CN107164246A - 一种耐高温酵母菌及其应用 - Google Patents
一种耐高温酵母菌及其应用 Download PDFInfo
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- CN107164246A CN107164246A CN201710293845.8A CN201710293845A CN107164246A CN 107164246 A CN107164246 A CN 107164246A CN 201710293845 A CN201710293845 A CN 201710293845A CN 107164246 A CN107164246 A CN 107164246A
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- 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
- C12N1/145—Fungal isolates
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- 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
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- 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
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- 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
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Abstract
本发明公开一株耐高温酵母菌株Kluyveromyces marxianus LS201,已保藏在中国典型培养物保藏中心,保藏号CCTCC NO:M 2017112,本发明提供了该菌株的形态学及其生理生化特征。根据形态学、生理生化、核苷酸序列及系统发育分析结果表明,Kluyveromyces marxianus LS201属于Kluyveromyces属的marxianus种,该菌株可以直接利用葡萄糖、果糖、半乳糖、甘露醇、山梨醇、木糖、甘油、纤维二糖、乳糖、蔗糖、乳酸、麦芽糖、棉籽糖、木质纤维素水解液、海藻水解液等物质,将其转化为乙醇。高温发酵生产乙醇节省冷却水的用量和能耗,可抑制杂菌污染,减少浓硫酸用量,降低发酵废液的酸性,实现发酵废液的资源化利用。
Description
技术领域
本发明涉及一种耐高温酵母菌及其应用,属于应用微生物领域。
背景技术
随着能源危机和环境污染的日趋严重,可再生新能源的开发已成为世界各国亟待解决的问题。生物乙醇作为一种可再生的绿色能源,对缓解石油危机,降低环境污染问题具有重大的战略意义。同时,生物乙醇是当前可行性最高的替代液体燃料,孕育着巨大市场前景。
作物来源的淀粉和蔗糖仍然是目前乙醇生产的主要原料。糖蜜是制糖工业的副产品,作为燃料乙醇的生产原料,不但可以提高糖厂的经济效益,减轻环境压力,还可以有效的降低乙醇生产的原料成本。
传统的糖蜜乙醇生产工艺包括将废糖蜜稀释到合适的浓度后加酸酸化,利用酵母菌进行乙醇发酵,最后通过加温蒸馏回收乙醇的过程。
目前的糖蜜乙醇生产工艺中,为了防止杂菌污染和提高酵母的乙醇产量,需在发酵液中加入大量的浓硫酸进行酸化,对发酵罐具有很强的腐蚀作用,增加发酵成本。同时,产生的发酵废液(废醪液)虽然富含可利用有机物质,但因含有高浓度的硫酸根离子(SO4²¯),强酸性(pH值 3.0~3.5)等原因,资源化利用非常困难,直接排放会造成严重的环境污染。
另外,糖蜜乙醇工业中通常利用酿酒酵母(Saccharomyces cereviviae)在30℃左右的温度条件下发酵生产乙醇。酵母在发酵过程中产热和环境温度的提高,往往造成发酵体系温度的升高,从而降低酿酒酵母的生长和发酵活性。所以生产过程中为了保持酵母菌的活性,必须使用大量的冷却水降低发酵罐的温度,对能源和水资源的消耗非常严重。
因此,高能耗、运行费用高和发酵废液无法再利用、环境污染严重是当前乙醇生产工艺中的重要难题。
发明内容
本发明克服了现有技术的不足,提供了一株耐高温酵母菌株Kluyveromyces marxianus LS201,该菌株已于2017年3月13日其在中国典型培养物保藏中心的保藏编号为:CCTCC NO:M 2017112。保藏地址为中国武汉武汉大学。
本发明所述耐高温酵母菌株Kluyveromyces marxianus LS201的培养温度为25~48℃。
本发明所述耐高温酵母菌株Kluyveromyces marxianus LS201,其26S rRNA D1/D2+ITS区域具有SEQ ID No.1所示的核苷酸序列。
本发明中所述耐高温酵母菌株LS201,其核糖体RNA(rRNA)序列比对结果显示,与已知的Kluyveromyces marxianus具有99%以上的相似性,以及系统发育分析结果表明其分类上属于Kluyveromyces属的marxianus种(如图1所示)。
本发明另一目的是将耐高温酵母菌株应用在发酵生产乙醇中。
本发明中所述发酵生产乙醇是利用葡萄糖、果糖、半乳糖、甘露醇、山梨醇、木糖、甘油、纤维二糖、乳糖、蔗糖、乳酸、麦芽糖、棉籽糖中的一种或几种为碳源生产乙醇。
利用木质纤维素水解液、海藻水解液生产乙醇。
本发明中所述耐高温酵母菌株在发酵生产乙醇的温度为30~48℃。
本发明中所述的耐高温酵母菌株Kluyveromyces marxianus LS201主具有以下微生物学特征(如表1所示):
本菌株分离及培养的基础培养基酵母浸出粉胨葡萄糖培养基(YPD培养基),培养基组分如下:2%葡萄糖,2%蛋白胨,1%酵母提取物,固体培养基需加入2%的琼脂粉,115℃高温高压灭菌20min备用。除温度、pH实验之外的其他实验均在45℃,自然pH的条件下培养;接种量除生理生化实验中为1%外,其他实验均为5%。所有实验都设3组平行实验。本发明中除特殊说明外,鉴定菌株特性的培养基采用YPD培养基(碳源为葡萄糖)。
1、形态学特征
本发明菌株LS201是中国科学院西双版纳热带植物园土壤样品中分离筛选获得,酵母菌LS201为卵圆形,以单端出芽方式进行繁殖。在温度25℃的产孢子培养基上培养5天后有子囊孢子产生,孢子呈卵圆形。在YPD固体培养基上涂布稀释后的LS201菌株,结果发现,酵母菌菌落呈圆台形,向上凸起,颜色均一为乳白色,质地均匀,边缘圆整,表面光滑,粘稠易挑取(如图2所示)。
2、生理生化特性
本发明的耐高温酵母LS201菌株能无维生素的培养基(硫酸铵,2.0g/L;硫酸镁,0.2 g/L;磷酸二氢钠,0.5 g/L;氯化钙,0.1 g/L;磷酸氢二钾,0.5 g/L;葡萄糖,2.0g/L)中生长;
本发明的耐高温酵母LS201菌株在YPD(2%葡萄糖,2%蛋白胨,1%酵母提取物)培养基中,25~48℃条件下生长;
本发明的耐高温酵母LS201菌株,在YPD(2%葡萄糖,2%蛋白胨,1%酵母提取物)培养基中,可在pH 3.0~7.0条件下正常生长;
本发明的耐高温酵母LS201菌株可耐受最高乙醇浓度为8%(w/w);
本发明的耐高温酵母LS201菌株能在含有0.01%放线菌酮的在基础氮源培养基((NH4)2SO4 2g/L,MgSO4 0.2g/L,NaH2PO4 0.5g/L,CaCl2 0.1g/L,K2HPO4 0.5g/L;pH 6.5~6.8)中生长;
本发明的耐高温酵母LS201菌株能在50%的D-葡萄糖培养基(50%的D-葡萄糖,2%蛋白胨,1%酵母提取物,2%琼脂粉)上生长,在60%的D-葡萄糖培养基(60%的D-葡萄糖,2%蛋白胨,1%酵母提取物,2%琼脂粉)上不生长;
本发明的耐高温酵母LS201菌株可利用葡萄糖、果糖、半乳糖、木糖醇、甘露醇、木糖、乙醇、甘油、纤维二糖、乳糖、蔗糖、乳酸、麦芽糖和棉籽糖等糖类,不能利用山梨糖、鼠李糖、木糖醇和可溶性淀粉;
表1:耐高温酵母菌株 LS201菌株的微生物学特性
注:表中“+”表示可以,“-”表示不可以。
本发明提供的耐高温酵母菌株Kluyveromyces marxianus LS201利用葡萄糖、果糖、半乳糖、甘露醇、山梨醇、木糖、甘油、纤维二糖、乳糖、蔗糖、乳酸、麦芽糖、棉籽糖中的一种或几种为碳源生产乙醇。其中,耐高温酵母菌株Kluyveromyces marxianus LS201在温度为30~48℃的条件下发酵生产乙醇时乙醇的产率较高。
本发明中所述耐高温酵母LS201菌株能够利用糖蜜发酵生产乙醇。
本发明中所述耐高温酵母LS201菌株能够利用木质纤维素水解液发酵生产乙醇。
本发明中所述耐高温酵母LS201菌株能够利用乳清发酵生产乙醇。
耐高温酵母LS201菌株淀粉和非淀粉多糖水解产生的单糖发酵生产乙醇。
本发明提供的耐高温酵母Kluyveromyces marxianus LS201菌株具有如下优点是:
(1)本发明提供的耐高温酵母LS201菌株可以利用淀粉和非淀粉多糖水解后的单糖发酵生产乙醇,为乙醇的工业发酵提供了更多可能;
(2)本发明提供的耐高温酵母LS201菌株可以利用糖蜜是制糖工业中的副产品糖蜜发酵乙醇,大大减少了工业乙醇生产的成本;
(3)本发明提供的耐高温酵母LS201菌株能够在高温条件下高效生产乙醇。不仅可以实现大幅度减少冷却水的用量和能耗,还能通过提高发酵温度有效抑制杂菌的污染,从而能减少浓硫酸的添加量,降低发酵废液的酸性,使废醪液的资源化利用变得可能。
附图说明
图1是本发明菌株Kluyveromyces marxianus LS201的RNA(26S rRNA D1/D2+ITS)基因序列的系统发育进化树;
图2 是本发明菌株Kluyveromyces marxianus LS201在生物显微镜下的形态示意图;
图3是本发明菌株Kluyveromyces marxianus LS201在不同温度下的生长示意图。
具体实施方式
以下结合附图和实施例对本发明进一步的详细说明,但是本发明的保护范围并不仅限于所述内容。
实施例1:耐高温酵母Kluyveromyces marxianus LS201菌株筛选分离
将从中国科学院西双版纳热带植物园土壤样品加入到添加有终浓度均为25µg/L的链霉素和四环素盐酸盐的YPD液体培养基中,在42℃,150rpm摇床富集培养24h后,抽取1mL用生理盐水进行10倍梯度稀释,取各梯度稀释菌液1mL涂布在添加有四环素盐酸盐和氯霉素的YPD琼脂培养基中,于42℃静止培养24h,长出菌落后,选择具有典型酵母菌落特征的单菌落进一步划线分离2~3次,镜检为纯种后转入YPD液体培养基中,4℃保存。将筛选得到的菌株接入到YPD液体培养基中42℃培养24h,通过测定提高菌株的生长温度后得到的菌株生长OD600nm值,比较各菌株生长情况,筛选出能在48℃下高温下生长的酵母,编号为LS201。通过分子系统发育学分析,命名为Kluyveromyces marxianus LS201,送“中国典型培养物保藏中心”(简称CCTCC)保存。
实施例2:不同温度条件对耐高温酵母LS201菌株生长的影响
将新鲜培养的耐高温酿酒酵母LS201菌株接种于装有10mLYPD培养基的试管中,分别在25℃、30℃、37℃、42℃、45℃、48℃和50℃不同温度下于150rpm/min转速的摇床中培养24小时后,测定其在600nm处的OD值,结果见图3所示,由图3可知,LS201菌株在25~48℃范围内均能正常生长,在30℃时,OD600nm为2.65,48℃时为2.35,50℃时该菌株的生长明显受到影响,该菌株的适宜生长温度为25~48℃。
实施例3:在不同温度条件下,菌株LS201发酵葡萄糖生产乙醇
LS201菌株接种于活化培养基中,该活化培养基pH为6.5,活化培养基的质量百分比的组分为:葡萄糖10%,蛋白胨1%,酵母提取物1%,在装有20mL的100mL摇瓶中于150rpm/min转速的摇床中在30℃培养16小时后,按5%的接种量加入到装有100mL发酵培养基的250mL三角瓶中,发酵培养基的pH为6.5,发酵培养基的质量百分比组分为:葡萄糖10%,蛋白胨1%,酵母膏1%。分别在30℃、37℃、42℃、45℃、48℃的摇床中,150rpm/min转速下培养24小时,采用分光光度法测定菌体的生物量,高效液相色谱法测定发酵液中葡萄糖和乙醇的浓度,测试结果如表2所示,
表2
从表2可知,LS201菌株在以10%的葡萄糖为碳源发酵24小时,发酵液中LS201菌株的OD600nm为3.1,乙醇浓度为44.55g/l,乙醇得率为0.47g/g;耐高温酵母菌株LS201,在30℃、37℃、42℃、45℃、48℃温度条件下均能发酵葡萄糖生产乙醇;在45℃温度条件下细胞生长量最大,OD(600nm )值为3.1,乙醇浓度为44.55g/l,乙醇得率为0.47g/g,24小时内的糖消耗速率最快,达到3.96 g/L/h,乙醇产率最快,达到1.86g/L/h,乙醇得率最高,达到0.47 g/L/h,是理论值的92.2%(理论值为0.51g/g)。
实施例4:菌株LS201发酵不同浓度甘蔗糖蜜生产乙醇
耐高温酵母LS201菌株接种于活化培养基中,该活化培养基pH为6.5,活化培养基的质量百分比的组分为:葡萄糖10%,蛋白胨1%,酵母膏1%,在装有20mL的100mL摇瓶中于150rpm/min转速的摇床中在37℃培养12小时作为接种源;
将甘蔗糖蜜原液用蒸馏水稀释成浓度为 15BXº、20BXº、25BXº、30BXº的甘蔗糖蜜培养基,分别装到100mL的250mL的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;
按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度;测试结果如表3所示,
表3
从表3可知,在24h发酵结束时,糖蜜的初始浓度对乙醇产量的影响显著,甘蔗糖蜜的锤度为20BXº时,发酵液中乙醇浓度最高,为49.01 g/L,此时的乙醇得率为0.42g(乙醇)/g(总糖),是理论值的83.1%,24小时的乙醇生产速率为2.04 g/L/h。
实施例5:在不同浓度的硫酸镁条件下,耐高温酵母LS201菌株在温度45℃发酵糖蜜生产乙醇
本实施例采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
用蒸馏水稀释甘蔗糖蜜至20BXº浓度,并添加不同浓度的硫酸镁,制备硫酸镁终浓度为0%、0.02%、0.04%、0.06%和0.08%的糖蜜培养基;分别取100mL分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度;测试结果如表4所示,
表4
从表4可知,硫酸镁浓度对乙醇产量的影响显著,当硫酸镁添浓度为0.02%时,乙醇产量最高为55.08g/L,乙醇得率为0.47g(乙醇)/g(总糖),是理论值的91.6%,24小时的乙醇生产速率为2.29 g/L/h。
实施例6:在不同浓度的酵母提取物条件下,耐高温酵母LS201菌株在温度45℃发酵糖蜜生产乙醇
本实施例采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
用蒸馏水稀释甘蔗糖蜜至20BXº浓度,并添加不同浓度的酵母提取物,制备酵母提取物浓度为0%、0.5%、1%、1.5%和2%的糖蜜培养基;分别取100mL分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度;测试结果如表5所示,
表5
从表5可知,酵母提取物浓度对乙醇产量的影响显著,当硫酸镁添浓度为2%时,乙醇产量最高为56.59g/L,乙醇得率(乙醇)/(总糖)为0.44g/g,是理论值的86.7%,24小时的乙醇生产速率为2.36 g/L/h;
LS201菌株发酵甘蔗糖蜜乙醇的最适发酵初始锤度为20BXº,营养物质添加量分别为酵母膏0.5%,硫酸镁为0.04%。在此优化的发酵条件下,进行糖蜜乙醇的摇瓶发酵试验,发酵24小时的乙醇产量为60.69g/L,乙醇得率为0.47g(乙醇)/g(总糖),是理论值的93.0%,24小时的乙醇生产速率为2.53 g/L/h。
实施例7 :蔗糖对耐高温酵母LS201菌株在45℃发酵生产乙醇的影响
采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
制备含蔗糖、酵母粉、蛋白胨的培养基,分别取100mL上述培养基分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;
按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度,测试结果显示,发酵液中的总糖含量降低,发酵液中有乙醇。
实施例8 :乳糖对耐高温酵母LS201菌株在45℃发酵生产乙醇的影响
采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
制备含乳糖、酵母粉、蛋白胨的培养基,分别取100mL上述培养基分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;
按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度,测试结果显示,发酵液中的总糖含量降低,发酵液中有乙醇。
实施例9:耐高温酵母LS201菌株在45℃发酵海藻水解液生产乙醇的影响
采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
机械粉碎法将干海带粉碎成颗粒状,用0.1mol/L的盐酸在110℃下处理45min,制备成海藻酸水解液,分别取100mL上述培养基分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;
按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度,测试结果显示,发酵液中有乙醇。
实施例10:耐高温酵母LS201菌株在45℃发酵木质纤维素水解液生产乙醇
本实施例碳源为玉米秸秆的木质纤维素;
采用与实施例1相同的方法活化制备耐高温酵母LS201菌株接种源;
木质纤维素用0.1mol/L的HCl处理后得到木质纤维素水解液,分别取100mL上述培养基分装到250mL容量的摇瓶中,灭菌(121℃,20分钟)后冷却至发酵温度;
按5%的接种量加入上述接种源,在45℃的摇床中,150rpm/min转速下培养24小时;
采用高效液相色谱法测定发酵液中乙醇和总糖的浓度,测试结果显示,发酵液中有乙醇。
<110> 昆明理工大学
<120> 一株耐高温酵母及其乙醇发酵的应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 721
<212> DNA
<213> 马克思克鲁维酵母(Kluyveromyces marxianus) LS201菌株
<400> 1
tccgtaggtg aacctgcgga aggatcatta aagattatga atgaatagat tactggggga 60
atcgtctgaa caaggcctgc gcttaattgc gcggccagtt cttgattctc tgctatcagt 120
tttctatttc tcatcctaaa cacaatggag ttttttctct atgaactact tccctggaga 180
gctcgtctct ccagtggaca taaacacaaa caatattttg tattatgaaa aactattata 240
ctataaaatt taatattcaa aactttcaac aacggatctc ttggttctcg catcgatgaa 300
gaacgcagcg aattgcgata tgtattgtga attgcagatt ttcgtgaatc atcaaatctt 360
tgaacgcaca ttgcgccctc tggtattcca gggggcatgc ctgtttgagc gtcatttctc 420
tctcaaacct ttgggtttgg tagtgagtga tactcgtctc gggttaactt gaaagtggct 480
agccgttgcc atctgcgtga gcagggctgc gtgtcaagtc tatggactcg actcttgcac 540
atctacgtct taggtttgcg ccaattcgtg gtaagcttgg gtcatagaga ctcataggtg 600
ttataaagac tcgctggtgt ttgtctcctt gaggcatacg gctttaacca aaactctcaa 660
agtttgacct caaatcaggt aggagtaccc gctgaactta agcatatcaa taagcggagg 720
a 721
Claims (5)
1.一株耐高温酵母菌株Kluyveromyces marxianus LS201,其在中国典型培养物保藏中心的保藏编号为:CCTCC No:M 2017112。
2.权利要求1所述的耐高温酵母菌株在发酵生产乙醇中的应用。
3.根据权利要求2所述的耐高温酵母菌株在发酵生产乙醇中的应用,其特征在于:利用葡萄糖、果糖、半乳糖、甘露醇、山梨醇、木糖、甘油、纤维二糖、乳糖、蔗糖、乳酸、麦芽糖、棉籽糖中的一种或几种为碳源生产乙醇。
4.根据权利要求2所述的耐高温酵母菌株在发酵生产乙醇中的应用,其特征在于:利用木质纤维素水解液、海藻水解液生产乙醇。
5.根据权利要求2所述的耐高温酵母菌株在发酵生产乙醇中的应用,其特征在于:耐高温酵母菌株在发酵生产乙醇的温度为30~48℃。
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