Isolation Of A PAH-degrading Strain With Plant Growth-promoting Ability And Its Colonization And Performance On Plant Root Surface

Soil is an indispensable material that supports plants life,human survival and development.More attention has been paid to soil quality,but the soil pollution status in China is not optimistic.Polycyclic aromatic hydrocarbons(PAHs)pollution is a common soil pollutant in our country.PAHs pose a threat to human health and crop safety,because they are structurally stable,difficult to biodegrade,easily absorbed by plants,and potentially carcinogenic,teratogenic and mutagenic.Therefore,PAHs pollution risk avoidance and reduction in crops are gradually being widely recognized.There are a large number of rhizobacteria in the rhizosphere of plants which can form bacterial biofilms on the root surface of plants.Most of these bacterial biofilms have the functions of promoting plant growth,improving plant stress resistance,adsorbing,enriching and even degrading organic pollutants.Based on this,this paper attempts to isolate and screen a PAHs-degrading strain with plant growth-promoting ability from plant root surface,and study the effect of bacterial colonization on plant root surface on plant PAHs-absorption and accumulation.This study proposed a new approach of combining bacterial biofilms and plants to regulate the absorption of organic pollutants by plants,to provide reference for the prevention and control of soil organic pollution and the reduction of organic pollution risks of crops.In this study,a functional strain PHE-2 capable of efficiently degrading phenanthrene was isolated and screened from the roots of healthy Alopecurus aequalis Sobol from a PAHs-contaminated area in Nanjing.The PAH-degrading spectrum,resistance to antibiotics and the environmental conditions on the phenanthrene-degradation by strain PHE-2 were studied.The metabolic pathway of phenanthrene,biofilm-forming ability and growth-promoting properties of strain PHE-2 were investigated.Furthermore,the effects of the colonization and biofilm formation of strain PHE-2 on the growth of water spinach and its phenanthrene-accumulation in water spinac were studied by pot experiment in a greenhouse.The main research results are as follows:(1)A functional strain PHE-2 which can efficiently degrade phenanthrene was obtained from the root surface of healthy plants(Alopecurus aequalis Sobol)collected from PAH-contaminated sites.The 16S rRNA gene sequence homology analysis,bacterial physiological and biochemical tests confirmed that strain PHE-2 is belonging to Paenibacillus sp.Strain PHE-2 can grow with phenanthrene as the sole carbon source and energy source,and efficiently degrade phenanthrene under laboratory culture conditions.When strain PHE-2 was inoculated into phenanthrene-containing inorganic salt medium(100 mg·L-1)with 5%inoculum(OD600nm=1.0),and cultured for 144 hours at 30?,150 r·min-1 rotation speed,the phenanthrene-degradation rate of strain PHE-2 can reach over 99%.Environmental conditions significantly affected the growth of strain PHE-2 and its phenanthrene-degradation,but overall,strain PHE-2 has strong heat and alkali resistance.At the temperature of 30-40? or the pH of 7.0-10.0,strain PHE-2 grows well and the phenanthrene-degradation rate is over 90%Increasing the inoculum can significantly increase the phenanthrene-degradation rate of strain PHE-2,and when the inoculum is more than 6%,the phenanthrene-degradation rate is more than 90%and stable at the optimal conditions rate;the substrate concentration also affects the growth of strain PHE-2 and its phenanthrene-degradation.When the concentration of phenanthrene is within 100 mg·L-1,the degradation rate of strain PHE-2 to phenanthrene is more than 90%within 6 days;and when the concentration of phenanthrene is greater than 200 mg·L-1,the growth of strain PHE-2 is obviously inhibited,and its degradation rate to phenanthrene is also significantly reduced.(2)The pathway of the metabolism of phenanthrene by strain PHE-2 was clarified,its biofilm-forming ability in various media was studied,and the growth-promoting ability of the strain was determined.The the phenanthrene intermediates of strain PHE-2 were qualitatively analyzed using HPLC and HPLC-MS,and the structure of the intermediates was analyzed utilizing Peak View.Combined with the reported literatures,the pathway of the metabolism of phenanthrene by strain PHE-2 was inferred through the phthalic acid pathway.Strain PHE-2 was inoculated with 5%inoculum into kinds of liquid medium of MSgg,phenanthrene-containing inorganic salt and LB for 6 days,and it would form bacterial biofilm.The maximum yield of their biofilm is 0.7,0.79 and 2.1(OD590nm),respectively.Strain PHE-2 can produce 21.10 mg·L-1 indole-3-acetic acid(IAA)in nitrogen-containing medium,which had the ability to produce siderophore,and the ability to dissolve inorganic phosphorus and organic phosphorus.At the same time,the strain can form a transparent phosphate-solubilizing ring on the phosphorus-containing solid plate.(3)Strain PHE-2 was successfully colonized on the water spinach root soaking with PHE-2-inoculation treatment.The distribution of strains PHE-2 in the water spinach root surface,culture solution and the effect of phenanthrene absorption on the water spinach were further studied.The results showed that in the hydroponic system,strain PHE-2 could colonize well on the root surface of water spinach and form a bacterial biofilm.The colonization and biofilm formation of strain PHE-2 in the water spinach roots can promote its growth,and effectively reduce the phenanthrene content in the roots and the accumulation of phenanthrene in the water spinach.For example,when cultured in the solution with low concentration of phenanthrene(1.0 mg·L-1)after 15 days,the fresh weight and dry weight of the plants inoculated with wild bacteria increased by 48.59%and 62.51%,respectively,compared with the plant inoculated with inactivated bacteria.The phenanthrene content in its shoots decreased by 46.35%and 5.26%,respectively.The accumulation of phenanthrene in plant roots and shoots decreased by 25.48%and 22.36%,respectively.In addition,functional strains colonized on the roots of water spinach are also releasing into the culture solution,thereby promoting degradation of phenanthrene in the culture solution.