| As a novel probiotics,Clostridium butyricum has attracted widespread attentions in the fields of feed industry,clinic and energy industry for its properties such as regulating intestinal balance,improving immunity,anti-tumor and producing hydrogen as well as 1,3-propanediol.In order to improve the fermentation biomass,the fed-batch strategies in 10-20 L bioreactor were optimized basing on the features of growth and metabolism of C.butyricum.The results were shown as follows:(1)The effect of sodium citrate on the growth and metabolism of C.butyricum in 10-L bioreactor has been investigated.The results showed that the addition of 4 g/L sodium citrate promoted cell growth with a maximum biomass of 5.62×10~8 CFU/m L at 6 h which was increased by 19% than the control(11 h,4.72×10~8 CFU/m L).Meanwhile,the maximum production of butyrate was decreased by 18.9% and the sporulation was delayed over 2 h.The pyruvate concentration was only 57%~97% of the control,while the succinate content was 1.23~1.94 times than the control during 4~8 h.In additon,the maximum α-ketoglutarate production was 10.9 mg/L which was not detected in the control.At the same time,aromatic amino acids,histidine,branched chain amino acids,glutamate and aspartate was increased by 1.79%~58.02%,9.52%~71.78%,1.98~110.22%,4.18%~151.58% and 8.88%~33.24% compared with the control,respectively.These results suggested that the cellular carbon metabolic flux was redistributed with citrate addition by inhibiting the pyruvate synthesis.Meanwhile,the productions of α-ketoglutaric acid and succinate were increased which was the product of TCA oxidation branch and reduced branch,respectively.The synthesis of aromatic amino acids,histidine,branched-chain amino acids,glutamate and aspartate was also increased,respectively.The batch culture with additions of 16 g/L glucose and 8 g/L citrate was investigated basing on the results above.The maximum biomass of C.butyricum reached 8.78×10~8 CFU/m L at 9 h which was increased by 43% and 56.23% than the single factor addition culture of glucose(6.13 × 108 CFU/m L)and citrate,respectively.Also,it was enhanced by 86.02% compare to the control.(2)In order to delay the sporulation and improve the growth efficiency of C.butyricum,the effects of guanine(0.2 g/L)and Ile(0.5 g/L)on the growth and acids metabolism of C.butyricum in 20 L bioreactor were investigated.The results showed that the maximum biomass with the addition of guanine and Ile reached 5.31×10~8 CFU/m L and 4.65×10~8 CFU/m L,respectively,which was increased by 22.91% and 7.64% than the control(4.32×10~8 CFU/m L).The coefficient of butyrate to maximum biomass(Yp/x)was decreased by 48.64% and 40.54% than the control(0.37 g/g),respectively.Guanine and Ile addition could reduce the specific butyrate productivity,enhance the growth efficiency and delay the sporulation of C.butyricum.The biomass reached 5.4×10~8 CFU/m L at 6 h when guanine(0.2 g/L)and Ile(0.5 g/L)were simultaneously added to the fermentation medium,which was 25% higher than the control.Therefore,both guanine and Ile additions could facilitate cell growth by reducing the butyrate synthesis efficiency,moreover,guanine addition was the major factor to enhance the growth efficiency and delay the sporulation of C.butyricum.(3)The effects of different ORP levels(-50 m V~-115 m V)and exponential feeding strategy on the growth and metabolism of C.butyricum were studied in the 20 L bioreactor.The results showed that the biomass controlling ORP at-75 m V was the highest among the batch cultures,reaching 6.65 g/L(5.11×10~8 CFU/m L)at 8 h,which was 16.87% higher than that of the ORP uncontrolled fermentation(5 h,5.69 g/L,4.29×10~8 CFU/m L).Meanwhile,the butyrate production was also reduced by 25.38% relative to the control.The exponential fed-batch culture with the μset between 0.07~0.24 h-1 during 5~9 h were conducted basing on the batch culture maintaining the ORP at-75 m V(the control).Besides the maximum biomass with the μset of 0.07 h-1 was decreased by 3.3% than the control,that of 0.12 h-1,0.18 h-1 and 0.24 h-1 was increased by 10.73%,35.94% and 20.6% than the control,respectively.The optimal μset was found to be to 0.18 h-1 whose maximum biomass reached 9.04 g/L(7.1×10~8 CFU/m L)at 9 h that was 58.88% higher than the control.During the exponential feeding period,the coefficient of cell to glucose consumed(Yx/s)was 0.55 g/g that was increased by 71.88%,3.8% and 10% than that of 0.12 h-1,0.18 h-1 and 0.12 h-1,respectively,which improved the utilization efficiency of glucose. |
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