Study On Metabolic Mechanism Of Clostridium Tyrobutyricum And Its Application For Butyric Acid Production

Butyric acid and its derivatives have been considered as important chemical raw materials,which had been widely used in chemical industry,foodstuffs,pharmaceutical,environmental,chemical energy fields and feed additive.Nowadays,chemical synthesis and fermentation are the mainly production methods of butyric acid.However,the demand for the bioproduction of butyrate via microbial fermentation is high because of the strong consumer and manufacturer preference for bio-based,natural ingredients in foods,cosmetics,and pharmaceuticals.The increasing severity of environmental problems along with the expense and resource constraints associated with petroleum use have also contributed to demand for bio-based butyrate.In this paper,the butyric acid production with Clostridium tyrobutyricum and synthesis mechanism has been studied by genome comparison and metabolic flux analysis.Addtionally,the probioties of Clostridium tyrobutyricum was prepared and the processes had been optimized.First,the butyric acid systhesis mechanism with Clostridiumtyrobutyricum by genome comparision and metabolic flux analysis was studied.Compare Clostridium tyrobutyricum ZJU 8235 with Clostridium tyrobutyricum ATCC 25755 genome,the results indicated that there is no change in the key genes of pathway for acetic acid systhesis,and the same results account for enzymes of pathway for butyric acid systhesis.The 13 C metabolic flux analysis show that glucose is mainly directed to pyruvate via the EMP pathway in both wild-type and mutant-type,it could promote butyric acid formation when the value of p H increase within limits,because it could promote the active of EMP pathway to increasing the production of NADH and the carbon flux fraction to butyrate,and thus increase the production of butyric acid.Compare with C.tyrobutyricum ATCC 25755,the production of butyric acid was significantly improved(p < 0.05)in C.tyrobutyricum ZJU 8235,but there was no significantly improved(p > 0.1)in central carbon metabolism between butyrate and acetate formation when knockout the gene of pta in C.tyrobutyricum ZJU 8235,it indicated that the formation of acetate and butyrate is related,but the association is not completely depend on the central carbon metabolism,which may be related to cofactor supplementary in the whole metabolism.Then,Carboxyl functioned magnetic nanoparticles(CMNPs)were prepared by chemical co-precipitation and oleate modification methods,and then immobilized with Clostridium tyrobutyricum for theproduction of butyric acid.The immobilization conditions were systematically optimized to achieve maximum efficiency and the highest cell relative activity,in which the mass ratio,immobilization temperature and time were determined as 0.72 g/g,35 oC and 30 min,respectively.The endurance of CMNPs-cells was also evaluated in a repeated-batch mode of eight continuous batches.Stable and reliable production of butyric acid was obtained in each batch with an average butyrate titer and yield of ~11.02 g L-1 and ~0.43 g g-1 utilized sugar.Compared with the free-cell fermentation(concentration: 9.2 g/L,yield: 0.41 g/g),higher butyrate yield and concentration were obtained with immobilized cells of C.tyrobutyricum in repeated-batch fermentation,suggesting that the coated cells did not experience a mass transfer problem.Finally,we explored the spray dry process of fermentation broth by spray dryer(Labplant,UK).We optimized the process parameters(concentration of cells,inlet temperature,feed rate)and protectant concentration(non-fat powdered milk,maltodextrin)and achieved the optimum conditions: concentration of cells was 24 g/L(dcw),inlet temperature was 110 oC,feed rate was 240 m L/h,the concentration of non-fat powdered milk and maltodextrin was 7% and 6%,respectively.The number of living bacterium in microcapsules was 7.52 ×108 CFU/g,then we tried the prepatpration of premix and table for application in feed.