产酒精热带假丝酵母突变株的五、六碳糖共代谢分析
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摘要
生物质水解产物五碳糖的利用一直是个难题,提高五碳糖的利用率既能减少木质纤维素的浪费,又能提高乙醇产量。本实验对热假丝酵母进行紫外诱变,通过杜氏小管法和TTC显色法获得一株能利用木糖的突变菌株,并对其进行发酵实验,测定发酵情况以及对五、六碳糖共发酵效率;进而对其发酵机理进行探讨,构建酵母菌代谢网络并建立方程,进行矩阵计算得到细胞的代谢通量。结果表明,原始菌和突变株对葡萄糖的利用情况均较好,利用率分别接近95%、96%;但原始菌木糖的利用率低,乙醇产量最终只有17.58 g/L;而突变株T70-2的乙醇产量达到22.85 g/L,木糖的利用率可达92.2%。因此,本研究所得突变菌株能够实现五、六碳糖的共发酵。
The use of biomass hydrolysate,pentose,has always been a difficult problem. Increasing the utilization of pentose can reduce the waste of lignocellulose and increase ethanol production. In this paper,a mutant strain of Candida tropicalis bred by UV mutagenesis capable of utilizing xylose was obtained by Dux tube method and TTC chromogenic method,and fermentation experiments were carried out to determine the fermentation and the efficiency of co-fermentation of hexose and pentose. Then,the fermentation mechanism was discussed through metabolic network and metabolic equations and mathematical matrix.The results showed that,the utilization of glucose by the original and mutant strains was good,and the utilization rates were close to 95% and 96%,respectively. However,the utilization rate of the original strain xylose was low,and the ethanol yield was only 17.58 g/L. The ethanol yield of the mutant strain T70-2 was 22.85 g/L,and the utilization rate of xylose was 92.2%. Therefore,the mutant strain obtained in this study was capable of achieving co-fermentation of pentose and xylose.
The use of biomass hydrolysate,pentose,has always been a difficult problem. Increasing the utilization of pentose can reduce the waste of lignocellulose and increase ethanol production. In this paper,a mutant strain of Candida tropicalis bred by UV mutagenesis capable of utilizing xylose was obtained by Dux tube method and TTC chromogenic method,and fermentation experiments were carried out to determine the fermentation and the efficiency of co-fermentation of hexose and pentose. Then,the fermentation mechanism was discussed through metabolic network and metabolic equations and mathematical matrix.The results showed that,the utilization of glucose by the original and mutant strains was good,and the utilization rates were close to 95% and 96%,respectively. However,the utilization rate of the original strain xylose was low,and the ethanol yield was only 17.58 g/L. The ethanol yield of the mutant strain T70-2 was 22.85 g/L,and the utilization rate of xylose was 92.2%. Therefore,the mutant strain obtained in this study was capable of achieving co-fermentation of pentose and xylose.
引文
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[19]沈煜,郑华军,王颖,等.木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响[J].生物化学与生物物理进展,2004,31(8):746-751.
[2]QuinteroJ A,Moncada J,CardonaC A.Techno-economic analysis of bioethanol production from lignocellulosic residues in Colombia:A process simulation approach[J].Bioresour Technol,2013,139,300-307.
[3]张强,孙立志,王一东.纤维质酒精预处理液中抑制物脱毒方法研究进展[J].化工进展,2012,31(1):53-56.
[4]Mohamed G.Enhanced enzymatic hydrolysis of waste paper for ethanol production using separate saccharification and fermentation[J].Appi Biotechnol,2015,175:25-42.
[5]Ebrahimi M,Caparanga A R,Ordono E E,et al.Effect of ammonium carbonate pretreatment on the enzymatic digestibility,structural characteristics of rice husk and bioethanol production via simultaneous saccharification and fermentation process with Saccharomyces cerevisiae Hansen 2055[J].Industrial crops and products,2017,101:84-91.
[6]闭帅珠,彭林才,陈克利.蔗渣纤维素乙醇的预处理技术研究进展[J].生物质化学工程,2016,50(2):53-60.
[7]Harinder S O,Praveen V V,Khushal B,et al.Enhanced ethanol production via fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803[J].Process Biochemistry,2010,45:1299-1306.
[8]Kamoldeen A A,Lee C K,Abdullah W N W,Leh C P.Enhanced ethanol production from mild alkali-treated oil-palm empty fruit bunches via co-fermentation of glucose and xylose[J].Renewable Energy,2017,107:113-123.
[9]张强,冀伟,王一东.五碳糖和六碳糖共发酵生产酒精菌株选育的研究进展[J].化工进展,2013,1(32):152-154.
[10]姚笛,王颖,王长远,等.紫外诱变季也蒙毕赤酵母原生质体筛选木糖醇高产菌株.中国生物制品学杂志,2015,28(8):861-864.
[11]姚小飞,吴晓慧,叶璐.高酒精耐受性产酒酵母菌的选育[J].化学与生物工程,2013,30(7):67-69.
[12]刘胜男,张燕,刘泰瑜,等.TTC比色法筛选高存活率的毕赤酵母突变株[J].化学与生物工程,2016,33(10):18-22.
[13]李勤.三种酵母菌生长曲线的对比研究[J].食品与发酵科技,2014,50(4):40-41.
[14]赖颖,赵锦慧.木糖发酵菌株生产乙醇工艺条件的研究[J].中国酿造,2011,231(6):135-136.
[15]Nielsen J.Metabolic engineering[J].Appl Microbiol Biotechnol,2001,55(3):263-283.
[16]Riascosa C A M,Gomberta A K,Pintoa J M.A global optimization approach for metabolic flux analysis based on labeling balan-ces[J].Comput Chem Eng,2005,29(3):447-458.
[17]董博宇,陈叶福,岳瑞雪,等.TTC 在发酵木糖高产乙醇的休哈塔假丝酵母选育中的应用[J].酿酒科技,2008,172(10):40-41.
[18]McKinlay J B,Shachar-Hill Y,Gregory Zeikusa J,et al.Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers[J].Metabolic Engineering,2007,9(2):177-192.
[19]沈煜,郑华军,王颖,等.木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响[J].生物化学与生物物理进展,2004,31(8):746-751.