Production Of Dihydroxyactone From Biodiesel Byproduct Glycerol By Biotechnological Method

Glycerol is available in abundance because of huge supply from biodiesel manufactory as the byproduct. It’s necessary to find new usage and markets for glycerol. 1,3-dihydroxyacetone, a value-added product from glycerol by biotechnological method, is applied extensively to the cosmetic industry and serves as a versatile building block for organic synthesis of a variety of fine chemicals.A high throughput screening method for dihydroxyacetone-producing bacterium by cultivation in a 96-well microtiter plate was developed in this work. About more than eighty strains were isolated form soil samples with this screening method. Four isolates were identified as Gluconobacter oxydans, Bacillus licheniformis, Clavispora lusitaniae, Pichia membranifaciens, respectively. They were stored at China Center for Type Culture Collection (CCTCC).A stable mutant strain G. oxydans ZJB09112 with high production yield was screened by UV mutangesis and Ion beam implantation. The results from shake flask fermentation showed that mannitol was the optimal carbon source for cell’s ability of dihydroxyacetone production. Yeast extract, peptone and corn steep were favorable nitrogen sources for dihydroxyacetone production, and yeast extract was the optimal nitrogen source. Low initial pH range (4.0-6.0) and high KLa might favor dihydroxyacetone production.Pulse-feeding fed batch fermentation was carried out in this paper, and the resulted showed that the optimal bioprocess for dihydroxyacetone was as following: 182.5 g/L of total glycerol concentration and four times of feeding were employed at experiment, and the maximal dihydroxyacetone concentration reached 161.9±5.9 g/L at 68 hour fermentation.The effects of dissolved oxygen (DO) and pH on dihydroxyacetone fermentation were investigated. DO-stat fed-batch fermentation with pH-shift control strategy was the optimal bioprocess for dihydroxyacetone production. The DO-stat fed-batch fermentation was carried out at 30°C with a DO concentration of 30 % of air saturation, and the culture pH was adjusted to pH 6.0 automatically within the first 20 h and then shifted to pH 5.0 until the end of the fermentation. The optimal dihydroxyacetone concentration of 175.9±6.7 g/L was obtained at 72 h of DO-stat fed-batch fermentation in a 15 L fermentor.According to the results of lab-scale fermentation, scale-up of dihydroxyacetone fed-batch fermentation in 500 L and 5.0 M3 fementors were carried out. It is successful to produce dihydroacetone firstly at large-scale in China, and the product of dihydroxyacetone is selling. 254.1 g/L of dihydroxyacetone was achieved after 72 h of fed-batch fermentation at 5.0 M3 fementor. The dihydroxyacetone concentration and product yield reported in this work are higher than those reported previously.The fermentation kinetics for dihydroxyacetone production was investigated in this paper. The bioprocess could be separated as three stages, and the kinetics models were as follows:Batch fermentation kinetics for the first stage (0-14 h): Fed-batch fermentation kinetics for the second stage (10-24 h):Fermentation kinetics for the third stage (24-72 h):After fermentation, whole cells were harvested by filtration with member, and used as biocatalysts for dihydroxyacetone production. The optimal temperature and pH for whole cell biocatalysis was 30°C and 5.0, respectively. The kinetic properties conformed to Michaelis-Menten, and Vmax=0.193 mmol/L/min,Km=21.89 mmol/L. The optimal process for dihydroxyacetone production by whole cell biocatalysis at bubble column was as follows: biomass content was 7.4 g/L, airflow aeration was 1.1 vvm (volumes of air/effective volume of reactor/minute), initial glycerol concentration was 10 g/L and fresh glycerol was feeding with the rate of about 0.4 mL/h, pH 5.0, temperature 30°C, dihydroxyacetone concentration of 61.2 g/L was achieved after 66 h biocatalysis with whole cell. Glycerol metabolism in G. oxydans was investigated in this work. Except of dihydroxyacetone, formic acid, acetic acid, succinic acid, pyruvate, malic acid, fumaric acid, and ketoglutaric acid were tested in broth, it means that parts of dihydoxyacetone can be metabolized by phosphokinase through glycolysis pathway and tricarboxylic acid cycle. However, 3-hydroxypropionaldehyde and 1,3-propanediol did not exist in broth, it means that glycerol dehyratase and 1,3-propanediol oxidoreductase do not exist in cell, and glycerol can’t be reduced.Metabolic flux was analyzed during fermentation, the results showed that glycerol was mainly used for cell growth coupling with formation of dihydroxyacetone, and some byproducts, formic acid, acetic acid, succinic acid and ethanol. These byproducts biosynthesis ceased after 30 h fermentation. Effects of airflow on metabolic flux were investigated, and the results showed that high airflow aeration benefit for formic acid and acetic acid formation, and unbenefit for succinic acid and ethanol formation. After cell growth cease, most glycerol was converted to dihydroxyacetone, and little glycerol was used for other metabolic pathways.