Isolation And Identification Of Benzo[A]Pyrene-degrading Strains And Its Degradation Characteristics In The Gut Of Earthworm(Eisenia Fetida)

Polycyclic aromatic hydrocarbons(PAHs)are typical persistent organic pollutants with widely distributed,biological toxicity and difficult to degrade.PAHs are potentially harmful to human health and ecological environment.The concentration of Benzo[a]pyrene(BaP)in soil has become an important indicator to environmental monitoring.With the in-depth research on PAHs pollution remediation,researchers found that earthworms have a good repair effect on PAHs-contaminated soil and are related to their intestinal microbes.In this study,BaP was used as the target pollutant.BaP-degrading bacteria were isolated and screened from the intestine tract and identified by morphological observation,physiological and biochemical tests and 16S rDNA sequencing analysis.The degradation characteristics of strain in shaker flask degradation system were investigated by single factor experiment,as well as preliminary research on the effect of strains and earthworm on artificial soil remediation of BaP contaminants.The results of the study are as follows:1.Using BaP as the sole carbon source and energy,the BaP-degrading strains were acclimating and isolated from the intestinal tract of earthworm,and their morphological characteristics,physiological and biochemical characteristics,and 16S rDNA identification were performed.The results showed that BaP(8 mg·L-1)was the only carbon source and energy source for acclimating of microorganisms in the intestine after 5 generations of acclimating.Two strains of BaP-degrading bacteria BJ-1 and BJ-7 were isolated by plating dilution method,all were the gram-negative bacteria.And they were identified by morphological observation,physiological and biochemical tests and 16S rDNA sequencing analysis.The strain BJ-1 was identified as Pseudomonas stutzeri,named Pseudomonas stutzeri BJ-1,BJ-7 was identified as an Elizabethkingia endophytica,named Elizabethkingia endophytica BJ-7.2.The degradation characteristics of strains BJ-1 and BJ-7 under different pH values,added nutrients and different concentrations of phenanthrene were explored.The results showed that the optimal degradation pH for P.stutzeri BJ-1 was 7,and the strain still had the ability to degrade BaP at pH5 and pH9,showing its broad spectrum of pH adaptability,for adding nutrients,glucose can promote the adsorption capacity of BaP in strains within 7 days.Starch can reduce the biotoxicity of BaP and maintain high degradation efficiency of Bap.Addition of yeast extract and peptone can significantly inhibit the degradation efficiency of BaP.The addition of inorganic carbon source sodium acetate will continue to inhibit the adsorption capacity of BJ-1,but BJ-1 still efficiently degrading BaP,adding phenanthrene as a co-metabolism substrate for degradation of BaP,showing a significant dose-effect correlation within a certain concentration range,and the strain showed resistance to high concentrations of phenanthrene.For the E.endophytica BJ-7,there is a degradation effect between pH 3 and 11,and the pH value has little effect on the strain degrading BaP,for adding nutrients,glucose,sucrose and starch all have inhibitory effects on BJ-7 degrading BaP,but BJ-7 still haved the high degradation rates on degrading BaP,which were all above 40%.After addition of peptone and sodium acetate,BJ-7 was inhibited from degrading BaP.However,the yeast extract could promote BJ-7 degrading BaP.For the addition of phenanthrene,BJ-7 showed the consistency of concentration,BJ-7 was promoted on degrading BaP,when added 40 mg·L-1 phenanthrene.In summary,the strains BJ-1 and BH-7 have good adaptability to various environmental factors,indicating that the BaP-degrading microorganisms isolated from the intestinal tract of earthworm have excellent degradation performance.3.In this study,BaP-contaminated artificial soil(8 mg/kg)was restored through earthworm,strain and the combination of earthworm and strain.After 28 days of restoration,the degradation rate of the earthworm group was 62.90%,the degradation rate of the strain BJ-1 group was 58.70%,the degradation rate of the strain BJ-7 group was 23.93%,the degradation rate of the(earthworm+BJ-1)group was 80.79%,and the degradation rate of the(earthworm+BJ-7)group was 54.59%.According to the results of degradation rate,the strain BJ-1 has a good repair effect on BaP-contaminated artificial soil,and has a synergistic effect with earthworm restoring BaP-contaminated artificial soil.The effect of strain BJ-7 on BaP-contaminated artificial soil was poor,there is no synergy effect in the process of repairing BaP-contaminated artificial soil with earthworms.