Enrichment of 1, 2-dichloroethane Degrading Bacterial Consortium, and Isolation and Identification of Ancylobacter sp. BL0 of a Key Degrading Bacterial Strain

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

Abstract

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

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.

Figures/Tables 11

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

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

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

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

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	-

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

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

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

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

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

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

References 34

[1]	赵兰辉. 化工园区废水中1,2-二氯乙烷去除工艺研究[D]. 上海: 华东理工大学, 2021.
	Zhao LH. Research on the removal process of 1, 2-dichloroethane in wastewater in chemical industry park[D]. Shanghai: East China University of Science and Technology, 2021.
[2]	王小春. 二氯乙烷降解菌的分离鉴定及降解特性研究[D]. 杭州: 浙江工业大学, 2013.
	Wang XC. Isolation, identification and degradation characteristics of dichloroethane degrading bacteria[D]. Hangzhou: Zhejiang University of Technology, 2013.
[3]	周佳伟, 施维林, 许伟, 等. 污泥生物炭硼掺杂改性及其对水中1, 2-二氯乙烷吸附行为和机制[J]. 环境科学, 2023, 44(5): 2671-2680.
	Zhou JW, Shi WL, Xu W, et al. Sludge biochar modified by B-doped and its adsorption behavior and mechanism of 1, 2-DCA in water[J]. Environ Sci, 2023, 44(5): 2671-2680.
[4]	张祎曼. 碳基表面衍生化材料的制备及其对1, 2-二氯乙烷的电催化脱氯研究[D]. 大连: 大连理工大学, 2022.
	Zhang YM. Preparation of carbon-based surface derived materials and its electrocatalytic dechlorination of 1, 2-dichloroethane[D]. Dalian: Dalian University of Technology, 2022.
[5]	黄煜韬, 孟宪荣, 许伟, 等. EDTA强化nZVI/PS降解地下水中1, 2-二氯乙烷的作用机制及影响因素[J]. 环境工程学报, 2022, 16(3): 885-893.
	Huang YT, Meng XR, Xu W, et al. Mechanism and influencing factors of 1, 2-dichloroethane degradation in groundwater by EDTA-enhanced nZVI/PS[J]. Chin J Environ Eng, 2022, 16(3): 885-893.
[6]	Janssen DB, Scheper A, Dijkhuizen L, et al. Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10[J]. Appl Environ Microbiol, 1985, 49(3): 673-677.
[7]	Olaniran AO, Pillay D, Pillay B. Aerobic dechlorination of cis- and trans-dichloroethenes by some indigenous bacteria isolated from contaminated sites in Africa[J]. J Environ Sci(China), 2004, 16(6): 968-972.
[8]	Mileva A, Sapundzhiev T, Beschkov V. Modeling 1, 2-dichloroethane biodegradation by Klebsiella oxytoca va 8391 immobilized on granulated activated carbon[J]. Bioprocess Biosyst Eng, 2008, 31(2): 75-85.
[9]	Maymó-Gatell X, Anguish T, Zinder SH. Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by “Dehalococcoides ethenogenes” 195[J]. Appl Environ Microbiol, 1999, 65(7): 3108-3113.
[10]	Xing ZL, Su X, Zhang XP, et al. Direct aerobic oxidation(DAO)of chlorinated aliphatic hydrocarbons: a review of key DAO bacteria, biometabolic pathways and in situ bioremediation potential[J]. Environ Int, 2022, 162: 107165.
[11]	Munro JE, Liew EF, Coleman NV. Adaptation of a membrane bioreactor to 1, 2-dichloroethane revealed by 16S rDNA pyrosequencing and dhlA qPCR[J]. Environ Sci Technol, 2013, 47(23): 13668-13676.
[12]	Davis GB, Patterson BM, Johnston CD. Aerobic bioremediation of 1, 2 dichloroethane and vinyl chloride at field scale[J]. J Contam Hydrol, 2009, 107(1/2): 91-100.
[13]	Hage JC, Hartmans S. Monooxygenase-mediated 1, 2-dichloroethane degradation by Pseudomonas sp. strain DCA1[J]. Appl Environ Microbiol, 1999, 65(6): 2466-2470.
[14]	Tardif G, Greer CW, Labbé D, et al. Involvement of a large plasmid in the degradation of 1, 2-dichloroethane by Xanthobacter autotrophicus[J]. Appl Environ Microbiol, 1991, 57(6): 1853-1857.
[15]	许文帅. 固定化Rhodococcus sp. W7处理焦化废水的研究及应用[D]. 天津: 天津科技大学, 2021.
	Xu WS. Study and application of coking wastewater treatment by immobilized Rhodococcus sp. W7[D]. Tianjin: Tianjin University of Science & Technology, 2021.
[16]	Strotmann U, Röschenthaler R. A method for screening bacteria: Aerobically degrading chlorinated short-chain hydrocarbons[J]. Curr Microbiol, 1987, 15(3): 159-163.
[17]	Lee SD, Kim ES, Hah YC. Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences[J]. FEMS Microbiol Lett, 2000, 182(1): 125-129. pmid: 10612743
[18]	张涛, 张锦, 史艳可, 等. 产酸克雷伯氏菌胞内粗酶液降解泰乐菌素[J]. 生物学杂志, 2022, 39(5): 65-71, 76.
	Zhang T, Zhang J, Shi YK, et al. Biodegradation of tylosin by intracellular crude enzyme solution of Klebsiella oxytoca[J]. J Biol, 2022, 39(5): 65-71, 76.
[19]	曹卫承. 三氯乙烯高效降解菌的筛选鉴定及降解特性研究[J]. 科技创新与应用, 2023, 13(36): 59-64.
	Cao WC. Screening, identification and degradation characteristics of trichloroethylene efficient degrading bacteria[J]. Technol Innov Appl, 2023, 13(36): 59-64.
[20]	De Marines F, Cruciata I, Di Bella G, et al. Degradation of 1, 2-dichloroethane in real polluted groundwater by using enriched bacterial consortia in aerobic and anaerobic laboratory-scale conditions[J]. Int Biodeterior Biodegrad, 2023, 183: 105644.
[21]	van den Wijngaard AJ, Wind RD, Janssen DB. Kinetics of bacterial growth on chlorinated aliphatic compounds[J]. Appl Environ Microbiol, 1993, 59(7): 2041-2048.
[22]	Torz M, Wietzes P, Beschkov V, et al. Metabolism of mono- and dihalogenated C1 and C2 compounds by Xanthobacter autotrophicus growing on 1, 2-dichloroethane[J]. Biodegradation, 2007, 18(2): 145-157.
[23]	Taylor DG, Bouwer EJ. Effect of CO₂ on oxidation of 1, 2-dichloroethane by Xanthobacter autotrophicus GJ10[J]. Environ Eng Sci, 2009, 26(11): 1577-1583.
[24]	Baptista IIR, Peeva LG, Zhou NY, et al. Stability and performance of Xanthobacter autotrophicus GJ10 during 1, 2-dichloroethane biodegradation[J]. Appl Environ Microbiol, 2006, 72(6): 4411-4418.
[25]	Hunkeler D, Aravena R. Evidence of substantial carbon isotope fractionation among substrate, inorganic carbon, and biomass during aerobic mineralization of 1, 2-dichloroethane by Xanthobacter autotrophicus[J]. Appl Environ Microbiol, 2000, 66(11): 4870-4876.
[26]	vander Ploeg J, van Hall G, Janssen DB. Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene[J]. J Bacteriol, 1991, 173(24): 7925-7933. pmid: 1744048
[27]	Hage JC, Van Houten RT, Tramper J, et al. Membrane-aerated biofilm reactor for the removal of 1, 2-dichloroethane by Pseudomonas sp. strain DCA1[J]. Appl Microbiol Biotechnol, 2004, 64(5): 718-725. pmid: 15034684
[28]	Hage JC, Kiestra FD, Hartmans S. Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1[J]. Appl Microbiol Biotechnol, 2001, 57(4): 548-554. pmid: 11762603
[29]	Preussger D, Giri S, Muhsal LK, et al. Reciprocal fitness feedbacks promote the evolution of mutualistic cooperation[J]. Curr Biol, 2020, 30(18): 3580-3590.e7. doi: S0960-9822(20)30986-6 pmid: 32707067
[30]	D’Souza G, Shitut S, Preussger D, et al. Ecology and evolution of metabolic cross-feeding interactions in bacteria[J]. Nat Prod Rep, 2018, 35(5): 455-488. doi: 10.1039/c8np00009c pmid: 29799048
[31]	D'Souza G, Kost C. Experimental evolution of metabolic dependency in bacteria[J]. PLoS Genet, 2016, 12(11): e1006364.
[32]	Oliveira NM, Niehus R, Foster KR. Evolutionary limits to cooperation in microbial communities[J]. Proc Natl Acad Sci USA, 2014, 111(50): 17941-17946. doi: 10.1073/pnas.1412673111 pmid: 25453102
[33]	Foster KR, Bell T. Competition, not cooperation, dominates interactions among culturable microbial species[J]. Curr Biol, 2012, 22(19): 1845-1850. doi: 10.1016/j.cub.2012.08.005 pmid: 22959348
[34]	Coyte KZ, Schluter J, Foster KR. The ecology of the microbiome: networks, competition, and stability[J]. Science, 2015, 350(6261): 663-666. doi: 10.1126/science.aad2602 pmid: 26542567