Investigation of the production possibilities of rare sugar L-sorbose by fermentation using D-sorbitol

L-sorbose is a ketose monosaccharide with the formula C6H12O6. The most common method to produce L-sorbose, which is found in very low amounts in natural sources as a rare sugar in nature, is the bio-oxidation of D-sorbitol to L-sorbose using Gluconobacter or Acetobacter species. The most important use of L-sorbose in the food industry is its use as a starting or intermediate material in the synthesis of vitamin C, and L-sorbose is mostly produced worldwide for use in the production of vitamin C. In this thesis, it was aimed to optimize the fermentation conditions that are effective on L-sorbose production by fermentation from D-sorbitol by using Gluconobacter oxydans ATCC 621 strain, and the optimization process was carried out in two stages, in a shaking incubator and bioreactor scale. In the first stage of the study, it was aimed to optimize the D-sorbitol concentration, initial pH value, temperature, and agitation speed parameters, which are effective on L-sorbose production in a shaking incubator, by using the response surface method. The limit values of the independent variables for the optimization of the fermentation conditions were determined as 90 to 170 g/L for the D-sorbitol concentration, 6 to 8 for the initial pH value, 25 to 35°C for the temperature, and 150 to 250 rpm for the agitation speed. A trial design of 27 productions, in which the midpoint was repeated 3 times, was used with the determined parameters and the central composite design statistical method. A trial design consisting of 27 productions, in which the midpoint was repeated 3 times, was used with the determined parameters and the central composite design statistical method. With the samples made during the fermentation, optical density, biomass, D-sorbitol, and L-sorbose amount analyzes were made. On the amount of L-sorbose production at the end of all fermentation experiments carried out in the shaking incubator; D-sorbitol concentration, initial pH value, temperature, D-sorbitol concentration-temperature, pH-temperature, and temperature-agitation speed interactions, and D-sorbitol concentration, pH value, and agitation speed squares were significant (p<0.01; 0.010.05) effect. As a result of shaking incubator experiments, optimum fermentation conditions for L-sorbose production were determined as 127.58 g/L D-sorbitol concentration, initial pH value of 7.20, 35°C temperature, and 224 rpm agitation speed. L-sorbose was produced with %89.95 conversion rate with 168 hours of fermentation carried out under the determined optimum conditions. In the second stage of the study, a new 1x3 factorial trial was carried out at 0.5, 1.0, and 1.5 vvm airflow conditions in the bioreactor, using the optimum conditions obtained from the fermentation experiments carried out in the shaking incubator. The kinetic parameters of the fermentations were calculated with the optical density, biomass, D-sorbitol, and L-sorbose amount data determined by the samples obtained during the 118 hours of fermentation. As a result of the experiments carried out at different airflow rates, it was determined that the airflow rate had a significant (p<0.01) effect on the amount of L-sorbose production. At the end of the bioreactor trials, the optimum airflow rate was determined as 1.5 vvm air flow rate, where the highest amount of L-sorbose production was realized with %98.33 yields. In all bioreactor experiments carried out, the pH value of the medium was followed, and it was determined that L-sorbose production did not start before the pH value of the fermentation medium fell below 5 and L-sorbose production started only after 72 to 95 hours. As a result, it was determined that optimum conditions for L-sorbose production by fermentation from D-sorbitol using G. oxydans ATCC 621 strain were 127.58 g/L D-sorbitol concentration, 7.2 initial pH value, 35°C temperature, 224 rpm agitation speed, and 1.5 vvm airflow rate, and L-sorbose was produced with a yield of %99.51 from the fermentation carried out under optimum conditions. The optimum conditions determined by considering the effect of the initial pH value of the medium on the fermentation time were confirmed by two different productions with the initial pH value of the medium 7.2 and 5.0. As a result of the fermentations, it was determined that the production yield of L-sorbose at both pH values was almost equal and the average yield was %99.36, but the production with the initial pH value of 7.2 was completed for 118 hours, the production with the initial pH value of 5.0 was completed in a shorter time, such as 56 hours. When all the results are evaluated, the optimum conditions to produce L-sorbose from D-sorbitol at 700 rpm agitation speed in the bioreactor are 127.58 g/L initial D-sorbitol concentration, 5 initial pH values, 35°C temperature, and 1.5 vvm air flow rate, and under these optimum conditions L-sorbose could be produced with a yield of %99.21 in 56 hours. With this study, L-sorbose production for the purpose of vitamin C production was also carried out for the first time in Turkey.