CN104003844A - Method for separating 2,3-butanediol from fermentation broth by coupling of extraction and fermentation - Google Patents

Method for separating 2,3-butanediol from fermentation broth by coupling of extraction and fermentation Download PDF

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CN104003844A
CN104003844A CN201410223050.6A CN201410223050A CN104003844A CN 104003844 A CN104003844 A CN 104003844A CN 201410223050 A CN201410223050 A CN 201410223050A CN 104003844 A CN104003844 A CN 104003844A
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butanediol
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sugar
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CN104003844B (en
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戴建英
刘春娇
修志龙
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Dalian University of Technology
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Abstract

The invention discloses a method for separating 2,3-butanediol from fermentation broth by coupling of extraction and fermentation. The method comprises the steps of adding a certain amount of sugar and/or inorganic salts into 2,3-butanediol fermentation broth and dissolving to obtain a mixed solution containing sugars; adding an organic solvent to the mixed solution containing sugars, mixing by shaking, standing the mixed solution at 15-40 DEG C or centrifuging until two phases are separated out, namely, the top phase is a 2,3-butanediol-enriched organic phase and the bottom phase is a sugar-enriched aqueous phase; distilling the top phase under a reduced pressure at a temperature below 105 DEG C to remove the organic solvent and water to obtain the crude 2,3-butanediol; removing the organic solvent from the bottom phase, adding water and diluting until the sugar concentration of the solution is 80-200g/L, inoculating a thallus and carrying out 2,3-butanediol fermentation. The method disclosed by the invention has the advantages of simple operation and low separation energy consumption, the coupling of fermentation and separation is achieved and the method is a method for separating 2,3-butanediol from fermentation broth with an industrial application prospect.

Description

The method of 2,3-butanediol in extraction and fermentation and coupling separate fermentation liquid
Technical field
The invention belongs to technical field of bioengineering, relate to the isolation technique of microbial fermentation solution, particularly a kind of method of utilizing 2,3-butanediol in abstraction technique coupled fermentation separate fermentation liquid.
Background technology
2,3-butanediol is a kind of important industrial chemicals and liquid fuel, is widely used in multiple fields such as chemical industry, medicine, food, fuel and aerospace.2,3-butanediol dehydrogenation obtains di-acetyl, is flavour of food products additive and antimicrobial additive.2,3-butanediol catalytic dehydration forms methylethylketone, is efficient liquid fuel additive, is also good low boiling point solvent, is applied to the industries such as resin, ink, glue, lubricant; Dehydration obtains 1,3-butadiene, can be used to the material such as synthetic rubber, polyurethane; Esterified form 2,3-butanediol can be applicable to medicine, makeup, washing composition etc.2,3-butanediol has higher fuel value and octane value very high, is the liquid fuel of high-quality, can be used for the fields such as aerospace.Because 2,3-butanediol has 2 chiral atoms, isomer is more, chemical method synthetic more difficult and process is loaded down with trivial details, not easy to operate, energy consumption is large, cost is high, so 2, the production of 3-butyleneglycol mainly, taking renewable resources as raw material, is converted into target product by microbial metabolism by monose.Biotransformation method produces 2,3-butyleneglycol, except target product, also has the byproducts such as acetic acid, ethanol, lactic acid, succsinic acid in fermented liquid, and the small molecules such as soluble proteins, nucleic acid, polysaccharide and a large amount of thalline, organic salt, inorganic salt, cause fermented liquid composition very complicated.2,3-butanediol boiling point is high, and has strongly hydrophilic, and in fermented liquid, production concentration is lower, causes its extraction process complexity, high expensive.Therefore, the separating-purifying of 2,3-butanediol is the bottleneck problem that hinders its mass-producing, suitability for industrialized production.
For the separate targets product from fermented liquid of economical and efficient, develop a lot of separation methods, comprise evaporation, distillation, membrane filtration, pervaporation, ion exchange chromatography, liquid-liquid extraction, reaction, extraction etc., wherein extraction is because of easily amplification, less energy-consumption, and industrial prospect is good.Traditional distillation and rectification method separating 2,3-butanediol energy consumption are huge, and in fermented liquid, the existence of biomacromolecule causes that heat transfer efficiency declines, product yield is lower.Adverse current vaporizing extract process was once considered to be best suited for the separating and extracting method of 2,3-butanediol, but this separation method energy consumption is larger.2,3-butanediol in salting out separate fermentation liquid, its rate of recovery is higher.Afschar etc. utilize salt of wormwood to saltout and separate the 2,3-butanediol in molasses fermented liquid, and its rate of recovery can reach 94%-96%.But the higher and fermented liquid of salt usage quantity need to carry out a series of pre-treatment in the method, operate too loaded down with trivial detailsly, be unsuitable for large-scale industrialization application.Reaction, extraction can reduce the wetting ability of product and improve partition ratio; But reagent also can react with by product and impurity.Organic solvent extractionprocess is the effective ways of separating 2,3-butanediol, but the rate of recovery is on the low side and solvent usage quantity is huge because the high-hydrophilic of 2,3-butanediol causes, and is only limited to laboratory scale and is not suitable for large-scale industrial production.Traditional high molecular polymer double-aqueous phase system extraction conditions gentleness, easily amplifies, can operate continuously; But because the most viscosity of water soluble polymer is larger, be difficult for quantitative work, be difficult to volatilization, need to strip, later separation is comparatively bothered, and superpolymer price general charged is more expensive, its industrial applications is restricted.We have developed 1,3-PD and 2,3-butanediol (Chinese patent: CN101012151A in the novel double-aqueous phase system salting-out extraction fermented liquid that utilizes organic solvent and inorganic salt composition; CN101012152A) technology, achieves satisfactory results.This technological operation is simple, mild condition, selectivity is high, energy consumption is little.Although the consumption of salt is lower than saltouing in this technology, concentration is still very high, and the salt of lower phase must be recycled to reduce separation costs.
At present, also there is no that separation method is simple, good separating effect, energy consumption be low, be applicable to the method for separating 2,3-butanediol from fermented liquid of suitability for industrialized production.
Summary of the invention
The object of the invention is to, for in above-mentioned current salting-out extraction method separate fermentation liquid 2, the 3-butyleneglycol at present high salt concentration of phase reclaims difficulty, the discharge of wastewater problems that wait more, propose in a kind of extraction and fermentation coupling separate fermentation liquid 2, the novel method of 3-butyleneglycol, the method will add sugar or sugar and a small amount of inorganic salt extraction 2 in fermented liquid, 3-butyleneglycol, extraction 2, after 3-butyleneglycol lower through dilution after be directly used in 2, thereby the fermentation of 3-butyleneglycol realizes the recycling of lower phase, avoid the problem that under salting-out extraction, phase salt need to reclaim, be a kind of have very much a prospects for commercial application from fermented liquid, separate 2, the method of 3-butyleneglycol.
For realizing object of the present invention, the technical solution used in the present invention is:
In extraction and fermentation coupling separate fermentation liquid, a method for 2,3-butanediol, is characterized in that, said method comprising the steps of:
1) by sugar or/and soluble inorganic salt is dissolved in 2,3-butanediol fermented liquid, obtain sugary mixed solution;
2) to step 1) sugary mixed solution in add organic solvent, vibration mixes, mixed solution leaves standstill or is centrifugal to phase-splitting at 15～40 DEG C, obtains the organic phase on upper strata and the water of the lower floor system that coexists;
3) step 2) in organic phase, carry out underpressure distillation under lower than 105 DEG C of conditions and remove organic solvent and water, obtain 2,3-butanediol crude product;
4) step 2) described in water, remove organic solvent, adding water to be diluted to sugared concentration is 80～200g/L, access thalline, carry out the fermentation of 2,3-butanediol.
In technique scheme, step 4) described in water remove and add water to be diluted to sugared concentration after organic solvent to be preferably 100～150g/L, more preferably 110～130g/L.
Further, in technique scheme, 2 of the 2,3-butanediol fermented liquid that described 2,3-butanediol fermented liquid is mycetome or removal thalline, 3-butyleneglycol fermentation clear liquid, in 2,3-butanediol fermented liquid, the concentration of 2,3-butanediol is 40～160g/L, be preferably 60～120g/L, more preferably 80～100g/L.
Further, in technique scheme, in step 1) the described sugar concentration in sugary mixed solution is 100～500g/L, is preferably 150～400g/L, more preferably 250～350g/L; The concentration of described soluble inorganic salt in sugary mixed solution is 1～100g/L, is preferably 10～50g/L, more preferably 15～25g/L.
Further, in technique scheme, in step 2) described in sugary mixed solution and the volume ratio of organic solvent be 0.5～2:1, be preferably 1～1.5:1, more preferably 1:1.
Further, in technique scheme, step 1) described in sugar be one or more mixtures that mix with any ratio in glucose, wood sugar, fructose, pectinose, sucrose, maltose, jerusalem artichoke, molasses, xylose mother liquid, be wherein preferably wood sugar, fructose, glucose, sucrose; More preferably glucose.In the present invention, step 1) described sugar is not limited to described sugar, and those skilled in the art can also adopt according to common practise can realize industrial by-products or the waste that other monose, disaccharides, polysaccharide and the main component of technical scheme of the present invention are above-mentioned carbohydrate.
Further, in technique scheme, described soluble inorganic salt is one or more the mixture in ammonium sulfate, dipotassium hydrogen phosphate, Secondary ammonium phosphate, is preferably Secondary ammonium phosphate.
Further, in technique scheme, step 2) described in organic solvent be methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol, amylalcohol, oleyl alcohol, ethyl acetate, butylacetate, methylcarbonate, diethyl carbonate, acetone, dioxane, one or more mixing solutions in tetrahydrofuran (THF), wherein be preferably n-propyl alcohol, Virahol, propyl carbinol, the trimethyl carbinol, the mixing solutions of one or more in amylalcohol, more preferably Virahol, the trimethyl carbinol, the mixing solutions of one or more in amylalcohol, most preferably be the trimethyl carbinol.
Further, in technique scheme, in step 4) described in the method for removal organic solvent be underpressure distillation or air lift method, wherein the temperature of underpressure distillation is 20～50 DEG C, pressure is-0.08～-0.1MPa; The gas that air lift method adopts is CO 2.
In the present invention, the extracting operation mode of utilizing sugar and organic solvent extraction 2,3-butanediol can be intermittently, can be also continuous; Can adopt the mode of multi-stage solvent extraction for the less system of partition ratio.
In the present invention, described 2,3-butanediol fermented liquid can carry out pre-treatment by flocculation, filtration, micro-filtration or centrifugal method, removes thalline; If directly carry out extracting operation without pre-treatment, thalline is distributed in lower phase.
The present invention has following beneficial effect with respect to prior art:
(1) sugared wide material sources and cheapness for extraction, sugar does not react with target component, and sugar is environment gentleness, if do not changed the pH of environment;
(2) be rich in the recyclable rear recycling of organic solvent in the organic phase of 2,3-butanediol, and low more than water of the specific heat of organic solvent, the energy consumption that reclaims 2,3-butanediol in still-process can greatly be reduced;
(3) compared with salt used in traditional salting-out extraction process, required sugared concentration is lower, has avoided the high salt concentration of traditional salt separation extracting process, and equipment is not corroded;
(4) the sugared reusable edible of water, in fermenting process, has reduced processing and the discharge of sewage, has improved the utilization ratio of residual substrate.
To sum up, the present invention adopts extraction to separate typical bio-based chemical-2 with the method for fermentation coupling, 3-butyleneglycol, the method is dissolved in sugar after 2,3-butanediol fermented liquid and obtains 2,3-butanediol with organic solvent extraction, percentage extraction reaches more than 80%, thereby the lower fermentation that is directly used in 2,3-butanediol after dilution realizes the recycling of lower phase, has avoided the problem that under salting-out extraction, phase salt need to reclaim.Method of the present invention is simple to operate, separating energy consumption is low, has realized fermentation and the coupling separating, and is a kind of method of separating 2,3-butanediol from fermented liquid with prospects for commercial application.
Brief description of the drawings
Fig. 1 is the phasor of glucose/trimethyl carbinol system.
Fig. 2 is glucose consumption and the impact of trimethyl carbinol consumption on 2,3-butanediol separating effect, and wherein Figure 1A is glucose consumption and the impact of trimethyl carbinol consumption on 2,3-butanediol partition ratio; Figure 1B is glucose consumption and the impact of trimethyl carbinol consumption on the 2,3-butanediol rate of recovery.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.In following embodiment, if no special instructions, the experimental technique using is ordinary method, and agents useful for same etc. all can be bought from chemistry or biological reagent company.
Describe the specific embodiment of the present invention in detail below in conjunction with technical scheme.
In following embodiment for 2, the bacterial strain of 3-butyleneglycol fermentation is enterobacter cloacae (Enterobacter cloacae), be this laboratory mutagenesis gained, be preserved in Chinese common micro-organisms DSMZ (CGMCC6053).
Seed culture medium (1L): glucose 80g, (NH 4) 2hPO 46.0g, KCl1.8g, EDTA0.51g, MgSO 47H 2o0.6g, trace element: FeSO 47H 2o0.0225g, ZnSO 47H 2o0.0075g, MnSO 47H 2o0.0038g, citric acid 0.21g, Trisodium Citrate 0.294g.
Fermention medium (1L): glucose 135g, (NH 4) 2hPO 418.0g, KCl1.8g, EDTA0.51g, MgSO47H 2o0.88g, trace element: FeSO 47H 2o0.0225g, ZnSO 47H 2o0.0075g, MnSO 47H 2o0.0038g, citric acid 0.21g, Trisodium Citrate 0.294g.
The preparation of 2.2,3-butyleneglycol fermentation liquid:
(1) enterobacter cloacae is seeded in seed culture medium, at 35～38 DEG C, 200～300rpm shaking table cultivation, 12～18h;
(2) bacteria culture fluid of step (1) is accessed in fermention medium by 10% inoculum size, at 37 DEG C, 200rpm shaking table cultivation 48～90h, obtain 2,3-butanediol fermented liquid, remaining sugar concentration 10～15g/L when fermentation ends.
In embodiment, bacteria culture fluid in step (1) also can adopt ferment tank, be specially: use Biotech5L automatic fermenter, bacteria culture fluid in step (1) is linked in 2L fermention medium by 10% inoculum size, controlling pH with 5mol/L NaOH is 5.8, at 37 DEG C, 250r/min condition bottom fermentation.In fermenting process, keep the pressurized air of logical 200ml/L ﹒ min, glucose initial concentration is 135g/L, in fermenting process, add solid glucose, keep in fermenting process in fermented liquid glucose concn in 20～100g/L left and right, cultivate 72～90h, obtain 2,3-butanediol fermented liquid, remaining sugar concentration 10～15g/L when fermentation ends.
In the 2,3-butanediol fermented liquid preparing according to the method described above, the concentration of 2,3-butanediol is 40～160g/L.
2,3-butanediol fermented liquid is through 8000rpm, and 10min is centrifugal, except thalline, obtains 2,3-butanediol fermentation clear liquid.
3. the method for separating 2,3-butanediol from 2,3-butanediol fermented liquid or 2,3-butanediol fermentation clear liquid, comprises the following steps:
1) by sugar or/and inorganic salt are dissolved in 2,3-butanediol fermented liquid or 2,3-butanediol fermentation clear liquid, obtain sugary mixed solution;
2) to step 1) sugary mixed solution in add organic solvent, vibration mixes, mixed solution mixes with XH-C eddy blending machine or electric blender at 15～40 DEG C, room temperature leaves standstill phase-splitting in 12 hours, obtain the two-layer liquid phase of water and the organic phase system that coexists, wherein go up phase (organic phase) and be rich in 2,3-butanediol, wherein organic solvent recovery is reusable, and lower phase (water) is rich in sugar; The concentration of 2,3-butanediol in sampling and measuring upper and lower phase, the partition ratio (K of calculating 2,3-butanediol bD) and the rate of recovery (Y); And measure and compare (R), specific formula for calculation is:
1. compare (R)=V on/ V underwherein V onfor the volume of upper phase, V underfor the volume of lower phase;
2. partition ratio (K bD)=C on/ C under, wherein C onfor upper middle 2,3-butanediol concentration (g/L) mutually, C underfor lower middle 2,3-butanediol concentration (g/L) mutually;
3. the rate of recovery (Y)=C onv on/ CV, wherein C onand V onconcentration and the upper phase volume of 2,3-butanediol in being respectively mutually, C and V are respectively above-mentioned steps 1) in 2, the concentration and 2 of the 2,3-butanediol in 3-butyleneglycol fermentation liquid or 2,3-butanediol fermentation clear liquid, the volume of 3-butyleneglycol fermentation liquid or 2,3-butanediol fermentation clear liquid.
3) get step 2) in organic phase, carry out underpressure distillation under lower than 105 DEG C of conditions and remove organic solvent and water, obtain 2,3-butanediol crude product;
4) step 2) described in water, remove organic solvent, adding water to be diluted to sugared concentration is 80～200g/L, access thalline, carry out the fermentation of 2,3-butanediol.
In aforesaid method, step 1) described sugar is one or more mixtures that mix with any ratio in glucose, wood sugar, fructose, pectinose, sucrose, maltose, jerusalem artichoke, molasses, xylose mother liquid, described in sugary mixed solution, the concentration of sugar is 100～500g/L; Described inorganic salt are one or more mixtures that mix with any ratio in ammonium sulfate, dipotassium hydrogen phosphate, Secondary ammonium phosphate, and the concentration of inorganic salt described in sugary mixed solution is 1～100g/L; Described organic solvent is one or more mixing solutionss that mix with arbitrary proportion in methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol, amylalcohol, oleyl alcohol, ethyl acetate, butylacetate, methylcarbonate, diethyl carbonate, acetone, dioxane, tetrahydrofuran (THF).
In aforesaid method, step 1) in can add separately sugar, also sugar and inorganic salt can be added simultaneously, salt add the phase time that shortens into of playing, improve in product partition ratio, minimizings mutually sugared measure and under the effects such as dosage that mix.
In aforesaid method, step 4) in, while removing the organic solvent in water, reduce as far as possible the content of residual organic solvents in water, avoid the toxicity of organic solvent to cell, be conducive to further fermentation, those skilled in the art can control the content of residual organic solvents in water as required.In the specific embodiment of the invention, remove organic solvent to the organic solvent content in final fermentation system lower than 5g/L.
2,3-butanediol fermented liquid, 2,3-butanediol fermentation clear liquid, on phase, measure 2 in mutually down, the method of 3-butyleneglycol content: adopt vapor-phase chromatography to measure the concentration of 2,3-butanediol, chromatographic column is capillary column, 30m × Φ 0.32mm, thickness is 0.25 μ m; Detector is flame ionization ditector, and detector temperature is 210 DEG C; The starting temperature of column temperature is 60 DEG C, is raised to 70 DEG C with 2 DEG C/s rank, then rises to 180 DEG C with 30 DEG C/s rank, keeps 5min; Rear sampler, injector temperature is 210 DEG C, sample size 2 μ L, sample be three every group parallel, average.
The impact of embodiment 1 different sugar on 2,3-butanediol separating effect
According to step 1 in above-mentioned 3 (methods of separating 2,3-butanediol from 2,3-butanediol fermented liquid or 2,3-butanediol fermentation clear liquid))-3) described method separating 2,3-butanediol from 2,3-butanediol fermentation clear liquid.Wherein, in 2,3-butanediol fermentation clear liquid 2, the concentration of 3-butyleneglycol is 85.54g/L, adds the sugar not of the same race (kind of sugar in 2,3-butanediol fermentation clear liquid, as table 1) dissolve after, add different organic solvents (organic solvent kind, as table 1), standing, phase-splitting under mixing, normal temperature, in sampling and measuring upper and lower phase 2, the concentration of 3-butyleneglycol, the partition ratio (K of calculating 2,3-butanediol bD) and the rate of recovery (Y); And measure and compare (R), result is as table 1.The volume ratio of organic solvent and 2,3-butanediol fermentation clear liquid is 1:1, and the consumption of sugar is fermentation clear liquid 30% (w/v).
The impact of the different types of sugar of table 1. on 2,3-butanediol separating effect
As can be seen from Table 1, the system that glucose, wood sugar, fructose, pectinose, sucrose, jerusalem artichoke form with n-propyl alcohol, Virahol and methylcarbonate is respectively to 2 in fermented liquid, 3-butyleneglycol all has good separating effect, the system that wherein said sugar and n-propyl alcohol form is to 2, the rate of recovery of 3-butyleneglycol all reaches more than 70%, the system forming with isopropylcarbinol all reaches more than 50% the rate of recovery of 2,3-butanediol.On the other hand, the system that same organic solvent and various sugar form, the rate of recovery difference of 2,3-butanediol is not very large; Because glucose is the conventional substrate of fermentation, therefore following experiment selects glucose as phase separation agent.
The impact of embodiment 2 different organic solvents on 2,3-butanediol separating effect
According to step 1 in above-mentioned 3 (methods of separating 2,3-butanediol from 2,3-butanediol fermented liquid or 2,3-butanediol fermentation clear liquid))-3) described method separating 2,3-butanediol from 2,3-butanediol fermentation clear liquid.Wherein, in 2,3-butanediol fermentation clear liquid, the concentration of 2,3-butanediol is 69.70g/L, adds 15% and 30% glucose of 2,3-butanediol fermentation clear liquid quality to dissolve in 2,3-butanediol fermentation clear liquid, obtains sugary mixed solution.In two kinds of sugary mixed solutions, add respectively different organic solvents (organic solvent kind, as table 2), mix, the volume ratio of organic solvent and 2,3-butanediol fermentation clear liquid is 1:1, standing, phase-splitting under normal temperature, in sampling and measuring upper and lower phase 2, the concentration of 3-butyleneglycol, the partition ratio (K of calculating 2,3-butanediol bD) and the rate of recovery (Y); And measure and compare (R), result is as table 2.
The impact of table 2. organic solvent on 2,3-butanediol separating effect
Note: "-": homogeneous phase.
As can be seen from Table 2, except oleyl alcohol, the rate of recovery of other alcohols is generally higher, and the rate of recovery of ester class system is generally low.Wherein, in glucose/Isopropanol Solvent, the rate of recovery of 2,3-butanediol is the highest, reaches 83.13%, but comparing of this system is too large; The rate of recovery of acetone system and n-propyl alcohol system is taken second place, and follows by trimethyl carbinol system.Consider the rate of recovery and the toxicity of lower phase residual solvent to cell, the trimethyl carbinol is to extract preferably organic solvent.On the one hand, the rate of recovery of the trimethyl carbinol/glucose system 2,3-butanediol is higher; In addition, when lower phase circulating fermentation, the trimethyl carbinol is less to the toxicity of cell, is conducive to the growth of cell.
The phasor of embodiment 3 glucose/trimethyl carbinol system
Adopt nephelometry to draw double-aqueous phase system phasor, experimentation is as follows: first take a certain amount of glucose (m 1) add a certain amount of solvent (m 2) dissolve; Then dropwise add the trimethyl carbinol (m 3) until muddiness appears in mixing solutions just, in equilibrium process, dripping a water, solution becomes clarification at once, then adds some trimethyl carbinols, and solution is at once muddy again, can judge that this composition point is stagnation point.With the massfraction (Y of the glucose that added sugar) be X-coordinate, with the massfraction (Y of the corresponding trimethyl carbinol alcohol) for ordinate zou drafting double-aqueous phase system phasor, as Fig. 1.Y sugarand Y alcoholcalculation formula as follows:
With distilled water and 40g/L and 150g/L2,3-butanediol solution is solvent drawing diagram respectively, and experimental result as shown in Figure 1.
The result of Fig. 1 shows, glucose/trimethyl carbinol system has wider phase-splitting scope, under glucose concn scope 8～45% (w/w), suitable determining alcohol, can form stable double-aqueous phase system.In the time that glucose concn is lower, the sugar of glucose analyse ability a little less than, therefore phase-splitting needs more organic solvent, along with the rising of glucose concn, the sugar of glucose is analysed also corresponding enhancing of effect, therefore the required trimethyl carbinol concentration of phase-splitting is reducing gradually.
The impact of the concentration of embodiment 4 sugar and organic solvent on 2,3-butanediol separating effect
According to the separating 2,3-butanediol from 2,3-butanediol fermentation clear liquid of the method described in above-mentioned 3.In 2,3-butanediol fermentation clear liquid, add the trimethyl carbinol and the glucose of appropriate amount, form the extraction system that contains the different concns trimethyl carbinol and glucose.Wherein, in this extraction system, t butanol content is respectively 30%, 40% and 50%, and glucose content is respectively 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%.Described different extraction system on the impact of 2,3-butanediol partition ratio as shown in Figure 2 A, on the impact of the 2,3-butanediol rate of recovery as shown in Figure 2 B.
As can be seen from Figure 2: the partition ratio of 2,3-butanediol and the rate of recovery increase along with the increase of glucose and t butanol content.When the massfraction of glucose is 26%, when the massfraction of the trimethyl carbinol is 40%, the partition ratio of 2,3-butanediol is 1.19, and the rate of recovery is 63.50%.At glucose content 20-34%, when t butanol content 30～40%, all there are good partition ratio (0.95-1.35), 2,3-to determine the rate of recovery of glycol all higher than 40%, reach as high as 63.50%.
Embodiment 5 glucose/trimethyl carbinol system extracts the effect 1 with fermented liquid
Get and contain the 2,3-butanediol fermented liquid 100mL that 2,3-butanediol concentration is 96.47g/L, add 30g glucose, after sugar is dissolved completely, then add the 100mL trimethyl carbinol, mixing, normal temperature leave standstill, phase-splitting, obtain phase 153mL, wherein the concentration of 2,3-butanediol is 44.44g/L, lower phase 55mL.The partition ratio of 2,3-butanediol is 0.86, and the rate of recovery is 70.48%.
Embodiment 6 glucose/trimethyl carbinol system extracts the effect 2 with fermented liquid
Get and contain the 2,3-butanediol fermented liquid 100mL that 2,3-butanediol concentration is 39.89g/L, add 30g glucose, after sugar is dissolved completely, then add the 100mL trimethyl carbinol, mixing, room temperature leave standstill, phase-splitting, obtain phase 145mL, wherein the concentration of 2,3-butanediol is 17.84g/L, lower phase 70mL.The partition ratio of 2,3-butanediol is 0.89, and the rate of recovery is 64.85%.
7 two kinds of sugar of embodiment or two kinds of organic solvents affect 1 to 2,3-butanediol separating effect
Get containing 108.02g/L2 the 2,3-butanediol fermentation clear liquid 4mL of 3-butyleneglycol, add 0.6g glucose and 0.6g sucrose, after sugar is dissolved completely, then add the 4mL trimethyl carbinol, stir, room temperature leaves standstill phase-splitting, upper mutually for being rich in the organic phase of 2,3-butanediol, volume is 7.1mL, the concentration of 2,3-butanediol is 47.13g/L; Lower mutually for to be rich in sugared water, volume is 1.6mL.The partition ratio of 2,3-butanediol is 0.92, and the rate of recovery is 80.31%.
8 two kinds of sugar of embodiment or two kinds of organic solvents affect 2 to 2,3-butanediol separating effect
Get containing 108.02g/L2 the 2,3-butanediol fermentation clear liquid 4mL of 3-butyleneglycol, add 1.2g glucose, after sugar is dissolved completely, then add 2mL n-propyl alcohol and the 2mL trimethyl carbinol, stir, room temperature leaves standstill phase-splitting, upper mutually for being rich in the organic phase of 2,3-butanediol, volume is 6.7mL, the concentration of 2,3-butanediol is 46.89g/L; Lower mutually for to be rich in sugared water, volume is 2.0mL.The partition ratio of 2,3-butanediol is 0.90, and the rate of recovery is 74.79%.
Embodiment 9 inorganic salt affect 1 to 2,3-butanediol separating effect
Get 2,3-butanediol fermentation clear liquid (concentration of 2,3-butanediol is 99.92g/L) 4mL, add 0.6g glucose, 0.048g Secondary ammonium phosphate, after dissolving it completely, add the 4mL trimethyl carbinol again, stir, room temperature leaves standstill, phase-splitting, obtain the two-layer liquid phase of water and the organic phase system that coexists, upper mutually for being rich in the organic phase of 2,3-butanediol, volume is 6mL, the concentration of 2,3-butanediol is 56.26g/L; Lower mutually for to be rich in sugared water, volume is 2.5mL.The partition ratio of 2,3-butanediol is 2.28, and the rate of recovery is 84.46%.
Embodiment 10 inorganic salt affect 2 to 2,3-butanediol separating effect
Get containing 99.54g/L2, the 2,3-butanediol fermentation clear liquid 4mL of 3-butyleneglycol, after adding 0.6g glucose and 0.12g Secondary ammonium phosphate that it is dissolved completely, add again the 4mL trimethyl carbinol, stir, room temperature leaves standstill phase-splitting, upper mutually for being rich in 2, the organic phase of 3-butyleneglycol, volume is 6.2mL, and the concentration of 2,3-butanediol is 49.64g/L; Lower mutually for to be rich in sugared water, volume is 2.2mL.The partition ratio of 2,3-butanediol is 1.22, and the rate of recovery is 75.28%.
Embodiment 11 inorganic salt affect 3 to 2,3-butanediol separating effect
Get containing 2, the fermentation clear liquid 4mL of 3-butyleneglycol 99.54g/L, after adding 0.6g glucose and 0.36g Secondary ammonium phosphate it being dissolved completely, then add the 4mL trimethyl carbinol, stir, room temperature leaves standstill phase-splitting, upper mutually for being rich in the organic phase of 2,3-butanediol, volume is 6mL, the concentration of 2,3-butanediol is 56.26g/L; Lower mutually for to be rich in sugared water, volume is 2.4mL.The partition ratio of 2,3-butanediol is 1.61, and the rate of recovery is 76.99%.
The fermentation again of the lower phase of embodiment 12 extractions
400mL2,3-butyleneglycol fermentation liquid, at the centrifugal 10min of 8,000rpm, obtains wet thallus and 2,3-butanediol fermentation clear liquid, and thalline is placed in 4 DEG C in order to recycling.
Get 2,3-butanediol fermentation clear liquid (concentration of 2,3-butanediol is 67.64g/L) 300mL, add 90g glucose, after 5.4g Secondary ammonium phosphate dissolves it completely, then add the 300mL trimethyl carbinol, stir, room temperature leaves standstill, phase-splitting, upper mutually for being rich in the organic phase of 2,3-butanediol, volume is 395ml, wherein the concentration of 2,3-butanediol is 38.16g/L; It is lower that mutually for to be rich in sugared water, volume is 240ml, wherein sugared concentration 235g/L, and trimethyl carbinol concentration is 121.53g/L.The partition ratio of 2,3-butanediol is 1.27, and the rate of recovery is 66.67%.The trimethyl carbinol is removed in lower phase low-temperature reduced-pressure distillation, and being diluted with water to glucose is 112g/L, and residual 2,3-butanediol concentration is 13.39g/L, and remaining trimethyl carbinol concentration is 3.77g/L.Lower thalline (the initial OD that access is reclaimed mutually after thin up 620be 3.8), shake-flask culture 90h under 37 DEG C, 200rpm condition, obtains Secondary Fermentation liquid, and wherein the concentration of 2,3-butanediol reaches 49.52g/L, the actual 36.13g/L that produces, inversion rate of glucose is 38.44%.
Embodiment 13
Again the ferment effect of extraction again of the fermented liquid producing of the lower phase that the present embodiment has been investigated extracting and separating.
Press the method for embodiment 12, obtain Secondary Fermentation liquid (fermented liquid that extracting phase ferments again), wherein containing 2,3-butyleneglycol 38.42g/L, gets Secondary Fermentation liquid 100mL, adds 30g glucose, after it is dissolved completely, add the 100mL trimethyl carbinol, mixing, room temperature leave standstill, phase-splitting, obtain phase 140mL again, lower phase 75mL, 2,3-butanediol partition ratio is 0.80, and the rate of recovery is 59.87%.

Claims (8)

1. a method for 2,3-butanediol in extraction and fermentation coupling separate fermentation liquid, is characterized in that, said method comprising the steps of:
1) by sugar or/and inorganic salt are dissolved in 2,3-butanediol fermented liquid, obtain sugary mixed solution;
2) to step 1) sugary mixed solution in add organic solvent, vibration mixes, mixed solution leaves standstill or is centrifugal to phase-splitting at 15～40 DEG C, obtains the organic phase on upper strata and the water of the lower floor system that coexists;
3) step 2) in organic phase, carry out underpressure distillation under lower than 105 DEG C of conditions and remove organic solvent and water, obtain 2,3-butanediol crude product;
4) step 2) described in water, remove organic solvent, adding water to be diluted to sugared concentration is 80～200g/L, access thalline, carry out the fermentation of 2,3-butanediol.
2. method according to claim 1, it is characterized in that, in step 1) described in 2,3-butyleneglycol fermentation liquid is mycetome 2, the 2,3-butanediol fermentation clear liquid of 3-butyleneglycol fermentation stoste or removal thalline, described 2, in 3-butyleneglycol fermentation liquid, the concentration of 2,3-butanediol is 40～160g/L.
3. method according to claim 1, is characterized in that, in step 1) described in the concentration of sugar in sugary mixed solution be 100～500g/L, the concentration of described inorganic salt in sugary mixed solution is 1～100g/L.
4. method according to claim 1, is characterized in that, in step 2) described in sugary mixed solution and the volume ratio of organic solvent be 0.5～2:1.
5. method according to claim 1, is characterized in that step 1) described in sugar be one or more the mixture in glucose, wood sugar, fructose, pectinose, sucrose, maltose, jerusalem artichoke, molasses, xylose mother liquid.
6. method according to claim 1, is characterized in that, described inorganic salt are one or more the mixture in ammonium sulfate, dipotassium hydrogen phosphate, Secondary ammonium phosphate.
7. method according to claim 1, it is characterized in that step 2) described in organic solvent be one or more mixing solutions in methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol, amylalcohol, oleyl alcohol, ethyl acetate, butylacetate, methylcarbonate, diethyl carbonate, acetone, dioxane, tetrahydrofuran (THF).
8. method according to claim 1, is characterized in that, in step 4) described in the method for removal organic solvent be underpressure distillation or air lift method, wherein the temperature of underpressure distillation is 20～50 DEG C, pressure is-0.08～-0.1MPa; The gas that air lift method adopts is CO 2.
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