Study Of 1,3-dihydroxyacetone Product By Biotechnology

With the depletion of petroleum resources, petroleum refining is being replaced by biorefining gradually. To solve the problems during the production of 1,3-dihydroxyacetone (DHA) from glycerol by biorefinery such as low initial substrate concentration, low production efficiency, complicated subsequent extraction process, low DHA yield and so on, the whole production technology of DHA from glycerol by Gluconobacter oxydans has been researched in this work.In order to increase the DHA production capacity of G. oxydans from glycerol, He-Ne laser was used to irradiate G. oxydans and among the dose range of 21 mW 19~21 min, higher positive mutant rate could be obtained. After the tolerance experiment of glycerol and DHA, mutant G. oxydans GM51 with high DHA production capacity and stable heredity was obtained finally. By comparison the performance of mutant GM51 and wild strain, the activity of glycerol dehydrogenase in GM51 was 75.17% higher than that in wild strain when the initial glycerol concentration was 100 g/L. The culture cyclic of mutant GM51 had been shortened by 10~15 hours and the yield of DHA had been increased by 77.6% during the culture in 7 L fermenter. The DHA productivity had been improved from 1.29 g·L-1·h-1 to 2.29 g·L-1·h-1.Based on the experimental results of single factor, response surface methodology was used to determine the optimal fermentation medium composition (g/L): yeast extract 2.39, peptone 2.18, glycerol 100.0, (NH4)2SO4 2.0, MgSO4·7H2O 1.0, K2HPO4·3H2O 2.62, MnSO4·1H2O 0.53, CaCO3 2.5, CaCl2 1.25; inoculum concentration 5% (v/v), initial pH 5.9, rotational speed of the cradle 210 rpm, culture time 45 h. Under the optimal conditions, the yield of DHA was 82.81 g/L by mutant GM51, which was 41% higher than that before optimization.The characteristics of batch, semi-fed-batch and fed-batch fermentation in a 7 L bioreactor were investigated. In the late phase of the batch fermentation, DHA biosynthesis would be repressed due to the exhaustion of nutrition substances. Semi-fed-batch could improve the substrate supply and increase DHA productivity. Fed-batch fermentation could prevent the dramatic transient phenomenon in fermentation environment aroused by semi-fed-batch culture; therefore the DHA yield was further increased by about 36% to 123.8 g/L. A proper kinetic model for cell growth, substrate consumption and DHA formation by mutant GM51 was established and it could predict the variation tendency of biomass, substrate and product correctly.Natural macromolecular flocculant—chitosan was used to remove cell and protein and absorbent charcoal was used to remove the pigment in the fermentation broth. Protein, nucleic acid, polyoses and salt were removed further by alcohol precipitation. The experimental results indicated that 100% cell and 98.6% protein were removed respectively through the above process, and most salt was separated and removed by crystallization with 96.4% decrease of the total conductivity. Meanwhile, DHA loss was 8.6% during the process. After crystallization in alcohol, the crystallization yield of DHA was 81%. By using the above separation technology, the yield of DHA was more than 72% and the purity of DHA was up to 99.8%. And the DHA product produced in this work has been met the quality requirements compared with DHA of commercial grade.