酿酒酵母甜高梁汁发酵生产乙醇研究
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摘要
甜高粱是一种低成本的生物乙醇生产原料。本文在分析甜高粱汁成分基础上,运用均匀设计实验优化了甜高粱汁发酵产乙醇的条件,包括磷酸、尿素、维生素B1、EDTA浓度以及初始pH值。并且运用5种回归分析方法分析均匀设计实验结果,包括二次多项式逐步回归分析、多因子及互作项逐步回归、多因子及平方项逐步回归、偏最小二乘(考虑互作项)回归、偏最小二乘(考虑平方项)回归分析方法,分别得到相应的数学模型。验证实验结果表明,由偏最小二乘(考虑平方项)回归分析方法得到的培养方案为最优组合：磷酸3g/l、尿素1g/l、EDTA1.2g/l、维生素B1 2mg/l pH4.4。2.51罐验证实验乙醇产量达到了13.5%(v／v),糖醇转化率达到92%,同时证明,维生素B1通过增强丙酮酸的分解代谢从而维持了发酵过程pH稳定。在前述所得最优培养条件基础上对于种子和通气策略等工艺条件也进行了初步研究,结果表明16h种龄的二级种子和阶段性通气策略较适合甜高粱汁乙醇的发酵。
本文还对于乙醇发酵酵母细胞中辅因子NAD+和NADH的提取和测定方法进行了研究。发现珠磨破碎细胞后加酸碱提取胞内辅因子,随后利用酶循环原理测定辅因子浓度是高效的胞内NAD+和NADH检测方法。
Sweet sorghum juice is a potential low-cost substrate for ethanol production. In this paper a uniform design method was applied to optimize the concentrations of phosphate, urea, thiamin, EDTA and pH of medium for ethanol production by Saccharomyces cerevisiae, based on analyzing components of sweet sorghum juice. Five predictive mathematical models were established according to different regression methods, including Multinomial Stepwise Regression, Multinomial Stepwise Regression (mutual), Multinomial Stepwise Regressions (square), Partial Least Squares Regression (mutual), Partial and Least Squares Regression (square). The results from respective validated experiments showed that the highest ethanol production was recorded with the optimized conditions based on the Partial and Least Squares Regression, which were phosphate 3 g/1, urea 1 g/1, thiamin 2 mg/1, EDTA 1.2 g/1 and pH 4.4. In the further validated experiments in a 2.51 fermentor, higher ethanol production 13.5%(v/v) and glucose-ethanol conversion 92%were observed; meanwhile it was verified that thiamin played a crucial role in maintaining the stability of pH in the ethanol fermentation process with the optimized conditions by enhancing the catabolism of pyruvate. The seed culture and aeration conditions have been discussed in the paper, which showed that the secondary seed culture of 16 h and changing aeration conditions in stages were suitable for ethanol production from sweet sorghum juice. The initial concentration of the total sugar in the optimum was also investigated and the results exhibited that the higher inhibition on yeast cells growth and ethanol production was obtained under the higher initial concentration of total sugar.
The methods for the determination of the concentrations of the NAD+and NADH in yeast cells have been investigated in the paper. We acquired a series of efficient methods including: yeast cells were disrupted by shaking bead, at the same time NAD+and NADH were extracted by acid and alkali respectively, which were then determined by enzymatic cycling.
本文还对于乙醇发酵酵母细胞中辅因子NAD+和NADH的提取和测定方法进行了研究。发现珠磨破碎细胞后加酸碱提取胞内辅因子,随后利用酶循环原理测定辅因子浓度是高效的胞内NAD+和NADH检测方法。
Sweet sorghum juice is a potential low-cost substrate for ethanol production. In this paper a uniform design method was applied to optimize the concentrations of phosphate, urea, thiamin, EDTA and pH of medium for ethanol production by Saccharomyces cerevisiae, based on analyzing components of sweet sorghum juice. Five predictive mathematical models were established according to different regression methods, including Multinomial Stepwise Regression, Multinomial Stepwise Regression (mutual), Multinomial Stepwise Regressions (square), Partial Least Squares Regression (mutual), Partial and Least Squares Regression (square). The results from respective validated experiments showed that the highest ethanol production was recorded with the optimized conditions based on the Partial and Least Squares Regression, which were phosphate 3 g/1, urea 1 g/1, thiamin 2 mg/1, EDTA 1.2 g/1 and pH 4.4. In the further validated experiments in a 2.51 fermentor, higher ethanol production 13.5%(v/v) and glucose-ethanol conversion 92%were observed; meanwhile it was verified that thiamin played a crucial role in maintaining the stability of pH in the ethanol fermentation process with the optimized conditions by enhancing the catabolism of pyruvate. The seed culture and aeration conditions have been discussed in the paper, which showed that the secondary seed culture of 16 h and changing aeration conditions in stages were suitable for ethanol production from sweet sorghum juice. The initial concentration of the total sugar in the optimum was also investigated and the results exhibited that the higher inhibition on yeast cells growth and ethanol production was obtained under the higher initial concentration of total sugar.
The methods for the determination of the concentrations of the NAD+and NADH in yeast cells have been investigated in the paper. We acquired a series of efficient methods including: yeast cells were disrupted by shaking bead, at the same time NAD+and NADH were extracted by acid and alkali respectively, which were then determined by enzymatic cycling.
引文
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[6]Lin Y, Tanaka S. Ethanol fermentation from biomass resources:Current state and prospects. Appl Microbiol Biotechnol 2006,69:627-642.
[7]苏小军,熊兴耀,谭兴和,刘明月.燃料乙醇发酵技术研究进展.湖南农业大学学报.2007,33：480-485.
[8]Malc-a J, Freire F. Renewability and life-cycle energy efficiency of bioethanol and bio-ethyl tertiary butyl ether (bioETBE):assessing the implications of allocation. Energy 2006,31:3362-3380.
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[14]Agbogbo FK, Coward-Kelly G. Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis. Biotechnology Letters. 2008,30:1515-1524.
[15]Zhang ZH, Qu YB, Zhang X, Lin JQ. Effects of oxygen limitation on xylose fermentation, intracellular metabolites, and key enzymes of Neurospora crassa AS3.1602. Applied Biochemistry and Biotechnology.2008,145:39-51.
[16]Agbogbo FK, Wenger KS. Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis. Journal of Industrial Microbiology& Biotechnology. 2007,34:723-727.
[17]Zhao L, Zhang X, Tan TW. Influence of various glucose/xylose mixtures on ethanol production by Pachysolen tannophilus. Biomass& Bioenergy.2008,32:1156-1161.
[18]Zhao XQ, Bai FW. Yeast flocculation:New story in fuel ethanol production. Biotechnology Advances.2009,27:849-856.
[19]Mobini-Dehkordi M, Nahvi I, Zarkesh-Esfahani H, Ghaedi K, Tavassoli M, Akada R. Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol. Journal of Bioscience and Bioengineering.2008,105:403-408.
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