Screening And Detection Of Polycyclic Aromatic Hydrocarbons Degrading Bacteria And Their Degradation Characteristics Of PAHs In Soil

Polycyclic aromatic hydrocarbons(PAHs),due to their high toxicity,hydrophobicity,and resistance to degradation,exhibit a strong"three-factor effect",and have gradually become a hot spot of widespread concern.Studies have shown that there are varying degrees of polycyclic aromatic hydrocarbon pollution in the soil in many areas of our country.Among them,the pollution of polycyclic aromatic hydrocarbons in the soil near oil extraction areas is particularly serious.Therefore,how to effectively control the soil contaminated by polycyclic aromatic hydrocarbons has become an urgent problem to be solved in the current ecological construction.Microorganisms play an important role in the bioremediation of polycyclic aromatic hydrocarbons due to their large numbers,wide distribution and rich diversity.Different types of microorganisms have different carbon sources.The selection of degrading strains from the contaminated system is the key link for the implementation of microbial remediation technology.In this paper,L9 was used as the culture medium,typical PAH pyrene was used as the sole carbon source,and 5 PAH-degrading bacteria were screened from the oil-contaminated soil in northern Shaanxi using traditional enrichment culture and plate streaking method(serial number:MT065748～MT065752).Physical and chemical identification of 5 strains and 16S rDNA sequence analysis were carried out.The results showed that the 5 strains selected were all Gram-positive bacteria,of which 4 were Bacillus and 1 was Myroides.All 5 strains and mixed strains can degrade pyrene in the liquid phase.The degradation efficiency of mixed strains is better than that of single strains,and the degradation rate of 50 mg/L pyrene reached 43.1% after 35 days of degradation.The addition of glucose as a co-metabolism matrix promoted the degradation of pyrene.The degradation rate of pyrene by the five mixed bacteria in the presence of glucose reached 55.2%.A method for rapid detection of active PAH-degrading bacteria in the liquid-phase degradation system was established by flow cytometry.Use SG/PI double staining method for staining,use FL1 and FL3 channels,the cell flow rate is 33 mL/min,the counting rate is kept below 1000 cells/s,the threshold is set to 600,the total number of degrading bacteria is determined,and the resulting degradation is The correlation coefficient between the number of bacteria and the plate counting method was 0.9742～0.9962.Use NaClO to inactivate the bacteria to divide the negative control area,and determine the upper right area as the dead bacteria area according to the position of the positive and negative controls in the flow cytometer detection panel,and the lower right area as the live bacteria area,which is determined by dividing the area The number of active bacteria.The results of the degradation experiment of pyrene in the liquid phase showed that the number of active degrading bacteria in each system increased after 7 days of degradation,and the number of mixed bacteria reached the maximum,which was 4.26×10~8 cells/mL.After that,it decreased and stabilized at 21 d,and the number of active bacteria was 1.54×10~8 cells/mL.The addition of glucose matrix has a clear promotion effect on the growth of PAHs degrading bacteria;the viable number of mixed bacteria reached 4.53×10~8 cells/mL after 7 days of degradation.Degrading bacteria can use polycyclic aromatic hydrocarbon pyrene as a carbon source for growth and metabolism.The correlation analysis between the total number of bacteria and the number of viable bacteria at 7 days of degradation shows that the total number of bacteria in each system is significantly positively correlated with the number of viable bacteria,and the total number of bacteria As the number of viable bacteria increases.The artificially prepared PAHs-contaminated soil and oil-contaminated soil were inoculated with mixed degrading strains for restoration treatment for 90 days.The degradation rates of PAHs in the soil reached 28.1%,40.5%,35.0%,and 16.6%,respectively.The degradation rate of PAHs in the soil without degrading bacteria was 20.0%,27.8%,26.1%and 9.4%.Inoculation of mixed degrading bacteria could promote the degradation of PAHs and total petroleum hydrocarbons in the soil.When glucose was added as a co-metabolic substrate,the degradation rates of PAHs in the soil were 29.2%,45.6%,39.6%,and 17.7%,respectively.Adding glucose as a co-metabolism matrix can promote the degradation of PAHs.In the initial stage of remediation(0-45 d),the degradation rate of the pollutants in each degradation system is relatively large,and gradually slows down with the extension of the remediation process,and finally stabilizes.The results of sterilization and unsterilized inoculation restoration experiments on artificially prepared PAHs contaminated soil(100 mg/kg)showed that there is no competition and synergy between indigenous microorganisms and foreign bacterial sources.The results of functional degradation gene determination showed that the more functional polycyclic aromatic hydrocarbon degradation genes in the soil,the better the degradation effect of PAHs.The changes of microbial activity in artificially prepared contaminated soil were analyzed,and the results showed that the metabolic activities of soil microbes all increased with the extension of the culture time within 0-24 h,and remained at a stable and high level thereafter.When bio-enhanced remediation of polycyclic aromatic hydrocarbons contaminated soil for 60 days,when polycyclic aromatic hydrocarbons are used as a substrate for metabolism:100 mg/kg pollution concentration of soil microbial activity>200 mg/kg pollution concentration of soil microbial activity>500mg/kg pollution concentration of soil microbes Activity,indicating that the degradation of PAHs has a certain correlation with the activity of degrading strains.