Studying On The Bioconversion Of Glycerol Into Dihydroxyacetone By Gluconobacter Oxydans

Dihydroxyacetone (DHA) is the simplest of all ketoses , and it is biodegradable and biocompatible. The normal form is a dimer which is slowly soluble in one part water and 15 parts ethanol. When freshly prepared, it reverts rapidly to the monomer in solution. The monomer is very soluble in water, ethanol, diethyl ether and acetone. DHA and its derivatives can be applied in several industral fields such as food, cosmetics and medicine. Therefore, it is interested in how to produce DHA by mico-organism fermentation.In this paper, we studied how to screen the Gluconobacter oxydans mutant of high yield of DHA, and it was investigated that the process of producing DHA by shaking flask and 10 L fermentator. The preliminary technique of isolation of DHA and characteristics of glycerol dehydrogenase was researched.Gluconobacter oxydans (G50) was mutagenized by ultraviolet-ray and ⁶⁰Coγ-irradiation. A high yield mutant GUR186 was isolated from irradiated mutant clones. Tested by several generation cultures the isolated mutant Bacillus GUR186 was genetic stability of producing DHA.Compared with the DHA yield of the original strain G50, the mutant GUR186 was increased 59.2 %.In the shaking inoculation the culture of GUR186 for ferment seed and conditions of shaking fermentation were studied. The single-factor design was used to determine the ranges of each related factor, which may effect on the productivity of DHA, then used it to evaluate the influence of related factors on the productivity of DHA. The result showed that the optimal culture medium was as follow: glycerol 14%, yeast extract 0.5%, CaCO₃ 0.7%. More than 12.4g of 100ml broth was produced by mutant GUR186 in the optimal cultivation condition for shaking incubation 2 days.In 10 L bioreactor, the maximum concentration of DHA were reached at 400 rpm of agitation speed, 1:0.75 per minute of aeration and 30℃fermentational tempreture after inoculating 44 hs. The DHA concentration was 13.2g of 100ml broth.The flocculating effect of the active carbon and an ion polyacrylamide on impurity such as bacteria and pigments in the broth was studied in this paper. Adding 50mg of flocculating agent into 1000 L broth and 3g of active carbon into 100 ml broth and the mixture was stirred at 20-40 rpm at 60°C for 10 min. By filtering the pure pre-treated broth was gotten. The result of extraction DHA test sindicated that it does not effectively separate DHA from pre-treated broth by the method of physical and dissolvant extraction does. However, a process coupling the reversible reaction of DHA with acetaldehyde and a synchronous extraction of the product by toluene can be effectively separated DHA in the acid resin catalysis condition. The collection rate of DHA was 82%.Glycerol dehydrogenase of Gluconobacter oxydans was purified by DEAE Sepharose—Fast Flow exchang chromatography and Sephacryl S—200 chromatography. The optimum temperature and pH of the enzyme activity were 30°C and pH 6 respectively.