Construction And Evaluation Of Genetic Engineered Strains For Biodesulfurization

Biodesulfurization is one of the most promising techniques for deep desulfurization of petroleum with advantages of high selectivity and mild reaction conditions. The limiting factor is the low activity of the biocatalysts for the commercialization of BDS. The aim of this thesis is to improve the desulfurization activity of the specific desulfurization strain, Pseudomonas delafieldii R-8, using the approaches of genic engineering and establish the base for further application of desulfurization.In this study, the dsz operon of Pseudomonas delafieldii R-8 was first cloned into the expressing plasmid (pPR9TT) to construction the recombinant plasmid pPR-dsz, and then reintroduced into strain R-8 to obtain a muti-copy dsz operon engineering strain R-8-1. Compared to the wild-type, strain R-8-1 showed a higher desulfurization activity for dibenzothiophene (DBT). Initial rates of DBT removal by strain R-8-1 were 6.25μmol /g dry cell/h, about 2-fold higher than that for wild-type strain. The recombinant cells were also applied in the desulfurization progress of diesel. It resulted in a 68% reduction of total sulfur from 310.8 mg/L to 100.1 mg/L, whereas only 53% of sulfur was removed by strain R-8. The stability of pPR-dsz in strain R-8-1 had been studied. It indicated that engineering strain has a high genetic stability. These results first obtain a muti-copy dsz operon engineering strain and are helpful for further advance of the biodesulfurization.To construct an engineering strain of higher desulfurization activity with Vitreoscilla hemoglobin gene (vgb) and study vgb application to the desulfurization of diesel,we constructed the vgb expressing plasmid, pPR-Pdsz-vgb and transformed by electroporation to Pseudomonas delafieldii R-8, then obtained a genetic engineering strain R-8-2. The results of CO-difference spectrum analysis indicated that the Vitreoscilla hemoglobin (VHb) having biological activity was expressed in strain R-8-2. Compared with the wild-type R-8, the density of strain R-8-2 increased by 20%, and its desulfurization activity is also higher in the lower deliquescence oxygen environment. In the end, about 69.9% of total sulfur in diesel oil was removed by strain R-8-2 from 320.5 mg/L to 96.6 mg/L, whereas only 57.2% of sulfur was removed by strain R-8. The results showed that the desulfurization activity of strain R-8-2 was higher than that of R-8 not only under the hypoxic conditions but also under normal aeration.In the thesis, we construct two engineering strains of higher desulfurization activity of P. delafieldii R-8. Furthermore, the work is helpful for further improvement of desulfurization strains and development in biodesulfurization.