脱卤杆菌介导的厌氧微生物富集菌群对1,2,4-三氯苯的降解特性

doi:10.13745/j.esf.sf.2022.10.41

地学前缘 ›› 2024, Vol. 31 ›› Issue (2): 472-480.DOI: 10.13745/j.esf.sf.2022.10.41

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脱卤杆菌介导的厌氧微生物富集菌群对1,2,4-三氯苯的降解特性

吕良华¹^,²^,³(), 乔文静³^,^*(), 张晗³, 叶淑君⁴, 吴吉春⁴, 王水¹^,², 蒋建东³

1.江苏省环境科学研究院江苏省环境工程重点实验室, 江苏南京 210036
2.江苏省土壤与地下水污染防控工程研究中心, 江苏南京 210036
3.南京农业大学生命科学学院农业农村部农业环境微生物重点实验室, 江苏南京 210095
4.南京大学地球科学与工程学院表生地球化学教育部重点实验室, 江苏南京210023

收稿日期:2022-10-13 修回日期:2022-11-10 出版日期:2024-03-25 发布日期:2024-04-18
通讯作者: *乔文静(1989—),女,博士,副教授,主要从事土壤和地下水中卤代有机污染物的厌氧微生物修复研究工作。E-mail: qiaowenjing@njau.edu.cn
作者简介:吕良华(1989—),男,博士,高级工程师,主要从事土壤与地下水污染修复研究工作。E-mail: lvlianghua2013@163.com
基金资助:
国家重点研发计划项目(2018YFC1802501);国家自然科学基金项目(42007214);国家自然科学基金项目(42077174);中国博士后科学基金项目(2021M691614);江苏省“双创博士”(JSSCBS20210278);江苏省“333高层次人才培养工程”科研项目(680803125);江苏省卓越博士后计划项目(2023ZB141);江苏省地下水环境状况调查项目

Degradation of 1,2,4-trichlorobenzene by an anaerobic enrichment culture mediated by Dehalobacter species

LÜ Lianghua¹^,²^,³(), QIAO Wenjing³^,^*(), ZHANG Han³, YE Shujun⁴, WU Jichun⁴, WANG Shui¹^,², JIANG Jiandong³

1. Jiangsu Provincial Key Laboratory of Environmental Engineering, Jiangsu Provincial Academy of Environmental Science, Nanjing 210036, China
2. Jiangsu Province Engineering Research Center of Soil and Groundwater Pollution Prevention and Control, Nanjing 210036, China
3. Key Laboratory of Agricultural and Environmental Microbiology (Ministry of Agriculture and Rural Affairs), College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
4. Key Laboratory of Surficial Geochemistry (Ministry of Education), School of Earth Sciences and Engineering, Nanjing University, Nanjing 210023, China

Received:2022-10-13 Revised:2022-11-10 Online:2024-03-25 Published:2024-04-18

摘要/Abstract

摘要：

1,2,4-三氯苯是我国工业污染场地土壤和地下水中典型的有机污染物,具有持久性、生物蓄积性和高毒性特点,对生态环境和人体健康危害巨大。1,2,4-三氯苯密度比水大,容易迁移至深部厌氧区域,因此,开展1,2,4-三氯苯厌氧微生物降解与修复研究具有重要实际应用价值。本文通过长期富集培养,获得一份可以稳定地将1,2,4-三氯苯还原脱氯至1,4-二氯苯,再进一步还原脱氯至氯苯的厌氧菌液。通过16S rRNA基因扩增子测序及引物特异性的定量PCR实验,证明厚壁菌门的脱卤杆菌属细菌(Dehalobacter species)是1,2,4-三氯苯和1,4-二氯苯厌氧还原脱卤的功能菌株,其生长率为(1.68±0.8)×10⁶ copies·μmol^-1(释放的氯离子)。通过PCR扩增,获得Dehalobacter菌株的16S rRNA基因序列,构建了系统发育树。本研究可为1,2,4-三氯苯污染场地开展原位厌氧微生物修复提供菌株资源和理论指导。

关键词: 1,2,4-三氯苯, 地下水污染, 生物修复, 脱卤杆菌属, 还原脱卤

Abstract:

1,2,4-Trichlorobenzene (TCB) is a typical organic contaminant in soil and groundwater at industrial plant in China. 1,2,4-TCB is recalcitrant, bio-accumulative and highly toxic, posing serious threats to the environment and human health. 1,2,4-TCB is denser than water and easily penetrates into an anaerobic environment. Therefore, understanding the anaerobic biotransformation of 1,2,4-TCB is of great practical significance to on-site remediation. In this study, an anaerobic enrichment culture, which can sustainably dechlorinate 1,2,4-TCB to MCB via 1,4-DCB, is obtained. 16S rRNA gene amplicon sequencing and qPCR demonstrated Dehalobacter is responsible for the observed reductive dechlorination with a growth yield of (1.68±0.8)×10⁶ copies per μmol released Cl^-. A complete 16S rRNA gene sequence was obtained through PCR and a corresponding phylogenetic tree was constructed. This study provides a reference for the enrichment culture technique as well as theoretical guidance for the in-situ anaerobic bioremediation of contaminated sites.

Key words: 1,2,4-trichlorobenzene, groundwater contamination, bioremediation, Dehalobacter, reductive dechlorination

中图分类号:

X523
X172

吕良华, 乔文静, 张晗, 叶淑君, 吴吉春, 王水, 蒋建东. 脱卤杆菌介导的厌氧微生物富集菌群对1,2,4-三氯苯的降解特性[J]. 地学前缘, 2024, 31(2): 472-480.

LÜ Lianghua, QIAO Wenjing, ZHANG Han, YE Shujun, WU Jichun, WANG Shui, JIANG Jiandong. Degradation of 1,2,4-trichlorobenzene by an anaerobic enrichment culture mediated by Dehalobacter species[J]. Earth Science Frontiers, 2024, 31(2): 472-480.

图/表 6

图1 实验组中1,2,4-三氯苯的厌氧微生物还原脱卤过程 Benzene、MCB、DCB和TCB分别代表苯、氯苯、二氯苯和三氯苯。

Fig.1 The dechlorination of 1,2,4-trichlorobenzene in three active bottles

图2 1,2,4-三氯苯厌氧脱卤菌液微生物群落结构组成

Fig.2 The microbial community composition of the 1,2,4-trichlorobenzene-dehalogeanting enrichment culture

图3 总细菌和Dehalobacter的qPCR结果

Fig.3 The qPCR results of the total bacteria and Dehalobacter

表1 专性脱卤呼吸菌Dehalobacter生长率

Table 1 Growth yield of obligate organohalide-respiring bacterium Dehalobacter

培养周期	平行样	释放的氯离子量/ μmol	Dehalobacter细胞数量/ (copies·bottle^-1)	Dehalobacter生长率(释放的氯离子)/ (copies·μmol^-1)
第0~30天	#1	32.11	5.01×10⁶	1.58×10⁵
	#2	31.73	8.78×10⁶	2.78×10⁵
	#3	24.97	1.47×10⁶	6.23×10⁴
第31~90天	#1	147.95	3.34×10⁸	2.18×10⁶
	#2	110.69	2.40×10⁸	2.09×10⁶
	#3	103.69	8.19×10⁷	7.61×10⁵

图4 PCR产物电泳图 M列为DNA Marker,0列是阴性对照,1~9的DNA模板分别为第0、30、90天实验组1、2、3号平行样的DNA。

Fig.4 Electrophoretic diagram of the PCR products

图5 Dehalobacter的系统发育树

Fig.5 The phylogenetic tree of Dehalobacter spp.

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脱卤杆菌介导的厌氧微生物富集菌群对1,2,4-三氯苯的降解特性

Degradation of 1,2,4-trichlorobenzene by an anaerobic enrichment culture mediated by Dehalobacter species

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