Study On High Concentration Dihydroxyacetone Synthesis With Gluconobacter Oxydans

1,3-Dihydroxyacetone (DHA) is a commercially important chemical, which is mainly produced from glycerol through a microbial fermentation process with Gluconobacter oxydans.In this study, a strain of capable of producing DHA was isolated and then identified as a stain of the genus Gluconobacter. It was firstly proved that the synthesis of DHA can be independent on viable cells. Subsequently, the fermentation conditions of this bacterium were optimized, and then the optimization of the activity and stability of GDH were carried out in this work. Based on this work, a two stage fermentation stratagy was determined and the enzymatic process of DHA biosynthesis in a5-L fermenter was studied.The results were showed as follows:1. The isolation and identification of DHA producing strain:A bacterium strain named3-9which can produce DHA was isolated after enrichment culture and streaking plate purification. Based on its morphological studies, physiochemical identification,16S rDNA sequencing and phylogenetic analysis, this isolate was identified to be Gluconobacter sp.2. The study on the relationship of alive cells and GDH:By the traditional fermentation process analysis, DHA tolerance study and ultrasonic broken test, it was verified that the GDH of dead cells can catalyze the synthesis of DHA and GDH was more stable than living cells under high concentration DHA.3. The enzyme fermentation conditions:Based on the studies for the conditions of glycerol dehydrogenase (GDH) production, the optimum fermentation conditions were established with the composition of the medium of glycerol50g/L, K2HPO40.1g/L, KH2PO40.9g/L, yeast extract7.5g/L at pH5.5and culture temperature of27.5℃.4. Optimization of the activity and stability of GDH:Response surface methodology was applied to optimize the conditions of GDH activity and stability.With the parameters of a pH of4.61, a temperature of33.3℃and the concentration of glycerol at80.0g/L, the optimum predicted GDH activity is2.24U/mL.To GDH stability, the interactive effects of the studied factors were obvious. GDH was stable at high concentrations of glycerol and low concentrations of DHA. However, at low temperatures, GDH was found to be relatively stable even in high concentrations of DHA. It was also found that the optimum pH was affected by the DHA concentration or temperature.5. The fermentation process of DHA biosynthesisA two-stage process of enzymatic transformation was developed in a5-L bioreactor that separated the growth of G. oxydans and the biotransformation process.This methodology showed high stability of GDH in a136-hour transformation process and elevated DHA production from approximately100g/Lobtained from traditional fermentation methods to as high as286.2g/L.