Characterization Of Microbial Communities And The Mechanisms Underlying The Anaerobic Reductive Dehalogenation Of Tetrabromobisphenol A

Tetrabromobisphenol A(TBBPA)is the most widely used brominated flame retardant all over the world.It often accumulates in anaerobic areas such as deep soil,river or lake sediments resulting from the characteristics of durability,high hydrophobicity and heavier than water.Researchers have detected TBBPA in water,soil,river sediments and even organisms such as fish,birds and human tissues.TBBPA affects the secretion of thyroxine,induces stress response,inhibits immune activity and has been classified as a potential human carcinogen(group 2 A),posing a serious threat to the health of animals,plants and humans.The clean-up of TBBPA includes physical,chemical and microbial degradations while the microbial degradation has the advantages of low cost,little impact on the environment,and no secondary pollution.TBBPA is recalcitrant under aerobic conditions while,it can be reductively dehalogenated to a more bioavailable metabolite,bisphenol A,which can be readily degraded by bacteria,fungi,algae and even higher plants under aerobic conditions.However,due to the extremely low water solubility of TBBPA,the low growth rate of anaerobic bacteria and the difficulty of molecular genetic manipulation,the discovery of organohalide-respiring bacteria responsible for TBBPA dehalogenation and the mechanism of reductive dehalogenation remain poorly understood.The process of microbial reduction dehalogenation mediated by humin remains unclear.Based on this,this thesis studied the key microorganisms and dehalogenation mechanism,and initially discussed the mechanism of humin in the electron transfer process during the reductive dehalogenation of tetrabromobisphenol A.The main results are as follows:1.Enrichment and characterization of tetrabromobisphenol A dehalogenating consortia.Through the anaerobic enrichment method,an enrichment that can continuously reductively dehalogenate TBBPA to bisphenol A was obtained.The anaerobic reductive dehalogenation of TBBPA relies on solid humin.The electrons produced by the electron donor(hydrogen)cannot be directly transferred to the reductive dehalogenase through the electron transport chain on the cell membrane of organohalide-respiring bacteria,thereby catalyzing the reductive dehalogenation of TBBPA.Instead,the electrons were firstly transfered to the oxidized solid humin,and the reduced humin transfers the electrons to the reductive dehalogenase,and finally catalyzes the reductive dehalogenation of TBBPA.The humin acts as an electron shuttle in the process of reductive dehalogenation of TBBPA.In addition,when sodium lactate was used as the electron donor,the dehalogenation rate of TBBPA is 1.00,1.12,1.30,and 2.23 times higher than that when sodium formate,sodium propionate,ethanol,and sodium acetate served as the electron donors,respectively.The dehalogenated substrate spectrum test of the TBBPA anaerobic enrichment found that the enrichment can also dehalogenated 1,2-dichlorobenzene,1,3-dichlorobenzene,2,4,6-tribromophenol to chlorobenzene and phenol,respectively.Whether the dehalogenation process depends on humus remains to be verified by further experiments.2.Microbial community and the key microorganisms responsible for the anaerobic reductive dehalogenation of tetrabromobisphenol A.Through 16S rRNA gene high-throughput sequencing,the community composition of TBBPA dehalogenating enrichment and its succession rule over time were studied.Sequencing results showed that the initial relative abundance of obligate organohalide-respiring bacteria Dehalobacter was 0.29%±0.05%.With the reductive dehalogenation of TBBPA,after 77 days,it increased to 8.29%±1.70%.Other microorganisms that stably exist in the community also play an indispensable role in the dehalogenating respiration process.For example,Desulfovibrio has the ability to synthesize cobalamin de novo and to ferment sodium lactate to produce hydrogen which be used by Dehalobacter directly.Although Methanosarcina competes electrons with Dehalobacter,it can also cooperate with organohalide-respiring bacteria through co-metabolism,providing necessary growth factors for organohalide-respiring bacteria.The existence of these microorganisms accelerates the electron transfer and the synthesis of amino acids in the microbial community,and provides de novo synthesis and salvage pathways for the cofactor cobalamin in reductive dehalogenase.In addition,using the Taqman probe method for q PCR experiments,it was found that during the reductive dehalogenation of TBBPA,Dehalobacter increased from 1.31±0.41×10~6 copies/ml culture to 7.63±2.96×10~6 copies/ml culture,and the yield was 6.60±1.60×10~4 copies/(μmol released Br~-).Combined with high-throughput sequencing results,it can be clear that Dehalobacter were the key organohalide-respiring bacteria responsible for the reductive dehalogenation of TBBPA.3.Metagenomic sequencing of TBBPA dehalogenating enrichment.Through metagenomic sequencing technology,a genome draft of Dehalobacter was obtained.The draft genome is relatively complete(96.88%)and the contamination is low(6.95%).The closest relative species at the phylogenetic level is Dehalobacter restrictus,with average nucleotide identity of 94.77%.According to the results of RAST annotations,the genome encodes 34 reductive dehalogenases(Rdh A),of which 23 dehalogenase genes(rdh A)have genes encoding transmembrane anchored proteins rdh B in their upstreams.The function of Rdh B is to anchor the reductive dehalogenase outside the cell membrane.The phylogenetic analyzes of Rdh A from Dehalobacter genome implied that there were new unknown function Rdh A encoded in the assembled Dehalobacter genome.