Study On Mechanism Of Tetrabromobisphenol A Biodegradation By Marine Bacteria Under Aerobic Cometabolic Conditions

Tetrabromobisphenol A(TBBPA)is one of the most widely used brominated flame retardants with large consumption all around the world has deleterious bioaccumulation,persistence,and biotoxicity effects,and has attracted extensive attention.It has been frequently distributed in various environmental compartments including coastal seawater,sediment,and organism.Thus far,prior studies on TBBPA biodegradation have been primarily focused on anaerobic reductive transformation.Aerobic studies have been exclusively focused on terrigenous microorganisms and the degradation mechanism is still lacking.Until now,information on its aerobic biodegradability in marine environments remains unknown.In view of TBBPA will be subjected to long-term aerobic environments before it deposits into anaerobic environments.Moreover,TBBPA generally coexists with various readily-degradable substances.Therefore,a comprehensive understanding of aerobic cometabolic TBBPA biodegradation is of great importance.Accordingly,the aerobic TBBPA-degrading functional marine microbes and the mechanism of TBBPA degradation were systematically investigated in this dissertation on the basis of marine bacterial consortia,pure strain,and functional gene.The following works have been done:Firstly,aerobic cometabolic TBBPA degradation by marine bacterial consortia was carried out.It was found that different substrate amendments enriched coastal marine microbial consortia(with treatments A-H)had TBBPA-degrading capabilities,with a combination of beef extract and peptone as cometabolic substrates yielding higher removal efficiency(approximately 60%after 7 days cultivation)than the others.Furthermore,the microbial community diversity and structure of 120 d enriched TBBPA-degrading microbial consortia with different cometabolic substrate treatments were analyzed using high-throughput sequencing technique.Results showed that Pseudoalteromonas,Alteromonas,Glaciecola,Thalassomonas,and Limnobacter were the dominant genera in enrichment cultures,and might be involved in TBBPA biodegradation.TBBPA degradation intermediates by marine bacterial consortium(with treatment H)were analyzed using HPLC-LTQ-Orbitrap MS and the degradation pathways were proposed,involving ?-scission and debromination routes.The characteristics of aerobic cometabolic TBBPA degradation by marine bacteria were investigated.Two pure strains capable of degrading TBBPA were isolated from an enrichment culture(with treatment H)with beef extract and peptone as cometabolic substrates under aerobic conditions.On the basis of 16S rRNA gene sequence analysis,the two pure strains were identified and termed as Alteromonas macleodii strain GCW and Pseudoalteromonas sp.strain GCY,respectively.TBBPA(10 mg/L)biodegradation by strain GCW and GCY followed pseudo-first-order kinetics.Among which,the degradation rate constants(k)were 0.27 and 0.15 d-1,which corresponded to half-lives(T1/2)of 2.6 and 4.7 days,respectively.Based on the physiological and biochemical analyses,it was found that the extracellular polymeric substances(EPS)could be stimulated with increasing TBBPA concentration,which might account for the alleviation of oxidative damage.The dehydrogenase activity(DHA)of strain GCW and GCY was increased by 3.2 and 1.6 folds when cells were cultivated with TBBPA(10 mg/L),respectively.Moreover,the electron transport system activity(ETSA)was also shown to be an obvious upward trend during cultivation.Based on active species localization,the experiment results showed that both the intracellular and extracellular active species of strain GCW played important roles in TBBPA degradation,while an extracellular process was mainly responsible for TBBPA degradation by strain GCY.Notably,the extracellular TBBPA-degrading active species of strain GCY were composed of enzymatic and nonenzymatic substances.TBBPA degradation routes by marine Pseudoalteromonas isolate under aerobic cometabolic conditions were analyzed.Considering the strain was facultative and the extracellular active species of Pseudoalteromonas sp.strain GCY mainly participated in TBBPA degradation,strain GCY was chosen as typical marine bacterium to further investigate the nonenzyamtic degradation process in detail.Based on CAS assay,reactive oxygen species(ROS)probe,and electron spin resonance(ESR)spectroscopy techniques,it was found that siderophore,hydrogen peroxide(H2O2),superoxide anion free radical(O2·-),and hydroxyl radical(·OH)were present in the extracellular fraction of this strain.Of which,the extracellular non-enzymatic low molecular mass component was capable of reducing Fe(?)and complexing Fe(?);the generation of extracellular H2O2 was derived from L-amino acid oxidase(LAAO)-catalyzed reaction;the formation of extracellular O2·-was associated with NAD(P)H oxidoreductase;the Fe(?),siderophore,O2·-,and H2O2 contributed to extracellular·OH generation.The intermediates of TBBPA biodegradation by strain GCY were analyzed using high-resolution HPLC-LTQ-Orbitrap MS,and the debromination and ?-scission were the main TBBPA degradation routes.The formation of 2,4,6-tribromophenol(TBP)as an intermediate implied reactive bromine species(RBS)formation during TBBPA degradation.The mechanism of extracellular TBBPA degradation by Pseudoalteromonas isolate was studied.Functional annotation analyses revealed that lysine-epsilon oxidase antimicrobial protein LodA(lodA gene)and Na+-translocating NADH:ubiquinone oxidoreductase(nqrA gene)coding for extracellular H2O2 and O2·-production were present in the draft genome of strain GCY.RT-qPCR analyses showed that the lodA gene expression was 61.0 and 93.4-folds up-regulated by incubating cells with/without TBBPA(10 mg/L)compared to the unexposed control,respectively.Meanwhile,the nqrA gene expression was separately 1.7 and 2.1-folds up-regulated when cells were incubated with/without TBBPA.These results confirmed that the lodA and nqrA genes were responsible for extracellular H2O2 and O2·-generation,respectively.On the basis of the conclusions and thermodynamical analyses,three principal mechanisms in TBBPA degradation via extracellular biogenic ROS by marine Pseudoalteromonas sp.isolate were proposed as follows:(?)Biogenic O2·-debrominated TBBPA directly.(?)Biogenic Fenton-like reaction:Fe(?)/Fe(?)-siderophores cycling by H2O2 could result in TBBPA degradation via Fenton-like reaction.(?)Biogenic Haber-Weiss mechanism:reaction of O2·-and H2O2 with Fe(?)/Fe(?)as catalyst could drive Haber-Weiss cycle and responsible for TBBPA degradation.Moreover,similar extracellular degradation mechanism was found in terrigenous microbes.Thus,aerobic cometabolic extracellular TBBPA degradation by a terrigenous bacterium(Pseudomonas sp.fz)was investigated.A biogenic Fenton-like reaction mediated via extracellular H2O2 and Fe(?)-oligopeptide complexes in TBBPA degradation by strain fz was proposed.Membrane integrity assays were carried out.Laser scanning confocal microscopy results showed that the membranes of strain fz were disrupted in the TBBPA-amended group,indicating that extracellular ROS could facilitate extreme cell membrane damage upon prolonged cultivation periods in the presence of TBBPA.Flow cytometry results showed that the increase of intracellular catalase(CAT)activity and the extracellular polymeric substances might account for the alleviation of oxidative damage of strain GCY.Based on the comparison of the extracellular degradation mechanism by marine bacterium GCY and terrigenous bacterium fz,it was found that the type of siderophore,pH variation of the incubation system,O2·,source of bromine ion and cell membrane integrity were the main difference.As for the common feature of marine and terrigenous bacteria,TBBPA was aerobically degraded via cometabolic process,the degradation was occurred by extracellular fraction,and TBBPA was degraded by extracellular active species(siderophore and ROS)via Fenton-like reaction.Collectively,all above results in this dissertation showed that TBBPA could be biodegraded by marine bacteria under aerobic cometabolic conditions.Meanwhile,a novel mechanism of extracellular cometabolic TBBPA degradation was found.These findings revealed that the extracellular ROS played important roles in the fate of this compound in the coastal environments.This study could provide insight into the aerobic biodegradation mechanism of TBBPA and other related organic pollutants in coastal and artificial bioremediation systems.Also,it could provide theoretical guidance for the development of novel and efficient bioremediation approaches.