Kinetic Analysis Of Curdlan Production By Alcaligenes Faecalis Based On Different Carbon Sources

Curdlan,a water-insoluble extracellular ?-(1,3)polysaccharide,is produced mostly by Agrobacterium sp or A.faecalis under nitrogen-limitated conditions.It has widely applied in food and pharmaceutical industries for its unique rheological and thermal gelling properties.The carbon sources have significant effects on curdlan production,but hardly any efforts has been focused on the biosynthesis mechanism of A.faecalis.Therefore,this study investgated the effects of carbon sources on the mechanism of curdlan biosynthesis from the metabolic kinetic analysis and micro-metabolism analysis,and optimized the fermentation technology.In this paper,we first analyzed the differences of four carbon sources(maltose,sucrose,glucose and fructose)from cell concentration,curdlan production,residual sugar concentration,specific growth rate(?),carbon dioxide emission rate(CER),and oxygen uptake rate(OUR),and we found the highest production(39.3 g/L)and productivity(0.5 g/L/h)with maltose as the carbon source.The maximum specific growth rate(0.44/h)and the growth-associated rate constant(2.05 g curdlan/g cell)were achieved under maltose condition according to kinetic parameters used by Logistic and Luedeking-Piret equations.The values of the growth-associated rate constant(a)and non-growth-associated rate constant(?)for curdlan production were not zero,suggesting that curdlan biosynthesis is partial growth-associated process.Further according to the changes of residual sugar on hydrolysis process of sucrose and maltose fermentation,fructose accumulation and low glucose concentration from maltose were observed.It is suggested that fructose is a poor-performance carbon source and slow-releasing glucose from maltose boost cell growth and curdlan production.Secondly,in order to understand the physiological metabolic properties of A.faecalis,chemostat cultivations were conducted with different dilution rates ranging from 0.05 h-1 to 0.32 h-1.Then kinetic analysis were carried out on the cell growth,curdlan biosynthesis,and glucose consumption by the classic model of Monod,Luedeking-Piret,and Herbert-pirt equations.In addition,the curdlan production characteristics under the batch culture and the chemostat condition were further compared by the specific production rate qp and the specific productivity DP.The results demonstrated that qp and DP of chemostant culture were almost 3 times higher than that of batch culture.A metabolic network of A.faecalis was further established to analyze the micro-metabolic flux distribution and the difference with maltose and glucose as the carbon source.The statistical analysis revealed that the EMP pathway originated from glucose and HMP+ED pathways from 6-P-G were 33.3%and 37.8%higher than that with maltose as the carbon source,respectively.While the G-6-P flux to curdlan biosynthesis was 31.4%lower than that of maltose condition.The change in the metabolic fluxes distribution of EMP,HMP,and ED could regulate the balance of coenzyme ?NADPH supply,so as to harmonize the precursor material generation for cell growth under different carbon substrates.With maltose as the substrate,the flux of NADPH,FADH2,NADH and other energy substances flow to the respiratory chain for ATP generation,which was 23%higher than that of glucose condition.The sufficient conversion ATP to UTP is the key bottleneck for rapid UTPG formation and high curdlan biosynthesis rate.