WO2016033886A1 - 一种嗜热厌氧杆菌及利用其生产乙醇的方法 - Google Patents

一种嗜热厌氧杆菌及利用其生产乙醇的方法 Download PDF

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WO2016033886A1
WO2016033886A1 PCT/CN2014/092862 CN2014092862W WO2016033886A1 WO 2016033886 A1 WO2016033886 A1 WO 2016033886A1 CN 2014092862 W CN2014092862 W CN 2014092862W WO 2016033886 A1 WO2016033886 A1 WO 2016033886A1
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fermentation
strain
fermentation medium
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ethanol
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李爽
赖志城
王菊芳
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华南理工大学
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    • 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
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    • 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
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    • 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
    • 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
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    • 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

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  • the present invention relates to a method for producing ethanol by fermentation using a mutant strain of thermoanaerobacterium aotearoense SCUT27/ ⁇ ldh using glucose and xylose as main raw materials.
  • Ethanol is a new type of clean fuel and an important direction for the development and utilization of renewable energy.
  • the development of bioethanol is the general trend.
  • the second generation of bioethanol is produced by pretreatment, inorganic acid or cellulase hydrolysis and then by biological fermentation.
  • Hydrogen energy attracts the attention of researchers because of its cleanliness, high energy density, and diverse production methods.
  • the main obstacle to fermentative production of ethanol from cellulosic materials is the high cost. This difficulty can be better solved by fermentation with Thermoanaerobacterium.
  • the production of chemicals for fermentation strains has the following advantages (O-Thong, S. et al., International Journal of Hydrogen Energy, 2008. 33(4): 1204-1214): (1) reduction of fibrous materials The amount of cellulase added during the fermentation. This is because the strain itself is a type of hemicellulose-decomposing microorganism capable of hydrolyzing xylan at a high temperature to produce a variety of chemicals, thereby reducing the demand for cellulase. (2) High-temperature continuous fermentation production process is conducive to the separation and purification of ethanol.
  • thermophilic anaerobic bacteria was screened in the applicant's previous study (Li, S., et al., Bioresource Technology, 2010, 101(22): 8718-8724, Cai, Y., et al., Enzyme and Microbial Technology, 2011.48(2): p.155-161), the strain can effectively utilize substrates such as glucose and xylose Fermentation accumulates ethanol, hydrogen, and the like.
  • substrates such as glucose and xylose Fermentation accumulates ethanol, hydrogen, and the like.
  • the strain is affected by high glucose osmotic stress, resulting in a long growth lag period, resulting in a long fermentation cycle and a significant increase in production costs.
  • the high concentration of sugar substrate tolerance is necessary to achieve the simultaneous production of ethanol by cellulosic biomass fermentation.
  • thermophilic anaerobic strain if a high-sugar-resistant thermophilic anaerobic strain can be screened, the tolerance of the thermophilic anaerobic sugar substrate concentration can be improved, and the trait is stable, which can be applied to the fermentation of high concentration sugar substrate. It can shorten the fermentation lag period and reduce the process amplification cost, so that it can be more suitable for industrial production.
  • the starting strain Thermoanaerobacterium aotearoense SCUT27/ ⁇ ldh P8G0 (erythromycin resistance) was cultured according to the method of gradually increasing the concentration of the sugar substrate, and was repeated for several generations until the bacteria were able to be inoculated at this concentration for 6 hours.
  • the body OD 600 is as long as 0.6 and the trait is stable.
  • 1 mL of bacterial solution was added to the separation medium, cultured in an anaerobic incubator for 2-3 days, and single colonies were picked. After 240 hours of fermentation in the fermentation medium, the final concentration of ethanol reached 28.11 g/L, and the conversion rate reached 1.22. Mol/mol, finally obtained a mutant strain P8G3#4 (erythromycin resistance) capable of normal growth and passage under a high concentration substrate.
  • Thermoanaerobacterium aotearoense P8G3#4 which has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC), and the deposit number is CGMCC NO.9000, and the deposit date is 2014. On April 3, the deposit address was No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.
  • the P8G3#4 strain is: Gram-positive bacteria, long rod-shaped, about 0.6-1.0 ⁇ m in diameter, about 2-15 ⁇ m in length, and can be tumbling motion under a microscope.
  • the optimum growth temperature is 55 ° C, pH 6.3, can be used to grow a variety of sugar substrates, such as glucose, xylose, mannose, fructose, galactose, arabinose, trehalose, xylan, Dextran and the like.
  • the genetic characteristic of the strain is: a lactic acid metabolism-blocking mutant strain having erythromycin resistance and an erythromycin resistance concentration of 20-100 ng/ ⁇ l.
  • UDP-Gal UDP- ⁇ -D-galactose
  • CAIR 1-(5-phosphate-D-ribose)-5-amino-4-imidazolecarboxylic acid
  • a method for producing ethanol by using the strain of claim 1 firstly preparing a seed solution of Thermoanaerobacter carbophilus CGMCC9000, and then transferring the Agrobacterium thermophilus CGMCC9000 seed solution to a fermentation medium at a dose of 10-15% w/w. In the middle, the culture is stirred under anaerobic conditions, the culture temperature is 45-60 ° C, the culture time is 50-250 hours, and finally the ethanol is separated from the fermentation liquid.
  • the fermentation medium has a total sugar concentration of 80-150 g/L.
  • the fermentation medium has an initial pH of 5.5-6.5 and the agitation speed is 120-200 rpm.
  • the initial pH of the fermentation medium was 6.3.
  • the fermentation medium components are: glucose 80-100, xylose 40-60, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
  • the fermentation medium further comprises the following components: tripotassium citrate 1-4, citric acid 0.5-3 monohydrate, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium hydrogencarbonate 1-4, hexahydrate
  • Triotassium citrate 1-4 citric acid 0.5-3 monohydrate, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium hydrogencarbonate 1-4, hexahydrate
  • Magnesium chloride 0.5-3 ferrous chloride tetrahydrate 0.05-0.5, calcium chloride dihydrate 0.05-0.4, monohydrate cysteine hydrochloride 0.5-3, pyridoxamine dihydrochloride 0-0.1, p-aminobenzoic acid 0 -0.01, D-biotin 0-0.01, vitamin B120-0.01, vitamin B10-0.01, unit g/L.
  • thermophilic anaerobic CGMCC9000 is placed in a seed culture medium at a temperature of 45-60 ° C, 100-250 rpm, and cultured for 8-24 hours.
  • the seed medium is: glucose 2-4, xylose 2-4, and the other components are the same as the fermentation medium.
  • the following components are further included: tripotassium citrate 1-4, citric acid 0.5-3 monohydrate, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium hydrogencarbonate 1-4, magnesium chloride hexahydrate 0.5 -3, ferrous chloride tetrahydrate 0.05-0.5, calcium chloride dihydrate 0.05-0.4, monohydrate cysteine hydrochloride 0.5-3, pyridoxamine dihydrochloride 0-0.1, p-aminobenzoic acid 0-0.01 , D-biotin (D-biotin) 0-0.01, vitamin B120-0.01, vitamin B10-0.01, unit g / L.
  • the present invention has the following beneficial effects:
  • thermophilic anaerobic bacterium P8G0 screened and constructed in this laboratory was used as a parent strain, and the high sugar substrate tolerance domestication was carried out according to a conventional method to obtain a mutant strain P8G3#4.
  • the mutant has the following genetic characteristics: L-lactate dehydrogenase deficiency, erythromycin resistance, sugar substrate concentration tolerance over 120-150 g/L, intracellular UDP-Gal (UDP- ⁇ -D-half The accumulation of lactose) and 1-(5-phosphate-D-ribose)-5-amino-4-imidazolecarboxylic acid (CAIR) increased; the expression of mtGDP in the glycerophospholipid metabolic pathway was down-regulated.
  • Figure 1 is a graph showing the growth curve of the domesticated high-sugar resistant strain P8G3#4 and the starting strain P8G0. a) Growth of P8G0 at different carbon source concentrations, - ⁇ -30g/L, - ⁇ -81g/L, - ⁇ -120g/L; b) Growth of P8G3#4 at different carbon source concentrations,- ⁇ -30 g/L, - ⁇ -81 g/L, - ⁇ -120 g/L; wherein the sugar substrate is glucose and xylose (2:1, g:g).
  • Figure 2 is a schematic diagram showing the significant enrichment of the cellular metabolic pathway function of P8G3#4 and P8G0.
  • thermophilic anaerobic strain of the present invention can be obtained as follows:
  • the starting strain for screening is a Thermoanaerobacterium aotearoense SCUT27/ ⁇ ldh, which is screened, mutated and preserved in the laboratory, and is referred to herein as P8G0.
  • the starting strain P8G0 was first cultured in an anaerobic shake flask of 81 g/L screening medium (glucose and xylose mass ratio of 2:1). After the growth absorbance OD 600 of the strain was 2.0 or above, a new anaerobic shake flask was transferred. The medium was repeated for several generations until a strain P8G1 capable of maintaining the OD 600 of the cells up to 0.6 and stable in character after 6 h of inoculation was obtained;
  • the strain P8G2 was obtained under conditions of 102 g/L glucose and xylose (2:1, g:g);
  • the strain P8G3 was obtained under the conditions of 120 g/L glucose and xylose (2:1, g:g);
  • transcriptomics was used to analyze the differential gene expression of P8G0 and P8G3#4, and the differential genes were clustered to identify the gene expression level leading to the difference in growth and metabolism between P8G0 and P8G3#4. s reason.
  • > 1 (ie, twice the difference), and the differentially expressed genes in the two samples were analyzed.
  • > 1 (ie, twice the difference), and the differentially expressed genes in the two samples were analyzed.
  • 35 and 24 genes were up-regulated and down-regulated in the logarithmic growth phase, respectively, and P8G3#4 was different from P8G0 in the transcriptome.
  • KEGG metabolic pathway analysis (Fig. 2), a total of 27 genes are annotated into 20 signaling pathways, annotated to the Purine metabolism, the ABC transporter system, the Galactose metabolism, Amino sugar and nucleotide sugar metabolism, Nitrogen metabolism, Phosphotransferase system, Chloroalkane and chloroalkene degradation, Alanine, aspartate and glutamate metabolism and Glycerophospholipid metabolism have more genes, 5, 5, 4, 4, 4, 4, respectively. 3, 3 and 2 articles.
  • the seed culture medium adopts MTC modified medium, and various combinations thereof can be divided into A, B, C, D, and E liquids, respectively, after high temperature sterilization, and then mixed in a ratio of 45:2:1:1:1. .
  • the specific composition of each liquid is shown in Table 1.
  • the seed culture medium is prepared by separately preparing A, B, C, and D liquids into a serum bottle, vacuuming, filling with nitrogen, sterilizing at 115 ° C for 20 min, and storing for use; E liquid filtering and sterilization directly injecting
  • the serum bottle that has been filled with nitrogen and sterilized is kept for use; the serum bottle containing the liquid A is taken, and the syringe is used.
  • the seed medium is obtained by injecting the corresponding volume of the above prepared B, C, D, E liquid into the working concentration.
  • the carbon source concentration in the solution A was 30 g/L, 81 g/L or 120 g/L, wherein the mass ratio of glucose to xylose was 2:1, and the remaining components were consistent with the composition of the seed medium.
  • the specific preparation process is consistent with the operation and processing methods such as seed medium preparation and sterilization.
  • Y is the dry cell weight (DCW), g/L; and X is OD 600 .
  • Fermentation tank culture compares the differences of metabolites between strains before and after acclimation
  • Fermentation conditions According to the 10% inoculation amount, the seed liquid was connected to a 5 L fully automatic reaction kettle containing 3 L medium (120 g/L mixed sugar substrate). Before inoculation, the reaction kettle was first purged with nitrogen for 30 min, and then inoculated with nitrogen for 30 min to ensure that the fermentation environment was oxygen-free.
  • the metabolites of ethanol, acetic acid, lactic acid and sugar consumption were measured by Waters 2695 high pressure liquid chromatography (HPLC).
  • Sample preparation 2 ml of the fermentation broth was added with 1 g of CaCO 3 , shaken for 1 min, centrifuged at 16,000 g for 5 min, and the supernatant was filtered through a 0.22 ⁇ m membrane. Used to detect residual sugar.
  • Sample preparation 1.9 ml of fermentation broth was added to 100 ⁇ l of 10% H 2 8O 4 , centrifuged at 16,000 g for 5 min, and the supernatant was filtered through a 0.22 ⁇ m membrane. Used to detect acid and ethanol. Injection volume: 10 ⁇ l
  • Detector Differential detector.

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Abstract

提供了一种嗜热厌氧杆菌及利用其生产乙醇的方法。长白云嗜热厌氧杆菌（Thermoanaerobacterium aotearoense）P8G3#4，已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC NO.9000，保藏日期为2014年4月3日。利用所述菌株生产乙醇的方法：首先配制嗜热厌氧杆菌CGMCC9000的种子液，然后将嗜热厌氧杆菌CGMCC9000种子液以10-15％w/w接种量转接到发酵培养基中，在厌氧条件下搅拌培养，最后从发酵液分离出乙醇。

Description

一种嗜热厌氧杆菌及利用其生产乙醇的方法 技术领域
一种生产乙醇的菌株和其高浓度糖耐受提高的驯化方法，以及利用该菌株生产乙醇的方法。本发明涉及以葡萄糖、木糖为主要原料，利用嗜热厌氧杆菌(Thermoanaerobacterium aotearoense SCUT27/Δldh)突变株发酵生产乙醇的方法。
背景技术
乙醇是一种新型清洁燃料，是可再生能源开发利用的重要方向。发展生物乙醇是大势所趋。第二代生物乙醇利用植物纤维经过预处理、无机酸或纤维素酶水解再通过生物发酵方式生产。而氢能因其清洁、能量密度高、制取方法多样等特点，吸引着研究者们的关注。
以纤维质类物质为原料发酵生产乙醇的主要障碍在于成本过高。采用嗜热厌氧菌(Thermoanaerobacterium)发酵能够较好的解决该困难。以其为发酵菌株生产化学品具有如下几方面的优势(O-Thong，S.et al.，International Journal of Hydrogen Energy，2008.33(4)：1204-1214)：(1)降低以纤维质类原料发酵过程中的纤维素酶添加量。这是因为该类菌株本身即是一类半纤维素分解微生物，其能够在高温下水解木聚糖发酵产生多种化学品，从而减少了纤维素酶的需求量。(2)高温连续发酵生产过程，有利于乙醇的分离纯化。(3)可以生料发酵生产乙醇。由于工业规模发酵生产乙醇过程中，纯种发酵对于设备的无菌要求较高，因此大幅增加了经济成本。但采用嗜热菌发酵生产，由于绝大部分常温微生物较难在高温下生产，因此对发酵过程的无菌要求不高，从而进一步降低了生产成本。(4)能够利用高温有机废水为发酵原料，这一方面降低了生产成本，另一方面也减轻了环境压力。
申请人前期研究中筛选到一株嗜热厌氧杆菌(Li，S.，et al.，Bioresource Technology，2010，101(22)：8718-8724，Cai，Y.，et al.，Enzyme and Microbial Technology，2011.48(2)：p.155-161)，该菌株能够有效利用葡萄糖、木糖等底物 发酵积累乙醇、氢气等。通过对其进行基因改造，敲除乳酸脱氢酶基因，代谢途径改造后，突变体H2和乙醇产量分别提高了2.7和2.4倍。但该菌株在高浓度糖底物情况下，受到高糖渗透应激的影响，导致生长迟滞期过长，导致发酵周期过久，生产成本大幅度增加。而高浓度糖底物耐受是实现纤维素类生物质同步糖化发酵生产乙醇所必须的。
从工业化角度来看，如果能够筛选到一株耐高糖的嗜热厌氧杆菌菌株，提高嗜热厌氧杆菌糖底物浓度耐受性，且性状稳定，可应用于高浓度糖底物发酵，缩短发酵迟滞期，减少工艺放大成本，这样才能更适合工业化生产。
发明内容
本发明的目的在于提供一种生产乙醇的菌株及利用该菌株生产乙醇的方法。
将出发菌株嗜热厌氧杆菌(Thermoanaerobacterium aotearoenseSCUT27/Δldh)P8G0(红霉素抗性)按照逐步提高糖底物浓度的方法进行培养，重复数代，直到得到在此浓度下能够在接种6h后菌体OD600长至0.6而且性状稳定。取1mL菌液加到分离培养基中，在厌氧培养箱中培养2-3天，挑取单菌落，在发酵培养基中培养240h后，乙醇终浓度达28.11g/L，转化率达1.22mol/mol，最后得到能在高浓度底物下能够正常生长、传代的突变株P8G3#4(红霉素抗性)。
上述突变株为长白云嗜热厌氧杆菌(Thermoanaerobacterium aotearoense)P8G3#4，已在中国微生物菌种保藏管理委员会普通微生物中心保藏(简称CGMCC)，保藏编号为CGMCC NO.9000，保藏日期为2014年4月3日，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
所述P8G3#4菌株为：革兰氏阳性菌，长杆状，直径约0.6-1.0μm，长约2-15μm，显微镜下观察其可翻滚运动。为严格厌氧菌，最适生长温度为55℃，pH 6.3，可利用多种糖底物生长，如葡萄糖、木糖、甘露糖、果糖、半乳糖、阿拉伯糖、海藻糖、木聚糖、葡聚糖等。该菌株的遗传特性为：乳酸代谢阻断型突变株，具有红霉素抗性，红霉素抗性浓度为20-100ng/μl。能耐受120-150g/L糖底物浓度，细胞内UDP-Gal(UDP-α-D-半乳糖)和1-(5-磷酸-D-核糖)-5-氨基-4-咪唑羧酸(CAIR)积累增加；甘油磷脂代谢途径中的mtGDP的表达下调。
利用权利要求1所述菌株生产乙醇的方法：首先配制嗜热厌氧杆菌CGMCC9000的种子液，然后将嗜热厌氧杆菌CGMCC9000种子液以10-15％w/w接种量转接到发酵培养基中，在厌氧条件下搅拌培养，培养温度45-60℃，培养时间为50-250小时，最后从发酵液分离出乙醇。
所述发酵培养基的总糖浓度为80-150g/L。
所述发酵培养基的初始pH为5.5-6.5，所述搅拌转速为120-200rpm。
所述发酵培养基的初始pH为6.3。
所述发酵培养基成分为：葡萄糖80-100，木糖40-60，尿素1-10，酵母抽提物1-5，氯化铵0.5-3，单位g/L。
所述发酵培养基还包括如下成分：柠檬酸三钾盐1-4，一水柠檬酸0.5-3，硫酸钠0.5-3，磷酸二氢钾0.5-3，碳酸氢钠1-4，六水氯化镁0.5-3，四水氯化亚铁0.05-0.5，二水氯化钙0.05-0.4，一水半胱氨酸盐酸0.5-3，二盐酸吡哆胺0-0.1，对氨基苯甲酸0-0.01，D-生物素(D-biotin)0-0.01，维生素B120-0.01，维生素B10-0.01，单位g/L。
所述种子液的配制：将嗜热厌氧杆菌CGMCC9000置于种子培养基中，在45-60℃，转速为100-250rpm，培养8-24h。
所述种子培养基为：葡萄糖2-4，木糖2-4，其它组分与发酵培养基相同。主要为尿素1-10，酵母抽提物1-5，氯化铵0.5-3，单位g/L。优选地，还包括如下成分：柠檬酸三钾盐1-4，一水柠檬酸0.5-3，硫酸钠0.5-3，磷酸二氢钾0.5-3，碳酸氢钠1-4，六水氯化镁0.5-3，四水氯化亚铁0.05-0.5，二水氯化钙0.05-0.4，一水半胱氨酸盐酸0.5-3，二盐酸吡哆胺0-0.1，对氨基苯甲酸0-0.01，D-生物素(D-biotin)0-0.01，维生素B120-0.01，维生素B10-0.01，单位g/L。
与现有技术相比，本发明具有如下有益效果：
(1)以本实验室筛选、构建得到的嗜热厌氧杆菌P8G0为亲株，按常规方法进行高糖底物耐受性驯化，得到突变株P8G3#4。该突变株具有以下遗传特性：L-乳酸脱氢酶缺陷型，红霉素抗性，糖底物浓度耐受范围超过120-150g/L，细胞内UDP-Gal(UDP-α-D-半乳糖)和1-(5-磷酸-D-核糖)-5-氨基-4-咪唑羧酸(CAIR)积累增加；甘油磷脂代谢途径中的mtGDP的表达下调。
(2)以120g/L葡萄糖、木糖混合糖为底物在5L发酵罐发酵生产乙醇，迟滞期缩短至原来的约1/4，乙醇终浓度达28g/L，转化率达1.2mol/mol。本发明 提供筛选的突变体，能够利用高糖底物快速发酵积累目标产品乙醇，且产生的副产物少，工艺放大较为容易，适合于工业化生产。
附图说明
图1为驯化后的耐高糖菌株P8G3#4与出发菌株P8G0的生长曲线图。a)P8G0在不同碳源浓度下的生长情况，-■-30g/L，-●-81g/L，-▲-120g/L；b)P8G3#4在不同碳源浓度下的生长情况，-□-30g/L，-○-81g/L，-△-120g/L；其中糖底物为葡萄糖和木糖(2∶1，g∶g)。
图2为P8G3#4与P8G0的细胞代谢通路功能显著性富集示意图。
A)ABC转运蛋白系统，右图为低聚糖和多元醇转运系统中乳糖/L-阿拉伯糖转运蛋白，左图为磷酸盐和氨基酸转运系统中的支链氨基酸转运蛋白；B)双组份调控系统(Two-component system)，GlnL调控系统；C)磷酸转移酶系统(PTS)中的Lac家族，右图为乳糖的转运，左图为纤维二糖的转运；D)DNA复制相关元件。灰色方框表示累积的代谢物。
具体实施方式
实施例1
菌株的驯化与分离
本发明的耐高糖嗜热厌氧杆菌菌株可以这样得到：
本发明中，用于筛选的出发菌株为实验室筛选、突变并保存的嗜热厌氧杆菌(Thermoanaerobacterium aotearoense SCUT27/Δldh)，此处记为P8G0。
筛选培养基：葡萄糖：80-100g·L-1；木糖：40-60g·L-1；(NH4)25O4：1-5g·L-1；MgCl2·6H2O：1-5g·L-1；KH2PO4：0.5-5g·L-1；K2HPO4：0.5-5g·L-1；CaCl·2H2O：0.1-2.0g·L-1；Na-β-glycerophosphate：2-10g·L-1；FeSO4·7H2O：0.0001-0.001g·L-1；酵母提取物：2-10g·L-1；一水半胱氨酸盐酸(C3H7NO2S·HCl·H2O)：0.1-2.0g·L-1；刃天青：0.001-0.01g·L-1；琼脂粉：5-20g·L-1；红霉素：20-100ng/μl。
出发菌株P8G0首先是经过81g/L筛选培养基(葡萄糖和木糖质量比为2∶1)厌氧摇瓶培养，待菌株生长吸光值OD600为2.0或以上，转接新的厌氧摇瓶培养基，重复数代，直到得到在此浓度下能够在接种6h后菌体OD600长至0.6，并且性状稳定的菌株P8G1；
与步骤(1)方法一样，在102g/L葡萄糖和木糖(2∶1，g∶g)条件下得到菌株P8G2；
与步骤(1)方法一样，在120g/L葡萄糖和木糖(2∶1，g∶g)条件下得到菌株P8G3；
通过平板培养，得到单菌落P8G3#4。
实施例2
P8G3#4驯化后菌株鉴定
为了鉴定驯化前后菌株的差异，采用转录组学方法分析P8G0和P8G3#4代谢基因差异，并对差异基因进行聚类分析，鉴定导致P8G0和P8G3#4两个菌株生长代谢差异的基因表达层面上的原因。
将出发菌株嗜热厌氧杆菌(Thermoanaerobacterium aotearoenseSCUT27/Δldh)P8G0以及筛选得到能在高浓度底物下正常生长的突变株P8G3#4，进行转录组测序分析。分析两个样本中的差异表达基因，筛选标准为|logFC|＞＝1(即两倍差异)，分析两个样本中的差异表达基因，P8G0和P8G3#4共有69个基因在表达丰度上存在显著差异，与出发菌株P8G0相比，分别有35和24个基因在菌株对数生长前期出现表达上调和下调，P8G3#4在转录组上与P8G0存在差异性。
通过KEGG代谢通路分析(图2)，共有27条基因注释到20个信号通路当中，其中注释到嘌呤代谢途径(Purine metabolism)、ABC转运蛋白系统(ABCtransporters)、半乳糖代谢途径(Galactose metabolism)、氨基糖和核苷酸糖代谢途径(Amino sugar and nucleotide sugar metabolism)、氮代谢途径(Nitrogen metabolism)、磷酸转移酶系统(Phosphotransferase system)、氯烷烃和氯烯烃的降解途径(Chloroalkane and chloroalkene degradation)、丙氨酸，天门冬氨酸和谷氨酸代谢途径(Alanine，aspartate and glutamate metabolism)以及甘油磷脂代谢途径(Glycerophospholipid metabolism)的基因较多，分别为5、5、4、4、4、4、3、3和2条。
根据差异基因的表达分析有以下发现：(1)相比于驯化前的出发菌株，突变株P8G3#4细胞内UDP-Gal(UDP-α-D-半乳糖)和1-(5-磷酸-D-核糖)-5-氨基-4-咪唑羧酸(CAIR)积累增加(图2B)；(2)P8G3#4细胞内甘油磷脂代谢途径中的mtGDP的表达下调(图2C)，导致胞内甘油含量增加(3)以及ABC转运蛋 白上调(图2A)，导致胞内几种小分子溶质，如乳糖和支链氨基酸等含量增加；(4)DNA复制能力降低(图2D)，磷酸转移酶系统(PTS)Lac家族中的纤维二糖的转运系统下调。这些差异基因表达是上调或下调，其生理基础是为了维持细胞内外渗透压平衡而作出的应激反应，通过提高细胞内水活度，使细胞体积和膨压达到正常水平，维持细胞的正常生理代谢。
实施例3
摇瓶发酵培养比较驯化后的耐高糖菌株与出发菌株的生长差异
种子液的配制：
种子培养基采用MTC改良培养基，其中的各种组合可分为A、B、C、D、E液，分别高温灭菌后，再按45∶2∶1∶1∶1的比例混合后使用。具体各液组成成分见表1。
Figure PCTCN2014092862-appb-000001
具体的，种子培养基的配制方法为：分别配制A、B、C、D液装于血清瓶中，抽真空，充氮气，115℃灭菌20min，保存备用；E液过滤除菌直接注射入已充好氮气并灭菌的血清瓶中保存备用；取已装有A液的血清瓶，采用注射器 分别注入相应体积上述配制好的B、C、D、E液至工作浓度，即得种子培养基。
发酵培养基的配制：
发酵培养基组成成分中，A液中碳源浓度分别为30g/L，81g/L或120g/L，其中葡萄糖与木糖质量比为2∶1，其余组成成分与种子培养基成分一致。其具体配制过程与种子培养基配制、灭菌等操作处理方法一致。
将过夜培养的P8G0以及P8G3#4分别按1∶10比例接种至含有50mL新鲜配制发酵培养基的血清瓶中。发酵液在温度55℃，转速150rpm的摇床里培养84h，每隔6-12小时取样测定菌体OD600，计算菌体细胞干重(图1)：
Y＝0.421X-0.006(R2＝0.99)
其中，Y为细胞干重(DCW)，g/L；X为OD600。
从图1a可以看出，在正常的培养基浓度下，出发菌株P8G0的DCW到0.5所需要的时间为8h，培养24h后DCW可以达到1.5，在60h达到最高值2.3。当底物浓度提升至81g/L时，菌株DCW到0.5所需要的时间延长至42h，同样在120g/L下需要更长的时间82h。相比较之下，耐高糖菌株P8G3#4在三个浓度下的生长(图1b)与出发菌株在正常的培养基浓度下生长趋势基本一致，表明了耐高糖菌株P8G3#4已经克服了在高浓度底物下生长迟滞期长的缺点。
实施例4
发酵罐培养比较驯化前后菌株代谢产物差异
发酵条件：按10％接种量，将种子液接入装有3L培养基(120g/L混合糖底物)的NBS公司5L全自动反应釜。在接种前，反应釜先通入氮气30min，接种后再通入氮气30min，以保证发酵环境无氧。
代谢产物乙醇、乙酸、乳酸及糖耗的检测采用Waters 2695高压液相色谱(HPLC)测定。
1.葡萄糖、木糖消耗的检测
色谱柱：Aminex HPX-87P(Biorad)
流动相：超纯水
流速：0.6ml/min
柱温：60℃
检测器温度：40℃
样品制备：2ml发酵液加入1g CaCO3，振荡1min，在16,000g下离心5min，取上清用0.22μm膜过滤。用于检测残糖。
进样量：10μl
检测器：示差检测器
2.代谢产物乙醇、乙酸、乳酸的检测
色谱柱：Aminex HPX-87H(Biorad)
流动相：5mM H2SO4
流速：0.6ml/min
柱温：60℃
检测器温度：40℃
样品制备：1.9ml发酵液加入100μl 10％H28O4，在16,000g下离心5min，取上清用0.22μm膜过滤。用于检测酸和乙醇。进样量：10μl
检测器：示差检测器。
3、测定发酵产物中的乳酸、乙酸和乙醇(表1)。结果表明，相比于出发菌株P8G0，驯化后的菌株P8G3#4的生长迟滞期从原来的100h缩短至24h，且发酵末期细胞干重也从1.98g/L增加至4.26g/L，提高了约2倍。且乙醇产量和转化率也都有相应的提高，即最终乙醇浓度达到28.11g/L，乙醇最大转化率达1.22mol/mol。
表1驯化前后菌株以120g/L混合糖为底物上罐发酵结果比较
Figure PCTCN2014092862-appb-000002

Claims (10)

  1. 一种嗜热厌氧杆菌，其特征在于，该菌株为长白云嗜热厌氧杆菌(Thermoanaerobacterium aotearoense)P8G3#4，已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC NO.9000，保藏日期为2014年4月3日。
  2. 一种利用权利要求1所述菌株生产乙醇的方法，其特征在于，首先配制嗜热厌氧杆菌CGMCC9000的种子液，然后将嗜热厌氧杆菌CGMCC9000种子液以10-15％w/w接种量转接到发酵培养基中，在厌氧条件下搅拌培养，培养温度45-60℃，培养时间为50-250小时，最后从发酵液分离出乙醇。
  3. 根据权利要求2所述的方法，其特征在于，所述发酵培养基的总糖浓度为80-150g/L。
  4. 根据权利要求2所述的方法，其特征在于，所述发酵培养基的初始pH为5.5-6.5，所述搅拌转速为120-200rpm。
  5. 根据权利要求4所述的方法，其特征在于，所述发酵培养基的初始pH为6.3。
  6. 根据权利要求2或3或4或5所述的方法，其特征在于，所述发酵培养基成分为：葡萄糖80-100，木糖40-60，尿素1-10，酵母抽提物1-5，氯化铵0.5-3，单位g/L。
  7. 根据权利要求6所述的方法，其特征在于，所述发酵培养基还包括如下成分：柠檬酸三钾盐1-4，一水柠檬酸0.5-3，硫酸钠0.5-3，磷酸二氢钾0.5-3，碳酸氢钠1-4，六水氯化镁0.5-3，四水氯化亚铁0.05-0.5，二水氯化钙0.05-0.4，一水半胱氨酸盐酸0.5-3，二盐酸吡哆胺0-0.1，对氨基苯甲酸0-0.01，D-生物素0-0.01，维生素B12 0-0.01，维生素B1 0-0.01，单位g/L。
  8. 根据权利要求2或3或4或5所述的方法，其特征在于，所述种子液的配制：将嗜热厌氧杆菌CGMCC9000置于种子培养基中，在45-60℃，转速为100-250rpm，培养8-24h。
  9. 根据权利要求8所述的方法，其特征在于，所述种子培养基为：葡萄糖2-4，木糖2-4，尿素1-10，酵母抽提物1-5，氯化铵0.5-3，单位g/L。
  10. 根据权利要求9所述的方法，其特征在于，所述种子培养基还包括如
    下成分：柠檬酸三钾盐1-4，一水柠檬酸0.5-3，硫酸钠0.5-3，磷酸二氢钾0.5-3，碳酸氢钠1-4，六水氯化镁0.5-3，四水氯化亚铁0.05-0.5，二水氯化钙0.05-0.4，一水半胱氨酸盐酸0.5-3，二盐酸吡哆胺0-0.1，对氨基苯甲酸0-0.01，D-生物素0-0.01，维生素B12 0-0.01，维生素B1 0-0.01，单位g/L。
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