Isolation And Characterization Of Phthalate-degrading Bacteria And Its Application In Soil Remediation

Owing to the agricultural practices such as the application of plastic films, sewage sludge, fertilizes and irrigation waste water, the amount of PAEs in the arable soil is increasing sharply. The pollution of PAEs in agricultural soil can have an adverse effect on the quality and production function of soil. More seriously, PAEs can be riched in human body through food chain, posing threats to human health. Therefore, the effective and advanced remediation technologies for removal of these contaminates are critically needed. Studies show that microbial degradation is believed to be one of the major processes that remediate soil contaminated by organic pollutants. So far, amounts of PAEs degradation bacteriums have been isolated from various environments such as activated sludge, mangrove sediment, wastewater, and municipal solid waste. But no studies have examined the degradation of PAEs by strains isolated from compost. In addition, the addition of compost has been demonstrated to be effective in biodegrading PAEs in soil, but the direct effect of compost on PAE-degrading bacteria remains unclear. In this study, manure samples contaminated by plastics chronically were collected as inoculum for isolation of efficient degrading microorganisms. The selected strains were identified by morphology, physio-biochemical characteristics, and 16 Sr DNA gene sequence analysis. The degradation characteristics were studied and the optimum degradation conditions were investigated on the basis of response surface methodology(RSM). The degradation products of DBP were identified by GC-MS and the key enzyme responsible for degradation was cloned or expessed, finally the biochemical degradation pathways were proposed. Then the isolated bacterial strain was added into the DBP contaminated soils to measure the degradation efficiency, The following results were obtained in this study:(1) Screening and identification of DBP-degrading strains. A strain was obtained from the landfill soil samples using the enrichment and domestication method. Based on the morphology, physio-biochemical characteristics, and 16 Sr DNA gene sequence analysis, the degrading strain were identified as Providencia sp. 2D.(2) Studies on the degradation characteristics and optimization of the degrading conditions. The effects of temperature, pH and inoculum size on degradation abilities of strain 2D was investigated through one-factor experiment. The optimum cultural conditions by 2D were estimated to be temperature 32.4℃, pH 8.3, and an inoculum size of 0.6(OD600) by response surface methodology using Central Composite Rotatable Design. Additionally, the DBP degradation kinetics experiment by Providencia sp. 2D under the optimal conditions was conducted. The results indicated that at the initial concentration of 50~1000 mg/L, more than 80% of the added DBP was degraded within 72h; the degradation reaction fitted the first-order kinetics and the half live(T1/2) ranged from 8 to 27 h.(3) The investigation of the degradation pathway. The degradation products of DBP were characterized by GC-MS. Based on the degradation products, the clone of related gene, the expression of key enzyme, and the degradation experiments under anaerobic condition, the biochemical degradation pathway for DBP were proposed.Firstly, DBP was hydrolyzed by esterase to MBP and then further to PA, or directly to PA;Then under different oxygen conditions, PA was degraded via different pathways by Providencia sp. 2D.: Under aerobic condition, PA was degraded via 4,5-dihydroxyphthalate to PCAwhich was ultimately transformed into CO2 and H2O; under anaerobic condition, PA initially was metabolized to BA.Similar to the degradation of PA by Providencia sp.2D, the degradation of BA by Providencia sp.2D also had two different pathways: Under anaerobic conditions, BA was ring cleaved and mineralized to CO2 and H2O; Under aerobic condition, BA was initially transformed into PCA, then to CO2 and H2 O.(4)Degradation kinetics of DBP in contaminated soil by Providencia sp. 2D. The results showed that Providencia sp. 2D was able to efficiently degrade DBP in the contaminated soil even at high concentration(100 mg·kg-1);the addition of compost indeed enhanced DBP degradation in soil; during degradation process, the stain 2D could degrade DBP probably synergically with soil indigenous microorganisms. As a result, Providencia sp. 2D showed the huge potential for the bioremediation of DBP-contaminated soil.