The Anaerobic Reductive Dechlorination Of 4-Hydroxychlorothalonil And Analysis Of The Associated Functional Microbial Communities And Metagenomics

4-Hydroxychlorothalonil(TPN-OH)is the main metabolite of chlorothalonil,a kind of organochlorine bactericide,via hydrolysis and dechlorination in the environment.4-Hydroxychlorothalonil is more toxic to fish,algae and other animals and plants than its parent chlorothalonil because of the higher water solubility.In addition,the residues of 4-hydroxychlorothalonil have serious damage for ecological environment and human health.4-Hydroxychlorothalonil is difficult to be biodegraded,leading to the higher persistence and stability in the natural environment.So far,there have been no reports on the microbial degradation of 4-hydroxychlorothalonil.This thesis aimed at the problem of 4-hydroxychlorothalonil riduces in deep soil,groundwater and other anaerobic environments,so we enriched anaerobic microorganism responsible for the reductive dechlorination of 4-hydroxychlorothalonil.We researched the reductive dichlorination mechanism and the characteristics of 4-hydroxychlorothalonil by the bacterial consortium.Additionally,we revealed the species composition and community structure of the dechlorination bacterial consortium.Finally,the reductive dechlorination functional genes from this consortium were analysed.The main results are as follows.1.Characteristics of the anaerobic 4-hydroxychlorothalonal-dechlorinating enrichment.By inoculating a 1,2,4-trichlorobenzene dechlorinating enrichment,we obtained the bacterial consortium capable of reductively dechlorinating 4-hydroxylchlorothalonil after continuous feedings of 4-hydroxylchlorothalonil.The optimal temperature and pH of reductive dechlorination of 4-hydroxychlorothalonil were 30℃ and 8.0,respectively.The dechlorination efficiency of 4-hydroxylchlorothaloni reached 82.13% within 14 days when sodium acetate served as an electron donor.Via the analysis of the LC-MS and the interaction force between the benzene ring substituent groups,it was found that the chlorine atom at position 6 of 4-hydroxychlorothalonil was substituted by hydrogen atom(Reductive dechlorination).2.Microbial communities of the 4-hydroxychlorothalonil dechlorinating enrichment.The 16 S rRNA genes amplicon paired-end sequencing was used to analyze the composition and changes of the bacterial community structure in the process of 4-hydroxychlorothalonil reductive dechlorination.After the addition of 0.08 m M 4-hydroxychlorothalonil,the abundance of the obligate dehalogenated respiratory bacteria,Dehalogenimonas,gradually increased from 7.01% to 18.98%.The qPCR results showed that the quantity of 16 S rRNA genes of Dehalogenimonas increased from 7.01% to 18.98% copies/m L.Combined the results of qPCR experiment and 16 S rRNA gene sequence,Dehalogenimonas was determined as the main bacterial genus,which was responsible for reductive dechlorination of 4-hydroxychlorothalonil(organohalogen dehalogenation respiration).In addition,in the late stage of enrichment culture,anaerobic Clostridium also occupied a dominant position in the communities,increased from 5.92% to 14.25%,while the abundance of Methanobacterium has reduced to 5.14%.Therefore,Clostridium had more obvious advantages than Methanobacterium in the mixed communities,and it could provide the hydrogen and other potential cofactors for Dehalogenimonas to reduce 4-hydroxychlorothalonil.3.Metagenomic analysis of the 4-hydroxychlorothalonil dechlorinating enrichment.The separation and purification of organohalide-respiring bacteria are difficult due to its harsh anaerobic culture conditions and slow growth rate.Metagenomic sequencing was used to conduct a deeper analysis for the dechlorination community.In the process of reductive dechlorination of 4-hydroxychlorothalonil,the abundance of clusters of orthologous groups(COG)related to cell division(D),cell synthesis(M)and energy conversion(C)increased by 1-2 times,and the corresponding functions of COG were significantly enhanced.But the abundance of COG related to transcription(K)and secondary metabolite synthesis(Q)significantly reduced,and the corresponding functions of COG gradually weakened.The abundance of a variety of enzymes(6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase,2-furan-CoA dehydrogenase,aromatic alcohol dehydrogenase,formaldehyde dehydrogenase,homogentisic acid 1,2-dioxygenase,4-hydroxybenzoic acid 3-monooxygenase,non-specific monooxygenase,homogentisic acid 1,2-dioxygenase,6,7-dihydropteridine reductase,benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase)increased by 1.5-6 times through the KEGG function annotation analysis.Combined with blast analysis,it was predicted that the key reductive dechlorinase gene(trdH)was responsible for the reductive dechlorination of 4-hydroxychlorothalonil.