Metabolic Profile Analysis And Adaptive Evolution Of Clostridium Butyricum For 1,3-propanediol Production

1,3-propanediol is an important industrial raw material,which can be used as monomer for synthesis of PTT and other polymerized materials.It has important applications in polymer,biodegradable materials,military industry,clothing,medicine,cosmetics and some other industries.Compared with chemical method,bio-conversion of crude glycerol to 1,3-propanediol is more efficient through microorganism.Acted as a natural producer of 1,3-propanediol,Clostridium butyricum has been widely studied in strain screening,fermentation process optimization due to the anaerobic culture system and biological safety.However,the low tolerance of strain to by-products,low product concentration and production efficiency still hinders the industrial production process of 1,3-propanediol.In this paper,metabolomics,fermentation kinetics model and adaptive evolution technique were used to analyze the effects of by-products on growth and enhance the concentration and productivity of 1,3-propanediol.The metabolomics analysis platform was first established to analyze the metabolic profile of Clostridium butyricum under the inhibition of by-products in repeated batch fermentation process.Second,a new kinetic model which suitable for anaerobic fermentation process of Clostridium butyricum was established and used to describe the kinetic behavior of 1,3-propanediol fermentation process.Third,by using the adaptive evolution technique,in which the gradually rising concentrations of glycerol were used as the stress inducer,the inhibition of glycerol on the growth of strain and the production of 1.3-propanediol were finally reduced.At last,butyric acid,acetic acid and lactic acid were also set as stress inducer respectively to select the strain with high tolerance to each acid.Adaptive evolution technique endows strains with the performance of rapid growth,high concentration of 1,3-propanediol and co-production of 1,3-propanediol and lactic acid.The main work of this paper includes four parts as follow:First,in the present study,repeated batch fermentation coupled with activated carbon adsorption strategy was first established which conduced not only to saving the time of seed cultivation and enhancing the productivity,but also to reducing the costs for the seed cultivation to achieve the purpose of 1,3-propanediol continuous production.The concentration of 1,3-propanediol from first to fourth cycle was 42.89,45.78,44.48,42.39(g/L),and the corresponding volumetric productivity was 2.14,1.91,1.85,2.12 g/(L·h)respectively.More importantly,the metabolomics analysis platform was first established to analyze the metabolic profile of Clostridium butyricum under the inhibition of by-products in repeated batch fermentation process.Then,a relatively complete schematic diagram of the proposed metabolic pathways was firstly mapped out based on the intracellular metabolites’ analysis through GC-MS.At the same time,metabolic pathway and principal components analyses were carried out to give us deep insight into metabolic state.Many metabolites occurred to response to the stress in Cycle Ⅱ.Owing to the worsening environment,changes of protein expression and lipid accumulation resulted in the formation of resting body in the group without activated carbon in Cycle Ⅲ.Thus,it demonstrated that activated carbon provided a favorable microenvironment for Clostridium butyricum in the repeated batch fermentation process to achieve the purpose of 1,3-propanediol continuous production.Second,the present study is aimed at establishing a novel kinetic model not only based on biomass,substrate and 1,3-propanediol,but also considering the by-product concentration to describe 1,3-propanediol fermentation process by Clostridium butyricum.The simulative result of the model fit well with that in the batch fermentation process.Compared with the model reported,this new model contained some new kinetic parameters.Furthermore,the model was also used to predict the result of fed-batch fermentation process after some modifications.The predicted result of model fit well with the data in experiment when glycerol was controlled at around 5-15 g/L.Thus,a new novel kinetic model was established which could not only serve as a tool for further optimization of the fermentation process,but also be improved for some other similar processes.Third,in order to obtain the excellent 1,3-propanediol producer from wild-type Clostridium butyricum.Adaptive evolution was carried out to select the strain for high glycerol tolerance.The most significant change was that fermentation time decreased from 36(Gen 1)to 20 hours(Gen 4)after adaptive evolution.Thus,it led to the corresponding volumetric productivity of 1,3-PDO increasing from 0.97(Gen 1)to 2.14 g/(L·h)(Gen 4)which increased by 114%.The consumption rate of glycerol,synthetic rate of 1,3-propanediol and growth rate were enhanced significantly.Thus,the fast-growing strain was successfully selected by using the adaptive evolution technique.Fourth,adaptive evolution was also applied to butyric acid tolerant strain selected based on the fast-growing one through a simple equipment.Compared with the fast-growing strain,1,3-PDO concentration increased from 40.28 g/L to 66.23 g/L through fed-batch fermentation by butyric acid tolerant one.Then,Clostridium butyricum Y,which grows rapidly and does not produce butyric acid,was set as the starting strain.Clostridium butyricum Y1 and Y2 are selected by using gradually rising concentrations of acetic acid as stress inducer.Then,the high concentration of lactic acid(30 g/L)was set as stress inducer to select Y2 strain from Y3 strain.The concentration of 1,3-propanediol and lactic acid produced by Clostridium butyricum Y2 were 65.33 g/L and 28.91 g/L through fed-batch fermentation.More importantly,the concentration of lactic acid 1,3-propanediol produced by Y3 strain reached up to 40.60 g/L and 63.74 g/L.The corresponding volumetric productivity of Y2 and Y3 strain were 1.81 and 1.59 g/(L·h)respectively.Thus,the adaptive evolution could achieve the purpose of co-production of 1,3-propanediol and lactic acid in high concentration.The pathway of acetic acid could provide a large amount of NADH for the rapid accumulation 1,3-propanediol.However,when the synthesis process of acetic acid stopped,the pathway of lactic acid could provide NADH for 1,3-propanediol formation sequentially.Compared with lactic acid,acetic acid and butyric acid had a greater inhibition on the growth of strain and the production of 1,3-propanediol.It demonstrated that,adaptive evolution was an excellent strategy which could help us select the fast-growing strain and reduce the negative effect from substrate and by-products inhibition.