Resistance And Degradation-Adsorption Mechanisms Of Bacillus Sp.M1 To Pyrene And Benzo[a]pyrene

Microbial remediation is a green and efficient method for the degradation of Polycyclic Aromatic Hydrocarbons(PAHs).It has been found that there are many strains degrading High Molecular Weight PAHs(HMW-PAHs),Bacillus sp.It has an important role in the degradation process due to its spore-forming properties and special functions.The Bacillus sp.M1 degrading HMW-PAHs,which was preferred in the previous group,had a removal rate of 5 ?g/mL benzo[a]pyrene in the culture solution as high as 90.13%.However,the strain was tolerant to the maximum concentration of HMW-PAHs,the way in which HMW-PAHs enter the cell(biodegradation and adsorption distribution)and the transmembrane transport mechanism are still unclear.Based on this,this paper takes Pyrene(Pyr)and benzopyrene(BaP)monomers as research objects,and Bacillus sp.M1 as a degrading strain to explore the tolerance of Pyr and BaP,and in-depth analysis of the dynamic changes of biodegradation and biosorption of Pyr and BaP monomers by M1,Combined qPCR technology to analyze the expression of functional gene degradation PAHs,and to explore the degradation mechanism of Pyr and BaP by M1.The main findings are as follows:1.The tolerance of Pyr and BaP in Bacillus sp.M1 was confirmed by high concentration PAHs stress test.M1 grew well in 200 ?g/mL Pyr and BaP inorganic salt solution under the stress of Pyr and BaP at 5 ~ 200 ?g/mL.Bacillus sp.M1 was highly tolerant to 200 ?g/mL Pyr and BaP in inorganic salt and LB medium.The removal rates of Pyr and BaP in inorganic salt culture were as high as 17.74% and 29.98%,respectively;the removal rates of Pyr and BaP in LB medium reached 45.52% and 91.99%,respectively.Further analysis of intracellular enzymes revealed that the intracellular enzyme activity associated with the removal of Pyr and BaP was more active in the LB medium than the inorganic salt medium.And the removal of Pyr and BaP may be related with the activity of catechol-1,2-dioxygenase and gentisic acid dioxygenase in different time periods.2.The dynamic changes of M1 degradation and adsorption of Pyr and BaP were studied by the degradation and adsorption tests of Pyr and BaP by Bacillus sp.M1.The biodegradation rate of Pyr and BaP in the culture cycle reached the peak at 120 h,which was 49.97% and 65.02%.The biosorption rate reached the peak at 168 h and 72 h,respectively,which was 19.72% and 23.53%.The removal of Pyr and BaP by M1 was carried out by biosorption and biodegradation.When the cell adsorption reached saturation,the degradation was dominant,and the equilibrium adsorption amount was 1.4929 ?g/mL and 1.7549 ?g/mL,respectively.3.The transmembrane transport mechanism of Pyr and BaP was elucidated by low temperature and sodium azide inhibition experiments.4 °C low temperature(inhibition of bacterial metabolism)and sodium azide inhibitor(inhibition of ATP anabolic)treatment significantly prevented the entry of Pyr into the M1 cell.It could be considered that the way of Pyr entering the cell was energy-dependent active transport.The two inhibition treatments significantly inhibited the degradation of BaP by M1,but did not significantly inhibit the adsorption.It was speculated that BaP enters the intracellular independent energy and was passive transport.4.The expression characteristics of different functional genes under BaP stress were determined by qPCR assay.The relative expression of the five genes of Bacillus sp.M1 on day 1 was higher than that of other time,and the relative expression of RS10855 was better than the other four genes.The relative expression of five genes was consistent with the change of BaP removal rate.The expression of RS10855 gene was consistent with the activity of gentisic acid dioxygenase and its degradation and adsorption characteristics.It was speculated that the activity of gentisic acid dioxygenase is mainly controlled by RS10855 gene,which needs further verification.In conclusion,Bacillus sp.M1 is highly resistant to 200 ?g/mL Pyr and BaP.The removal of Pyr and BaP by M1 is coordinated by biosorption and biodegradation.It was confirmed that the way of Pyr entering M1 cells was active transport.BaP was passive transport,and the removal of BaP may be related to the regulation of five functional genes.This result provides a scientific theoretical basis for the microbial remediation of polycyclic aromatic hydrocarbons.