WO2015135302A1 - 一株高耐受性拜氏梭菌及其应用 - Google Patents
一株高耐受性拜氏梭菌及其应用 Download PDFInfo
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- WO2015135302A1 WO2015135302A1 PCT/CN2014/085470 CN2014085470W WO2015135302A1 WO 2015135302 A1 WO2015135302 A1 WO 2015135302A1 CN 2014085470 W CN2014085470 W CN 2014085470W WO 2015135302 A1 WO2015135302 A1 WO 2015135302A1
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- 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
- C12N1/20—Bacteria; 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
- C12N1/20—Bacteria; 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
- C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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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
- C12P7/16—Butanols
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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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
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- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the invention relates to a highly tolerant C. beijedii which can ferment and produce butanol by using undetoxified bagasse acid hydrolyzing sugar liquid and application thereof, and belongs to the technical field of biological fermentation.
- Butanol is a colorless liquid, has a wine taste, and is miscible with ethanol/diethyl ether and various other organic solvents. It has been widely used in the manufacture of various fine chemicals such as plasticizers. Butanol has the advantages of high energy density, can be mixed with gasoline at any ratio, can be directly used in internal combustion engines, and is convenient to transport. As a new type of renewable liquid fuel, more and more attention has been paid.
- Clostridium beijerii 13-2 strain has high fermentation efficiency for bagasse hydrolysate, and it is used in 0.5% sulfuric acid. Alcohol fermentation can reach up to 4.5g / L.
- Bagasse is an important renewable wood fiber raw material.
- the use of bagasse to produce butanol not only saves resources, but also promotes energy saving and emission reduction and industrial upgrading of the sugar industry.
- inhibitors such as organic acids, furfural, and phenols are produced. These inhibitors have high removal cost and have a certain inhibitory effect on microbial growth.
- Thaddeus Ezeji et al. Biotechnology & Bioengineering) 2007, 97(6): 1460-1469
- It was found that inhibitors such as organic acids and furfural did not affect the fermentation of butanol, while phenolic inhibitors had a significant inhibitory effect on butanol fermentation.
- the toxin inhibitors (especially phenolic compounds) in the sugarcane acid hydrolysate severely inhibit the fermentation performance of Clostridium butyricum; and the improvement of the strain is to improve the tolerance of the strain to the inhibitor and the fermentation economy.
- the present invention obtains a high-efficiency utilization rate not only for the sugarcane sulphate glycolysis but also for the toxin inhibitor (especially phenolic compound) by inducing mutation and screening. Sexual mutant strain.
- the object of the present invention is to provide a new strain: Clostridium beijerinckii (C/ ytr M beijerinckii) M17.
- Another object of the present invention is to provide the use of the above strains in the production of butanol.
- the applicant deposited the strain in the China Center for Type Culture Collection at Wuhan University, Wuhan, Wuchang, Wuhan.
- the deposit center received the strain provided by the applicant on September 14, 2013.
- the deposit number assigned to the culture is CCTCC ⁇ M 2013425, and the proposed classification is named Clostridium beijerinckii ⁇ .
- the strains deposited on September 23, 2013 were found to be viable.
- a method for producing butanol comprising: the following steps:
- Clostridium beijerinckii C/oWr 63 ⁇ 4 M17 was inoculated onto the plate medium, and subjected to anaerobic culture, the culture temperature was 33 to 37 ° C, and the culture time was 12 to 24 h to activate the strain;
- Seed culture Inoculate the plate culture activated C. typhimurium (Ctotr i beijerinckii) M17 into the seed culture medium, the lOOmL anaerobic bottle liquid volume 40 ⁇ 60mL, nitrogen gas for 3 ⁇ 5min, culture temperature 33 ⁇ 37 V, culture time 12 ⁇ 24h;
- the plate medium described in step 1) comprises the following mass percentage components: carbon source 0.3% ⁇ 1%, nitrogen source 0.5% ⁇ 1%, inorganic salt 0.5% ⁇ 0.8%, agar 1.5% ⁇ 2 %, the rest is water; wherein, the carbon source is selected from at least one of glucose, starch, bagasse solubilized sugar solution, corncob acid solubilizing solution; nitrogen source is selected from the group consisting of ammonium acetate, ammonium chloride, peptone, yeast powder, At least one of corn steep liquor; the inorganic salt is at least one selected from the group consisting of a sodium salt, a potassium salt, a magnesium salt, a phosphate salt, and a ferrous salt.
- carbon source is selected from at least one of glucose, starch, bagasse solubilized sugar solution, corncob acid solubilizing solution
- nitrogen source is selected from the group consisting of ammonium acetate, ammonium chloride, peptone, yeast powder, At least one of corn steep liquor
- the inorganic salt is
- the seed culture medium according to step 2) comprises the following mass percentage components: a carbon source of 0.5% to 1%, a nitrogen source of 0.5% to 1%, an inorganic salt of 0.5% to 0.8%, and the balance being water;
- the carbon source is selected from at least one of starch and glucose;
- the nitrogen source is at least one selected from the group consisting of ammonium acetate, ammonium chloride, peptone, yeast powder, and corn steep liquor;
- the inorganic salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, and phosphoric acid. At least one of salt and ferrous salt.
- the fermentation medium described in step 3) comprises the following components by mass percentage: carbon source 3% to 6%, nitrogen source 0.1% to 0.3%, inorganic salt 0.1% to 0.2%, growth factor 0.05% ⁇ 0.1%, the rest is water; wherein, the carbon source is selected from At least one of glucose, xylose, bagasse solubilized sugar solution, corncob acid solubilizing solution; nitrogen source selected from at least one of ammonium acetate, ammonium chloride and yeast powder; inorganic salt selected from sodium salt and potassium salt At least one of a magnesium salt, a calcium salt, a phosphate salt, and a ferrous salt; the growth factor is selected from at least one of p-aminobenzoic acid, vitamin B1, biotin, and corn steep liquor.
- the carbon source is selected from At least one of glucose, xylose, bagasse solubilized sugar solution, corncob acid solubilizing solution
- nitrogen source selected from at least one of ammonium acetate, ammonium chloride and yeast powder
- the strain of the present invention Clostridium beijerinckii Ml 7 is highly resistant to toxins in the undetoxified lignocellulosic acid solubilizing liquid, and can be fermented very well in a fermentation broth having a phenol concentration of up to 2.0 g/L. Butanol, its total solvent yield and butanol yield reached 10.9 g / L and 7.9 g / L, respectively, even if the phenol concentration is as high as 2.4 g / L, the strain of the present invention still has considerable ability to produce butanol, indicating that the strain of the present invention Clostridium sp. M17 is highly resistant to toxins, has high solvent yield and high butanol yield, and is highly reproducible. It is an excellent strain suitable for fermenting butanol by bagasse.
- Clostridium beijerinckii M17 has high utilization rate of bagasse acidolysis solution, and is highly resistant to toxin inhibitors (especially phenolic compounds), which is not only beneficial to butanol
- toxin inhibitors especially phenolic compounds
- Figure 1 is a plasma mutagenesis survival curve of Clostridium beijerinckii.
- the first step of plasma mutagenesis of the original strain of Clostridium beijerinckii is as follows:
- the original strain of Clostridium beijerii NCIMB 8052 was activated and cultured at a temperature of 33 ⁇ 37 ° C.
- the volume of 50 ml SCHOTT anaerobic bottle was 15 ⁇ 20ml, nitrogen was filled for 3min, and the culture time was 12 ⁇ 18h.
- Dilute the freshly cultured cells to a cell concentration of OD 6 (K) 0.1 to 1.0, add dropwise to the sterilized and cooled slides, and blow dry with sterile air; use xenon as the discharge gas, and use 100 W as the RF power.
- the strain was subjected to plasma mutagenesis with 10 SLM as the gas flow rate and 10 to 240 s as the irradiation time. After the mutagenesis, the bacterial membrane on the carrier was eluted, and the survival rate was calculated.
- the experimental results are shown in Figure 1. As can be seen from Figure 1, 180s is the optimal mutagenesis irradiation time.
- Bagasse solubilized sugar solution plate medium yeast powder 0.3%, peptone 0.5%, soluble starch 1%, ammonium acetate 0.2%, sodium chloride 0.2%, magnesium sulfate heptahydrate 0.3%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate heptahydrate 0.01%, agar 1.5%, resazurin 0.02%, bagasse The acid hydrolysate is 25% (v/v), the rest is water, pH 6.
- Seed medium yeast powder 0.3%, peptone 0.5%, soluble starch 1%, ammonium acetate 0.2%, sodium chloride 0.2%, magnesium sulfate heptahydrate 0.3%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1 %, ferrous sulfate heptahydrate 0.01%, the rest is water, pH 6.
- Shake flask fermentation initial screening medium glucose 3%, ammonium acetate 0.22%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium chloride 0.001%, magnesium sulfate heptahydrate 0.02%, sulfuric acid heptahydrate Iron 0.001%, manganese sulfate monohydrate 0.001%, corn syrup 0.1%, the rest is water, pH 6.6.
- Shake flask fermentation rescreening medium 3% of bagasse solubilized sugar solution, 0.22% ammonium acetate, 0.05% potassium dihydrogen phosphate, 0.05% dipotassium hydrogen phosphate, 0.001% sodium chloride, 0.02% magnesium sulfate heptahydrate , ferrous sulfate heptahydrate 0.001%, manganese sulfate monohydrate 0.001%, corn syrup 0.1%, the rest is water, pH 6.6.
- the mutagenized slide was placed in a stoppered test tube containing 1 ⁇ 2 ml of physiological saline, violently shaken, and the strain on the slide was eluted and diluted to a different concentration and applied to the bagasse solubilized sugar liquid plate medium.
- anaerobic culture was carried out for 12 to 36 hours. 50 colonies with clear circles and large colonies were selected, numbered M1 to M50, respectively, and they were separately cultured with seed medium and preserved.
- the 50 strains of the primary cultured expanded colonies and the original strain were inoculated into the shake flask fermentation initial screening medium, the inoculation amount was 10% (v/v), the lOO mL Schott anaerobic bottled liquid volume was 50 mL, and the fermentation temperature was 35. °C, the total solvent yield and butanol yield of each strain were measured after 72 h fermentation time. The results showed that strains M17, M39 and M48 could ferment higher yields of butanol, as shown in Table 1.
- the total solvent yield and butanol yield of the three mutant strains obtained by combined screening were higher than those of the original strain during fermentation. Yield, of which M17 also has the highest total solvent yield and butanol production.
- the strain M17 and the original strain with the highest total solvent and butanol yield obtained by the initial screening of the fermentation were inoculated into the shake flask fermentation screening medium, the inoculation amount was 10% (v/v), and the 100 mL SCHOTT anaerobic bottle volume was 50.
- the total solvent yield and butanol yield of the two strains were measured after the fermentation temperature was 35 °C, and the fermentation time was 72 h. The results showed that the strain M17 could ferment higher yield of butanol, as shown in Table 2.
- Fermentation medium 1 ammonium acetate 0.22%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium chloride 0.001%, magnesium sulfate heptahydrate 0.02%, ferrous sulfate heptahydrate 0.001%, manganese sulfate monohydrate 0.001 %, corn syrup 0.1%, prepared with acid-free sugar solution of undetoxified bagasse (total reducing sugar is 3%), the rest is water, pH 6.6, and the total soluble phenol content in the medium is 1.5 g/L.
- Fermentation medium 2 ammonium acetate 0.22%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium chloride 0.001%, magnesium sulfate heptahydrate 0.02%, ferrous sulfate heptahydrate 0.001%, manganese sulfate monohydrate 0.001 %, corn syrup 0.1%, prepared with acid-free sugar solution of undetoxified sugar cane bagasse (total reducing sugar is 4%), the rest is water, pH 6.6, and the total soluble fraction in the medium is 2.0 g/L.
- Fermentation medium 3 ammonium acetate 0.22%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium chloride 0.001%, magnesium sulfate heptahydrate 0.02%, ferrous sulfate heptahydrate 0.001%, manganese sulfate monohydrate 0.001 %, corn syrup 0.1%, with undetoxified corncob acid solubilized sugar solution (total reducing sugar is 4.7%), the rest is water, pH 6.6, and the total soluble phenol content in the medium is 2.4 g/L.
- Plate medium yeast powder 0.3%, peptone 0.5%, soluble starch 1%, ammonium acetate 0.2%, sodium chloride 0.2%, magnesium sulfate heptahydrate 0.3%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%,
- the ferrous sulfate heptahydrate is 0.01%, the agar is 1.5%, and the rest is water, pH 6.
- Seed medium yeast powder 0.3%, peptone 0.5%, soluble starch 1%, ammonium acetate 0.2%, sodium chloride 0.2%, magnesium sulfate heptahydrate 0.3%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%,
- the ferrous sulfate heptahydrate is 0.01%, and the rest is water, pH 6.
- Plate culture The original bacteria C. beijingii NCIMB 8052, Clostridium beijerincki IB4 (CCTCC NO: M2010310), and Clostridium beijerinckii M17 were inoculated onto the plate medium, anaerobic culture, culture temperature 35 ° C, culture time 12 h, the strain was activated.
- Seed culture The three strains activated by the above plate culture were inoculated into the seed culture medium respectively, 250 mL of Schott's anaerobic bottle was filled with 150 mL, nitrogen was filled for 3 min, culture temperature was 35 ° C, and culture time was 12 h. Seed culture solution.
- Fermentation culture Inoculum of the original cultured Clostridium beijerii NCIMB 8052, Clostridium beijerinckii IB4 (CCTCC NO: M2010310) and Clostridium beijerinckii M17 was inoculated to a fermentation medium containing 1 L. Medium 2, fermentation culture In the 2L fermenter of Nutrient 3, as shown in Table 4, the inoculation amount was 10% (v/v), the fermentation temperature was 35 °C, and nitrogen gas was continuously supplied at a flow rate of 0.3 L/min under the same experimental conditions. After 72 h of fermentation, the total solvent yield and butanol production in the fermentation products of each group were measured.
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Abstract
提供了一株高耐受性拜氏梭菌(Clostridium beijerinckii)M17 CCTCCNO: M 2013425及其在生产丁醇中的应用。该菌株通过常温等离子体诱变和筛选获得,对甘蔗渣酸解糖液具有高效利用率,且对其中的酚类化合物具有高度耐受性,适合用于甘蔗渣发酵产丁醇。
Description
一株高耐受性拜氏梭菌及其应用 技术领域
本发明涉及一株能利用未脱毒甘蔗渣酸解糖液发酵产丁醇的高耐受性拜氏梭菌及其应 用, 属于生物发酵技术领域。
背景技术
丁醇是无色液体, 有酒味, 与乙醇 /乙醚及其他多种有机溶剂混溶; 已广泛应用于增塑剂 等各种精细化学品的制造。 丁醇具有能量密度大、可与汽油任意比混合、可直接用于内燃机、 运输方便等优点; 作为可再生的新型液体燃料受到越来越多的重视。
随着化石能源的日益枯竭和价格飞涨, 利用可再生的木质纤维质原料 (如秸秆、 甘蔗渣 等)发酵生产丁醇, 成为研究的重点和热点之一。 Thaddeus Ezeji等 (Bioresource Technology. 2008, 99: 5915-5922) 利用拜氏梭菌突变株 BA101, 以 XAD-4 resin脱毒的玉米芯酸解和酶解 糖液为底物发酵, 总溶剂产量为 9.30 g/L; 但突变株 BA101不能利用未脱毒的酸解糖液发酵 产丁醇。庞浩等(生物技术, 2011, 21(5) , 79-82)报道拜氏梭菌 13-2株对甘蔗渣水解液具有较 高的发酵效率, 在 0.5%硫酸用量条件下, 它的丁醇发酵量最高可达到 4.5g /L。 中国专利 ZL 201110020102.6报道: 通过粒子束诱变育种得到的拜氏梭菌 (C/oWr i<¾ beijerinckii) IB4对 酚类化合物具有较高的抗逆性,其能以未脱毒的玉米芯酸解糖液作为碳源,在 2L发酵罐中总 溶剂产量和丁醇产量分别达到了 10.3g/L和 7.1 g/L; 同时, 以未脱毒的甘蔗渣酸解糖液作为 碳源,在 2L发酵罐中总溶剂产量和丁醇产量分别达到了 10.6 § 和7.3 § 。然而, 郭亭等(J Ind Microbiol Biotechnol, 2012, 39(3), 401-407)研究发现, 当玉米芯和甘蔗渣酸解糖液中的酚 类化合物的浓度提升到 1.5 g/L以上时,
beijerinckii) IB4基本不生长。
甘蔗渣是一种重要的可再生木质纤维原料, 利用蔗渣生产丁醇, 不仅节约资源, 还促进 制糖产业节能减排, 产业升级。 但是, 甘蔗渣等原料经稀酸处理后, 会产生有机酸、 糠醛、 酚类等抑制物, 这些抑制物的去除成本较高、 并对微生物生长有一定的抑制作用; Thaddeus Ezeji等 ( Biotechnology &Bioengineering. 2007, 97(6): 1460-1469) 研究发现有机酸、 糠醛等抑 制物不影响丁醇的发酵, 而酚类抑制物对丁醇发酵有明显的抑制效果。 基于此, 甘蔗渣酸解 糖液中的毒素抑制物 (尤其是酚类化合物) 严重抑制产丁醇梭菌的发酵性能; 而菌种改良是 提高菌株对抑制物的耐受性、 发酵经济性的关键手段之一。
发明内容
为了解决上述存在的问题, 本发明通过诱导突变和筛选的方法获取一株不仅对甘蔗渣酸 解糖液具有高效利用率, 且对其中的毒素抑制物 (尤其是酚类化合物) 具有高度耐受性的突 变菌株。
本发明的目的在于提供一株新菌种: 拜氏梭菌 (C/ ytr M beijerinckii) M17。
本发明的另一个目的在于提供上述菌株在丁醇生产中的应用。
本发明所采取的技术方案是:
申请人将菌株保藏在位于武汉市武昌珞珈山武汉大学的中国典型培养物保藏中心, 保藏 中心于 2013年 9月 14日收到申请人提供的菌株。保藏中心给予该培养物的保藏号为 CCTCC ΝΟι M 2013425, 提议的分类命名为 Clostridium beijerinckii ΜΙΊ, 已于 2013年 9月 23日鉴 定保藏的菌株是存活的。
一种生产丁醇的方法, 其特征在于: 包括以下步骤:
2 ) 种子培养: 将平板培养活化的拜氏梭菌 (Ctotr i beijerinckii) M17接种到种子培养 基中, lOO mL的厌氧瓶装液量 40〜60mL, 充氮气 3〜5min, 培养温度 33〜37 V, 培养时间 12〜24h;
3 ) 发酵产丁醇: 将种子培养后的拜氏梭菌 (Ctotr i beijerinckii) M17菌液接种到发酵 培养基中, 接种量按 5〜15%的体积百分比, 充氮气 3〜5min, 发酵温度 33〜37°C, 发酵培养 时间为 70〜90 h, 即可发酵产出丁醇。
进一步的, 步聚 1 )所述的平板培养基包含如下质量百分比的组分: 碳源 0.3%〜1%、 氮 源 0.5%〜1%、 无机盐 0.5%〜0.8%、 琼脂 1.5%〜2%、 其余为水; 其中, 碳源选自葡萄糖、 淀粉、 甘蔗渣酸解糖液、 玉米芯酸解糖液中至少一种; 氮源选自乙酸铵、 氯化铵、 蛋白胨、 酵母粉、 玉米浆中的至少一种; 无机盐选自钠盐、 钾盐、 镁盐、 磷酸盐、 亚铁盐中的至少一 种。
进一步的, 步聚 2 )所述的种子培养基包含如下质量百分比的组分: 碳源 0.5%〜1%、 氮 源 0.5%〜1%、 无机盐 0.5%〜0.8%、 其余为水; 其中, 碳源选自淀粉、 葡萄糖中至少一种; 氮源选自乙酸铵、 氯化铵、 蛋白胨、 酵母粉、 玉米浆中至少一种; 无机盐选自钠盐、 钾盐、 镁盐、 磷酸盐、 亚铁盐中至少一种。
进一步的, 步聚 3 ) 所述的发酵培养基包含如下质量百分比的组分: 碳源 3%〜6%、 氮 源 0.1%〜0.3%、 无机盐 0.1%〜0.2%、 生长因子 0.05%〜0.1%、 其余为水; 其中, 碳源选自
葡萄糖、 木糖、 甘蔗渣酸解糖液、 玉米芯酸解糖液中至少一种; 氮源选自乙酸铵、 氯化铵、 酵母粉中至少一种; 无机盐选自钠盐、 钾盐、 镁盐、 钙盐、 磷酸盐、 亚铁盐中至少一种; 生 长因子选自对氨基苯甲酸、 维生素 B l、 生物素和玉米浆中至少一种。
本发明的有益效果是:
本发明菌株拜氏梭菌 Clostridium beijerinckii Ml 7对未脱毒木质纤维酸解糖液中的毒素物 质具有高度耐受性, 在酚浓度高达 2.0 g/L的发酵液中, 能非常好地发酵生产丁醇, 其总溶剂 产量和丁醇产量分别达到了 10.9 g/L和 7.9 g/L, 即使酚浓度高达 2.4 g/L, 本发明菌株仍具有 可观的产丁醇能力, 说明本发明菌株拜氏梭菌 M17对毒素物质耐受性强、溶剂产量和丁醇产 量高、 重复性好, 是一种适合利用甘蔗渣发酵产丁醇的优良菌种。
本发明菌株拜氏梭菌 Clostridium beijerinckii M17对甘蔗渣酸解糖液具有高效的利用率, 且对其中的毒素抑制物(尤其是酚类化合物)具有高度的耐受性, 这不仅有利于丁醇的生产, 还有利于资源的节约, 促进制糖产业的节能减排, 促进产业升级。
附图说明
图 1为拜氏梭菌的等离子体诱变存活率曲线。
具体^ ½方式
根据下述实施例, 可以更好地理解本发明。 然而, 本领域的技术人员容易理解, 实施例 所描述的具体的物料配比、 工艺条件及其结果仅用于说明本发明, 而不应当也不会限制权利 要求书中所详细描述的本发明。
鎌例
一、等离子体诱变原始拜氏梭菌株
拜氏梭菌原始菌株进行第一步等离子体诱变的方法如下:
将拜氏梭菌 NCIMB 8052原始菌株活化培养, 培养温度 33〜37°C, 50ml 肖特厌氧瓶装 液量为 15〜20ml, 充氮气 3min, 培养时间 12〜18 h, 得到生长旺盛的菌液; 取新鲜培养的细 胞稀释至细胞浓度 OD6(K)=0.1〜1.0, 滴加在灭菌冷却后的载片上, 用无菌空气吹干; 以氦气 为放电气体, 以 100W作为射频功率, 以 10SLM作为气体流量, 以 10〜240s作为辐照时间 对菌株进行等离子体诱变, 诱变后, 将载体上的菌膜洗脱下来, 计算存活率。 实验结果如附 图 1所示; 由图 1可知, 180s是最佳的诱变辐照时间。
二、 筛选诱变后的目的拜氏梭菌株
( 1 ) 培养基配方 (%为质量百分比):
1. 甘蔗渣酸解糖液平板培养基: 酵母粉 0.3%, 蛋白胨 0.5%, 可溶性淀粉 1%, 乙酸铵
0.2%, 氯化钠 0.2%, 七水合硫酸镁 0.3%, 磷酸二氢钾 0.1%, 磷酸氢二钾 0.1%, 七水合硫酸 亚铁 0.01%, 琼脂 1.5%, 刃天青 0.02%, 甘蔗渣酸解糖液 25% (v/v), 其余为水, pH 6。
2. 种子培养基:酵母粉 0.3%,蛋白胨 0.5%,可溶性淀粉 1%,乙酸铵 0.2%,氯化钠 0.2%, 七水合硫酸镁 0.3%, 磷酸二氢钾 0.1%, 磷酸氢二钾 0.1%, 七水合硫酸亚铁 0.01%, 其余为 水, pH 6。
3. 摇瓶发酵初筛培养基: 葡萄糖 3%, 乙酸铵 0.22%, 磷酸二氢钾 0.05%, 磷酸氢二钾 0.05%,氯化钠 0.001%,七水合硫酸镁 0.02%,七水合硫酸亚铁 0.001%,一水合硫酸锰 0.001%, 玉米浆 0.1%, 其余为水, pH 6.6。
5. 摇瓶发酵复筛培养基: 甘蔗渣酸解糖液 3%, 乙酸铵 0.22%, 磷酸二氢钾 0.05%, 磷酸 氢二钾 0.05%, 氯化钠 0.001%, 七水合硫酸镁 0.02%, 七水合硫酸亚铁 0.001%, 一水合硫酸 锰 0.001%, 玉米浆 0.1%, 其余为水, pH 6.6。
(2) 筛选步骤:
1、 甘蔗渣酸解糖液平板培养基培养
将诱变后的载片置于装有 l〜2ml生理盐水的具塞试管中, 剧烈震荡,将载片上的菌株洗 脱, 稀释成不同浓度涂布于甘蔗渣酸解糖液平板培养基上, 33〜37°C厌氧培养 12〜36h, 挑 选出透明圈和菌落较大的菌落 50株, 分别编号 M1〜M50, 将其分别用种子培养基进行扩大 培养, 并保种。
2、 摇瓶发酵初筛
将初筛的扩大培养后的 50株菌落和原始菌株接种到摇瓶发酵初筛培养基中,接种量 10% (v/v), lOO mL肖特厌氧瓶装液量 50 mL, 发酵温度 35°C, 发酵时间 72 h后检测各菌株的总 溶剂产量和丁醇产量, 结果显示菌株 M17、 M39和 M48可发酵产出较高的丁醇产量, 如表 1 所示。
表 1 各菌株的总溶剂产量和丁醇产量
总溶剂产量 丁醇产量
菌号
(g L) (g L) 原始菌株 NCIMB 8052 10.8 7.4
M17 11.4
M39 11.1 7.4
M48 11.2
经过组合筛选获得的三株突变株在发酵过程中总溶剂产量和丁醇产量均高于原始菌株的
产量, 其中 M17的总溶剂产量和丁醇产量也最高。
3、 摇瓶发酵复筛
将发酵初筛获得的总溶剂和丁醇产量最高的菌株 M17和原始菌株接种到摇瓶发酵复筛培 养基中, 接种量 10% (v/v), lOO mL肖特厌氧瓶装液量 50 mL, 发酵温度 35 °C, 发酵时间 72 h后检测两个菌株的总溶剂产量和丁醇产量,结果显示菌株 M17可发酵产出较高的丁醇产量, 如表 2所示。
表 2 原始菌株和 M17的总溶剂产量和丁醇产量 总溶剂产量 丁醇产量
菌号
(g L) (g L) 原始菌株 NCIMB 8052 1 1
M17 9.7 6.5
从摇瓶发酵复筛结果可知, 原始菌株拜氏梭菌 NCIMB 8052 基本不生长; Clostridium beijerinckii M17的总溶剂产量和丁醇产量分别 9.4g/L和 6.5 g/L,可见 M17在发酵过程中总溶 剂产量和丁醇产量明显高于原始菌株的产量。 这与平板培养及发酵初筛的结果是一致的。
三、 目的拜氏梭菌株的传代稳定性
在以葡萄糖为碳源的发酵培养基中, 检测突变株拜氏梭菌 M17的传代稳定性, 菌株 M17 传代发酵试验结果如表 3所示:
表 3 拜氏梭菌 M17的传代稳定性
拜氏梭菌 M17
传代次数 丁醇产量 总溶剂产量
(g L) (g L)
1 7.7 11.4
2 7.5 11.2
3 7.8 11.6
4 7.7 11.5
5 7.6 11.3
6 7.9 11.7
7 7.8 11.4
从实验结果可知, 经过 7次连续传代, 突变株 M17、 M39和 M48的总溶剂产量和丁醇
产量都较稳定, 具有较好的传代稳定性, 可作为进一步研究和开发的生产菌株。 四、 检测目的拜氏梭菌对未脱毒木质纤维酸解糖液中毒素物质的耐受性
( 1 ) 培养基配方 (%为质量百分比):
发酵培养基 1 : 乙酸铵 0.22%, 磷酸二氢钾 0.05%, 磷酸氢二钾 0.05%, 氯化钠 0.001%, 七水合硫酸镁 0.02%, 七水合硫酸亚铁 0.001%, 一水合硫酸锰 0.001%, 玉米浆 0.1%, 用未 脱毒的甘蔗渣的酸解糖液(总还原糖为 3 %)配制, 其余为水, pH 6.6, 培养基中可溶性总酚 含量为 1.5 g/L。
发酵培养基 2: 乙酸铵 0.22%, 磷酸二氢钾 0.05%, 磷酸氢二钾 0.05%, 氯化钠 0.001%, 七水合硫酸镁 0.02%, 七水合硫酸亚铁 0.001%, 一水合硫酸锰 0.001%, 玉米浆 0.1%, 用未 脱毒甘蔗渣的酸解糖液(总还原糖为 4 %)配制, 其余为水, pH 6.6, 培养基中可溶性总分含 量为 2.0 g/L。
发酵培养基 3 : 乙酸铵 0.22%, 磷酸二氢钾 0.05%, 磷酸氢二钾 0.05%, 氯化钠 0.001%, 七水合硫酸镁 0.02%, 七水合硫酸亚铁 0.001%, 一水合硫酸锰 0.001%, 玉米浆 0.1%, 用未 脱毒的玉米芯酸解糖液 (总还原糖为 4.7 %) 配置, 其余为水, pH 6.6, 培养基中可溶性总酚 含量为 2.4 g/L。
平板培养基: 酵母粉 0.3%, 蛋白胨 0.5%, 可溶性淀粉 1%, 乙酸铵 0.2%, 氯化钠 0.2%, 七水合硫酸镁 0.3%,磷酸二氢钾 0.1%,磷酸氢二钾 0.1%,七水合硫酸亚铁 0.01%,琼脂 1.5%,, 其余为水, pH 6。
种子培养基: 酵母粉 0.3%, 蛋白胨 0.5%, 可溶性淀粉 1%, 乙酸铵 0.2%, 氯化钠 0.2%, 七水合硫酸镁 0.3%, 磷酸二氢钾 0.1%, 磷酸氢二钾 0.1%, 七水合硫酸亚铁 0.01%, 其余为 水, pH 6。
(2) 实验过程、 结果和结论
1. 菌种的平板培养、 种子培养和发酵培养
平板培养: 将原始菌拜氏梭菌 NCIMB 8052、 拜氏梭菌 IB4 (CCTCC NO: M2010310)、 拜氏梭菌 M17分别接种至平板培养基上, 厌氧培养, 培养温度 35°C, 培养时间 12 h, 使菌种 活化。
种子培养:将上述平板培养活化后的三种菌种分别接种到种子培养基中, 250 mL 肖特厌 氧瓶装液量 150 mL, 充氮气 3min, 培养温度 35°C, 培养时间 12 h, 获得种子培养液。
发酵培养: 将种子培养后的原始菌拜氏梭菌 NCIMB 8052、 拜氏梭菌 IB4 (CCTCC NO: M2010310)、拜氏梭菌 M17的菌液分别接种至装有 1 L发酵培养基 1、发酵培养基 2、发酵培
养基 3的 2L发酵罐中, 如表 4所示, 接种量为 10% (v/v) , 发酵温度 35 °C, 连续通入氮气, 流速为 0.3 L/min, 在同样的实验条件下发酵培养 72 h后, 分别检测各组发酵产物中的总溶剂 产量和丁醇产量。
表 4 各菌种在含不同酚浓度的发酵培养基中的总溶剂产量和丁醇产量
2. 实验结果
检测结果如表 4所示, 从中可以看出, 当发酵培养基中的总酚含量为 1.5 g/L时, 原始菌 种拜氏梭菌 NCIMB 8052均基本不生长, 而拜氏梭菌 IB4 ( CCTCC NO: M2010310 ) 和拜氏 梭菌 M17均能良好的生长,且拜氏梭菌 IB4总溶剂产量和丁醇产量分别为 10.3g/L和 7.1 g/L, 拜氏梭菌 M17的产量分别为 10.5g/L和 7.2 g/L, 二者丁醇产量相差并不显著; 当发酵培养基 中的总酚含量为 2.0g/L和 2.4g/L时,拜氏梭菌 NCIMB 8052和拜氏梭菌 IB4均不能正常生长, 而拜氏梭菌 M17仍可以良好地生长, 且当总酚含量为 2.0g/L时, M17产丁醇的能力还有所 上升, 其总溶剂产量和丁醇产量分别为 10.9 g/L和 7.9 g/L, 当总酚含量高达 2.4g/L时, M17 产丁醇的能力受到一定的影响, 其总溶剂产量和丁醇产量分别降至 8.7 g/L和 5.9 g/L。
3. 实验结论
上述的检测结果说明本发明获得的拜氏梭菌 (C/O M beijerinckii) M17能直接利用未 脱毒甘蔗渣酸解糖液发酵产丁醇, 且其对毒性的耐受能力远高于目前的拜氏梭菌 (C/a trW M beijerinckii) IB4 (最高耐受的总酚浓度约为 1.5g/L), 即使在高达 2.4g/L的总酚浓度下, 仍具 有较好的产丁醇能力。
Claims
1. 一株拜氏梭菌, 其分类命名为 Clostridium beijetinckii ΜΙΊ, 已保藏于中国典型培养物保 藏中心 CCTCC, 保藏编号: CCTCC NO; M 2013425。
2. 权利要求 1所述的 Clostridium beijerinckii M17在生产丁醇中的应用。
3. 一种生产丁醇的方法, 其特征在于: 包括以下步骤:
2 ) 种子培养: 将平板培养活化的拜氏梭菌 (Ctotn'ife M17接种到种子培养 基中, lOO mL的厌氧瓶装液量 40〜60mL, 充氮气 3〜5min, 培养温度 33〜37 V, 培养 时间 12〜24h;
3 ) 发酵产丁醇: 将种子培养后的拜氏梭菌 (C/oWrW M fe^n'wc ') M17菌液接种到发酵 培养基中, 接种量按 5〜15%的体积百分比, 充氮气 3〜5min, 发酵温度 33〜37°C, 发酵 培养时间为 70〜90 h, 即可发酵产出丁醇。
4. 根据权利要求 3所述的一种生产丁醇的方法, 其特征在于: 步聚 1 )所述的平板培养基包 含如下质量百分比的组分: 碳源 0.3%〜1%、 氮源 0.5%〜1%、 无机盐 0.5%〜0.8%、 琼脂 1.5%〜2%、 其余为水; 其中, 碳源选自葡萄糖、 淀粉、 甘蔗渣酸解糖液、 玉米芯酸解糖 液中至少一种; 氮源选自乙酸铵、 氯化铵、 蛋白胨、 酵母粉、 玉米浆中的至少一种; 无 机盐选自钠盐、 钾盐、 镁盐、 磷酸盐、 亚铁盐中的至少一种。
5. 根据权利要求 3所述的一种生产丁醇的方法, 其特征在于: 步聚 2 )所述的种子培养基包 含如下质量百分比的组分: 碳源 0.5%〜1%、 氮源 0.5%〜1%、 无机盐 0.5%〜0.8%、 其余 为水; 其中, 碳源选自淀粉、 葡萄糖中至少一种; 氮源选自乙酸铵、 氯化铵、 蛋白胨、 酵母粉、 玉米浆中至少一种; 无机盐选自钠盐、 钾盐、 镁盐、 磷酸盐、 亚铁盐中至少一 种。
6. 根据权利要求 3所述的一种生产丁醇的方法, 其特征在于: 步聚 3 )所述的发酵培养基包 含如下质量百分比的组分: 碳源 3%〜6%、 氮源 0.1%〜0.3%、 无机盐 0.1%〜0.2%、 生长 因子 0.05%〜0.1%、 其余为水; 其中, 碳源选自葡萄糖、 木糖、 甘蔗渣酸解糖液、 玉米 芯酸解糖液中至少一种; 氮源选自乙酸铵、 氯化铵、 酵母粉中至少一种; 无机盐选自钠 盐、 钾盐、 镁盐、 钙盐、 磷酸盐、 亚铁盐中至少一种; 生长因子选自对氨基苯甲酸、 维 生素 B l、 生物素和玉米浆中至少一种。
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US20130149757A1 (en) * | 2011-12-09 | 2013-06-13 | Optinol, Inc. | Method for Producing Butanol and Isopropanol |
CN103898010A (zh) * | 2014-03-10 | 2014-07-02 | 广州甘蔗糖业研究所 | 一株高耐受性拜氏梭菌及其应用 |
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CN101748114A (zh) * | 2008-12-12 | 2010-06-23 | 中国科学院微生物研究所 | 一种获得丁醇产生菌突变株的方法 |
US20130149757A1 (en) * | 2011-12-09 | 2013-06-13 | Optinol, Inc. | Method for Producing Butanol and Isopropanol |
CN102703523A (zh) * | 2012-07-03 | 2012-10-03 | 广西科学院 | 以甘蔗渣和糖蜜为原料混合发酵生产丁醇的方法 |
CN103898010A (zh) * | 2014-03-10 | 2014-07-02 | 广州甘蔗糖业研究所 | 一株高耐受性拜氏梭菌及其应用 |
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