| As an important C4 platform compound,succinic acid plays an increasingly important role in people's life and industrial production.E.coli,with its rapid growth and clear genetic background,is currently the object of more research on succinic acid producing strains.E.coli is subjected to mixed acid fermentation under anaerobic conditions.After removing other competing metabolic pathways,on the one hand,there is a node selection problem in producing succinic acid,that is,whether glucose enters the tricarboxylic acid cycle as phosphoenolpyruvate has a greater effect on the production of succinic acid,or as pyruvate into the tricarboxylic acid cycle;on the other hand,although high concentration of succinic acid was obtained by microbial fermentation,the cost of culture medium was still high.Demand for Chinese herbal medicines(CHMs)is increasing due to the interest in CHMs for disease prevention and treatment.Functional ingredients are extracted using water or organic solvents,leaving her-bal-extraction residues.The increasing output of residues from herbal-extraction processes has already become a social problem due to environmental pollution and may restrict the sustainable.development of the Chinese herbal medicine industry.At present,alternative carbon sources for succinic acid production by microbial fermentation are mainly concentrated in crop stalks,wheat or corn stalks but not herbal-extraction.In this paper,we first used glucose as carbon source and strain ZW310(E.coli MG1655 knockout lactate dehydrogenase gene)as parent strain.Bacillus subtilis pyruvate carboxylase gene(PYC.)and phosphoenolpyruvate carboxykinase gene(PCK),Bacillus subtilis 168,Aclinobacillus succinate 130Z and Escherichia coli ATCC8739,were introduced into ZW310 respectively,named ZW333,ZW330,ZW331 and ZW332 respectively,the succinic acid yield of each strain was increased by 7,2.17,17 and 5.92 times.Therefore,the knock of PYC gene had a great effect on the yield of succinic acid,and then the strain ZW333 was used as the fermentative strain for further experiment.The sugar-producing ability of 10 kinds of herbal-extraction was preliminarily studied under the unoptimized conditions,and the cellulose content was determined by 6 kinds of herbal-extraction with high sugar production.Combined with the preliminary sugar production ability and the determination of cellulose content,Panax ginseng C.A.Mey was used to optimize the enzymatic hydrolysis conditions.Before and after pretreatment,Arnebia euchroma(Royle)Johns residue has the lowest cellulose content,which was 17.722%and 25.62%,while the two highest cellulose content of Morus alba L.and Panax ginseng C.A.Mey residue before and after acid treatment was 47%,52.5%and 35.33%,49.52%,respectively.The content of cellulose was high,which indicated that the residue of traditional Chinese medicine had the potential to produce carbon source of succinic acid,and selected different amount of enzyme to hydrolyze Panax ginseng C.A.Mey residue by comparing the amount of sugar produced and the rate of enzymatic hydrolysis.The optimal enzyme dosage was 40 FPU/g DW.Under this condition,the concentration of glucose,xylose and arabinose in single batch enzymatic hydrolysis of was 41.51 g/L,5.14 g/L and 4.01 g/L,and the enzymatic hydrolysis rate was 83.8%.The sugar yield of the residue was 65.76 g/L and the final yield of succinic acid was 56.28 g/L.The residue of traditional Chinese medicine has high content of cellulose and hemicellulose and has the potential to produce succinic acid and other high added value chemicals and biofuel,which can be converted into monosaccharides by enzymatic hydrolysis.Fermentation can not only produce high yield succinic acid,other high value-added chemicals,biofuels and so on can also be used as a carbon source for fermentation.So as to realize the sustainability and greening of the pharmaceutical production process of traditional Chinese medicine. |
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