WO2017054748A1 - 一种拜氏梭菌及其应用和生产丁醇的方法 - Google Patents

一种拜氏梭菌及其应用和生产丁醇的方法 Download PDF

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WO2017054748A1
WO2017054748A1 PCT/CN2016/100764 CN2016100764W WO2017054748A1 WO 2017054748 A1 WO2017054748 A1 WO 2017054748A1 CN 2016100764 W CN2016100764 W CN 2016100764W WO 2017054748 A1 WO2017054748 A1 WO 2017054748A1
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fermentation
butanol
culture
medium
nitrogen source
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PCT/CN2016/100764
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English (en)
French (fr)
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张全
曹长海
高慧鹏
关浩
王领民
乔凯
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中国石油化工股份有限公司
中国石油化工股份有限公司抚顺石油化工研究院
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Priority to KR1020187012328A priority Critical patent/KR102568703B1/ko
Priority to JP2018516840A priority patent/JP6942697B2/ja
Priority to EP16850372.0A priority patent/EP3358001B1/en
Priority to SG11201802596XA priority patent/SG11201802596XA/en
Priority to DK16850372.0T priority patent/DK3358001T3/da
Publication of WO2017054748A1 publication Critical patent/WO2017054748A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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 in particular relates to a butanol-producing Clostridium beijerinckii and an application thereof and a method for producing butanol by using the Clostridium beijerinckii.
  • Butanol is an important organic chemical raw material and has a wide range of uses in the chemical, pharmaceutical and petroleum industries. And because it has two more methylene groups than ethanol, butanol has higher hydrophobicity, lower volatility, can be mixed with gasoline in any ratio, and has a calorific value comparable to gasoline. As a potential renewable bio-energy alternative to gasoline, butanol is receiving increasing attention from countries around the world.
  • the production process of butanol mainly includes two kinds of chemical synthesis methods and microbial fermentation methods.
  • the method of producing butanol by petroleum-based propylene oxo process has been difficult, and due to backward technology, the equipment is too small and the production capacity is insufficient, resulting in insufficient long-term supply of the butanol market in China, which cannot meet the domestic market.
  • the preparation of butanol by microbial fermentation has its unique advantages. The development of biobutanol will greatly alleviate the current shortage of butanol supply.
  • the main products of acelone-butanol fermentation are acetone, butanol and ethanol, and the concentration ratio of the aforementioned products is 3:6:1.
  • the traditional acetone-butanol fermentation generally has the following problems: (1) The traditional acetone-butanol fermentation strain needs to be cultured under strict anaerobic conditions. If the operation is careless, it is easy to enter the air, causing the bacteria to grow normally. Therefore, in the fermentation process, it is usually necessary to pass an inert gas such as N 2 to ensure an oxygen-free environment, and the energy consumption is high; (2) the yield of butanol is low, only about 20% by weight, so that the raw material cost of the butanol fermentation process is high. It restricts the development of butanol fermentation industry; (3) 40% by weight of by-products such as acetone and ethanol in addition to butanol, which increases the difficulty of product separation and improves energy consumption.
  • strains and raw materials have been a bottleneck that plagues the fermentation of butanol.
  • the research focuses on the breeding of strains, the selection of suitable fiber raw materials, the preparation of sugar liquid, the optimization of fermentation process conditions and solvent extraction.
  • the strains currently used in industrial butanol production are mainly Clostridium acetobutylicum and Clostridium beijerii, which have similar metabolic pathways.
  • the products are mainly divided into three categories: 1) solvents (acetone, ethanol and butanol); Organic acids (acetic acid, lactic acid, and butyric acid); 3) gases (including carbon dioxide, hydrogen, etc.).
  • Recovering the hydrogen in the product can further increase the economic competitiveness of ABE (ie, solvent).
  • ABE ie, solvent
  • by-products such as acetone, ethanol, etc. consumes a limited carbon metabolic flux, reduces the proportion of butanol in the product, and increases the difficulty in recovering butanol.
  • Cipheral patent application CN 102162001A provides a method for producing butanol by anaerobic fermentation of Clostridium acetobutylicum, Clostridium acetobutylicum XY16, Clostridium acetobutylicum AS1.134, or acetone butanol
  • Clostridium acetobutylicum AS1.135 is a fermenting strain (strict anaerobic bacteria) with glucose as the substrate and an initial glucose concentration of 60 g/L. In the case of a strict anaerobic environment in which the fermenter is kept under N 2 , the acetone is diced. The pH of the acid-producing period and the alcohol-producing period of Clostridium alcoholicum were regulated.
  • the total solvent and butanol yields were 19.20-19.65g/L and 11.43-12.30g/L, respectively, and the butanol selectivity was 58.2-63.1%.
  • the yield was 32.0-32.8%, and the butanol yield was 19.1-20.5%.
  • the method needs to be fermented under a strict anaerobic environment, and there is a risk that the cells do not grow due to unclean oxygen, and there are more acetone and ethanol by-products in the fermentation product, resulting in a higher pressure of subsequent butanol recovery.
  • the separation energy consumption is higher.
  • Chinese patent application CN 102719371A discloses a butanol-producing Clostridium beijerinckii Y-3, which is a mutant obtained by mutagenesis of the starting strain Clostridium beijerinckii NCIMB8052 with ethyl methanesulfonate (EMS).
  • EMS ethyl methanesulfonate
  • biobutanol can be prepared from xylose residue. When the xylose residue is used as the carbon source, the total solvent yield is 16g/L, the butanol yield is 8.2g/L, and the acetone content is It was 6.8 g/L and the ethanol content was 1.0 g/L.
  • the fermentation process also needs to be carried out under strict anaerobic conditions, and due to the formation of more acetone and ethanol by-products in the fermentation product, there are still defects such as large recovery pressure of the butanol and high energy consumption of separation.
  • the object of the present invention is to overcome the above-mentioned drawbacks in the prior art, to provide a novel butanol-producing Clostridium beijerinckii and an application thereof, and a method for producing butanol by using the Clostridium beijerinckii.
  • the strain can be grown under facultative conditions and fermented to produce butanol, which has the characteristics of high yield and yield of butanol and less by-products of acetone and ethanol.
  • the inventors of the present invention conducted a large number of experiments to obtain a strain which is strong in oxygen resistance when fermentatively produced butanol, can grow under facultative conditions, has high yield and yield of butanol, and simultaneously produces a fermentation product. There is almost no Clostridium beijerii produced by acetone and ethanol by-products.
  • the present invention provides a Clostridium beijerincki, which is deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No. 9124.
  • the invention provides the use of the above Clostridium beijerincii for the production of butanol.
  • the present invention provides a method for producing butanol, which comprises inoculating the above-mentioned Clostridium beijerii in a fermentation medium for fermentation culture to produce butanol.
  • the Clostridium beijieri of the present invention with the accession number CGMCC No. 9124 belongs to a facultative bacterium, and can grow well under the conditions of pH 4-9 and temperature of 20-42 ° C, and produces butanol in fermentation.
  • the oxygen-resistance is strong, no oxygen scavenger is added to the medium, and no N 2 and/or inert gas is required to maintain the anaerobic environment, which reduces the oxygen removal step in the traditional anaerobic fermentation process. It avoids the phenomenon that the bacteria do not grow due to the de-oxygenation and reduces the energy consumption.
  • the fermentation of the C is a facultative bacterium, and can grow well under the conditions of pH 4-9 and temperature of 20-42 ° C, and produces butanol in fermentation.
  • the oxygen-resistance is strong, no oxygen scavenger is added to the medium, and no N 2 and/or inert gas is required to maintain the anaerobic environment, which reduces the
  • the C. beijerincii of the present invention under facultative conditions yields high yield and yield of butanol, and the obtained fermentation product contains almost no acetone and ethanol by-products, thereby reducing the pressure of subsequent butanol recovery and reducing the pressure. Separate energy consumption.
  • the C. beijerinckii can also ferment to produce butanol under anaerobic conditions.
  • the Clostridium beijerinckii of the present invention was deposited on May 4, 2014 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (abbreviated as CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China) Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101), the deposit number is CGMCC No. 9124.
  • the invention provides a Clostridium beijerinckii, the accession number of which is CGMCC No. 9124.
  • Clostridium beijerinckii of the present invention is a Gram-positive bacterium, and the cell morphology is rod-shaped, and spores can be formed.
  • Physiological and biochemical identification was negative for contact enzyme, negative for oxidase, and different for nitrate. The specific physiological and biochemical characteristics are shown in Table 1.
  • the total DNA of the strain of Clostridium beijerincii was subjected to 16S rRNA sequence analysis, and the 16S rDNA sequence thereof was as shown in SEQ ID NO: 1, and the Clostridium beijerinckii was named XH0906 and preserved in Chinese microbial bacteria.
  • the Clostridium beijieri of the present invention belongs to a facultative bacterium (ie, a facultative anaerobic bacterium), and can grow well under the conditions of a pH of 4-9 and a temperature of 20-42 ° C, and is fermented under facultative conditions.
  • the culture has a high yield of butanol, and the fermentation product contains almost no acetone and ethanol; in addition, the C. beijerinckii can also ferment to produce butanol under anaerobic conditions (strict anaerobic conditions).
  • the present invention provides the use of the C. beijerinckii in the production of butanol, in particular The use of fermentative production of butanol under facultative conditions.
  • the present invention provides a method for producing butanol, which comprises: inoculating the C. beijerinckii of the present invention into a fermentation medium for fermentation culture to produce butanol.
  • the fermentation culture may be carried out under anaerobic conditions (strict anaerobic conditions) or under anaerobic conditions, preferably under anaerobic conditions.
  • the embodiment of the facultative condition includes: no oxygen scavenging agent is added to the medium, and N 2 and/or an inert gas is not introduced during the culture to maintain the anaerobic environment.
  • the facultative condition means that in the culture process (including seed culture and fermentation culture), it is not necessary to add an oxygen scavenger such as sodium dithionite (also called a powder) to the culture medium, and it is not necessary.
  • an oxygen scavenger such as sodium dithionite (also called a powder) to the culture medium, and it is not necessary.
  • An anaerobic environment is maintained by introducing N 2 and/or an inert gas or the like.
  • the change of oxygen partial pressure in the culture environment under facultative conditions is as follows: the pressure of the tank in the fermentation process is atmospheric pressure, and the partial pressure of oxygen in the initial state of fermentation in the fermenter is 20% ⁇ 1%, which is generated in large quantities as the fermentation proceeds.
  • the CO 2 and hydrogen, the oxygen partial pressure will drop and can be as low as 0, but as the gas production decreases in the later stage of the fermentation, the oxygen partial pressure will rise again.
  • the conditions for the fermentation culture include a fermentation temperature of 20 to 42 ° C, further preferably 28 to 42 ° C, and still more preferably 32 to 38 ° C.
  • the conditions for the fermentation culture further include: the fermentation time is 48-120 h, and more preferably 60-80 h.
  • the specific fermentation time can be determined according to the specific fermentation conditions.
  • the fermentation culture is carried out under stirring, and the stirring speed is further preferably from 50 to 200 rpm.
  • the conditions for the fermentation culture further include a pH of 4 to 9, more preferably 5 to 7.
  • the initial pH of the fermentation medium is 5.5-8.5, preferably 6-8.
  • the inventors of the present invention have also found in the research that during the fermentation process, as the fermentation progresses, the pH of the fermentation liquid first drops (can be reduced to 4-5) and then rises, and the pH of the fermentation liquid is increased.
  • the initial pH value of the medium is lowered to any of 5.5 to 6.5
  • the pH of the fermentation liquid in this stage from the control time to the end of the fermentation is 5.5 to 6.5, which further increases the yield and yield of butanol.
  • the pH of the fermentation broth is controlled from the moment to the end of the fermentation. 5.5-6.5 (Control within the aforementioned range or control to any value within the range).
  • the fermentation culture conditions include: the fermentation temperature is 28-42 ° C, further preferably 34-38 ° C; the initial pH of the fermentation is natural, and the pH is controlled after the log phase is 4-7, further It is preferably 5-6; the fermentation time is 72-120h.
  • the C. beijerinckii of the present invention can enter the log phase by culturing for 12 to 36 hours under the aforementioned fermentation culture conditions.
  • the logarithmic phase can be judged by periodically detecting the OD 600 value of the fermentation broth (i.e., the absorbance at a wavelength of 600 nm).
  • the logarithmic phase has an OD 600 value of 1-5.
  • the specific process for the fermentation culture is not particularly limited, and as long as the conditions of the aforementioned fermentation culture are satisfied, various fermentation processes commonly used in the art may be used, for example, batch fermentation, batch compensation
  • various fermentation processes commonly used in the art may be used, for example, batch fermentation, batch compensation
  • the specific operation steps of each process are well known to those skilled in the art, and will not be described herein.
  • the method further comprises: sequentially performing strain activation culture and seed culture before performing the fermentation culture.
  • the activated culture is for inoculating the cultured seed in a suitable medium to restore the fermentation performance.
  • Seed culture is to obtain a pure and strong culture, that is, a culture of a fermented strain that is vigorous and inoculated in a sufficient amount.
  • the strain activation culture and the seed culture are not particularly limited, and various culture conditions commonly used in the art may be used.
  • the cultured culture of the present invention is carried out under anaerobic conditions, and the seed culture is carried out under anaerobic conditions.
  • the method of producing butanol comprises:
  • step (3) The seed liquid obtained in the step (2) is inoculated to the fermentation medium at an inoculation amount of 2 to 20% by volume, and fermentation culture is carried out under facultative conditions.
  • the conditions of the specific fermentation culture are described in the foregoing description, and are not described herein again.
  • the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt and a vitamin, and the fermentation medium has an initial pH of 5.5 to 8.5, further preferably 6 to 8.
  • the carbon source is not particularly limited and may be at least one of various carbon sources commonly used in the art, such as sugar, starch, and lignocellulosic raw materials, and preferably, the carbon source is glucose, xylose, galactose, and nectar.
  • the carbon source is glucose and/or xylose, and further preferably glucose.
  • the nitrogen source is not particularly limited and may be various nitrogen sources commonly used in the art.
  • the nitrogen source includes an organic nitrogen source and an inorganic nitrogen source, and the organic nitrogen source is beef paste, yeast extract, peptone, corn syrup and soybean meal hydrolysis.
  • the organic nitrogen source is one or more of soybean meal hydrolysate, peptone and beef extract, and further preferably peptone and Beef cream.
  • the inorganic nitrogen source is one or more of ammonium acetate, sodium nitrate and ammonium sulfate.
  • the inorganic nitrogen source is ammonium sulfate and/or ammonium acetate. Still more preferably ammonium sulfate.
  • the inorganic salt is not particularly limited and may be various inorganic salts commonly used in the art.
  • the inorganic salt is dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sulfuric acid.
  • ferrous iron, iron sulfate, magnesium sulfate, calcium chloride, and manganese sulfate are examples of ferrous iron, iron sulfate, magnesium sulfate, calcium chloride, and manganese sulfate.
  • the introduction of phosphate can play a role in buffering the pH.
  • the vitamin is one or more of vitamin B1, biotin and p-aminobenzoic acid.
  • the carbon source is used in an amount of 20-60 g, more preferably 40-50 g per L of the fermentation medium; the organic nitrogen source is used in an amount of 1-20 g, further preferably 10-18 g; and the amount of the inorganic nitrogen source is used. It is 0.1 to 10 g, more preferably 0.5 to 5 g; the inorganic salt is used in an amount of 0 to 10 g, more preferably 2.5 to 5 g; and the vitamin is used in an amount of 0 to 0.2 g, more preferably 0.04 to 0.12 g.
  • the inventors of the present invention further found through a large number of experiments that the medium includes 40-50 g of glucose, 8-12 g of peptone, 4-8 g of beef extract, and 0.6-1.2 g of ammonium sulfate per L of fermentation medium.
  • the fermentation medium comprises 40-50 g of glucose, 8-12 g of peptone, 4-8 g of beef extract, 0.6-1.2 g of ammonium sulfate, 0.25-0.75 g of sodium chloride, 0.05- 0.2 g of iron sulfate, 0.15-0.45 g of magnesium sulfate, 0.05-0.15 g of calcium chloride, 0.5-4 g of potassium dihydrogen phosphate
  • the seed medium may be the same as the composition of the fermentation medium, and the solid medium may be prepared by adding 1-2% by weight of agar to a liquid medium such as a fermentation medium, but is not limited thereto.
  • a liquid medium such as a fermentation medium
  • the preparation method of the soybean meal hydrolysate comprises: weighing a proper amount of soybean meal, adding 5 times of mass of water, and mixing uniformly. Add concentrated sulfuric acid with a mass concentration of 98% according to 2% of the volume of water, mix quickly and uniformly, and do not carbonize the material locally. Steam is introduced to raise the temperature of the material to 100 ° C for 20 hours. Stir for 5 minutes every 1 hour. After the completion of the hydrolysis, a soy sauce hydrolyzate having a red sauce and a fruity aroma was obtained.
  • the products and by-products in the fermentation broth were analyzed by liquid chromatography to calculate the concentration of the main components: liquid chromatograph (Agilent 1200), the column was Bole HPX-87H (300 mm ⁇ 7.8 mm), mobile phase It is 0.005 mol/L H 2 SO 4 , the flow rate is 0.6 ml/min, the column oven is 65 ° C, the detector is a differential detector (Agilent 1200), the detector temperature is 45 ° C, and the injection volume is 5 ⁇ L.
  • liquid chromatograph Agilent 1200
  • the column was Bole HPX-87H (300 mm ⁇ 7.8 mm)
  • mobile phase It is 0.005 mol/L H 2 SO 4
  • the flow rate is 0.6 ml/min
  • the column oven is 65 ° C
  • the detector is a differential detector (Agilent 1200)
  • the detector temperature is 45 ° C
  • the injection volume is 5 ⁇ L.
  • Butanol yield butanol concentration obtained by fermentation / (pre-fermentation glucose concentration - post-fermentation glucose concentration).
  • This example is intended to illustrate a method for fermentative production of butanol under facultative conditions using Clostridium beijerii by CGMCC No. 9124 of the present invention.
  • Liquid medium (including seed medium and fermentation medium) components: peptone 10g / L, beef extract 6g / L, glucose 45g / L, sodium chloride 0.5g / L, ammonium sulfate 0.9g / L, iron sulfate 0.1g /L, magnesium sulfate 0.3g / L, calcium chloride 0.1g / L, potassium dihydrogen phosphate 1g / L, disodium hydrogen phosphate 2g / L, p-aminobenzoic acid 0.04g / L, vitamin B1 0.04g / L and Biotin 0.004 g/L, pH 7.0, sterilized at 121 ° C for 15 min.
  • the solid medium was added with 1 wt% agar in the liquid medium.
  • the method for producing butanol by the oxy-fermentation of Clostridium beijerinckii comprises: (1) inoculating the C. beijerinckii on a solid medium inclined surface, placing it in an anaerobic environment, and incubating at 30 ° C for 24 hours; (2) Step (1) cultured Clostridium beijerii is scraped from the inclined surface and inserted into the seed culture medium of the shake flask.
  • the pH of the fermentation broth was controlled at 6.0 by the addition of 10 M NaOH solution to the end of the fermentation.
  • the residual glucose concentration in the fermentation broth was 9.67 g/L.
  • the concentration was 0.04 g/L
  • the concentration of ethanol was 0.02 g/L
  • the concentrations of butanol, acetic acid and butyric acid were 10.17 g/L, 0.56 g/L and 0. 42 g / L
  • butanol yield was 28.79%.
  • the difference is that different carbon sources and nitrogen sources are used to replace the glucose, peptone or ammonium sulfate in the medium, and after testing, the acetone concentration in the fermentation liquid is less than 0.1 g/L after 72 hours.
  • the fermentation results of the residual glucose concentration (residual sugar), acetic acid, butyric acid and butanol were all less than 0.1 g/L.
  • the composition of the liquid medium is: peptone 10 g / L, beef extract 6 g / L, glucose 45 g / L, sodium chloride 0.5 g / L, ammonium sulfate 0.9 g / L, ferric sulfate 0.1 g/L, magnesium sulfate 0.3 g/L, calcium chloride 0.1 g/L, potassium dihydrogen phosphate 1 g/L, and disodium hydrogen phosphate 2 g/L, pH 7.0.
  • the residual glucose concentration in the fermentation broth was 10.95g/L after 72h, and the acetone concentration was 0.09 g/L, ethanol concentration was 0.05 g/L, butanol, acetic acid and butyric acid concentrations were 9.33 g/L, 0.55 g/L and 0.47 g/L, respectively, and the yield of butanol was 27.4%.
  • the pH of the whole fermentation process is not controlled.
  • the pH of the fermentation liquid first decreases and then rises, and the pH value during the fermentation is the lowest. Dropped to 4.5.
  • the residual glucose concentration in the fermentation broth was 13.36g/L
  • the acetone concentration was 0.05g/L
  • the ethanol concentration was 0.03g/L
  • the concentrations of butanol, acetic acid and butyric acid were 8.61g/L and 0.496g, respectively.
  • /L and 0.326 g/L the butanol yield was 27.21%.
  • step (3) when the pH of the fermentation broth is lowered from the original pH 7.0 to 5.5, the time is controlled by adding 10 M NaOH solution to the end of the fermentation.
  • the pH of the fermentation broth was 5.5.
  • the residual glucose concentration in the fermentation broth was 9.55 g/L
  • the acetone concentration was 0.05 g/L
  • the ethanol concentration was 0.03 g/L
  • the butanol, acetic acid and butyric acid concentrations were 9.92 g/L and 0.54 g, respectively.
  • /L and 0.32 g / L, butanol yield was 27.98%.
  • step (3) when the pH of the fermentation broth is lowered from the original pH 7.0 to 6.5, the time is controlled by the addition of 10 M NaOH solution to the end of the fermentation.
  • the pH of the fermentation broth was 6.5.
  • the residual glucose concentration in the fermentation broth was 10.41 g/L
  • the acetone concentration was 0.07 g/L
  • the ethanol concentration was 0.04 g/L
  • the butanol, acetic acid and butyric acid concentrations were 9.67 g/L and 0.54 g, respectively.
  • /L and 0.9 g/L the butanol yield was 27.96%.
  • step (3) the pH of the fermentation broth From the initial pH 7.0 to 5, the pH of the fermentation broth was controlled by the addition of 10 M NaOH solution to the end of the fermentation. After 72 hours, the residual glucose concentration in the fermentation broth was 11.15g/L, the acetone concentration was 0.08g/L, the ethanol concentration was 0.04g/L, and the butanol, acetic acid and butyric acid concentrations were 8.54g/L and 0.51g, respectively. /L and 0.23 g/L, the butanol yield was 25.23%.
  • the seed culture is carried out using a fully enclosed anaerobic bottle, the medium is deaerated in an anaerobic tank, and sodium dithionite is added to make the oxygen in the anaerobic bottle.
  • the partial pressure is reduced to 0, and then inoculated and cultured in an anaerobic tank; in the step (3), the fermenter medium is added with sodium dithionite to remove oxygen, and the whole process is passed through N 2 to maintain an oxygen-free environment, and the whole fermentation process does not control the pH. value.
  • the residual glucose concentration in the fermentation broth was 5.0g/L
  • the acetone concentration was 0.21g/L
  • the ethanol concentration was 0.07g/L
  • the concentrations of butanol, acetic acid and butyric acid were 9.98g/L and 1.22g, respectively.
  • /L and 1.46 g/L the butanol yield was 24.95%.
  • Liquid medium components peptone 10g / L, beef extract 6g / L, glucose 40g / L, sodium chloride 0.5g / L, ammonium sulfate 0.9g / L, ferric sulfate 0.1g / L, magnesium sulfate 0.3g / L, Calcium chloride 0.1 g/L, pH 7.0, was sterilized at 121 ° C for 15 min. The solid medium was added with 1 wt% agar in the liquid medium.
  • the method for producing butanol by using the method of CGMCC No. 9124 of the present invention is: (1) inoculating the C. beijerinckii on a solid medium inclined surface and placing it in an anaerobic environment. , cultured at 30 ° C for 24 h; (2) the step (1) cultured Clostridium beijingii scraped from the inclined surface 2 ring into the shake flask seed culture medium, no need to add oxygen scavenger or need to pass N 2 to maintain Anaerobic environment, static culture at 35 ° C for 24 h, to obtain seed liquid; (3) the seed liquid cultured in step (2) was added to the liquid fermentation medium at an inoculation amount of 10% by volume, without adding an oxygen scavenger There is no need to pass N 2 to maintain an anaerobic environment, the pH is natural, and fermentation is carried out at 37 ° C for 88 h.
  • the concentration of butanol in the fermentation broth was 7.5 g/L, no acetone and ethanol products, and the concentrations of acetic acid and butyric acid were 1.0 g/L and 0.6 g/L, respectively.
  • Example 9 According to the method of Example 9, except that different carbon sources and nitrogen sources were used to replace glucose, peptone or ammonium sulfate in the medium, and the fermentation results are shown in Table 3.
  • the fermentation was carried out in a 5 L fermentor, and glucose was used as a substrate, and the initial medium had a glucose concentration of 40 g/L. Fermentation medium components were added to 0.1% potassium dihydrogen phosphate (1 g / L), 0.2% disodium hydrogen phosphate (2 g / L), 0.004% p-aminobenzoic acid (0.04 g / L), 0.004%. Vitamin B1 (0.04 g/L) and 0.0004% biotin (0.004 g/L). The liquid loading was 3L, the stirring rate was 150r/min, and the pH was not controlled at 16h before fermentation, and it naturally decreased. After 16h, the pH was adjusted to 6.0 by adding 10M NaOH solution, and the glucose was completely consumed after 72 hours. The concentrations of butanol, acetic acid and butyric acid in the fermentation broth were determined to be 8.25 g/L, 1.2 g/L and 0.8 g/L, respectively.
  • Example 1 Comparing Example 1 with Example 2, it can be seen that in the process of fermentative production of butanol by the Clostridium beijerii of CGMCC No. 9124 of the present invention under the facultative conditions, the carbon source of the medium is Portuguese.
  • the sugar and organic nitrogen sources are peptone and beef extract, and the inorganic nitrogen source is ammonium sulfate, the yield and yield of butanol can be further improved.
  • Example 1 Comparing Example 1 with Example 3, it can be seen that in the process of fermentative production of butanol under the facultative conditions by the Clostridium beijerii of CGMCC No. 9124 of the present invention, when the medium further contains a specific type of vitamin It can further increase the yield and yield of butanol.
  • Example 1 Comparing Example 1 with Example 4-7, it can be seen that in the process of fermentative production of butanol under the facultative conditions using the C. hyopnium strain of CGMCC No. 9124 of the present invention, the fermentation is carried out as the fermentation proceeds.
  • the pH of the liquid falls to any of 5.5 to 6.5
  • the pH of the fermentation liquid is controlled to be 5.5 to 6.5 at this stage from the time of the end of the fermentation, and the yield and yield of butanol can be further increased.
  • Example 8 From the results of Example 8, it is understood that the fermentation of the butanol under anaerobic conditions using the C. hyopnium strain of the present invention having the accession number CGMCC No. 9124 still has a high yield and yield of butanol.
  • the present invention has the accession number CGMCC No. 9124, Clostridium beijerii, can grow under anaerobic conditions and ferment to produce butanol, has strong oxygen tolerance, and does not need to add an oxygen scavenger in the medium. There is no need to pass an inert gas such as N 2 to maintain the anaerobic environment, reduce the oxygen removal step in the traditional anaerobic fermentation process, avoid the phenomenon of non-growth caused by de-oxygenation, and reduce energy consumption.
  • an inert gas such as N 2
  • the fermentation of the present invention by Clostridium beijerii has a high butanol yield, and the obtained fermentation product has almost no acetone and ethanol by-products, which reduces the pressure of subsequent butanol recovery and reduces the separation energy consumption.

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Abstract

提供了一种拜氏梭菌，其保藏编号为CGMCC No.9124，及其在生产丁醇中的应用和生产丁醇的方法。

Description

一种拜氏梭菌及其应用和生产丁醇的方法 技术领域
本发明属于微生物技术领域，具体地，涉及一种产丁醇的拜氏梭菌(Clostridium beijerinckii)及其应用和一种利用该拜氏梭菌生产丁醇的方法。
背景技术
丁醇是一种重要的有机化工原料，在化工、医药和石油等工业部门有广泛的用途。而且由于比乙醇多两个亚甲基，丁醇具有更高的疏水性，更低的挥发性，可与汽油以任意比例混合，并具有与汽油相当的热值。作为一种有潜力的可以替代汽油的可再生生物能源，丁醇越来越受到世界各国的关注。
丁醇的生产工艺主要有化学合成法和微生物发酵法两种。随着石油资源的日益枯竭，采用以石油为原料丙烯羰基合成法生产丁醇的方法已举步维艰，而且由于技术落后，装置偏小导致产能不够，致使中国丁醇市场长期供应不足，不能满足国内市场的需求。微生物发酵法制备丁醇有其独到的优势，发展生物丁醇将极大地缓解丁醇供应不足的现状。
丙酮-丁醇发酵(acelone-butanol fermentation，ABF)的主要产物是丙酮、丁醇和乙醇，前述产物的浓度比为3:6:1。传统丙酮-丁醇发酵普遍存在以下问题：(1)传统的丙酮-丁醇发酵菌株需在严格厌氧条件下培养发酵，稍有操作不慎，很容易进入空气，造成菌体不能正常生长，因此在发酵过程中通常需要通入惰性气体如N2来保证无氧环境，能耗较高；(2)丁醇产率低，仅20重量％左右，使得丁醇发酵过程原料成本偏高，制约了丁醇发酵产业的发展；(3)发酵产物中除了丁醇外还有40重量％的丙酮和乙醇等 副产物，增加了产品分离难度，提高了能耗。
其中，菌株和原料问题一直是困扰丁醇发酵的瓶颈。近年来，国内外对纤维原料发酵生产丁醇的研究很多，主要围绕菌种诱变选育、寻找合适的纤维原料及其糖液制备、发酵工艺条件优化和溶剂提取等方面进行研究。目前工业上用于丁醇生产的菌株主要是丙酮丁醇梭菌和拜氏梭菌，它们具有相似的代谢路径，产物主要分为3类：1)溶剂(丙酮、乙醇和丁醇)；2)有机酸(乙酸、乳酸和丁酸)；3)气体(包括二氧化碳、氢气等)。将产物中的氢气回收可以进一步提高ABE(即溶剂)的经济竞争力。然而，副产物如丙酮、乙醇等的生成消耗了有限的碳代谢流，降低了产物中丁醇所占的比例，增加了回收丁醇的难度。
中国专利申请CN 102162001A提供了一种利用丙酮丁醇梭菌厌氧发酵产丁醇的方法，以丙酮丁醇梭菌Clostridium acetobutylicum XY16，丙酮丁醇梭菌Clostridium acetobutylicum AS1.134，或者丙酮丁醇梭菌Clostridium acetobutylicum AS1.135为发酵菌株(为严格厌氧菌)，以葡萄糖为底物，初始葡萄糖浓度为60g/L，在通入N2保持发酵罐严格厌氧环境的情况下，对丙酮丁醇梭菌产酸期和产醇期的pH进行调控，总溶剂和丁醇的产量分别为19.20-19.65g/L和11.43-12.30g/L，丁醇选择性为58.2-63.1％，溶剂收率为32.0-32.8％，丁醇收率为19.1-20.5％。但是，该方法需在严格厌氧环境下进行发酵，存在因除氧不干净造成菌体不生长的风险，且发酵产物中有较多的丙酮和乙醇副产物，导致后续丁醇回收压力较大，分离能耗较高。
中国专利申请CN 102719371A公开了一株产丁醇的拜氏梭菌Clostridium beijerinckii Y-3，该菌是通过对出发菌株Clostridium beijerinckii NCIMB8052采用甲基磺酸乙酯(EMS)诱变获得的突变株(为严格厌氧菌)，可以木糖渣为原料制备生物丁醇，在以木糖渣酶解液为碳源时，总溶剂产量为16g/L，丁醇产量为8.2g/L，丙酮含量为6.8g/L，乙醇含量为1.0g/L。该菌株虽解决了传统生物发酵生产丁醇菌种能力和原料不足的问题，但该 发酵过程同样需在严格厌氧环境下进行，且因发酵产物中有较多的丙酮和乙醇副产物生成，仍存在后续丁醇回收压力较大、分离能耗较高等缺陷。
发明内容
本发明的目的是为了克服现有技术中的上述缺陷，提供一种新的产丁醇的拜氏梭菌(Clostridium beijerinckii)及其应用和一种利用该拜氏梭菌生产丁醇的方法，该菌株可在兼氧条件下生长并发酵生产丁醇，具有丁醇产量和收率高，丙酮和乙醇副产物少等特点。
为了实现上述目的，本发明的发明人进行了大量的实验，得到一株在发酵生产丁醇时，耐氧能力强，能够在兼氧条件下生长，丁醇产量和收率高，同时发酵产物中几乎无丙酮和乙醇副产物生成的拜氏梭菌。
因此，第一方面，本发明提供了一种拜氏梭菌(Clostridium beijerinckii)，所述拜氏梭菌保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.9124。
第二方面，本发明提供了上述拜氏梭菌在生产丁醇中的应用。
第三方面，本发明提供了一种生产丁醇的方法，所述方法包括：将上述拜氏梭菌接种至发酵培养基中进行发酵培养以生产丁醇。
本发明的保藏编号为CGMCC No.9124的拜氏梭菌，属于兼氧细菌，在pH值为4-9，温度为20-42℃的条件下均能良好生长，且在发酵产丁醇的过程中，耐氧能力强，培养基中不需要加入除氧剂，全程也无需通入N2和/或惰性气体等来保持厌氧环境，减少了传统厌氧菌发酵过程中的除氧步骤，避免了因除氧不干净造成的菌体不生长现象，降低了能耗。另外，利用本发明的拜氏梭菌在兼氧条件下进行发酵，丁醇产量和收率高，且得到的发酵产物中几乎无丙酮和乙醇副产物，减轻了后续丁醇回收的压力，降低了分离能耗。同时，该拜氏梭菌也可以在厌氧条件下发酵生产丁醇。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说 明。
生物保藏
本发明的拜氏梭菌(Clostridium beijerinckii)于2014年5月4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(缩写为CGMCC，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，邮政编码：100101)，保藏编号为CGMCC No.9124。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，
这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
第一方面，本发明提供了一种拜氏梭菌(Clostridium beijerinckii)，该拜氏梭菌的保藏编号为CGMCC No.9124。
本发明所述的拜氏梭菌为革兰氏阳性菌，细胞形态为杆状，可以形成芽孢。经生理生化鉴定为接触酶阴性，氧化酶阴性，不同化硝酸盐。具体的生理生化特性如表1所示。
表1
Figure PCTCN2016100764-appb-000001
Figure PCTCN2016100764-appb-000002
注：“+”表示阳性反应或能利用；“－”表示阴性反应或不能利用。
对该拜氏梭菌提取菌株总DNA，进行16S rRNA序列分析，其16S rDNA序列如SEQ ID NO：1所示，将该拜氏梭菌(Clostridium beijerinckii)命名为XH0906，并保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.9124。
本发明的拜氏梭菌，属于兼氧细菌(即兼性厌氧菌)，在pH值为4-9，温度为20-42℃的条件下均能良好生长，在兼氧条件下进行发酵培养，丁醇产率高，且发酵产物中几乎不含丙酮和乙醇；另外，该拜氏梭菌也可以在厌氧条件(严格厌氧条件)下发酵生产丁醇。
第二方面，本发明提供了所述拜氏梭菌在生产丁醇中的应用，特别是 在兼氧条件下发酵生产丁醇的应用。
第三方面，本发明提供了一种生产丁醇的方法，该方法包括：将本发明所述的拜氏梭菌接种至发酵培养基中进行发酵培养以生产丁醇。
本发明的生产丁醇的方法中，所述发酵培养可以在厌氧条件(严格厌氧条件)下进行，也可以在兼氧条件下进行，优选在兼氧条件下进行。
本发明的生产丁醇的方法中，优选情况下，兼氧条件的实施方式包括：在培养基中不加入除氧剂，培养过程中也不通入N2和/或惰性气体以保持厌氧环境。也就是说，本发明中，兼氧条件是指在培养过程(包括种子培养和发酵培养)中，不需要向培养基中加入连二亚硫酸钠(也称保险粉)等除氧剂，也不需要通入N2和/或惰性气体等来保持厌氧环境。一般地，兼氧条件下培养环境的氧分压的变化情况如下：发酵过程罐压为大气压，发酵罐中发酵初始状态时的氧分压为20％±1％，随着发酵的进行产生大量的CO2和氢气，氧分压会下降并可低至0，但随着发酵后期气体产量减少，氧分压会再次上升。
本发明的生产丁醇的方法中，优选情况下，发酵培养的条件包括：发酵温度为20-42℃，进一步优选为28-42℃，更进一步优选为32-38℃。
优选情况下，发酵培养的条件还包括：发酵时间为48-120h，进一步优选为60-80h。具体的发酵时间可以根据具体的发酵条件进行确定。
优选情况下，发酵培养在搅拌下进行，搅拌速度进一步优选为50-200rpm。
优选情况下，发酵培养的条件还包括：pH为4-9，进一步优选为5-7。其中，发酵培养基的初始pH值为5.5-8.5，优选为6-8。进一步地，本发明的发明人在研究中还发现，在发酵过程中，随着发酵的进行，发酵液的pH值会先下降(可降至4-5)后上升，待发酵液的pH值从培养基初始pH值降至5.5-6.5中的任一值时，控制该时刻至发酵结束这一阶段内发酵液的pH值为5.5-6.5，能够进一步提高丁醇的产量和收率。因此，为了进一步提高 丁醇的产量和收率，优选情况下，在发酵过程中，待发酵液的pH值降至5.5-6.5中的任一值时，控制该时刻至发酵结束这一阶段内发酵液的pH为5.5-6.5(控制在前述范围内或控制为该范围内的任一值)。
根据本发明的一种优选实施方式，发酵培养的条件包括：发酵温度为28-42℃，进一步优选为34-38℃；发酵初始阶段pH自然，对数期后控制pH为4-7，进一步优选为5-6；发酵时间为72-120h。一般地，在前述发酵培养条件下培养12-36h，本发明的拜氏梭菌能够进入对数期。其中，对数期可以通过定期检测发酵液的OD600值(即在600nm波长处的吸光值)进行判断，一般地，对数期的OD600值为1-5。
本发明的生产丁醇的方法中，对于发酵培养的具体工艺没有特别的限定，只要满足前述发酵培养的条件，可以为本领域常用的各种发酵工艺，例如可以为批次发酵、批次补料发酵、连续发酵、原位萃取发酵或气体原位抽提发酵等，各工艺的具体操作步骤为本领域技术人员所熟知，在此不再赘述。
本发明的生产丁醇的方法中，该方法还包括：在进行发酵培养之前，依次进行菌种活化培养和种子培养。活化培养是为了将保藏状态的菌种接种至适宜的培养基中培养，使其恢复发酵性能。种子培养是为了得到纯而壮的培养物，即获得活力旺盛、接种数量足够的发酵菌种的培养物。其中，对于菌种活化培养和种子培养没有特别的限定，可以分别为本领域常用的各种培养条件。优选地，本发明的菌种活化培养在厌氧条件下进行，种子培养在兼氧条件下进行。
优选地，生产丁醇的方法包括：
(1)将拜氏梭菌接种至固体培养基，在厌氧条件下，28-42℃培养12-48h；
(2)将步骤(1)培养的拜氏梭菌接种至种子培养基，在兼氧条件下，28-42℃静置培养12-48h，得到种子液；
(3)将步骤(2)得到的种子液以2-20体积％的接种量接种至发酵培养基，在兼氧条件下进行发酵培养。具体的发酵培养的条件参见前述说明，在此不再赘述。
本发明的生产丁醇的方法中，优选情况下，发酵培养基包括碳源、氮源、无机盐和维生素，所述发酵培养基的初始pH为5.5-8.5，进一步优选为6-8。
对于碳源没有特别的限定，可以为本领域常用的各种碳源，如糖、淀粉和木质纤维素原料等中的至少一种，优选地，碳源为葡萄糖、木糖、半乳糖、甘露糖、果糖、麦芽糖、纤维二糖、低聚葡萄糖和低聚木糖中的一种或多种。为了进一步提高丁醇的产量和收率，进一步优选地，碳源为葡萄糖和/或木糖，更进一步优选为葡萄糖。
对于氮源没有特别的限定，可以为本领域常用的各种氮源，优选地，氮源包括有机氮源和无机氮源，有机氮源为牛肉膏、酵母膏、蛋白胨、玉米浆和豆粕水解液中的一种或多种，为了进一步提高丁醇的产量和收率，进一步优选地，有机氮源为豆粕水解液、蛋白胨和牛肉膏中的一种或多种，更进一步优选为蛋白胨和牛肉膏。优选地，无机氮源为醋酸铵、硝酸钠和硫酸铵中的一种或多种，为了进一步提高丁醇的产量和收率，进一步优选地，无机氮源为硫酸铵和/或醋酸铵，更进一步优选为硫酸铵。
对于无机盐没有特别的限定，可以为本领域常用的各种无机盐，优选地，无机盐为磷酸氢二钾、磷酸二氢钾、磷酸氢二钠、磷酸二氢钠、氯化钠、硫酸亚铁、硫酸铁、硫酸镁、氯化钙和硫酸锰中的一种或多种。其中，磷酸盐的引入可以起到缓冲pH值的作用。
优选情况下，维生素为维生素B1、生物素和对氨基苯甲酸中的一种或多种。
优选地，以每L发酵培养基计，碳源的用量为20-60g，进一步优选为40-50g；有机氮源的用量为1-20g，进一步优选为10-18g；无机氮源的用量 为0.1-10g，进一步优选为0.5-5g；无机盐的用量为0-10g，进一步优选为2.5-5g；维生素的用量为0-0.2g，进一步优选为0.04-0.12g。
本发明的发明人经过大量的实验进一步发现，当以每L发酵培养基计，培养基包括40-50g的葡萄糖、8-12g的蛋白胨、4-8g的牛肉膏、0.6-1.2g的硫酸铵、0.25-0.75g的氯化钠、0.05-0.2g的硫酸铁、0.15-0.45g的硫酸镁、0.05-0.15g的氯化钙、0.5-4g的磷酸二氢钾、1-4g的磷酸氢二钠、0.01-0.06g的对氨基苯甲酸、0.01-0.06g的维生素B1和0.001-0.006g的生物素时，能够为了进一步提高丁醇的产量和收率，因此，优选情况下，以每L发酵培养基计，所述发酵培养基包括40-50g的葡萄糖、8-12g的蛋白胨、4-8g的牛肉膏、0.6-1.2g的硫酸铵、0.25-0.75g的氯化钠、0.05-0.2g的硫酸铁、0.15-0.45g的硫酸镁、0.05-0.15g的氯化钙、0.5-4g的磷酸二氢钾、1-4g的磷酸氢二钠、0.01-0.06g的对氨基苯甲酸、0.01-0.06g的维生素B1和0.001-0.006g的生物素。
本发明的生产丁醇的方法中，种子培养基可以和发酵培养基的成分相同，固体培养基可以通过在液体培养基如发酵培养基中加入1-2wt％的琼脂制得，然而并不限于此，本领域技术人员能够理解的是：其它常规可用于活化培养和种子培养的培养基也在本发明的范围内。
实施例
以下的实施例将对本发明作进一步的说明，但并不因此限制本发明。
以下实施例中的实验方法，如无特殊说明，均为本领域常规方法。下述实施例中所用的实验材料，如无特殊说明，均从常规生化试剂商店购买得到。
豆粕水解液的制备方法包括：称取适量豆粕，加入5倍质量的水，混合均匀。按水量体积的2％加入质量浓度为98％的浓硫酸，迅速混合均匀，勿使物料局部碳化。通入蒸汽，使物料温度升至100℃，保温20小时，其 间，每隔1小时搅拌5分钟。水解结束后，得到呈酱红色、具果香味的豆粕水解液。
通过液相色谱仪对发酵液中的产物和副产物进行分析，以计算其中主要成分的浓度：液相色谱仪(安捷伦1200)，色谱柱为伯乐HPX-87H(300mm×7.8mm)，流动相为0.005mol/L H2SO4，流速为0.6ml/min，柱温箱为65℃，检测器为示差检测器(安捷伦1200)，检测器温度为45℃，进样量为5μL。
丁醇收率＝发酵得到的丁醇浓度/(发酵前葡萄糖浓度-发酵后葡萄糖浓度)。
实施例1
本实施例用于说明利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌在兼氧条件下发酵生产丁醇的方法。
液体培养基(包括种子培养基和发酵培养基)成分：蛋白胨10g/L、牛肉膏6g/L、葡萄糖45g/L、氯化钠0.5g/L、硫酸铵0.9g/L、硫酸铁0.1g/L、硫酸镁0.3g/L、氯化钙0.1g/L、磷酸二氢钾1g/L、磷酸氢二钠2g/L、对氨基苯甲酸0.04g/L、维生素B1 0.04g/L和生物素0.004g/L，pH 7.0，在121℃下灭菌15min。固体培养基在液体培养基中加入1wt％的琼脂。
利用该拜氏梭菌兼氧发酵生产丁醇的方法包括：(1)将所述拜氏梭菌接种在固体培养基斜面上，置于厌氧环境中，30℃培养24h；(2)将步骤(1)培养的拜氏梭菌从斜面上刮下2环接入置于摇瓶的种子培养基中，不需要加入除氧剂也不需要通入N2保持厌氧环境，35℃静置培养24h，得到种子液；(3)将步骤(2)培养的种子液以10体积％的接种量接种到置于5L发酵罐的3L液体发酵培养基中，不需要加入除氧剂也不需要通入N2保持厌氧环境，在37℃进行发酵培养，搅拌速率为150r/min，随着发酵的进行，发酵液的pH值会先下降后上升，待发酵液的pH值由原始pH 7.0降至6.0 时，通过流加10M的NaOH溶液控制该时刻至发酵结束这一阶段内发酵液的pH值为6.0，经检测，72h后发酵液中残余葡萄糖浓度为9.67g/L，丙酮浓度为0.04g/L，乙醇浓度为0.02g/L，丁醇、乙酸和丁酸浓度分别为10.17g/L、0.56g/L和0.42g/L，丁醇收率为28.79％。
实施例2
按照实施例1的方法，不同的是，分别采用不同的碳源和氮源替换培养基中的葡萄糖、蛋白胨或硫酸铵，经检测，72h后发酵液中丙酮浓度均小于0.1g/L，乙醇浓度均小于0.1g/L，残余葡萄糖浓度(残糖)、乙酸、丁酸和丁醇的发酵结果见表2。
表2
Figure PCTCN2016100764-appb-000003
实施例3
按照实施例1的方法，不同的是，液体培养基成分为：蛋白胨10g/L、牛肉膏6g/L、葡萄糖45g/L、氯化钠0.5g/L、硫酸铵0.9g/L、硫酸铁0.1g/L、硫酸镁0.3g/L、氯化钙0.1g/L、磷酸二氢钾1g/L和磷酸氢二钠2g/L，pH7.0。
经检测，72h后发酵液中残余葡萄糖浓度为10.95g/L，丙酮浓度为 0.09g/L，乙醇浓度为0.05g/L，丁醇、乙酸和丁酸浓度分别为9.33g/L、0.55g/L和0.47g/L，丁醇收率为27.4％。
实施例4
按照实施例1的方法，不同的是，在步骤(3)中，整个发酵过程均不控制pH值，随着发酵的进行，发酵液的pH值会先下降后上升，发酵过程中pH值最低降至4.5。经检测，72h后发酵液中残余葡萄糖浓度为13.36g/L，丙酮浓度为0.05g/L，乙醇浓度为0.03g/L，丁醇、乙酸和丁酸浓度分别为8.61g/L、0.496g/L和0.326g/L，丁醇收率为27.21％。
实施例5
按照实施例1的方法，不同的是，在步骤(3)中，待发酵液的pH值由原始pH 7.0降至5.5时，通过流加10M的NaOH溶液控制该时刻至发酵结束这一阶段内发酵液的pH值为5.5。经检测，72h后发酵液中残余葡萄糖浓度为9.55g/L，丙酮浓度为0.05g/L，乙醇浓度为0.03g/L，丁醇、乙酸和丁酸浓度分别为9.92g/L、0.54g/L和0.32g/L，丁醇收率为27.98％。
实施例6
按照实施例1的方法，不同的是，在步骤(3)中，待发酵液的pH值由原始pH 7.0降至6.5时，通过流加10M的NaOH溶液控制该时刻至发酵结束这一阶段内发酵液的pH值为6.5。经检测，72h后发酵液中残余葡萄糖浓度为10.41g/L，丙酮浓度为0.07g/L，乙醇浓度为0.04g/L，丁醇、乙酸和丁酸浓度分别为9.67g/L、0.54g/L和0.9g/L，丁醇收率为27.96％。
实施例7
按照实施例1的方法，不同的是，在步骤(3)中，待发酵液的pH值 由原始pH 7.0降至5时，通过流加10M的NaOH溶液控制该时刻至发酵结束这一阶段内发酵液的pH值为5。经检测，72h后发酵液中残余葡萄糖浓度为11.15g/L，丙酮浓度为0.08g/L，乙醇浓度为0.04g/L，丁醇、乙酸和丁酸浓度分别为8.54g/L、0.51g/L和0.23g/L，丁醇收率为25.23％。
实施例8
按照实施例1的方法，不同的是，在步骤(2)中种子培养时采用全封闭的厌氧瓶，培养基在厌氧箱中除氧，并加入连二亚硫酸钠使厌氧瓶中的氧分压降低到0，然后在厌氧箱中接种和培养；步骤(3)中发酵罐培养基加入连二亚硫酸钠除氧，并全程通入N2保持无氧环境，整个发酵过程均不控制pH值。经检测，72h后发酵液中残余葡萄糖浓度为5.0g/L，丙酮浓度为0.21g/L，乙醇浓度为0.07g/L，丁醇、乙酸和丁酸浓度分别为9.98g/L、1.22g/L和1.46g/L，丁醇收率为24.95％。
实施例9
液体培养基成分：蛋白胨10g/L、牛肉膏6g/L、葡萄糖40g/L、氯化钠0.5g/L、硫酸铵0.9g/L、硫酸铁0.1g/L、硫酸镁0.3g/L、氯化钙0.1g/L，pH 7.0，在121℃下灭菌15min。固体培养基在液体培养基中加入1wt％的琼脂。
利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌兼氧发酵生产丁醇的方法包括：(1)将所述拜氏梭菌接种在固体培养基斜面上，置于厌氧环境中，30℃培养24h；(2)将步骤(1)培养的拜氏梭菌从斜面上刮下2环接入摇瓶种子培养基中，不需要加入除氧剂也不需要通入N2保持厌氧环境，35℃静置培养24h，得到种子液；(3)将步骤(2)培养的种子液以10体积％的接种量接入到液体发酵培养基中，不需要加入除氧剂也不需要通入N2保持厌氧环境，pH自然，37℃发酵88h。发酵88h后，发酵液中的 丁醇浓度为7.5g/L，无丙酮和乙醇产物，乙酸和丁酸浓度分别为1.0g/L和0.6g/L。
实施例10
按照实施例9的方法，不同的是，分别采用不同的碳源和氮源替换培养基中的葡萄糖、蛋白胨或硫酸铵，发酵结果如表3所示。
表3
Figure PCTCN2016100764-appb-000004
实施例11
利用5L发酵罐发酵，以葡萄糖为底物，初始培养基中葡萄糖浓度为40g/L。发酵培养基成分在实施例9基础上加入0.1％磷酸二氢钾(1g/L)、0.2％磷酸氢二钠(2g/L)、0.004％对氨基苯甲酸(0.04g/L)、0.004％维生素B1(0.04g/L)和0.0004％生物素(0.004g/L)。装液量为3L，搅拌速率为150r/min，发酵前16h不控制pH，任其自然下降，16h后通过流加10M的NaOH溶液调节pH为6.0，72h后葡萄糖完全消耗。经检测，发酵液中的丁醇、乙酸和丁酸浓度分别为8.25g/L、1.2g/L和0.8g/L。
将实施例1与实施例2比较可知，利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌在兼氧条件下发酵生产丁醇的过程中，培养基的碳源为葡 萄糖、有机氮源为蛋白胨和牛肉膏、无机氮源为硫酸铵时，能够进一步提高丁醇的产量和收率。
将实施例1与实施例3比较可知，利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌在兼氧条件下发酵生产丁醇的过程中，培养基中进一步含有特定种类的维生素时，能够进一步提高丁醇的产量和收率。
将实施例1与实施例4-7比较可知，利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌在兼氧条件下发酵生产丁醇的过程中，随着发酵的进行，待发酵液的pH值降至5.5-6.5中的任一值时，控制该时刻至发酵结束这一阶段内发酵液的pH为5.5-6.5，能够进一步提高丁醇的产量和收率。
从实施例8的结果可知，利用本发明的保藏编号为CGMCC No.9124的拜氏梭菌在厌氧条件下发酵生产丁醇，仍具有较高的丁醇的产量和收率。
综上所述，本发明的保藏编号为CGMCC No.9124的拜氏梭菌，可在兼氧条件下生长并发酵生产丁醇，耐氧能力强，培养基中不需要加入除氧剂，全程也无需通入N2等惰性气体来保持厌氧环境，减少了传统厌氧菌发酵过程中的除氧步骤，避免了因除氧不干净造成的菌体不生长现象，降低了能耗。另外，利用本发明的拜氏梭菌进行发酵，丁醇产率高，且得到的发酵产物中几乎无丙酮和乙醇副产物，减轻了后续丁醇回收的压力，降低了分离能耗。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (11)

  1. 一种拜氏梭菌(Clostridium beijerinckii)，该拜氏梭菌的保藏编号为CGMCC No.9124。
  2. 权利要求1所述的拜氏梭菌在生产丁醇中的应用。
  3. 一种生产丁醇的方法，其特征在于，该方法包括：将权利要求1所述的拜氏梭菌接种至发酵培养基中进行发酵培养以生产丁醇。
  4. 根据权利要求3所述的方法，其中，所述发酵培养在兼氧条件下进行。
  5. 根据权利要求3或4所述的方法，其中，所述发酵培养的条件包括：发酵温度为20-42℃，优选为28-42℃；发酵时间为48-120h，优选为60-80h；pH为4-9，优选为5-7。
  6. 根据权利要求5所述的方法，其中，发酵培养基的初始pH值为5.5-8.5，优选为6-8；在发酵过程中，待发酵液的pH值降至5.5-6.5中的任一值时，控制该时刻至发酵结束这一阶段内发酵液的pH为5.5-6.5。
  7. 根据权利要求3-6中任意一项所述的方法，其中，所述发酵培养基包括碳源、氮源、无机盐和维生素，所述发酵培养基的初始pH值为5.5-8.5，优选为6-8。
  8. 根据权利要求7所述的方法，其中，所述碳源为葡萄糖、木糖、半乳糖、甘露糖、果糖、麦芽糖、纤维二糖、低聚葡萄糖和低聚木糖中的一种 或多种，优选为葡萄糖和/或木糖；
    所述氮源包括有机氮源和无机氮源，所述有机氮源为牛肉膏、酵母膏、蛋白胨、玉米浆和豆粕水解液中的一种或多种，优选为蛋白胨和牛肉膏；所述无机氮源为醋酸铵、硝酸钠和硫酸铵中的一种或多种，优选为硫酸铵；
    所述无机盐为磷酸氢二钾、磷酸二氢钾、磷酸氢二钠、磷酸二氢钠、氯化钠、硫酸亚铁、硫酸铁、硫酸镁、氯化钙和硫酸锰中的一种或多种；
    所述维生素为维生素B1、生物素和对氨基苯甲酸中的一种或多种；
    优选地，以每L发酵培养基计，所述碳源的用量为20-60g，进一步优选为40-50g；所述有机氮源的用量为1-20g，进一步优选为10-18g；所述无机氮源的用量为0.1-10g，进一步优选为0.5-5g；所述无机盐的用量为0-10g，进一步优选为2.5-5g；所述维生素的用量为0-0.2g，进一步优选为0.04-0.12g。
  9. 根据权利要求8所述的方法，其中，以每L发酵培养基计，所述发酵培养基包括40-50g的葡萄糖、8-12g的蛋白胨、4-8g的牛肉膏、0.6-1.2g的硫酸铵、0.25-0.75g的氯化钠、0.05-0.2g的硫酸铁、0.15-0.45g的硫酸镁、0.05-0.15g的氯化钙、0.5-4g的磷酸二氢钾、1-4g的磷酸氢二钠、0.01-0.06g的对氨基苯甲酸、0.01-0.06g的维生素B1和0.001-0.006g的生物素。
  10. 根据权利要求3-9中任意一项所述的方法，其中，该方法还包括：在进行发酵培养之前，依次进行菌种活化培养和种子培养，
    优选地，生产丁醇的方法包括：
    (1)将所述拜氏梭菌接种至固体培养基，在厌氧条件下，28-42℃培养12-48h；
    (2)将步骤(1)培养的拜氏梭菌接种至种子培养基，在兼氧条件下，28-42℃静置培养12-48h，得到种子液；
    (3)将步骤(2)得到的种子液以2-20体积％的接种量接种至发酵培养基，在兼氧条件下进行所述发酵培养。
  11. 根据权利要求4或10所述的方法，其中，所述兼氧条件的实施方式包括：在培养基中不加入除氧剂，培养过程中也不通入N2和/或惰性气体以保持厌氧环境。
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