Biotransfermation Of Glycerol For 1,3-dihydroxyacetone Production By Resting Cells

1,3-Dihydroxyacetone(DHA)is an commercially important chemical used as raw material of chemistry,pharmaceuticals,and food additive.In this paper,the culture conditions of Gluconobacter oxydans were optimized to obtain resting cells with high specific glycerol dehydrogenase activity.Then the condition of DHA production technics biocatalysized by resting cells(including free cells and immobilized cells)was optimized.And we got better production technology.Single factor and response surface analysis were used to determine the optimal nutrient media components(g/L):mannitol 41.9,glycerol 4.6,yeast extract 5.6,K2HP04 0.5,KH2PO4 0.5,CaCO3 2.0,initial pH 4.9;culture condition:temperature 30℃,rotational speed 150rpm,culture time 20h.Under the above conditions,dry biomass was 1.14g/L,and the glycerol dehydrogenase activity was 184.±1.1 U/g,which was 208.9%higher than that before optimization.The influences of biocatalyzing conditions of free cells on DHA yield were investigated.In the process of biocatalyzing to produce DHA,alkali(NaOH)was continuously added to the reaction system,to adjust the pH of the reaction.At different pH and airflow rates,the consumption of alkali and the formation of organic acids were analyzed.It showed that the higher of pH and airflow,the more consumption of alkali and the yield of organic acids,which indicated that the biocatalytic reaction preferred to form organic acids.It was not beneficial to the formation of the target product DHA.Therefore,it was suitable for the formation of DHA at pH 5.0,airflow 0.8 L/min.The optimum biocatalyzing conditions by free cells were obtained,when the assay mixture contained 36g/L G.oxydans,50mM of potassium phosphate buffer(pH 5.0),airflow rate was 0.8L/min and the reaction was monitored at 30 ℃,it showed the highest productivity.Under the optimal conditions,glycerol was continuing added,after conversion for 70h,total adding glycerol was 190g/L,DHA yield was 163.0g/L,the conversion of glycerol reached 89.1%,and DHA productivity was 2.3g/L/h.The free cells could be reused for three cycles;after the second and third cycles,DHA yield respectively was 77.1%、47.4%of that of the first cycle(163.1g/L).We focused on entrapment method of sodium alginate and the adsorption of porous ceramic.Biocatalytic process of immobilized whole cells in sodium alginate was as follow:beads diameter 3mm,wet biomass 6%;and biocatalytic conditions:pH 5.0,airflow rate 0.9L/min.Under these conditions,the immobilized cells could be reused for five cycles,and after five cycles,DHA yield was 81.4%of that of the first batch.Immobilization of whole cells on porous ceramics was also studied.To obtain high DHA yield,some basic conditions were investigated.The preparation of immobilized cells:size of porous ceramics was 40μm,300mL medium A with 162g carriers,temperature was 30℃,airflow was 0.8 L/min,culture time for 23h;the conditions of biocatalytic conditions were as follows:phosphate buffer 200ml,pH 5.0,glycerol was continuing added,initial glycerol 20g/L,airflow 0.8L/min.Under these conditions,DHA yield was 52.5%of the first batch after five cycles.Since excessive cells were adsorbed on porous ceramics,the reuse of this method was not better than sodium alginate entrapment method,but DHA yield of each batch was more than sodium alginate entrapment method.After biotransformation bioprocess,activity of the resting cells reduced significantly,cells were regenerated through cultivation.And the regeneration conditions were as follows:mannitol 25g/L,yeast extract 7g/L,calcium ion 29/L;cells were regeneration cultured in above medium at 150rpm for 10h.Immobilized cells were regenerated,sodium alginate immobilized cells could be reused for five batches,the average DHA yield and average conversion rate of glycerol to DHA were 79.0g/L and 91.1%,respectively;porous ceramics immobilized cells were reused for eight batches,the average DHA yield and average conversion of glycerol to DHA reached 113.5g/L and 90.9%.