Çeşitli Doğal Substratların Yerel Bir Aureobasidium Pullulans Suşunun Pullulan Üretimine Etkilerinin İncelenmesi

Abstract

In this thesis, effects of the usage of various natural substrates as fermentation media on production of pullulan by domestic A. pullulans AZ-6 strain were investigated. For this purpose various citrus peels (orange, lemon, grapefruit, bitter orange), grape pomaces, the hydrolysates of hazelnut and chestnut shells, sugarcane molasses residue, dried and fresh hazelnut husks and pumpkin peel were used. The batch fermentations were performed in a dark place at shaking water bath having temperature of 24.2°C and 100 rpm constant agitation speed. The fermentation media prepared from natural substrates were preprocessed and pH of the media were adjusted to 6.48. At specific time intervals, cultures were taken from the fermentation media and the concentrations of biomass, exopolysaccharide (EPS), pullulan and substrates were determined. During the experiments, the values of the specific growth rate of microorganism and maximum specific product formation rate were calculated. Among the natural substrates, the dried hazelnut husk promoting the best pullulan production were chosen. In the fermentation medium prepared from dried hazelnut husk, the maximum EPS and pullulan concentrations were found as 30.36 and 30.02 g/L, respectively. In this medium, the maximum biomass concentration was 3.54 g/L. Furthermore, in logarithmic phase of the growth curve, the specific growth rate of microorganism was calculated as 0.016 h-1, and the maximum specific product formation rate was found as 1.56 g pullulan/(g mo.h). In the hazelnut husk medium, the initial concentrations of the xylose and glucose were 2.71 ve 8.46 g/L. These substrates in the medium were totally consumed by A. pullulans AZ-6 at the end of the fermentation. At the second part of this research, a study was performed by response surface methodology for the optimization of the fermentation medium prepared from dried hazenut husk. By the help of central composite design method in optimization software, an experimental design consisting of 20 experiments with 6 runs at the center point, was created. In the experimental design, while the independent variables were (NH4)2SO4 concentration, the volume of concentrated H2SO4 and the amount of ground hazelnut husk; the dependent variables were maximum pullulan concentration, maximum EPS concentration and specific growth rate of microorganism. The effects of independent variables on the dependent ones were evaluated by multi-response regression analysis by using the model equations derived from software. The convenience of the models was analysed by variance analysis (ANOVA). As a result of the optimization experiment, the optimal conditions for (NH4)2SO4 concentration, the volume of H2SO4 and the amount of ground hazelnut husk were determined as 7.2 g /L, 2.5 mL and 20 g, respectively. In optimal conditions, the maximum pullulan concentration, maximum EPS concentration and specific growth rate of A. pullulans AZ-6 were found as in order of 74.39 g/L, 75.95 g/L and 0.097 h-1. In addition, the desirability function was 0.915. Lastly, molecular structure of produced EPS samples were analysed by FT-IR spectrometer. At the spectrum obtained for the EPS samples, the representative peaks for the functional groups of pullulan molecule were similar with those of pure pullulan sample. Keywords: Pullulan, exoploysaccaharides, Aureobasidium pullulans, fermentation, natural substrate, optimization