Study On Immobilization And Degradation Mechanism Of Composite Petroleum-degrading Bacteria

With the increasing frequency of offshore oil exploitation,oil spills occur frequently,which not only causes huge economic losses,but also endangers marine ecosystems.The use of biotechnology to repair oil-contaminated marine ecosystems still has problems such as the lack of stable micro-environment of microorganisms.In this paper,the high-efficiency petroleum-degrading bacteria and biosurfactant-producing bacteria were isolated and screened.The immobilized composite petroleum-degrading bacteria was prepared by using sodium alginate and straw as immobilized carriers.The preparation method of immobilized composite petroleum-degrading bacteria was optimized,and the degradation performance.and diesel degradation kinetics were studied.Fourteen petroleum-degrading bacteria were isolated from oil-contaminated seas.HDMP1 was selected as a high-efficiency petroleum-degrading bacteria by determining the degradation rate of diesel.HDMP1 had the highest rate(50.52%)for degradation of diesel after 5days.HDMB2 was selected as the best biosurfactant-producing bacteria by screening.Its emulsification index was 81%,the foaming rate was 25%,and there was obvious droplet collapse.HDMB2 produced lipopeptide biosurfactants by analysis of Fourier Transform Infrared(FTIR)spectroscopy.The free cells used in the experiment were obtained by mixing the bacteria HDMP1 and HDMB2 in equal proportions.Sodium alginate was used as the embedding carrier,straw was used as the adsorption carrier.The immobilized composite petroleum-degrading bacteria was prepared by the equal mixing of two carriers to adsorb-embed HDMP1 and HDMB2 bacteria,and immobilized cells in straw-alginate beads were obtained.Mechanical properties and degradation performance were used as indicators.The multi-factor analysis of variance showed that the concentration of CaCl2,sodium alginate and straw had a great influence on the mechanical properties and degradation performance of beads.With the diesel degradation rate as the response value,the optimum condition was obtained by Response surface methodology:the concentration of CaCl2 was 3g/L,the concentration of SA was 4.59 g/L,and the concentration of straw was 1g/L.Under this condition,the degradation rate of diesel can reach 66.71%.The differences between the immobilized cells by various methods and free cells were compared by diesel degradation rate and SEM images.The result indicated that the degradation performance of the four methods from large to small is adsorption-embedding method>adsorption method>free bacteria method>embedding method.The immobilized cells in straw-alginate beads prepared by the adsorption-embedding method had the highest degradation rate(68.68%),and it had a good pore structure.Further,the degradation kinetics were analyzed by fitting the Monod equation.The vmax(maximum specific degradation rate of diesel)and Ks(half saturation rate constant)of free bacteria were 1.42 d-1 and 4.64 g/L,respectively.The vmax and Ks of immobilized cells in straw-alginate beads were 1.84 d-1 and 3.23 g/L,respectively.It was also proved from the degradation kinetics equation that the degradation efficiency of straw-alginate beads on diesel was higher than that of free bacteria.A bioremediation simulation experiment of marine oil spill contamination of straw-alginate beads was conducted.High-throughput sequencing analysis showed that after the oil spill occurred,the species diversity of the community showed a downward trend,while the relative abundance of the species was higher.The content of Pseudoalteromonas and Thalassospira in the microbial community after bioremediation was higher than that of the original seawater,and the abundance of related biodegradation functional genes increased significantly.In this paper,sodium alginate and straw were selected as immobilized carriers,and the degradation performance of diesel on immobilized composite petroleum-degrading bacteria prepared by immobilized carrier was analyzed.It provides a basis for the application of immobilized bioremediation technology to the treatment of marine oil pollution.