究为三苯甲烷类染料的微生物降解发掘了新的微生物和酶资源，也为解析微生物降解三苯甲烷类染料的机理研究提供了参考。