Research On The Mutation Breeding Of High Citric Acid Producing Strains And The Production Of Citric Acid By High-density Fermentation

In this paper, using the Aspergillus Niger CS-217 as starting strains, a high citric acid producing strain UNT-55 was obtained through composite mutation and a series of screening. Then the conditions of batch fermentation and high-density fermentation were optimized.The high citric acid producing strain UNT-55 was breeding by composite mutation (the ultraviolet, high temperature treatment and NTG), obtained through a series of screening including the BCG indication screening medium and high citric acid-hypertonic medium (ASPDA).The strain of genetically stable performance, after five ubcultivation citric acid production changed little. By drawing acid production and consumption of sugar curve, the optimum time of inoculation and fermentation period of UNT-55 were determined by drawing curve of citric acid production and Sugar consumption.The optimization of batch fermentation conditions including temperature, initial pH, nitrogen, inoculation and speed were investigated by single factor experiments. The optimal fermentation condition is that:culture temperature35℃, initial pH control at 4.5, ammonium sulfate as nitrogen source, adding an amount of 0.5%,10% inoculation quantity,250 r/min, liquid volume 50 mL/250 mL. The important factors including the amount of ammonium sulfate added, temperature and speed influencing the citric acid production were identified by Plackett-Burman factorial design. The steepest ascent experiment was adopted to approach the optimal response value region. The results were0.5%ammonium sulfate added, fermentation temperature at 35℃,260 r/min. Finally, the optimal fermentation conditions were obtained by Box-Behnken design and regression analysis as follows:0.5% ammonium sulfate added, fermentation temperature at 35±1℃,190 r/min. Under these conditions, citric acid production was reached 13.88 g/100 mL, which was increased by 6.7% than that before the optimization.The article studied the effect of high cell density cultivation and through batch fermentation to improve the yield of citric acid. By single factor experiment, we studied several chief factors that could impact on the yield of citric acid, including feed-batching time, quantity, batches and the concentration of methanol as promoter. The optimized conditions are as follows:feeding once after about 30 h, feed-batching quantity is 40% of the initial broth volume, the concentration of methanol is 0.2%(v:v). Through orthogonal experiments, the result indicate that feed-batching time is the most influential factor of the yield of citric acid, follows the concentration of methanol and feed-batching quantity. The optimized operational conditions were feeding after fermentation 30 h, feed-batching quantity is 20 mL and the concentration of methanol is 0.2%(v:v).This is consistent with single factor experiment. After using the optimized conditions the yield of citric acid improved markedly, the yield of citric acid is increased to 16.72%, compared to the yield increased by 19.43%. The equipment utilization rate increased significantly.Taking into account the "magnified effect" of microbial fermentation production, experimental design shaking flask fermentation and 10 L fermentation tank fermentation contrast test, the test results show that the existence of "magnified effect" in UNT-55.10 L fermentation tank fermentation maximum yield was 15.7%, which was decreased by 5.42% than shaking flask fermentation. But 10 L fermentation tank fermentation period was 68 h which was less than 70 h (shaking flask fermentation). The purity of fermented liquid citric acid were tested by using high performance liquid chromatography (HPLC) method, the results showed that citric acid content of 15.24%, the ratio of citric acid of total acid was 96.66% more than 95%.