The Oxidation Of Glycerol And Benzyl Alcohol By Gluconobacter Oxydans Based On The Characteristic Of The Membrane-bound Dehydrogenase

Gluconobacter oxydans is known for its incomplete oxidation of a wide range of carbohydrates and alcohols in a bioprocess. The corresponding oxidative products are secreted almost completely into the medium. In this paper, the processes have researched for production of dihydroxyacetone (DHA) and benzaldehyde by the resting cell of G. oxydans. The DHA was be used widely in the cosmetic as sunless tanning tangent, and the benzaldehyde is the most important aromatic aldehyde in the industry.In the first section, the oxidation of glycerol to DHA by G. oxydans DSM 2003 based on the membrane-bound glycerol dehydrogenase was performed.A gas chromatographic method that accurately measured glycerol and DHA from fermentation broth is described. The method incorporates a sample derivatization reaction using N-methylimidazole as catalyst in the presence of acetic anhydride. The resulting derivatives were separated on a DB-5 capillary column and flame ionization detector. The present method exhibited a good linearity at the concentration range from 1 to 100 g/1 with regression coefficients of 0.9997 and 0.9998 for DHA and glycerol, respectively. The limits of detection were 0.025 and 0.013g/l. The recoveries of DHA and glycerol for fermentation sample were in the range of 95.37-98.76% with RSD below 1%.A comparative experiment has done for production of DHA between fermentation and resting cell bioconversion from the obtained dry cell weight (DCW), productivity and the cell physiological state. The results showed the conversions have the advantage than fermentation in the all aspects by resting cell.In the shake flask reaction, the productivity of DHA was no more than 30g/l under the optimization condition. To enhance DHA production, the process of bioconversion was improved in 3.7L bioreactor based on the optimized parameters in shaking flasks, by the optimization,100-120g/l of glycerol was converted completely to DHA by 5-8g/l DCW in 8-16h. The inhibition concentration of DHA was founded beyond 100g/l, and the critical concentration of DHA of reaction ceased was about 140g/l.The scale up process was operated in the 300L fermentor for cell culture and 30L fermentor for bioconversion of glycerol. It could be obtained the 8.5g/l DCW in the 300L fermentor, compared to the shaking flask and 30L seeds fermentor, the 164.5and 13.1%DCW could be improvement. In the 30L bioreactor for resting cell conversion, the initial velocity reached 15.2g/l, compared to the 3.7L, the 17.4% of improvement has been obtained. The nine batch bioconversion was completed in the 30L bioreactor, the conversion rate reached about 98% and the total concentration of glycerol converted was 850g/l.The resting cell physiological state was studied by flow cytometry, the results showed the influence of DHA on the cell physiological was smaller than catalytic activity of glycerol dehydrogenase In the second section, bioconversion of benzyl alcohol to benzaldehyde by G. oxydans M5 based on the membrane-bound alcoholdehydrogenasewas studied.The selective oxidation of primary alcohols to the corresponding aldehydes is one of the most important transformations in fundamental and industrial research owing to the carbonylic group as a versatile building block.The mutant strain defective in membrane-bound alcohol dehydrogenase (ADH) gene (GOX1067-1068) and the complementary strain were constructed and the results of oxidation of benzyl alcohol showed that the ADH was the key enzyme responsible for oxidation of benzyl alcohol to benzaldehyde in G. oxydans M5. The mechanism of oxidation benzyl alcohol by ADH is sequential ordered Bi Bi, and O2 is the leading substrate.The mutant strain with disruption of membrane-bound aldehyde dehydrogenase (ALDH) gene (GOX0585-0587) was be used for production of benzaldehyde. The gene strain (67.3%) showed obvious improvement for the selective toward benzaldehyde than the wild strain (2.8%), the 5g/l benzyl alcohol was be converted completely to benzaldehyde in the aqueous/isoctane biphasic system within 1h, the selectively toward benzaldehyde reached 100%.To avoid the organic solvents toxicity to resting cell, the immobilized cell was applied in the biphasic system for production of benzaldehyde. The response surface methodology was used for optimization the factors of alginate concentration, the cell load, diameters, the best results were 2.55% (w/v),49.26mg/ml,2mm, respectively. By the immobilized, the tolerance of cells has marked improvement. By ten recycles, the activity of immobilized cell has still retained 53.2%, but the free cell has only 15.7%.The oxidation benzyl alcohol was performed by resting cell of G. oxydans M5 under the anaerobic conditions using the p- benzoquinone accepted hydrogen. The results indicated the selectivity for benzaldehyde was reached 100% in the aqueous.