| Chlorobenzoates are important organic compounds,which are widely used as intermediates for pesticides,pharmaceuticals,preservatives,dyes,coating and fungicide.Chlorobenzoates are intermediate metabolites of the degradation of various chlorinated aromatic compounds(including organochlorine pesticides)and considered to be refractory compounds like other chlorinated compounds such as chlorobenzene and organochlorine solvents.Chlorobenzoates are highly water-soluble,so they can easily permeate into the ground with the rainwater and cause groundwater pollution.Chlorobenzoates are often detected in the environment.Although microbial degradation of chlorobenzoate has been reported,the studies generally include only certain chlorobenzoate,but the degradation of multiple chlorobenzoates in one same chlorobenzoates contaminated soil has not been studied.Therefore,the screening of chlorobenzoate degrading strains to rich the resources of degrading strains,exploring the degradation of genes and pathways have great significance for repairing chlorobenzoate contaminated soil.The purpose of this study is to reveal the microbial community distribution and the genes and pathways involved in the degradation of pollutants in situ chlorobenzoates contaminated soils;isolating the chlorobenzoate degrading strains,researching degradation characteristics;cloning of diverse gene clusters involved in the chlorobenzoates degradation;further revealing the function of the gene and the complete pathways of chlorobenzoate degradation to provide the theoretical basis and enriching resources for the repair of chlorobenzoates.Soil metagenome data were obtained by sequencing soil metagenome DNA.KEGG PATHWAY analysis of soil samples revealed some degradation pathways,which are inferred to be responsible for chlorobenzoates degradation.Gene cluster benABCD and gene cluster catAB are invovled in the pathway in the degradation of 3-chlorobenzoate,gene cluster cbdABC is invovled in the pathway in the degradation of 2-chlorobenzoate,gene cluster fcbABC is invovled in the degradation pathway of 4-chlorobenzoate,suggesting that there are abundant cholrobenzoate-degrading microbial resources as well as diverse cholrobenzoates metabolic pathways in this abandoned chemical plant soils.Three chlorobenzoate-degrading strains(strain 2-CBA,strain 3-CBA and strain 4-CBA)were isolated from this soil contaminated with chlorobenzoates by continuous subculture method.Strain 2-CBA was able to degrade 2-chlorobenzoate,2-bromobenzoate,3chlorobenzoate and 3-bromobenzoate.Strain 3-CBA was able to degrade 3-chlorobenzoate and 3-bromobenzoate.Strain 4-CBA was able to degrade 4-chlorobenzoate and 4bromobenzoate.The strain 2-CBA and strain 3-CBA were identified as Pseudomonas sp,strain 4-CBA was identified as Hvdrogenophaga sp.according to the colony morphology and 16S rRNA gene sequence analysis.At the same time,a new species of Aquamicrobium was isolated from chlorobenzoates contaminated soil.Cells were aerobic,Gram-stain-negative,short rod-shaped,non-sporeforming(approx.2.0 μm long and 0.4 μm wide),oxidase-and catalase-positive.Strain grew well on LB,R2A and TSA agars.Colonies were smooth,beige and circular after incubation for 48 h at 30℃ on LB agar.The optimal growth temperature and pH were 30℃ and pH 7.0.Cells grew in the NaCl concentration range from 0%to 4%,cells grew best in the absence of NaCl.The major cellular fatty acids were C19:0 cyclo ω8c(45.6%)and C18:1ω7c(33.4%).The respiratory quinone was ubiquinone Q-10.The major polar lipids were phosphatidylcholine,diphosphatidylglycerol and moderate amounts of phosphatidylethanolamine,phosphatidylmonomethylethanolamine,aminolipid and phospholipid.DNA G+C content of the type strain is 65.5 mol%.The type strain was identified as Aquamicrobium soli NK-8T(=KCTC 52165T=ACCC AB2016045T).The strain Pseudomonas sp.2-CBA was capable to degrade 2-chlorobenzoate and 3chlorobenzoate and release the same amount of chloride ions at the same time.The strain Pseudomonas sp.2-CBA was able to degrade 0.5 mM 2-chlorobenzoate within 27 h,degraded 0.5 mM 3-chlorobenzoate within 60 h.The optimum temperature for the growth and degradation of 2-chlorobenzoate was 37℃.The optimum pH was 7.0.When the inoculation amount was 5%,the degradation effect reached the highest.1 mM Co2+,Ni2+,Zn2+ and Cd2+ inhibited the degradation of 2-chlorobenzoate.Pseudomonas sp.3-CBA was capable to degrade 0.5 mM 3-chlorobenzoate within 24 h as a carbon source and energy source.The optimum temperature for the growth and degradation of 3-chlorobenzoate was 30℃.The optimum pH was 7.0.When the inoculation amount was 5%,the degradation effect reached the highest.1 mM Co2+,Ni2+,Zn2+and Cd2+inhibited the degradation of 3chlorobenzoate.The strain Hydrogenophaga sp.4-CBA was able to degrade 0.5 mM 4chlorobenzoate within 24 h as a carbon source and energy source.The optimum temperature for the growth and degradation of 4-chlorobenzoate was 30℃ and the optimum pH was 6.0.When the inoculation amount was 5%,the degradation effect reached the highest.1 mM Co2+,Ni2+and Cd2+inhibited the degradation of 4-chlorobenzoate,1 mM Mg2+ promoted the degradation of 4-chlorobenzoate.The genes and pathways involved in the degradation of three kind chlorobenzoates are completely different.Strain 3-CBA converted 3-chlorobenzoate to chlorocated catechol by benzoate 1,2-dioxygenase encoded by the gene cluster benABCD.Chlorinated catechol benzoate ring was cleavaged by gene catA-encoded catechol 1,2-dioxygenase and formed chloromuconate,then the product went into the TCA cycle.Strain 2-CBA converted 2chlorobenzoate to catechol by the combined action of the 2-chlorobenzoate 1,2-dioxygenase subunit encoded by the gene cluster cbdAB and the reductase encoded by the gene benC.Gene catA-encoded catechol 1,2-dioxygenase decomposed catechol into muconate,then the product went into the TCA cycle.Strain 4-CBA converted 4-chlorobenzoate to 4hydroxybenzoate by a hydrolase encoded by the gene cluster fcbABC. |
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