Selective Bio-oxidation Technology Research Of Glucose From Lignocellulosic Enzymatic Hydrolysis

Gluconobacter oxydans can incompletely oxidize a variety of substrates relying on membrane-bound dehydrogenases.It has been widely used in the industrial production of a variety of chemicals.Due to its well resistance to lignocellulosic degradation products,Gluconobacter oxydans has attracted wide attention in the field of lignocellulosic-based biorefinery.In this study,the high value utilization of lignocellulosic resources was taken as the goal,and Gluconobacter oxydans was used as the fermentation microorganism to achieve the directed bio-oxidation of lignocellulosic hydrolysate to produce saccharic acid through the regulation of condition factors and environmental factors.Moreover,the regulation mechanism was analyzed by enzymology,and the main research results were obtained as follows.(1)Effects of p H on glucose metabolism of Gluconobacter oxydans.Three different p H neutralizers were selected to explore their effects on glucose metabolism.It was found that the types of p H neutralizer had no significant difference in the experiment.By studying the effects of different p H environments on Gluconobacter oxydans glucose metabolism,it was found that p H had no significant effect on the growth of Gluconobacter oxydans,but low p H environment could inhibit glucose metabolism,and the activity of gluconic acid dehydrogenase was significantly inhibited under low p H environment.The p H of the fermentation broth decreased rapidly during the glucose metabolism of Gluconobacter oxydans,therefore,the production of2-ketogluconic acid can be effectively inhibited by not adjusting the p H.Gluconic acid production by fermentation with corncob enzymatic hydrolysate,without the addition of p H neutralizer,could be fermented at a maximum concentration of 100 g/L with a yield of 90.9%.(2)Study on the strategy of producing gluconic acid by fermentation of high concentration glucose enzymatic hydrolysate The optimization of the initial inoculum was concluded that 2OD was the optimal inoculum.In the optimization experiment of initial glucose concentration,the higher substrate concentration would inhibit the biocatalytic effect of Gluconobacter oxydans.Three kinds of bioreactor models were established.The gluconic acid production efficiency was significantly improved in the compressed-oxygen-supply sealed-stirredbioreactor system,and there was no large quantities of oxygen consumption problem in the pure oxygen-supply aerated-stirred-bioreactor system.When fermentation was carried out with lignocellulosic materials,the viscosity increased sharply with the increase of the concentration of the enzymatic hydrolysate.When the glucose concentration of the enzymatic hydrolysate exceeded 240 g/L and the viscosity exceeded 4 m Pa·s,the catalytic efficiency decreased significantly and the reaction rate decreased by more than 20%.This study pretreated the concentrated corncob enzymatic hydrolysate(glucose concentration 240 g/L)with 6% activated carbon,which effectively reduced its viscosity(from 4.17 m Pa·s to 3.52 m Pa·s),and finally fermented the enzymatic hydrolyssate with activated carbon pretreatment in a compressedoxygen-supply sealed-stirred-bioreactor for 36 h.The gluconic acid yield reached 231.2 g/L with a yield of 95.3%,and the average volume productivity was 9.63 g/L/h.(3)Production of 2-ketogluconic acid by acetic acid-mediated enzymatic hydrolysate fermentation.HAc,the typical inhibitor in the lignocellulosic hydrolysate,reduces the metabolic rate of glucose,but at the same time,inhibits the intracellular transport of gluconic acid and increases the yield of 2-ketogluconic acid.It was found that the addition of acetic acid had little effect on the enzyme activity of glucose dehydrogenase,but significantly increased the activity of gluconic acid dehydrogenase.Through the optimization experiment,it was concluded that it was more feasible to use 2 OD inoculum in the production of 2-ketogluconic acid.The optimal experiment of acetic acid concentration concluded that adding 5.0 g/L acetic acid was the most suitable for 2-ketogluconic acid fermentation with corncob enzymatic hydrolysate.Under regulation of acetic acid,after 48 h fermentation,the concentration of 2-ketogluconic acid was 44.6 g/L with the yield of 83.5%,which was similar to that of pure sugar medium.It has important theoretical significance for the directed production of 2-ketogluconic acid from lignocellulosic materials.