Optimization Of Fermentation Conditions And Biosynthesis Of 1,3-propanediol By Clostridium Beijerinckii

1,3-propanediol(1,3-PD)is an important value-added platform compound and has extensive applications in foods,pharmaceuticals and other industrial sectors.Microbial biosynthesis of 1,3-PD from glycerol has proven to be an eco-friendly,safe and effective approach.However,sustainable biosynthesis of this value-added material is challenged by high production cost and low yield.This study was done with the aim of addressing these issues using Clostridium beijerinckii CCIC 22954 as a novel 1,3-PD producer.The main research contents and results of this study are summarized as follows:(1)Fermentation conditions were optimized for C.beijerinckii CICC 22954 to reduce the medium cost and enhance the 1,3-PD production.The results showed that replacing the costly ingredient,yeast extract,from the reinforced clostridial medium(RCM)with low-cost corn steep liquor(RCM-CSL)neither significantly affected the final yield of 1,3-PD nor increased the formation of the by-products.Subsequently,the fermentation conditions were optimized using the single-factor-at-a-time approach.The optimal conditions were as follows:pure glycerol(PG)concentration 25 g/L,temperature 37°C,p H 7.5 and CSL concentration 7.5 g/L,under anaerobic condition and without agitation for 48 h.Eventually,batch fermentation using the optimal conditions led to a final 1,3-PD titer of 14.3 g/L,with the yield of 0.57 g/g glycerol and productivity of 0.3 g/L/h.The final titer of 1,3-PD obtained from RCM-CSL showed a 28%increase when compared with that obtained from RCM(11.1 g/L).(2)Next,an attempt was made to further reduce the cost of the fermentation process by replacing PG with the low-cost crude glycerol(CG).C.beijerinckii CCIC 22954 was able to convert 25 g/L glycerol in CG into 15.2 g/L of 1,3-PD with a yield of 0.61 g/g glycerol,showing an increase of 6.2%and 7.0%,respectively,when compared to PG.The cause of this increase was further investigated by developing a mixture that had similar composition to that of the CG.This mixture was obtained by individually adding methanol,Na Cl or oleic acid into PG.Addition of low concentration of Na Cl(?10 g/L)or oleic acid(2.5 g/L)could promote cell growth and increase the titer of 1,3-PD.Combination of 10 g/L Na Cl and 2.5 g/L oleic acid increased the 1,3-PD titer to 15.2 g/L,whereas the addition of high concentrations of methanol(?10 g/L)decreased cell growth and 1,3-PD production.However,the combination of methanol with the other impurities led to the alleviation of its inhibition effect due to the promoting effect of the other impurities.Therefore,it was preliminarily shown that CG with lower concentrations of methanol impurities could be used to replace PG in the biosynthesis of 1,3-PD.(3)The fermentation conditions were further optimized using the response surface methodology(RSM).The results showed that the statistical significance of the quadratic regression equation was highly significant(P?0.0001),the lack of fit was not significant(P?0.05).The R-squared(R~2)value was shown to be 0.9766,indicating that the equation was a reflection of the relationship between each factor and the response value.The optimal conditions for each factor were temperature 37°C,p H 7.0 and 30 g/L glycerol in CG,which led to the actual final 1,3-PD titer of 19.8 g/L and yield of 0.66 g/g glycerol.The titer of 1,3-PD showed an increase of 30.2%,when compared with the titer obtained from the CG under the previous optimum conditions(15.2g/L).(4)Finally,the co-fermentation of glycerol with individual sugars(glucose,arabinose,xylose and sucrose)was done to increase the concentration of 1,3-PD.The results showed that using an appropriate proportion of CG and individual sugars could improve the biosynthesis of 1,3-PD.It was observed that a concentration(30 g/L)of glycerol in CG and a glucose concentration of 10g/L proved to be the best co-substrates,leading to a 1,3-PD titer of 23.3 g/L and yield of 0.78 g/g glycerol.When compared to the 1,3-PD titer of CG alone obtained from the optimization by RSM,the co-fermentation approach increased the 1,3-PD titer and yield by 17.7%and 18.2%,respectively.