WO2006133637A1 - Procédé de préparation du 1,3-propanediol et du 2,3-butanediol à partir de matériau d'amidon brut - Google Patents

Procédé de préparation du 1,3-propanediol et du 2,3-butanediol à partir de matériau d'amidon brut Download PDF

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WO2006133637A1
WO2006133637A1 PCT/CN2006/001291 CN2006001291W WO2006133637A1 WO 2006133637 A1 WO2006133637 A1 WO 2006133637A1 CN 2006001291 W CN2006001291 W CN 2006001291W WO 2006133637 A1 WO2006133637 A1 WO 2006133637A1
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fermentation
medium
seed
crude
propanediol
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PCT/CN2006/001291
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English (en)
French (fr)
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Dehua Liu
Keke Cheng
Hongjuan Liu
Rihui Lin
Jian Hao
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Tsinghua University
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Priority to AT06752933T priority Critical patent/ATE538207T1/de
Priority to AU2006257543A priority patent/AU2006257543B2/en
Priority to US11/917,682 priority patent/US7968319B2/en
Priority to EP06752933A priority patent/EP1897955B1/en
Priority to BRPI0613714-8A priority patent/BRPI0613714A2/pt
Publication of WO2006133637A1 publication Critical patent/WO2006133637A1/zh

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the present invention relates to a process for producing 1, 3-propanediol and 2,3-butanediol, and more particularly to a process for producing 1, 3-propanediol and 2,3-butanediol from a raw material of a crude starch.
  • 1,3-propanediol is an important chemical raw material that can be used as an organic solvent in inks, printing and dyeing, coatings, lubricants, antifreeze and other industries.
  • 3-propanediol is as a monomer for the synthesis of polyester and polyurethane, especially polybutylene terephthalate (PTT) formed by polymerization with terephthalic acid, showing a ratio of 1,2- Propylene glycol, butanediol, and ethylene glycol are superior properties of polymers synthesized from monomers.
  • PTT polybutylene terephthalate
  • PTT polyethylene terephthalate
  • PTT fiber has the advantages of abrasion resistance, low water absorption and low static electricity, which can compete with nylon in the carpet field. It can also be used for non-woven fabrics, engineering plastics, clothing, home décor, linings, fabrics, etc. with excellent properties.
  • PTT was named one of the six major petrochemical products in the United States in 1998 and is considered to be an upgraded product of PET.
  • 2, 3-butanediol A by-product of the production of 1,3-propanediol as a fermentation process is also an important chemical raw material. It is a colorless, odorless liquid that can be used as a fuel to prepare polymers, inks, perfumes, antifreezes, fumigants, moisturizers, softeners, plasticizers, explosives, and chiral carriers for pharmaceuticals. 2, 3-butanediol can also be used as a valuable chemical raw material to synthesize other chemicals, such as 2, 3-butanediol dehydration can produce methyl ethyl ketone, A The application of ethyl ketone is quite extensive, and further dehydration can form 1,3-butadiene.
  • 3-butanediol can be polymerized by Diels-Alder reaction to form styrene.
  • 3-butanediol is condensed with methyl ethyl ketone and hydrogenated to form octenant, which can be used to produce high quality flying materials.
  • 3-butanediol reacts with acetic acid to form 2,3-butanediol diacetate, which can be added to the cream to improve flavor.
  • 1, 3-propanediol it is generally not isolated and purified as a product.
  • the production methods of 1, 3-propanediol mainly include chemical methods and biological methods. Compared with chemical synthesis methods, the production of 1, 3-propanediol by microbial fermentation has mild production conditions, good selectivity, few by-products, and easy separation and purification. Significant advantages such as no environmental pollution are getting more and more attention.
  • Anaerobic bacteria such as Klebsiella are fermentatively produced under anaerobic conditions to produce 1, 3-propanediol (Ruch et al. Regulation of glycerol catabolisra in Klebsiella aerogenes. J Bacteriol. 1974, 119(1): 50-56; Streekstra et al. Overflow metabolism during anaeric growth of Klebsiella pneumoniae NCTC418 on glycerol and dihydroxyacetone in chemostat culture. Arch Microbiol. 1987, 147 : 268-275 ; Zeng et al. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: Regulation of reducing equivalent balance And product formation. Enzyme
  • Microbial two-stage fermentation process produces 1,3-propanediol and 2,3-butanediol from glycerol (Andy Lau et al., Patent Application No.: 200410037692. 3) 6.
  • Method for producing 1, 3-propanediol by two-step microbial fermentation Xiu Zhilong et al., Chinese Patent No.: ZL01138769. 6)
  • Method 4 proposed simultaneous fermentation of 1, 3-propanediol and 2,3-butanediol, but the fermentation level was very low due to process conditions.
  • Method 5 adopts a new process of aerobic aerobic period in the early stage, which greatly increases the concentration of 1,3-propanediol and 2,3-butanediol in the fermentation products, which reduces the production cost to some extent, but because of its fermentation, Glycerin is a substrate, so the cost is still high.
  • the glycerin concentration in the preferred embodiment is only 49.9 g/L.
  • the glycerol concentration in the preferred embodiment is only 49.9 g/L. 8% ⁇
  • the molar yield is only 39.1%, 1, 3-propanediol concentration is also very low, only 13.18g / L, the molar yield is only 22.8%.
  • the method for producing 1, 3-propanediol and 2,3-butanediol from a raw material of crude starch comprises the following steps:
  • the yeast cells removed by filtration in the step 1) can be directly recovered for fermentation in the next batch; the cell recycling can save the seed culture time of the next batch of fermentation.
  • the Candida krusei or Arabidopsis angustifolia is derived from a primary seed or a secondary seed; the primary seed is prepared as follows - picking Candida krusei or arabinose Into a species containing a crude starch saccharification solution
  • the sub-culture medium is obtained by incubating for 18-20 hours at 30-35 ° C, a shake flask having a volume of 1/5 volume, a rotation radius of 25, and a rotation speed of 200-250 rpm.
  • the second-stage seed is prepared according to the following method: the first-stage seed is connected to the fermenter of the seed culture medium with the crude saccharification solution as the carbon source, and the aeration speed is 300-500 rpm at 30-35 ° C. 2 ⁇ 0. 5vvm, cultured for 5 ⁇ 7h.
  • the fermentation medium with the crude mashing solution as a carbon source has a pH of 4-5, and further contains corn paddle and urea; the content of the crude mashing solution is to treat all the reducing sugar in the crude mashing solution as glucose. calculating the glucose in the medium content of 260- 350 g / L; corn steep liquor is the content of 2- 3 g / L; the amount of urea 2. 5- 4 g / L;
  • the seed medium containing the crude mashing solution has a pH of 4-5, and further contains corn syrup and urea; the content of the crude mashing solution is calculated by using all the reducing sugar in the crude mashing solution as glucose.
  • the glucose content in the medium reaches 80-100 g/L; the content of the corn paddle is 2-3 g/L; and the content of the urea is 2-3 g/Lo.
  • the crude starch in the step 1) may be a starch raw material such as sweet potato powder, corn flour or tapioca powder; and the crude starch saccharified solution has a DE value of 90-110.
  • the crude mashing solution can be prepared according to the following method:
  • the starch syrup is prepared by using the crude starch and water in a mass ratio of 1:1800-2000, and the liquefying enzyme is added twice at 80-85 Torr and 90-95 ⁇ , respectively.
  • 3-5U/g crude starch liquefy for 40-50 minutes, then warm to 110 ⁇ 120 °C to inactivate the enzyme, cool, add saccharification enzyme 150 ⁇ 200U/g starch, saccharification at 50 ⁇ 60 °C for 8 ⁇ 12 hours, A crude mashing solution having a DE value of 90-110 was obtained.
  • Klebsiella, Clostridium butyricum or Clostridium baumannii are derived from primary or secondary seeds; the primary seed is prepared as follows - Klebsiella, Clostridium butyricum or Bath
  • the seed culture medium prepared by the glycerol fermentation broth obtained in the step 1) is placed at a culture temperature of 30-33 ° C, a volume of 1/5 volume of the shake flask, a radius of rotation of 25 ⁇ , a shaker
  • the first-stage seed is obtained by culturing at a rotation speed of 130-150 rpm and aerobic conditions for 18-20 hours; the first-stage seed is prepared according to the following method: the first-stage seed is inserted into the seed culture containing the glycerin fermentation liquid obtained in the step 1) In the fermenter of the base, at a temperature of 30-33 ° C, a stirring speed of 60 to: L50 rpm, aeration of 0. 2 ⁇ 0. 5vvm, the culture is carried out for 5 to 10 hours to obtain
  • the pH of the fermentation medium in which the glycerin fermentation liquid obtained in the step 1) is a carbon source is 6.8-8.0, the content of the glycerin fermentation liquid is glycerin content in the glycerol to reach 20-80 g / L ; the glycerol fermentation broth as a carbon source also contains ⁇ 2 ⁇ 0 4 ⁇ 3H 2 0 2.225 - 3.5g/L, (N3 ⁇ 4) 2 S0 4 2.0— 4.0g/L, KH 2 P0 4 0.65-1.2g/ MgS0 4 ⁇ 73 ⁇ 40 0.1-0.2g/L, yeast powder 1 1.5g/L, trace elements Solution 2—3IIIL/L and defoaming agent 0. lmL / L; the composition of the trace element solution is ZnCl 2 70mg / L, MnCl 2 ⁇ 4H 2 0
  • PH glycerol broth seed medium in said step 1) is obtained as a carbon source is 6.8-8.0, the content of the glycerol to glycerol fermentation broth was calculated that the content of glycerol in the medium 20g / L; glycerol fermentation
  • the medium in which the liquid is a carbon source further contains ⁇ 2 ⁇ 0 4 ⁇ 3H 2 0 4.45- 5.6 g/L, (NH 4 ) 2 S0 4 2.0 - 4.0 g/L, KH 2 P0 4 1.3 - 2.6 g/L, MgS0 4 ⁇ 7H 2 0 0.1 — 0.2g/L, yeast powder 1.0—2.0 g/L, CaC0 3 1.0—2.0 g/L, trace element solution 2-3 mL/L; the composition of the trace element solution is ZnCl 2 70mg/L, MnCl 2 ⁇ 4H 2 0 100mg/L, 3 ⁇ 4B0 3 60mg/L, CoCl
  • the fermentation temperature in the fermentation process of the step 1) is 30-35 ⁇ ;
  • the aerobic condition in the step 1) is that the air is ventilated during the fermentation, and the aeration amount is 0.5-2v Vm (L/L/min, the ratio of the volume of air introduced into the fermentor per minute to the volume of the fermentation broth in the fermenter);
  • the anaerobic condition in the step 1) is that nitrogen is passed through the fermentation process, and the aeration amount is 0.2-2vvm.
  • the glycerin fermentation liquid obtained in the step 1) is further added to maintain the glycerin content in the medium at 20-80 g/L.
  • the fermentation process of the step 2) is supplemented with two nitrogen sources, and yeast powder and ammonium sulfate are added in an amount of 0.8 g of yeast powder/L medium and lg ammonium sulfate/L medium each time.
  • the pH of the fermentation in the step 2) is 6.8-8.0, and the fermentation temperature is 30-37 °C.
  • the method further comprises the steps of purifying the 1, 3-propanediol and 2,3-butanediol, that is, collecting the fermentation liquid, filtering and removing the bacteria, collecting the filtrate for desalting, distillation, and vacuum distillation.
  • the starch milk was placed in a ratio of 1475 g of corn crude starch and 2722 L of water, heated, and liquefied by adding liquefied enzyme (5 U/g starch) to the mixture at 50 ° C and 95 ° C for 50 min, respectively, and then warmed to 110 ⁇ to inactivate the enzyme. .
  • the sugar liquid glucose concentration was 31.55%
  • Candida krusei 2. 1048 (purchased from the Institute of Microbiology, Chinese Academy of Sciences).
  • Inclined medium (g/L): glucose (prepared with the saccharification solution of step 1) 200; corn syrup 3; urea 3; agar 20;
  • Seed medium (g/L): glucose (prepared with the saccharification solution of step 1) 100 ; corn syrup 3 ; urea 3 ;
  • Fermentation medium (g/L): glucose (prepared with the saccharification solution of step 1) 315; corn syrup 3; urea 2. 5.
  • the pH of the above medium was adjusted to 4 - 4. 5, and sterilized at 110 Torr for 15 minutes before use.
  • Candida krusei 2.1048 was placed in a slant medium and cultured for 35 hours at 35 , for strain activation.
  • the activated Candida krusei 2.1048 was added to the seed medium containing the crude saccharification solution, and the first-stage seed was cultured in a shake flask (500 mL flask, liquid volume 100 mL), 35 ° C, 200 rpm (rotation) A radius of 25 galls) was incubated for 20 h to obtain a first-grade seed.
  • the first-stage seed is added to the fermenter containing the seed culture medium containing the crude mashing solution to culture the second-stage seed, and the agitation speed is 3, 2 ⁇ 0. 5vvm, and the culture is 5 ⁇ 7h at 35 ° C, stirring speed 300 ⁇ 500 rpm.
  • Fermentation is carried out in any of the following ways A, B and C:
  • A. Fermentation is carried out using a 5L fermentor and primary seed. The first stage seed is added to the fermentation medium in a volume ratio of 10%, and is fermented in a 5L fermenter, the air is ventilated for the first 60 hours, the aeration is 2.0 vvm, after 60 hours, the nitrogen is ventilated, the aeration is 0. 5vvm, the stirring speed is 500 rpm, After fermentation for 70 h, the fermentation temperature during the whole fermentation was 30 °C. 4% ⁇ The glycerol yield was 165 g / L, the concentration of residual sugar was 5g / L, the yield of glycerol to glucose was 52.4%.
  • the secondary seed is connected to the fermentation medium at a volume ratio of 10%, fermented in a 500L fermenter, and the air is ventilated for the first 60 hours.
  • Fermentation is carried out using a 75000L fermentor and secondary seeds.
  • the second stage seed is added to the fermentation medium in a volume ratio of 10%, and is fermented in a 75000L fermenter, the air is ventilated for the first 60 hours, the aeration is 0. 5vvm, after 60h, the nitrogen is ventilated, the aeration is 0. 2vvm, the stirring speed is 300rpm, After fermentation for 72 h, the fermentation temperature during the whole fermentation was 35 °C.
  • the glycerol yield was determined to be 158 g/L, the residual sugar was 5 g/L, and the yield of glycerol to glucose was 53.3%.
  • the pH of the above medium is 6.8-8. 0.
  • Klebsiella 1.117 was added to the seed medium prepared with the glycerol fermentation broth of step 2 (500 mL flask, liquid volume lOOmL), culture temperature 30 ° C, shaker speed 150 rpm (with a radius of 25). 0 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2. 0. 5vvm culture for 5 to 10 hours to obtain secondary seeds.
  • Fermentation culture - Fermentation is carried out by any of the following methods C, D and E.
  • a and B are controls: A. 5L fermentor, culture temperature 37 ° C, pH from hydroxide Potassium was adjusted to 6.8.
  • the seed liquid was placed in a fermentation medium (glycerin concentration: 50 g/L) prepared by using the glycerin fermentation liquid of the second step, and no glycerin fermentation liquid was added during the fermentation.
  • the fermenter was rotated at 150 rpm. 5 ⁇ 5. 0vvm nitrogen. Fermentation for 30 h, 1, 3-propanediol concentration of 21 g / L, 2, 3-butanediol concentration of 2 ⁇ a few.
  • the pH was adjusted to 6.8 by potassium hydroxide, the pH was adjusted to 6.8.
  • the seed liquid is connected to the fermentation medium (glycerol concentration 80 g/L) prepared by using the glycerin fermentation liquid of the step 2, No glycerol fermentation broth was added during the fermentation.
  • the fermenter was rotated at 150 rpm. Access to 0. 5vvra air. 5 ⁇ / /
  • the concentration of 1,3 - propylene glycol is 35g / L
  • the concentration of 2,3 - butanediol is 6. 6 ⁇ /
  • the pH was adjusted to 6.8 by potassium hydroxide.
  • the first-stage seed liquid is connected to the fermentation medium (glycerin concentration: 30 g/L) prepared by the glycerin fermentation liquid of the second step, and the glycerin fermentation liquid is added during the fermentation to control the flow rate to maintain the glycerin concentration in the fermentation liquid at 30 g/L. .
  • the fermenter was rotated at 150 rpm. 5vvm ⁇ The gas was introduced into the air, the ventilation was 0. 5vvm. Nitrogen was added twice during the fermentation period at 16h and 30h (the yeast powder and ammonium sulfate were added in an amount of 0.8 g of yeast powder/L medium and lg ammonium sulfate/L medium).
  • 64h Fermentation is over.
  • the fermentation broth was collected, the cells were removed by filtration, and the filtrate was subjected to desalting, distillation, and vacuum distillation to obtain the product 1, 3-propanediol and 2,3-butanediol.
  • the second seed liquid is connected to the fermentation medium (glycerol concentration 30g / L) prepared by the glycerin fermentation liquid of step 2, the fermentation tank speed is 60 rpm, and the aeration amount is 0.3 vvm.
  • the glycerol fermentation broth was added during the fermentation, and the flow rate was controlled to maintain the glycerol concentration in the fermentation broth at 30 g/L.
  • Other control conditions are the same as 5L cans.
  • the concentration of 1,3-propanediol in the fermentation broth is 72 g/L
  • the concentration of 2,3-butanediol is 25.4 g/L
  • the molar yield of 1, 3-propanediol is 55.38 %
  • the total diol yield is 71. . 85 %.
  • the secondary 500L seed solution is placed in the initial fermentation medium containing the glycerol fermentation broth (glycerol concentration 30g/L) as described above.
  • Glycerol fermentation broth was added during the fermentation to control the flow rate to maintain the glycerol concentration in the fermentation broth at 30 g/L.
  • the concentration of 1,3-propanediol in the fermentation broth is 66.6 g/L
  • the concentration of 2,3-butanediol is 30. 4 g/L
  • the molar yield of 1, 3-propanediol is 59.1%. 2% ⁇
  • Cell reuse The cells obtained by filtering the glycerol fermentation broth are directly used for the next batch of glycerol fermentation.
  • the fermentation conditions are the same as those of the first batch. After reuse of more than 10 batches, the cell viability remains basically unchanged, and the glycerol concentration is basically stable.
  • Medium - slant medium glucose (prepared with the saccharification solution of step 1) 200; corn syrup 2; urea 2; agar 20;
  • Seed medium (g/L): glucose (prepared with the saccharification solution of step 1) 100; corn syrup 2; urea 2;
  • Fermentation medium (g/L): glucose (prepared with the saccharification solution of step 1) 268; corn syrup 2; urea 4.
  • the pH of the above medium was adjusted to 4 - 4. 5, and sterilized at 110 ° C for 15 minutes before use.
  • the activated arabinose Hansenula 2.887 was added to the seed medium containing the crude saccharification solution, and the first-stage seed was cultured in a shake flask (500 mL flask, liquid volume 100 mL), 30 ° C, 200 rpm (rotation) A radius of 25 let) culture for 20 h to obtain a first-grade seed.
  • the seed is added to the fermenter of the seed medium containing the crude mashing solution to culture the second seed, 30 ° C, stirring speed 300 ⁇ 500 rpm ventilation 0. 2 ⁇ 0. 5vvm, culture 5 ⁇ 7h.
  • Fermentation is carried out using a 5L fermentor and primary seed.
  • the first stage seed is added to the fermentation medium in a volume ratio of 10%, and is fermented in a 5L fermenter, the air is ventilated for the first 60 hours, the aeration is 2.0 vvm, after 60 hours, the nitrogen is ventilated, the aeration is 0. 5vvm, the stirring speed is 500 rpm, After fermentation for 70 h, the fermentation temperature during the whole fermentation was 30 °C.
  • the glycerol yield was measured and the results showed that the glycerol concentration was 140 g/L.
  • the residual sugar concentration is 4. 8g / L
  • the yield of glycerol to glucose is 53%
  • the secondary seed is connected to the fermentation medium at a volume ratio of 10%, fermented in a 500L fermenter, and the air is ventilated for the first 60 hours.
  • Fermentation is carried out using a 75000L fermentor and secondary seeds.
  • the second stage seed is connected to the fermentation medium in a volume ratio of 10%, and is fermented in a 75000L fermenter.
  • the air is ventilated for the first 60 hours, and the aeration is 0. 5 60h, then the nitrogen is supplied, the aeration is 0. 2vvm, the stirring speed is 300rpm, and the fermentation is performed.
  • 72h The fermentation temperature during the whole fermentation was 35 °C.
  • the glycerol yield was determined to be 142 g/L, the residual sugar was 4 g/L, and the yield of glycerol to glucose was 54.1%.
  • the glycerin fermentation broth of the previous step was filtered to remove Hansenula sinensis 2. 887 cells, and the cells obtained by filtration were directly used for the next batch of glycerol fermentation, and the fermentation conditions were the same as those of the first batch. The filtrate obtained was used for the fermentation of 1 3-propanediol and 2 3-butanediol.
  • the pH of the above medium is 6.8-8. 0.
  • Seed culture Connect Clostridium pasteurianuw 1. 208 into the seed medium prepared with the glycerol fermentation broth of step 2 (500 mL flask, liquid volume 100 mL), culture temperature 33 ° C, shaker speed 130 rpm (rotation) The radius is 25mm). 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2. 0. 5vvm culture for 5 to 10 hours to obtain secondary seeds.
  • the pH was adjusted to 6.8 by potassium hydroxide.
  • the seed liquid was placed in a fermentation medium (glycerin concentration: 50 g/L) prepared by using the glycerin fermentation liquid of the second step, and no glycerin fermentation liquid was added during the fermentation.
  • the fermenter was rotated at 150 rpm. 5 ⁇ 5. 0vvm nitrogen.
  • the fermentation was carried out for 30 h, the concentration of 1,3-propanediol was 24 g/L, and the concentration of 2,3-butanediol was 1. 7 ⁇ .
  • the pH was adjusted from potassium hydroxide to 6.8.
  • the seed solution was placed in a fermentation medium (glycerol concentration: 80 g/L) prepared by using the glycerin fermentation liquid of the second step, and no glycerin fermentation liquid was added during the fermentation.
  • the fermenter was rotated at 150 rpm. Access to 0. 5v V ra air. Fermentation for 30h, 1, 3-propanediol concentration of 38g / L, 2,3-butanediol concentration of 5. 6 ⁇ / ⁇
  • the pH was adjusted to 6.8 by potassium hydroxide.
  • the first-stage seed liquid is connected to the fermentation medium (glycerol concentration: 30 g/L) prepared by the glycerin fermentation liquid of the second step, and the glycerin fermentation liquid is added during the fermentation to control the flow rate to maintain the glycerin concentration in the fermentation liquid.
  • the fermenter was rotated at 150 rpm. 5vvm ⁇ The gas was introduced into the air, the ventilation was 0. 5vvm. Nitrogen supplementation was carried out twice at 16 and 30 h during the fermentation (your yeast powder and ammonium sulfate were added in an amount of 0.8 g of yeast powder/L medium and lg ammonium sulfate/L medium).
  • the molar number is a percentage of the moles of glycerol consumed), the total diol molar yield is 70% (the percentage of moles of 1, 3-propanediol and 2,3-butanediol to the moles of glycerol consumed).
  • the ventilating rate is 0. 3vvm.
  • the fermenter is rotated at 60 rpm, and the aeration is 0. 3vvm.
  • the glycerol fermentation broth was added during the fermentation, and the flow rate was controlled to maintain the glycerol concentration in the fermentation broth at 30 g/L. Other control conditions are the same as 5L cans.
  • the concentration of 1,3-propanediol in the fermentation broth was 54 g/L
  • the concentration of 2,3-butanediol was 22 ⁇
  • the yield of 1, 3-propanediol was 55 %
  • the yield of total diol was 73%.
  • the secondary 500L seed solution is placed in the initial fermentation medium containing the glycerol fermentation broth (glycerol concentration 30g/L) as described above.
  • Glycerol fermentation broth was added during the fermentation to control the flow rate to maintain the glycerol concentration in the fermentation broth at 30 g/L.
  • the concentration of 1, 3-propanediol in the fermentation broth at the end of the 500Lo fermentation is 57. 6g / L, 2, 3 - butanediol concentration 27. 3g / L, 1, 3-propanediol molar yield 56%, total two 2 ⁇
  • the molar yield of alcohol was 76.2%.
  • Cell reuse The cells obtained by filtering the glycerol fermentation broth are directly used for the next batch of glycerol fermentation.
  • the fermentation conditions are the same as those of the first batch. After reuse of more than 10 batches, the cell viability remains basically unchanged, and the glycerol concentration is basically stable.
  • the method of the present invention can significantly increase the concentration and yield of glycerol and 1, 3-propanediol in the production of 1, 3-propanediol by the two-step fermentation process, and also obtain high added value of 1, 3-propanediol and 2 , 3-butanediol, which effectively improves the utilization of raw materials and reduces production costs.
  • the method of the invention has good effects on 5L, 500L, 5000L fermenters, wherein the concentration of glycerol obtained by fermentation can reach 158 - 179g / L, and the concentration of 1, 3-propanediol obtained by fermentation can reach 66-72g. /L, 2, 3 monobutanediol concentration 16 - 30. 4g / L.

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Description

一种由粗淀粉原料生产 1, 3-丙二醇和 2, 3-丁二醇的方法 技术领域
本发明涉及一种生产 1, 3-丙二醇和 2, 3-丁二醇的方法, 特别涉及一种 由粗淀粉原料生产 1, 3-丙二醇和 2, 3-丁二醇的方法。
背景技术
1,3-丙二醇 (PD0)是一种重要的化工原料, 可作为有机溶剂应用于 油墨、 印染、 涂料、 润滑剂、 抗冻剂等行业。 1, 3-丙二醇最主要的用途 是作为聚酯和聚氨酯合成的单体, 特别是与对苯二甲酸聚合生成的聚对 苯二甲酸丙二酯 (PTT) , 显示了比以 1,2-丙二醇、 丁二醇、 乙二醇为 单体合成的聚合物更优良的性能。 目前全球每年消费数千万吨聚对苯二 甲酸乙二酯(PET), 而 PTT的化学稳定性、 生物可降解性等与 PET相当, 但耐污染性、 韧性和回弹性及抗紫外性能等更优越。 此外 PTT纤维还具 有耐磨、 吸水性低、 低静电等优点, 可在地毯领域与尼龙竞争。 它还可 用于具有优良性能的无纺布、 工程塑料、 服装、 家庭装饰、 垫衬料、 织 物等方面。 PTT被评为美国 98年六大石化新产品之一, 被认为将是 PET 的升级产品。
PTT的优越性能及市场潜力早在 50年前就被人们所认识, 只因原料 1, 3-丙二醇生产技术难度大、成本高而导致 PTT很难大规模工业化生产, 迄今为止,只有 Dupont和 Shell两家跨国公司采用传统的化学合成路线, 以环氧乙烷或丙烯为原料生产仅供它们合成 PTT自用的 1,3-丙二醇。 化 学合成法的缺点是副产物多, 选择性差, 操作条件需高温高压, 设备投 资巨大, 原料为不可再生资源, 且环氧乙烷和另一路线的中间产物丙烯 醛分别是易燃易爆或剧毒的危险品。 由于发酵法生产 1, 3-丙二醇选择性 高, 操作条件温和, 因此近年来受到特别的重视。
2, 3-丁二醇 ( 2, 3-butanediol ) 作为发酵法生产 1, 3-丙二醇的一种 副产物也是一种重要的化工原料。 它是一种无色无味液体, 可作为燃料, 可用来制备聚合物、 油墨、 香水、 防冻剂、 熏蒸剂、 增湿剂、 软化剂、 增塑剂、 炸药以及药物的手性载体等。 2, 3-丁二醇还可作为一个很有价 值的化工原料来合成其他化学品, 如 2, 3-丁二醇脱水可产生甲乙酮, 甲 乙酮的应用相当广泛, 再进一步脱水可形成 1, 3-丁二烯。 2, 3-丁二醇 可通过 Diels— Alder反应聚合生成苯乙烯。 2, 3-丁二醇与甲乙酮缩合并 进行加氢反应生成辛垸, 辛烷可用来产生高质量的飞行原料。 2, 3-丁二 醇与乙酸反应生成 2, 3-丁二醇二乙酸酯, 此酯类可添加到奶油中改善风 味。但由于其在 1, 3-丙二醇发酵中产量较低,一般不作为产物进行分离、 提纯。
目前, 1, 3-丙二醇的生产方法主要有化学法和生物法, 与化学合成 法相比, 微生物发酵法生产 1, 3-丙二醇具有生产条件温和、 选择性好、 副产物少、 易于分离纯化、 无环境污染等显著优点越来越受到重视。
目前, 由生物法生产 1, 3-丙二醇主要有以下几条途径:
1. 采用肠道细菌厌氧条件下将甘油歧化为 1, 3-丙二醇(USP5254467, EP0373230 A1 ) 。
2. 克雷伯氏杆菌等厌氧菌在厌氧条件下发酵生产 1, 3-丙二醇(Ruch et al. Regulation of glycerol catabolisra in Klebsiella aerogenes. J Bacteriol. 1974, 119 (1) : 50-56 ; Streekstra et al. Overflow metabolism during anaeric growth of Klebsiella pneumoniae NCTC418 on glycerol and dihydroxyacetone in chemostat culture. Arch Microbiol. 1987, 147 : 268-275 ; Zeng et al. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: Regulation of reducing equivalent balance and product formation. Enzyme
Microbiol Technol. 1993, 15 : 770-779. ) 。
3. 采用克雷伯氏杆菌在微氧条件下发酵生产 1,3_丙二醇(王剑锋 等, 克雷伯氏菌微氧发酵生产 1, 3-丙二醇的研究, 现代化工, 2001, 21
( 5 ) : 28— 31。 修志龙等, 一种微生物微氧发酵生产 1, 3-丙二醇的方 法, 中国专利公开号: CN1348007)
4. 采用克雷伯氏杆菌在厌氧条件下发酵产 1, 3-丙二醇和 2, 3-丁二 醇 ( Biebl et al. Fermentation of glycerol to 1, 3- propanediol and 2, 3- butanediol. Appl Microbiol Biotechnol, 1998, 50 : 24-29 ) .
5. 微生物两段发酵法由甘油生产 1, 3-丙二醇和 2, 3-丁二醇 (刘德 华等, 专利申请号: 200410037692. 3 ) 6. 两步微生物发酵生产 1, 3-丙二醇的方法 (修志龙等, 中国专利 号: ZL01138769. 6)
上述方法中 1一 3均是采用甘油为底物发酵产单一的 1, 3-丙二醇产 品, 且发酵液中 1, 3-丙二醇浓度很低, 因此生产成本很高。 方法 4提出 了同时发酵产 1, 3-丙二醇和 2, 3-丁二醇, 但由于工艺条件限制, 其发酵 水平很低。 方法 5采用了前期厌氧后期有氧的新工艺, 大大提高了发酵 产品 1, 3-丙二醇和 2, 3-丁二醇的浓度, 从一定程度上降低了生产成本, 但由于其发酵仍以甘油为底物, 因此成本依然较高。 方法 6提出了两步 发酵法由淀粉等原料生产 1, 3-丙二醇的方法, 理论上可大大降低生产成 本, 但由于其工艺限制, 其最佳实施例中甘油浓度仅有 49. 9g/L, 摩尔 产率仅为 39. 1%, 1, 3-丙二醇浓度也很低, 仅为 13. 18g/L, 摩尔产率仅 为 22. 8% .
发明公开
本发明的目的是提供一种由粗淀粉原料生产 1, 3-丙二醇和 2, 3-丁二 醇的方法。
本发明所提供的由粗淀粉原料生产 1, 3-丙二醇和 2, 3-丁二醇的方法, 包括以下步骤:
1 )将克鲁氏假丝酵母或阿拉伯糖醇汉逊酵母接入以粗淀粉糖化液为 碳源的发酵培养基中,采用先有氧后厌氧的工艺制备甘油, 即在有氧条件 下培养至葡萄糖消耗速率明显降低后进行厌氧发酵至糖浓度为 5- 10g/L, 收集发酵液,过滤除去发酵液中的酵母细胞,得到的滤液即为甘油发酵液;
2) 将克雷伯氏菌、 丁酸梭菌或巴斯德梭菌接入以步骤 1 ) 得到的甘 油发酵液为碳源的发酵培养基中,厌氧发酵 30- 32小时, 至 1, 3-丙二醇生 产强度明显降低时进行有氧发酵至甘油浓度降至 10g/L以下结束发酵, 得 到 1, 3-丙二醇和 2, 3-丁二醇。
其中,所述步骤 1 )中过滤除去的酵母细胞可直接回收用于下个批次 的发酵; 细胞回用可节省下一批次发酵的种子培养时间。
所述克鲁氏假丝酵母或阿拉伯糖醇汉逊酵母来自于一级种子或二级 种子; 所述一级种子按照以下方法制备- 将克鲁氏假丝酵母或阿拉伯糖醇汉逊酵母接入含粗淀粉糖化液的种 子培养基, 在 30— 35°C、 装液量为 1/5体积的摇瓶、 旋转半径为 25讓、 转速为 200— 250rpm的条件下, 培养 18— 20小时得到的。 所述二级种子 按照以下方法制备:将一级种子接入以粗淀粉糖化液为碳源的种子培养基 的发酵罐中, 在 30— 35 °C、 搅拌转速 300〜500 rpm通气量 0. 2〜0. 5vvm, 培养 5〜7h。
所述以粗淀粉糖化液为碳源的发酵培养基的 pH为 4- 5,还含有玉米桨 和尿素; 所述粗淀粉糖化液的含量为将粗淀粉糖化液中还原性糖全部当作 葡萄糖计算, 使培养基中葡萄糖含量达到 260- 350 g/L; 所述玉米浆的含 量为 2— 3 g/L; 所述尿素的含量为 2. 5— 4 g/L;
所述含粗淀粉糖化液的种子培养基的 pH为 4-5, 还含有玉米浆和尿 素; 所述粗淀粉糖化液的含量为将粗淀粉糖化液中还原性糖全部当作葡萄 糖计算, 使培养基中葡萄糖含量达到 80— 100 g/L; 所述玉米桨的含量为 2- 3 g/L; 所述尿素的含量为 2— 3 g/Lo
所述步骤 1 ) 中所述粗淀粉可为红薯粉、 玉米粉或木薯粉等淀粉原 料; 所述粗淀粉糖化液的 DE值为 90-110。
所述粗淀粉糖化液可按照下述方法制备: 用粗淀粉与水按照 1 : 1800-2000的质量比配置淀粉乳, 分别在 80-85 Ό和 90 - 95 Ό两次加入 液化酶,每次 3-5U/g 粗淀粉,液化 40-50分钟,然后升温至 110〜120°C 使酶失活, 冷却, 加入糖化酶 150〜200U/g淀粉, 50〜60 °C糖化 8〜12 小时, 得到 DE值为 90-110的粗淀粉糖化液。
克雷伯氏菌、 丁酸梭菌或巴斯德梭菌来自于一级种子或二级种子; 所述一级种子按照以下方法制备- 将克雷伯氏杆菌、 丁酸梭菌或巴斯德梭菌接入以步骤 1 ) 得到的甘油 发酵液配制的种子培养基中, 在培养温度 30— 33°C, 装液量为 1/5体积的 摇瓶、 旋转半径为 25匪、 摇床转速 130— 150rpm、 好氧条件下培养 18— 20 小时得到一级种子; 所述一级种子按照以下方法制备: 将一级种子接入装 有以步骤 1 ) 得到的甘油发酵液配制的种子培养基的发酵罐中, 在 30— 33°C、 搅拌转速 60〜: L50rpm、 通气量 0. 2〜0. 5vvm的条件下, 培养 5〜10 小时得到二级种子。
所述以步骤 1 ) 得到的甘油发酵液为碳源的发酵培养基的 PH为 6.8-8.0, 所述甘油发酵液的含量为以甘油计算使培养基中甘油含量达到 20-80g/L; 以甘油发酵液为碳源的培养基中还含有 Κ2ΗΡ04 · 3H20 2.225- 3.5g/L、 (N¾)2S04 2.0— 4.0g/L、 KH2P04 0.65-1.2g/ MgS04 · 7¾0 0.1-0.2g/L, 酵母粉 1一 1.5g/L、 微量元素溶液 2— 3IIIL/L和消泡剂 0. lmL/L; 所述微量元素溶液的组成为 ZnCl2 70mg/L、 MnCl2 · 4H20
100mg/L、 H3B03 60mg/L、 CoCl2 · 6H20 200mg/L、 NiCl2 · 6H20 25 mg/L、 NiCl2 · H20 27.64 mg/L、 Na2Mo04 · 2H20 35 mg/L、 CuCl2 · H20 20 mg/L、 CuS04 · 5H20 29.28 mg/L和浓盐酸 0.9mL/L;
所述以步骤 1) 得到的甘油发酵液为碳源的种子培养基的 PH为 6.8-8.0, 所述甘油发酵液的含量为以甘油计算使培养基中甘油含量达到 20g/L; 以甘油发酵液为碳源的培养基中还含有 Κ2ΗΡ04 · 3H20 4.45- 5.6g/L、 (NH4)2S04 2.0 - 4.0g/L、 KH2P04 1.3— 2.6g/L、MgS04 · 7H20 0.1 — 0.2g/L、 酵母粉 1.0— 2.0 g/L、 CaC03 1.0— 2.0g/L、 微量元素溶液 2-3 mL/L; 所述微量元素溶液的组成为 ZnCl2 70mg/L、 MnCl2 · 4H20 100mg/L、 ¾B03 60mg/L、 CoCl2 · 6H20 200mg/L、 NiCl2 · 6H20 25 mg/L, NiCl2 · ¾0 27.64 mg/L, Na2Mo04 · 2¾0 35 mg/L, CuCl2 · ¾0 20 mg/L、 CuS04 · 5H20 29.28 mg/L和浓盐酸 0.9mL/L。
所述步骤 1) 的发酵过程中的发酵温度为 30-35Ό;
所述步骤 1) 中有氧条件是发酵过程中通空气, 通气量为 0.5- 2vVm (L/L/min, 每分钟通入发酵罐的空气体积与发酵罐中发酵液体积的比 值) ; 所述步骤 1) 中厌氧条件是发酵过程中通氮气, 通气量为 0.2- 2vvm
(L/L/min, 每分钟通入发酵罐的氮气体积与发酵罐中发酵液体积的比 值) 。
所述步骤 2) 的发酵过程中还流加所述步骤 1) 得到的甘油发酵液, 使培养基中的甘油含量保持在 20- 80g/L。
所述步骤 2) 的发酵过程中还补充两次氮源, 每次按 0.8g酵母粉 /L 培养基和 lg硫酸铵 /L培养基的量加入酵母粉和硫酸铵。
所述步骤 2) 的发酵过程中 pH为 6.8- 8.0, 发酵温度为 30- 37°C。 所述方法中还包括纯化 1, 3-丙二醇和 2, 3-丁二醇的步骤, 即收集发 酵液, 过滤除去菌体, 收集滤液进行脱盐、 蒸馏、 真空精馏。 实施发明的最佳方式
下述实验方法, 如无特别说明, 均为常规方法。
实施例 1、 由玉米粗淀粉生产 1, 3-丙二醇和 2, 3-丁二醇
1、 粗淀粉原料的液化1糖化
以玉米粗淀粉 1475g, 水 2722L的比例配置淀粉乳,加热, 分别在温 度升至 80°C和 95°C两次加入液化酶 (5U/g 淀粉) 液化 50min, 然后升 温至 110Ό使酶失活。 冷却, 加入糖化酶 (200U/g淀粉) 6CTC糖化 9h, 测定结果表明该糖化液的葡萄糖值 (DE值, 糖化液中还原性糖全部当作 葡萄糖计算, 占干物质的百分比称为 DE值) 103. 46, 糖液葡萄糖浓度为 31. 55%
2、 糖化液发酵产甘油
( 1 ) 菌种: 克鲁氏假丝酵母 2. 1048, (购自中科院微生物所) 。
( 2) 培养基:
斜面培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 200 ; 玉米 浆 3 ; 尿素 3 ; 琼脂 20;
种子培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 100 ; 玉米浆 3 ; 尿素 3 ;
发酵培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 315 ; 玉米 浆 3 ; 尿素 2. 5。
以上培养基 pH均调为 4-4. 5, 使用前均于 110Ό灭菌 15分钟。
( 3 ) 种子培养
将克鲁氏假丝酵母 2. 1048接入斜面培养基, 35Ό培养 24h,进行菌种 活化。
将活化的克鲁氏假丝酵母 2. 1048接入含粗淀粉糖化液的种子培养基, 在摇瓶 (500mL三角瓶, 装液量 lOOmL) 中培养一级种子, 35°C、 200rpm (旋转半径为 25膽) 培养 20h, 得到一级种子。
将一级种子接入含粗淀粉糖化液的种子培养基的发酵罐培养二级种 子, 35°C、 搅拌转速 300〜500 rpm通气量 0, 2〜0. 5vvm,培养 5〜7h。
( 4) 发酵
釆用下述 A、 B和 C中任一种方式进行发酵: A. 采用 5L发酵罐和一级种子进行发酵。将一级种子以 10 %的体积比 接入发酵培养基中, 在 5L发酵罐中发酵, 前 60h通空气, 通气量 2. 0vvm, 60h后通氮气, 通气量 0. 5vvm, 搅拌转速 500rpm, 发酵 70h, 整个发酵过 程中的发酵温度为 30°C。 测定甘油产量, 结果表明甘油浓度为 165 g/L, 残糖浓度为 5g/L, 甘油对葡萄糖质量得率为 52. 4%。
B. 采用 500L发酵罐和二级种子进行发酵。将二级种子以 10 %的体积 比接入发酵培养基中, 在 500L发酵罐中发酵, 前 60h通空气, 通气量
0. 8vvm, 60h后通氮气, 通气量 0. 2vvm, 搅拌转速 300rpm, 发酵 72h, 整 个发酵过程中的发酵温度为 33 °C。 测定甘油产量, 结果表明甘油浓度为 179g/L, 残糖浓度为 5g/L, 甘油对葡萄糖质量得率为 55. 38 %。
C. 采用 75000L发酵罐和二级种子进行发酵。将二级种子以 10 %的体 积比接入发酵培养基中, 在 75000L发酵罐中发酵, 前 60h通空气, 通气 量 0. 5vvm, 60h后通氮气, 通气量 0. 2vvm, 搅拌转速 300rpm, 发酵 72h, 整个发酵过程中的发酵温度为 35 °C。测定甘油产量, 结果表明甘油浓度为 158g/L, 残糖为 5g/L, 甘油对葡萄糖质量得率为 53. 3 %。
3. 甘油发酵液发酵生产 1, 3-丙二醇和 2, 3-丁二醇
(1) 将上一步甘油发酵液过滤除去克鲁氏假丝酵母 2. 1048,过滤所得 细胞直接用于下一批甘油发酵, 发酵操作条件均与第一批次相同。 所得滤 液用于 1, 3-丙二醇和 2, 3-丁二醇的发酵。
(2) 菌种: 克雷伯氏杆菌
Figure imgf000008_0001
應 1734 (购自 中科院微生物所)
(3) 1, 3-丙二醇种子培养基和发酵培养基如表 1所示, 其中微量元 素溶液组成如表 2所示
Figure imgf000008_0002
MgS(V7¾0 0. 2g 0. 2g 酵母粉 l. Og 1. 5g
微量元素溶液 2mL 2mL
CaCOa 2. Og
消泡剂 0. linL
表 2. 微量元素溶液组成
Figure imgf000009_0001
上述培养基的 pH为 6. 8-8. 0。
( 4)种子培养:将克雷伯氏杆菌 1. 1734接入用步骤 2的甘油发酵液 配制的种子培养基中 (500mL三角瓶, 装液量 lOOmL) , 培养温度 30°C, 摇床转速 150rpm (旋转半径为 25麵) 。 好氧培养 18h得到一级种子; 将 该摇瓶中的发酵液按 2%的体积比接入装有种子培养基的发酵罐中 30°C、 搅拌转速 60〜150rpm、通气量 0. 2〜0. 5vvm培养 5〜10小时得到二级种子。
( 5 ) 发酵培养- 采用下述 C、D和 E中任一种方式进行发酵,下述方法中 A和 B为对照: A. 采用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。 将种 子液接入用步骤 2的甘油发酵液配制的发酵培养基(甘油浓度 50g/L)中, 发酵过程中不流加甘油发酵液。 发酵罐转速 150rpm。 通入 0. 5vvm氮气。 发酵 30h, 1, 3—丙二醇浓度为 21g/L, 2, 3—丁二醇浓度为 2§几。
B. 釆用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。 将种 子液接入用步骤 2的甘油发酵液配制的发酵培养基(甘油浓度 80g/L)中, 发酵过程中不流加甘油发酵液。 发酵罐转速 150rpm。 通入 0. 5vvra空气。 发酵 30h, 1,3—丙二醇浓度为 35g/L, 2,3〜丁二醇浓度为 6. 6§/
C. 采用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。 将一 级种子液接入用步骤 2的甘油发酵液配制的发酵培养基 (甘油浓度 30g/L) 中, 发酵过程中流加甘油发酵液, 控制其流速使发酵液中甘油浓度维持 在 30g/L。 发酵罐转速 150rpm。 发酵过程中先通入氮气, 32h后通入空 气, 通气量均为 0. 5vvm。 发酵过程中 16h和 30h补氮两次(每次按 0. 8g 酵母粉 /L培养基和 lg硫酸铵 /L培养基的量加入酵母粉和硫酸铵) 。 64h 发酵结束。 收集发酵液, 过滤除去菌体, 收集滤液经脱盐、 蒸馏、 真空 精馏, 得到产品 1, 3-丙二醇和 2, 3-丁二醇。 测定结果表明发酵结束时发 酵液中 1,3—丙二醇浓度为 70g/L, 2,3—丁二醇浓度为 16§/ , 1, 3—丙 二醇摩尔得率 51 % ( 1, 3—丙二醇摩尔数与所消耗甘油摩尔数的百分比), 总二醇摩尔得率 71. 85 % ( 1, 3—丙二醇和 2, 3-丁二醇摩尔数与所消耗甘 油摩尔数的百分比) 。
D. 采用 500L发酵罐, 将 50L二级种子液接入用步骤 2的甘油发酵液 配制的发酵培养基 (甘油浓度 30g/L) 中, 发酵罐转速 60rpm, 通气量 0. 3vvm。 发酵过程中流加甘油发酵液, 控制其流速使发酵液中甘油浓度 维持在 30g/L 。 其他控制条件均同 5L罐。 发酵结束时发酵液中 1, 3—丙 二醇浓度 72g/L,2, 3—丁二醇浓度 25. 4g/L, 1, 3—丙二醇摩尔得率 55. 38 % , 总二醇摩尔得率 71. 85 %。
E. 采用 5000L发酵罐, 将二级 500L种子液接入含前面所述甘油发酵 液(甘油浓度 30g/L)配制的初始发酵培养基中。 发酵过程中流加甘油发 酵液, 控制其流速使发酵液中甘油浓度维持在 30g/L。 发酵条件控制同 500Lo 发酵结束时发酵液中 1, 3—丙二醇浓度 66. 6g/L, 2, 3—丁二醇浓 度 30. 4g/L, 1, 3—丙二醇摩尔得率 59. 1 %, 总二醇摩尔得率 83. 2 %。
4. 细胞回用: 将甘油发酵液过滤所得细胞直接用于下一批甘油发酵, 发酵操作条件均与第一批次相同, 回用 10批次以上, 细胞活力基本保持 不下降, 甘油浓度基本稳定。
实施例 2、 由红薯粗淀粉生产 1, 3-丙二醇和 2, 3-丁二醇
1、 粗淀粉原料的液化糖化 以红薯粗淀粉 1475g, 水 2722L的比例配置淀粉乳,加热, 分别在温 度升至 80Ό和 95Ό两次加入液化酶 (5U/g 淀粉) 液化 50min, 然后升 温至 110°C使酶失活。 冷却, 加入糖化酶 (200U/g淀粉) 60Ό糖化 9h, 测定结果表明该糖化液的葡萄糖值 (DE值, 糖化液中还原性糖全部当作 葡萄糖计算, 占干物质的百分比称为 DE值) 95. 5, 糖液葡萄糖浓度为 26. 8%。
2、 糖化液发酵产甘油
( 1 ) 菌种: 阿拉伯糖醇汉逊酵母 2. 887, 购自中科院微生物所。
( 2 ) 培养基- 斜面培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 200; 玉米 浆 2; 尿素 2; 琼脂 20;
种子培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 100; 玉米浆 2; 尿素 2;
发酵培养基 (g/L) : 葡萄糖 (用步骤 1的糖化液配制) 268; 玉米 浆 2; 尿素 4。
以上培养基 pH均调为 4-4. 5, 使用前均于 110°C灭菌 15分钟。
( 3) 种子培养
将阿拉伯糖醇汉逊酵母 2. 887接入斜面培养基, 35°C培养 24h, 进行 菌种活化。
将活化的阿拉伯糖醇汉逊酵母 2. 887接入含粗淀粉糖化液的种子培 养基, 在摇瓶 (500mL三角瓶, 装液量 lOOmL) 中培养一级种子, 30°C、 200rpm (旋转半径为 25讓) 培养 20h, 得到一级种子。
将一级种子接入含粗淀粉糖化液的种子培养基的发酵罐培养二级种 子, 30°C、 搅拌转速 300〜500 rpm通气量 0. 2〜0. 5vvm,培养 5〜7h。
( 4) 发酵
釆用下述4、 B和 C中任一种方式进行发酵:
A. 釆用 5L发酵罐和一级种子进行发酵。将一级种子以 10 %的体积比 接入发酵培养基中, 在 5L发酵罐中发酵, 前 60h通空气, 通气量 2. 0vvm, 60h后通氮气, 通气量 0. 5vvm, 搅拌转速 500rpm, 发酵 70h, 整个发酵过 程中的发酵温度为 30°C。 测定甘油产量, 结果表明甘油浓度为 140 g/L, 残糖浓度为 4. 8g/L, 甘油对葡萄糖质量得率为 53%
B. 采用 500L发酵罐和二级种子进行发酵。将二级种子以 10 %的体积 比接入发酵培养基中, 在 500L发酵罐中发酵, 前 60h通空气, 通气量
0. 8vvm, 60h后通氮气, 通气量 0. 2vvm, 搅拌转速 300rpm, 发酵 72h, 整 个发酵过程中的发酵温度为 33°C。 测定甘油产量, 结果表明甘油浓度为 146g/L, 残糖浓度为 4. 7g/L, 甘油对葡萄糖质量得率为 55. 7 %
C. 采用 75000L发酵罐和二级种子进行发酵。将二级种子以 10 %的体 积比接入发酵培养基中, 在 75000L发酵罐中发酵, 前 60h通空气, 通气 量 0. 5 60h后通氮气, 通气量 0. 2vvm, 搅拌转速 300rpm, 发酵 72h 整个发酵过程中的发酵温度为 35°C。测定甘油产量, 结果表明甘油浓度为 142g/L, 残糖为 4g/L, 甘油对葡萄糖质量得率为 54. 1 %
3. 甘油发酵液发酵生产 1 3-丙二醇和 2 3-丁二醇
(1) 将上一步甘油发酵液过滤除去阿拉伯糖醇汉逊酵母 2. 887细胞, 过滤所得细胞直接用于下一批甘油发酵, 发酵操作条件均与第一批次相 同。 所得滤液用于 1 3-丙二醇和 2 3-丁二醇的发酵。
(2) 菌种 巴氏梭菌 Clostridium pasteurianum)\. 208,购自中科 院微生物所)
(3) 1 3-丙二醇种子培养基和发酵培养基如表 1所示, 其中微量元 素溶液组成如表 2所示
表 1. 种子培养基和发酵培养基组成
培养基组分 种子培养基 ( / L) 发酵培养基 ( / L) 甘油 (用步骤 2 的甘油 20g 20-80g
发酵液配制)
Κ2ΗΡ04·3¾0 4. 45g 2. 225g
(匪 4) 2S04 2. 0g 2. 0g
K¾P04 1. 3g 0. 65g
MgS0„-7H20 0. 2g 0. 2g
酵母粉 l. Og 1. 5g
微量元素溶液 2mL 2mL
CaC03 2. Og
消泡剂 0. lniL 表 2. 微量元素溶液组成
Figure imgf000013_0001
上述培养基的 pH为 6. 8-8. 0。
( 4 ) 种子培养: 将 Clostridium pasteurianuw 1. 208接入用步骤 2 的甘油发酵液配制的种子培养基中 (500mL三角瓶, 装液量 lOOmL) , 培 养温度 33°C, 摇床转速 130rpm (旋转半径为 25mm) 。 好氧培养 18h得到 一级种子; 将该摇瓶中的发酵液按 2 %的体积比接入装有种子培养基的发 酵罐中 33°C、 搅拌转速 60〜150rpm、 通气量 0. 2〜0. 5vvm培养 5〜10小 时得到二级种子。
( 5 ) 发酵培养:
采用下述 C、 D和 E中任一种方式进行发酵, 以 A和 B为对照:
A. 采用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。 将种 子液接入用步骤 2的甘油发酵液配制的发酵培养基(甘油浓度 50g/L)中, 发酵过程中不流加甘油发酵液。 发酵罐转速 150rpm。 通入 0. 5vvm氮气。 发酵 30h, 1,3—丙二醇浓度为 24g/L, 2,3—丁二醇浓度为 1. 7§
B . 采用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。将种 子液接入用步骤 2的甘油发酵液配制的发酵培养基(甘油浓度 80g/L )中, 发酵过程中不流加甘油发酵液。 发酵罐转速 150rpm。 通入 0. 5vVra空气。 发酵 30h, 1,3—丙二醇浓度为 38g/L, 2,3—丁二醇浓度为 5. 6§/^
C. 釆用 5L发酵罐, 培养温度 37°C, pH值由氢氧化钾调至 6. 8。 将一 级种子液接入用步骤 2的甘油发酵液配制的发酵培养基(甘油浓度 30g/L) 中, 发酵过程中流加甘油发酵液, 控制其流速使发酵液中甘油浓度维持 在 30g/L。 发酵罐转速 150rpm。 发酵过程中先通入氮气, 32h后通入空 气, 通气量均为 0. 5vvm。 发酵过程中 16和 30h补氮两次 (每次按 0. 8g 酵母粉 /L培养基和 lg硫酸铵 /L培养基的量加入酵母粉和硫酸铵) 。 64h 发酵结束。 收集发酵液, 过滤除去菌体, 收集滤液经脱盐、 蒸馏、 真空 精馏, 得到产品 1, 3-丙二醇和 2, 3-丁二醇。 测定结果表明发酵结束时发 酵液中 1, 3—丙二醇浓度为 50g/L, 2, 3—丁二醇浓度为 18g/L, 1,3—丙 二醇摩尔得率 54% ( 1, 3—丙二醇摩尔数与所消耗甘油摩尔数的百分比), 总二醇摩尔得率 70 % ( 1, 3—丙二醇和 2, 3-丁二醇摩尔数与所消耗甘油 摩尔数的百分比) 。
D . 釆用 500L发酵罐, 将 50L二级种子液接入用步骤 2的甘油发酵液 配制的发酵培养基 (甘油浓度 30g/L) 中, 发酵罐转速 60rpm, 通气量 0. 3vvm。 发酵过程中流加甘油发酵液, 控制其流速使发酵液中甘油浓度 维持在 30g/L 。 其他控制条件均同 5L罐。 发酵结束时发酵液中 1,3—丙 二醇浓度 54g/L, 2,3—丁二醇浓度 22§ , 1, 3—丙二醇摩尔得率 55 %, 总二醇摩尔得率 73 %。
E. 采用 5000L发酵罐, 将二级 500L种子液接入含前面所述甘油发酵 液(甘油浓度 30g/L)配制的初始发酵培养基中。 发酵过程中流加甘油发 酵液, 控制其流速使发酵液中甘油浓度维持在 30g/L。 发酵条件控制同 500Lo 发酵结束时发酵液中 1, 3—丙二醇浓度 57. 6g/L, 2, 3—丁二醇浓 度 27. 3g/L, 1, 3—丙二醇摩尔得率 56 %, 总二醇摩尔得率 76. 2 %。
4. 细胞回用: 将甘油发酵液过滤所得细胞直接用于下一批甘油发酵, 发酵操作条件均与第一批次相同, 回用 10批次以上, 细胞活力基本保持 不下降, 甘油浓度基本稳定。
工业应用
实验表明, 本发明的方法可显著提高两步发酵法生产 1, 3-丙二醇过程中 甘油和 1, 3-丙二醇的浓度和得率, 同时还可得到高附加值的 1, 3-丙二醇和 2, 3-丁二醇, 从而有效提高了原料利用率, 降低了生产成本。 本发明的方法 应用于 5L, 500L, 5000L发酵罐上都取得了良好的效果, 其中, 发酵得到的甘 油浓度可达到 158 - 179g/L, 发酵得到的 1, 3—丙二醇浓度可达 66- 72g/L, 2, 3 一丁二醇浓度 16- 30. 4g/L。

Claims

权利要求
1. 一种由粗淀粉原料生产 1, 3-丙二醇和 2, 3-丁二醇的方法, 包括以 下步骤- 1 ) 将克鲁氏假丝酵母或阿拉伯糖醇汉逊酵母接入以粗淀粉糖化液为 碳源的发酵培养基中,在有氧条件下培养至葡萄糖消耗速率明显降低后进 行厌氧发酵至糖浓度为 4-10g/L, 收集发酵液, 过滤除去发酵液中的酵母 细胞, 得到的滤液即为甘油发酵液;
2 ) 将克雷伯氏菌、 丁酸梭菌或巴斯德梭菌接入以步骤 1 ) 得到的甘 油发酵液为碳源的发酵培养基中, 厌氧发酵 30-32小时, 至 1, 3-丙二醇生 产强度明显降低时进行有氧发酵至甘油浓度降至 10g/L以下结束发酵, 得 到 1, 3 -丙二醇和 2, 3-丁二醇。
2、 根据权利要求 1所述的方法, 其特征在于: 所述步骤 1 ) 中过滤 除去的酵母细胞直接回收用于下个批次的发酵。
3、 根据权利要求 1所述的方法, 其特征在于: 所述克鲁氏假丝酵母 或阿拉伯糖醇汉逊酵母来自于一级种子或二级种子; 所述一级种子按照 以下方法制备:将克鲁氏假丝酵母或阿拉伯糖醇汉逊酵母接入含粗淀粉糖 化液的种子培养基, 在 30— 35°C、 装液量为 1/5体积的摇瓶、 旋转半径为 25醒、 转速为 200- 250rpm的条件下, 培养 20小时得到的; 所述二级种子 按照以下方法制备:将一级种子接入以粗淀粉糖化液为碳源的种子培养基 的发酵罐中, 在 30— 35°C、 搅拌转速 300〜500 rpm通气量 0. 2〜0. 5vvm, 培养 5〜7h。
4、 根据权利要求 1所述的方法, 其特征在于: 所述以粗淀粉糖化液 为碳源的发酵培养基的 PH为 4-5, 还含有玉米浆和尿素; 所述粗淀粉糖化 液的含量为将粗淀粉糖化液中还原性糖全部当作葡萄糖计算, 使培养基中 葡萄糖含量达到 260-350 g/ 所述玉米浆的含量为 2-3 g/L; 所述尿素 的含量为 2. 5-4 g/Lo
5、 根据权利要求 3所述的方法, 其特征在于: 所述含粗淀粉糖化液 的种子培养基的 PH为 4-5, 还含有玉米浆和尿素; 所述粗淀粉糖化液的含 量为将粗淀粉糖化液中还原性糖全部当作葡萄糖计算, 使培养基中葡萄糖 含量达到 80-100 g/L; 所述玉米浆的含量为 2-3 g/L; 所述尿素的含量为
Figure imgf000016_0001
6、 根据权利要求 1所述的方法, 其特征在于: 克雷伯氏菌、 丁酸梭 菌或巴斯德梭菌来自于一级种子或二级种子; 所述一级种子按照以下方 法制备: 将克雷伯氏杆菌、 丁酸梭菌或巴斯德梭菌接入以步骤 1) 得到的 甘油发酵液配制的种子培养基中, 在培养温度 30-33°C, 装液量为 1/5体 积的摇瓶、旋转半径为 25謹、摇床转速 130- 150rpm、好氧条件下培养 18-20 小时得到一级种子; 所述一级种子按照以下方法制备: 将一级种子接入装 有以步骤 1)得到的甘油发酵液配制的种子培养基的发酵罐中,在 30-33°C、 搅拌转速 60〜: I50rpm、通气量 0.2〜0.5vvm的条件下, 培养 5〜10小时得 到二级种子。
7、 根据权利要求 1所述的方法, 其特征在于: 所述以步骤 1) 得到 的甘油发酵液为碳源的发酵培养基的 pH为 6.8-8.0,所述甘油发酵液的含 量为以甘油计算使培养基中甘油含量达到 20-80g/L;以甘油发酵液为碳源 的培养基中还含有 K2HP04 * 3H202.225— 3.5g/L、 (NH4)2S 04 2.0 - 4. Og/L, KH2P04 0.65—1.2g/L、 MgS04 ' 7 0 0.1— 0.2g/L、酵母粉 1— 1.5g/L、 微量元素溶液 2— 3mL/L和消泡剂 0. ImL/L; 所述微量元素溶液的组成为 ZnCl2 70mg/L、 MnCl2 · 4H20 100mg/L、 ¾B03 60mg/L、 CoCl2 · 6H20 200mg/L、 NiCl2 · 6H20 25 mg/L、 NiCl2 · H20 27.64 mg/L、 Na2Mo04 · 2H20 35 mg/ CuC · H20 20 mg/L、 CuS04 · 5H20 29.28 mg/L和浓盐酸 0.9mL/L。
8、 根据权利要求 6所述的方法, 其特征在于: 所述以步骤 1) 得到 的甘油发酵液为碳源的种子培养基的 pH为 6.8-8.0,所述甘油发酵液的含 量为以甘油计算使培养基中甘油含量达到 20-25g/L;以甘油发酵液为碳源 的培养基中还含有 Κ2ΗΡ04 · 3¾0 4.45— 5.6g/L、 (NH4)2S04 2.0— 4. Og/ KH2P04 1.3— 2.6g/L、 MgS(WH20 0.1— 0.2g/L、 酵母粉 1.0-2.0 g/L、 CaC03 1.0-2.0g/L 微量元素溶液 2- 3mL/L; 所述微量元素溶液的 组成为 ZnCl2 70mg/L、 MnCl2 · 4H20 lOOmg/L, ¾B03 60mg/L、 CoCl2 · 6H20 200mg/L、 NiCl2 · 6H20 25 mg/L、 NiCl2 · H20 27.64 mg/L、 Na2Mo04 · 2H20 35 mg/ CuCl2 · H20 20 mg/ CuS04 · 5H20 29.28 mg/L和浓盐酸
Figure imgf000017_0001
9、 根据权利要求 1-8中任一所述的方法, 其特征在于: 所述步骤 1 ) 中所述粗淀粉为红薯粉、 玉米粉或木薯粉; 所述粗淀粉糖化液的 DE值为 90-110。
10、 根据权利要求 9所述的方法, 其特征在于: 所述粗淀粉糖化液 按照下述方法制备: 用粗淀粉与水按照 1 : 1800-2000 的质量比配置淀粉 乳, 分别在 80-85°C和 90-95°C两次加入液化酶, 每次 3_5U/g 粗淀粉, 液化 40-50分钟, 然后升温至 110〜120°C使酶失活, 冷却, 加入糖化酶 150〜200U/g淀粉, 50〜60°C糖化 8〜12小时, 得到 DE值为 100〜110 的粗淀粉糖化液。
11、根据权利要求 1-8中任一所述的方法,其特征在于:所述步骤 1 ) 的发酵过程中的发酵温度 30- 35°C ; 所述步骤 1 ) 中有氧条件是发酵过程 中通空气, 通气量为 0. 5- 2VVm; 所述步骤 1 ) 中厌氧条件是发酵过程中通 氮气, 通气量为 0. 2- 2vvm。
12、 根据权利要求 1-8中任一所述的方法, 其特征在于: 所述步骤
2 ) 的发酵过程中还流加所述步骤 1 ) 得到的甘油发酵液, 使培养基中的 甘油含量保持在 20- 80g/L。
13、 根据权利要求 1-8中任一所述的方法, 其特征在于: 所述步骤 2 ) 的发酵过程中还补充两次氮源, 每次按 0. 8g酵母粉 /L培养基和 lg 硫酸铵 /L培养基的量加入酵母粉和硫酸铵。
14、 根据权利要求 1-8中任一所述的方法, 其特征在于: 所述步骤 2 ) 的发酵过程中 pH为 6. 8- 8. 0, 发酵温度为 30- 37°C ; 所述步骤 2 ) 中 厌氧发酵是发酵过程中通氮气, 通气量为 0. 1- 0. 5vvm; 所述步骤 2 ) 中有 氧发酵是发酵过程中通空气, 通气量为 0. 1-0. 5vvm。
15、 根据权利要求 1-8中任一所述的方法, 其特征在于: 所述方法 中还包括纯化 1, 3-丙二醇和 2, 3-丁二醇的步骤, 即收集发酵液, 过滤除 去菌体, 收集滤液进行脱盐、 蒸馏、 真空精熘。
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