| Polycyclic aromatic hydrocarbons(PAHs)are ubiquitous environmental pollutants in contaminated soils.The effect and fate of PAHs in soil is of great environmental and human health concern because of the carcinogenic,mutagenic,and teratogenic properties of PAHs,as well as their high concentrations and frequent observation in soil.PAHs accumulate in a variety of plant species.As the basis of human and animal food webs,plants could be routes by which harmful PAHs enter human and animal populations.There are numerous bacterial cells existing in the rhizosphere soil and many of them usually form biofilms on root surfaces,thus protecting plants from harsh external environments and promoting plant growth.PAH-degrading bacteria with the ability to form biofilms on root surfaces assist in reducing the plant PAH contamination risk and enhance the PAH-removal efficiency from contaminated soil.In this work,two PAH-degrading bacterial strains,Diaphorobacter sp.Phe15 and Mycobacterium sp.Pyr9,were isolated from the root surface of healthy plants(Eleusine indica(L.)Gaertn.)grown in a PAH-polluted site.The influences of environmental conditions on PAH biodegradation and bacterial growth were investigated.Strain Phe15 was marked with antibiotic resistance,and strain Pyr9 was marked with the gfp gene.The colonization on root surface of clover and PAH-degradation performance of the two strains were elucidated with greenhouse experiments.The effects of a nonionic surfactant Tween 80 on the uptake of test PAHs by plant were evaluated.The main results are as follows.(1)Two PAH-degrading bacterial strains,Phe15 and Pyr9,were isolated from the root surface of healthy plants(Eleusine indica(L.)Gaertn.)grown in a PAH-polluted site.The phenanthrene-degrading bacterial strain Phe15 was identified as Diaphorobacter sp.and the pyrene-degrading strain Pyr9 was identified as Mycobacterium sp.based on physiological characteristics and 16S rRNA gene sequence homology analysis.Strain Phe15 can use phenanthrene as the sole carbon source,and strain Pyr9 can use pyrene as the sole carbon source.At 30?,pH 7.0 and 150 r·min-1,93%of phenanthrene(initial concertration of 100 mg·L-1)in mineral medium is degraded by strain Phe15 in 24 hours,and all the phenanthrene in medium can be degraded totally within 48 hours.The optimum temperature for bacterial growth and phenanthrene degradation by strain Phe15 is 30?,and the optimum pH is between 7.0-10.0.With the inoculum density increases,the phenanthrene-degrading rate increases correspondingly.While for strain Pyr9,the pyrene in mineral medium(initial concertration of 50 mg·L-1)can be degraded completely after 10 d at 30?,pH 7.0 and 150 r·min-1.The optimum temperature for bacterial growth and pyrene degradation by strain Pyr9 is 30?,and the optimum pH is 7.0-8.0.When the inoculum density increases,the pyrene-degrading rate increases correspondingly.(2)Strain Phe15 and strain Pyr9 are marked with antibiotic resistance and gfp gene,respectively,and then the colonization on root surface of clover and PAHs degradation performance of the two strains were investigated with greenhouse experiments.Strain Phe15 is resistant to ampicillin(75 mg·L-1)and chloramphenicol(25 mg·L-1),so the two antibiotics are selected to marke strain Phe15 after colonization on root surface of clover with root irrigation or seed soaking.Strain Phe15 can promote the plant growth and reduce the phenanthrene concertration and accumulation inner plant significantly.Furthermore,after root irrigation or seed soaking,strain Phe15 can also enhance the phenanthrene removel from the rhizosphere soil.In addition,compared to seed soaking,more phenanthrene removel from soil was detected with root irrigation by strain Phe15.Strain Pyr9 is resistant to ampicillin(75 mg·L-1)and spectinomycin(25 mg·L-1),it is successfully marked with gfp gene by electric transformation and the positive transformant with gfp-label is named as Pyr9-gfp.The pyrene-degrading ability of Pyr9-gfp does not show any significant difference with the wild strain Pyr9.Pyr9-gfp can actively colonize on clover root surface by root irrigation and form bacterial biofilm observed by confocal laser scanning microscope(CLSM).Strain Pyr9-gfp can grow and propagate inner plant tissues,the number of Pyr9-gfp increased with the increase of pyrene concentration in contaminated soil.The biomass of plant in Pyr9-gfp-innoculated treatment was significantly improved compared to the Pyr9-gfp-free treatment.The colonization on root surface by strain Pyr9-gfp decreased the pyrene concentration,accumulation and plant concentration factor(PCF)in plant tissues,as well as the pyrene residual in rhizosphere soil.(3)Surfactant Tween 80 was added into the natural PAH-contaminated soil to improve the bio-availability of pyrene,and then the effects on pyrene uptake by clover and pyrene removel from soil after colonization on root surface by strain Pyr9 were investigated.Between the Tween 80 concentration of 100-1000 mg·kg-1,with the concentration increased,the number of strain Pyr9 inner plant tissues doesn't change significantly,while the number of strain Pyr9 in soil increases at first and then decreases when the concentration of Tween 80 is more than 500 mg·kg-1.Similarly,the biomass of plants increases at first and then decreases when the concentration of Tween 80 is more than 250 mg·kg-1.In addition,after root irrigation with strain Pyr9,the plant growth of clover improves siganificantlyWith the addition of Tween 80(100-1000 mg·kg-1),the plant uptake of pyrene is significantly promoted.For example,when 500 mg·kg-1 of Tween 80 is added,the pyrene concentration in shoot or root increases by 38%and 23%,respectively.Furthermore,root irrigation by strain Pyr9 can significantly reduce the pyrene concentration and accumulation in plant tissues.For example,under 500 mg·kg-1 of Tween 80,the concentration of pyrene in clover root is reduced by 19.1%after root irrigation.A certain amount of Tween 80 can enhance the pyrene removal from soil.In the range of 0-500 mg·kg-1,the pyrene residual concentration in soil decreases gradually with the increase of Tween 80.In addition,the pyrene concentration in soil decreases significantly after root irrigation.For example,when the Tween 80 concentration is 100 mg·kg-1,the pyrene concentration in soil of Pyr9-gfp-innoculated treatment decreases by 12.8%and 19.2%,respectively,compared to Pyr9-gfp-free treatment and Pyr9-gfp-soil treatment.When the added amount of Tween 80 was 500 mg·kg-1,the final residue of pyrene in soil was 2.17 mg·kg-1 in Pyr9-gfp-innoculated treatment,reduced by 49.4%compared to the 4.28 mg·kg-1 of pyrene in the contaminated soil control. |
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