WO2010072093A1 - 一种纤维素乙醇的生产方法 - Google Patents

一种纤维素乙醇的生产方法 Download PDF

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Publication number
WO2010072093A1
WO2010072093A1 PCT/CN2009/074249 CN2009074249W WO2010072093A1 WO 2010072093 A1 WO2010072093 A1 WO 2010072093A1 CN 2009074249 W CN2009074249 W CN 2009074249W WO 2010072093 A1 WO2010072093 A1 WO 2010072093A1
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Prior art keywords
ethanol
fermentation
cellulose
production method
cellulase
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PCT/CN2009/074249
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English (en)
French (fr)
Inventor
俞学锋
李知洪
余明华
姚鹃
李志军
刘代武
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安琪酵母股份有限公司
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Priority to ES09834046T priority Critical patent/ES2931305T3/es
Priority to PL09834046.6T priority patent/PL2369004T3/pl
Priority to US13/054,851 priority patent/US20110171710A1/en
Priority to EP09834046.6A priority patent/EP2369004B1/en
Publication of WO2010072093A1 publication Critical patent/WO2010072093A1/zh
Priority to ZA2011/00479A priority patent/ZA201100479B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the field of bioenergy development technology, and specifically relates to a method for producing cellulosic ethanol. Background technique
  • Fuel ethanol a renewable and clean energy source, was introduced in the United States and Brazil in the 1970s. China's ethanol production capacity has reached more than 5 million tons / year, of which the annual output of fuel ethanol is maintained at about 1.1 million tons, second only to Brazil, the United States, ranking third in the world.
  • Lignocellulosic raw materials are the most abundant renewable resources on the earth, including forest trees, crop straws, and agricultural and sideline products processing wastes. They occupy an important position in the energy flow and material circulation of natural ecosystems, and are potential resources for the production of fuel alcohol. , has great potential for solving future energy problems.
  • the current pilot technology of cellulosic ethanol has very prominent problems.
  • commercial industrial yeasts appearing on the market can ferment glucose and maltose, but not cellobiose.
  • expensive ⁇ -glucosidase other than cellulase is added to avoid cellobiose inhibition.
  • Cellulase is used to hydrolyze cellulose into simple sugars.
  • One of these simple sugars is cellobiose, which consists of two molecules of glucose that can be broken down into glucose.
  • cellulase does not have enough ⁇ -glucosidase to turn cellulose hydrolyzed and decomposed cellobiose into glucose.
  • Cellobiose inhibits endoglucanase and exoglucanase, which hinders the rate and yield of the entire ethanol during the simultaneous saccharification and fermentation process, and also requires a large supplement of expensive beta-glucosidase. The process cost is very high.
  • yeast fermenting cellobiose has been found, but the tolerance to alcohol is 4%.
  • the simultaneous saccharification and fermentation technology cannot guarantee the coordination of the enzymatic hydrolysis temperature and the fermentation temperature.
  • the enzymatic hydrolysis temperature is 40-50 °C
  • the fermentation temperature of Saccharomyces cerevisiae is 28-35 °C, so the process can only be compromised, resulting in excessive use of the enzyme or excessive use of yeast, simultaneous saccharification and fermentation time. Generally it takes more than 100 hours and the fermentation efficiency is low.
  • the domestic patent (CN 101 1 30792A) reports on the method of enzymatic hydrolysis of straw to prepare fuel ethanol.
  • Patent CN 101230359 reports a cellulosic ethanol process using a modified polydithiosiloxane polymer membrane to solve the problem of too low a concentration of fermented alcohol and high distillation cost, but it cannot avoid enzymatic hydrolysis. The problem of low fermentation efficiency. Summary of the invention
  • An object of the present invention is to provide a method for producing cellulosic ethanol which ultimately improves the production rate and yield of cellulosic ethanol by increasing the efficiency of the simultaneous saccharification and fermentation process.
  • a method for producing cellulosic ethanol comprising the following steps:
  • the source of the cellulose and/or hemicellulose raw material in step 1) is a renewable lignocellulosic biomass.
  • a renewable lignocellulosic biomass Such as wheat straw, rice straw and fast-growing forest, etc., can be used as raw materials for cellulosic ethanol.
  • This type of biomass contains 20-70% cellulose and 10-40% hemicellulose of the plant dry weight, as well as some lignin. It also includes a variety of biomass-rich biomass that is discarded in the environment and biology. 5-6.
  • the pH is 3. 5-6.
  • the pH is in the range of 3. 5-6. 0.
  • the medium may be added with Tween 80, ergosterol or the like depending on the nature of the liquid.
  • the cellulase in step 2) contains an endoglucanase, an exoglucanase and a small amount of ⁇ -glucosidase.
  • Candida albicans is preferably a yeast acclimated by ethanol or cellobiose.
  • the medium for domestication contains cellobiose and an appropriate amount of ethanol.
  • the domesticated cockroaches were separated by three concentration gradients of cellobiose 5%, 10%, 15%, and then domesticated with 1%, 2%, 3% ethanol gradient, and the dominant strain was isolated.
  • the pH condition of the simultaneous saccharification fermentation in step 3) is H value 3. 5-6.
  • the reaction temperature is 35-45 ° C, preferably 40-45 ° C.
  • the Candida utilis used in the simultaneous saccharification and fermentation in the invention has high ethanol tolerance and high ethanol production rate, and can simultaneously synthesize saccharification and fermentation under the action of cellulase using cellulose and hemicellulose as raw materials. Efficient preparation of cellulosic ethanol. Experiments have shown that by comparing the fermentation of Candida utilis with other yeasts using glucose and cellobiose, it is found that it has a higher rate and yield of ethanol production; in addition, Candida utilis has tolerance to ethanol compared to other yeasts. Strong ability. DRAWINGS
  • Figure 1 shows the ethanol production capacity of different yeasts and Candida brevifolia during simultaneous saccharification and fermentation.
  • Figure 2 shows the comparison of ethanol tolerance in different yeasts.
  • the fermentation raw material involved in the present invention includes cellulose and/or hemicellulose.
  • Its source is renewable lignocellulosic biomass.
  • Such as wheat straw, rice straw and fast-growing forest, etc. can be used as raw materials for cellulosic ethanol.
  • This type of biomass contains 20-70% cellulose and 10-40% hemicellulose of the plant dry weight, as well as some lignin. It also includes a variety of biomass-rich biomass that is discarded in the environment and biology.
  • the cellulase used in the present invention contains an endoglucanase, an exoglucanase, and a small amount of ⁇ -glucosidase.
  • Cellulase is used to hydrolyze cellulose into simple sugars such as glucose.
  • One of these simple sugars is cellobiose, which consists of two molecules of glucose that can be broken down into glucose.
  • ⁇ _ Glucosidase can convert cellobiose hydrolyzed and decomposed into glucose, but only cellulase Containing a small amount of ⁇ -glucosidase, the cellulase itself cannot fully utilize cellobiose.
  • Candida lus i taniae used in the present invention may utilize cellulose hydrolysate cellobiose and glucose, and may also utilize mannose and galactose hydrolyzed from hemicellulose, the above combination of sugar or other related Sugar can also be used for fermentation.
  • available disaccharides such as sucrose, maltose, lactose and cellobiose, but excluding sucrose and trehalose, can also utilize polysaccharides hydrolyzed by monosaccharides such as starch and cellulose after hydrolysis of glucose and other singles.
  • Sugar if sugar, sorbose, mannose and galactose.
  • Candida utilis was isolated from the soil samples and enriched with potato agar medium. This yeast is biologically pure, was identified as Candida lus i taniae (CLAS 5566) 0 a sample collection at Three Gorges University Institute of Renewable Energy Allen Collection.
  • the medium for domestication contains cellobiose and an appropriate amount of ethanol.
  • the domesticated carp was separated by 5%, 10%, and 15% concentration gradients, and then lined with 1%, 2%, and 3% ethanol, and the dominant strain was isolated.
  • the fermentation process requires a wide range of dissolved oxygen requirements.
  • the dissolved oxygen may be either micro-oxygen which is intermittently fermented, or a small amount of air may be introduced into the inoculated substrate in continuous fermentation. It can also be anaerobic fermentation.
  • the technique depends on the initial cell density, substrate concentration and inoculation conditions.
  • This medium for fermentation is a common medium containing appropriate sources of nitrogen, minerals, vitamins and carbon. These carbon sources include hexoses (glucose, galactose and mannose) and disaccharides (cellobiose).
  • the fermentation inoculating medium mainly comprises 18% cellulose, 1% Angel yeast extract, 2% peptone, 2 mg/L antibiotic, and the pH is 3. 5-6.
  • the medium may be added with Tween 80, ergosterol or the like depending on the nature of the liquid.
  • the fermentation process temperature can range from 28 °C to 45 °C, however the optimum temperature is 40-45 °C.
  • saccharification involves the hydrolysis process of cellulose.
  • Cellulase is used to hydrolyze cellulose into simple sugars.
  • One of these simple sugars is cellobiose, which consists of two molecules of glucose that can be broken down into glucose.
  • the yeast used in the present invention is Candida lusitaniae, which can be used at 42 ° C [fermented fermented cellobiose, which can be used in simultaneous saccharification fermentation to provide faster wine production rate and higher ethanol production. The rate is superior to other known yeasts for cellobiose fermentation.
  • Table 1 compares the effects of simultaneous saccharification and intermittent saccharification on the final wine production, indicating that the simultaneous saccharification and fermentation process has a 50% increase in cellulose utilization and a two-fold increase in fermentation efficiency. The detailed results are shown in the table below. Effect of simultaneous saccharification and intermittent saccharification on final wine production
  • Figure 1 depicts different yeast simultaneous saccharification fermentation data. Compared with other yeast Saccharomyces cerevisiae and high temperature resistant active dry yeast, CLAS5566 used yeast CLAS 5566 ethanol concentration to reach about 90g/L after 6 days. It is clear that it is superior to other yeasts in terms of rate and yield compared with other yeasts.
  • Figure 2 shows that CLAS 5566 obtained in 25% cellulose at 40 ° C, the ethanol concentration can reach 100g / L, ethanol tolerance is significantly better than Saccharomyces cerevisiae, high temperature resistant active dry yeast, slightly lower than An Qi Company Super Wine Yeast. Recording preparation example 1
  • the cellulose-containing medium was added to a 6 L reactor containing 2 L of water and the loading amount was 2.5 L to ensure sufficient space for inoculation.
  • the medium was mixed in a fermenter containing lipids (30 ml/L oleic acid) and sterilized at 120 ° C for 30 to 40 min.
  • the antibiotic contained 500 mg of 10 mg/L penicillin and 500 mg (10 mg/L) of streptomcycin, H at 4.5_5.0.
  • the cellulase was added, and the cellulosic ethanol-specific yeast CLAS 5566 was inoculated, and the cell density was 2 ⁇ 10 7 , diluted to 3 L, and diluted with water.
  • the cellulose-containing medium was added to a 20 L reactor containing 4 L of water and the loading amount was 5.5 L to ensure sufficient space for inoculation.
  • the medium was mixed in a fermenter containing lipids (ergosterol 5 mg/L) and sterilized at 120 ° C for 30 to 40 min.
  • the antibiotic contained 500 mg of 10 mg/L penicillin and 500 mg (10 mg/L) of streptomcycin, H at 4.0_4.5.
  • the cellulase was added, and the cellulosic ethanol-specific yeast CLAS 5566 was inoculated, and the cell density was 2 ⁇ 10 8 , diluted to 5 L, and diluted with water.
  • the enzyme destroys cellulose into glucose and cellobiose, and the yeast is fermented to ethanol.
  • the temperature is controlled at 42 ° C, and simultaneous saccharification and fermentation is carried out for 96 hours to obtain 98 g/L of ethanol, and then ethanol is fractionated from the fermentation substrate to obtain cellulose ethanol.
  • the production method of the cellulosic ethanol provided by the present invention has been described in detail above. It should be noted that the specific embodiments described are preferred embodiments for better implementing the present invention, and the scope of protection of the present invention is not limited to the technical solutions described in the above embodiments, but should be described in the claims. The substance of the present invention is subject to the scope of the present invention as long as it does not depart from the substance of the claims of the present invention.

Description

一种纤维素乙醇的生产方法
技术领域
本发明涉及生物能源开发技术领域， 具体一种涉及纤维素乙醇的生产方 法。 背景技术
随着石油资源的枯竭、 石油价格的上涨和环境的恶化， 寻找新型可再生 资源替代化石资源和开发洁净能源已成为工业生物技术领域的研究热点。 生 物质能的开发， 可以緩解对石油资源的依赖、 控制二氧化碳的排放， 并促进 农业新型产业链的发展， 对推动我国社会主义新农村建设具有重要意义。
燃料乙醇作为一种可再生的洁净能源， 从上世纪 70年代在美国、 巴西 开始推行。我国乙醇的生产能力已达到 500多万吨 /年，其中燃料乙醇的年产 量保持在 110万吨左右， 仅次于巴西、 美国， 居世界第三位。
由于大量使用玉米为原料开发生物能源导致玉米价格持续增长，所以在 经过了将粮食转化为乙醇的热潮之后， 纤维素乙醇技术的开发和纤维素乙醇 的商业化生产正渐成气候和规模。
木质纤维素原料是地球上产量最大的可再生资源， 包括林木、农作物秸 秆、 农副产品加工下脚料等， 在自然生态系统的能量流与物质循环流中占有 重要位置， 是生产燃料酒精的潜在资源， 对解决未来能源问题有着巨大的潜 力。
目前的纤维素乙醇的中试技术存在非常突出的问题。 一是市场上出现的 商业化的工业酵母可以发酵葡萄糖和麦芽糖， 但不是纤维二糖， 生产中要添 加除纤维素酶外的价格昂贵的 β -葡萄糖苷酶， 以避免纤维二糖抑制。利用纤 维素酶将纤维素水解为简单糖。 这些简单糖中有一种糖是纤维二糖， 包括两 个分子的葡萄糖，可以分解为葡萄糖。但纤维素酶没有足够的 β -葡萄糖苷酶， 使纤维素水解分解的纤维二糖变为葡萄糖。 纤维二糖会抑制内切葡聚糖酶和 外切葡聚糖酶， 在同步糖化发酵工艺中会阻碍整个乙醇的速率和产率， 而且 由于需要大量补充价格昂贵的 β _葡萄糖苷酶，导致工艺成本非常高。另一方 面， 发酵纤维素二糖的酵母已经找到， 但对酒精的耐受力 4艮低。 二是同步糖化发酵技术无法保证酶解温度和发酵温度的协调统一。 一般 酶水解温度在 40-50 °C , 而酿酒酵母的发酵温度在 28-35 °C , 因此工艺上只能 折中进行， 导致酶的用量过大或酵母的用量过大， 同步糖化发酵时间一般要 超过 1 00小时， 发酵效率低下。 三是在高底物含量的条件下， 大量的纤维素 未被转化，酶解效果显著降低， 乙醇的产率也很低，一般只能达到 3-5% ( v/v ) 目前乙醇的发酵制备速率和产率不能满足同步糖化工艺的要求， 没有经 济性。 国内专利（CN 101 1 30792A )报道了酶解秸杆制备燃料乙醇的方法， 发 酵时间 1 0-15天， 酶解效率十分低下， 没有工业化的经济可行性。 国内另一 篇专利 （CN1 01270372A )报道了一种纤维素乙醇发酵液中糖液的浓缩方法， 目的也是想解决酶解后的糖浓度过低的问题， 这表明现在酶解技术不能满足 工业化要求。 虽然釆用这个浓缩专利技术可以改变已经酶解糖的浓度， 但对 纤维素的酶解率低下的问题并没有解决， 会导致原料利用率低， 生产成本高 的问题。专利 CN 101230359报道一种利用改性聚二曱基硅氧烷高分子膜提浓 的纤维素乙醇工艺， 也是想解决发酵酒精浓度过低， 蒸馏成本过高的问题， 但也无法回避酶解和发酵效率低的问题。 发明内容
本发明的目的是提供一种纤维素乙醇的生产方法， 通过提高同步糖化发 酵工艺的效率， 最终提高纤维素乙醇的生产速率和产率。
为实现本发明的发明目的， 提供的技术方案如下：
一种纤维素乙醇的生产方法， 包括如下步骤：
1 )将含有纤维素和 /或半纤维素原料的培养基加入到发酵反应釜中；
2 ) 向发酵反应釜中加入纤维素酶， 并接种葡萄牙假丝酵母 （Candida lus i taniae );
3 )在纤维素酶和葡萄牙假丝酵母的共同作用下进行同步糖化发酵， 分离 得到纤维素乙醇。
其中， 步骤 1 ) 中纤维素和 /或半纤维素原料的来源为可再生的木质纤维 素类生物质。 如麦杆， 稻杆和速生林等， 均可以作为纤维素乙醇的原料。 该 类生物质含有植物干重的 20-70%的纤维素和 10-40%的半纤维素，以及部分木 质素。 同时也包括环境和生物中废弃的富含纤维素的各种生物质。 其中， 步骤 1 ) 中所述培养基包括 15% - 30%的纤维素和 /或半纤维素原 料， 还包括少量的酵母抽提物、 蛋白胨、 抗生素， 控制 pH值在 3. 5-6. 0。 培 养基可以根据液体性质添加吐温 80 , 麦角笛醇等。
其中， 步骤 2 ) 中纤维素酶含有内切葡聚糖酶、 外切葡聚糖酶和少量 β _ 葡萄糖苷酶。
其中， 步骤 2 ) 中葡萄牙假丝酵母优选经过乙醇、 纤维二糖驯化的酵母。 驯化用的培养基含有纤维二糖和适量乙醇。 驯化釆用纤维二糖 5%、 1 0%、 15% 三个浓度梯度， 逐代划线分离， 然后釆用 1 % , 2% , 3%乙醇梯度驯化， 分离得 到优势菌株。
其中， 步骤 3 ) 中同步糖化发酵的酸碱度条件是 H值 3. 5-6. 0 , 反应温 度 35-45 °C , 优选 40-45 °C。
本发明中用于同步糖化发酵的葡萄牙假丝酵母具有较高的乙醇耐受力 和较高的乙醇生产速率， 能够以纤维素、 半纤维素为原料在纤维素酶的共同 作用下同步糖化发酵高效制备纤维素乙醇。 实验表明， 通过比较葡萄牙假丝 酵母与其他酵母利用葡萄糖和纤维二糖发酵， 发现其具有较高的生产乙醇的 速率和产率； 另外， 葡萄牙假丝酵母与其他酵母相比对乙醇的耐受能力较强。 附图说明
图 1为不同酵母与葡萄牙假丝酵母同步糖化发酵产乙醇能力比较。
图 2为不同酵母对乙醇耐受力的比较。 具体实施方式
下面通过实施例对本发明的内容做详细描述。
本发明中所涉及的发酵原料包括纤维素和 /或半纤维素。 其来源为可再生 的木质纤维素类生物质。 如麦杆， 稻杆和速生林等， 均可以作为纤维素乙醇 的原料。 该类生物质含有植物干重的 20-70%的纤维素和 1 0-40%的半纤维素， 以及部分木质素。同时也包括环境和生物中废弃的富含纤维素的各种生物质。
本发明中所使用的纤维素酶含有内切葡聚糖酶、 外切葡聚糖酶和少量 β -葡萄糖苷酶。 利用纤维素酶将纤维素水解为简单糖， 如葡萄糖。 这些简单糖 中有一种糖是纤维二糖， 包括两个分子的葡萄糖， 可以分解为葡萄糖。 β _ 葡萄糖苷酶能够使纤维素水解分解的纤维二糖变为葡萄糖， 但纤维素酶中只 含有少量 β -葡萄糖苷酶， 因此纤维素酶本身不能充分利用纤维二糖。
本发明中使用的葡萄牙假丝酵母 ( Candida lus i taniae )可能利用纤维 素水解物纤维二糖和葡萄糖，也能利用从半纤维素中水解的甘露糖和半乳糖， 以上组合的糖或其他相关糖也可以利用其发酵。 其中， 可利用的二糖， 如蔗 糖， 麦芽糖， 乳糖和纤维二糖， 但不包括密二糖和海藻糖， 也能利用多糖水 解后的单糖如淀粉和纤维素水解后的葡萄糖和其他单糖如果糖， 山梨糖， 甘 露糖和半乳糖。
下面具体说明本发明涉及的同步糖化发酵高效制备纤维素乙醇的步骤 和过程。
一、 葡萄牙假丝酵母的培养及驯化
从土样中分离出来葡萄牙假丝酵母， 釆用土豆琼脂培养基富集。 这种酵 母菌在生物学上是纯种， 被鉴定为 Candida lus i taniae (CLAS 5566 ) 0 一 个样收藏在三峡大学艾伦再生能源研究所保藏中心。 驯化用的培养基含有纤 维二糖和适量乙醇。 驯化釆用 5%、 10%、 15%三个浓度梯度， 逐代划线分离， 然后釆用 1%, 2% , 3%乙醇梯度驯化， 分离得到优势菌株。
二、 发酵条件
1、 发酵工艺溶氧控制
发酵工艺的溶氧要求范围宽。 溶氧既可以是间歇发酵的微氧， 也可以是 连续发酵中的接种的底物通入少量空气。 而且也可以是厌氧发酵。 技术取决 于最初的细胞密度， 底物浓度和接种条件。
2、 发酵培养基的配方
这种用于发酵的培养基是常见的培养基， 含有适当的氮源、 矿物质、 微 生素和碳源。 这些碳源包括己糖（葡萄糖， 半乳糖和甘露糖）和二糖（纤维 二糖）。 发酵接种用培养基主要包括 18%的纤维素， 1%的安琪酵母抽提物， 2% 的蛋白胨， 2mg/L抗生素， pH值在 3. 5-6. 0。 培养基可以根据液体性质添加 吐温 80 , 麦角笛醇等。
3、 发酵工艺的温度控制
本发酵工艺温度可以从 28 °C到 45 °C , 然而最适温度为 40-45 °C。
三、 同步糖化发酵 在同步糖化发酵工艺中， 糖化涉及纤维素的水解过程。 利用纤维素酶将 纤维素水解为简单糖。 这些简单糖中有一种糖是纤维二糖， 包括两个分子的 葡萄糖， 可以分解为葡萄糖。
本发明使用的酵母菌是葡萄牙假丝酵母 Candida lusitaniae , 在 42 °C下可以 [艮好的发酵纤维二糖， 可以用在同步糖化发酵中， 提供更快的产 酒速率和更高的乙醇产率， 优于目前的已知的纤维二糖发酵的其他酵母。
表 1通过比较同步糖化发酵与间歇糖化发酵对最终产酒的影响，说明同 步糖化发酵工艺比间歇糖化发酵工艺纤维素利用率提高 50%,发酵效率提高 约两倍， 详细结果见下表。 同步糖化发酵与间歇糖化发酵对最终产酒的影响
编号 同步糖化发酵工艺 间歇发酵工艺
糖化率 85.1% 58.5%
残还原糖（v/v) 0.15% 0.235%
酒精度（v/v, 20°C ) 5.3% 图 1描述不同酵母同步糖化发酵数据。 CLAS5566与其他酵母菌酿酒酵母， 耐高温活性干酵母比较 6天后， 利用酵母菌 CLAS 5566 乙醇浓度达到 90g/L 左右， 清楚看到与其他酵母相比在速率和产率方面优于其他酵母。
图 2显示， 在 40°C, 25%的纤维素中得到的 CLAS 5566, 耐乙醇浓度可达 到 100g/L, 乙醇耐受力明显优于酿酒酵母， 耐高温活性干酵， 略低于安琪公 司超级酒酵母。 錄制备例 1
将含有纤维素的培养基加入到一个 6L的反应釜中含水 2L, 装样量达到 2.5L, 以确保接种时有足够的空间。 培养基混合在发酵器中， 含有脂类物质 ( 30ml/L油酸） , 灭菌 120°C 30到 40min。 抗生素含有 500mg的 10mg/L的 penicillin 和 500mg ( 10mg/L ) 的 streptomcycin, H 在 4.5_5.0。 接着, 将纤维素酶加入， 接种纤维素乙醇专用酵母 CLAS 5566, 细胞密度 2X107,稀 释至 3L, 用水兌稀。 酶破坏纤维素为葡萄糖和纤维二糖， 酵母发酵为乙醇， 控制温度 38°C, 同步糖化发酵 72小时， 得到 89g/L乙醇， 接着从发酵底物 中分馏出乙醇， 得到纤维素乙醇。 錄制备例 2
将含有纤维素的培养基加入到一个 20L的反应釜中含水 4L,装样量达到 5.5L, 以确保接种时有足够的空间。 培养基混合在发酵器中， 含有脂类 （麦 角甾醇 5mg/L ), 灭菌 120°C 30到 40min。 抗生素含有 500mg的 10mg/L的 penicillin 和 500mg ( 10mg/L ) 的 streptomcycin, H 在 4.0_4.5。 接着, 将纤维素酶加入， 接种纤维素乙醇专用酵母 CLAS 5566, 细胞密度 2X108,稀 释至 5L, 用水兌稀。 酶破坏纤维素为葡萄糖和纤维二糖， 酵母发酵为乙醇， 控制温度 42°C, 同步糖化发酵 96小时， 得到 98g/L乙醇， 接着从发酵底物 中分馏出乙醇， 得到纤维素乙醇。 以上对本发明所提供纤维素乙醇的生产方法进行了详细介绍。 需要指出 的是，具体实施方式所描述的内容是为更好的实施本发明而优选的实施方式， 本发明的保护范围不限于上述实施方式所述的技术方案， 而应以权利要求书 所述的实质内容为准， 任何可能的工艺上的改变只要不脱离本发明权利要求 的实质内容均属于本发明所保护的范围。

Claims

权 利 要 求 书
1、 一种纤维素乙醇的生产方法， 其特征在于包括如下步骤：
1 )将含有纤维素和 /或半纤维素原料的培养基加入到发酵反应釜中；
2 ) 向发酵反应釜中加入纤维素酶， 并接种葡萄牙假丝酵母 （Candida lus i taniae );
3 )在纤维素酶和葡萄牙假丝酵母的共同作用下进行同步糖化发酵， 分离 得到纤维素乙醇。
2、 根据权利要求 1 所述的生产方法， 其特征在于步骤 1 ) 中纤维素和 / 或半纤维素原料的来源为可再生的木质纤维素类生物质。
3、 根据权利要求 1所述的生产方法， 其特征在于步骤 1 ) 中所述培养基 包括 15% - 30%的纤维素和 /或半纤维素原料，
4、 根据权利要求 3所述的生产方法， 其特征在于步骤 1 ) 中所述培养基 还包括少量的酵母抽提物、 蛋白胨和抗生素。
5、 根据权利要求 1所述的生产方法， 其特征在于步骤 1 ) 中所述培养基 控制 pH值在 3. 5-6. 0。
6、 根据权利要求 1所述的生产方法， 其特征在于步骤 2 ) 中纤维素酶含 有内切葡聚糖酶、 外切葡聚糖酶和少量 β _葡萄糖苷酶。
7、 根据权利要求 1所述的生产方法， 其特征在于步骤 2 ) 中葡萄牙假丝 酵母为经过纤维二糖和乙醇驯化的酵母。
8、 根据权利要求 7所述的生产方法， 其特征在于驯化釆用纤维二糖 5%、 10%、 15%三个浓度梯度， 逐代划线分离， 然后釆用 1% , 2% , 3%乙醇梯度驯化， 分离得到优势菌株。
9、 根据权利要求 1所述的生产方法， 其特征在于步骤 3 ) 中同步糖化发 酵的反应温度 35-45 °C
10、 根据权利要求 9所述的生产方法， 其特征在于步骤 3 ) 中同步糖化 发酵的反应温度 40-45 °C。
PCT/CN2009/074249 2008-12-24 2009-09-27 一种纤维素乙醇的生产方法 WO2010072093A1 (zh)

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