Isolation,Identification And Biodegradation Characteristics Of PAH-degrading Bacteria

Five pyrene-degrading bacterial strains were isolated from paddy soil sampling in Fuzhou County of Minhou.1)Five strains were characterized and identified based on Gram staining,biochemical tests and 16S rDNA gene sequence analysis 2)The pyrene-degrading consortium was further analyzed to determine the predominant bacteria responsible for pyrene degradation.3)The impact of artificially added pyrene as PAHs pollution was Investigated on soil microorganism.4)Biodegradation characteristics of identified strains were studied in liquid cultures to select high efficient pyrene-degrading strains.5)The kinetic biodegradation process of high efficient pyrene-degrading strains was studied Based on microorganism growth models and substrate consumption models.The main experiments results are summarized as follows:(1)The pyrene-degrading consortium was screened from soil sample and five pyrene degrading stains(designated as PyB-02,PyB-06,PyB-09,PyB-10 and PyB-13)were isolated from pyrene-degrading consortium.PyB-02 and PyB-13 were identified as species of Rhizobium and Sphingomonas respectively.PyB-09 were characterized and identified as a novel strain of the genus Bacillus,based on Gram staining,biochemical tests and 16S rDNA gene sequence analysis.Two high efficient pyrene-degrading strains,PyB-06 and PyB-10,were identified as species of Escherichia and Agromyces respectively.This is the first report of a representative of the genus Agromyces and wild Escherichia capable of degrading PAHs with such degradation efficiency.The pyrene-degrading consortium was further analyzed by PCR-DGGE indicating that PyB-02,PyB-06,PyB-13 consisted of the main bacterial population and PyB-06 was the predominant bacteria responsible for pyrene degradation.(2)The impact of artificial simulation experiment added pyrene as PAHs pollution was Investigatsd on soil microorganism found that the total PLFAs content of soil added pyrene were higher than that of control soil over the first 15days and lower than that of control soil at 30days.The PLFAs of bacteria,gram positive bacteria,gram negative bacteria,fungi and actinomycetes had the similar trend during incubation.The bacterial PLFAs accounted for 79%-83%of the total PLFAs in the samples,indicating that the soil microbial community was dominated by bacteria,followed by actinomycetes and fungi.The addition of pyrene finally changed the structure of soil microbial community PLFAs and the change continued over time.The analysis of PCR-DGGE showed that PyB-02,PyB-06,Pyb-09 and PyB-13 were the dominant species of indigenous bacterial communities in tested soil.During culture time,the status of the above four strains continue to change and to the end of the incubation,PyB-06 and PyB-02 were still the dominant species in bacterial community structure in soil added pyrene.Compared with the control soil,the bacterial community structure of soil added pyrene did not change significantly at 1day and 7days.However,bacterial community structure had changed a lot at 15days and 30days.Bacterial community structure had most changes at 15days when the diversity index is the lowest and evenness index is higher than that of control soil.At the end of incubation,the diversity index of soil added pyrene compared with the control soil had no significant change,but the evenness index decreased significantly.(3)Biodegradation characteristics of identified strains were studied in liquid cultures containing 50 mg/L of pyrene.Strain PyB-06 and PyB-10 can remove 48mg/L of pyrene in 12 days and 47.5mg/L of pyrene in 15 days respectively.There was no significant difference between the two strains to degrade pyrene in the concentration of 50mg/L,which was significantly higher than that of strain PyB-09 removing l0mg/L of pyrene in 15 days.Strain PyB-06 and PyB-10 can remove 57mg/L and 72mg/L of pyrene in 23 days respectively.Therefor strain PyB-10 has higher degradation efficiency.Compared with the the previous reportes,PyB-06 and PyB-10 have a high degradation efficiency of pyrene and the strain PyB-10 has higher degradation ability and the tolerance range of pyrene.When supplemented with 50mg/L of pyrene,the degradation curve of strain PyB-06 and PyB-10 on pyrene showed an excellent fit to Logistic model and Quiroga-Sales-Romero model,and the degradation curve of strain PyB-09 fitted Quiroga-Sales-Romer model.When supplemented with 50mg/L of pyrene,the degradation curve of strain PyB-06 and PyB-10 accorded with the Logistic model and First order model.Therefor the kinetic model that best represents the behavior observed in 50mg/L and 100mg/L pyrene of biodegradation is the Logistic model for comparable and convenient.According to the degradation characteristics of isolated strains,graphical calculation of the values of the lag time(tL)and time starting from the end of the lag phase to reach 50%of biodegradation(t50)were introduced in research.The values of tL and t50 were calculated in accordance with Logistic model to compare degradation characteristics of strains PyB-06 and PyB-10 in different concentrations of pyrene.The results showed that tL of the two strains calculated under the condition of 50mg/L pyrene were significantly less than tL calculated in the concentration 100mg/L pyrene.So during the earlier stage of incubation,PyB-06 and PyB-10 have a higher degradation rate in the high.concentration(100mg/L)of pyrene.The values of t1/2 was calculated in accordance with Logistic model,showed that the degradation rate of strain PyB-10 in liquid cultures containing 100mg/L of pyrene was significantly higher than that in liquid cultures supplemented with 50mg/L of pyrene.Indicating that in a certain degree,degradation rate of strain PyB-10 increased with the increasing concentration of pyrene.