Degradation Characteristics And Proteomic Study Of Anthracene By A Petroleum Source Bacillus Firmus

Oil pollution caused by human production activities poses a serious threat to the environment and ecosystems.Among the main components of petroleum hydrocarbons,polycyclic aromatic hydrocarbons(PAHs)in aromatic hydrocarbons are the most harmful.Microorganisms have the advantages of high efficiency,environmental friendliness and low cost in degrading PAH pollution.Although many strains that can degrade PAHs have been screened and isolated,the degradation mechanism of PAHs by strains remains to be explored..In this study,a strain of Bacillus firmus isolated from petroleum sources was firstly selected,and the growth kinetics of the strain was firstly analyzed to determine the initial degradation rate of four PAHs:naphthalene,anthracene,phenanthrene and fluorene.Strong PAHs-anthracene,strain domestication experiments were carried out to determine the degradation rate of anthracene by strains after domestication and analyze the degradation kinetics.The intermediate products of anthracene degradation were determined by GC-MS,and the molecular change mechanism of the strain during the degradation process was analyzed by i TRAQ proteome,and the mechanism of anthracene degradation by the strain was analyzed,providing theoretical reference and help for the microbial remediation of PAHs..The results of the PAHs screening experiments showed that:(1)Comparative analysis showed that the SGompertz model could better fit the growth curve of Bacillus firmus in LB medium at 30°C and 150 rpm.The error is small;(2)After 7 days of incubation at 30?and 150 rpm,Bacillus firmus was able to degrade naphthalene,anthracene,phenanthrene and fluorene as the sole carbon source and energy source,respectively,and the degradation rates of naphthalene(50 mg/L),anthracene(50 mg/L),phenanthrene(50 mg/L)and fluorene(50 mg/L)were 54.68%,60.64%,53.89%and44.28%,respectively,and the bacterial growth increased by 7.7,11.77,7.77 and 7.26times,and the combination of growth and degradation rate concluded that Bacillus firmus was effective in the degradation of green onion.The results of anthracene degradation experiments showed that:(1)The domesticated Bacillus firmus could degrade 100 mg/L anthracene well under the culture conditions of 30?and 150 rpm.After the adaptation period,the growth amount and degradation rate were significantly improved.After 72 h of culture,the growth amount of the strain reached 0.98,and the degradation rate of anthracene reached 84.41%.In the blank control group without bacteria,these indicators basically(2)The increase of extraction times and concentration and dehydration steps can effectively improve the recovery rate of anthracene in the pretreatment operation.Among them,n-hexane is used as the extractant for 3 times of extraction,and the recovery rate of the operation sample that is concentrated and dehydrated after merging the organic phases reaches 91.02%,which meets the requirement of sample analysis recovery rate.(3)Kinetic analysis of the degradation of anthracene by Bacillus firmus showed that the kinetic model of the degradation of anthracene by Bacillus firmus was in good agreement with the first-order kinetic model,and the rate constants k and R2of anthracene degradation were-0.117 and 0.982,respectively.The degradation kinetics equation is:C=100×e^0.11755t(R~2=0.982);(4)According to the analysis results of intermediate products,it is inferred that the metabolic pathway of Bacillus firmus degrading anthracene is as follows:The enzyme secreted by Bacillus firmus first oxidizes anthracene to anthracene-9,10-dihydrodiol is then dehydrogenated to form dihydroxyanthracene,C9 and C10 are oxidized to form anthraquinone,and the ring is cleaved to form phthalic acid.Phthalic acid is converted into benzoic acid,and finally metabolized by pyruvate to form small molecular substances that enter the TCA cycle and metabolize to CO₂.The results of proteomic analysis can preliminarily determine the anthracene metabolism mechanism of the strain,that is,in the process of anthracene degradation,the ABC transporter family mediates the transmembrane transport of anthracene to maintain the intracellular physiological activity,and the periplasmic dipeptide ABC transporter Dpp A(ead?OV14?1655)and flagellar motor protein Mot B(mdi?METDI3141)maintain the balance between degrading anthracene and being poisoned by anthracene.When anthracene enters the cell,dioxygenase 1,2-dioxygenase(cti?RALTA?B1481)first catalyzes anthracene The aromatic epoxidation produced dihydrodiol,which was unstable and rapidly converted into anthraquinone,which was subsequently processed by a series of aldol dehydrogenase(mch?Mchl?4576),hydrolase(meu?ACJ67?14400),lyase(nwi?Nwi?2929)The lower ring is cleaved to form phthalate,which is finally metabolized to pyruvate,which is converted into acetyl-Co A under the action of pyruvate dehydrogenase(meh?M301?0669,saga?M5M?05837,mea?Mex?1p2986)and malate dehydrogenase(mch?Mchl?5152).The citric acid cycle(TCA cycle)produces energy and synthesizes NADH.