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题名	一株耐高温东方伊萨酵母IPE100高温发酵乙醇的研究
作者	YONG-JIN KWON
学位类别	博士
答辩日期	2011-07-24
授予单位	中国科学院研究生院
授予地点	北京
导师	刘春朝
关键词	耐热酵母 分离鉴定 东方伊萨酵母
其他题名	High-Temperature Ethanol Fermentation by Newly Isolated Thermotolerant Issatchenkia orientalis IPE 100
学位专业	生物化工
中文摘要	随着化石燃料的日益紧缺以及温室气体排放对环境造成的破坏，人类急需寻找可再生的燃料替代品。如今，最常见的就是由生物质生产燃料乙醇，而且纤维素生物乙醇能够满足长期供应的需要。然而，纤维素乙醇的生产仍然存在一些技术难题，包括预处理过程、水解、发酵、蒸馏及规模放大，因此，需要寻找既能耐受抑制物和环境压力，又能利用原料中不同碳源高产乙醇的优良菌株。高温发酵技术具有较高的产物生成和回收速率，同时又能避免杂菌污染并节省冷却费用，是燃料乙醇生产的优良途径。为了研究高温发酵纤维素乙醇体系，该研究在不同环境条件下对酵母进行筛选并对高温发酵特性进行了研究，最终，分离得到了一株高效产乙醇的耐高温酵母菌株，在42 ℃下的乙醇产量能够达到理论值的85%，通过表型和基因型的鉴定，命名为东方伊萨酵母IPE 100。首先，研究了该酵母菌在不同温度下的菌体生长、产乙醇速率等动力学参数和热稳定性。随着温度的升高，IPE100的最大比生长速率、最大比乙醇生成速率及比衰亡速率也随之增加，这与Arrhenius函数的描述相符。菌体生长活化能、乙醇生成速率及衰亡速率分别为0.399×104、0.150×104 和3.223×105 KJ/Kmol。在不同通气量条件下，高温乙醇发酵实验在5 L反应器中进行。当转速为150 rpm，最初培养12 h的通气量为0.2 vvm时，乙醇产量达到最高，为理论值的85.54%，产率为1.56 g/L/h。接着，研究了环境压力包括乙醇浓度及纤维素抑制物对酵母菌体生长和乙醇生产的影响。高温乙醇发酵的主要目标是提高菌体对高温和乙醇的双重耐受性。在30-45℃的温度范围内，研究了外源添加乙醇（0-100 g/L）对菌体生长的影响。采用产物抑制型Monod方程来计算乙醇耐受性，结果表明，在实验温度下，该方程能够很好的预测乙醇对菌体生长的抑制作用。在最适生长温度42 ℃条件下，最大乙醇抑制浓度（Pm）和毒性因子（n）分别为96.65 g/L 和 1.23。在较低的温度30 ℃下，乙醇耐受性更高（其中Pm=103.25 g/L， n=0.41）。IPE100在40-45℃的乙醇耐受性在目前报道的耐热酵母中是最高的。对于纤维素抑制物的影响，在添加了糠醛、5-羟甲基糠醛和香草醛的YPG培养基和汽爆秸秆酶水解液中进行，最大的抑制浓度分别为5.56、7.81和3.17 g/L。汽爆秸秆酶水解液的最大乙醇产量为45.92 g/L，为理论产量的93.8%，产率为0.91 g/L/h。最后，研究了耐热酵母IPE100甜高粱高层床固态发酵产乙醇过程。250 mL摇瓶中的最佳操作条件为42 ℃，含水量75%（w/w），颗粒大小2 mm，接种量3%。结果表明热积累作用在发酵床中造成的温度梯度变化取决于床层高度和操作温度。在10 L反应器中，最高乙醇产量（0.25 1 g-乙醇/g-干秸秆，相当于甜高粱中可溶性糖发酵理论产量的91.3%）在温度为40℃，床层高度为10 cm和温度为37 ℃，床层高度为20 cm时获得。由上述结果，本文中的东方伊萨耐热酵母IPE100可以作为纤维素乙醇生产的高效生物催化剂应用于高温发酵和高床层固态发酵中。
英文摘要	The increased concerns for the security of fossil fuel supply and its negative impact on the environment, particularly greenhouse gas emissions, have put pressure to find renewable fuel alternatives. Today, one of the most common renewable fuels is the ethanol produced from biomass, in which lignocellulosic bioethanol production has a great potential for long-term supply of fuel. However, there are still challenges for lignocellulosic ethanol production. The major technological challenges are included in the processes of pretreatment, hydrolysis, fermentation and distillation as well as extending integration. Therefore, it is necessary to develop robust fermenting microorganisms, which should be more tolerant to inhibitors, environmental stresses but also more suitable for different carbon sources in the raw material at high productivity. High temperature fermentation technology has been developed as an effective process for fuel ethanol production because of its advantages such as high rate of product formation and recovery, reducing contamination and cooling cost etc. In order to develop an efficient system for ethanol fermentation from lignocellulosic biomass at high temperature, a thermotolerant ethanologenic strain with high efficiency of 85% theoretical yield at 42 °C was isolated to be identified as Issatchenkia orientalis IPE100 based on phenotype and genotype characterization. Kinetics and thermostability of I. orientalis IPE 100 were investigated with respect to microbial growth and ethanol production at different temperatures. The maximum specific growth rate, the maximum specific ethanol production rate and the specific death rate of I. orientalis IPE 100 increased with increasing temperature. The estimated activation energies for the cell growth, ethanol production and death rate were 0.399×104, 0.150×104 and 3.223×105 KJ/Kmol, respectively. Ethanol fermentation experiments in a 5-L bioreactor at 42 °C were conducted under various aeration conditions. The highest concentration of ethanol was obtained at 150 rpm throughout the fermentation with aeration at 0.2 vvm for the first 12 h, which was corresponding to 85.54% of theoretical yield and 1.56 g/L/h of ethanol productivity. The cross-tolerance of thermal and ethanol was a major issue for high-temperature fuel ethanol fermentation. The maximum inhibitory concentration of ethanol (Pm) and toxic power (n) for the cell growth at optimal growth temperature of 42 °C were estimated to be 96.65 g/L and 1.23, respectively. A higher ethanol tolerance (Pm=103.25 g/L, n=0.41) was found at lower temperature (30 °C). Inhibitory effects of lignocellulosic inhibitory compounds were evaluated in furfural, 5-HMF and vanillin-supplemented YPG media and enzymatic hydrolysates of steam exploded corn stalk. The maximum inhibition terms of furfural, 5-HMF and vanillin for the cell growth were 5.56, 7.81 and 3.17 g/L, respectively. Ethanol production from enzymatic hydrolysates of steam-exploded corn stalk reached 45.92 g/L, equivalent to 93.8% of theoretical yield and 0.91 g/L/h of productivity at 42 °C. A solid state fermentation (SSF) of dry sweet sorghum stalk to ethanol was conducted statically using newly isolated strain IPE 100. The optimal operation parameters for SSF were determined as 42 °C, 75% (w/w) water content, 2 mm particle size and 3% (w/w) inoculation rate in 250-mL conical flask. The results of deep-bed SSF showed that temperature gradient in the bed due to heat accumulation was dependent on both substrate depths and operation temperatures. In a 10-L bioreactor, the highest ethanol yield of 0.25 g-ethanol/g-dry stalk, equivalent to 91.3% of theoretical yield based on soluble sugar in sweet sorghum stalk, was obtained at the operation temperature of 37 °C with 20 cm substrate depth. From the above results, I. orientalis IPE100 showed a great potential for larger-scale SSF with deep-bed.
公开日期	2013-09-24
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1760]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	YONG-JIN KWON. 一株耐高温东方伊萨酵母IPE100高温发酵乙醇的研究[D]. 北京. 中国科学院研究生院. 2011.

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