Optimization Of Glutamate Acid Fermentation Process And Regulation Of Biotin On Glutamate Acid Fermentation

Among all amino acids, glutamate acid possesses large production in the world.Glutamate acid production in China occupies70%total production of the world. In China,glutamate acid was mainly produced with “biotin limitation” process, therefore, biotin is a keyfactor that must be strictly controlled in glutamate acid fermentation. Biotin in fermentationmedium was completely derived from sugar beet molasses and corn steep liquor, but biotin insugar beet molasses and corn steep liquor varies with originating sources and even productionbatches. It is hard to accurately measure because of slight level of biotin concentration （5-10μg/L）, which caused the performance of glutamate acid fermentation. In this paper, biotincontent in sugar beet molasses and corn steep was measured by an established method, andthe repeatable glutamate acid fermentation process was optimized and achieved. To explorethe regulation of biotin on glutamate acid fermentation, the effects of “nominal biotinconcentration” on intracellular and extracellular glutamate acid concentrations duringglutamate acid fermentation were investigated. The main results are as follows:The biotin in sugar beet molasses and corn steep liquor was determined withmicrobiological method. The cell absorbance related biotin concentration was as followequation: Y （）=0.24446X （）-0.000107143, R²=0.9986, which indicating that cellabsorbance showed a good linear relationship with biotin concentration. It is showed thatbiotin concentration of sugar beet molasses and corn steep liquor reaches levels of2500μg/Kg and549μg/Kg, respectively. Recovery experiments indicated that biotin recovery ratioof sugar beet molasses and corn steep liquor reaches102.44%and118.70%, respectively. Thefinal content of biotin in sugar beet molasses and corn steep liquor was2441μg/Kg and462μg/Kg, respectively.The optimization experiments were carried out both in shake flask and5-L fermentor.The optimal conditions in flask （g/L）: glucose160, sugar beet molasses0.5, corn steep liquor4, Na₂HPO₄·12H₂O1, KCl2.2, MgSO₄·7H₂O1, FeSO₄·7H₂O0.002, MnSO₄·H₂O0.002,initial urea100μL（500g/L）, pH7.0⁷.2. The condition of fermentation was loaded volume15mL/500mL,6%inoculation, rotate speed200r/min, temperature was from32℃to36℃.The production of glutamate acid reached up to82g/L. The optimal condition in5Lfermentor （g/L）: sugar beet molasses1.5, corn steep liquor6, glucose and salts were as sameas flask medium, pH7.0⁷.2. Fermentation conditions:20%inoculation, DO10%, pH7.0,temperature was from34℃to39℃.The production of glutamate acid reaches to142g/L.Because DO level in5L fermenter was better than that in flask, which lead to the amountof bacteria in5L fermenter increase, more amount of sugar beet molasses and corn steep wasneeded in order to increase the biotin content in fermentation medium.To investigate the effect of biotin on regulation of glutamate acid fermentation, theglutamate acid fermentation was carried out with different initial biotin concentration. It wasindicated that biotin concentration was the key factor for controlling glutamate acid secretion.Glutamate acid started to secrete when the concentration of biotin was1.97².29μg/g dry cell weight of “nominal biotin-limitation”. The glutamate acid secretion was immediatelyterminated when20μg/L biotin was supplemented during glutamate acid secretion phase.After that the intracellular glutamate acid began to increase, which further indicated thatglutamate acid secretion was controlled by biotin concentration. A new assumed secretionmode—“biotin concentration trigger secretion” was proposed through the comparison ofintracellular and extracellular glutamate acid concentrations.