CN108017514A - The method of 1,3- propane diols, acetic acid and butyric acid in two step salting-out extraction separation and fermentation liquids - Google Patents
The method of 1,3- propane diols, acetic acid and butyric acid in two step salting-out extraction separation and fermentation liquids Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, there is provided a kind of method of 1,3 propane diols, acetic acid and butyric acid in two step salting-out extraction separation and fermentation liquids.This method is：Soluble acid inorganic salts are dissolved in 1,3 propanediol fermentation liquors, add hydrophobic organic solvent, vibration mixing, stands split-phase at room temperature, carries out first step salting-out extraction, upper phase is the organic phase rich in butyric acid, and lower phase is the salt phase rich in 1,3 propane diols and acetic acid；With the butyric acid in aqueous slkali or the upper phase of basic mineral salting liquid back extraction；Hydrophilic organic solvent is added to downwards, carries out second step salting-out extraction, extracts 1,3 propane diols and acetic acid.The present invention solves current fermentation method and produces the problems such as 1,3 propane diols present in 1,3 propane diols separating technologies are difficult to separate with accessory substance, are of high cost.This method technique is simple, and disengaging time is short, and the rate of recovery is high, and separation costs are low, are a kind of separation methods for having very much prospects for commercial application.
Description
Technical field
The invention belongs to technical field of bioengineering, is related to the isolation technics of microbial fermentation solution, more particularly to a kind of sharp
With the method for 1,3- propane diols, acetic acid and butyric acid in salting-out extraction technology separation and fermentation liquid.
Background technology
1,3-PD, acetic acid and the butyric acid industrial chemicals and intermediate wide as prospect of production, in many fields all
There is wide application.1,3- propane diols can be used as the list of solvent, adhesive, cosmetics, antistaling agent and synthesizing polyester, polyurethane
Body, wherein the polypropylene terephthalate (PTT) synthesized with terephthalic acid (TPA), due to being not likely to produce electrostatic, uvioresistant, good
, there be huge answer in the advantages that resilience well, resistance to soiling and biodegradability in industries such as carpet, weaving and engineering plastics
Use prospect.Acetic acid is mainly for the production of vinyl acetate, acetate, aceticanhydride, ketene dimer, monoxone, acetate fiber etc., or conduct
Solvent or raw material in the industrial processes such as terephthalic acid (TPA), pesticide, medicine and fuel.Butyric acid can be used for manufacturing butyrate
Class, cellulose butyrate, also serve as emulsifying agent, fungicide and extractant, and butyric acid and its derivative can be applied in food, doctor
The field such as medicine and feed.
Traditionally, 1,3-PD is mainly produced by chemical method, petering out and thus bringing with fossil energy
Environmental and safety problems cause people more and more to worry, therefore biotransformation method producing 1,3-propanediol through fermentation becomes
The hot spot of research.The methods that 1,3-PD is converted glycerol into using klebostiella pneumoniae much more domestic at present, used in the method
Bacterium be conditioned pathogen, and accessory substance has 2,3-butanediol, acetic acid, ethanol, lactic acid, butanedioic acid, citric acid, formic acid etc.,
Product separation is relatively difficult.Clostridium butyricum can also convert glycerol into 1,3-PD in nature, which is probiotics, and secondary
Product only has acetic acid and butyric acid.If recycle organic acid while 1,3-PD in zymotic fluid is separated, then be beneficial to
Separation costs are reduced, are increased economic efficiency.
At present, the method for 1,3-PD being separated from zymotic fluid be mainly zymotic fluid by centrifugation, flocculation treatment or
Membrane filtration removes thalline and part biological macromolecular, then by organic solvent deposit, organic solvent extraction, reaction, extraction or
Electrodialysis desalination etc. carries out crude separation, removes partial impurities, 1,3-PD is obtained finally by rectifying.To the zymotic fluid of concentration
Middle addition ethanol can separate out a large amount of precipitations, washing precipitation, and cleaning solution and supernatant rectifying can obtain 1,3-PD, still
This step needs to consume a large amount of organic solvents, and the readily volatilized loss of solvent.1,3-PD can be extracted with ethyl acetate, its
Distribution coefficient is 0.22, and distribution coefficient can be brought up to 0.33 by adding a small amount of ethanol and making cosolvent, but the rate of recovery is still very low.
The reason is that the hydrophily of 1,3-PD is too strong, it is efficiently extracted it is difficult to find suitable solvent.Reaction, extraction can incite somebody to action
1,3-PD changes into hydrophobicity product, so that extracted by organic solvent, but complicated component in zymotic fluid, a variety of side reactions
With progress, while catalyst also easily inactivates, and reduces catalytic efficiency.If zymotic fluid ultrafiltration is first removed into partial impurities, then
Small molecule salt is removed using electrodialysis, then contributes to reaction, extraction, but electrodialysis energy consumption is big, and film is easy to pollute, and cost is higher.By
The salting-out extraction system of hydrophilic organic solvent and inorganic salts composition one step can efficiently extract 1,3- propane diols from zymotic fluid
Take out, but organic acid (such as acetic acid, lactic acid) while be also extracted into upper phase, later separation is still had any problem.
Common are machine acid separation method mainly has salt forming method, absorption method, membrane separation process, esterification process and liquid-liquid extraction method
Deng.Salt forming method is that a large amount of alkali or inorganic salts are added into organic acid soln, forms the salt of precipitation or heat endurance, then is passed through
Filter, thermal dehydration, acidifying, distillation obtain sterling, salt forming method technical maturity, but need to consume a large amount of soda acids, easily produce secondary
Pollution.Contain a large amount of impurity in zymotic fluid, complicated component, this causes ion exchange and membrane separating process is faced with resin and film is easy
Pollution, regeneration is frequent, produces the problem of a large amount of waste liquids, and industrialized production has certain difficulty at present.With immobilized lipase (such as
Novozym 435) catalysis, the butyric acid in zymotic fluid is changed into ethyl butyrate, then with trioctylamine (TOA)-hexamethylene be extraction
Agent extract ethyl butyrate, but there are esterification yield it is not high, technics comparing is complicated the problem of.Extracted with medium chain fatty acid in aqueous solution
Acetic acid, then obtains acetic acid by fractionation, while aliphatic acid can recycle, but the distribution coefficient of acetic acid is not high, recycling
Rate is too low, needs to consume abundant fatty acid using multitple extraction.Saltout and be applied to the separation of butyric acid, but system needs are relatively low
PH and higher concentration butyric acid, therefore usually require that and concentrate zymotic fluid；And salting-out extraction can be used for lactic acid, butanedioic acid decile
From the former can carry out in alkaline conditions, and the latter then needs acid condition.
Salting-out extraction technology is using organic solvent as extractant, and inorganic salts are salting-out agents, under both comprehensive functions, from
A kind of separation method of hydrophily target product is extracted in aqueous solution.Organic solvent uses hydrophilic short chain alcohol, ketone etc. more, because
This kind of salting-out extraction system is often known as double-aqueous phase system by this.Salting-out extraction technology is with distribution coefficient is big, the rate of recovery is high, organic
Solvent dosage is few, mild condition, the plurality of advantages such as low to equipment corrosion, has been used for butyric acid, lactic acid, butanedioic acid, 1,3- the third two
The separation of a variety of biological-based chemicals such as alcohol, 2,3-butanediol and acetone and butanol, also has two step salting-out extractions to separate 1,3-
The report of propane diols and lactic acid.Since the organic solvent used in two step salting-out extractions is hydrophilic, the total recovery of lactic acid is only
For 73.8%, and second step needs pH being adjusted to 6.5 from 10.0, causes K2CO3Loss.Do not ferment at present from clostridium butyricum still
The report of 1,3- propane diols, acetic acid and butyric acid is separated in liquid.
The content of the invention
It is an object of the present invention to at present from zymotic fluid extract 1,3-PD during existing accessory substance
It is difficult to recycle, the problems such as separation costs are high, thalline and protein salvage are difficult, it is proposed that carry using two step salting-out extraction technologies
Take the new method of 1,3- propane diols, acetic acid and butyric acid in separation and fermentation liquid.
To achieve the object of the present invention, technical scheme is as follows：
A kind of method of 1,3-PD, acetic acid and butyric acid in two steps salting-out extraction separation and fermentation liquid, the described method includes
Following steps：
1) first step salting-out extraction：Soluble acid inorganic salts are dissolved in 1,3-PD zymotic fluid, are added hydrophobic
Property organic solvent, vibration mixing stands split-phase at room temperature, and upper phase be the organic phase rich in butyric acid, and lower phase is rich in 1,3- the third two
The salt phase of alcohol and acetic acid；
2) aqueous slkali or basic mineral salting liquid are added in the organic phase obtained to step 1), is stripped butyric acid；
3) second step salting-out extraction：Add hydrophilic organic solvent in the salt phase obtained to step 1), 1,3- of extraction the third two
Alcohol and acetic acid.
Further, in the above-mentioned technical solutions, the 1,3-PD zymotic fluid is sent out for the 1,3-PD of mycetome
Ferment stoste or the 1,3-PD fermentation clear liquid for removing thalline, in the 1,3-PD zymotic fluid, 1,3-PD concentration
For 50~100g/L, acetic acid concentration is 6~15g/L, and butyric acid density is 10~20g/L.
Further, in the above-mentioned technical solutions, the soluble acid inorganic salts described in step 1) are ammonium sulfate, phosphorus
One kind in acid dihydride sodium or potassium dihydrogen phosphate, is preferably sodium dihydrogen phosphate.
Further, in the above-mentioned technical solutions, the hydrophobic organic solvent described in step 1) is ethyl acetate, second
One kind in acid butyl ester, methyl tertiary butyl ether(MTBE) or hexone, is preferably butyl acetate.
Further, in the above-mentioned technical solutions, the addition of the soluble acid inorganic salts described in step 1) is
The 15%~27.5% of extraction system quality, is preferably 25%.
Further, in the above-mentioned technical solutions, the addition of the hydrophobic organic solvent described in step 1) is extraction
The 15%~35% of system quality is taken, is preferably 30%.
Further, in the above-mentioned technical solutions, the alkali in the aqueous slkali described in step 2) is sodium hydroxide, hydrogen-oxygen
Change one kind in potassium, the bases inorganic bases in the basic mineral salting liquid are sodium carbonate, sodium acid carbonate, potassium carbonate or carbonic acid
One kind in hydrogen potassium.
Further, in the above-mentioned technical solutions, when the hydrophobic organic solvent used in step 1) be ethyl acetate or
During butyl acetate, in step 2), basic mineral salting liquid is added in the organic phase, the alkaline, inorganic salts are carbonic acid
One kind in sodium, sodium acid carbonate, potassium carbonate or saleratus；When the hydrophobic organic solvent used in step 1) is methyl- tert fourth
During a kind of in base ether or hexone, in step 2), aqueous slkali or basic mineral are added in the organic phase
Salting liquid, the one kind of the alkali for sodium hydroxide, in potassium hydroxide, the alkaline, inorganic salts are sodium carbonate, sodium acid carbonate,
One kind in potassium carbonate or saleratus, preferably sodium carbonate.
Further, in the above-mentioned technical solutions, the organic acid in the organic phase described in step 2) and the alkali or
The molar ratio of alkaline, inorganic salts is 5:1~8；Organic phase is 1~5 with the volume ratio of aqueous slkali or basic mineral salting liquid:1, it is excellent
Elect 2 as:1.Wherein described organic acid refers to the summation of acetic acid and butyric acid.
Further, in the above-mentioned technical solutions, the hydrophilic organic solvent described in step 3) for acetone, methanol,
One kind in ethanol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, dimethyl carbonate or tetrahydrofuran, is preferably second
Alcohol.
Further, in the above-mentioned technical solutions, the addition of the hydrophilic organic solvent described in step 3) is extraction
The 20%~52% of system volume is taken, is preferably 50%.
In the present invention, the 1,3-PD zymotic fluid refers to using conventional biotransformation method fermenting and producing 1,3- third
The zymotic fluid containing 1,3-PD during glycol, mainly contains 1,3-PD, acetic acid and butyric acid, can also contain a small amount of first
Acid, lactic acid, butanedioic acid and ethanol.It is preferred that zymotic fluid when 1,3-PD is converted glycerol into using clostridium butyricum, the fermentation
Liquid contains 1,3- propane diols, acetic acid and butyric acid.
In the present invention, the extracting operation mode of 1,3-PD, acetic acid and butyric acid can be interval or continuous
's；Can be by the way of multitple extraction for the less system of distribution coefficient.
In the present invention, the 1,3-PD zymotic fluid can be pre-processed with the method for flocculation, micro-filtration or centrifugation, be removed
Remove thalline and Partial Protein；If not preprocessed directly carry out extracting operation, thalline and albumen are distributed in centre.
Beneficial effects of the present invention：
The method of the present invention, exists when solving by two step salting-out extractions and separate 1,3-PD from zymotic fluid at present
Byproduct organic acids be difficult to recycling the problem of.Compared with inorganic salts/hydrophilic organic solvent system, in first step salting-out extraction
Inorganic salts used/hydrophobic organic solvent system directly handles zymotic fluid, while butyric acid is extracted, to thalline and albumen
Removal effect is more preferable.The butyric acid and acetic acid in organic phase are stripped using alkali (or basic mineral salting liquid), the rate of recovery is high, You Jirong
Agent almost free of losses, can direct circulation utilize, and butyrate value be higher than butyric acid.In second step salting-out extraction, 1,3-PD
Upper phase (organic phase) is assigned to acetic acid, can be isolated by conventional separation methods such as distillations.This method technique is simple, is dividing
Organic acid is recycled while from 1,3-PD, achievees the purpose that to reduce cost, increase economic efficiency, is before one kind has industry
The separation method of scape.
Brief description of the drawings
Fig. 1 is the salting-out extraction system that is formed of different organic solvents and sodium dihydrogen phosphate to butyric acid and acetic acid, 1,3- the third two
The influence of alcohol separating degree.
Fig. 2 for salinity to butyric acid in sodium dihydrogen phosphate/butyl acetate salting-out extraction system separation and fermentation clear liquid, acetic acid and
The influence of 1,3-PD, wherein Fig. 2A are the influence to butyric acid distribution coefficient and the rate of recovery；Fig. 2 B are to acetic acid distribution coefficient
With the influence of the rate of recovery；Fig. 2 C are the influence to 1,3- propane diols distribution coefficient and the rate of recovery.
Fig. 3 for organic solvent concentration to butyric acid in sodium dihydrogen phosphate/butyl acetate salting-out extraction system separation and fermentation clear liquid,
The influence of acetic acid and 1,3-PD, wherein Fig. 3 A are the influence to butyric acid distribution coefficient and the rate of recovery；Fig. 3 B are to acetic acid point
The influence of distribution coefficient and the rate of recovery；Fig. 3 C are the influence to 1,3- propane diols distribution coefficient and the rate of recovery.
Fig. 4 is the influence that concentration of sodium carbonate is stripped butyric acid, acetic acid and 1,3- propane diols yield.
Fig. 5 is volume fraction of ethanol to acetic acid in simulated fermentation broth and 1,3- propane diols distribution coefficient and the shadow of the rate of recovery
Ring.
Fig. 6 is influence of the volume fraction of ethanol to acetic acid in fermentation clear liquid and 1,3- propane diols distribution coefficient and the rate of recovery.
Embodiment
Following non-limiting examples can make those of ordinary skill in the art be more fully understood the present invention, but not with
Any mode limits the present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, institute
Reagent etc. can chemically or biological reagent company purchase.
Describe the embodiment of the present invention in detail below in conjunction with technical solution.
1.1,3- the preparation of propanediol fermentation liquor
1,3-PD zymotic fluid, is the hair containing 1,3-PD, acetic acid and butyric acid being prepared in conventional manner
Zymotic fluid.
Following embodiments use the mixed bacterium fedbatch culture crude glycerine based on clostridium butyricum (Clostridium butyricum)
What fermentation obtained, according to adjustment concentration of substrate or feed supplement time, the 1,3-PD zymotic fluid containing appropriate constituent content is obtained,
Mixed bacterium composition and fermentation method be shown in CN106399204A, wherein 1,3-PD (1,3-PD), acetic acid (HAc) and butyric acid (BA)
Concentration is respectively 50~100g/L, 6~15g/L and 10~20g/L.By 1,3-PD zymotic fluids through doughnut membrane filtration, remove
Thalline and Partial Protein, obtain fermentation clear liquid.
2. separating the method for 1,3-PD from 1,3-PD zymotic fluids or fermentation clear liquid, comprise the following steps：
1) soluble acid inorganic salts being dissolved in 1,3-PD zymotic fluids, adds hydrophobic organic solvent, vibration mixes,
Stand split-phase at room temperature, carry out first step salting-out extraction, upper phase is the organic phase rich in BA, lower phase be rich in 1,3-PD and
The salt phase of HAc；
2) aqueous slkali or basic mineral salting liquid are added in the organic phase obtained to step 1), is stripped BA；
3) hydrophilic organic solvent is added in the salt phase obtained to step 1), carries out second step salting-out extraction, vibration mixes,
Split-phase is stood at room temperature, and upper phase is the organic phase for containing 1,3-PD and HAc, and lower phase is salt phase.
In step 2), butyrate and a small amount of acetate and 1,3- are rich in the anti-stripping agent (salt phase) being stripped
PD, anti-stripping agent obtain butyrate (sodium butyrate or potassium butyrate) through distillation water removal, drying.
The organic phase obtained for step 3), according to the difference of boiling point substance, can use the conventional separation methods such as distillation
Separate 1,3-PD and HAc.Preferably, organic phase pH is adjusted to 7.0 with sodium hydroxide or potassium hydroxide, is filtered to remove the phosphorus of precipitation
Hydrochlorate, filtrate is distilled, and 110 DEG C of overhead fractions 1,3-PD are collected under conditions of 0.094~0.096MPa of vacuum, several
Whole acetic acid are retained in tower reactor in the form of acetate (sodium salt or sylvite).
In the above-mentioned methods, the Acidic inorganic salts described in step 1) are in ammonium sulfate, sodium dihydrogen phosphate or potassium dihydrogen phosphate
One kind, the additions of inorganic salts is the 15~27.5% of extraction system quality；The hydrophobic organic solvent for ethyl acetate,
One kind in butyl acetate, methyl tertiary butyl ether(MTBE) or hexone, the addition of organic solvent is extraction system quality
15~35%.
In the above-mentioned methods, the one kind of the alkali described in step 2) for sodium hydroxide, in potassium hydroxide, the alkaline nothing
Machine salt is one kind in sodium carbonate, sodium acid carbonate, potassium carbonate or saleratus.
In the above-mentioned methods, the hydrophilic organic solvent described in step 3) is acetone, methanol, ethanol, normal propyl alcohol, isopropyl
One kind in alcohol, n-butanol, isobutanol, the tert-butyl alcohol, dimethyl carbonate or tetrahydrofuran, the addition of the hydrophilic organic solvent
Measure as the 20~52% of extraction system volume.
Each step is separately sampled, measures 1 in phase up and down, the concentration of 3-PD, HAc and BA.Calculation of distribution coefficient (K), recycling
Rate (Y) and removal rate (R).
Distribution coefficient (K), compared to (P), the calculation formula of the rate of recovery (Y) and removal rate (R) is as follows：
Ri=100%-Yi(formula 4)
In formula (1)~formula (4), i represents 1,3-PD, HAc or BA, CtAnd CbFor phase mass concentration (g/ up and down after balance
L), VtAnd VbFor upper and lower phase volume (mL) after balance.Separating degree is the ratio between distribution coefficient of two kinds of materials.
By whole separation process, 1,3-PD, HAc and BA ult recs (Yf) calculation formula it is as follows：
YF, 1,3-PD=RIYIII(formula 5)
YF, HAc=YIYII+RIYIII(formula 6)
YF, BA=YIYII(formula 7)
In formula (5)~formula (7), I, II and III represents first step salting-out extraction, back extraction and second step salting-out extraction respectively.
The removal rate of cell and protein (R) is defined as follows in each step salting-out extraction：
First step salting-out extraction：
Second step salting-out extraction：
In formula (8)~formula (9), behalf cell or protein, MmRepresent the matter of cell or albumen at boundary up and down
Amount, MtRepresent the quality of cell or albumen in upper phase, M1、M2Represent total matter of cell or albumen in the step salting-out extraction system
Amount.
3. analysis method
In salt phase, 1,3-PD, HAc and BA contents are detected using liquid chromatography.AminexHPX-87H chromatographies
Column, 300mm x7.8mm；Mobile phase 5mM sulfuric acid；Flow velocity 0.6mL/min, Composition distribution detect at 410nm, 20 μ of sample size
L, 65 DEG C of column temperature, detection time 23min.
In hydrophobic organic solvent, 1,3-PD, HAc and BA contents be detected using gas chromatography, chromatographic condition
For：BGB-174 capillary columns (30m × 0.25mm I.D.0.25 μm df)；Fid detector, 220 DEG C of detector temperature；Sample introduction
210 DEG C of temperature of mouth, split ratio 1:8；Carrier gas is High Purity Nitrogen；Using external standard method；2 μ L of sample size.
The measure of thalline uses spectrophotometry, and turbidity is measured under 650nm.Protein determination uses Coomassie Brilliant Blue.
The selection of inorganic salts and organic solvent in 1 first step salting-out extraction of embodiment
The concentration of 1,3-PD, HAc and BA are respectively 67.76g/L, 7.07g/L, 11.94g/L in 1,3-PD fermentation clear liquids.
First inorganic salts are dissolved in 1,3-PD fermentation clear liquids, NaCl, (NH will be separately added into4)2SO4、NaH2PO4Fermentation clear liquid pH
4.5 are adjusted to, is separately added into Na2CO3And K3PO4Fermentation clear liquid keep natural ph；Add organic solvent (inorganic salts and
Organic solvent species, such as table 1).Vibration mixing, stands split-phase, upper phase is organic phase, and lower phase is salt phase at room temperature.Purpose be by
BA is extracted into phase, and 1,3-PD and HAc stays in lower phase.The concentration of each material in upper and lower phase is measured by sampling, calculates its distribution
Coefficient (K) and the rate of recovery (Y), as a result such as table 1.The quality of inorganic salts, 1,3-PD fermentation clear liquids and organic solvent in extraction system
Percentage composition is respectively 10%, 60% and 30%.
The distribution behavior of table 1. 1,3-PD, HAc and BA in different salting-out extraction systems
As can be known from the results of Table 1, in Na2CO3And K3PO4In salting-out extraction system Deng basic salt composition, except hydrophily
Normal propyl alcohol system, BA divide equally fits over the lower phase (rate of recovery<30%).This is because when pH ＜ 4.82 (pKa value of BA), BA master
To exist with molecular conformation, it is easier to be extracted to organic phase；During pH ＞ 4.82, BA mainly exists with ionic forms, is more prone to
In distribution in water phase.In basic salt/normal propyl alcohol system, the separating degree of BA and 1,3-PD may each be about 1, and separating effect is poor, institute
With Na2CO3And K3PO4The alkaline salting-out extraction system of formation is unsuitable for the separation of BA and 1,3-PD, HAc.
In NaCl, (NH4)2SO4And NaH2PO4In the acid salting-out extraction system of composition, BA is mainly distributed in (the recycling of upper phase
Rate is between 65.30%~94.10%).Wherein, NaCl's is poor into phase ability, is difficult to form salting-out extraction system with ethanol,
Influence second step salting-out extraction.NaH2PO4During as salting-out agents, (NH is better than to the effect of extracting of BA4)2SO4, while 1,3-PD and
The effect of extracting of HAc approaches.Zymotic fluid is a big buffer system, wherein comprising plurality of inorganic salt, organic salt, protein and
Nucleic acid etc., adjusting pH needs addition substantial amounts of sour, and NaH2PO4It is acid stronger ackd salt (50g/L NaH2PO4Solution
PH value is between 4.2-4.6), acid can not be added during BA is extracted and reaches relatively low pH.
Then, with NaH2PO4As salting-out agents, in the salting-out extraction system that it is formed with five kinds of organic solvents, body is observed
It is the effect of extracting to BA, the effect of extracting according to system organic solvent is：Normal propyl alcohol ＞ isobutanol ＞ n-butanol ＞ methyl is different
Butyl ketone ＞ methyl tertiary butyl ether(MTBE) ＞ ethyl acetate ＞ butyl acetates, this is almost consistent with the hydrophily order of solvent.
When the organic solvent selection stronger alcohols of hydrophily, the distribution coefficient and the rate of recovery of BA is higher, but at the same time also at least 29%
1,3-PD and HAc be extracted to organic phase, be unsuitable for BA and 1,3-PD separation.When organic solvent selects hydrophobic solvent
When, the distribution coefficient and the rate of recovery of BA can decrease to some degree, but 1,3-PD distribution coefficient and the rate of recovery drastically under
Drop.NaH is shown in Fig. 12PO4The salting-out extraction system formed with different organic solvents is to BA in fermentation clear liquid and HAc, 1,3-
The influence of PD separating degrees, ethyl acetate, butyl acetate, methyl tertiary butyl ether(MTBE) and hexone and NaH2PO4The salt of composition
Analysis system is better than polar organic solvent normal propyl alcohol, isobutanol, the system of saltouing of n-butanol to BA and 1, the separating degree of 3-PD, wherein
The boiling point of methyl tertiary butyl ether(MTBE) is too low (lower 55 DEG C of normal pressure), and volatility is stronger.In addition, and NaH2PO4/ ethyl acetate and
NaH2PO4/ hexone salting-out extraction system is compared, NaH2PO4Separation of/butyl acetate the system to BA and 1,3-PD
The separating degree of degree higher, BA and HAc are slightly higher.
The salting-out extraction (first step salting-out extraction) of 2 butyric acid of embodiment
1. prepared according to the method described in embodiment 1 by various concentrations butyl acetate and NaH2PO4And 1,3- propane diols hair
The extraction system of ferment clear liquid composition, the mass percentage of butyl acetate is respectively 25%, 30% and 35%, NaH2PO4Quality
Percentage composition is between 15~27.5%, 1 in 1,3-PD fermentation clear liquid, the concentration of 3-PD, HAc and BA be respectively 79.36g/L,
9.30g/L、14.22g/L.Extraction system stirs evenly, and stands split-phase, investigates NaH2PO4Concentration changes to three kinds of dispensed materials systems
The influence of number and the rate of recovery, the results are shown in Figure 2, wherein, Fig. 2A is the influence to BA distribution coefficients and the rate of recovery, and Fig. 2 B are pair
The influence of HAc distribution coefficients and the rate of recovery, Fig. 2 C are the influence to 1,3-PD distribution coefficients and the rate of recovery.In Fig. 2 as it can be seen that with
The mass fraction increase of salt, the distribution coefficient and the rate of recovery of BA, HAc and 1,3-PD are all being continuously improved.BA is mainly distributed upper
Phase, maximum recovery can reach 96.89%；HAc and 1,3-PD are mainly distributed is in lower phase, the maximum recovery of HAc
40.12%, and 1, the 3-PD rate of recovery is below 6%.
2. prepared according to the method described in embodiment 1 by various concentrations NaH2PO4Sent out with butyl acetate and 1,3- propane diols
The extraction system of ferment clear liquid composition, NaH2PO4Mass percentage be respectively 20% and 25%, the quality percentage of butyl acetate
Content is between 15~35%, and 1 in 1,3-PD fermentation clear liquid, the concentration of 3-PD, HAc and BA are respectively 79.36g/L, 9.30g/
L, 14.22g/L, investigates influence of the butyl acetate concentration change to three kinds of dispensed materials coefficients and the rate of recovery, as a result such as Fig. 3 institutes
Show, wherein, Fig. 3 A are the influence to BA distribution coefficients and the rate of recovery；Fig. 3 B are the influence to HAc distribution coefficients and the rate of recovery；Figure
3C is the influence to 1,3-PD distribution coefficients and the rate of recovery.As it can be seen that on the whole in Fig. 3, the distribution coefficient of three kinds of materials and recycling
Rate is all improved with the increase of organic solvent mass fraction, only for the distribution coefficient of 1,3-PD, when butyl acetate concentration increases
When big, it may appear that the phenomenon of reduction, this is because, excessive ester concentration causes the concentration of 1,3-PD in phase to reduce, and makes distribution
Coefficient declines.
As 25% (w/w) NaH of use2PO4During/35% (w/w) butyl acetate system, the rate of recovery of BA is up to
97.48%；The rate of recovery of HAc and 1,3-PD is respectively 42.11%, 5.13%.
As 25% (w/w) NaH of use2PO4During/30% (w/w) butyl acetate salting-out extraction system, the distribution coefficient of BA and
The rate of recovery is 42.21 and 96.42%, and the rate of recovery of corresponding HAc and 1,3-PD are respectively 33.21%, 3.71%, it is contemplated that are carried
While the high BA rate of recovery, principal product 1 should be reduced, the loss of 3-PD is more suitable when avoiding using excessive organic solvent again
Close and select the system.
The back extraction of 3 butyric acid of embodiment and acetic acid
1. according to the method described in embodiment 2, in 25% (w/w) NaH2PO4/ 30% (w/w) butyl acetate salting-out extraction body
To 1,3-PD fermentation clear liquids under the conditions of system (concentration of 1,3-PD, HAc and BA are respectively 79.36g/L, 9.30g/L, 14.22g/L)
First step salting-out extraction is carried out, extracts BA, BA is enriched in organic phase, and the concentration of wherein BA, HAc and 1,3-PD are respectively
16.24g/L、3.96g/L、3.64g/L.The organic acid being stripped using aqueous slkali or basic mineral salting liquid in organic phase, due to
Alkali can result in ester hydrolysis as catalyst and reactant, therefore select sodium carbonate liquor to make back washing agent.Since this step will have
Concentrated effect, back extraction are fixed tentatively as 2 compared to (organic phase and the volume ratio of alkali (or alkaline, inorganic salts) solution):1, concentration of sodium carbonate becomes
Change scope is 0~0.8mol/L, investigates the influence that concentration of sodium carbonate is stripped material.As shown in figure 4, with concentration of sodium carbonate
Rise, the rate of recovery of three kinds of materials all gradually increasing, wherein that influenced maximum by concentration of sodium carbonate is BA, HAc takes second place, and 1,3-
PD is minimum.Concentration of sodium carbonate from 0 increase to 0.3mol/L when, the BA rate of recovery increases to 91.28% by 9.98%, at this time, continues to increase
Add concentration of sodium carbonate, the rate of recovery change of BA is little.HAc is similar to the changing rule of BA, and 0.3mol/L sodium carbonate liquors can take
Obtain the 93.96% HAc rate of recovery.1,3-PD hydrophily is most strong, its existence in the solution is hardly influenced by pH value,
Therefore its rate of recovery maintains 90% or so with the change unobvious of concentration of sodium carbonate.Therefore it is molten using the sodium carbonate of 0.3mol/L
Liquid energy obtains preferable back extraction effect, and the molar ratio of sodium carbonate and organic acid (HAc and BA) in butyl acetate solution is 3 at this time:
5。
2. according to the method described in embodiment 2, in 25% (w/w) NaH2PO4/ 30% (w/w) butyl acetate salting-out extraction body
To 1,3-PD fermentation clear liquids under the conditions of system (concentration of 1,3-PD, HAc and BA are respectively 79.36g/L, 9.30g/L, 14.22g/L)
First step salting-out extraction is carried out, extracts BA, BA is enriched in organic phase, and the concentration of wherein BA, HAc and 1,3-PD are respectively
16.12g/L、3.22g/L、4.96g/L.Molar ratio according to organic acid in sodium carbonate and butyl acetate solution is 3:5 ratio,
The influence that butyl acetate solution compares back extraction with sodium carbonate liquor initial phase is investigated, since the back extraction of BA will have concentrated effect,
So the initial of selection is in a ratio of 2:1,2.4:1,3:1 and 4:1, the results are shown in Table 2.With compared to increase, three kinds of materials
Concentration in lower phase constantly raises, it is shown that good concentrated effect, but its rate of recovery also decreases.This is because compare
Increase influences intermolecular contact and alternate mass transfer, and higher compared to remaining concentration of sodium carbonate is raised, and causes certain journey
The salting-out effect of degree.Butyl acetate solution and the initial of sodium carbonate liquor are in a ratio of 2 in the present embodiment:Can be obtained when 1 compared with
The BA and HAc of high-recovery, the rate of recovery more than 90%；Preferable effect equally can also be received when described other are compared, is returned
Yield more than 85%, but compared to no more than 5:1, it cannot be come into full contact with compared to excessive stripping solution with organic phase, reduce production
The product rate of recovery, causes damages, it is impossible to reaches preferable separation purpose.Lower phase solution after back extraction is concentrated, drying obtains butyric acid
The mixture of sodium and a small amount of sodium acetate, BA and HAc are the important short chain fatty acids of enteron aisle, play a variety of physiological actions, therefore fourth
Sour sodium and sodium acetate can be used as feed addictive, applied to animal feed.
The influence to BA, HAc and 1,3-PD distribution behavior is compared in the back extraction of table 2.
The extraction (second step salting-out extraction) of 4 1,3- propane diols of embodiment and acetic acid
1. by the dissolving of HAc and 1.3-PD standard items in deionized water, prepare the simulated solution of HAc and 1,3-PD, HAc and
The concentration of 1.3-PD is respectively 8.05g/L, 61.60g/L.First step salting-out extraction uses hydrophobicity organic in embodiment 2
Solvent, most water and inorganic salts are retained in lower phase after extraction, therefore use 25% (w/w) NaH2PO4/ 30% (w/w) second
After acid butyl ester salting-out extraction system extraction 1,3-PD fermentation clear liquids, water and NaH in lower phase2PO4Mass ratio be about 9:5, by this
Ratio, NaH is added into simulated solution2PO4, it is to be dissolved, then the ethanol that purity is 95% is added thereto, form salting-out extraction body
System.In extraction system, volume fraction of ethanol excursion is 20~50%, investigates it to 1,3-PD in simulated solution and HAc points
The influence matched somebody with somebody.As shown in figure 5, on the whole, the distribution coefficient and the rate of recovery of HAc and 1,3-PD are with the increasing of the volume fraction of ethanol
Add and increase, when the volume fraction of ethanol is 40%, the rate of recovery of HAc and 1,3-PD can reach 89.59%, 92.32%.
2. according to the method described in embodiment 2, in 25% (w/w) NaH2PO4/ 30% (w/w) butyl acetate salting-out extraction body
To 1,3-PD fermentation clear liquids under the conditions of system (concentration of 1,3-PD, HAc and BA are respectively 78.62g/L, 6.41g/L, 12.93g/L)
First step salting-out extraction is carried out, 1,3-PD and HAc distribution is respectively 6.41g/L, 78.62g/L in lower phase, its concentration.Downward phase
95% ethanol is added, the volume fraction excursion of ethanol is 40%~52%, investigates it to 1,3-PD in fermentation clear liquid and HAc
The influence of extraction.The results are shown in Figure 6, and two kinds of objects are similar with the rate of recovery in fermentation clear liquid in simulated solution, but are fermenting
In clear liquid, its distribution coefficient is obvious than big in simulated solution, between the distribution coefficient of HAc maintains 6.71~7.74,1,3-
PD's is maintained at 9.3 or so, this is because the material such as residual protein present in fermentation clear liquid, nucleic acid and salt increases effect of saltouing
Should.In 50% (v/v) ethanol system, the rate of recovery of HAc and 1,3-PD are respectively 94.40%, 95.50%.
Two step salting-out extractions of 5 zymotic fluid of embodiment
The concentration of 1,3-PD, HAc and BA are respectively 87.21g/L, 6.66g/L and 17.64g/L in zymotic fluid.To 225g's
125g NaH are added in zymotic fluid2PO4, it is to be dissolved, 150g butyl acetates are added, is uniformly mixed, is stored at room temperature, carry out first
Salting-out extraction is walked, is divided into three-phase, upper phase is the organic phase rich in BA, and lower phase is the salt phase rich in 1,3-PD and HAc, interphase master
To be thalline and albumen.The distribution coefficient and the rate of recovery of BA is respectively 23.34,93.78%；95.20% 1,3-PD and
69.04% HAc is distributed in salt phase；Thalline and albumen removal rate are respectively 99.63%, 95.78%.
Add 79mL 0.76mol/L sodium hydroxide solutions into organic phase, the back extraction yield of BA and HAc is 87.33%,
The rate of recovery of 181.44%, HAc are considerably beyond theoretical value, this is because the sodium hydroxide solution of strong basicity causes butyl acetate water
Solution, caused by producing a large amount of acetic acid, so when the hydrophobic organic solvent that first step salting-out extraction uses is esters, back extraction
It cannot be sodium hydroxide or potassium hydroxide solution to take solution；79mL 0.38mol/L sodium carbonate liquors, BA are added into organic phase
Back extraction yield with HAc is 88.72%, 82.70%.
95% ethanol of 251mL, which is added to, to salt carries out second step salting-out extraction, 1,3-PD yields are 96.29% in upper phase,
HAc yields are 97.26%, and whole thalline and 77.89% albumen are removed.The recycling of 1,3-PD, HAc and BA in whole process
Rate is respectively 91.67%, 92.75%, 83.20%, and whole thalline and 97.16% albumen are removed.Three kinds of materials are each
The rate of recovery in step is as shown in table 3.
The distribution of 1,3-PD, HAc and BA in different units operation in 3. zymotic fluid of table
I, II and III represents first step salting-out extraction, back extraction and second step salting-out extraction respectively in table 3.
BA in anti-stripping agent can obtain more than 94% rate of recovery through evaporation water removal, drying, finally obtain sodium butyrate and
The mixture of a small amount of sodium acetate.
The 1,3-PD and HAc of ethanol phase can pass through separated.Organic phase pH is adjusted to 7.0 with sodium hydroxide, crosses and filters out
The phosphate separated out is removed, filtrate is distilled, 110 DEG C of overhead fractions are collected under conditions of vacuum 0.094-0.096MPa
1,3-PD, more than 92% 1,3-PD is recovered, and almost all acetic acid is retained in tower reactor with acetic acid na form.
Claims (7)
1. a kind of method of 1,3-PD, acetic acid and butyric acid in two steps salting-out extraction separation and fermentation liquid, it is characterised in that described
Method comprises the following steps：
1) first step salting-out extraction：Soluble acid inorganic salts are dissolved in 1,3-PD zymotic fluid, adding hydrophobicity has
Solvent, vibration mixing, stands split-phase at room temperature, and upper phase be the organic phase rich in butyric acid, lower phase be rich in 1,3-PD with
The salt phase of acetic acid；
2) aqueous slkali or basic mineral salting liquid are added in the organic phase obtained to step 1), is stripped butyric acid；
3) second step salting-out extraction：Add hydrophilic organic solvent in the salt phase obtained to step 1), extraction 1,3-PD and
Acetic acid.
2. according to the method described in claim 1, it is characterized in that, the 1,3-PD zymotic fluid is 1, the 3- third of mycetome
Glycol fermenation raw liquid or the 1,3-PD fermentation clear liquid for removing thalline, 1,3-PD is dense in the 1,3-PD zymotic fluid
It is 6~15g/L to spend for 50~100g/L, acetic acid concentration, and butyric acid density is 10~20g/L.
3. according to the method described in claim 1, it is characterized in that, the soluble acid inorganic salts described in step 1) are sulphur
One kind in sour ammonium, sodium dihydrogen phosphate or potassium dihydrogen phosphate, addition are the 15%~27.5% of extraction system quality.
4. according to the method described in claim 1, it is characterized in that, the hydrophobic organic solvent described in step 1) is acetic acid
One kind in ethyl ester, butyl acetate, methyl tertiary butyl ether(MTBE) or hexone, addition are the 15% of extraction system quality
~35%.
5. according to the method described in claim 4, it is characterized in that, when the hydrophobic organic solvent used in step 1) is acetic acid
When ethyl ester or butyl acetate, in step 2), alkaline, inorganic salts are added in the organic phase, the alkaline, inorganic salts are carbon
One kind in sour sodium, sodium acid carbonate, potassium carbonate or saleratus；When the hydrophobic organic solvent used in step 1) is methyl- tert
During a kind of in butyl ether or hexone, aqueous slkali or alkaline nothing are added in step 2), in the organic phase
Machine salting liquid, the alkali is sodium hydroxide, one kind in potassium hydroxide, and the alkaline, inorganic salts are sodium carbonate, bicarbonate
One kind in sodium, potassium carbonate or saleratus.
6. according to the method described in claim 1, it is characterized in that, organic acid and alkali in organic phase described in step 2)
Solution or alkali in basic mineral salting liquid or the molar ratio of alkaline, inorganic salts are 5:1~8, organic phase and aqueous slkali or alkaline nothing
The volume ratio of machine salting liquid is 1~5:1.
7. according to the method described in claim 1, it is characterized in that, the hydrophilic organic solvent described in step 3) is third
One kind in ketone, methanol, ethanol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, dimethyl carbonate or tetrahydrofuran,
Addition is the 20%~52% of extraction system volume.
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| CN112920021A (en) * | 2021-02-01 | 2021-06-08 | 大连理工大学 | Method for extracting 1, 3-propylene glycol from fermentation liquor by using alcohol mixed solvent |
| CN113203720A (en) * | 2021-06-15 | 2021-08-03 | 山西农业大学 | Method for detecting residual amount of dichlorvos in aquatic product by fluorescence analysis method and detection kit |
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| CN109574802A (en) * | 2018-11-27 | 2019-04-05 | 大连理工大学 | A method of separating 1,3- propylene glycol, acetic acid and butyric acid from fermentation liquid |
| CN112920021A (en) * | 2021-02-01 | 2021-06-08 | 大连理工大学 | Method for extracting 1, 3-propylene glycol from fermentation liquor by using alcohol mixed solvent |
| CN113203720A (en) * | 2021-06-15 | 2021-08-03 | 山西农业大学 | Method for detecting residual amount of dichlorvos in aquatic product by fluorescence analysis method and detection kit |
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