GENETIC MODIFICATION OF PAENIBACILLUS UTILIZING VITREOSCILLA HEMOGLOBIN TO IMPROVE BIODESULFURIZATION
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Biodesulfurization (BDS) is a type of microbial biodegradation involving the removal of organic sulfur compounds from fossil fuels that is accomplished by utilizing the metabolism of certain microorganisms (McFarland et al., 1999). In past decades, many related genetic modifications or transformations have been made to improve biodesulfurization. In this study, Vitreoscilla hemoglobin (VHb) was used to enhance the ability of sulfur metabolism and growth in Paenibacillus naphthalenovorans. Paenibacillus naphthalenovorans wild type (32O-Y) is a thermophilic bacterium isolated in our lab that has also been transformed with recombinant plasmid pnW33N-vgb (producing strain 32O-Y[pNW33N-vgb]). This plasmid encodes both VHb and chloramphenicol (Chl) resistance. The Chl resistance gene provides a method to distinguish from wild type and select for transformed 32O-Y[pNW33N-vgb]. Presence of the plasmid in the transformed strain was verified utilizing PCR. Both 32O-Y and 32O-Y[pNW33N vgb] grew in minimal medium (CDM) that contained dibenzothiophene (DBT) as the sole sulfur source (while Chl was added to 32O-Y[pNW33N-vgb] cultures). DBT is a common organic sulfur-containing molecule in petroleum. It is metabolized by the dsz pathway, present in a number of soil bacteria, which produces 2-hydroxybiphenol (2-HBP) as the final product. Growth (OD600mm) of 32O-Y and 32O-Y[pNW33N-vgb] at 37°C, 45°C, 50°C were compared. The desulfurization activities of wild type and transformed Paenibacillus naphthalenovorans strains were monitored by the Gibbs assay, which measures the concentration of 2-HBP through absorbance at 610 nm. The results demonstrate that the expression of VHb in strain 32O-Y[pNW33N-vgb] correlated with increased growth, especially at 37°C. The desulfurization activities of 32O-Y[pNW33N-vgb] at various temperatures were also improved, compared to untransformed 32O-Y, as VHb was expressed. Efficiency of the VHb-related improvements at the three temperatures was calculated as the ratio between maximum 2-HBP produced and OD600nm at this point. The efficiency values indicated that when compared to 32O-Y, 32O-Y[pNW33N-vgb] had maximum increased productivity at 37°C and 50°C, and minimum at 45°C.