Impact Of Pollutants As Selective Pressure On Bioaugmentation For Dioxins Removal With Rhodococcus Sp.strain P52

Microorganisms can degrade a wide variety of organic contaminants and can adapt to different inhospitable environments.However,there are a variety of pollutants,both man made(xenobiotic)and nature produced,that are not easy to degrade biologically,even over long periods of time.They are recalcitrant substances,including persistent organic pollutants(POPs)of concern.Poor conventional bioreactor performance may be due to the lack of a sufficient number of a specific microorganism harboring a key metabolic pathway to transform the target contaminant into less harmful end products.Plasmid mediated bioaugmentation has application potential for recalcitrant pollutants cleanup in environment.Although functional redundancy(i.e.different strains that can carry out the same or similar functions),microbial diversity of a community confer sufficient recipients/donors for conjugative transfer of catabolic plasmid.The isolates of biodegradable strains capable of surviving in harsh ecological environments provides an opportunity for bioaugmentation.In the previous study.Rhodococcus sp.strain p52 can degrade dioxins,and its catabolic plasmids can conjugative transfer within bacteria.In this study,the influence of different category and concentration of pollutants(as selection pressure)on conjugation transfer of catabolic plasmid were examined.The three pollutants used in the experiment were phenanthrene,dibenzothiophene and dibenzo-p-dioxin,which were the substrates of dioxygenases encoded by the catabolic plasmid.The research contents and results are as follows:When Rhodococcus sp.strain p52 was bioaugmented into the activated sludge reactors containing different pollutants,the specific removal rates in the reactors for phenanthrene,dibenzothiophene,and dibenzo-p-dioxin elevated in turn.The three contaminants exerted different influence on the activated sludge bacteria bearing the catabolic plasmids.Transconjugants,totally affiliated to nine genera,were isolated distinctively from activated sludge of different reactors.The difference in genomic G+C content(the highest was 27.54%)between transconjugants and strain p52 indicated that the catabolic plasmid could transfer crossing phylogenetic boundaries.Among the activated sludge reactors with different category or concentration of pollutants(as the selective pressure),the amount of transconjugants was the highest in dibenzo-p-dioxin-treating reactor,and the increase of concentration also increased the number of transconjugants.Quantitative PCR was used to monitor the community changes in the reactors.Compared with the other pollutants,the total amount of bacteria in dibenzo-p-dioxin-treating reactor was the lowest.The amount of Rhodococcus sp.strain p52 showed an increase under the treatment of contaminants,which proved that strain p52 had a strong ability to degrade the three contaminants was adaptive in the reactors.The three contaminants exerted different influence on the activated sludge bacteria bearing the catabolic plasmid.The highest ratio of transconjugants of sludge bacteria was detected in the reactor fed with dibenzo-p-dioxin.Additionally,as dibenzo-p-dioxin from 10 to 80 mg/L was fed into the reactors,the ratios of transconjugants increased.Film mating tests demonstrated the effects of pollutants on catabolic plasmid conjugative transfer varied for different recipients.The mechanism of the effect of selective pressure on catabolic plasmid conjugative transfer was preliminarily investigated.The changes of ROS and LDH in the reactors treated with different kinds of pollutants and different concentrations of dibenzo-p-dioxin were determined.It is speculated that the pollutants may affected conjugative transfer of the catabolic plasmid by triggering bacteria to produce ROS and reducing membrane permeability.The up-regulation of transcriptional activity of conjugative transfer-related genes also supported the hypothesis that the presence of contaminants promoted conjugative transfer of catabolic plasmid.The stability of catabolic plasmid in host bacteria was investigated and can stably maintain in Pseudomonas sp.for 145 generations.At the same time,different selection pressure had an impact on the fitness cost of the catabolic plasmids in the host.The results of this study will be helpful to understand the mechanism of contaminants as selective pressure to modulate catabolic plasmid transfer,so as to better apply bioaugmentation to removal of recalcitrant pollutants in the environment.