Optimization Of Bacterial Cellulose Fermentation And Regulation Of Metabolic Pathways

As a new kind of biological nano structured material,compared with botanical cellulose,bacterial cellulose has better advantages in chemical purity,crystallinity,water holding capacity,biocompartibility,biodegrability,adjustable physical property,and shape plasticity.Accordingly,bacterial cellulose has a spreading application prospect in food,paper making,petroleum extraction,medical,tissue engineering and fuel cells.Furthermore,as one of the hot topics on microbial synthetic material,the research on bacterial cellulose has attracted the attention of more and more scientists.Until now,the application of bacterial cellulose is only used in some production with high value added because of its low production rate and high price,which limited its commercial process and popularization.To overcome the current problems and improve the production of bacterial cellulose,we used Acetobacter gluconicum Hansenii identified by isolation and screening to optimize the culture mediums and analyze the factors for the fermentation of strains to produce bacterial cellulose,combining with the coupled genetic algorithm on artificial neural network.The optimized culture medium improves the production efficiently and reduces the cost,besides minimizing the workload and rotation period.Based on the metabolic analysis,the mechanism of the synthesis of bacterial cellulose was discussed,and the metabolic model was constructed.Also,to control the fermentation,the mechanism of the fermentation was studied by using metabolic flux analysis,protomics and metabolomics based the pathway of the strainsFirst,by using strains cultured in 24h,the culture medium was concluded by using one-way analysis and orthogonal test:sucrose 2%,yeast extract 0.3%,peptone 0.5%,KH2PO4 0.2%,MgSO4 0.015%.By one-way analysis,the fermentation status was concluded:strains cultured time 24h,inoculated concentration 7%,rotation speed 150r/min,the culture temperature 30?,and the starting pH 6.5.The orthonormal and uniform designed models by using DPS software were compared,and the latter was found better.Based on the correlation coefficient and path coefficient analysis,it was found that glucose and ethanol contribute mainly the production.The left path coefficient is 0.2488,which showed that there should be other important factors need to be considered.According to the number of the culture medium components and index optimization,the designed neural network model has a 6-5-1 topology structure.The average relative error resulted from the model test based on 5 sets of data is 0.2594%,suggesting the higher generalization capability,less error and a better convergence and fitting precision compared with the traditional regression model.Using this model as the fitness function to optimize the genetic algorithm,resulting in the best culture medium:glucose 3.9789%,peptone 0.3396%,yeast extract 0.1933%,Na2HP04 0.2230%,K2HP04 0.4565%,ethanol 2.2255%.The best production is 2.915g/L.The production by fermentation test is 2.87g/L,1.42 times higher than the original medium.The metabolic flux balance model based on the biosynthesis of bacterial cellulose was tested.With the comparsion of the flux distribution before and after the optimization,the key regulatable flexible points were found:G6P and Acetate.The flux banlance analysis suggested the reduction of HMP pathway will be necessary for the efficient production of cellulose,so how to control the ratio of r2/r5 will be the focus for the future research.The too much accumulation of acetate resulted in the waste of C source,and the analysis suggested that the byproduct may be because of the control status.The comparsion of the fermentation suggested that increasing N source or changeing C source as well as improving other growth factors could be the way for the further optimization.According to the analysis for the pathway and metabolic control points,Mg2+,sodium acetate,sodium pyruvate and sodium citrate were used to adjust the metabolic flux.The results indicated the add of 2g/L sodium citrate resulted in as high as 7%improvement of the production of cellulose,besides the final PH value as high as 3.66.The amount decreased for acetate from 5.86g to 5.22g,glycerol from 0,82g to 0.54g,and citrate from 1.21g to 1.113g.In the opposite,pyruvate increased from 1.92g to 2.41g.The phylogeny tree suggested that the adding of sodium citrate resulted in an earlier fermentation.PCA indicated that the energy and pyruvate kinase in the early fermentation affected the fermentation obviously.The enzymatic analysis indicated that the indirect biosynthesis of bacterial cellulose is mainly controled by pyruvate kinase and pyruvate dehydrogenase.The complex analysis supported the TCA cycle should be weakened,and the dissolved oxygen,pH and the substrates were the main limiting factors for the fermentation.