WO2012167525A1 - 一种二元醇与有机酸联产与分离方法 - Google Patents

一种二元醇与有机酸联产与分离方法 Download PDF

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WO2012167525A1
WO2012167525A1 PCT/CN2011/080589 CN2011080589W WO2012167525A1 WO 2012167525 A1 WO2012167525 A1 WO 2012167525A1 CN 2011080589 W CN2011080589 W CN 2011080589W WO 2012167525 A1 WO2012167525 A1 WO 2012167525A1
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fermentation
acid
phase
extraction
butanediol
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French (fr)
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孙亚琴
魏搏超
李志刚
戴建英
修志龙
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Dalian University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
    • C07C29/86—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
    • 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; 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
    • 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
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

Definitions

  • the invention belongs to the field of bioengineering technology, relates to microbial fermentation co-production and carbon dioxide recycling technology, and particularly relates to a method for co-production and separation of glycol and organic acid.
  • 1,3-propanediol and 2,3-butanediol are important chemical raw materials and are used in a wide range of applications such as solvents, synthetic pharmaceutical intermediates, automotive antifreeze, lubricants, additives and polymer monomers. Wait. Due to the differences in the structure and nature of the two, there is a slight difference in application.
  • the main use of 1,3-propanediol is in the production of high quality polyester materials - polytrimethylene terephthalate (PTT).
  • 2,3-butanediol can be used to synthesize chiral pharmaceutical intermediates, which can be converted into important compounds such as methyl ethyl ketone, 2-butene, 1,3-butadiene, acetoin and diacetyl.
  • a mixture of 1,3-propanediol and 2,3-butanediol and terephthalic acid to synthesize new polyester materials have also attracted attention.
  • Organic acids (formic acid, acetic acid, lactic acid, succinic acid, citric acid, malic acid, fumaric acid, etc.) are widely used as chemical raw materials and intermediates in many fields.
  • lactic acid can be used as a preservative, seasoning and sour agent
  • lactic acid as a monomeric polylactic acid (PLA) can be widely used in medicine, plastics, cosmetics and agriculture
  • succinic acid is an important C 4 platform compound.
  • 1,3-propanediol is mainly synthesized by petroleum cracking of ethylene and propylene as raw materials, and the price is high; and chiral 2,3- The chemical synthesis of butanediol is quite difficult, so the microbial fermentation process produces these two glycols with considerable advantages.
  • DuPont completed the conversion of glucose into 1,3- in genetic engineering E. coli.
  • a pilot study of propylene glycol, Tsinghua University and Dalian University of Technology in Germany and China have also completed pilot tests using natural strains to convert glycerol to 1,3-propanediol.
  • Glycerol biotransformation fermentation production in recent years The research of propylene glycol process mainly includes two-step fermentation method, glucose-assisted fermentation, mixed-fermentation fermentation, micro-oxygen fermentation, and direct utilization of crude glycerin by-product of biodiesel.
  • Microbial production 2,3- Butylene glycol has achieved industrial production in Germany during World War II, and has attracted the attention of the business community in recent years.
  • 1,3-propanediol 2,3- The butanediol strain has a wider substrate recipe, and hexose, pentose, specific disaccharide, and uronic acid can be used as substrates for non-grain materials such as sugar cane juice ( Marilia et al, 2001). ), the use of lignocellulose (Yu et al, 1985; Frazer and McCaskey, 1991), Jerusalem artichoke, etc.
  • Each ton of 1,3-propanediol produced will produce about 0.3-0.5 tons of CO 2 , with glycerol fermentation emitting about 0.1 tons of CO 2 and the remainder coming from the energy consumption of fermentation and separation.
  • carbon dioxide emissions are actually a waste of raw materials. If the CO 2 emissions can be recycled for the production of succinic acid, and the production of 1,3-propanediol and succinic acid can be achieved, the utilization of raw materials can be improved and the greenhouse can be reduced. Gas emissions.
  • succinic acid by CO 2 usually uses high-yield succinic acid species (such as Anaerobiospirillum succiniproducens and Actinobacillus succinogenes ) or genetically engineered bacteria (such as Escherichia coli). It requires different culture systems and is more cumbersome to implement. How to achieve the recycling of CO 2 and the co-production of the above diols and organic acids in the same culture system?
  • high-yield succinic acid species such as Anaerobiospirillum succiniproducens and Actinobacillus succinogenes
  • genetically engineered bacteria such as Escherichia coli
  • succinic acid acts as a tricarboxylic acid intermediate and a terminal reduction product of anaerobic metabolism, and other by-products include acetic acid. , lactic acid, ethanol, 2,3-butanediol, and the like. From the perspective of economic efficiency, acetic acid and ethanol are relatively inexpensive, but they are the main by-products, while succinic acid, lactic acid and 2,3-butanediol are economically good, but the yield of succinic acid is low.
  • Klebsiella pneumoniae can be used to produce 1,3-propanediol and its own CO 2 can be reused for the synthesis of succinic acid through metabolic regulation, then the co-production of glycols and organic acids can be achieved, and cheap by-products can be reduced and improved.
  • the overall economic benefits of the glycerol biotransformation process From the perspective of NADH 2 and ATP equilibrium, the succinic pathway can completely replace the acetic acid and ethanol pathways. By raising the pressure in the fermentor and switching to carbonate to adjust the pH, it is expected to metabolize the succinic acid pathway.
  • Increasing the tank pressure means increasing the partial pressure of CO 2 and H 2 in the fermenter, thereby inhibiting the activity of the formate lyase, which in turn affects the further metabolism of pyruvate, which causes a series of metabolic changes.
  • the growth process of K. pneumoniae shows the metabolic physiology of alcohol production after acid production. If artificially added some metabolic organic acids, it will change the metabolic process and facilitate the formation of 1,3-propanediol.
  • glycerin When co-fermentation of glycerin with sugars (glucose, fructose, xylose or arabinose, etc.) is advantageous for increasing the conversion of glycerol, and also for forming by-products such as lactic acid, 2,3-butanediol, and succinic acid.
  • sugars glucose, fructose, xylose or arabinose, etc.
  • by-products such as lactic acid, 2,3-butanediol
  • succinic acid When the sugar (glucose, Jerusalem artichoke tuber hydrolyzate, Jerusalem artichoke and other plant straw hydrolysate) is used as the fermentation substrate, the main metabolite of Klebsiella pneumoniae is 2,3 -butanediol, and the by-products are acetic acid, ethanol, lactic acid, Succinic acid, etc. It is also possible to achieve the co-production of 2,3-butane
  • the salting-out extraction or the two-aqueous phase extraction technology can well solve the outstanding problems of the prior bisdiol and organic acid separation and extraction methods, such as many steps, low yield and high cost.
  • the salt digestive extraction technique is used to separate the dihydric alcohol and the organic acid, that is, the inorganic salt and the organic solvent are simultaneously added to the fermentation liquid, and the fermentation liquid can form two phases within a certain range.
  • the upper phase is the solvent phase or the extract phase rich in the target product
  • the lower phase is the salt-rich phase or the raffinate phase.
  • the fermentation broth containing the cells is subjected to salting-out extraction to form a solid phase layer between the two phases, and is composed of cells, proteins, nucleic acids, polysaccharides, and the like.
  • the inorganic salts selected in the salting out extraction system include soluble phosphates, sulfates, carbonates, and the like, and the organic solvents include methanol, ethanol, propanol, butanol, acetone, and the like.
  • the separation and the extraction of the product are combined into one, which is a simple and practical separation and integration technology.
  • the first step is to extract 1, 3-propanediol and / or 2, 3-butanediol in the upper phase, lactic acid and succinic acid in the lower phase
  • the second step The organic acid is then extracted into the upper phase so that the separation of the glycol and the organic acid can be achieved.
  • the first step of extracting the 1, 3-propanediol and/or 2,3-butanediol in the upper phase can be recovered by distillation, rectification, etc., and the 1, 3-propanediol and 2, 3-butadiene can be recovered.
  • the alcohol is separated; the second step of extracting the lactic acid and succinic acid in the upper phase can separate different organic acids by crystallization, electrodialysis or esterification rectification.
  • the lower phase or the raffinate phase of the soluble carbonate and the organic solvent forming the extraction system can be used to adjust the pH of the fermentation, and the CO 2 in the fermentation tail gas can be used to recover the carbonate in the raffinate phase, of course, the CO 2 and the corresponding alkali in the exhaust gas.
  • the product (carbonate) formed by the oxidation reaction can also be used to adjust the pH or as a salting-out agent. In short, fermentation, extraction, CO 2 recycling can be combined.
  • the invention proposes a new idea of pressure fermentation and carbonate adjustment pH-salting extraction-CO 2 immobilization coupling to produce diol and organic acid, in order to establish a green, efficient and low-cost co-production new process.
  • the invention provides a novel aqueous two-phase system formed by a hydrophilic low molecular organic substance/salt and a hydrophobic organic substance/hydrophilic low molecular organic substance/salt/the problem of long production cycle and large solvent consumption.
  • the water three-liquid phase system simultaneously extracts and separates various active ingredients from the sea cooking cooking liquor.
  • a method for salting out extraction and separating active ingredients of sea cucumber the operation steps are as follows: In the sea cucumber extract, some electrolytes and common organic solvents are directly added, and the hydration of the salt ions is used to form an organic solvent. Salt multiphase salting out extraction system. According to the difference in solubility, the distribution of the active ingredients with different polarities in the sea cucumber is different in each phase, thereby achieving separation and concentration of the active ingredients.
  • salt and hydrophilic low molecular organics Adding to sea cucumber extract in a certain proportion, stirring evenly, standing or centrifuging to form a double aqueous phase: sea cucumber alcohol soluble active ingredient is concentrated in the upper phase, protein and polysaccharide are mainly distributed to the solid phase between the upper phase and the lower phase Medium, heavy metals are enriched in the lower phase;
  • the hydrophilic low molecular organic substance and the hydrophobic organic substance are added to the sea cucumber extract liquid in a certain proportion, stirred uniformly, and left standing or centrifuged to form a three-liquid salting out extraction system: Sea cucumber oil is enriched in the upper phase, and a small amount of soluble protein and polysaccharide are distributed in the middle phase. Most of the protein and polysaccharide are distributed in the solid phase between the middle phase and the lower phase, while heavy metals are enriched in the lower phase.
  • the mass percentage of the salt in the aqueous two-phase system is 10 ⁇ 30%, and the mass percentage of the hydrophilic low molecular organic substance is 15 ⁇ 40%.
  • the extraction temperature is 15 ⁇ 40 °C
  • the time of stirring and standing phase separation is 0.1 ⁇ 4.0 h, or it can be centrifuged at 2000 ⁇ 8500 rpm for 5 ⁇ 20 min.
  • the mass percentage of the salt in the three liquid phase system is 10 ⁇ 30%
  • the mass percentage of the hydrophilic low molecular organic matter is 15 ⁇ 40%
  • the mass percentage of the hydrophobic organic matter is 10 ⁇ 40%.
  • the applicable sea cucumber extract refers to a liquid containing the active ingredient of sea cucumber, including fresh sea cucumber juice or a concentrated liquid thereof, or a sea cooking cooking liquid or a concentrated liquid thereof.
  • the sea cooking cooking liquid refers to the waste liquid obtained by cooking sea cucumber.
  • the hydrophilic low molecular organic substances selected are ethanol, ethylene glycol, n-propanol, isopropanol, 1,2-propanediol, 1 , 3- One of propylene glycol, n-butanol, isobutanol, tert-butanol, 1,2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2, 3-butanediol, acetonekind or several combinations;
  • the selected salts are sodium chloride, lithium chloride, ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium citrate, citric acid.
  • the hydrophobic organic material selected is methyl acetate, ethyl a
  • the extraction operation may be one extraction, multiple extraction, continuous extraction or countercurrent extraction.
  • the invention has the beneficial effects that compared with the prior art extraction and separation method, the present invention utilizes a multi-phase salting-out extraction system to simultaneously extract and separate a plurality of active components with different polarities, and obtains sea cucumber oil and alcohol-soluble active ingredients ( With ethanol as extractant, a variety of products such as sea cucumber wine, crude protein and polysaccharide can be prepared to complete the preliminary purification of the target product.
  • the content of harmful heavy metal ions (arsenic, mercury, lead, cadmium) in the solid phase lyophilized powder obtained after extraction is significantly reduced. Most of the beneficial ions (calcium, iron, magnesium, zinc) remain.
  • the whole production process is simple in operation, low in production cost, short in cycle, mild in extraction conditions, cheap and easy to recover organic solvent, low in mass transfer, fast in mass separation, and easy to industrialize.
  • the product meets the requirements of food safety.
  • the invention aims at the problems of carbon dioxide emission, low utilization rate of raw materials and single fermentation product in the fermentation of 1, 3-propanediol and 2,3-butanediol, and proposes pressure fermentation/carbonate adjustment pH-salting extraction-CO 2 A new method of immobilized coupling to produce glycols and organic acids.
  • the invention utilizes Klebsiella pneumoniae to carry out fermentation under different raw materials by increasing the pressure of the fermenter to 0.01-0.5 atm, the pH of the fermentation to adjust the pH of 6.0 or 7.0, and the regulation of the ventilation rate of 0.01-0.8 vvm.
  • the gas is CO 2 , fermentation tail gas or a mixed gas containing CO 2 , and the carbon dioxide recovery and utilization of the tail gas and the co-production of the glycol and the organic acid are realized in the same culture system.
  • the diol is separated from the organic acid by a two-step salting out extraction.
  • the fermentation raw material can be 95% industrial glycerin, 60-85%
  • One or more of the biodiesel by-products such as glycerin, glucose, Jerusalem artichoke tubers, straw hydrolysate, and the like are mixed in proportion.
  • the carbonate is sodium carbonate, potassium carbonate, ammonium carbonate, calcium carbonate, magnesium carbonate, etc., which may be CO 2 in the fermentation tail gas and the corresponding alkali or oxide (sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide or oxidation).
  • the product of the reaction of calcium, magnesium hydroxide or magnesium oxide, or a soluble carbonate (sodium carbonate, potassium carbonate or ammonium carbonate) and an organic solvent form a raffinate phase of the salting out extraction system.
  • the diol is 1,3-propanediol, 2,3-butanediol, and the organic acid is lactic acid or succinic acid.
  • the fermentation pH is adjusted to 7.0; the diol is 2,3- When butanediol is the main product, the fermentation pH is adjusted to 6.0.
  • the invention can be produced by batch fermentation, batch flow addition or continuous fermentation.
  • the salting-out agent selected for use in the salting out extraction system includes a soluble phosphate, a sulfate, a carbonate, or a combination thereof, and the organic solvent includes methanol, ethanol, propanol, butanol, acetone, or a combination thereof.
  • the first step in the two-step extraction will be 1 . 3-propanediol and / or 2, 3- The butanediol is extracted in the upper or organic phase, the lactic acid and succinic acid are extracted in the lower phase or the salt-rich phase, and the second step is to extract the organic acid from the salt-rich phase of the first step to the upper phase to realize the diol and the organic acid. Separation.
  • the extraction can be carried out in a single-stage, multi-stage or continuous extraction mode.
  • the co-production of glycols and organic acids usually requires two different strains, which are carried out under different fermenters and different culture conditions, which undoubtedly increases the difficulty of implementation.
  • Klebsiella pneumoniae to produce 1,3-propanediol and/or 2,3-butanediol by converting glycerol or glucose while producing by-products of succinic acid and lactic acid, by adjusting the pressure of the tank, adjusting the pH of the carbonate, and recycling the by-product organic acid.
  • Such measures regulate cell metabolism, increase the production of succinic acid and lactic acid, and the unused CO 2 is reused in the carbonate absorption cycle to achieve zero CO 2 emissions and full utilization of raw materials.
  • the first extraction can directly separate the diol from the fermentation broth, and the second extraction will The organic acid is separated from the salt-rich phase of the first step.
  • the salting out extraction system formed by the soluble inorganic salt and the organic solvent can integrate the multi-step processes such as solid-liquid separation, concentration, protein removal and organic acid into one, greatly simplifying the separation process.
  • a two-step extraction strategy was used to separate the two types of products. The extraction operation is easy to scale up and industrialize, and the two-step extraction is closely connected before and after, making it easy to implement.
  • Salting out extraction can effectively overcome the problem of difficult recovery of extractant by distillation and rectification, but the amount of salt is large, and the recovery of salt is hindering salting out - One of the most important factors in the industrialization of extraction.
  • the raffinate phase extracted by the soluble carbonate and the organic solvent is used to adjust the fermentation pH, regulate the co-production of the diol and the organic acid, and realize the integrated integration of the fermentation-separation-CO 2 fixation.
  • part of the raffinate phase can be reused for pH adjustment of the fermentation process to realize the integration of fermentation-separation-CO 2 immobilization.
  • the partial lower carbonate-rich lower phase is used to replace the existing sodium hydroxide or potassium hydroxide lye to adjust the pH of the fermentation process, saving the salt removal cost and facilitating the production of succinic acid; Containing the remaining substrate of fermentation and by-products such as acetic acid and succinic acid, the addition of organic acids such as succinic acid helps to increase the concentration and conversion of 1,3-propanediol and 2,3-butanediol, thus saving separation. The cost is also conducive to improving the utilization rate of raw materials.
  • Example 1 Adding sodium carbonate solution to adjust fermentation pH to achieve 1,3-propanediol, lactic acid and succinic acid co-production
  • Glycerol 20 g ; KH 2 PO 4 : 1.3 g ; CaCO 3 : 2 g ; K 2 HPO 4 ⁇ 3H 2 O : 4.454 g ; ( NH 4 ) 2 SO 4 : 2.0 g ; MgSO 4 ⁇ 7H 2 O : 0.2 g ; Yeast powder: 1 g; trace element A: 2 mL; Ca 2+ solution: 1 mL; Fe 2+ solution: 1 mL.
  • Glycerol 40 g; KH 2 PO 4 : 1.36 g; citric acid: 0.42 g; MgCl 2 ⁇ 6H 2 O : 0.26 g; (NH 4 ) 2 SO 4 : 6.61 g; yeast powder: 1 g; trace element B: 5 mL.
  • Fe 2+ solution composition (100 ml ): saturated hydrochloric acid: 0.4 ml ; FeSO 4 ⁇ 7H 2 O : 0.5 g ;
  • Fermentation control 5L fermenter, batch type fed fermentation, liquid volume 3L, fermentation temperature 37 °C, tank pressure control 0.05atm, 85% biodiesel by-product glycerol, initial concentration of 40g / L, inoculum volume of 10% (v / v), stirring speed of 300 r / min, The air flow rate was 0.04 vvm, and the pH was adjusted to 7.0 by using a 5 mol/L sodium hydroxide solution and a 2.5 mol/L sodium carbonate solution, respectively. Fermentation begins 4 After the hour, the glycerol concentration was measured, and glycerol was added to control the glycerin concentration to about 20 g / L, and the fermentation was completed until 33 hours.
  • the concentration of 1,3-propanediol and 2,3-butanediol decreases slightly with respect to sodium hydroxide to adjust the fermentation pH, but the concentrations of succinic acid and lactic acid are increased, respectively. At 175% and 86%, the total conversion of glycerol increased by 13%.
  • Example 2 Adjusting the pH by adding sodium carbonate solution to achieve the joint production of 1,3-propanediol, succinic acid and lactic acid
  • Fermentation control 5L fermenter, batch type fed fermentation, liquid volume 3L, fermentation temperature 37 °C, flow plus 95% Industrial glycerin, the initial concentration is 40g / L, the inoculum is 10% (v / v), the stirring speed is 300 r / min, the air volume is 0.02vvm, respectively
  • the pH was adjusted to 7.0 by a 5 mol/L sodium hydroxide solution and a 2.5 mol/L sodium carbonate solution.
  • the glycerol concentration was measured 4 hours after the start of fermentation, and glycerol was added to control the glycerol concentration. Fermentation to about 36 hours ends at around 15-20g/L.
  • the final concentrations of the products 1,3-propanediol, succinic acid, lactic acid, 2,3-butanediol and ethanol are respectively 65.50, 9.65, 43.76, 10.79 and 6.86g / L; the mass conversion rates of 1,3-propanediol, succinic acid, lactic acid, 2,3-butanediol and ethanol are respectively 36%, 5%, 24%, 6% and 4%, the total mass conversion of glycerol is 75%.
  • Example 3 Sodium carbonate / ethanol raffinate phase adjustment pH 1,3-propanediol batch fed-batch fermentation
  • Fermentation control 5L fermenter, batch type fed fermentation, liquid volume 3L, fermentation temperature 37 °C, stirring speed 200r/min, the inoculation amount is 10% (v/v), the air volume is 0.02vvm, and 5mol/L sodium hydroxide solution and sodium carbonate are used in the fermentation process respectively.
  • the pH of the raffinate phase of the ethanol salting-out extraction system was adjusted to 7.0.
  • the residual glycerol concentration was 18.40 g after 36 h of fermentation. /L, the maximum cell OD value is 12.4, and the final concentrations of 1,3-propanediol, succinic acid, lactic acid and 2,3-butanediol are 64.29, 10.34, 57.92 and The mass conversion rate of 1,3-propanediol, succinic acid, lactic acid and 2,3-butanediol was 46%, 4%, 31% and 5%, respectively, and the total mass conversion rate of glycerol was 8.08 g / L; 86%.
  • Example 4 Sodium carbonate / ethanol raffinate phase adjustment pH 2,3-butanediol batch flow fermentation
  • Seed medium (1 L): Glucose: 80 g; (NH 4 ) 2 HPO 4 : 6.0 g ; KCl : 1.8 g ; EDTA : 0.51 g ; MgSO 4 ⁇ 7H 2 O : 0.6 g ; FeSO 4 ⁇ 7H 2 O : 0.0225g; MnSO 4 ⁇ 7H 2 O : 0.0038g ; ZnSO 4 ⁇ 7H 2 O : 0.0075g ; Citric acid: 0.21g ; Sodium citrate: 0.294g
  • Fermentation medium (1L): glucose: 50g; (NH 4 ) 2 SO 4 : 6.61g; KH 2 PO 4 : 1.36g; yeast powder: 1g; trace element B: 5ml; MgCl 2 ⁇ 6H 2 O : 0.26 g; citric acid: 0.42g;
  • Fermentation control 5L fermenter, batch type fed fermentation, liquid volume 3L, fermentation temperature 37 °C, stirring speed 300 r/min, inoculum volume is 5% (v/v), air volume is 0.1vvm, and 5mol/L sodium hydroxide solution and sodium carbonate are used in the fermentation process respectively.
  • the pH of the raffinate phase of the ethanol salting-out extraction system was adjusted to 6.0.
  • the glucose concentration is lower than 50 g / L, a certain amount of solid glucose is added, and the concentration is maintained by periodically adding glucose. Between 30-50g/L.
  • Fermentation results Fermentation 52h when pH is adjusted with 5mol/L sodium hydroxide solution At the time of product concentration, the final residual sugar concentration was 43.43g / L, and the maximum bacterial OD value reached 15.91; the final concentrations of 2,3-butanediol, acetoin, lactic acid and succinic acid were respectively 62.63, 15.62, 14.26 and 4.13g / L; the mass conversion rates of 2,3-butanediol, acetoin, lactic acid and succinic acid were 36%, 9%, 8% and 2%, the total mass conversion of glucose is 55%.
  • the concentration of 2,3-butanediol, lactic acid and succinic acid is higher than that of sodium hydroxide lye. 25% , 28% and 97%, the conversion rate of the three increased by 14%, 17% and 79% respectively.
  • Example 5 Salting out extraction of 1,3-propanediol, 2,3-butanediol with lactic acid and succinic acid
  • Salting and extracting the fermentation broth of 1,3-propanediol, 2,3-butanediol, lactic acid and succinic acid, 1,3- in the fermentation broth The concentrations of propylene glycol, 2,3-butanediol, lactic acid and succinic acid were 46.67, 8.36, 56.13 and 22.67 g / L, respectively, and 1.0 g of the fermentation broth was added to 7.0 g. Sodium carbonate and 2.0 g of ethanol are stirred uniformly and allowed to stand at room temperature to form two phases.
  • the upper phase is an alcohol phase containing 1,3-propanediol and 2,3-butanediol
  • the lower phase is a salt rich in lactic acid and succinic acid.
  • phase. 1,3-
  • the partition coefficient of propylene glycol was 7.0, the yield was 90%, the partition coefficient and yield of 2,3-butanediol were 8.0 and 94%, respectively; 74% lactic acid and 95% Succinic acid is partitioned in the salt phase.
  • a second extraction was carried out by adding 0.5 g of dipotassium hydrogen phosphate and 2.5 g of ethanol to the salt phase, and the yield of lactic acid in the upper phase was 75%, and the yield of succinic acid was 68%.

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Abstract

本发明属于生物工程技术领域，提供了一种二元醇与有机酸联产与分离方法。其特征是以生物转化甘油或葡萄糖等产二元醇的Klebsiella pneumoniae为研究对象，通过加压发酵和碳酸盐调节发酵pH-盐析萃取-CO2固定化耦合生产二元醇和有机酸，通过两步盐析萃取将1，3-丙二醇和2，3-丁二醇与乳酸和琥珀酸分离。本发明既可以通过控制不同的发酵条件，在同一培养体系中实现不同二元醇和有机酸的联产，提高原料利用率；同时对发酵中产生的尾气二氧化碳进行回收利用，解决了发酵行业二氧化碳排放的共性问题。本发明建立了高效、低成本的发酵-分离-CO2固定化集成的新工艺，为CO2回用联产其它化学品提供借鉴。

Description

一种二元醇与有机酸联产与分离方法
技术领域
本发明属于生物工程技术领域，涉及到微生物发酵联产和二氧化碳回收利用技术，特别涉及一种二元醇与有机酸联产与分离的方法。
背景技术
在能源需求不断扩大、石油资源供应不稳定的情况下，以可再生生物质资源为原料的生物炼制技术越来越受到国内外研究学者的高度重视，尤其是利用工业生物技术生产的生物质能源和生物基大宗化学品，如燃料乙醇、生物柴油、生物气、生物氢气以及 1,3- 丙二醇、 2,3- 丁二醇、乳酸、琥珀酸、丁醇 / 丙酮等。其中， 1,3- 丙二醇、 2,3- 丁二醇、乳酸和琥珀酸等都备受关注、也是最有工业化前景的二元醇和二元酸类发酵产物。 1,3- 丙二醇和 2,3- 丁二醇都是重要的化工原料，具有广泛的应用领域，如用作溶剂、合成药物中间体、汽车抗冻剂、润滑剂、添加剂以及聚合物单体等。由于二者结构和性质存在差异，应用上也稍有区别。 1,3- 丙二醇主要用途是生产高品质的聚酯材料 -- 聚对苯二甲酸丙二酯（ PTT ）。 2,3- 丁二醇可以用于合成手性药物中间体，可以转化生成甲乙酮、 2- 丁烯、 1,3- 丁二烯、乙偶姻和二乙酰等重要化合物。近几年 1,3- 丙二醇与 2,3- 丁二醇的混合物与对苯二甲酸合成新的聚酯材料也已引起人们的关注。有机酸（甲酸、乙酸、乳酸、琥珀酸、柠檬酸、苹果酸、延胡索酸等）作为生产前景广阔的化工原料和中间体，在很多领域都有着广泛的应用。其中乳酸可用作防腐剂、调味料及酸味剂，而以乳酸为单体的聚乳酸（ PLA ）可广泛应用于医药、塑料、化妆品及农业中；琥珀酸是一种重要的 C4 平台化合物，可以衍化生成 1,4- 丁二醇、四氢呋喃、 γ- 丁内酯等大宗化学品，琥珀酸也广泛用于医药、食品、农药、香料、橡胶、防护涂料、染料和照相材料等工业中。
目前 1,3- 丙二醇主要以石油裂解的乙烯和丙烯为原料化学合成，价格较高；而具有手性的 2,3- 丁二醇化学合成相当困难，因此微生物发酵法生产这两种二元醇具有相当大的优势。 2006 年杜邦公司完成了以基因工程大肠杆菌将葡萄糖一步转化为 1,3- 丙二醇的中试研究，德国和国内的清华大学、大连理工大学也相继完成了用天然菌株将甘油转化为 1,3- 丙二醇的中试试验。 近几年甘油生物转化发酵生产 1,3- 丙二醇工艺研究主要包括两步发酵法、葡萄糖辅助发酵、混菌发酵 、 微氧发酵、生物柴油副产粗甘油的直接利用等。 微生物法生产 2,3- 丁二醇在二战期间的德国就实现了工业化生产，近几年又引起企业界的关注。相对于产 1,3- 丙二醇的菌种，用于生产 2,3- 丁二醇的菌株具有更宽的底物食谱，己糖、戊糖、特定的二糖、糖醛酸都可以作为底物，适合于非粮原料，如甘蔗汁（ Marilia et al, 2001 ）、木质纤维素 (Yu et al, 1985; Frazer and McCaskey, 1991 ) 、菊芋等的利用。
与化学法相比，生物法生产二元醇和有机酸利用的是绿色资源，是一条绿色化工路线，生产过程中排放的 CO2 较少，并且琥珀酸生产还消耗 CO2, 理论上每生成 l mol 琥珀酸要消耗 l mol CO2 。 尽管如此，微生物发酵过程中排放 CO2 也是一个不容忽视的问题。仅乙醇、 1,3- 丙二醇、 2,3- 丁二醇、乳酸、丁醇 / 丙酮、谷氨酸等几种大宗发酵产品，二氧化碳的排放量就将达到 3.5-6 亿吨 / 年。每生产 1 吨 1,3- 丙二醇将产生约 0.3-0.5 吨 CO2 ，其中甘油发酵约排放 0.1 吨 CO2 ，其余部分来自发酵和分离的能源消耗。就发酵自身而言，二氧化碳排放实际上是浪费了原料，如果能将排放的 CO2 回收用于生产琥珀酸，实现 1,3- 丙二醇与琥珀酸联产，就能提高原料利用率， 减少温室气体的排放 。利用 CO2 生产琥珀酸通常采用高产琥珀酸的菌种（如 产琥珀酸厌氧螺菌 (Anaerobiospirillum succiniproducens) 和 产琥珀酸放线杆菌 (Actinobacillus succinogenes) ）或基因工程菌（如大肠杆菌），这样就需要不同的培养体系，实施起来比较麻烦。如何在同一培养体系中实现 CO2 的回收利用和上述二元醇和有机酸的联产？已有的研究表明，甘油在 Klebsiella pneumoniae 内代谢的过程中，除 1,3- 丙二醇作为主要代谢产物外，琥珀酸作为三羧酸 中间产物以及厌氧代谢的终端还原产物，其它副产物包括 乙酸、乳酸、乙醇、 2,3- 丁二醇等。从经济效益角度看，乙酸、乙醇比较廉价，但却是主要副产物，而琥珀酸、乳酸和 2,3- 丁二醇经济性较好， 但 琥珀酸 产量较低。如果能通过代谢调控， 利用 Klebsiella pneumoniae 生产 1,3- 丙二醇的同时 将自身排放的 CO2 再用于合成琥珀酸，那么就能实现二元醇和有机酸的联产，减少廉价的副产物，提升甘油生物转化过程的整体经济效益。从 NADH2 和 ATP 平衡的角度分析，琥珀酸途径完全可以取代乙酸和乙醇途径。通过升高发酵罐压力、改用碳酸盐调节 pH ，有望使代谢倾向琥珀酸途径。调高罐压就意味着增加发酵罐内 CO2 和 H2 的分压，从而抑制甲酸裂解酶的活性，进而影响丙酮酸的进一步代谢，丙酮酸积累将引发一系列代谢变化。 K. pneumoniae 的生长过程表现出先产酸后产醇的代谢生理特征，如果人为补加一些代谢有机酸，将会改变代谢的进程，有利于 1,3- 丙二醇的形成。甘油与糖类（葡萄糖、果糖、木糖或阿拉伯糖等）共发酵时有利于提高甘油的转化率，也有利于 乳酸、 2,3- 丁二醇、琥珀酸等副产物的形成。单独以糖类（葡萄糖、菊芋块茎水解液、菊芋等植物秸秆水解液等）为发酵底物时， Klebsiella pneumoniae 的主要代谢产物是 2 ， 3- 丁二醇，副产物是 乙酸、乙醇、乳酸、琥珀酸等。通过代谢调控同样有可能实现 2 ， 3- 丁二醇与乳酸和琥珀酸的联产。
产物分离是二元醇和有机酸联产面临的新问题。 盐析萃取或双水相萃取技术能够很好地解决现有的双二元醇和有机酸分离提取方法中存在的步骤多、收率低、成本高等突出问题。采用盐析萃取技术分离双二元醇和有机酸，即将无机盐和有机溶剂同时加入发酵液，在一定范围内发酵液可形成两相。上相即为富含目标产物的溶剂相或萃取相，下相为富盐相或萃余相。含有菌体的发酵液经盐析萃取后在两相之间形成固相层，由细胞、蛋白、核酸、多糖等组成。盐析萃取体系中所选用的无机盐包括可溶性磷酸盐、硫酸盐和碳酸盐等，有机溶剂包括甲醇、乙醇、丙醇、丁醇和丙酮等。前期的研究结果表明，盐析萃取体系适合于亲水性产物的提取分离，特别是二元醇和有机酸等生物基化学品，一步萃取收率通常在 90% 以上，而且可以将细胞和发酵液的分离与产物的萃取合二为一，是一种简单实用的分离集成技术。合理地设计萃取策略，采取不同的盐析萃取体系组合，第一步将 1 ， 3- 丙二醇和 / 或 2 ， 3- 丁二醇萃取在上相，乳酸和琥珀酸萃取在下相，第二步再将有机酸萃取到上相，这样就可以实现二元醇和有机酸的分离。第一步萃取上相中的 1 ， 3- 丙二醇和 / 或 2 ， 3- 丁二醇可以通过蒸馏、精馏等方法将有机溶剂回收，并将 1 ， 3- 丙二醇和 2 ， 3- 丁二醇分离开来；第二步萃取上相中的乳酸和琥珀酸可通过结晶、电渗析或酯化精馏等方法将不同有机酸进行分离。可溶性碳酸盐与有机溶剂形成萃取体系的下相或萃余相可以用来调节发酵的 pH ，发酵尾气中的 CO2 可以用来回收萃余相中的碳酸盐，当然尾气中 CO2 与相应碱或氧化物反应形成的产物（碳酸盐）也可以用来调节 pH 或者用作盐析剂。总之，发酵、萃取、 CO2 回收利用可以藕合在一起。
本发明提出加压发酵和碳酸盐调节 pH- 盐析萃取 -CO2 固定化耦合生产二元醇和有机酸的新思路，以期建立一个绿色、高效、低成本的联产新工艺。
发明内容
为解决现有海参有效成分提取中存在的目标产物单一、其它有效成分损失大、成本高、操作过程 繁琐 、生产周期长、溶剂耗量大等问题，本发明提供了一种亲水性低分子有机物 / 盐形成的新型双水相体系及疏水性有机物 / 亲水性低分子有机物 / 盐 / 水三液相体系从海参加工蒸煮液中同时提取分离多种有效成分的方法。
本发明的技术方案如下：
一种盐析萃取分离海参有效成分的方法，其操作步骤如下： 向海参提取液中，直接加入某些电解质和普通有机溶剂，利用盐离子的水化作用，形成有机溶剂 / 盐多相盐析萃取体系。根据溶解度差异，海参中极性不同的有效成分在各相中的分配不同，从而实现有效成分的分离与浓缩。如：将盐与 亲水性低分子有机物 按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成双水相：海参醇溶性有效成分富集在上相，蛋白质和多糖主要分配到上相和下相之间的固相中，重金属则被富集在下相；另外将盐、 亲水性低分子有机物以及疏水性有机物 按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成三液相盐析萃取体系 ： 海参油脂富集在上相，少量可溶性蛋白和多糖分配在中相， 绝大部分 蛋白和多糖分配在中相与下相之间的固相中，同时重金属被富集在下相。
其中双水相体系中盐的质量百分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ， 萃取温度为 15~40℃，搅拌、静置分相的时间为0.1~4.0 h ， 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ；三液相体系盐的质量百分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ，疏水性有机物的质量百分数为 10~40% ，搅拌均匀、静置分相 0.1~4.0 h 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ， 萃取温度为 15~40 ℃。
所适用的海参提取液是指含有海参有效成分的液体，包括鲜海参汁液或其浓缩液，也可以是海参加工蒸煮液或其浓缩液。海参加工蒸煮液是指蒸煮海参所得废液。
所选用的 亲水性低分子有机物 为 乙醇、乙二醇、正丙醇、异丙醇、 1 ， 2- 丙二醇、 1 ， 3- 丙二醇、正丁醇、异丁醇、叔丁醇 、 1, 2- 丁二醇、 1, 3- 丁二醇、 1, 4- 丁二醇、 2, 3- 丁二醇、 丙酮中的一种或几种组合； 所选用的盐为氯化钠、氯化锂、硫酸铵、硫酸钠、碳酸钠、碳酸钾、醋酸钠、醋酸钾、磷酸钾、磷酸二氢钾、磷酸氢二钾、柠檬酸钠、柠檬酸钾、草酸钠、草酸钾中的一种或几种组合； 所选用的疏水性有机物为乙酸甲酯、乙酸乙酯、正己烷。
其中 萃取操作可以是一次萃取、多次萃取、连续萃取或逆流萃取。
本发明的有益效果是和现有的提取分离方法相比较，本发明利用多相盐析萃取体系对极性不同的多种有效成分进行同步提取与分离，获得了海参油、醇溶性有效成分（以乙醇为萃取剂可制作海参酒）、粗蛋白和多糖等多种产品，完成目标产物的初步纯化。经过萃取后获得的固相冷干粉中有害重金属离子（砷、汞、铅、镉）的含量显著降低， 有益离子（钙、铁，镁、锌）大部分保留。 整个生产工艺操作简单、生产成本低、 周期短、萃取条件温和、 有机溶剂廉价易回收、粘度低传质分相快、易于产业化 、产品符合食品安全的要求 。
本发明针对目前 1 ， 3- 丙二醇和 2 ， 3- 丁二醇发酵中二氧化碳排放、原料利用率低、发酵产品单一等问题，提出加压发酵 / 碳酸盐调节 pH- 盐析萃取 -CO2 固定化耦合生产二元醇和有机酸的新方法。
本发明的技术方案如下：
本发明利用克雷伯氏杆菌（ Klebsiella pneumoniae ）在不同的原料下进行发酵，通过升高发酵罐压力为 0.01-0.5atm 、碳酸盐调节发酵 pH 6.0 或 7.0 、调控通气量 0.01-0.8vvm ，气体为 CO2 、发酵尾气或含 CO2 的混合气，在同一培养体系中实现尾气二氧化碳的回收利用与二元醇和有机酸的联产。采用两步盐析萃取将二元醇和有机酸分开。
其中发酵原料可以为 95% 工业甘油、 60-85% 生物柴油副产甘油、葡萄糖、菊芋块茎、秸秆水解液等中的一种或者几种按比例混合。
碳酸盐为碳酸钠、碳酸钾、碳酸铵、碳酸钙、碳酸镁等，它们可以是发酵尾气中 CO2 与对应碱或氧化物（氢氧化钠、氢氧化钾、氨水、氢氧化钙或氧化钙、氢氧化镁或氧化镁）反应的产物，或是可溶性碳酸盐（ 碳酸钠、碳酸钾或碳酸铵）与有机溶剂形成盐析萃取体系的萃余相。
二元醇是 1,3- 丙二醇、 2,3- 丁二醇，有机酸是乳酸、琥珀酸。
二元醇以 1,3- 丙二醇为主要产物时，调节发酵 pH 为 7.0 ；二元醇以 2,3- 丁二醇为主要产物时，调节发酵 pH 为 6.0 。
本发明可以采用间歇发酵、批式流加或连续发酵等方式生产。
盐析萃取体系中所选用的盐析剂包括可溶性磷酸盐、硫酸盐、碳酸盐或者它们的组合，有机溶剂包括甲醇、乙醇、丙醇、丁醇、丙酮或者它们的组合。两步萃取中第一步将 1 ， 3- 丙二醇和 / 或 2 ， 3- 丁二醇萃取在上相或有机相，乳酸和琥珀酸萃取在下相或富盐相，第二步再将有机酸从第一步的富盐相中萃取到上相，实现二元醇和有机酸的分离。
萃取可以采用单级、多级或连续萃取方式。
本发明的效果和益处：
（一）同一株菌完成二元醇和有机酸的联产，实现 CO2 零排放。
按照以往的工艺要完成二元醇和有机酸的联产通常需要两株不同的菌种，在不同的发酵罐和不同的培养条件下进行，这无疑增加了实施的难度。利用 Klebsiella pneumoniae 在转化甘油或葡萄糖生产 1,3- 丙二醇和 / 或 2 ， 3- 丁二醇的同时副产琥珀酸和乳酸，通过提高罐压、碳酸盐调节 pH 、回用副产有机酸等措施调控细胞代谢，增加琥珀酸和乳酸的产量，未被利用的 CO2 再用碳酸盐吸收循环，达到 CO2 零排放和原料充分利用的目的。
（二）采用两步盐析萃取分离二元醇和有机酸，第一步萃取可以直接从发酵液中分离出二元醇，第二步萃取将 有机酸从第一步的富盐相中分离出来。
可溶性无机盐与有机溶剂形成的盐析萃取体系可将固液分离、浓缩、除蛋白和有机酸等多步工序整合为一，极大地简化分离工艺。合理地利用二元醇与有机酸在不同盐析萃取体系中的不同分配行为，采取两步萃取的策略将两类产物分离开来。萃取操作易于放大和工业化，两步萃取前后紧密衔接，极易实施。
盐析萃取可以通过蒸馏和精馏的方式有效地克服萃取剂难回收的问题，但是其盐的用量较大，而盐的回收是阻碍盐析 - 萃取工业化的最重要因素之一。我们提出采用二氧化碳反应沉淀方法回收该盐析萃取体系中的碳酸盐，不仅可以降低分离能耗和成本，而且可以将生产过程中产生的大部分二氧化碳回收，极大的缓解了环境压力。
（三）可溶性碳酸盐与有机溶剂盐析萃取的萃余相用于调节发酵 pH ，调控二元醇和有机酸联产，实现了发酵 - 分离 -CO2 固定的集成一体化。
用可溶性碳酸盐与有机溶剂盐析萃取时，其部分萃余相可回用于发酵过程的 pH 调节，实现发酵 - 分离 -CO2 固定化的集成。一方面，利用部分富含碳酸盐的下相代替现有的氢氧化钠或氢氧化钾碱液调节发酵过程的 pH ，节省除盐成本并有利于琥珀酸的生产；另一方面萃余相中还含有发酵剩余的底物和乙酸、琥珀酸等副产物，琥珀酸等有机酸的添加有助于提高 1,3- 丙二醇和 2 ， 3- 丁二醇的浓度和转化率，这样既可以节约分离成本，又有利于提高原料利用率。
具体实施方式
以下结合技术方案详细叙述本发明的具体实施方式。
实施例 1 ：流加碳酸钠溶液调节发酵 pH 实现 1,3- 丙二醇、乳酸和琥珀酸联产
（一） 菌种：克雷伯氏杆菌（ Klebsiella pneumoniae CGMCC 2028 ）
（二）培养基组成：
① 种子培养基（ 1L ）：
甘油： 20g ； KH2PO4 ： 1.3g ； CaCO3 ： 2g ； K2HPO4·3H2O ： 4.454g ； (NH4)2SO4 ： 2.0g ； MgSO4·7H2O ： 0.2g ；酵母粉： 1g ；微量元素 A ： 2mL ； Ca2+ 溶液： 1mL ； Fe2+ 溶液： 1mL 。
② 发酵培养基（ 1L ）：
甘油： 40g ； KH2PO4 ： 1.36g ；柠檬酸： 0.42g ； MgCl2·6H2O ： 0.26g ； (NH4)2SO4 ： 6.61g ；酵母粉： 1g ；微量元素 B ： 5mL 。
③ Fe2+ 溶液组成（ 100 ml ）：饱和盐酸： 0.4 ml ； FeSO4·7H2O ： 0.5 g ；
④ Ca2+ 溶液（ 100 ml ）： CaCl2 ： 2g 。
⑤ 微量元素 A 组成（ 1L ）：
饱和盐酸： 0.9mL ； MnCl2·4H2O ： 100mg ； NiCl2·6H2O ： 25mg ； H3BO3 ： 60mg ； ZnCl2 ： 70mg ； NaMoO4·2H2O ： 35mg ； CuCl2·2H2O ： 20mg ； CoCl2·6H2O ： 200mg 。
⑥ 微量元素 B 组成（ 1L ）：
饱和盐酸： 10mL ； NaMoO4·2H2O ： 0.005g ； FeCl3·6H2O ： 5.4g ； CoCl2·6H2O ： 0.47g ； H3BO3 ： 0.06g ； MnCl2·4H2O ： 0.17g ； ZnCl2·6H2O ： 0.68g ； CuCl2·2H2O ： 0.47g 。
（三）发酵控制： 5L 发酵罐，批式流加发酵，装液量 3L ，发酵温度 37℃ ，罐压控制为 0.05atm ，流加 85% 的生物柴油副产甘油，初始浓度为 40g /L ，接种量为 10% （ v/v ），搅拌转速为 300 r/min ， 通空气量为 0.04vvm ，分别采用 5mol/L 的氢氧化钠溶 液和 2.5mol/L 碳酸钠溶液调节 pH 为 7.0 。发酵开始 4 小时后检测甘油浓度，流加甘油，使甘油浓度控制在 20g /L 左右，发酵至 33 小时结束。
（四）发酵结果：采用 5mol/L 氢氧化钠溶液调节 pH 时，发酵 33h ，菌种在 8h 时生长最为旺盛， OD 为 8.26 。产物 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的终浓度分别为 52.32 、 8.24 、 30.23 和 10.02g /L ； 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的质量转化率分别为 37% 、 6% 、 22% 和 7% ，甘油的总质量转化率为 72% 。
采用 2.5mol/L 碳酸钠溶液调节 pH 时，发酵 33 小时，菌种在 9h 时生长最为旺盛， OD 为 9.77 。产物 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的终浓度分别为 46.67 、 22.67 、 56.13 和 8.09g /L 。 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的质量转化率分别为 30% 、 14% 、 36% 和 5% ，甘油的总质量转化率为 85% 。碳酸钠溶液调节发酵 pH 相对于氢氧化钠调节发酵 pH 而言， 1,3- 丙二醇和 2,3- 丁二醇的浓度略有下降，但琥珀酸和乳酸的浓度则分别提高了 175% 和 86% ，甘油总转化率提高了 13% 。
实施例 2 ：流加碳酸钠溶液调节 pH 实现 1,3- 丙二醇、琥珀酸和乳酸联产
（一）菌种：克雷伯氏杆菌（ Klebsiella pneumoniae CGMCC 2028 ）
（二）培养基组成：发酵培养基酵母粉： 2g ，其它 同实施例 1 。
（三）发酵控制： 5L 发酵罐，批式流加发酵，装液量 3L ，发酵温度 37℃ ，流加 95% 工业甘油，初始浓度为 40g /L ，接种量为 10% （ v/v ），搅拌转速为 300 r/min ， 通空气量为 0.02vvm ，分别采用 5mol/L 氢氧化钠溶液和 2.5mol/L 碳酸钠溶液调节 pH 为 7.0 。发酵开始 4 小时后检测甘油浓度，流加甘油，使甘油浓度控制在 15-20g/L 左右，发酵至 36 小时结束。
（四）发酵结果：采用 5mol/L 氢氧化钠溶液调节 pH 时，发酵 36h ，菌种在 14h 时生长最为旺盛， OD 为 9.47 ，发酵结束 OD 降至 6.51 。产物 1,3- 丙二醇、琥珀酸、乳酸、 2,3- 丁二醇和乙醇的最终浓度分别为 65.50 、 9.65 、 43.76 、 10.79 和 6.86g /L ； 1,3- 丙二醇、琥珀酸、乳酸、 2,3- 丁二醇和乙醇的质量转化率分别为 36% 、 5% 、 24% 、 6% 和 4% ，甘油的总质量转化率为 75% 。
采用 2.5mol/L 碳酸钠溶液调节 pH 时，发酵 36h ，菌种在 16h 时生长最为旺盛， OD 为 13.2 ，发酵结束 OD 降至 8.56 。产物 1,3- 丙二醇、琥珀酸、乳酸、 2,3- 丁二醇和乙醇的最终浓度分别为 65.20 、 40.47 、 60.97 、 13.15 和 10.04g /L ； 1,3- 丙二醇、琥珀酸、乳酸、 2,3- 丁二醇和乙醇的质量转化率分别为 26% 、 16% 、 25% 、 5% 和 4% ，甘油的总质量转化率为 76% 。碳酸钠溶液调节发酵 pH 相对于氢氧化钠调节发酵 pH 而言更有利于琥珀酸的生产，琥珀酸的浓度和转化率分别提高了 319% 和 207% ，乳酸的浓度提高了 39% ， 1,3- 丙二醇浓度相差不大， 2,3- 丁二醇浓度略有提高。
实施例 3 ：碳酸钠 / 乙醇萃余相调节 pH 值的 1,3- 丙二醇批式流加发酵
（一）菌种：克雷伯氏杆菌（ Klebsiella pneumoniae CGMCC 2028 ）
（二）培养基组成：同实施例 1 。
（三）发酵控制： 5L 发酵罐，批式流加发酵，装液量 3L ，发酵温度为 37℃ ，搅拌转速为 200r/min ，接种量为 10%(v/v) ， 通空气量为 0.02vvm ，发酵过程中分别采用 5mol/L 氢氧化钠溶液和碳酸钠 / 乙醇盐析萃取体系萃余相调节 pH 为 7.0 。 流加 95% 工业甘油，初始浓度为 40g /L ，待发酵液中甘油消耗至一定浓度时 (20g/ 左右 ) 开始流加甘油，发酵过程中控制甘油浓度在 15-25g/L ， 发酵至 36 h 结束。
（四）发酵结果：采用 5mol/L 氢氧化钠溶液调节 pH 时，发酵 36h 后，产物浓度达到最佳，最终残余甘油浓度为 18.42g /L, 最大菌体 OD 值为 8.1 ，最终 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的浓度分别为 55.41 、 6.85 、 25.63 和 7.68g /L ； 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的质量转化率分别为 49% 、 4% 、 19% 和 6% ，甘油的总质量转化率为 78% 。
采用碳酸钠 / 乙醇盐析萃取体系萃余相调节 pH 时， 发酵 36 h 后，残余甘油浓度为 18.40g /L, 最大菌体 OD 值为 12.4 ，最终 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的浓度分别为 64.29 、 10.34 、 57.92 和 8.08g /L ； 1,3- 丙二醇、琥珀酸、乳酸和 2,3- 丁二醇的质量转化率分别为 46% 、 4% 、 31% 和 5% ，甘油的总质量转化率为 86% 。碳酸钠 / 乙醇盐析萃取体系萃余相回用于 1,3- 丙二醇发酵过程相对于 氢氧化钠碱液调节 pH 而言， 1,3- 丙二醇和琥珀酸的转化率略有下降，但是二者的浓度分别上升了 16% 和 49% ；乳酸的浓度和转化率更是分别提高了 126% 和 60% ，浓度达到了 57.92g /L ；而甲酸和乙酸等其他主要杂质酸浓度分别下降了 123% 和 122% 。
实施例 4 ：碳酸钠 / 乙醇萃余相调节 pH 值的 2,3- 丁二醇批式流加发酵
（一）菌种：克雷伯氏杆菌（ Klebsiella pneumoniae CICC 10011 ）
（二）培养基组成：
① 种子培养基（ 1L ）： 葡萄糖： 80g ； (NH4)2HPO4 ： 6.0g ； KCl ： 1.8g ； EDTA ： 0.51g ； MgSO4·7H2O ： 0.6g ； FeSO4·7H2O ： 0.0225g ； MnSO4·7H2O ：0.0038g ； ZnSO4·7H2O ： 0.0075g ； 柠檬酸 ： 0.21g ； 柠檬酸钠 ： 0.294g
② 发酵培养基（ 1L ）： 葡萄糖： 50g ； (NH4)2SO4 ： 6.61g ； KH2PO4 ： 1.36g ；酵母粉： 1g ；微量元素 B ： 5ml ； MgCl2·6H2O ： 0.26g ；柠檬酸： 0.42g ；
③ 微量元素 B 组成（ 1L ）： 饱和盐酸： 10mL ； NaMoO4·2H2O ： 0.005g ； FeCl3·6H2O ： 5.4g ； CoCl2·6H2O ： 0.47g ； H3BO3 ： 0.06g ； MnCl2·4H2O ： 0.17g ； ZnCl2·6H2O ： 0.68g ； CuCl2·2H2O ： 0.47g 。
（三）发酵控制： 5L 发酵罐，批式流加发酵，装液量 3L ，发酵温度为 37℃ ，搅拌转速为 300 r/min ，接种量为 5%(v/v) ， 通空气量为 0.1vvm ，发酵过程中分别采用 5mol/L 的氢氧化钠溶液和碳酸钠 / 乙醇盐析萃取体系萃余相调节 pH 为 6.0 。 当葡萄糖浓度低于 50 g /L 时补加一定量固体葡萄糖， 并通过定时补加葡萄糖使其浓度保持在 30-50g/L 之间 。
（四）发酵结果：采用 5mol/L 氢氧化钠溶液调节 pH 时，发酵 52h 时，产物浓度达到最佳，最终残糖浓度为 43.43g /L, 最大菌体 OD 值达到了 15.91 ； 2,3- 丁二醇、乙偶姻、乳酸和琥珀酸的终浓度分别为 62.63 、 15.62 、 14.26 和 4.13g /L ； 2,3- 丁二醇、乙偶姻、乳酸和琥珀酸的质量转化率分别为 36% 、 9% 、 8% 和 2% ，葡萄糖的总质量转化率为 55% 。
采用碳酸钠 / 乙醇盐析萃取体系萃余相调节 pH 时， 发酵 66 h 后，最终残糖浓度为 49.49g /L, 最大菌体 OD 值达到了 15.73 ， 2,3- 丁二醇、乙偶姻、乳酸和琥珀酸的终浓度分别为 78.18 、 10.23 、 18.32 和 8.13g /L ； 2,3- 丁二醇、乙偶姻、乳酸和琥珀酸的质量转化率分别为 41% 、 5% 、 10% 和 4% ，葡萄糖的总质量转化率为 60% 。碳酸钠 / 乙醇盐析萃取体系萃余相 回用于 2,3- 丁二醇发酵时， 2,3- 丁二醇、乳酸和琥珀酸的浓度分别比选用氢氧化钠碱液调节 pH 时提高了 25% 、 28% 和 97% ，三者的转化率分别提高了 14% 、 17% 和 79% 。
实施例 5 ： 1,3- 丙二醇、 2,3- 丁二醇与乳酸和琥珀酸的盐析萃取
对 1,3- 丙二醇、 2,3- 丁二醇、乳酸和琥珀酸联产的发酵液进行盐析萃取，发酵液中 1,3- 丙二醇、 2,3- 丁二醇、乳酸和琥珀酸浓度分别为 46.67 、 8.36 、 56.13 和 22.67g /L ，向 7.0g 的发酵液中加入 1.0g 碳酸钠和 2.0g 乙醇，搅拌均匀，室温静置，形成两相，上相为含有 1,3- 丙二醇和 2,3- 丁二醇的醇相，下相为富含乳酸和琥珀酸的盐相。 1,3- 丙二醇的分配系数为 7.0 ，收率为 90% ， 2,3- 丁二醇的分配系数和收率分别为 8.0 和 94% ； 74% 乳酸和 95% 琥珀酸分配在盐相。向盐相中加入 0.5g 磷酸氢二钾和 2.5g 乙醇进行第二次萃取，上相中乳酸的收率为 75% ，琥珀酸收率为 68% 。

Claims (1)

1. 一种二元醇与有机酸联产与分离方法，是以工业甘油、生物柴油副产的甘油、葡萄糖、菊芋块茎、秸秆水解液中的一种或几种为原料，在同一培养体系中实现二元醇和有机酸的联产与 CO2 的回收利用，采用两步盐析萃取将二元醇和有机酸分开，其特征是：
（ 1 ）菌种：克雷伯氏菌属（ Klebsiella pneumoniae ）；
（ 2 ）联产调控方式：利用碳酸盐调节发酵 pH 6.0 或 7.0 ，通 0.01-0.8vvm CO2 、发酵尾气或含 CO2 的混合气，发酵罐压力为 0.01-0.5atm ；
（ 3 ）产品分离方法：第一步盐析萃取将二元醇萃取在上相或有机相，有机酸萃取在下相或富盐相；第二步再将有机酸从第一步的富盐相中萃取到上相，从而实现二元醇和有机酸的分离。
2. 根据权利要求 1 所述的方法，其特征是发酵原料为 95% 工业甘油、 60-85% 生物柴油副产甘油、葡萄糖、菊芋块茎、秸秆水解液中的一种或者几种混合。
3. 根据权利要求 1 所述的方法，其特征是调节 pH 的碳酸盐为碳酸钠、碳酸钾、碳酸铵、碳酸钙、碳酸镁。
4 根据权利要求 1 、 2 或 3 所述的 方法 ，其特征是二元醇是 1,3- 丙二醇、 2,3- 丁二醇，有机酸是乳酸、琥珀酸。
5. 根据权利要求 1 、 2 或 3 所述的 方法 ，其特征是发酵方式是间歇发酵、批式流加或连续发酵。
6. 根据权利要求 4 所述的 方法，其特征是 二元醇以 1,3- 丙二醇为主要产物时，调节发酵 pH 为 7.0 ；
7. 根据权利要求 4 所述的 方法，其特征是 二元醇以 2,3- 丁二醇为主要产物时，调节发酵 pH 为 6.0 。
8. 根据权利要求 1 、 2 或 3 所述的 方法 ，其特征是 盐析萃取体系所选用的无机盐是可溶性磷酸盐、硫酸盐和碳酸盐的一种或几种组合。
9. 根据权利要求 1 、 2 或 3 所述的 方法 ，其特征是 有机溶剂是甲醇、乙醇、丙醇、丁醇和丙酮的一种或几种组合。
10. 根据权利要求 3 所述的方法，其特征是碳酸盐是发酵尾气中 CO2 与对应碱或氧化物反应的产物，或是可溶性 碳酸盐与有机溶剂形成萃取体系的萃余相。
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