| L-lactic acid is one of the important natural organic acids.It is widely used in food,medicine,chemical industry and other fields.L-lactic is used as an important acid agent and preservative in food industry.In recent years,the biodegradable polylactic acid,which is synthesized by lactic,has led to the emerging field of lactic acid consumption and resulting in a flourishing market demand for high optical purity lactic acid,especially for L-lactic acid.Compared with the traditional lactic acid production method,biofilm method has great advantages,such as simple immobilization process,high cell density,and strong tolerance.Therefore,it has been considered as one of the most promising tool for lactic acid production.Fiber membranes with high specific surface area and high porosity are ideal materials for bacterial adhesion and biofilm formation due to the perfect three-dimensional space which is benefit for bacteria living and reproduction.Lactobacillus paracasei(LPA)is widely existing in human gut and traditional cheese products.It is a good strain for producing L-lactic acid.In this study,the LPA biofilms on the Cellulose acetate nanofiber(NCA)were studied and the LPA biofilm reactor was constructed.The culture condition,cell activity,biofilm stability,resistance properties,and L-lactic acid producticity of the LPA biofilm were characterized in detail.The relative work was divided into the following aspects:1.Regulation of lactobacillus paracasei biofilmThe influence of fiber membranes on the formation of LPA biofilm were studied and the most suitable material,NCA,was screened from different materials.The effects of culture time,inoculation amount,and nutrient on the formation of LPA biofilm on NCA were characterised.The results demonstrated that the dry weight of biofilm increased to the highest value after three day culture,and then kept constant.There was no significant effect of inoculation amount on the dry weight of biofilm.The sucrose and Mg2+ had a little improving effect on the formation of LPA biofilms on the NCA.2.Structure and physiological characteristics of lactobacillus paracasei biofilmVarious methods were adopted to characterize the chemical and physical structures of LPA biofilms on NCA.The number of viable cells was studied via viable count method.The viable rate of LPA in different layers of LPA biofilm was characterized by laser confocal microscopy.The content of polysaccharides and proteins in EPS secreted by bacteria with different culture time was quantitatively analyzed through chemical method.The results demonstrated that the LPA biofilm formed on the NCA possesses typical morphology and structure of biofilm.The number of viable cells in the biofilm remained at 1012 CFU/g during all the culture time.The survival rate of bacteria in bottom of the biofilm was lower than that of the inner layer and surface layer in biofilm.However,the average survival rate of bacteria in different layers in biofilm remained above 80%.The contents of polysaccharides and proteins in EPS increased with the increasing culture time,and the content of protein was always higher than that of polysaccharides.3.Stability and stress resistance of lactobacillus paracasei biofilmThe stability of LPAbiofilms on NCA was studied by vibration treatment and ultrasound treatment.The stress resitance of LPA biofilms on NCA was studied under high temperature,low pH,high pH,and antibiotics treatments.All the results showed that the LPA biofilms on the NCA show extremely high stability and stress resistance.As compared with the suspended bacteria,the LPA in biofilm showed stronger stress resistance after treatment with 50?,100 ?,200 ?,pH 1.2,pH 2.0,pH 10 and ampicilin with three concentrations(0.05 mg/mL,0.10 mg/mL,and 0.15 mg/mL).Based on these results,the gene expression analysis of LPA biofilm and suspended bacteria was carried out by RNA sequencing.The results showed that the gene expression pattern of LPA biofilm was different from suspended bacteria.In addition,the carbon fixation and sulfur metabolism were also found to play an important role in bacteria adhesion and biofilm formation.4.Production of L-lactic acid by lactobacillus paracasei biofilm reactorFirstly,the fermentation abilities of LPA biofilm on NCA and the suspened bacteria were compared by shake flask fermentation.Then the lactobacillus biofilm reactor was constructed.The effects of concentration of initial glucose,pH,and flow rate on the L-lactic acid production performance were studied.Besides,the long-term fermentation stability of the lactobacillus biofilm reactor was also evaluated.The results showed that the lag phase of fermentation almost not exists in the biofilm reator,and the fermentation efficiency of biofilm reactor is higher than that of suspended bacteria.When the initial glucose concentration was 80 g/L,pH was 6.5,and the flow rate was 0.3 L/h,the L-lactic acid yield and conversion efficiency of the biofilm reactor achieved to the highest value.During the 9 batches of 30 days fermentation,the average conversion rate of L-lactic acid in the biofilm reactor was over 75%.All the results demonstrated the biofilm reactor not only has good L-lactic acid fermentation performance,but also has stable production.This work provides a new idea for production of L-lactic acid by high-density continuous fermentation,and also provides a new approach for industrial production of L-lactic acid. |
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