WO2015035734A1 - 一种表面活性剂改进的乙醇发酵法 - Google Patents

一种表面活性剂改进的乙醇发酵法 Download PDF

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WO2015035734A1
WO2015035734A1 PCT/CN2014/000398 CN2014000398W WO2015035734A1 WO 2015035734 A1 WO2015035734 A1 WO 2015035734A1 CN 2014000398 W CN2014000398 W CN 2014000398W WO 2015035734 A1 WO2015035734 A1 WO 2015035734A1
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surfactant
fermentation
fermentation method
ethanol fermentation
ethanol
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PCT/CN2014/000398
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English (en)
French (fr)
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张宗超
刘秀梅
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中国科学院大连化学物理研究所
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Priority claimed from CN201310416058.XA external-priority patent/CN104450793B/zh
Priority claimed from CN201310660369.0A external-priority patent/CN104694584B/zh
Application filed by 中国科学院大连化学物理研究所 filed Critical 中国科学院大连化学物理研究所
Priority to KR1020167007341A priority Critical patent/KR20160052584A/ko
Priority to US14/909,801 priority patent/US10066243B2/en
Priority to EP14844323.7A priority patent/EP3045539B1/en
Priority to CA2923932A priority patent/CA2923932C/en
Priority to JP2016541768A priority patent/JP6229864B2/ja
Priority to AU2014321058A priority patent/AU2014321058B2/en
Publication of WO2015035734A1 publication Critical patent/WO2015035734A1/zh

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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
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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 ethanol production by fermentation processes, especially ethanol fermentation processes with improved surfactants under VHG fermentation conditions.
  • 20110201093A1 reports that the addition of carbonate in the fermentation medium can increase the ethanol yield and reduce Less fermentation time; US20100143993A1 and WO2010/065595A2 use ionic liquids as extractants to continuously extract ethanol from the fermentation system in situ to ensure yeast activity.
  • Ultra-high concentration (VHG) fermentation is a technology that can improve the economic index of ethanol fermentation.
  • Ultra-high concentration ethanol fermentation has the following advantages: 1. Improve the productivity and utilization of unit equipment; 2 Reduce water consumption; 3 Increase the unit volume of sputum Ethanol, reducing the energy consumption of cooking, fermentation, distillation and DDGS concentration and drying process; 4 inhibiting the growth of bacteria.
  • the ultra-high concentration ethanol fermentation also brings some problems because the initial sugar concentration is obviously increased.
  • the yeast cells are subjected to high osmotic pressure under the action of high sugar concentration, resulting in decreased growth and survival rate, prolonged fermentation time and no fermentation.
  • the invention provides an improved ethanol fermentation method for surfactants, which uses high-activity Saccharomyces cerevisiae and uses glucose as a raw material to add nonionic surfactant in fermentation culture to reduce alcohol inhibition and reduce high sugar concentration.
  • Osmotic pressure increase the survival rate and growth rate of Saccharomyces cerevisiae cells under special circumstances, and finally increase the end point ethanol concentration, effectively reduce the water consumption during the fermentation process and the energy consumption of the rectification process; and ferment yeast and surfactant from the fermentation
  • the mixture is separated and reused in the next batch of fermentation.
  • the object of the present invention is to provide an improved ethanol fermentation process for a surfactant, relating to an ultrahigh concentration (VHG) fermentation ethanol production process, in particular to an ultrahigh concentration ethanol fermentation which is improved by a surfactant, in a fermentation medium.
  • a surfactant improves the endpoint ethanol concentration.
  • fuel ethanol is mainly produced by a fermentation process comprising a carbohydrate and a sugar source, a Saccharomyces cerevisiae, a fermentation medium having an initial pH of 3.5 to 5.3, and a fermentation temperature of 25 to 40 °C.
  • the invention proposes to add a nonionic surfactant to the fermentation medium to improve the ethanol concentration and the yeast cell viability, and the ethanol concentration can be increased from 110 g/L without adding a surfactant to after adding a surfactant.
  • the invention provides an improved ethanol fermentation method of a surfactant, wherein a fermentable carbohydrate is used as a carbon source, a surfactant-water mixture is used as a fermentation medium, a pH adjuster is added to adjust the pH of the fermentation medium, and the Saccharomyces cerevisiae cells are inoculated. Perform ultra-high concentration ethanol fermentation.
  • the surfactant provided by the present invention is an improved ethanol fermentation method, wherein the fermentable carbohydrate is glucose, and the fermentable carbohydrate concentration is 270 to 500 g/L.
  • the surfactant provided by the present invention is an improved ethanol fermentation method in which the mass ratio of the surfactant to water in the fermentation medium is from 0.001 to 0.5 (preferably from 0.125 to 0.375).
  • the surfactant-proven ethanol fermentation process comprises: (a) a fermentation medium comprising Saccharomyces cerevisiae; (b) a nonionic surfactant polyethylene glycol (PEG), At least one of polyethylene glycol monomethyl ether (MPEG), polyethylene glycol monomethyl ether (DMPEG), polydimethylsiloxane (PDMS), the addition of surfactant can be increased under VHG fermentation conditions Yeast cell life and endpoint ethanol concentration ⁇ ' (c) Yeast cells can be recovered from the fermentation mixture for reuse; (d) Surfactants and pH adjusters can be recycled and reused simultaneously.
  • PEG polyethylene glycol
  • MPEG polyethylene glycol monomethyl ether
  • DMPEG polyethylene glycol monomethyl ether
  • PDMS polydimethylsiloxane
  • PEG is at least one of PEG-200, 400, 600, 800, 1000;
  • the structural formula of PEG is:
  • the structural formula of MPEG is:
  • DMPEG The structural formula of DMPEG is: , n OM
  • the fermentation temperature is generally selected from 28 ° C to 44 ° C, especially from 30 ° C to 38 ° C (most preferably 33 ° C)
  • the pH is generally selected from 3.0 to 6.5, preferably.
  • the pH ranges from 4.0 to 5.0
  • the yeast cell concentration is from 10 7 to 10 9 /L
  • the fermentation time is from 12 to 120 h (preferably 60 h).
  • the invention provides an improved ethanol fermentation method for a surfactant, wherein the pH adjusting agent of the fermentation medium is a sulfuric acid solution, a citric acid-sodium citrate buffer, a phosphate buffer solution, a carbonate buffer solution, and an acetic acid-sodium acetate buffer solution.
  • the pH adjusting agent of the fermentation medium is a sulfuric acid solution, a citric acid-sodium citrate buffer, a phosphate buffer solution, a carbonate buffer solution, and an acetic acid-sodium acetate buffer solution.
  • the tolerance of yeast cells to high concentrations of glucose osmotic pressure and tolerance to high concentrations of ethanol are key factors to consider.
  • the addition of a nonionic surfactant to the fermentation medium is effective to increase the concentration of ethanol.
  • this phenomenon may be caused by the surfactant directly or indirectly acting as a cytoprotective agent to improve the yeast cells. Survivability thus increases ethanol concentration.
  • the surfactant used is a nonionic surfactant.
  • nonionic surfactants polyethylene glycol (PEG), especially PEG-200, PEG-400 and PEG-600.
  • PEG polyethylene glycol
  • the mass ratio of surfactant to water is from 0 to 2/3, and the mixed fermentation medium of surfactant and water can increase the concentration of ethanol, which is consistent with the idea of the present invention.
  • the fermentation medium prepared by mixing the surfactant to water in a mass ratio of from 1/5 to 2/3 and from 1/5 to 1/4 can also increase the ethanol concentration.
  • the fermentation bath for producing ethanol proposed by the present invention comprises an ethanol producing microorganism (for example, yeast), a fermented carbon source (for example, a carbohydrate, a monosaccharide, a polysaccharide, etc.), a pH adjuster (for example, H 2 S0 4 , citric acid-lemon Sodium acetate buffer, phosphate buffer, carbonate buffer, acetic acid-sodium acetate).
  • the fermentation substrate concentration is 275g/L to 500g/L, the fermentation substrate is composed of fermentable carbohydrates, and glucose, cellulose and starch are especially suitable as carbon sources.
  • the present invention provides an improved ethanol fermentation method for yeast.
  • the yeast cells can be recovered and reused, and the fermentation broth is recovered by freeze centrifugation to directly use the yeast cells for the next fermentation process.
  • the fermentation of the yeast cells is carried out by freeze centrifugation using a standard centrifugation technique at a centrifugal speed of 4000 to 15000 rpm and a centrifugation time of 1 to 30 minutes.
  • the surfactant provided by the present invention is an improved ethanol fermentation method.
  • the surfactant and the pH adjuster can be recycled and reused, and the yeast is removed by freeze centrifugation, and the residue is distilled to replace the fresh surfactant for the next fermentation process.
  • the recovery process of the surfactant and the pH adjuster is a standard vacuum distillation technique, and the distillation temperature is 40 ° C to 100 ° C under a pressure of 0 Mpa to 0.09 Mpa, and the distillation time is 20 min to 120 min.
  • the present invention provides an improved ethanol fermentation method for yeast, yeast cells and surfactants can be simultaneously recovered and reused, and the yeast cells are recovered by freeze centrifugation, and the supernatant is distilled.
  • the surfactant both of which are used in the next batch of fermentation process.
  • the surfactant provided by the invention has improved ethanol fermentation method, and the surfactant is recycled and reused, and the yeast cells are removed by freezing and centrifugation after the fermentation, and the surfactant and the pH adjuster are recovered by standard vacuum distillation technology, and the next fermentation process can be carried out.
  • Direct addition of fermentable carbohydrates, deionized water, and recycled surfactants and pH adjusters eliminates the need to readjust the pH of the fermentation medium.
  • Surfactants not only have a low vapor pressure, but also have an adjustable water solubility, and fine-tuning of their physical properties can be achieved by the choice of structure and molecular weight. This range of properties determines the surfactant's broad biocompatibility for many microorganisms and can be separated from the fermentation mixture by techniques such as centrifugation and distillation.
  • yeast cell recovery proposed by the present invention: yeast cells can be recovered and reused from the fermentation mixture of surfactant and water by centrifugation, and the specific process steps are shown in FIG.
  • the present invention provides a surfactant recovery process: After the fermentation is complete, the surfactant can be recovered from the fermentation mixture by standard distillation techniques and the surfactant can be recycled multiple times. The specific process steps are shown in Figure 2.
  • Saccharomyces cerevisiae Hubei Angel Yeast Company. Other reagents and medicines, Sinopharm Group.
  • a certain amount of Saccharomyces cerevisiae was weighed and placed in a lOOmL Erlenmeyer flask, and activated at 60 °C with ultrapure water for 20 min to be used as a wine master. Then add glucose, surfactant, pre-configured pH solution, seal with plastic wrap, put into a shaker and shake culture, the speed of the shaker is
  • the product sample was diluted with deionized water.
  • the content of each component in the fermentation broth was determined by high performance liquid chromatography (Agilent 1260), and the conversion rate was calculated according to the glucose dosage.
  • the ethanol yield is calculated from the ethanol mass in the fermentation broth, the activated water and the pH liquid volume.
  • the chromatographic conditions were: ion exchange column, column temperature 65 ° C, parallax refractometer, detector 50 ° C; mobile phase: 5Mm H 2 S0 4 , flow rate 0.6ml / min, injection volume 25uL.
  • FIG. 1 Process steps for recycling and recycling solid yeast powder
  • FIG. 1 Process steps of simultaneous recovery and reuse of surfactant and sulfuric acid
  • Figure 12 Effect of surfactant cycle/fermentation 48h fermentation cycle on glucose conversion;
  • Figure 13 Effect of surfactant/water fermentation 48h yeast cycle on ethanol yield;
  • Figure 14 Effect of surfactant/water fermentation 48h yeast cycle on ethanol concentration;
  • Figure 15 Effect of surfactant/water fermentation 36-72h yeast cycle on glucose conversion;
  • Figure 16 Surfactant/water fermentation 36-72h yeast cycle Effect on ethanol yield;
  • Figure 17 Effect of surfactant/water fermentation 36-72h yeast cycle on ethanol concentration;
  • Figure 18 Surfactant/sulfuric acid recovery and reuse.
  • Example 1-9 Effect of the ratio of PEG-200 to water on the efficiency of ethanol fermentation
  • Example 10-17 Effect of the ratio of PEG-400 to water on the efficiency of ethanol fermentation
  • the end point ethanol concentration decreases as the temperature rises; However, the endpoint ethanol concentration also decreased when the temperature dropped to 30 °C.
  • the optimum fermentation temperature should be selected at 33 °C.
  • the ethanol concentration at the end of the fermentation can reach 160 gi- 1 , and the fermentation system has no residual glucose.
  • yeast powder 0.5g is rehydrated with 4mL of ultrapure water for 15-30 minutes under 25-38 ⁇ conditions, then 6.5 g of glucose and 12 mL of a pre-configured pH 4.3 sulfuric acid solution were added, shaken in a shaker, and fermented at 33 ° C for 48 hours. After the fermentation, the fermentation was centrifuged at 8000 rpm for 5 minutes. The fermentation broth after the fermentation was removed, leaving solid yeast powder, and 4 mL of ultrapure water, 6.5 g of glucose, 12 mL of sulfuric acid solution were added again, and fermentation was carried out at 33 ° C for 48-72 hours. This cycle 2 times. The glucose conversion rate, ethanol yield and concentration are shown in Fig. 9, Fig. 10, and Fig. 11, respectively. As can be seen from the data in FIG, when the fermentation system no surfactant is added, the recovery efficiency is very low yeast, yeast recovered first recycling achieved only after 48 hours of fermentation 60g Shang - 1 of ethanol concentration.
  • Example 29 Surfactant - Fermentation in a Water Mixture System 48h Yeast Cycle Reuse
  • yeast powder 0.5g was rehydrated with 4mL of ultrapure water at 25-38 ° C for 15-30 minutes, then 6.5g of glucose, PEG-400 4.0g, pre-formulated pH of 4.3 sulfuric acid solution 12mL, put The mixture was shaken in a shaker and fermented at 33 ° C for 48 hours. After the end of the fermentation, the mixture was centrifuged at 8000 rpm for 5 minutes, and the fermentation broth after the fermentation was removed, leaving a solid yeast powder, and 4 mL of ultrapure water, 6.5 g of glucose, 4.0 g of PEG-400, and 12 mL of sulfuric acid solution were added again, 33 ° C. Fermentation for 48 hours, this cycle 3 times.
  • the glucose conversion rate, ethanol yield and concentration are shown in Fig. 12, Fig. 13, and Fig. 14, respectively.
  • the recovery efficiency of the yeast is high, and the first cycle of recovery of the recovered yeast after 48 hours of fermentation can obtain an ethanol concentration of 118 g.
  • Example 30 Surfactant - Fermentation in a Water Mixing System 36-72h Yeast Cycle Recycling
  • yeast powder 0.5g was rehydrated with 4mL of ultrapure water for 15-30 minutes under 25-38 ⁇ conditions, then 6.5g of glucose, PEG-400 4.0g, pre-formulated pH of 4.3 sulfuric acid solution 12mL, put into shake The bed was shaken and fermented at 33 ° C for 36-72 hours. 8000 rpm after fermentation The mixture was centrifuged for 5 minutes, and the fermentation broth after the fermentation was removed, leaving a solid yeast powder, and 4 mL of ultrapure water, 6.5 g of glucose, 4.0 g of PEG-400, and 12 mL of sulfuric acid solution were added again, and fermentation was carried out at 33 ° C for 48 hours. This cycle 3 times.
  • the glucose conversion rate, ethanol yield, and concentration are shown in Fig. 15, Fig. 16, and Fig. 17, respectively. It can be seen from the data in the figure that when PEG-400 is added to the fermentation system, the recovery efficiency of the yeast is high, and the ethanol concentration of llSgl 1 can be obtained by recycling the yeast for the first time after 48 hours of fermentation.
  • 0.5g of yeast powder was rehydrated with 4mL of ultrapure water at 25-38 ° C for 15-30 minutes, then 7.5g of glucose, 4.0g of PEG-400, pre-configured pH of 4.3 sulfuric acid solution 12mL, put The mixture was shaken in a shaker and fermented at 33 ° C for 72 hours. After the end of the fermentation, the mixture was centrifuged at 8000 rpm for 5 minutes to remove the solid yeast powder, and the supernatant was distilled. After the distillation, the residue was transferred to the next fermentation process, and 0.5 g of yeast powder was again added to recover 4 mL of ultrapure water under the conditions of 25-38 Torr.
  • the water was fermented for 15-30 minutes, 6.5 g of glucose, 12 mL of sulfuric acid solution, and cultured at 33 ° C for 72 hours, and thus cycled 4 times.
  • the glucose conversion rate, ethanol yield and concentration are shown in Fig. 18.
  • the surfactant PEG-400 can be recycled and has no significant effect on the fermentation efficiency.

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Abstract

一种表面活性剂改进的乙醇发酵法，包括在发酵培养基中添加非离子表面活性剂，酵母细胞和表面活性剂可以回收再利用。该方法提高了超高浓度乙醇发酵液中酵母细胞的存活率和终点乙醇浓度，降低了生产成本。

Description

说 明 书
一种表面活性剂改进的乙醇发酵法
技术领域
本发明涉及发酵工艺生产乙醇领域，尤其是在 VHG发酵条件下表面活 性剂改进的乙醇发酵法。
背景技术
随着世界经济的飞速发展， 人们对化石资源的依赖程度逐渐增加， 化 石资源的高强度开采与消耗排放大量 C02等有害气体 [Progress In Chemistry, 2006, 18(2-3): 131-141],造成严重的环境污染，影响了人类社会的生存与发 展。 近年来， 为了缓解能源日益短缺与环境因温室气体排放的增加而导致 全球温度提升的危机， 人们纷纷致力于发展清洁 "绿色"可替代能源。 燃 料乙醇作为新型可再生能源拥有清洁、 可再生等特点， 可以降低汽车尾气 中一氧化碳和碳氢化合物的排放的特点， 也是有效缓解上述能源危机的主 要对策之一。
生物乙醇的生产主要是通过微生物的发酵将各种生物质转化为燃料酒 精。降低生产成本是提高生产燃料乙醇经济效益、 尤其是纤维素乙醇的生 产， 使其可以得到更广泛应用的关键因素。 为了有效节约生产成本， 目前 人们主要致力于改善发酵时间、 提高酵母循环利用效率、 提高终点乙醇浓 度。 Kwang Ho Lee等（Bioresource Technology, 102, 2011： 8191-8198 )报 道了一种以藻酸钙为载体将酵母固定化的方法。 与游离的细胞相比较固载 酵母可获得更高的乙醇收率， 并且有效縮短发酵时间 10小时； 美国专利 US20110201093A1 报道了在发酵介质中加入碳酸盐可提高乙醇收率并减 少发酵时间； 专利 US20100143993A1及 WO2010/065595A2采用离子液体作 为萃取剂从发酵体系原位连续萃取乙醇以保证酵母活性。
超高浓度 (VHG)发酵是一种能够提高乙醇发酵经济指标的技术， 超高 浓度乙醇发酵具有如下优点： ①提高单位设备的生产率和利用率； ②减少 水耗； ③增加单位体积醪液中的乙醇， 降低蒸煮、发酵、蒸馏和 DDGS浓缩 干燥过程的能耗； ④抑制杂菌的生长。 但是， 超高浓度乙醇发酵因为起始 糖浓度明显提高也带来了一些问题， 一方面高糖浓度作用下酵母细胞受到 高渗透压的胁迫， 导致生长和存活率下降， 发酵时间延长和发酵不完全； 另一方面酵母细胞受到高浓度乙醇的强产物抑制； 还有营养不足也会抑制 发酵的进行。文献（Biomass and Bioenergy 39， 2012 :48-52)和（Energies 2012, 5, 3178-3197)探讨了在超高浓度乙醇发酵（VHG)条件下加入不同种类的 氮源改善终点乙醇浓度。
本发明提出了一种表面活性剂改进的乙醇发酵法， 采用高活性酿酒酵 母并以葡萄糖为原料， 在发酵培养中添加非离子表面活性剂， 用以减小酒 精抑制， 降低高糖浓度引起的渗透压， 提高酿酒酵母细胞在特殊环境下的 成活率及生长速率， 最终提高终点乙醇浓度， 有效降低了发酵过程中水的 消耗和精馏过程的能源消耗； 而且将酵母和表面活性剂从发酵混合物中进 行分离， 在下一批发酵过程中得到再利用。
发明内容
本发明的目的是提供一种表面活性剂改进的乙醇发酵法， 涉及一种超 高浓度 （VHG) 发酵乙醇生产过程， 尤其是涉及表面活性剂改进的超高浓 度乙醇发酵， 通过在发酵介质中加入表面活性剂改善终点乙醇浓度。 众所 周知， 燃料乙醇主要通过发酵法生产， 发酵介质包括碳水化合物和糖源、 酿酒酵母、 发酵介质的初始 pH为 3.5-5.3、 发酵温度 25°C到 40°C。 本发明提 出在发酵介质中添加非离子表面活性剂改善乙醇浓度和酵母细胞生存能力， 乙醇浓度可由未添加表面活性剂的 110g/L增加到添加表面活性剂后的
Figure imgf000004_0001
本发明提供了一种表面活性剂改进的乙醇发酵法， 以可发酵碳水化合 物为碳源， 以表面活性剂-水混合物为发酵介质，加入 pH调节剂调节发酵介 质 pH值， 接种酿酒酵母细胞， 进行超高浓度乙醇发酵。
本发明提供的表面活性剂改进的乙醇发酵法， 所述可发酵碳水化合物 为葡萄糖， 可发酵碳水化合物浓度为 270〜500g/L。
本发明提供的表面活性剂改进的乙醇发酵法， 所述发酵介质中表面活 性剂与水的质量比为 0.001〜0.5 (优选为 0.125〜0.375)。
本发明提供的表面活性剂改进的乙醇发酵法，在一个实施方案中包括： (a)包含酿酒酵母的发酵介质； （b)发酵介质中包含非离子表面活性剂聚 乙二醇（PEG)、 聚乙二醇单甲醚（MPEG)、 聚乙二醇单甲醚 （DMPEG)、 聚二甲基硅氧垸 (PDMS)中的至少一种， 表面活性剂的添加可以在 VHG发 酵条件下增加酵母细胞寿命及终点乙醇浓度 ·' (c)酵母细胞可以从发酵混合 物中回收再利用； （d)表面活性剂和 pH调节剂可以同步回收再利用。
PEG为 PEG-200, 400, 600, 800, 1000中的至少一种；
PEG的结构式为：
Figure imgf000004_0002
MPEG的结构式为：
Figure imgf000005_0001
DMPEG的结构式为： 。、 n OM
PDMS的结构式为：
Figure imgf000005_0002
其中， n为 1-25; M为垸基。
根据本发明， 发酵过程或者至少一部分发酵过程包含发酵介质 H20和 表面活性剂、 发酵产物乙醇、 发酵材料碳水化合物和酿酒酵母。 在一个实 施方案中， 发酵温度一般选择在 28°C至 44°C，尤其是从 30°C到 38°C (最优 选为 33°C )， pH—般选择在 3.0到 6.5， 最好的 pH范围为 4.0到 5.0， 酵母细胞 浓度为 107〜109/L， 发酵时间为 12〜120h (优选为 60h) 。
本发明提供的表面活性剂改进的乙醇发酵法，所述发酵介质 pH值调节 剂为硫酸溶液、 柠檬酸 -柠檬酸钠缓冲液、 磷酸缓冲液、 碳酸盐缓冲液、 醋 酸 -醋酸钠缓冲液中的一种。
在 VHG发酵过程中， 酵母细胞对高浓度葡萄糖渗透压的承受能力以及 对高浓度乙醇的耐受性是需要考虑的关键因素。 在本发明中， 向发酵介质 中添加非离子表面活性剂可有效提高乙醇浓度， 我们猜测出现这种现象的 原因可能是由于表面活性剂直接或间接的作为细胞保护剂改善酵母细胞的 生存能力从而提高乙醇浓度。
所用的表面活性剂是非离子型表面活性剂。 例如， 非离子表面活性剂 聚乙二醇 （PEG) ， 尤其是 PEG-200, PEG-400和 PEG-600。 表面活性剂与 水的质量比为从 0到 2/3， 表面活性剂与水的混合发酵介质可以增加乙醇浓 度， 与本发明提出观点相一致。表面活性剂与水的质量比为从 1/5到 2/3、从 1/5到 1/4混合配制的发酵介质也能够增加乙醇浓度。
本发明提出的生产乙醇的发酵浴包含生产乙醇的微生物（例如，酵母）、 发酵碳源（例如，碳水化合物、单糖、多糖等）、 pH调节剂 (例如 H2S04,柠 檬酸 -柠檬酸钠缓冲液、 磷酸缓冲液、 碳酸盐缓冲液、 醋酸-醋酸钠） 。 发 酵底物浓度为 275g/L到 500g/L，发酵底物是可发酵碳水化合物构成，葡萄糖、 纤维素和淀粉尤其适合做碳源。
本发明提供的表面活性剂改进的乙醇发酵法， 酵母细胞可以回收再利 用， 发酵液通过冷冻离心回收酵母细胞直接用于下一次发酵过程。 所述酵 母细胞回收再利用是应用标准的离心技术对发酵液进行冷冻离心， 离心速 度为 4000〜15000rpm， 离心时间为 l〜30min。
本发明提供的表面活性剂改进的乙醇发酵法， 表面活性剂与 pH调节剂 可以回收再利用， 冷冻离心除去酵母， 蒸馏上清液后剩余物代替新鲜表面 活性剂用于下一个发酵过程。 所述表面活性剂和 pH值调节剂的回收过程是 应用标准的真空蒸馏技术， 在 0Mpa〜0.09Mpa压力下蒸馏温度为 40°C〜 100°C， 蒸馏时间为 20min〜120min。
本发明提供的表面活性剂改进的乙醇发酵法， 酵母细胞和表面活性剂 可以同步回收再利用， 通过冷冻离心回收酵母细胞、 蒸馏上清液后回收表 面活性剂， 二者同时用于下一批发酵过程。
本发明提供的表面活性剂改进的乙醇发酵法， 表面活性剂回收再利用 是发酵结束后冷冻离心除去酵母细胞， 通过标准的真空蒸馏技术回收表面 活性剂及 pH值调节剂，在下一发酵过程可直接添加可发酵碳水化合物、去 离子水及回收的表面活性剂和 pH调节剂， 无需重新调整发酵介质 pH值。
表面活性剂不仅蒸汽压低、 而且具有可调整的水溶性、 通过对结构及 分子量的选择能够实现对其物理性质的精细调控。 这一系列的特性决定了 表面活性剂对很多微生物具有广泛的生物相容性， 而且可通过离心、 蒸熘 等技术将其从发酵混合物中进行分离。 本发明提出的一个酵母细胞回收的 过程： 酵母细胞可以通过离心分离从表面活性剂与水的发酵混合物中回收 再利用， 具体工艺步骤如图 1所示。
在一个实施方案中， 本发明提出了一种表面活性剂的回收过程： 发酵完 成后， 表面活性剂可以从发酵混合物中通过标准的蒸馏技术进行回收再利 用， 表面活性剂可以循环利用多次。 具体工艺步骤如图 2所示
材料和发酵分析过程
酿酒酵母， 湖北安琪酵母公司。 其它试剂及药品， 国药集团。 首先， 称取一定量的酿酒酵母置于 lOOmL三角烧瓶中， 在 37°C条件下用超纯水复 水活化 20min后即可做酒母使用。然后加入葡萄糖、表面活性剂、预先配置 好 pH的溶液， 用保鲜膜封口， 放入摇床中进行震荡培养， 摇床转速为
Figure imgf000007_0001
发酵结束后， 产物样品用去离子水稀释， 发酵液中各组分物的含量测 定采用高效液相色谱仪 (Agilent 1260), 依据葡萄糖投料量计算其转化率、 乙醇收率， 依据发酵液中乙醇质量、 活化水与 pH液体积计算乙醇浓度。 色 谱条件为： 离子交换柱， 柱温为 65°C， 视差折光检测器， 检测器为 50°C ; 流动相： 5Mm H2S04， 流速 0.6ml/min， 进样量 25uL。
= (籠投料量 -葡萄謹量 (mol)) X讓
葡萄糖投料量 (mol)
乙薩 c i^^ 附图说明
图 1 固体酵母粉的回收再利用工艺步骤；
图 2表面活性剂与硫酸同步回收再利用工艺步骤；
图 3 发酵温度对葡萄糖转化率的影响；
图 4发酵温度对乙醇收率的影响；
图 5 发酵温度对乙醇浓度的影响；
图 6 pH值对葡萄糖转化率的影响；
图 7 pH值对乙醇收率的影响；
图 8 pH值对乙醇浓度的影响；
图 9纯水中发酵 48h酵母循环对葡萄糖转化率的影响；
图 10纯水中发酵 48h酵母循环对乙醇收率的影响；
图 11纯水中发酵 48h酵母循环对乙醇浓度的影响；
图 12表面活性剂 /水中发酵 48h酵母循环对葡萄糖转化率的影响; 图 13表面活性剂 /水中发酵 48h酵母循环对乙醇收率的影响； 图 14表面活性剂 /水中发酵 48h酵母循环对乙醇浓度的影响； 图 15表面活性剂 /水中发酵 36-72h酵母循环对葡萄糖转化率的影响； 图 16表面活性剂 /水中发酵 36-72h酵母循环对乙醇收率的影响； 图 17表面活性剂 /水中发酵 36-72h酵母循环对乙醇浓度的影响； 图 18表面活性剂 /硫酸的回收再利用。
具体实施方式
下面结合具体实施例对本发明进行进一步描述， 但本发明的保护范围 不受实施例的限制， 如果该领域的技术熟练人员根据上述本发明内容对本 发明做出一些非本质的改进和调整， 仍属于本发明的保护范围。
实施例 1-9 PEG-200与水的比例对乙醇发酵效率的影响
首先， 称取 0.4g酿酒酵母置于 lOOmL三角烧瓶中， 在 38°C条件下用 4mL超纯水复水活化 20min后即可做酒母使用。 然后加入 6.0g葡萄糖， PEG-200(按表 1用量） （l-8g)， 预先配置好的 pH=3.4的 H2S04溶液 (按表 1 用量)， 用保鲜膜封口， 放入摇床中在 36°C条件下进行震荡培养 48h， 摇床 转速为 160r/min。由表 1数据可见，当发酵体系中没有添加 PEG-200时（实 施例 1 )， 葡萄糖转化率为 96%， 乙醇收率为 81%， 乙醇浓度为 112 g'l 1, 而当采用 l.Og PEG-200取代 pH液时， 葡萄糖转化率提高到 99%, 乙醇收 率提高到 88%， 乙醇浓度提高到 129 g*!/1, 随着 PEG-200量得增加， 葡萄 糖转化率、 乙醇收率及浓度均呈现先上升后下降的趋势。 这些结果初步说 明了 PEG可在一定比例范围内取代水作为发酵介质， 提高发酵终点产物乙 醇浓度、 减少发酵终点残余葡萄糖含量， 减少水的消耗、 降低生产成本、 提高发酵效率。 表 1. PEG-200与水的比例对乙醇发酵效率的影响
Figure imgf000010_0001
实施例 10-17 PEG-400与水的比例对乙醇发酵效率的影响
称取 0.4g酵母置于 lOOmL三角烧瓶中， 在 38°C条件下用 4mL超纯水 复水活化 20min后即可做酒母使用。 然后加入 6.0g葡萄糖， PEG-400(按表 2用量）（l-8g)， 预先配置好的 pH=3.4的 H2S04溶液 (按表 2用量)， 用保鲜 膜封口，放入摇床中在 36Ό条件下进行震荡培养 48h，摇床转速为 160r/min。 由表 2数据可见， 而当采用 4.0g PEG-400取代 pH液时， 乙醇浓度可达到 150 g-L"1,而且在发酵体系几乎没有残糖保留。这些结果进一步证明了 PEG 可在一定比例范围内取代水作为发酵介质， 提高发酵终点产物乙醇浓度、 减少发酵终点残余葡萄糖含量， 减少水的消耗、 降低生产成本、 提高发酵 效率。
表 2. PEG-400与水的比例对乙醇发酵效率的影响
Figure imgf000010_0002
10 1.0 19 1/19 99.4 88.1 129.3
11 2.0 18 2/18 99.1 90.0 139.4
12 3.0 17 3/17 99.5 89.3 146.5
13 4.0 16 4/16 99.3 86.8 151.3
14 5.0 15 5/15 93.9 79.6 148.0
15 6.0 14 6/14 84.3 69.6 138.7
16 7.0 13 7/13 43.0 26.0 55.7
17 8.0 12 8/12 19.6 4.0 9.3 实施例 18-25 PEG-600与水的比例对乙醇发酵效率的影响
称取 0.4g酵母置于 lOOmL三角烧瓶中， 在 38°C条件下用 4mL超纯水 复水活化 20min后即可做酒母使用。 然后加入 6.0g葡萄糖， PEG-600(按表 3用量）（l-8g)， 预先配置好的 pH=3.4的 H2S04溶液 (按表 3用量)， 用保鲜 膜封口，放入摇床中在 36°C条件下进行震荡培养 48h，摇床转速为 160r/min。 由表 3数据可见，而当采用 PEG-600取代 pH液时，乙醇浓度随着 PEG-600 量的增加逐渐上升、 然后下降， 最高乙醇浓度可达到 153 这些结果 充分证明了 PEG可在一定比例范围内取代水作为发酵介质， 提高发酵终点 产物乙醇浓度、 减少发酵终点残余葡萄糖含量， 减少水的消耗、 降低生产 成本、 提高发酵效率。
表 3. PEG-600与 pH液的比例对乙醇发酵效率的影响
Figure imgf000011_0001
23 6.0 14 6/14 83.3 77.3 153.7
24 7.0 13 7/13 41.8 38.0 81.5
25 8.0 12 8/12 10.1 6.3 14.6 实施例 26发酵温度影响
称取 0.5g酵母置于 lOOmL三角烧瓶中， 在 38°C条件下用 4mL超纯水 复水活化 20min后即可做酒母使用。然后加入 6.5g葡萄糖， 4.0g PEG-400, 12mL pH=3.4的 H2S04溶液，用保鲜膜封口， 放入摇床中在 32 °C -42 °C条件 下进行震荡培养 12-72h， 摇床转速为 160r/min。 由图 3、 图 4、 图 5可以看 出，不同温度导致发酵过程中葡萄糖转化率、乙醇收率与浓度有较大差异， 发酵温度超过 35°C时随着温度上升， 终点乙醇浓度下降； 但是温度降到 30°C 时终点乙醇浓度也降低。 最佳的发酵温度应该选在 33 °C附件， 发酵终点乙 醇浓度最高可达到 160 gi—1 , 而且发酵体系没有剩余的葡萄糖。
实施例 27发酵 pH的影响
称取 0.5g酵母置于 lOOmL三角烧瓶中， 在 37°C条件下用 4mL超纯水 复水活化 20min后即可做酒母使用。然后加入 6.5g葡萄糖， 4.0g PEG-400, 12mL pH=3.4-5.1的 H2S04溶液， 用保鲜膜封口， 放入摇床中在 35°C条件 下进行震荡培养 12-72h，摇床转速为 160r/min。由于 pH值会影响酵母细胞 的通透性， 进而影响酵母细胞对营养物质的吸收和乙醇的排泄， 因此适宜 的 pH对发酵的影响至关重要。 由图 6、 图 7、 图 8可以看出， 当发酵液 PH 值在 4.3-5.1的范围内时， 60h可以达到发酵终点， 终点乙醇浓度最高可达 到 WO g'L—1, 而且发酵葡萄糖发酵完全。
实施例 28纯水中发酵 48h酵母循环再利用
将 0.5g酵母粉在 25-38Ό条件下用 4mL超纯水复水 15-30分钟， 然后 加入 6.5g葡萄糖、 预先配置好的 pH值为 4.3硫酸溶液 12mL， 放入摇床中 进行震荡， 在 33°C条件下发酵 48小时。 发酵结束后 8000rpm冷冻离心分 离 5分钟， 将发酵结束后的发酵液去除清夜， 留下固体酵母粉， 再次加入 4mL超纯水、 6.5g葡萄糖、 12mL硫酸溶液， 33 °C发酵 48-72小时， 如此循 环 2次。 葡萄糖转化率、 乙醇收率及浓度分别如图 9、 图 10、 图 11所示。 从图中数据可以看出， 当在发酵体系中没有加入表面活性剂时， 酵母的回 收效率很低， 发酵 48小时后回收酵母第一次循环再利用只能取得 60g丄—1 的乙醇浓度。
实施例 29表面活性剂 -水混合体系中发酵 48h酵母循环再利用
将 0.5g酵母粉在 25-38°C条件下用 4mL超纯水复水 15-30分钟， 然后 加入 6.5g葡萄糖、 PEG-400 4.0g、预先配制好的 pH值为 4.3硫酸溶液 12mL， 放入摇床中进行震荡， 在 33°C条件下发酵 48小时。 发酵结束后 8000rpm 冷冻离心分离 5分钟，将发酵结束后的发酵液去除清夜，留下固体酵母粉， 再次加入 4mL超纯水、 6.5g葡萄糖、 4.0g PEG-400、 12mL硫酸溶液， 33 °C 发酵 48小时， 如此循环 3次。 葡萄糖转化率、 乙醇收率及浓度分别如图 12、图 13、图 14所示。从图中数据可以看出，当在发酵体系中加入 PEG-400 时， 酵母的回收效率很高， 发酵 48小时后回收酵母第一次循环再利用可获 得 118g 的乙醇浓度。
实施例 30表面活性剂 -水混合体系中发酵 36-72h酵母循环再利用
将 0.5g酵母粉在 25-38Ό条件下用 4mL超纯水复水 15-30分钟， 然后 加入 6.5g葡萄糖、 PEG-400 4.0g、预先配制好的 pH值为 4.3硫酸溶液 12mL， 放入摇床中进行震荡，在 33°C条件下发酵 36-72小时。发酵结束后 8000rpm 冷冻离心分离 5分钟，将发酵结束后的发酵液去除清夜，留下固体酵母粉， 再次加入 4mL超纯水、 6.5g葡萄糖、 4.0g PEG-400、 12mL硫酸溶液， 33°C 发酵 48小时， 如此循环 3次。 葡萄糖转化率、 乙醇收率及浓度分别如图 15、图 16、图 17所示。从图中数据可以看出，当在发酵体系中加入 PEG-400 时， 酵母的回收效率很高， 发酵 48小时后回收酵母第一次循环再利用可获 得 llSgl 1的乙醇浓度。
实施例 31 表面活性剂回收再利用
将 0.5g酵母粉在 25-38°C条件下用 4mL超纯水复水 15-30分钟， 然后 加入 7.5g葡萄糖、4.0g PEG-400、预先配置好的 pH值为 4.3硫酸溶液 12mL， 放入摇床中进行震荡， 在 33°C条件下发酵 72小时。 发酵结束后 8000rpm 冷冻离心分离 5分钟， 除去固体酵母粉， 将上清液进行蒸馏， 蒸馏后剩余 物进入下一个发酵过程， 再次加入 0.5g酵母粉在 25-38Ό条件下用 4mL超 纯水复水 15-30分钟、 6.5g葡萄糖、 12mL硫酸溶液， 33 °C发酵 72小时， 如此循环 4次。葡萄糖转化率、 乙醇收率及浓度如图 18所示， 表面活性剂 PEG-400可以循环再利用， 对发酵效率没有明显影响。

Claims

权 利 要 求 书
一种表面活性剂改进的乙醇发酵法， 其特征在于： 以可发酵碳水化 合物为碳源， 以表面活性剂-水混合物为发酵介质， 加入 pH调节剂调节发 酵介质 pH值， 接种酿酒酵母细胞， 进行超高浓度乙醇发酵。
2、 按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述可发酵碳水化合物为葡萄糖， 可发酵碳水化合物浓度为 270〜500g L。
3、 按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述发酵介质中表面活性剂与水的质量比为 0.001〜0.5。
4、 按照权利要求 3所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述发酵介质中表面活性剂与水的质量比为 0.125〜0.375。
5、 按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述表面活性剂为非离子型表面活性剂聚乙二醇（PEG)、 聚乙二醇单甲醚
(MPEG)、聚乙二醇单甲醚（DMPEG)、聚二甲基硅氧垸 (PDMS)中的至少 一种或其任意比例组合；
PEG的结构式为：
Figure imgf000015_0001
MPEG的结构式为:
Figure imgf000015_0002
DMPEG的结构式为:
Figure imgf000016_0001
PDMS的结构式为:
Figure imgf000016_0002
其中， n为 1-25; M为垸基。
6、 按照权利要求 5所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述 PEG为 PEG-200, 400, 600, 800, 1000中的至少一种。
7、 按照权利要求 1所述表面活性剂改迸的乙醇发酵法， 其特征在于： 所述的酿酒酵母细胞浓度为 107〜109 L。
8、 按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 酵母细胞可以回收再利用， 发酵液通过冷冻离心回收酵母细胞直接用于下 一次发酵过程。
9、 按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 表面活性剂与 pH调节剂可以回收再利用，冷冻离心除去酵母，蒸馏上清液 后剩余物代替新鲜表面活性剂用于下一个发酵过程。
10、按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于 : 酵母细胞和表面活性剂可以同步回收再利用，通过冷冻离心回收酵母细胞、 蒸熘上清液后回收表面活性剂， 二者同时用于下一批发酵过程。
11、按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于: 所述发酵温度为 28〜44 C。
12、按照权利要求 11所述表面活性剂改进的乙醇发酵法，其特征在于: 所述发酵温度为 33°C。
13、按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于: 所述发酵时间为 12〜120h。
14、按照权利要求 13所述表面活性剂改进的乙醇发酵法，其特征在于： 所述发酵时间为 60h。
15、按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于: 所述发酵介质 pH为 3.0〜6.5。
16、按照权利要求 1所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述发酵介质 pH值调节剂为硫酸溶液、 柠檬酸 -柠檬酸钠缓冲液、 磷酸缓 冲液、 碳酸盐缓冲液、 醋酸 -醋酸钠缓冲液中的一种。
17、按照权利要求 8所述表面活性剂改进的乙醇发酵法， 其特征在于： 所述酵母细胞回收再利用是应用标准的离心技术对发酵液进行冷冻离心， 离心速度为 4000〜15000rpm， 离心时间为 l〜30min。
18、按照权利要求 9所述表面活性剂改进的乙醇发酵法， 其特征在于： 表面活性剂回收再利用是发酵结束后冷冻离心除去酵母细胞， 通过标准的 真空蒸馏技术回收表面活性剂及 pH值调节剂，在下一发酵过程可直接添加 可发酵碳水化合物、去离子水及回收的表面活性剂和 pH调节剂，无需重新 调整发酵介质 pH值。
19、按照权利要求 9所述表面活性剂改进的乙醇发酵法， 其特征在于: 表面活性剂和 pH值调节剂的回收过程是应用标准的真空蒸馏技术， 在 0Mpa〜0.09Mpa压力下蒸馏温度为 40°C〜； 100°C， 蒸馏时间为 20mir！〜 120min。
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