Biodegradation Of Phthalic Acid Esters Under Aerobic Conditions

Partâ… PAEs pollution in source water and drinking water by qualitativeinvestigationObjective: To qualitatively investigate the kinds of PAEs in source water and drinkingwater of the Yangtze River and Hanjiang River (Wuhan section) by GC/MS. Toqualitatively investigate the kinds of PAEs in source water and effluents treated bydifferent advanced water treatment processes including ozone binding biologicalactivated carbon.Method: The source water and drinking water of Zongguan and Pinghumen waterplants in Wuhan city were collected in March, July and December in the year of 2007and 2008. The volume of each sample was between 100～120 L. The organiccompounds in water were extracted with XAD-2 resin and eluted by acetone anddichloromethane. The eluate was concentrated (1:5000) and qualitatively analyzed byGC/MS. The water samples of a pilot-scale water plant in the south of China werecollected in April, 2006 and the methods of the sample pretreatments were identicalwith the others.Results: All the water samples have been polluted by PAEs. The main PAEs in sourcewater and drinking water in Wuhan were DBP and DEP.Part of samples had DMP, DOP, and BBP.Conclusion: The pollution of PAEs in source water and drinking water was universal.The present water treatment processes regardless of conventional or advancedtechniques were hard to remove the PAEs completely.Partâ…¡Acclimation, screen and identification for dominant PAEsdegradation bacteriaObjective: To obtain dominant PAEs degradation bacteria and identify it.Method: Activated sludge was collected at a dyeing plant of Wuhan, an industrial citylocated in central region of China. Eight L were mixed with 24 L of the inorganic saltsolution in an aerated basin. The acclimation process was conducted at roomtemperature. The DMP, DEP and DBP were equally added as sole carbon and energysource. The total concentration of PAEs was increased gradually from 30 to 420 mg/L.After eight weeks of acclimation, the activated sludge was used to inoculate nutrientagar plates under aseptic conditions. The plates were incubated at 37℃and differentcolonies grew after 36 h. The pure clones were obtained by plate streaking repeatedly.Then, each isolated bacterial strain was put into PAEs waste water in order to screendominant bacterium. All the flasks were put in swing bed with speed of 140 rpmunder 37℃. After one week degradation, the dominant PAEs-degrading bacteriumcould be confirmed in term of turbidity and degradation rate. After this screening test,we obtained a high-performance strain named strain L4. The dominant bacterial strainwas identified though analysis with its morphology, physiochemical characteristicsand 16S rDNA sequence. The characteristics of growth including environmentaltemperature and osmotic pressure of strain L4 were studied.Results: Seven strains have been isolated form the aerated basin, named strain L1～L7. The screen test showed the degradation rates of DMP, DEP and DBP by strainL1～L5 were 87.5%, 99.1%, 98.3%; 31.9%, 10.2%, 0.0%, 19.1%, 14.2%, 16.9%; 99.0%, 98.6%, 99.2% and 28.9%, 20.0%, 6.5%, respectively。L6 and L7 can notutilize DMP, DEP or DBP as sole carbon source. The strain L4 was recognized asdominant biodegradation bacterium. Strain L4 was identified as Rhocococcus ruber.This strain can grow at temperature between 16 and 42℃, but the optimaltemperature was 37℃. The best salinity for strain L4 growth was lower than 0.5%,but it also grew well with salinity of 0.5%～3%.Conclusion: The Rhodococcus ruber strain L4 had great ability in PAEs degradation.This strain had strong environmental adaptability.Partâ…¢The characteristics of Rhodococcus ruber strain L4about PAEs degradationObjective: To explore the optimal environmental conditions (pH, temperature andinitial concentration of PAEs) for strain L4 degradaion and to study the kinetics andsome important characteristics related with degradation.Method: The optimal environmental conditions were explored by orthogonal test(three factors and four levels). The kinetic equations were studied by curve fittingthrough detection the degrading residue of PAEs with different initial concentrations.The PAEs synthetic water samples (300 mg/L) with non-emulsification and completedemulsification were prepared to investigate their effects on PAEs degradation rate.The biosurfactant produced by strain L4 during PAEs degradation was measured bythe repellent size of oil cycle. Frequent aromatic compounds including phenol, 2,4-dinitrophenol, p-nitrophenol, sodium benzoate and naphthalene were prepared withMSM solution (4 retool/L) to explore the utilization spectrum of strain L4. The strainL4 was inoculated to ethanol, acetdimethylamide, octane and toluene solution withdifferent concentrations to investigate the environmental tolerance throughcomparison of survive rate. Results: The degradation batch tests of DMP, DEP and DBP by the Rhodococcusruber strain L4 showed the optimal pH value, temperature and substrate concentration:pH 7.0～8.0, 30～37℃and PAEs concentration≤450 mg/L. Kinetics studies showedthat the half-life of degradation was about 1.30d when the concentration of PAEsmixture was lower than 300 mg/L. The degradation rates of DMP, DEP and DBP insame system were different, DMP＞DEP＞DBP during degradation process. Littledifference between the above two sample preparations was observed in terms ofultimate degradation rate. Biosurfactant can be produced by strain L4, which candecrease the surface tension between water and oil like 69.2 mg/L SDS solution. Thisstrain can also grow on phenol, sodium benzoate or naphthalene solution as solecarbon source and energy, but cannot utilizing 2, 4-dinitrophenol, p-nitrophenol. Themetabolism velocity of sodium benzoate was faster than that of glucose, phenol,sodium benzoate according to OD₆₀₀ monitoring. Strain L4 can survive in ethanol andacetdimethylamide solution if their concentrations were lower than 10%; alkane waslitter harm to strain L4 as it can tolerate 60% octane solution; strain L4 was fragilewhen tested by 4% toluene as most bacteria were killed after 24 h exposure.Conclusion: Strain L4 had adaptability for various environments, but optimalenvironmental factors were benefit for biodegradation. The kinetics of PAEsdegradation by strain L4 can be described with exponential model under optimalconditions. During degradation, the PAE with shorter chain was prior to be utilized.Production of biosurfactant and mycolic acid by strain L4 were dominance fordegradation. That broad-spectrum utilization of organic compounds andenvironmental tolerance suggested strain L4 can be used as a potential candidate forremedying PAEs containing wastes.Partâ…£The mechanism of PAEs degradationby Rhodococcus ruber strain L4Objective: To approach the mechanism of PAEs degradation by strain L4 based on empirical methods of analytical chemistry and molecular biology.Method: The DEP solution with concentration of 300 mg/L was degraded by strainL4. The intermediate products after 24 h, 48 h, 72 h, 96 h were collected and extractedby C18 columnella, subsequently eluted by dichloromethane and detected by GC/MS.The plasmids of strain L4 were extracted with alkaline lysis method. The MW wasdetermined by agarose gel electrophoresis (AGE). The strain L4 was transferred 5times in nutrient broth culture, which then collected as control group. The abovebacteria were put into 300 mg/L PAEs solution and cultured at 37℃in 24 h, whichthen collected as induction group. The total proteins of strain L4 before and afterPAEs induction were extracted through ultrasound. Their concentrations weremeasured by Bradford method. The activity of C120 and C23O was determined bycolorimetry. The bands of total protein of strain L4 before and after PAEs induction inSDS-PAGE were compared.Results. Two intermediate products including phthalic acid and monoethyl phthalatewere detected. Three plasmids have been extracted from strain L4, whose MW were65 kb, 75 kb and 100 kb. The activities of C12O and C23O after PAEs induction werehigher than control group (P＜0.05). In induced group, the activity of C12O(8.78±0.98 U/mg) was higher than that of C23O (3.97±0.55 U/mg). The SDS-PAGEshowed two more bands were found in induced group.Conclusion: Phthalic acid was the main intermediate product during DEP degradation.Strain L4 contains three big plasmids. C12O and C23O were critical enzymes forPAEs degradation.Partâ…¤Degradaion of PAEs in water by immobilized bacteriaObjective: To compare the effect of immobilized cells and free cells of strain L4 inPAEs degradation. Method: The balls with immobilized bacteria were made by 10% PVA, 0.5% sodiumpolymannuronate, with adding 4% activated carbon powder. The balls was made byadding the mixture form injector, and cross linked in saturate boracic acid solution inabout 20 h. The DMP, DEP and DBP mixture (300 mg/L) in water were degraded byimmobilized bacteria and free cells. The removal rates of PAEs were compared.Results: The degradation rates of PAEs by immobilized bacteria were DMP 72.6%,DEP 70.1%, DBP68.3% in 48 h and 79.2%, DEP 74.9%, DBP 72.1% in 72 h。Thedegradation rates of PAEs by free cells were DMP 48.2%, DEP 41.1%, DBP27.3% in48 h and 72.2%, DEP 71.3%, DBP 58.8% in 72 h。Conclusion: The immobilized bacteria with good ability in removal of PAEs in watercan be made by 10% PVA, 0.5% sodium polymannuronate and 4% activated carbonpowder. Comparing the degradation effects, the immobilized bacteria were superior tofree cells.Partâ…¥Estrogenic activity of PAEs before and after biodegradationObjective: To evaluate the safety of the residuals of PAEs after degradation bydetermination of estrogenic activity with yeast estrogen screen (YES).Method: The estrogenic activities of DMP, DEP and DBP or their mixture weredetected by YES. The PAEs in water (300 mg/L) were degraded by strain L4. Theresiduals after 24 h, 48 h, 72 h and 96 h degradation were collected by Liquid-liquidextraction. The estrogenic activity of the residual was detected by YES subsequently.Results: The estrogenic activity of DEP can be detected, which was like the level of1/2000000 compared to 17β-E2. No obvious estrogenic activity can be found in DMPand DBP. The estrogenic activity of PAEs residues after degradation can be detectedby YES. The biggest activity of PAEs mixture were 1.81, 1.41, 1.25 and 1.15 after 24h, 48 h, 72 h and 96 h degradation, which were lower than the activity of PAEs mixture before degradation (2.97).Conclusion: The estrogenic activity of DMP, DEP and DBP can be detected by YES.The estrogenic activity of DEP was obvious, and stronger than DMP and DBP. Theestrogenic activity of PAEs mixture was caused by DEP and decreased afterdegradation by strain L4.