Screening Aspergillus Oryzae For Production Of Ethanol From Isoflavone Waste Water By Simultaneous Saccharification And Fermentation

600 thousand tons of soybean isoflavone waste water was produced every year in China and dealing with it became an economic burden which enterprises can't ignore. According to the request of enterprises, this thesis aimed to ferment ethanol using soybean isoflavone waste water, implement the comprehensive use of soybean resources and reduce the pollution of waste water to environment.Soybean isoflavone waste water was the main liquid byproduct of isoflavone production processe, and it's suitable for bioethanol production. However, as the soybean isoflavone waste water contains high concentration of raffinose and stachyose, which can not be completely fermented by Saccharomyces cerevisiae to produce ethanol. The aim of this dissertation was to screen out a strain with highα-galactosidase activity and to develop an effective fermentation process to maximize the ethanol production using soybean isoflavone waste water.Aspergillus oryzae L-09, a strain with highα-galactosidase activity was obtained through selection after U.V., and NTG treatment. The enzyme formed by Aspergillus oryzae L-09 was increased from 12.5 to 36.5 U/mL.The optimization of the medium component and the culture condition showed that the optimum medium was composed of (g/L) soybean isoflavone waste water 80, soybean flour 48 and wheat brans 48. Theα-galactosidase activity of 52.57 U/mL was obtained in the optimum medium. The results also signified that the optimum culture condition was inoculation 0.6×106 yeast cells per mL, 50 mL medium in 250-mL flask bottles, culture temperature 30°C, initial pH 4.9-5.5 and the rotate speed 140 r/min.The character of the raw enzyme was studied. It was found that optimum catalyzing temperature, pH, and substrate concentration were 50°C, 5.0 and 12%, respectively. Meanwhile, the substrate inhibition occurred significantly in the process of hydrolysis, therefore, in the fermentation process, it is necessary to control the concentration of theα-galactose, fructose and glucose, which should be lower than 3%. Especially, the glucose concentration should be lower than 1%.In order to improve the ethanol production, the simultaneous saccharification and fermentation process was used in the process. The optimum fermentation condition was: soybean isoflavone waste water dilution ratio 1:1, the rotate speed 50 r/min. It was also found that the defoamer affected the ethanol production and conversion efficiency dramatically, and the optimum concentration of the defoamer was 0.2‰. Aeration was unfavorable to the ethanol production, and fed-batch culture was superior to batch culture. Under these optimum fermentation conditions, ethanol concentration of 15% (V/V) was obtained in 60 h, and the conversion efficiency was 96.3% of the maximum theoretical yield.Experiments were carried out to verify the result obtained in the flask fermentation. The results signified that after 60 h of fermentation, the concentration of ethanol and residual sugar were 15 % (V/V) and 3.2% (w/w), respectively, which was consistent with the result obtained from flask test. Meanwhile, the content of the raffinose and stachyose were decreased from 12.0 % to 0.24% and 5 % to 0.06 %, respectively, and the conversion efficiencies of which were 98.0 % and 98.8 %, respectively.