超微细菌降解不同六溴环十二烷对映异构体的途径及机理

Hexabromocyclododecane (HBCD) is a kind of persistent organic pollutant. It has been reported to be accumulated in many different environmental matrix and could be detected in a variety of commercial foods. Hence the effective treatments are urgently needed. The industrial grade HBCD mainly consists of 6 enantiomers. Previous studies have highlighted the difference of different isomer’s toxicity towards animals. To date, most studies focused on the degradation of HBCD were directed against several mixtures of isomers. Furthermore, limited degradation bacteria are available, largely due to the fact that most bacteria are “uncultivable” with conventional methods. Ultramicrobacteria have been reported to be an important part of the microbial community in various environments and play an important ecological role. This project will focus on the degradation of each single HBCD isomer under both aerobic and anaerobic conditions using ultramicrobacteria. The main aims are to analysis the ability and efficiency of ultramirobacterial degradation of different HBCD isomers, explore the phenomenon of mutual conversion of different HBCD isomers and identify the degradation products of each HBCD isomers and determine their degradation pathways. The molecular mechanism differences in the degradation of different HBCD isomers will be elucidated by RT-qPCR analysis of the degradation gene. Meanwhile, the structural biological methods will be applied to solve the 3D structure of key degradation enzymes. The results of the project would provide more microbial resource with HBCD degrading ability and new microbial degradation methods for different HBCD isomers in practical application.

六溴环十二烷（HBCD）作为持久性有机污染物，亟需有效的治理方法。工业级别HBCD主要由六种对映异构体构成，不同HBCD异构体具有不同的生理毒性，且在生物体中的代谢转化都有差异。目前针对HBCD不同异构体的微生物降解研究十分有限，同时在菌种的筛选培养方式上存在一定的局限性。本项目将围绕超微细菌对不同HBCD对映异构体及非对映异构体的降解途径及降解分子机理进行研究。筛选在好氧及厌氧条件下降解HBCD的超微细菌菌株，比较不同异构体在降解过程中的转化、代谢路径差异，对比不同超微降解菌株对几种异构体的降解效果。分析不同异构体的代谢产物，阐明其降解途径。解析关键性降解酶三维结构，阐明降解过程的分子机理。研究成果将促进对持久性有机污染物微生物降解的整体认识，为HBCD污染环境的修复提供理论依据。

六溴环十二烷(HBCD)作为持久性有机污染物，造成的环境问题与生态风险亟待解决。HBCD不同异构体具有差异性的生物毒性以及代谢转化过程，迄今针对HBCD不同异构体的微生物降解过程与机理研究仍十分匮乏。本项目HBCD异构体单体为研究对象，系统地研究了超微细菌对不同HBCD异构体的降解途径及机理。项目成功优化了HBCD异构体单体的制备方法，筛选与分离出两株好氧(Acinetobacter haemolyticus sp. strain HW-2、Pseudomonas sp. GJY)与一株厌氧(Citrobacter sp. strain ZYD-1)HBCD降解菌株。针对不同条件对菌株HW-2降解HBCD效率影响的研究发现，在35 ℃、pH=7、初始浓度为1 mg/L的条件下能够达到最高降解率。且添加1/100和2/100 LB培养基作为外加碳源能够促进菌株的降解效率。在对不同菌株对HBCD异构体降解过程中发现，降解过程具有异构体选择性。菌株HW-2在9天的降解过程中对α-HBCD、β-HBCD表现出了手性选择性，(−)HBCD的降解效率低于(+)HBCD。菌株GJY在8天的降解后对α-HBCD、β-HBCD和γ-HBCD的降解率分别可达85.38%、82.64%和75.50%，且γ-HBCD的对映体分数(EFs)降至0.4以下，表明同样存在异构体选择性过程。厌氧菌ZYD-1对HBCD非对映异构体的降解效率同样存在差异，异构体的降解效率排序为：β-HBCD＞γ-HBCD＞α-HBCD。菌株HW-2对HBCD的降解路径较为单一，而在菌株GJY对三种异构体的降解产物丰富度上，存在α-HBCD<β-HBCD<γ-HBCD的关系。此外，结合菌株HW-2全基因组信息，筛选出4个关键降解基因并对其在降解过程中的表达情况进行了研究，发现基因表达情况在不同异构体降解过程中存在区别。对4种关键蛋白进行了表达纯化并进行了体外实验，四种蛋白对底物的降解率为β-HBCD＞γ-HBCD＞α-HBCD，与前期实验结果相吻合。结合以上理论基础，建立了实验室水/土污染体系并进行了微生物修复，实验发现菌株HW-2的添加使土壤中HBCD的降解效率提升16.27%，且菌株的添加同样增强了菌群对HBCD异构体的差异选择性，在降低HBCD浓度的同时改变其异构体组成，完成对土壤的减毒和修复。