| Polycyclic aromatic hydrocarbons(PAHs)are a class of refractory organics that have carcinogenicity,teratogenicity and mutagenicity.Due to their high octanol-water partition coefficients and low water solubility,PAHs often accumulate in the soil,which has potential harm to human health and environmental ecological safety,and has caused concern of the researchers in environmental field.In this thesis,two PAHs-degrading bacteria capable of using phenanthrene(Phe),fluoranthene(Flua),and pyrene(Pyr)as carbon sources were purified and isolated from contaminated soil near the coking furnace of steel Plant.The morphological observations,physiological and biochemical characteristics and molecular biological identification were carried out.In order to enhance the degradation effect of high molecular weight PAHs,the strain KL(Klebsiella sp.)isolated in our early research,which was constructed as mixed strains system,and polyvinyl alcohol(PVA)-sodium alginate(SA)-nano-alumina(ALNPs)composite materials were used to immobilize functional bacteria.Biodegradation characteristics of PAHs between free mixed strains,immobilized single strains and immobilized mixed strain were researched in simulated soil washing effluent.The degradation effects of PAHs by immobilized mixed strains were discussed in actual soil washing effluent.The main research contents and conclusions of this thesis are as follows:(1)Isolation,purification and identification of characteristic PAHs-degrading bacteriaTwo characteristic PAHs-degrading bacteria(named as Z1 and Z2)using Phe,Flua,and Pyr as carbon sources were purified and isolated.The observation of apparent forms and scanning electron microscope,experiments of growth curve,physiological and biochemical characteristics,molecular biology identification of Strains Z1 and Z2 were carried out.Strains Z1 and Z2 were identified and named as Pseudomonas sp.,Sphingobacterium sp.,the Gen Bank accession numbers were: MN647530,MN647529,respectively.(2)Preparation and characterization of immobilized mixed strainsThe isolated PAHs-degrading bacterium KL by our group in early research,which was used to construct the mixed strains system.The PVA-SA-ALNPs mixed gel as embedded materials were used to immobilize mixed strains.The appearance of embedding beads was white gel,whose average size was about 3 mm,and the beads' mass transfer properties and mechanical strength were good.Scanning electron microscopy showed that the inside of the embedded beads was reticular staggered structure with various pores,which could provide a large number of adhesion area for microorganisms,and also was conducive to mass transfer.(3)Biodegradation characteristics of PAHs in simulated soil washing effluentThe free mixed strains(experimental group 1),immobilized single strain(experimental group 2),and immobilized mixed strains(experimental group 3)were transferred to simulated soil washing effluent for degradation experiments.The initial concentrations of Phe,Flua,and Pyr were 15 mg/L in the simulated soil washing effluent.The experimental results showed that the degradation rates of Phe,Flua,and Pyr in group 1 were 78.84%,75.28%,and 75.63%,respectively,on the 30 th day at 25?,and the degradation rates of Phe,Flua,and Pyr in group 3 were 91.56%,88.59% and88.52%,respectively.The degradation rates of the three kinds of PAHs in immobilized strains group increased by 12.72%,13.31% and 12.89%,respectively.The results of first-order kinetic showed that the PAHs reaction rates of the immobilized mixed strains were greater than the free mixed strains,the half-life was decreased by 4d,5d,5.1d,respectively,compared with free mixed strains group,whose reaction times were significantly reduced.Moreover,compared with group 2,the degradation rates of high molecular PAHs that Flua and Pyr in group 3 were significantly increased,it could be speculated to be the results of Co-metabolic pathway.(4)Factors affecting PAHs degradation efficiency in simulated soil washing effluentThe certain concentration of Tween 80 significantly affected the PAHs degradation effect of immobilized mixed strains in the simulated soil washing effluent.In the non-Tween 80 system,the degradation rates of Phe,Flua,and Pyr on the 30 th day were reduced by 16.38%,22.25% and 21.29%,respectively.The presence of Tween 80 promoted the PAHs degradation.The single factor experiments showed that optimal dosing ratio of the immobilized mixed strains was determined as 10%,and microbial amount embedded in immobilized beads was determined as 20%.When the embedded beads were used for three cycles,the degradation rates of the three kinds of PAHs were still higher than the free mixed strains group,which could be reused.(5)Biodegradation characteristics of PAHs in actual soil washing effluentThe non-ionic surfactant Tween 80 was used as the eluent to leach the spiked soil contaminated with Phe,Flua,and Pyr,whose concentrations were 200 mg/kg.On the30 th day,the degradation rates of the three kinds of PAHs by the immobilized mixed strains were 92.21%,87.97%,and 88.81%,respectively.The degradation rates were slightly higher than the simulated soil washing effluent group,which may be due to the role of indigenous bacteria in the degradation process of Phe.In this thesis,the research that biodegradation characteristics of Phe,Flua,and Pyr between simulated soil washing effluent and actual soil washing effluent was carried out,which was based on immobilized mixed strains.The research results can provide theoretical supports for PAHs bioremediation technology in soil washing effluent. |
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