Breeding Of High Pullulan Producing Strain Utilizing Xylose And Optimization Of Its Fermentation Conditions

The utilization efficiency of xylose during the biotransformation of lignocellulosic materials, including cellulosic ethanol production, is still quite low. Biotransformation of xylose to other high-value product can provide an alternative way for efficient lignocellulose utilization. Aureobasidium pullulans is capable of producing a high-value exopolysaccharide pullulan from xylose, and pullulan has been widely used in the food industry, chemical industry as well as pharmerceutical industry, due to its property of nontoxic, adhensive and good film-forming ability. In this study, a UV mutant strain AY82 capable of producing high content of pullulan from xylose was selected. Then a series of experiments were undertaken in shake flask and 5 L fermenter in order to optimize pullulan production. Subsequently, the potential of hemicellulsoe hydrolysate for pullulan production was examined.Firstly, the structure of the exopolysaccharide produced by A.pullulans with xylose was confirmed to be pullulan by Fourier Transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). Aiming to solve the problem of low utilization efficiency of xylose by A.pullulans, a higher pullulan producing mutant strain was selected, and the fermentation medium composition and fermentation conditions were optimized by single factor experiment and response surface methodology. The optimized medium composition were:xylose of 55 g/L, yest extract of 0.8 g/L, (NH4)2SO4 of 0.7 g/L, NaCl of 2.0 g/L, MgSO4 of 0.4 g/L, K2HPO4 of 5.8 g/L. The optimized fermentation conditions were:initial pH of 6.5, temperature of 28℃,rotation rate of 200 r/min, and inoculation size of 8%. Under the optimized conditions,16.25 g/L of pullulan was obtained, resulting in a 27.55% of increase compared with the result before optimization.The pullulan fermentation of AY82 was further optimized in 5 L fermenter. Effects of DL-dithiothreitol (DTT) addition, pH, aeration rate, dissolved oxygen level, and feeding of nitrogen source on pullulan production was extensively studied. According to the results, additon of 1.0mM of DTT at 24 h after cultivation, controlled pH at 5.0, aeration rate of 1.5 (V/V-m) and with feeding of nitrogen source (a total amout of 1.0 g/L yeast extract and 1.0 g/L sulfate ammonium was added) was most suitable for pullulan production. Under this condition, pullulan concentration was 21.65 g/L, and the convertion rate was 43.3%.Under the optimized conditions but without the feeding of nitrogen source, the potential of sugarcane hemicellulose hydrolysates for pullulan production was examined. Compared with pure xylose, less pullulan was produced from sugarcane hemicellulose hydrolysate but with increased amount of pigment. After cultivation of 7 d,12.65 g/L of pullulan was produced, and the structure of the pullulan obtained was indicated to be identical with pullulan standard by FTIR and HPLC.