WO2013138998A1 - 一种丙酮丁醇梭菌及其应用 - Google Patents

一种丙酮丁醇梭菌及其应用 Download PDF

Info

Publication number
WO2013138998A1
WO2013138998A1 PCT/CN2012/072649 CN2012072649W WO2013138998A1 WO 2013138998 A1 WO2013138998 A1 WO 2013138998A1 CN 2012072649 W CN2012072649 W CN 2012072649W WO 2013138998 A1 WO2013138998 A1 WO 2013138998A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
butanol
concentration
hydroxybutanone
medium
Prior art date
Application number
PCT/CN2012/072649
Other languages
English (en)
French (fr)
Inventor
应汉杰
柳东
陈勇
李安
周涛
吴菁岚
林晓清
陈晓春
谢婧婧
柏建新
Original Assignee
南京工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京工业大学 filed Critical 南京工业大学
Priority to PCT/CN2012/072649 priority Critical patent/WO2013138998A1/zh
Priority to US14/386,286 priority patent/US9249433B2/en
Publication of WO2013138998A1 publication Critical patent/WO2013138998A1/zh

Links

Classifications

    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P7/16—Butanols
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • 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 invention belongs to the technical field of microorganisms, and relates to a 3-hydroxybutanone-producing Clostridium acetobutylicum and application thereof. Background technique
  • Acetone, butanol and ethanol are collectively referred to as ABE. They are important raw materials for medicines, pesticides, plastics, rubber and light industry, and are also important chemical solvents. Therefore, ABE research is important for the development of modern industry. effect.
  • Acetone also known as dimercaptopurone, is a colorless, transparent liquid that is volatile. Acetone is an important organic solvent and a very important chemical raw material. It is used in the industries of explosives, plastics, fiber, leather, paint, etc. It can also be used to synthesize ketene, acetic anhydride, iodoform, olefin rubber, epoxy resin and so on.
  • Butanol also known as 1-butanol, is a colorless, transparent liquid with a strong alcoholic taste, slightly soluble in water, a relative density of 0.81 and a boiling point of 117.7 °C. It is a secondary flammable product. Butanol is an important organic raw material and chemical solvent. It is widely used in various plastics, rubber products, resin manufacturing, and light industry such as leather and paper. Another very important role of butanol can be used as a new biofuel of today's potential, known as second-generation biofuels.
  • butanol Compared with ethanol, butanol has a higher combustion value, can support the car to walk 30% more, and the properties of hydrocarbons are close, no need to modify the car cylinder; and low volatility; not hydrophilic; no corrosion; high Octane number - good antiknock. Therefore, the research and development of butanol has rapidly become a new hot spot in the context of the depletion of global stone resources.
  • 3-hydroxybutanone also known as acetoin, acetyl sterol, usually a pale yellow liquid or crystal, naturally found in corn, grapes, strawberries, cheese, meat, etc.
  • FEMA American Food and Extract Association
  • 3-hydroxybutanone can also be used as an important raw material in chemical synthesis, such as for the synthesis of chiral smectic materials and nematic materials.
  • Hummel et al. obtained the method of culturing bacteria to obtain diacetyl reductase in lactobacillus or yeast, and then used the reductase and coenzyme NADPH to catalyze the diacetylation at pH 5 and 70 °C. Hydroxybutanone, yield up to 100%.
  • RH Blom of the US Department of Agriculture synthesized diacetyl and 3-hydroxybutanone by oxidative dehydrogenation with 2,3-butanediol. 2, 3-butanediol was heated at 140 °C and passed through a Pyrex tubular reactor with air.
  • the reactor was filled with copper shavings at a reaction temperature of 315 °C to obtain the product diacetyl (yield 33%) and 3-hydroxyl. Butanone (yield 25%).
  • the separator was an ion exchange membrane, and the electrodes in the reaction were all reversible hydrogen electrodes.
  • the anode is Pt-Pb
  • the porous Pt/Ir ((10%) is the corresponding electrode
  • the electrolyte is HC10 4
  • the solvent is ultrapure water
  • the reaction temperature is 40 °C
  • the battery voltage is 0.8V.
  • 3-hydroxybutanone can be produced by microbial fermentation.
  • two molecules of pyruvic acid synthesize one molecule of acetolactate under the action of acetolactate synthase, and acetolactate can form 3-hydroxybutanone by acetolactate decarboxylase.
  • Acetyl lactic acid can also be naturally oxidatively decarboxylated under aerobic conditions to form diacetyl, which is then reduced to form 3-hydroxybutanone.
  • 3-hydroxybutanone can be further reduced to form 2,3-butanediol, and the reduction of diacetyl to 3-hydroxybutanone and 3-hydroxybutanone can be reduced to form 2,3-butanediol from the same enzyme.
  • (2,3-butanediol dehydrogenase) so in many microorganisms 3-hydroxybutanone is often used as an intermediate in the production of 2,3-butanediol, accompanied by the formation of diacetyl, which affects its Yield and separation.
  • 3-hydroxybutanone such as: Lactococcus lactis, Lactobacillus casei, Saccharomyces cerevisiae, or the like.
  • the fermentation strain was fermented, but the yield of 3-hydroxybutanone was lower than lg/L.
  • Klebsiella pneumoniae, Enterobacter aerogenes and Bacillus subtUi are also capable of fermentative production of 3-hydroxybutanone with higher yields, but these strains are mainly used Production of 2,3-butanediol, 3-hydroxybutanone is only a by-product thereof.
  • Olson and Johnson used oxytobacillus to convert 226 g/L of glucose to 14 g/L of 3-hydroxybutanone and 97 g/L of 2,3-butanediol.
  • Ma Cuiqing and others from Shandong University used chiral 3-hydroxybutanone and 2,3-butanediol to prepare chiral 3-hydroxyl groups using recombinant Escherichia coli containing the 2,3-butanediol dehydrogenase gene and NADH oxidase gene. The concentration of butanone reached 36 g/L.
  • Another object of the present invention is to provide a process for the co-production of butanol and 3-hydroxybutanone.
  • the object of the present invention is achieved by the following technical solutions.
  • the present invention provides a Clostridium acetobutylicum for fermentation of butanol and 3-hydroxybutanone, the preservation number of which is CGMCC No. 5234, which has been deposited with Chinese organisms on September 9, 2011. Common Microbiology Center of the Culture Collection Management Committee (CGMCC), the address of the depository: No. 3, No.1, Beichen West Road, Chaoyang District, Beijing, China. Institute of Microbiology, Chinese Academy of Sciences.
  • CGMCC Culture Collection Management Committee
  • the present invention also provides the use of the above Clostridium acetobutylicum for the simultaneous fermentation of butanol and 3-hydroxybutanone.
  • the present invention provides a method for the simultaneous production of butanol and 3-hydroxybutanone by fermentation, which comprises subjecting the above-mentioned Clostridium acetobutylicum to fermentation culture in a liquid fermentation medium to produce butanol and 3-hydroxyl Butanone.
  • the method provided by the present invention comprises the following steps: 1) culturing the Clostridium acetobutylicum on a solid plate medium for 12-36 hours; 2) introducing the Clostridium acetobutylicum cultured in step 1) into the seed culture medium Incubate at 5-39 ° C for 10-20 hours; 3) Add the Clostridium acetobutylicum cultured in step 2) to the liquid fermentation medium at a dose of 5%-15%, and stand still at 24-40 °C. Incubate for 40-85 hours.
  • the liquid fermentation medium comprises a carbon source, a nitrogen source and/or an inorganic salt.
  • the carbon source is one or more selected from the group consisting of glucose, glycerin, corn flour, fructose, starch, xylose, and the like, and has a concentration of 20 g/L to 80 g/L.
  • the nitrogen source is selected from the group consisting of ammonium sulfate, ammonium acetate, corn syrup, yeast powder, yeast extract, One or more of urea and the like, and the concentration is from 0.1 g/L to 10 g/L.
  • the inorganic salt is one or more selected from the group consisting of a sodium salt, a potassium salt, an iron salt, a ferrous salt, a manganese salt, a phosphate salt, a sulfate salt, and the like, and has a concentration of 0.001 g/L to 5 g/L.
  • the liquid fermentation medium further comprises trace elements.
  • the trace element is selected from the group consisting of vitamin B1 (thiamine or its hydrochloride), vitamin H
  • vitamin B7 biotin, vitamin B7
  • vitamin Bx p-aminobenzoic acid, vitamin HI
  • the liquid fermentation medium further comprises one or more metabolic or growth regulators.
  • the metabolic or growth regulator is selected from one or more of the group consisting of acetate, branched chain amino acid, and glycerin.
  • the acetate comprises one or more of sodium acetate, ammonium acetate, magnesium acetate, calcium acetate, potassium acetate, etc., and the concentration after adding the medium is 0.5 g/L to 8 g/L, and the addition time is It is 0-50 hours after the start of fermentation.
  • the addition of acetate can be used as a raw material for the synthesis of acetone, which directly increases the yield of acetone. Therefore, the acetone produced by the sugar is reduced and the 3-hydroxybutanone is increased under the regeneration requirement of NAD+.
  • the addition of acetate can also accelerate the sugar consumption. Rate, shorten the fermentation cycle. If acetate is added in the early stage of growth, the growth of the cells is inhibited, but the yield of butanol is increased.
  • the branched chain amino acid comprises one or more of leucine, isoleucine, valine, etc., and the concentration after adding the medium is 0.05 g/L to 5 g/L, and the addition time is fermentation. 0-20 hours after the start.
  • the branched chain amino acid and the 3-hydroxybutanone share the same precursor acetolactate.
  • the invention can feedback inhibition of the synthesis of acetolactate by adding a branched chain amino acid, which can reduce the yield of 3-hydroxybutanone and further increase the yield of butanol, however,
  • the addition of chain amino acids has a certain inhibitory effect on the growth of the cells.
  • the concentration of the glycerol after the addition of the medium is 2-20 g/L, and the addition time is 0-60 hours after the start of fermentation.
  • the degree of reduction of glycerol is higher than that of glucose, and glycerol metabolism can provide more NADH.
  • the present invention can provide NADH for acetic acid and butyric acid in the late stage by adding glycerin to the fermentation, thereby increasing the yield of butanol. .
  • the liquid fermentation medium further comprises a carrier medium capable of adsorbing butyric acid or butanol or facilitating growth of the cells, including one or more of activated carbon, fibers, resins, emulsifiers and the like.
  • Butyric acid and butanol are the main inhibitors of cell growth.
  • the concentration of butyric acid in the early stage limits the concentration of bacteria, and the rate and yield of alcohol in the late butanol-restricted cells.
  • the present invention adds a resin or an adsorption carrier during the fermentation process. It can strengthen the growth of the bacteria, reduce the inhibition of the product, enhance the resistance of the bacteria, greatly increase the fermentation rate, decrease the cycle, and greatly increase the yield of 3-hydroxybutanone.
  • the fermentation culture is a two-stage culture mode.
  • the two-stage culture mode comprises controlling the fermentation temperature, pH or fermentation sugar concentration in two stages.
  • the fermentation temperature is controlled as follows: 0-30 hours after the start of fermentation, the control temperature is 24-40 °C, and then the control temperature is 32-37 °C.
  • Clostridium acetobutylicum pre-growth consumes a small amount of acetic acid, butyric acid, and then enters the alcohol production period to produce acetone, butanol, etc.
  • the growth and metabolism of the two stages have different behaviors at different temperatures.
  • the invention coordinates the growth and production of alcohol and the metabolism of the early stage and the late metabolism by temperature, which is beneficial to increase the production of metabolites.
  • the fermentation pH is controlled as follows: 0-30 hours after the start of fermentation, the pH is controlled to pH 4-5.5, and the pH is not controlled after the start of alcohol production.
  • the pH is very important in the fermentation of Clostridium acetobutylicum. It can reflect and affect the dissociation form of organic acids such as acetic acid and butyric acid in the fermentation broth, which in turn affects the utilization of acid by the bacteria. In some cases, due to the problem of acid utilization, fermentation is likely to delay in the acid production period for a long time. Therefore, during the period from the acid production period to the alcohol production period, the pH is determined to determine the success or failure of production. The key factor.
  • the present invention ensures that the cells are successfully and rapidly transitioned to the alcohol phase by controlling the pH of the acid producing phase at a suitable level.
  • the fermentation sugar concentration is controlled as follows:
  • the initial sugar concentration of the fermentation is 30-50 g/L, and when the sugar concentration is reduced to 10-30 g/L, the sugar carbon source is added to maintain the sugar concentration in the medium. At 10-30 g/L, when the total sugar concentration reaches 60-90 g/L, the addition is stopped.
  • the above-mentioned mutagenesis of Clostridium acetobutylicum has growth inhibition at a higher initial sugar concentration, and at the same time, the bacteria exhibit different carbon-sulfur distribution at different sugar concentrations.
  • the invention controls the concentration of sugar in the fermentation process through the strategy of adding sugar, and at the same time achieves the purpose of promoting the growth of the cells and maintaining the cells in a state of better carbon and sulfur distribution.
  • a strain of Clostridium acetobutylicum B3 is subjected to ultraviolet mutagenesis, and a strain of Clostridium acetobutylicum which can produce high-butanol and 3-hydroxybutanone is screened.
  • the mutagenized Clostridium acetobutylicum obtained by the present invention has strong acetolactate synthase activity and acetolactate decarboxylase activity, and lacks 2,3-butanediol dehydrogenase activity and isopropanol dehydrogenase activity.
  • Clostridium acetobutylicum has strong oxygen tolerance, and the seed culture and the fermentation culture do not need to remove residual air by anaerobic gas, and the seed culture and the fermentation culture can be ventilated without stirring and stirring.
  • the invention ferments this bacteria to produce butanol, ethanol, acetone and 3-hydroxybutanone (acetoin),
  • the coupling regeneration of NAD+ is realized, and the carbon metabolism flow is regulated at the same time.
  • the method for co-production of 3-hydroxybutanone by ABE fermentation by using the above-mentioned mutagenized Clostridium acetobutylicum comprises: culturing the above-mentioned mutagenized Clostridium acetobutylicum on a solid plate for 12-36 hours, and then accessing the seed culture.
  • Clostridium acetobutylicum is used in the early stage of ABE fermentation (acid production period). About 30%-40% of the sugar is converted into acetic acid and butyric acid. After the bacteria enter the alcohol production period, acetic acid and butyl The reuse efficiency of the acid which can be re-reduced to produce ethanol and butanol, acetic acid and butyric acid determines the production efficiency of butanol.
  • the mutagenized strain in the present invention produces 3-hydroxybutanone which can provide NADH for the reuse of acetic acid and butyric acid to form butanol, realize the coupling regeneration of NAD+, improve the sugar utilization rate and solvent yield, and does not produce Common by-products 2, 3-butanediol, isopropanol and the like.
  • the ABE production pathway and the 3-hydroxybutanone production pathway are in different branches and have different correlations with the growth of the cells, by adding small molecule effectors or growth regulators acting on each branch pathway, Effectively realize the flow distribution of carbon flow in these two ways, and flexibly adjust the output of both to meet market demand.
  • the production strain obtained by mutagenesis has strong oxygen resistance, which greatly reduces the oxygen avoidance measures in the conventional anaerobic fermentation process. Static fermentation, no ventilation, no agitation, energy saving and emission reduction.
  • the fermentation does not produce by-products such as 2,3-butanediol, isopropanol, citric acid and lactic acid, which improves the yield of the product and reduces the subsequent separation pressure.
  • the NADH provided by 3-hydroxybutanone can be used for the reduction of acetic acid and butyric acid to form ethanol and butanol, which realizes the coupling regeneration of NAD+ and improves the carbon yield.
  • 3-hydroxybutanone and ABE are on different metabolic branches, and the metabolic flux of the two can be flexibly adjusted to better meet market demand.
  • Clostridium acetobutylicum B3 was deposited on September 9th, 201, 1st at the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • Figure 1 is a schematic diagram of NAD+ coupled regeneration and co-production of ABE and 3-hydroxybutanone.
  • Figure 2 is a gas chromatogram prepared by sampling in a 50 L fermenter for 30 h, in the order of peak order: 3.752 min, acetone; 4.199 min, ethanol; 5.501 min, butanol; 6.867 min, 3-hydroxyl Butanone; 8.060 min, acetic acid; 9.705 min, butyric acid.
  • the best way to implement the invention is a gas chromatogram prepared by sampling in a 50 L fermenter for 30 h, in the order of peak order: 3.752 min, acetone; 4.199 min, ethanol; 5.501 min, butanol; 6.867 min, 3-hydroxyl Butanone; 8.060 min, acetic acid; 9.705 min, butyric acid. The best way to implement the invention
  • the medium components and culture conditions of the plate culture and the seed culture are as follows:
  • Plate medium glucose 10g / L, yeast extract 5g / L, peptone 3g / L, magnesium sulfate heptahydrate 3g / L, ammonium acetate 2g / L, potassium dihydrogen phosphate lg / L, dipotassium hydrogen phosphate lg / L, Agar 15g/L, sterilized at 121 ° C for 15 min.
  • Clostridium butyrate B3 was screened on the plate and placed in a Bugbox anaerobic chamber (Ruskinn, UK) for 24 hours at 37 ° C. The plate grew white irregular colonies.
  • Seed medium The medium is the same as the above plate medium without agar.
  • Seed culture conditions The bacterial sludge on the plate was scraped to the seed culture medium, and fermented in a 100 mL blue cap reagent bottle or a shake flask. The liquid volume was 50%, and the culture was statically cultured at 37 ° C for 15 hours, and a large amount of foam floated on the liquid surface.
  • the analytical method used was gas chromatography (GC) with the following conditions: Flame Ion Detector (FID), Agilent HP-INNO WAX 19091N-236 Capillary Column (60 m X 0.25 mm x 0.25 urn) , N 2 is carrier gas, flow rate 2mL / min, split ratio 90: 1 , ⁇ 2 flow rate 30ml / min, air flow rate 300ml / min, inlet temperature 180 ° C, detector 220 ° C, column temperature (programming temperature ): Retained at 70 ° C for 0.5 min, then ramped to 190 ° C at a rate of 20 ° C / min for 4 min.
  • a representative map of the detected fermentation product is shown in Figure 2.
  • Example 1 Mutagenesis of Clostridium acetobutylicum
  • Clostridium acetobutylicum was used as the original strain, and it was transferred to the plate for 24 hours after activation, and one ring of bacteria was picked up in a 60 mM lithium chloride sterile aqueous solution with 100 glass beads. 100ml, shaking in a shake flask at 200rpm for 1Omin The body was evenly dispersed, and then 1 mL of the bacterial suspension was placed in a sterilized plate in a 254 nm ultraviolet mutagenesis box for 90 s, diluted 100 times and applied to a bromophenol purple plate containing 20 mg/L, and anaerobic at 37 ° C. Cultivate for 3 days.
  • Colonies with large colonies, early discoloration, and large discoloration were selected as 120 strains of primary strains.
  • the culture was expanded and fermented to verify yield and stability.
  • a strain of Clostridium acetobutylicum B3 (C/ay rWM izcetobM y CM B3 ) was obtained and deposited on September 9, 2011 at the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • Clostridium acetobutylicum B3 has the following physiological characteristics:
  • Fermentation medium glucose 60g/L, ammonium acetate 3g/L, corn syrup 2ml/L, sterilization at 121 °C 15min
  • the seed solution was mixed and then inoculated into a 5 L fermentor at a 10% inoculation amount, and the liquid volume was 60%, and the culture was statically cultured at 37 ° C for 80 hours.
  • Fermentation medium 6% corn flour, gelatinized in boiling water for 60 min, and sterilized at 121 °C for 30 min. Mix the seed solution and put it into the 5L fermenter with a 5% inoculum. The liquid volume is 3L, at 37! Static culture for 50h.
  • Fermentation medium glucose 60g/L, ammonium acetate 2.5g/L, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, ferrous sulfate heptahydrate 0.01g /L, sodium chloride 0.01g/L, sterilized at 121 °C for 30min.
  • the seed solution was placed in a 5 L fermentor at a 10% inoculation amount, and the liquid volume was 3 L, and cultured at 37 ° C for 80 hours.
  • Example 4 The same procedure as in Example 4 except that when the fermentation was carried out for 30 hours, 3 g/L of sodium acetate was added and fermentation was carried out for 72 hours.
  • the resulting fermentation broth was acetone 5.2 g/L, ethanol l. lg/L, butanol 12.5 g/L, 3-hydroxybutanone 1.9 g/L.
  • Example 4 The same procedure as in Example 4 was carried out except that 0.5 g/L of proline and 0.5 g/L of leucine were added at the initial stage of fermentation, and fermentation was carried out for 85 hours.
  • Example 4 The same procedure as in Example 4 except that 5 g/L of glycerin was added for 40 hours of fermentation and fermented for 80 hours.
  • Example 9 The same as the method of Example 4, except that when the butanol concentration reaches about 5 g/L, the resin is added to adsorb the butanol, and the amount of the resin added is calculated by the adsorption capacity being half of the maximum amount of butanol produced, and stirred every 10 hours. 5min. Fermentation for 65 h. In the finally obtained fermentation broth, acetone was 3.0 g/L, ethanol was 1.lg/L, butanol was 11.8 g/L, and 3-hydroxybutanone was 3.5 g/L.
  • Example 9 Example 9
  • Example 4 The same procedure as in Example 4 except that the temperature was maintained at 38 ° C for 0-15 h at the beginning of the fermentation, then the temperature was maintained at 34 ° C and fermentation was carried out for 72 h.
  • Example 4 The procedure was the same as in Example 4 except that the temperature was maintained at 27 ° C for 0-35 h at the beginning of the fermentation, then the temperature was maintained at 34 ° C, and fermentation was carried out for 80 h.
  • Example 4 The same procedure as in Example 4 except that the pH of the medium was controlled at 4.5 ° with a 1 M sodium hydroxide solution at 0 to 30 h after fermentation, and the pH was not controlled after the start of alcohol production, and fermentation was carried out for 80 hours.
  • the method is the same as the method of Example 4, except that the initial sugar concentration of the medium is 40 g/L.
  • the concentration of the starting sugar is reduced to 15 g/L, 10 g/L of sugar is added every 10 hours for a total of 3 times.
  • the sugar concentration reached 70g/L and the fermentation was carried out for 80 hours.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供一种丙酮丁醇梭菌及其应用。本发明提供的丙酮丁醇梭菌的保藏编号为CGMCC No.5234。本发明提供的丙酮丁醇梭菌可以用于发酵联产丙酮、丁醇、乙醇和3-羟基丁酮，提高了丁醇发酵的经济效益。可以通过添加代谢或生长调节物实现NAD+的偶联再生，提高了产物得率，同时可以灵活调节联产产物的产量，适应市场需求。

Description

一种丙酮丁醇梭菌及其应用 技术领域
本发明属于微生物技术领域， 涉及一种产 3-羟基丁酮的丙酮丁醇梭菌 及其应用。 背景技术
丙酮（acetone ) 、 丁醇（ butanol )和乙醇（ ethanol )统称为 ABE, 是 医药、 农药、 塑料、 橡胶以及轻工业的重要原料， 也是重要的化工溶剂， 因此 ABE的研究对现代工业的发展有重要作用。
丙酮又称为二曱基曱酮， 无色透明液体， 易挥发。 丙酮既是重要的有 机溶剂也是非常重要的化工原料， 用于炸药、 塑料、 纤维、 制革、 喷漆等 行业中， 也可用于合成烯酮、 醋酐、 碘仿、 烯橡胶、 环氧树脂等。
丁醇又称为 1-丁醇， 无色透明液体， 有强烈的酒味， 稍溶于水， 相对 密度 0.81 , 沸点 117.7 °C , 属于二级易燃品。 丁醇是一种重要的有机原料 和化工溶剂， 广泛用于各种塑料、 橡胶制品、 树脂制造以及皮革、 造纸等 轻工行业中。 丁醇的另一个非常重要的作用可以作为当今极具潜力的新型 生物燃料，被称为第二代生物燃料。 与乙醇相比， 丁醇具有较高的燃烧值， 可支持汽车多走 30%的路程，和烃类性质接近，无需对汽车气缸进行改造； 并且低挥发性； 不亲水； 无腐蚀； 高辛烷值 -抗爆性好。 因此， 丁醇的研究 开发在全球化石资源日益枯竭的形势下， 迅速成为新的热点。
3-羟基丁酮， 又名乙偶姻 （acetoin ) 、 乙酰曱基曱醇， 通常为淡黄色 液体或晶体， 天然存在于玉米、 葡萄、 草莓、 干酪、 肉类等食品中， 是一 种应用广泛的香料，我国国家标准 GB2760-86规定其为允许使用的食用香 料， 美国食品和萃取协会（FEMA ) 安全号为 2008。 此外， 3-羟基丁酮还 可以作为化学合成中的重要原料， 比如可用于合成手性近晶材料和向列材 料。
3-羟基丁酮的传统化工制备主要是采用化学法或者酶法转化， 其原料 主要是双乙酰（丁二酮） 和 2,3-丁二醇。 1998年， 英国 Witwatersrand大 学的 Martin Studer等应用经过改性的铂作为催化剂选择性地加氢还原双乙 酰， 其产率为 30%。 英国 Hull大学的 Slipszenko也开展了铂催化双乙酰加 氢还原生成 3-羟基丁酮的研究， 产率为 85%。 但催化加氢非均相反应通常 在高压下进行，设备要求高，并且所用催化剂昂贵。 1992年，美国的 Hummel 等采用培养菌体的方法获得乳杆菌或者酵母菌中的双乙酰还原酶， 然后在 pH5、 温度 70 °C的条件下应用该还原酶及辅酶 NADPH催化双乙酰合成 3- 羟基丁酮， 产率最高达 100%。 1945年美国农业部的 R. H. Blom通过氧化 脱氢以 2 , 3-丁二醇合成双乙酰及 3-羟基丁酮。 2 , 3-丁二醇在 140 °C加热 后与空气一起通过 Pyrex管式反应器，反应器中装填铜刨花，反应温度 315 °C ,得到产品双乙酰（产率 33% )及 3-羟基丁酮（产率 25% )。法国 De Poitiers 大学的 A. Hilmi应用电化学氧化制备 3-羟基 -2-丁酮，其方法是在电解槽中 进行反应， 隔膜为离子交换膜， 反应中的电极均为可逆氢电极。 阳极为 Pt-Pb , 多孔的 Pt/Ir( ( 10% ) 为对应电极， 电解液为 HC104 , 溶剂为超纯 水， 反应温度为 40 °C , 电池电压为 0.8V。 应用这种电解氧化法， 其产物 除了 3-羟基丁酮外还有双乙酰和二氧化碳， 产率为 94%。 但是， 化学法存 在环境污染较严重和产品质量的问题， 并且原料主要来源于不可再生的化 石资源， 从长远来看限制了其发展。
另外，还可以采用微生物发酵法生产 3-羟基丁酮。在大多数微生物中， 两分子丙酮酸在乙酰乳酸合成酶作用下合成一分子乙酰乳酸， 乙酰乳酸经 乙酰乳酸脱羧酶作用即可生成 3-羟基丁酮。 乙酰乳酸也可以在有氧的条件 下自然氧化脱羧生成双乙酰， 双乙酰再还原生成 3-羟基丁酮。 但是， 3-羟 基丁酮可以被进一步还原生成 2,3-丁二醇， 并且双乙酰还原生成 3-羟基丁 酮和 3-羟基丁酮还原生成 2,3-丁二醇可以由同一个酶（ 2,3-丁二醇脱氢酶） 所催化， 因此在很多微生物中 3-羟基丁酮往往是作为 2,3-丁二醇生产中间 产物， 同时伴有双乙酰的生成， 影响了其产量和分离。 目前人们已经发现 4艮多可以产生 3-羟基丁酮的菌种，例如：乳酸乳球菌（ Lactococcus lactis )、 干酸 H干菌 ( Lactobacillus casei ) 、 酉良酒酵母 ( Saccharomyces cerevisiae ) 等奶制品或者酿酒发酵菌株， 但是 3-羟基丁酮的产量都低于 l g/L。 肺炎克 雷 4白氏菌 ( Klebsiella pneumoniae )、 产气节 干菌 ( Enterobacter aerogenes ) 和枯草芽孢杆菌 Bacillus subtUi 等也能够发酵生产 3-羟基丁酮， 并且 有较高产量， 但是这些菌株主要是用来生产 2,3-丁二醇， 3-羟基丁酮仅仅 是作为其副产物。 Olson和 Johnson利用产气节杆菌将 226 g/L的葡萄糖转 化为 14g/L的 3-羟基丁酮和 97 g/L的 2,3-丁二醇。 山东大学的马翠卿等人 利用含有 2,3-丁二醇脱氢酶基因和 NADH氧化酶基因的重组大肠杆菌制备 手性 3-羟基丁酮和 2,3-丁二醇， 手性 3-羟基丁酮的浓度达到 36g/L。 在中 国专利申请 CN101008019A中，公开了一株枯草芽孢杆菌在制备 3-羟基丁 酮中的应用， 其中以葡萄糖为主要原料， 利用枯草芽孢杆菌 （Bacillus subtilis ) SFA-H31( CGMCC1869)在 50 L发酵罐中发酵培养 52 h, 转化率 达到 48.26 %, 3-羟基丁酮发酵产率达 55.67 g/L, 并证实该菌株不产生副 产物丁二酮和 2,3-丁二醇。 然而， 枯草芽孢杆菌等通常好氧生长或发酵， 并且由于每分子葡萄糖到 3-羟基丁酮会产生 2个 NADH,专一生产 3-羟基 丁酮则造成 NADH的浪费。 同时， 由于 2,3-丁二醇和 3-羟基丁酮处于同一 支路的上下游，常规的联产很难做到两者代谢流量的单独调控和 NADH的 有效利用。 发明内容
本发明的目的是，提供一种用于联产丁醇和 3-羟基丁酮的诱变丙酮丁 醇梭菌。
本发明的另一个目的是， 提供一种联产丁醇和 3-羟基丁酮的方法。 本发明的目的是通过以下技术方案来实现的。 一方面， 本发明提供一 种用于发酵联产丁醇和 3-羟基丁酮的丙酮丁醇梭菌， 其保藏编号为 CGMCC No.5234, 该菌株已于 2011年 9月 9 日保藏于中国 生物菌种保 藏管理委员会普通微生物中心 （筒称 CGMCC ) , 保藏单位地址： 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。
本发明还提供了上述丙酮丁醇梭菌在发酵联产丁醇和 3-羟基丁酮中 的应用。
另一方面， 本发明提供一种发酵联产丁醇和 3-羟基丁酮的方法， 所述 方法包括采用上述丙酮丁醇梭菌在液体发酵培养基中进行发酵培养， 以生 产丁醇和 3-羟基丁酮。
本发明提供的方法包括以下步骤： 1 )将所述丙酮丁醇梭菌于固体平 板培养基上培养 12-36小时； 2 )将步骤 1 )培养的丙酮丁醇梭菌接入种子 培养基中， 于 5-39°C培养 10-20小时； 3 )将步骤 2 )培养的丙酮丁醇梭菌 以 5%-15%的接种量接入液体发酵培养基中，于 24-40 °C静止培养 40-85小 时。
优选地， 所述液体发酵培养基中包括碳源、 氮源和 /或无机盐。
优选地， 所述碳源选自葡萄糖、 甘油、 玉米粉、 果糖、 淀粉、 木糖等 中的一种或几种， 浓度为 20g/L-80g/L。
优选地， 所述氮源选自硫酸铵、 醋酸铵、 玉米浆、 酵母粉、 酵母膏、 尿素等中的一种或几种， 浓度为 0.1g/L-10g/L。
优选地， 所述无机盐选自钠盐、 钾盐、 铁盐、 亚铁盐、 锰盐、 磷酸盐、 硫酸盐等中的一种或几种， 浓度为 0.001g/L-5g/L。
优选地， 所述液体发酵培养基中还包括微量元素。
优选地， 所述微量元素选自维生素 B1 (硫胺或其盐酸盐 )、 维生素 H
(生物素， 维生素 B7 ) 、 维生素 Bx (对氨基苯曱酸， 维生素 HI )等中的 一种或几种。
优选地， 所述液体发酵培养基中还包括一种或多种代谢或生长调节 物。
优选地， 所述代谢或生长调节物选自乙酸盐、 支链氨基酸、 甘油中的 一种或几种。
更优选地， 所述乙酸盐包括乙酸钠、 乙酸铵、 乙酸镁、 乙酸钙、 乙酸 钾等中的一种或几种， 加入培养基后的浓度 0.5g/L-8g/L, 加入时间为发酵 开始后的 0-50小时。外加乙酸盐能作为丙酮的合成原料， 直接提高丙酮的 产量， 因此由糖生成的丙酮减少而 3-羟基丁酮在 NAD+的再生要求下则增 多； 另外乙酸盐的加入还能加快糖耗速率， 缩短发酵周期。 如果在生长前 期加入乙酸盐则会抑制菌体生长， 但是却能提高丁醇得率。
更优选地， 所述支链氨基酸包括亮氨酸、 异亮氨酸、 缬氨酸等中的一 种或几种， 加入培养基后的浓度 0.05g/L-5g/L, 加入时间为发酵开始后的 0-20小时。 支链氨基酸和 3-羟基丁酮共享同一个前体乙酰乳酸， 本发明通 过加入支链氨基酸能反馈抑制乙酰乳酸的合成， 可以降低 3-羟基丁酮产量 而进一步提高丁醇产量， 但是， 支链氨基酸的加入对菌体生长有一定的抑 制作用。
更优选地， 所述甘油加入培养基后的浓度为 2-20g/L, 加入时间为发 酵开始后 0-60小时。甘油的还原度比葡萄糖的还原度高，甘油代谢能提供 更多的 NADH, 本发明通过在发酵中加入甘油， 能为菌体后期回用乙酸和 丁酸提供 NADH, 因此提高了丁醇得率。
优选地， 所述液体发酵培养基中还包括能吸附丁酸或丁醇或利于菌体 生长的载体介质， 包括活性炭、 纤维、 树脂、 乳化剂等中的一种或几种。 丁酸、 丁醇是菌体生长的主要抑制因素， 前期的丁酸限制菌体的浓度， 后 期丁醇限制菌体产醇的速率和产量， 本发明通过在发酵过程中加入树脂或 吸附载体等， 能够强化菌体的生长， 减轻产物抑制作用， 增强菌体的抗逆 能力， 发酵速率大大提高， 周期下降， 3-羟基丁酮产量大幅提高。 优选地， 所述发酵培养为两阶段培养方式。
优选地， 所述两阶段培养方式包括分两阶段控制发酵温度、 pH或发 酵糖浓度。
更优选地， 所述发酵温度的控制方式如下： 发酵开始后的 0-30小时， 控制温度为 24-40 °C , 之后控制温度为 32-37 °C。 丙酮丁醇梭菌前期生长， 消耗初糖产生大量的乙酸、 丁酸， 然后进入产醇期产生丙酮、 丁醇等， 这 两个阶段菌体的生长和代谢在不同的温度下有不同的行为， 本发明通过温 度来协调菌体的生长与产醇以及前期代谢与后期代谢， 有利于提高代谢产 物产量。
更优选地， 所述发酵 pH的控制方式如下： 发酵开始后 0-30小时， 控 制 pH为 pH 4-5.5 , 产醇开始后不控制 pH。 pH在丙酮丁醇梭菌的发酵中 非常重要， 它能反映出并且影响到乙酸、 丁酸等有机酸在发酵液中的解离 形式， 进而影响到菌体对酸的利用。 在某些情况下， 因为酸利用的问题， 发酵很有可能会在产酸期延滞很长一段时间， 因此， 在菌体从产酸期向产 醇期过渡的时段， pH表现为决定生产成败的关键因素。 本发明通过控制 产酸期的 pH在某一合适的水平， 能够保证菌体成功并且迅速地过渡到产 醇期。
更优选地， 所述发酵糖浓度的控制方式如下： 发酵初始糖浓度为 30-50g/L , 待糖浓度降至 10-30g/L时， 补加糖碳源， 使得培养基中的糖浓 度维持在 10-30g/L , 待总糖浓度达到 60-90g/L时， 停止补加。 上述诱变丙 酮丁醇梭菌在较高的初糖浓度下有生长抑制的现象， 同时在不同的糖浓度 下菌体表现出不同的碳硫分配。 本发明通过补加糖的策略， 控制发酵过程 中的糖浓度， 同时达到促进菌体生长和保持菌体处在较佳碳硫分配的状态 下发酵的目的。
本发明通过将一株丙酮丁醇梭菌 B3 ( Clostridium acetobutylicum ) 经 紫外诱变后， 筛选出一株能高产丁醇和 3-羟基丁酮的丙酮丁醇梭菌。 本发 明获得的诱变丙酮丁醇梭菌有较强的乙酰乳酸合成酶活性和乙酰乳酸脱 羧酶活性， 同时缺失 2 , 3-丁二醇脱氢酶活性和异丙醇脱氢酶活性。 能高 产丁醇、 丙酮、 乙醇 （ABE ) 和 3-羟基丁酮， 并且没有 2 , 3-丁二醇、 异 丙醇等副产物。 此外， 上述诱变丙酮丁醇梭菌， 具有很强的耐氧性能， 种 子培养和发酵培养均无需通厌氧气体驱除残留空气， 种子培养和发酵培养 不通气不搅拌静止发酵即可。
本发明以此菌进行发酵生产丁醇、乙醇、丙酮和 3-羟基丁酮（乙偶姻）， 实现 NAD+的偶联再生， 同时对碳代谢流进行定向调控。 具体地， 本发明 利用上述诱变丙酮丁醇梭菌进行 ABE发酵联产 3-羟基丁酮的方法包括： 将上述诱变丙酮丁醇梭菌在固体平板上培养 12-36h后接入种子培养基， 25-39 °C培养 10-20h, 以 5%- 15%的接种量接入含碳源、 氮源、 无机盐以及 微量元素的培养基中培养， 添加代谢或生长调节物， 进行厌氧发酵生产丁 醇和 3-羟基丁酮， 发酵时间 40-85h。
如图 1所示， 丙酮丁醇梭菌进行 ABE发酵的前期 （产酸期） 约有 30%-40%的糖被转化生成乙酸和丁酸等， 菌体进入产醇期后， 乙酸和丁酸 可以被重新还原生成乙醇和丁醇、 乙酸和丁酸的回用效率决定着丁醇的生 产效率。 本发明中的诱变菌株生产 3-羟基丁酮可以为乙酸和丁酸的回用提 供 NADH进而生成丁醇， 实现了 NAD+的偶联再生，提高了糖利用率和溶 剂得率， 并且不产生常见副产物 2 , 3-丁二醇、 异丙醇等。 同时， 由于 ABE 产生途径和 3-羟基丁酮产生途径处于不同的分支， 并且与菌体生长有不同 的相关性， 因此通过添加作用于各支路途径的小分子效应物或生长调节 物， 可以有效实现碳流在这两条途径中的流量分配， 灵活调节两者产量以 适应市场需求。
综上所述， 本发明的有益效果在于以下几方面：
( 1 )通过诱变获得的生产菌种有很强的耐氧性能， 大大减少了常规 厌氧发酵过程中的避氧措施。 静止发酵， 不通气不搅拌， 节能减排。
( 2 )发酵不产生 2 , 3-丁二醇、 异丙醇、 曱酸和乳酸等副产物， 提高 了产物得率和减轻了后续分离压力。
( 3 ) 3-羟基丁酮产生提供的 NADH能用于乙酸、 丁酸的还原生成乙 醇和丁醇， 实现了 NAD+的偶联再生， 提高了碳得率。
( 4 ) 3-羟基丁酮和 ABE处于不同的代谢支路上， 两者的代谢流量能 够灵活调节， 可以更好地适应市场需求。 生物材料保藏信息
丙酮丁醇梭菌 （ Clostridium acetobutylicum ) B3 , 已于 201 1年 9月 9 日保藏于中国微生物菌种保藏管理委员会普通微生物中心 （筒称
CGMCC ) , 保藏单位地址： 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， 保藏编号为 CGMCC No.5234。 附图说明 以下， 结合附图来详细说明本发明的实施方案， 其中：
图 1为 NAD+偶联再生联产 ABE和 3-羟基丁酮的示意图。
图 2为在 50L发酵罐中发酵 30h时的取样做出的气相色谱图谱，按出 峰先后顺序依次为： 3.752min, 丙酮； 4.199min, 乙醇； 5.501min, 丁醇； 6.867min, 3-羟基丁酮； 8.060min, 乙酸； 9.705min, 丁酸。 实施发明的最佳方式
根据下述实施例可以更好地理解本发明， 然而实施例中所描述的具体 的物料配比， 工艺条件及结果仅用于说明本发明， 而不应当对本发明进行 限制。
以下各实施例中， 平板培养和种子培养的培养基成分和培养条件如 下：
平板培养基： 葡萄糖 10g/L, 酵母膏 5g/L, 蛋白胨 3g/L, 七水合硫酸 镁 3g/L, 乙酸铵 2g/L, 磷酸二氢钾 lg/L, 磷酸氢二钾 lg/L, 琼脂 15g/L, 于 121°C灭菌 15min。
平板培养条件： 将诱变筛选得到的丙酮丁酮梭菌 B3划线在平板上， 置于 Bugbox厌氧箱 （英国 Ruskinn) 37°C培养 24h, 平板长出白色不规则 菌落。
种子培养基： 培养基同以上平板培养基， 不加琼脂。
种子培养条件： 将平板上的菌泥刮至种子培养基， 以 lOOmL蓝盖试剂 瓶或者摇瓶进行发酵， 装液量为 50%, 37°C静止培养 15h, 有大量泡沫浮 于液面。
以下各实施例中， 所采用的分析方法为气相色谱（GC) , 条件如下： 火焰离子检测器 （FID) , Agilent HP-INNO WAX 19091N-236毛细管色谱 柱（60 m X 0.25mm x 0.25 urn), N2为载气， 流速 2mL/min, 分流比 90: 1 , Η2流速 30ml/min, 空气流速 300ml/min, 进样口温度 180°C , 检测器 220 °C, 柱温 （程序升温） ： 70°C保留 0.5min, 然后以 20°C/min的速率升温 到 190°C, 保留 4min。 所检测的发酵产物的代表性图谱见图 2。 实施例 1 丙酮丁醇梭菌的诱变
将丙酮丁醇梭菌（ Clostridium acetobutylicum )作为原始菌株， 在平板 上活化培养 24h后转接一次， 挑取一环菌泥于加了 100颗玻璃珠的 60mM 的氯化锂无菌水溶液中， 体积 100ml , 在摇瓶中 200rpm摇晃 1 Omin将菌 体均匀打散， 然后取 lmL菌悬液于无菌平板中置于 254nm紫外诱变箱中 照射 90s后稀释 100倍涂布于含有 20mg/L的溴曱酚紫平板中， 37°C厌氧 培养 3天。 挑选菌落大、 变色时间早且变色圏较大较亮的菌落作为初选菌 株共 120株， 扩大培养后进行发酵， 验证产量以及稳定性。 最终获得一株 丙酮丁醇梭菌 B3 ( C/ay rWM izcetobM y CM B3 ) , 已于 2011年 9月 9 日保藏于中国微生物菌种保藏管理委员会普通微生物中心 （筒称
CGMCC ) ,其保藏编号为 CGMCC No.5234。该菌株丁醇平均产量 11.3g/L, 3-羟基丁酮平均产量 2.8g/L,总溶剂量 19.6g/L且传代 8次后产量不低于平 均产量的 10%。 所筛选的丙酮丁醇梭菌 B3具有以下的生理特征：
( 1 )在平板中培养， 菌落圓形， 直径 4-8mm, 白色或灰白色， 突起， 边缘不规则， 菌落较厚容易挑起。
( 2 )在液体中培养， 会产生大量泡沫， 除此之外还会有絮状粘膜或 丝状物， 发酵结束后瓶底或者罐底有白色粉末状沉淀。
( 3 )革兰氏染色阳性； 细胞长杆状， 在发酵后期出现端生孢子； 厌 氧， 但发酵过程中无需通厌氧气体， 发酵容器无需密闭。
( 4 ) 具有较高的乙酰乳酸合成酶活性， 且该酶活性很容易受到支链 氨基酸的抑制。
( 5 ) 其发酵样品中无可检测到的曱酸， 乳酸， 柠檬酸及甘油， 但可 以利用这些物质。 实施例 2
发酵培养基： 葡萄糖 60g/L, 乙酸铵 3g/L, 玉米浆 2ml/L, 121 °C灭菌 15min„
将种子液混合后以 10%的接种量接入 5L发酵罐中， 装液量 60%, 于 37°C静止培养 80h。
最终得到的发酵液中， 丙酮 3.9g/L, 乙醇 0.8g/L, 丁醇 11.9g/L, 3-羟 基丁酮 2.4g/L。 实施例 3
发酵培养基： 6%的玉米粉， 沸水糊化 60min, 121 °C灭菌 30min。 将种子液混合后以 5%的接种量接入 5L发酵罐中， 装液量 3L, 于 37 ！静止培养 50h。
最终得到的发酵液中， 丙酮 4.2g/L, 乙醇 l. lg/L, 丁醇 11.8g/L, 3-羟 基丁酮 2.1g/L。 实施例 4
发酵培养基： 葡萄糖 60g/L, 乙酸铵 2.5g/L, 七水合硫酸镁 0.5g/L, 磷酸二氢钾 0.5g/L, 磷酸氢二钾 0.5g/L, 七水合硫酸亚铁 0.01g/L, 氯化钠 0.01g/L, 121 °C灭菌 30min。
将种子液以 10%的接种量接入 5L发酵罐中， 装液量 3L, 于 37°C静止 80h培养。
最终得到的发酵液中， 丙酮 4.1g/L, 乙醇 l. lg/L, 丁醇 12.2g/L, 3-羟 基丁酮 2.3g/L。 实施例 5
与实施例 4的方法相同， 所不同的是当发酵进行到 30h时， 加入 3g/L 的乙酸钠， 发酵 72h。
最终得到的发酵液中， 丙酮 5.2g/L, 乙醇 l. lg/L, 丁醇 12.5g/L, 3-羟 基丁酮 1.9g/L。 实施例 6
与实施例 4的方法相同，所不同的是发酵初期加入 0.5g/L的缬氨酸和 0.5g/L的亮氨酸， 发酵 85h。
最终得到的发酵液中， 丙酮 4.8g/L, 乙醇 l. lg/L, 丁醇 12g/L, 3-羟基 丁酮 1.6g/L。 实施例 7
与实施例 4的方法相同，所不同的是发酵 40h时加入 5g/L的甘油，发 酵 80h。
最终得到的发酵液中， 丙酮 3.9g/L, 乙醇 1.5g/L, 丁醇 12.8g/L, 3-羟 基丁酮 2.0g/L。 实施例 8
与实施例 4的方法相同，所不同的是在丁醇浓度达到 5g/L左右时，加 入树脂吸附丁醇， 树脂加入量以其吸附容量为最大丁醇产生量的一半计 算， 每隔 10h搅拌 5min。 发酵 65h。 最终得到的发酵液中， 丙酮 3.0g/L, 乙醇 l . lg/L, 丁醇 11.8g/L, 3-羟 基丁酮 3.5g/L。 实施例 9
与实施例 4的方法相同，所不同的是发酵初期 0-15h维持温度在 38 °C , 然后温度维持在 34°C , 发酵 72h。
最终得到的发酵液中， 丙酮 3.1g/L, 乙醇 l . lg/L, 丁醇 11.4g/L, 3-羟 基丁酮 2.5g/L。 实施例 10
与实施例 4的方法相同，所不同的是发酵初期 0-35h维持温度在 27°C , 然后温度维持在 34°C , 发酵 80h。
最终得到的发酵液中， 丙酮 3.2g/L, 乙醇 lg/L, 丁醇 11.8g/L, 3-羟基 丁酮 2.5g/L。 实施例 11
与实施例 4的方法相同， 所不同的是在发酵 0-30h, 以 1M的氢氧化 钠溶液控制培养基的 pH在 4.5 , 待产醇开始后不控制 pH, 发酵 80h。
最终得到的发酵液中， 丙酮 3.0g/L, 乙醇 0.9g/L, 丁醇 11.2g/L, 3-羟 基丁酮 2.5g/L。 实施例 12
与实施例 4的方法相同， 所不同的是培养基初糖浓度 40g/L, 待初糖 浓度降至 15g/L时， 每隔 10h补加 10g/L的糖， 共补加 3次， 总糖浓度达 到 70g/L, 发酵 80h。
最终得到的发酵液中， 丙酮 5.0g/L, 乙醇 1.4g/L, 丁醇 13.8g/L, 3-羟 基丁酮 3.6g/L。 参考文献：
Studer M, Okafor V, Blaser HU, 1998. Hydrogenation of butane - 2,
3-dione with heterogeneous cinchona modified platinum catalysts： a
combination of an enantioselective reaction and kinetic resolution[J ] . Chem Commun, 1053 - 1 054. Slipszenko JA, Griffiths SP, Simons KE, et al. , 1998. Enantioselective hydrogenation[J ] . Journal of Catalysts , 179: 267 ~ 276.
Hummel W, 1992. Microbiologically prepared diacetyl reductase [ P] . USP: 5164 314, 1992-17-11.
Blom RH, 1945. Configuration of acetylmethylcarbinol [ J ] . Am Chem
Soc, 67: 494-498.
Hilmi A, Belgsir EM, Leger JM, et al. 1997. Electrocatalytic oxidation of aliphatic diols. Part V. Electro - oxidation of butanediols on platinum based electrodes [J ] . Journal of Electroanalytical Chemistry, 435 : 69 - 75.
Olson BH, Johnson MJ, 1948. The production of 2,3-butylene glycol by
Aerobacter aerogenes. J bacterial. 55: 209-222.
马翠卿， 吕传娟， 肖梓军， 秦加阳， 徐平. 一株基因重组菌及在手性 纯乙偶姻和 2 , 3-丁二醇生产中的应用 [P] , CN101565685A, 2009-10-28。
刘建军， 赵祥颖， 田延军， 等. 一株枯草芽孢杆菌在制备 3-羟基丁酮 中的应用 [ P ] , CN101008019 , 2007-8-1。

Claims

权 利 要 求
1. 一种用于发酵联产丁醇和 3-羟基丁酮的丙酮丁醇梭菌，其保藏编号 为 CGMCC No.5234。
2. 权利要求 1所述的丙酮丁醇梭菌在发酵联产丁醇和 3-羟基丁酮中 的应用。
3. 一种发酵联产丁醇和 3-羟基丁酮的方法， 其特征在于，所述方法包 括采用权利要求 1所述丙酮丁醇梭菌在液体发酵培养基中进行发酵培养， 以生产丁醇和 3-羟基丁酮。
4. 根据权利要求 3所述的方法，其特征在于，所述方法包括以下步骤：
1 )将权利要求 1所述丙酮丁醇梭菌于固体平板培养基上培养 12-36 小时；
2 )将步骤 1 )培养的丙酮丁醇梭菌接入种子培养基中， 于 5-39 °C培养 10-20小时；
3 )将步骤 2 )培养的丙酮丁醇梭菌以 5-15%的接种量接入液体发酵培 养基中， 于 24-40 °C静止培养 40-85小时。
5. 根据权利要求 3或 4所述的方法， 其特征在于， 所述液体发酵培养 基中包括碳源、 氮源和 /或无机盐；
优选地， 所述碳源选自葡萄糖、 甘油、 玉米粉、 果糖、 淀粉、 木糖中 的一种或几种， 浓度为 20g/L-80g/L;
优选地， 所述氮源选自硫酸铵、 醋酸铵、 玉米浆、 酵母粉、 酵母膏、 尿中的一种或几种， 浓度为 0.1g/L-10g/L;
优选地， 所述无机盐选自钠盐、 钾盐、 铁盐、 亚铁盐、 锰盐、 磷酸盐、 石克酸盐中的一种或几种， 浓度为 0.001g/L-5g/L。
6. 根据权利要求 3至 5中任一项所述的方法， 其特征在于， 所述液体 发酵培养基中还包括微量元素；
优选地， 所述微量元素选自维生素 B1或其盐酸盐、 维生素 H、 维生 素 Bx中的一种或几种， 浓度 0.0001g/L-3g/L。
7. 根据权利要求 3至 6中任一项所述的方法， 其特征在于， 所述液体 发酵培养基中还包括一种或多种代谢或生长调节物；
优选地， 所述代谢或生长调节物选自乙酸盐、 支链氨基酸、 甘油中的 一种或几种；
更优选地， 所述乙酸盐包括乙酸钠、 乙酸铵、 乙酸镁、 乙酸钙、 乙酸 钾中的一种或几种， 加入培养基后的浓度 0.5g/L-8g/L, 加入时间为发酵开 始后的 0-50小时；
更优选地， 所述支链氨基酸包括亮氨酸、 异亮氨酸、 缬氨酸中的一种 或几种，加入培养基后的浓度 0.05g/L-5g/L,加入时间为发酵开始后的 0-20 小时；
更优选地， 所述甘油加入培养基后的浓度为 2-20g/L, 加入时间为发 酵开始后 0-60小时。
8. 根据权利要求 3至 7中任一项所述的方法， 其特征在于， 所述液体 发酵培养基中还包括能吸附丁酸或丁醇或利于菌体生长的吸附载体如树 脂、 活性炭、 纤维、 乳化剂中的一种或几种。
9. 根据权利要求 3至 8中任一项所述的方法， 其特征在于， 所述发酵 培养为两阶段培养方式；
优选地， 所述两阶段培养方式包括分两阶段控制发酵温度、 pH和 /或 发酵糖浓度。
10. 根据权利要求 9所述的方法， 其特征在于， 所述发酵温度的控制 方式如下： 发酵开始后的 0-30小时， 控制温度为 24-40°C , 之后控制温度 为 32-37 °C ;
优选地， 所述发酵 pH的控制方式如下： 发酵开始后 0-30小时， 控制 pH为 pH 4-5.5 , 产醇开始后不控制 pH;
优选地， 所述发酵糖浓度的控制方式如下： 发酵初始糖浓度为
30-50g/L, 待糖浓度降至 10-30g/L时， 补加糖碳源， 使得培养基中的糖浓 度维持在 10-30g/L, 待总糖浓度达到 60-90g/L时， 停止补加。
PCT/CN2012/072649 2012-03-20 2012-03-20 一种丙酮丁醇梭菌及其应用 WO2013138998A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2012/072649 WO2013138998A1 (zh) 2012-03-20 2012-03-20 一种丙酮丁醇梭菌及其应用
US14/386,286 US9249433B2 (en) 2012-03-20 2012-03-20 Clostridium acetobutylicum and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/072649 WO2013138998A1 (zh) 2012-03-20 2012-03-20 一种丙酮丁醇梭菌及其应用

Publications (1)

Publication Number Publication Date
WO2013138998A1 true WO2013138998A1 (zh) 2013-09-26

Family

ID=49221799

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/072649 WO2013138998A1 (zh) 2012-03-20 2012-03-20 一种丙酮丁醇梭菌及其应用

Country Status (2)

Country Link
US (1) US9249433B2 (zh)
WO (1) WO2013138998A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3230459B1 (en) 2014-12-08 2020-09-16 LanzaTech New Zealand Limited Recombinant microorganisms exhibiting increased flux through a fermentation pathway
CN107400646B (zh) * 2017-08-29 2018-07-27 汕头大学 一株高产丁醇梭菌及其筛选与应用
CN111269869B (zh) * 2020-02-10 2021-08-17 南京工业大学 一种重组丙酮丁醇梭菌的构建方法以及在发酵半纤维制备丁醇中的应用
US20210261987A1 (en) 2020-02-21 2021-08-26 Braskem S.A. Production of ethanol with one or more co-products in yeast
CN111500486B (zh) * 2020-03-19 2022-04-15 南京工业大学 一株可以利用菊粉作为唯一碳源直接合成丁醇的菌株及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101440381A (zh) * 2008-12-26 2009-05-27 中国科学院广州能源研究所 一种以戊糖和己糖共发酵生产丙酮丁醇的方法
WO2011090985A2 (en) * 2010-01-19 2011-07-28 University Of Delaware Generation of asporogenous solventogenic clostridia

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101440381A (zh) * 2008-12-26 2009-05-27 中国科学院广州能源研究所 一种以戊糖和己糖共发酵生产丙酮丁醇的方法
WO2011090985A2 (en) * 2010-01-19 2011-07-28 University Of Delaware Generation of asporogenous solventogenic clostridia

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KANOUNI, A. E. ET AL.: "Clostridium acetobutylicum Mutants Isolated for Resistance to the Pyruvate Halogen Analogs", CURRENT MICROBIOLOGY, vol. 18, 1989, pages 139 - 144 *
LI, SHUBO ET AL.: "Progress in microbial production of 3-hydroxybutanone", CHINESE JOURNAL OF BIOPROCESS ENGINEERING, vol. 9, no. 6, November 2011 (2011-11-01), pages 63 - 68 *
YUAN, ZHONGDI ET AL.: "Screening of Clostridium acetobutylicum for Fermentation of Corn Fiber Hydrolysate by Complex Mutagenesis", JOURNAL OF ANHUI AGRI. SCI., vol. 39, no. 24, 2011, pages 14778 - 14781 AND 14796 *

Also Published As

Publication number Publication date
US20150093796A1 (en) 2015-04-02
US9249433B2 (en) 2016-02-02

Similar Documents

Publication Publication Date Title
Sun et al. Enhanced ethanol production by Clostridium ragsdalei from syngas by incorporating biochar in the fermentation medium
AU2009258344B2 (en) Production of butanediol by anaerobic microbial fermentation
CA2786751C (en) Alcohol production process
Lamed et al. Effects of stirring and hydrogen on fermentation products of Clostridium thermocellum
Valdez-Vazquez et al. Semi-continuous solid substrate anaerobic reactors for H2 production from organic waste: mesophilic versus thermophilic regime
CN103320335B (zh) 一种丙酮丁醇梭菌及其应用
Zhang et al. Hydrogen production in batch culture of mixed bacteria with sucrose under different iron concentrations
Kundiyana et al. Effect of nutrient limitation and two-stage continuous fermentor design on productivities during “Clostridium ragsdalei” syngas fermentation
Pawar et al. Biohydrogen production from wheat straw hydrolysate using Caldicellulosiruptor saccharolyticus followed by biogas production in a two-step uncoupled process
Kopsahelis et al. Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses
Singla et al. Enrichment and optimization of anaerobic bacterial mixed culture for conversion of syngas to ethanol
Wu et al. Effect of ozone pretreatment on hydrogen production from barley straw
WO2013138998A1 (zh) 一种丙酮丁醇梭菌及其应用
Somda et al. Effect of minerals salts in fermentation process using mango residues as carbon source for bioethanol production
Mitchell et al. Continuous hydrogen and butyric acid fermentation by immobilized Clostridium tyrobutyricum ATCC 25755: Effects of the glucose concentration and hydraulic retention time
Xu et al. Buffering action of acetate on hydrogen production by Ethanoligenens harbinense B49
WO2011078709A1 (en) Alcohol production process
de la Cueva et al. Optimization of biohydrogen production by the novel psychrophilic strain N92 collected from the Antarctica
Kwon et al. Acetate-assisted carbon monoxide fermentation of Clostridium sp. AWRP
Jeon et al. Electrochemical and biochemical analysis of ethanol fermentation of zymomonas mobilis KCCM11336
Du et al. Improved sequential production of hydrogen and caproate by addition of biochar prepared from cornstalk residues
CN102296093A (zh) 一种氧化还原电位调控厌氧发酵生产丁醇的方法
Liu et al. 12, Patent Application Publication o Pub. No.: US 2015/0093796 A1
CN115873906A (zh) 一种垃圾联产氢和丁醇的方法
ROMÃO et al. INFLUENCE OF pH, AMMONIUM AND IRON ON THE HYDROGEN PRODUCTION BY MICROBIAL CONSORTIUM

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12871697

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 14386286

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 12871697

Country of ref document: EP

Kind code of ref document: A1