Biofilm Formation Of Phenanthrene-Degrading Bacteria On Root Surface And Its Infulences On Uptake Of Phenanthrene By Plants

Soil contamination has become a global environmental problem in recent decades.Polycyclic aromatic hydrocarbons(PAHs)are a group of persistent organic pollutants found in contaminated soil with high concentrations.Owing to their low aqueous solubility,hydrophobic nature and persistence,PAHs can be absorbed and accumulated by plants,which is a major pathway for them to reach the food chain,thereby posing serious threats to animal and human health.Rhizobacteria associated with plant root surfaces are known to benefit plant yields by mechanisms such as improved mineral uptake,phytohormone production and competitive suppression of pathogens by production of antibiotics and induction of secondary metabolite-mediated systemic resistance.However,whether rhizobacteria associated with plant root surfaces could be used to mediate the uptake and biodegradation of PAHs in plants.This issue is hitherto still under elucidation.In this work,two phenanthrene-degrading bacteria were isolated from the root surface of plants(Conyza canadensis(L.)Cronq.,Plantago depressa Willd and Salvia plebeia R.Brown)grown in a PAH-polluted site.The influences of environmental conditions on phenanthrene biodegradation by these strains were investigated.After being marked with gfp gene,the abilities of phenanthrene degradation of strain 45-RS2(gfp-labeled RS2)and its biofilm formation on root surface were evaluated.The influences of strain 45-RS2 colonization with plant root surface on plant growth and phenanthrene uptake and metabolism in plants were clarified using greenhouse experiments.The main results were shown as follows:(1)Both strain RS1 and RS2 were identified as Sphingobium sp.,based on physiological characteristics and 16S rRNA sequence homology analysis.The experiments on phenanthrene-degrading features and growth characteristics of the strain showed that strain RS1 and RS2 can utilize phenanthrene as the sole carbon and energy source.At 30℃and 150 r·min-1,the degradation ratio of phenanthrene(100 mg·L-1)in PMM medium were 88.9%and 98.3%by RS1 and RS2 separately after 72 h rotary culture and the maximum growth of RS1 and RS2 were 9.53 and 10.05 log cfu·mL-1.Effects of environmental conditions on phenanthrene biodegradation showed that,the best conditions for strain RSI to degrade phenanthrene was 30℃ and the suitable value of pH was 5.0-7.0,while RS2 was 370C and the suitable value of pH was 5,0～7.0.Between the tested phenanthrene concentration of 50 to 250 mg·L-1,the phenanthrene-degrading rates of both strains decreased while the phenanthrene concentration increased.In addition,between the tested inoculum densities of 1%to 15%,when the inoculum density increased,the phenanthrene-degrading rates of both strains increased correspondingly.Both strain RS1 and RS2 could utilize other PAH(such as acenaphthene,anthracene,pyrene and benzo[a]pyrene)as the sole carbon source to grow and degrade them to a certain extent.Strain RS1 and RS2 had the same ability to resist antibiotics,such as ampicillin(200 mg·L-1),kanamycin(25 mg·L-1),streptomycin(200 mg·L-1)and chloramphenicol(10 mg·L-1),and a slight difference of erythrocin.(2)The ability of both strains to form biofilms were studied,and strain RS2 was marked with gfp gene.Both strain RS1 and RS2 were able to form dense biofilms in microcentrifuge tube cultured at 30℃ without shaking.Both strain RS1 and RS2 were successfully marked with gfp gene by triparental conjugation and named 45-RS1 and 45-RS2.After being marked with the gfp gene,the biochemical characteristics of strain RS2 were slightly interfered.At 30℃ and 150 r·min-1,the degradation ratio of phenanthrene(100 mg·L-1)in PMM medium were 99.2%by strain RS2 and 45-RS2 after 72 h rotary culture and the maximum growth of RS2 and 45-RS2 were 8.67 and 9.62 log cfu·mL-1 separately.And gfp gene had no effect on the abilities of biofilm formation and phenanthrene degradation by 45-RS2.(3)Inoculating alfalfa(Medicago sativa L.)plants with strain 45-RS2(gfp-labeled)by irrigation or seed soaking revealed that strain 45-RS2 could actively colonize plant root-surface,form dense biofilm,and then transfer to the internal tissues of plant roots and shoots.Strain 45-RS2 can induce the absorption of phenanthrene and promote the growth of alfalfa.With irrigation or seed soaking of strain 45-RS2,phenanthrene in alfalfa root were significantly lower than the control group(P<0.05).It was indicated that inoculating alfalfa roots with strains 45-RS2 could promote the metabolism of the phenanthrene.(4)Inoculating alfalfa(Medicago sativa L.)plants with strain 45-RS2(gfp-labeled)influenced the activities of peroxidase(POD),catalase(CAT)and phenol oxidase(PPO)in plants.Without strain 45-RS2,the activities of POD increased in alfalfa under phenanthrene-contaminated soils,the activities of PPO decreased in roots and increased in shoots,the activities of CAT had no obvious changes in both roots and shoots.The activities of POD and PPO decreased when colonized with strain 45-RS2 and with phenanthrene contamination-free,while the activities of CAT decreased.When exposed to phenanthrene contamination and colonized with strain 45-RS2,the activities of PPO and CAT in alfalfa roots decreased,and the activities of PPO increased in shoots while the activities of CAT had no obvious changes.Exposed to phenanthrene contamination,the activities of POD enhanced in alfalfa with strain 45-RS2 inoculated by seed soaking,while the activities of POD had no significant influence in alfalfa with strain 45-RS2 inoculated by irrigation.Thus,the amount of bacteria in inner plants may have an effect on enzyme activities in plants.