| Fermenting very high concentration of glucose to ethanol by yeast, an approach called very high gravity (VHG) fermentation, has aroused considerable interest since it was proposed due to its potential higher final ethanol concentration in fermentative broth and thus economic importance. But there are challenging problems needed to tackle before a successful VHG ethanol fermentation can be performed. One of the most difficult problems is the incomplete fermentation resulting from the decreased growth or even death of the yeast cells subjected to the severe VHG fermentative conditions, such as high osmotic pressure and increased product inhibition. As a result, how to enhance yeast activity under such stress conditions is critically important. To this end, both optimizing fermentative conditions and understanding alterations in physiological and biochemical aspects of the yeast cells exposed to the stress conditions were emphasized in this study. The research was conducted employing a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae (termed fusant SPSC).Firstly, based on the previous experimental data from our laboratory, the research was carried out to optimize the conditions for VHG fermentation by Reponse Surface Methodology (RSM). An optimal conditions for 300 g·L-1 glucose fermentation were eventually established to be 16.82 g·L-1 (NH4)2SO4, 8.24 g·L-1 KH2PO4,0.50 g·L-1 CaCl2,2.00 g·L-1 MgSO4,0.08 g·L-1 ZnSO4,6.00 g·L-1 yeast extract,6.00 g·L-1 peptone,30℃and 11% of inoculum size, which were experimentally validated and resulted in a final ethanol of 137.7 g·L-1 in fermentative broth, representing an over 28% increase. The predicted value of final ethanol concentration was 137.9 g·L-1, showing that there was a very good accuracy between the experimental and predicted data. Meanwhile, the time required to end fermentation under the optimal conditions was shorten from 72 to 60 h, thus ethanol production rate was strikingly increased from 1.49 to 2.30 g·L-1·h-1. To the best of our knowledge, VHG ethanol fermentation based on initial glucose up to 300 g·L-1 usually led to incompletion with large amounts of residual sugar and low level of final ethanol. But our final ethanol concentration was as high as 137.7 g·L-1 with around 96.6% of initial glucose consumed, which was relatively satisfactory.The research was further conducted in a stirred tank fermentor based on the above-mentioned optimization results. The fermentor was flushed continuously with air in order to test the effect of aeration on the VHG ethanol fenmentation. An optimal condition for 300 g·L-1 glucose batch fermentation was eventually determined to be air at a flow rate of 10 L·h-1 throughout the fermentation, which resulted in a final ethanol of 127.3 g·L-1 in fermentative broth, representing about 10% increase compared to anaeration fermentation. Meanwhile, the fermentation time under the optimal condition was shorten to 39 h, thus ethanol production rate was strikingly increased from 2.42 to 3.26 g·L-1·h-1. In addition, it was found that the moderate amount of glycerol generated by yeast during the fermentation had some protective effect on yeast cells subjected to the high concentration of glucose or ethanol.Finally, the experiments were performed to develop aeration coupling fed-batch process. The effect of the initial concentration of glucose on the fed-batch fermentation was investigated, with the results indicating that glucose at 100 g·L-1 was better than 150 g·L-1 and 200 g·L-1. Then, the effects of aeration and active substances on fermentation were studied. The data suggested that both aeration and active substances played an important role in fermentation enhancement by improving the fenmentative activity and ethanol tolerance of the yeast cells. Thus, the best strategy for aeration and fed-batch fermentation was determined: it was conducted with 100 g·L-1 initial glucose and 10 L·h-1 air flow rate, Which led to a final ethanol concentration of 143.3 g·L-1, representing 13% increase compared to the control. |
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