Screening Of The Endophytic Bacterial Strain Stenotrophomonas Maltophilia PX1 And Its Pyrene Degradation Characteristics

Polycyclic aromatic hydrocarbons（PAHs）have attracted popular attention due to easy accumulation,high content and wide distribution.Many PAHs are toxic,mutagenic or carcinogenic.The accumulation of PAHs in soil and crops poses a threat to human health and ecological security.Therefore,how to efficiently and safely remove PAHs from soil environment and crops is an urgent problem to be solved.Endophytic bacteria with PAHs degradation function had been isolated and screened out,and the PAHs pollution in the plant body and its growth environment could be reduced by means of colonization.However,there was not much research on the mechanism of efficiently recolonizing functional endophytes into crops and reducing the risk of PAHs pollution in crops and their growth environment.In this study,a pyrene-degrading endophyte PX1 with growth promoting ability was screened out from a healthy Eleusine indica（L.）Gaertn.grown near the sewage outlet of a petrochemical enterprise in Nanjing.The taxonomic status of endophyte PX1 was identified to species.It was identified as Stenotrophomonas maltophilia.The degradation characteristics of PAHs were investigated,and the optimal growth and environmental factors of PAHs degradation were optimized.The degradation pathway of pyrene was preliminarily speculated.At the same time,through plant hydroponic experiments,it was verified that the strain S.maltophilia PX1 can efficiently colonize in Ipomoea aquatica Forsk.,Triticum aestivum L.,Vigna radiate（L.）R.Wilczak.and Fagopyrum esculentum Moench.and reduce pyrene accumulation.Through morphological observation,physiological and biochemical identification,and16S r RNA gene sequence analysis,the taxonomic status of the screened strain PX1 could be identified to the species.The results showed that the endophyte PX1 was Stenotrophomonas maltophilia.The endophyte PX1 was an aerobic bacteria.The optimum growth conditions of endophyte PX1 were 30℃,p H 6.0～9.0,and the liquid content was10%.Under these conditions,when the inoculation amount was 5～11%,the degradation efficiency of pyrene by endophyte PX1 could reach 75%within 10 days after exposure to pyrene at low concentration(10 mg·L^-1).The endophyte PX1 can degrade many single PAHs.After 7 days of culture,100.0 mg·L^-1 naphthalene was almost completely degraded by endophyte PX1.The degradation rates of phenanthrene(50.0 mg·L^-1),pyrene(20.0mg·L^-1),fluoranthene(20.0 mg·L^-1)and benzo[a]pyrene(10.0 mg·L^-1)were 72.6%,50.7%,31.9%and 12.9%after 10 d incubation,respectively.Liquid-mass spectrometry（HPLC-MS）had been used to qualitatively analyze the metabolites produced during the degradation of pyrene by strain S.maltophilia PX1.The results showed that 4,5-epoxidation pyrene,gentisate or protocatechuate,4,5-dihydroxy pyrene,1-hydroxy-2-naphthoic acid,salicylicaldehyde,cis,cis-2,4-hexadienedioic acid or2-hydroxymuconate semialdehyde,salicylic acid and cis-2’-benzene methyl pyruvate had been produced during the degradation of pyrene.Meanwhile,the activities of six enzymes in the process of pyrene degradation were also measured.Finally,it was preliminarily concluded that strain S.maltophilia PX1can open pyrene through the two pathways of dioxygenase and monooxygenase oxidation,and complete mineralization of pyrene could be achieved by the downstream pathway of"salicylate pathway"and"phthalate pathway".In this study,Ipomoea aquatica Forsk.,Triticum aestivum L.,Vigna radiate（L.）R.Wilczak.and Fagopyrum esculentum Moench.were selected as representatives of crops,and the colonization of strain S.maltophilia PX1 in crops was verified to reduce pyrene contaminations.The results showed that the strain S.maltophilia PX1 could efficiently colonize these four crops and reduce the pyrene contaminations in crops and the surrounding environment.At the same time,S.maltophilia PX1 could promote the growth of crops.After inoculation with strain S.maltophilia PX1,the growth of Ipomoea aquatic Forssk.and Triticum aestivum L.respectively increased by 9.8%～41.6%and 9.4～12.1%;the pyrene residue in Ipomoea aquatica Forsk.,Triticum aestivum L.,Vigna radiate（L.）R.Wilczak.and Fagopyrum esculentum Moench.respectively decreased by 29.8%～50.7%,52.4～67.1%,23.0%～36.4%and 21.2%～50.3%;and the pyrene residue in the nutrient solutions were reduced by 8.0%～13.9%,8.8%～15.3%,6.5%～9.5%and 12.0%～18.5%,respectively.The colonization of strain S.maltophilia PX1 effectively reduced the risk of pyrene contamination of crops and their surrounding environment.In conclusion,endophytic bacteria PX1 can be isolated from PAHs contaminated areas and re-colonized in target crops to reduce PAHs pollution in crops.The results of this study provide a new idea for ensuring the safety of agricultural products.