Removal And Mechanism Of PAHs In Leafy Vegetables By The Inoculating Of Composite Functional Endophytic Bacteria

Polycyclic aromatic hydrocarbons（PAHs）,a class of persistent organic pollutants（POPs）,are commonly found in organic contaminated soils.Crops can absorb PAHs from the soil through active or passive uptake,which causes crop contamination,and adversely affecting the safety of agricultural products and human health.As leafy vegetables are consumed in people’s daily life,the question of how to remove PAHs from leafy vegetables in contaminated areas has received considerable attention.In recent years,studies have shown that PAH contents in plants can be reduced through the colonization of PAH-degrading endophytic bacteria（or bacterial endophytes）.However,previous studies mostly used single bacterial endophytes with a narrow degradation spectrum,which can only degrade specific PAHs in plants;therefore,it is difficult to effectively deal with complex PAH contamination.Screening,domestication,and obtaining a composite of endophytic bacteria with the ability to degrade complex PAHs,and inoculating the multifarious bacteria in leafy vegetables is expected to effectively reduce the risk of PAH contamination in vegetables.However,a dearth of research studies exists in this aspect of endophytic microbe-assisted phytoremediation.In this dissertation,two endophytes were inoculated to remove phenanthrene and pyrene in water spinach（Ipomoea aquatica Forsk.）through a hydroponic experiment to analyze the possibility of reducing the complex PAHs in plants by inoculating composite endophytes.Furthermore,a composite of endophytic bacteria capable of degrading 16 EPA priority PAHs was constructed,and their degradation efficiency on PAHs was characterized under the conditions of pure culture and soil culture.On this basis,through the soil culture experiment,the efficiency of inoculating this composite to remove PAHs in water spinach,Chinese cabbage（Brassica chinensis L.）,and pakchoi（Brassica campestris L.）was studied.The possible mechanisms of inoculating the composite to remove PAHs were elucidated through the analysis of the changes in activities and abundances of PAH-degrading genes and enzymes in the plants after inoculating composite endophytes.The main research results are as follows:（1）It was found that the simultaneous inoculation of Sphingobium sp.RS2 and Mycobacterium sp.Pyr9 could effectively reduce the concentrations of phenanthrene and pyrene in water spinach,which provided the possibility for using composite endophytes to reduce 16 PAHs in leafy vegetables.Sphingobium sp.RS2 and Mycobacterium sp.Pyr9 were simultaneously inoculated into water spinach growing in the medium containing phenanthrene and pyrene through root soaking.Both endophytes could colonize in water spinach in large quantities,and the number of the endophytes in each part was root surface>inner roots>inner shoots.After 12 d of colonization,the concentrations of ∑PAHs（phenanthrene and pyrene）in shoots and roots of water spinach decreased by 35.27%and 73.46%,respectively,compared with CK.NidA and bphA1f genes were detected in water spinach inoculated with these endophytes,while none of these genes were detected in CK.Furthermore,the correlation between the abundances of these genes and the numbers of these endophytes was significantly positive（p<0.05）.The results showed that inoculated endophytes carrying PAH-degrading genes could introduce the gene into the host plant,and the simultaneous inoculation of two endophytes could reduce the contents of phenanthrene and pyrene in water spinach,which also provided the premise for the application of composite endophytes to reduce 16 PAHs in leafy vegetables.（2）A composite of endophytic bacteria with the ability to degrade 16 EPA priority PAHs was constructed,which could degrade complex PAHs efficiently in pure culture and soil culture.Through the domestication of endophytic bacteria with PAH-degrading ability obtained in previous studies,six endophytes that can degrade PAHs efficiently were selected and combined into a composite.The composite endophytic bacteria（CEB）were inoculated in MSM（v/v=1/20）containing 16 PAHs,and the degradation rate of ∑PAHs in MSM was up to 80.87%after 336 h.Under the soil culture system,the colonization of CEB by different colonization methods can effectively reduce the contents of ∑PAHs in Chinese cabbage,after 50 d of cultivation,the PAH contents in shoots and roots of plants decreased by 4.69%～30.06%and 8.04%～58.23%,respectively.Among these,the inoculation of CEB with the OD600nm=1.0 by seed soaking with leaf painting,and root irrigating had the best effect.These results indicated that it is feasible to reduce PAH contaminations by inoculating CEB.（3）It was found that the inoculated composite endophytes could efficiently degrade 16 EPA priority PAHs in different plants.After inoculation with CEB,the activities of PAH-degrading enzymes and the abundances of PAH-degrading genes in plants significantly increased,and the abundances of PAH-degrading genes in plants negatively correlated with the residual PAH contents.After 30 d,the contents of ∑PAHs in shoots of CEB inoculated water spinach,Chinese cabbage,and pakchoi decreased by 76.16%,87.25%,66.48%,and in roots decreased by 68.84%,77.02%,60.95%,respectively,compared with no CEB control.After colonization,activities of peroxidase in shoots of pakchoi significantly increased,and activities of polyphenol oxidase in shoots and roots of all three vegetables also increased.The colonization of CEB increased the copy numbers and relative abundances of nidA and bphA1f gene in the three vegetables,as well as the abundance of phe gene.The correlation between the residual contents of PAHs and the abundances of these genes was significantly negative（p<0.05）.These results revealed the effectiveness and possible mechanisms of CEB in reducing complex PAH contaminations in plants.