1,2-二氯乙烷降解菌群的富集及关键降解菌Ancylobacter sp. BL0的分离鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-0108

摘要/Abstract

摘要：

【目的】1,2-二氯乙烷（1,2-DCA）的污染会危害到人体的生命健康和环境生态安全。为获得高效的1,2-DCA生物降解种质资源，研究菌群特征并探索从中分离筛选高效1,2-DCA降解菌株的规律和方法。【方法】从受1,2-DCA污染的土壤中富集培养能以1,2-DCA为唯一碳源和能源的降解菌群，通过气相色谱、紫外分光光度仪考察了不同批次菌群的生长和对1,2-DCA的降解情况，利用高通量测序分析不同批次富集液的物种多样性和相对丰度。通过16S rDNA基因序列分析鉴定菌株，利用气相色谱-质谱联用测定降解产物，分析菌株的降解途径。【结果】得到了降解菌群BG1，且实验结果显示第6-11批和15批富集菌群在24 h内对12.5 mg/L 1,2-DCA的降解率呈先增加后降低趋势，菌群生物量（OD₆₀₀）可从0.03增长至0.095，但菌群中Ancylobacter sp.的相对丰度随着持续富集而降低。从第9批富集液中筛选出一株菌命名为BL0，鉴定为Ancylobacter sp.，能在6 h内降解120 mg/L 1,2-DCA，推测BL0降解1,2-DCA的途径为1,2-DCA水解为2-氯乙醇，2-氯乙醇再被氧化为氯乙酸，然后被彻底降解利用。【结论】本研究得到了高效降解1,2-DCA的菌群BG1和菌株Ancylobacter sp. BL0，同时发现1,2-DCA降解菌株的筛选分离需要的样品富集过程周期较长且微生物生长量较低，不适于过度富集。菌株Ancylobacter sp. BL0与富集菌群中的其他菌株之间以竞争关系为主。

关键词: 1,2-二氯乙烷, 生物降解, 菌群富集, 降解菌群, Ancylobacter, 群落互作, 代谢途径

Abstract:

【Objective】The pollution of 1, 2-dichloroethane（1,2-DCA）will seriously harm human health and environmental ecological safety. In order to obtain efficient 1,2-DCA biodegrading microorganism resources, the characteristics of bacterial flora were studied, and then the rules and methods for separating and screening efficient 1,2-DCA degrading strains were explored.【Method】The bacteria utilizing 1,2-DCA as the sole carbon source and energy were enriched and cultured from soil contaminated by 1,2-DCA. The growth of bacteria and degradation of 1,2-DCA in different batches were investigated by ultraviolet spectrophotometry and gas chromatography respectively. The species diversity and relative abundance of different enriched solution were analyzed by high-throughput sequencing. The strain was identified by 16S rDNA gene sequence analysis. The degraded products were determined by gas chromatography-mass spectrometry, and the degradation pathway was analyzed.【Result】Bacterial consortium BG1 for degradation was gained, and the experimental results showed that the degrading rate of 12.5 mg/L 1,2-DCA by enriched bacterial consortium in batch 6-11 and 15 increased at the beginning and then decreased within 24 h, and the microbial biomass（OD₆₀₀）increased from 0.03 to 0.095, but the relative abundance of Ancylobacter sp. in the flora decreased with continuous enrichment process. A strain named BL0 was screend from the batch 9 of enrichment solution and identified as Ancylobacter sp., which degraded 120 mg/L 1,2-DCA within 6 h. It was inferred that the metabolic pathway of degradation of 1,2-DCA by BL0 was hydrolysis of 1,2-DCA to 2-chloroethanol at first, and 2-chloroethanol was oxidized to chloroacetic acid, which was then completely degraded and utilized.【Conclusion】Highly efficient 1,2-DCA degrading bacterial consortium BG1 and strain Ancylobacter sp. BL0 are obtained, and it is found that a long period of sample enrichment is required for screening and separation of 1,2-DCA degrading strains during the process of sample enrichment and low microbial growth is observed, which is not suitable for over-enrichment. Strain Ancylobacter sp. BL0 is competitive with the other strains in the enrichment of bacterial consortium.

Key words: 1,2-dichloroethane, biodegradation, enrichment of bacterial consortium, degrading consortium, Ancylobacter, community interaction, metabolic pathway

张志梅, 张彦猛, 谢东明, 杨秀云, 王浪, 左梓涵, 吴志国. 1,2-二氯乙烷降解菌群的富集及关键降解菌Ancylobacter sp. BL0的分离鉴定[J]. 生物技术通报, 2024, 40(8): 288-298.

ZHANG Zhi-mei, ZHANG Yan-meng, XIE Dong-ming, YANG Xiu-yun, WANG Lang, ZUO Zi-han, WU Zhi-guo. Enrichment of 1, 2-dichloroethane Degrading Bacterial Consortium, and Isolation and Identification of Ancylobacter sp. BL0 of a Key Degrading Bacterial Strain[J]. Biotechnology Bulletin, 2024, 40(8): 288-298.

图/表 11

图1 第6-11及15批BG1富集液降解效果

Fig. 1 Degradation effect of batch 6-11 and 15 BG1 enrichment solution

图2 BG1的生长及对1,2-DCA的降解 A：BG1的生长；B：BG1对1,2-DCA的降解

Fig. 2 Growth of BG1 and degradation of 1,2-DCA by it A: Growth of BG1. B: Degradation of 1,2-DCA by BG1

表1 第6批和第15批BG1的α多样性指数统计

Table 1 α diversity index statistics of batch 6 and 15 of BG1

样本Sample	Ace	Chao1	Shannon	Simpson	Goods_coverage
第6批 Batch 6	83.415	76	2.066	0.656	1
第15批Batch 15	103.572	100.750	3.021	0.766	1

图3 属水平的微生物群落结构

Fig. 3 Microbial community structure of enrichment culture at genus level

表2 菌株BL0生理生化实验结果

Table 2 Physiological and biochemical experiment results of strain BL0

项目 Item	结果 Result	项目 Item	结果 Result
革兰氏染色 Gram stain	-	接触酶试验 Catalase test	+
淀粉水解 Starch hydrolysis	-	氧化酶试验 Oxidase test	-
明胶液化 Gelatin hydrolysis	+	V-P试验 V-P test	-
甲基红试验 Methyl red test	-	柠檬酸盐试验 Citrate test	-

图4 BL0扫描电镜照片与菌落形态 A：菌株BL0扫描电镜照片（19 000×）；B：菌落形态；

Fig. 4 Scanning electron microscopy and colony morphology of strain BL0 A: Scanning electron microscope photos of strain BL0（19 000×）; B: colony morphology

图5 菌株BL0的16S rDNA序列进化树

Fig. 5 Phylogenetic analysis of strain BL0 based on the 16S rDNA

图6 菌株BL0的生长和对1,2-DCA的降解 A：BL0的生长曲线；B：BL0对1,2-DCA的降解和过程中的菌量变化

Fig. 6 Growth of BL0 and degradation of 1,2-DCA by it A: Growth curve of BL0. B: Degradation of 1,2-DCA and growth by BL0

图7 BL0降解1,2-DCA途径分析 A：BL0在氯乙酸为唯一碳源条件下的生长情况；B：BL0降解1,2-DCA的溴百里酚蓝变色实验（a：对照；b：反应10 h；c：反应24 h）；C：BL0降解产物气相色谱图；D：BL0降解产物质谱图

Fig. 7 Analysis of 1,2-DCA metabolic pathway by BL0 A: Growth of BL0 utilizing chloroacetic acid as sole carbon source. B: Bromothymol blue discoloration experiment in degrading 1,2-DCA by BL0（a: Contrast. b: Biodegradation for 10 h. c: Biodegradation for total 24 h）. C: GC diagram of degrading product by BL0. D: Mass spectrum of degrading product

图8 菌株BL0与由BL0和BG1组成的混合菌群对1,2-DCA的降解对比

Fig. 8 Degradation of 1,2-DCA by strain BL0 or consortium composed of BL0 and BG1

图9 BL0的1,2-DCA降解途径

Fig. 9 1,2-DCA degradation pathway of BL0

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