Microbial Production Of Dihydroxyacetone

Glycerol, a byproduct of biodiesel, was used as substrate for producing value-added chemical, dihdroxyacetone. And a high throughput screening method for dihydroxyacetone-producing bacterium was developed in this work, and it can make the screening procedure more quick and efficient. Moreover, two strains, H004 and H005, were isolated from many soil samples which was obtained around China, and were identificated as Pichia membranifaciens and Clavispora lusitaniae, respectively. They were stored at China Center for Type Culture Collection (CCTCC).The effects of temperature, liquid volume in flask, initial pH, glycerol on dihydroxyacetone fermentation by P. membranifaciens were investigated. According to the results of single-factor experiments, initial pH, temperature, fermentation time were selected as factors for response surface methodology to optimize the production condition of dihydroxyacetone. And the optimized condition for dihydroxyacetone shake flask fermentation was as follows: medium was consisted of 40 g/L glycerol, 5 g/L yeast extract, 2 g/L peptone, initial pH 6.74. 12.88 g/L dihydroxyacetone was achieved at 48.8 h of fermentation at 29.6℃, 200 rpm with 50 mL liquid medium in 250-mL flask. Furthermore, 13.57 g/L of dihydroxyacetone was obtained at 15-L fementor.Separation of dihydroxyacetone from fermentation by silica gel column chromatography was studied. At first, elution solutions in column chromatography were selected, then, the effects of the height and diameter proportion of column, sample amounts and velocity of flow on separation result were studied. The optimal separate condition was obtained that ethyl acetate and ethanol(95:5) was selected as elution solution, the height and diameter proportion of column equals to 35, sample amounts equals to 5% of column volume, and velocity of flow equals to 0.55 BV/h. Under the condition, the resolution (R_s) between glycerol and dihydroxyacetone amount to 0.922. Then dihydroxyacetone product was obtained by condensing, and crystallizing from the elution.