Study On Screening Of Succinate Producing Strains And Optimization Conditions Of Fermentation Techniques

Succinic acid is a dicarboxylic acid which is produced as an intermediate of tricarboxylic acid cycle and also as one of the fermentation products of anaerobic metabolism. It is an important intermediate for chemical synthesis and it is widely used in food, chemical, and pharmaceutical industries. The fermentative production of succinic acid can be regarded as a green technology not only because renewable substrates are used for its production, but also because CO2, a greenhouse gas, is incorporated into succinic acid during the fermentation. Therefore, the fermentative production of succinic acid becomes more and more attractive. This paper focused on the study of screening procedure for identification of Succinate producing strains and fermentation technology. The main research contents and results are as follows.The method of chemical fluorescent ring was established. It was a semi-quantitative method and successfully applied to the analysis succinic acid. It was a simple and cost effective method that could be utilized to screen anaerobic strains producing succinic acid. The condition to analyze organic acid of fermentation broth with HPLC was studied. The chromatographic conditions were as follows.Chromatograph was agilent1100 and the chromatographic column was agilent ZORBAX SB-Aq(150 mm×4.6 mm,5μm).The buffer consisted of acetonitrile/ potassium dihydrogen phosphate ((0.02) mol/L)(0.5∶99.5,ph=2.0, adjusted by phosphoric acid ) and the mobile phase flow rate was 0.5 ml/min. The uv detection wavelength was 210nm at 30℃column temperature and the injection volume was 10μl.The strain JNUBE0709 could produce 9.45 g/L succinic acid which was isolated from bovine rumen. The organic acids which was produced by the strain were quite few which mainly were acetic acid and formic acid. Therefore JNUBE0709 was chosen for further study.According to physiological and biochemical characteristics of strain JNUBE0709,it was similar to Actinobacillus succinogenes. This strain was primarily identified as Actinobacillus succinogenes.Three significant factors were determined by single factor method. The concentration of three significant factors were analysised with response surface method furtherly. The optical fermentation medium was as follows: maltose 51.1 g/L, yeast extract 24.5 g/L,KH2PO4 3 g/L,KCl 3 g/L,NaCl 3 g/L,MnCl2 0.06 g/L,MgCO3 40.4 g/L,natural pH. At the optimal culture condition, the succinate concentration was increased by 74.49 % and reached 33.45 g/l. The fermentation conditions of JNUBE0709 were investigated for succinate production. The optimum values of liquid volume, inoculation volume, shaking speed, reaction temperature and reaction time were 25 mL/100 mL anaerobic flask, 5 %, 200rpm, 37℃and 48 h, respectively.