CN102617325B - Method for decoloration of succinic acid fermentation broth by ion exchange resin - Google Patents
Method for decoloration of succinic acid fermentation broth by ion exchange resin Download PDFInfo
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- CN102617325B CN102617325B CN201110029752.7A CN201110029752A CN102617325B CN 102617325 B CN102617325 B CN 102617325B CN 201110029752 A CN201110029752 A CN 201110029752A CN 102617325 B CN102617325 B CN 102617325B
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- exchange resin
- succinic acid
- ion exchange
- acid fermentation
- fermentation liquor
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Abstract
The invention relates to a method for decoloration of a succinic acid fermentation broth by an ion exchange resin. The method provided by the invention comprises a process of carrying out the decoloration of a succinic acid fermentation broth by an ion exchange resin and the process comprises 1, carrying out centrifugation impurity-removal of the succinic acid fermentation broth to obtain a solid-free succinic acid fermentation broth, 2, connecting a cation exchange resin and an anion exchange resin in series and carrying out pretreatment, 3, at a temperature of 30 to 50 DEG C, adding the solid-free succinic acid fermentation broth obtained by the step 1 into the ion exchange resin treated by the step 2, and carrying out decoloration so that colored matters and impurities are adsorbed by the ion exchange resin and thus a decolored succinic acid fermentation broth is obtained. Compared with the prior art, the method has the advantages that the ion exchange resin has good selective adsorbability of pigments and thus the pigments can be dissociated easily so that only water-washing is needed; and the ion exchange resin can be regenerated by acid-washing and alkali-washing.
Description
Technical field
The present invention relates to succinic acid separation and purification field, particularly, the present invention relates to a kind of method utilizing ion exchange resin to decolour to succinic acid fermentation liquor.
Background technology
Succinic acid (Succinic Acid) has another name called succsinic acid, belong to slightly acidic binary organic carboxyl acid, it is the important meta-bolites of microorganism tricarboxylic acid cycle and glycolytic pathway, microbe fermentation method can be adopted to produce, because of structure and the chemical property of its uniqueness, succinic acid has purposes widely in a lot of field, can be used for synthesized degradable plastics, the tranquillizer of field of medicaments, the foodstuff additive of foodstuffs industry, tensio-active agent and other etc.Except the exploration to aspects such as zymotechnique (US7455997, CN101389752, US6596921) and strain improvements (CN101531972), the separation and purification of succinic acid is also the important research content that succinic acid produced by fermented liquid.
A large amount of pigment molecular is had, the glycoprotein produced as some microbial metabolisms and polypeptide in microbial fermentation solution, and the pigment molecular existed in carbon source, nitrogenous source in substratum; If effectively pigment molecular can not be removed, not only can affect the separation and purification unit operation of upstream and downstream, also can affect product purity.The preparation cost of succinic acid is primarily of fermenting process and separation costs composition, and the cost of wherein separation and purification accounts for more than 60% of total cost of production, and how reducing separation costs, improving purity is study hotspot both domestic and external, has occurred the patent of some amount.The separation purification method of the succinic acid reported in current patent and document mainly contains calcium salt method (CN101643400, US5143834), liquid-liquid extraction method (US5773653), absorption method (CN101348428, CN101348429, CN101811953A), electroosmose process (CN101486637A), the methods such as membrane sepn (CN101748161A), wherein widely used discoloring agent is wood activated charcoal, be divided into powder activated carbon and granulated active carbon, although granulated active carbon decolorizing effect is fine and can regenerate, but the price of about 150,000 yuan per ton is too expensive.In use there is many drawbacks in powder activated carbon, as lost efficacy fast (< 5 months), and not reproducible utilization, dust is there is in use procedure, cause aftertreatment difficulty, fermented liquid raises by causing specific conductivity after activated carbon, affects subsequent unit operation.
Summary of the invention
The object of the invention is to ion exchange resin is applied in succinic acid fermentation liquor decolouring field.
Another object of the present invention there are provided a kind of method utilizing ion exchange resin to decolour to succinic acid fermentation liquor.
According to the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor of the present invention, described method comprises the step that spent ion exchange resin is decoloured to succinic acid fermentation liquor.
According to the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor of the present invention, said method comprising the steps of:
1) by centrifugal for succinic acid fermentation liquor removal of impurities, obtain not containing the succinic acid fermentation liquor of solid substance;
2) by positively charged ion and anionite-exchange resin series connection, and pre-treatment is carried out;
3) at 30 ~ 50 DEG C by step 1) in succinic acid fermentation liquor add step 2) in process after ion exchange resin, decolour, coloring matter and impurity absorption on ion exchange resin, obtain decolour after succinic acid fermentation liquor.
According to the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor of the present invention, described step 2) ion exchange resin comprises: ZGA455, ZGD301, ZGA331, NKD301, NKD380, XUS40285, Reillex 425, Reillex 402, IRA-93, IRA-35, AG-3 or AG-1.
According to the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor of the present invention, described step 2) pre-treatment of ion exchange resin is soaked by ion exchange resin, and successively with 4wt%HCl, water, 4wt%NaOH and water carry out processing to effluent liquid be neutrality.
According to one embodiment of the invention, utilize concrete the comprising the following steps of method that ion exchange resin decolours to succinic acid:
1) producing succinic acid by microbial fermentation is utilized, when in fermented liquid, the concentration of succinic acid reaches 10 ~ 100g/L, put tank, after putting tank, the fermented liquid containing microbial cells and substratum solid substance is carried out centrifugal pre-treatment, obtain not containing the succinic acid fermentation liquor of clarification;
2) water content is used to be 50 ~ 60%, wet volume density 0.65 ~ 0.85g/mL, wet true density 1.03 ~ 1.13g/mL, granularity is greater than 95%, after mill, rate of small round spheres is greater than 98%, succinic acid adsorptive capacity is less than to the ion exchange resin of 0.01mg/g dried resin, by in resin filling to the positively charged ion and anion chromatography post of series connection, when positively charged ion and anionite-exchange resin are connected, sequencing can exchange as required, Zeo-karb is mainly used in metal biosorption and coloring matter, and anionite-exchange resin is mainly used in the absorption of other anionic impurity and coloring matter, wherein resin Intake Quantity is according to 60 ~ 90% of calculating formula column volume after resin expansion, packed bed chromatography column is first just being washed rear backwash with deionized water,
3) by clarified broth 30 DEG C ~ 50 DEG C time, pump into ion exchange column with 1.5 ~ 4.5BV/h flow to decolour, when to wear liquid transmittance be 60 ~ 80% of sample introduction liquid to stream, be considered as resin and reach pigment molecular maximal absorptive capacity, adsorb saturated, stop sample introduction, when stream wear liquid transmittance be greater than 95% time, other succinic acid separation and purification operating unit can be entered and to purify further succinic acid;
4) resin after decolouring uses 0.1 ~ 10M NaOH, KOH, Na
2cO
3or K
2cO
3solution carries out zeolite regeneration with the flow of 2 ~ 5BV/h to the resin being in pigment separated saturated adsorption, and the resin after regeneration uses deionized water rinsing to pH6.0 ~ 8.5, and deionized water drip washing total amount is 3BV.
Instant invention overcomes the inertial thinking that conventional ion exchange resin can only be used for carrying out ion-exchange, separation and purification, ion exchange resin is applied in the decolouring field of succinic acid fermentation liquor, find that answering the fermented liquid of spent ion exchange resin to succinic acid to decolour has good effect, can also play the object to succinic acid fermentation liquor purifying simultaneously.In addition, operational condition when the present inventor is also decoloured to succinic acid to ion exchange resin is investigated, find the flow velocity relative to succinic acid fermentation liquor, the capacity of resin and eluent, the temperature of its decolouring is key factor, when temperature is crossed lower than 30 DEG C, fermented liquid is very slow through the speed of ion exchange resin, efficiency is very low, do not reach good economic benefit, when temperature is 50 DEG C, decolorizing effect reaches the highest, because resin reduces coloring matter adsorptive power when temperature is too high, therefore its percent of decolourization reduces, therefore best net effect can be reached when temperature is at 30 ~ 50 DEG C.
Compared with prior art, the present invention has the following advantages:
1, resin has good selective adsorption to pigment, and the easy wash-out of pigment, washes, and pickling and alkali cleaning can realize the regeneration of resin.
2, resin succinic acid is not adsorbed or adsorptive capacity little, in elutriant, contained succinic acid concentration is less than 5g/L.
3, resin physical strength is large, can improve sample introduction flow velocity.
4, there is filling gap in particulate resins, and back-pressure is low, and it is little that fermentation broth stream wears resistance.
5, fermented liquid is by while resin decolorization, can also play desalting effect, reduces fermented liquid specific conductivity.
6, resin regeneration rate is greater than 98%, reuses 30 times and is no more than 20% to the reduction of pigment adsorptive capacity, can Reusability.
7, spent ion exchange resin is decoloured to succinic acid fermentation liquor, effect is better than activated carbon decolorizing, can save separation costs.
Accompanying drawing explanation
Fig. 1 varies with temperature schematic diagram with NKD301 resin to the percent of decolourization that succinic acid decolours in the embodiment of the present invention 2.
Embodiment
Below by way of specific embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention, and be not used in restriction scope of the present invention.
Embodiment 1
1, the fermentation of succinic acid
The selected microorganism of fermentation adopts Actinobacillus succinogenes (Actinobacillus succinogenes), has another name called 130Z
t, be stored in American Type Culture Collecti (ATCC), numbering ATCC55618.The preparation of substratum: Storaged media and activation medium are nutritious TSB substratum; Seed culture based formulas (unit: g/L) glucose 10, yeast powder 6, NaCl 1, MgCl
22, CaCl
21.5, Na
2hPO
42.9, NaH
2pO
42.2; Seed culture based formulas (unit: g/L) glucose 30, yeast powder 15, urea 2, KCl 4, MgCl
22, MnCl
20.07, CaCl
21.5, Na
2hPO
44.4, NaH
2pO
43.3.Adjust ph is 6.4 ~ 6.9,121 DEG C of sterilizings 30 minutes.
Under aseptic technique, seed liquor is added in fermented liquid, the volume percent of inoculum size is 5 ~ 7%, now succsinic acid amount is 0, open and stir, the rotating speed of bio-reactor stirring rake is 125rpm ~ 150rpm, passes into the carbon dioxide of filtration sterilization, gas flow rate >=0.2vvm, at 37 ~ 39 DEG C of condition bottom fermentations.
Table 1 succinic acid-producing actinobacillus batch fermentation result
2, the choosing of ion exchange resin
The source that chromatography column potting resin is selected and type as shown in table 2, resin first soaks 24 hours with deionized water, then successively through 4 " 4wt%HCl, water, 4wt%NaOH and water " pre-treatment, plastic resin treatment is-OH type, finally use aseptic water washing bed, be neutral to effluent liquid pH value, sample introduction fermented liquid can be prepared; After the adsorptive capacity that reaches capacity, select 0.1 ~ 10MNaOH solution as eluent, selected resin in the effect in expanding bed after primary sorption, wash-out, reprocessing cycle fermented liquid in table 2.
Table 2 decolorizing resin type selecting
3, the evaluation method of ion-exchange resin decolorization effect
Adopt GB colour method (GB317-1998), measure the absorbancy change of pretreated fermentation liquid at 420nm place.Percent of decolourization and desorption efficiency method of calculation as follows:
The absorbancy of fermented liquid to be measured before percent of decolourization (%)=(before decolouring the absorbancy one of fermented liquid to be measured decolour after the absorbancy of fermented liquid to be measured)/decolouring
Desorption efficiency (%)=stripping liquid absorbancy/(decolouring primary fermentation liquid absorbancy one decolour secondary fermentation liquid absorbancy)
In selecting step 2, NKD301 resin carries out static decolorization experiment, investigate the impact of differing temps on the static percent of decolourization of NKD301 resin and accurately measure the pretreated wet resin of 10ml in 50ml triangular flask, add pH 7, the centrifugal pretreated fermented clear liquid of 30mL, respectively at 30,40,50,60,70 DEG C of stirred in water bath 4h, for reducing the error introduced, three Duplicate Samples are done in each experiment, survey the rear absorbancy of effluent liquid at 420nm place of absorption, calculate percent of decolourization.Fig. 1 is that the temperature that obtains of the static Decolorant Test of NKD301 resin is to percent of decolourization change curve, can find out, the impact of temperature on the absorption property of ion exchange resin is larger, and temperature increases gradually at 30 ~ 50 DEG C of static percent of decolourizations, declines comparatively obvious more than 50 DEG C of rear decoloring rates.
4, ion exchange resin is utilized to carry out desolventing technology to succinic acid fermentation liquor
With the fermented liquid putting tank in step 1 for 54 hours for sample decolours, wherein succinic acid concentration is 85g/L, and formic acid concn is 10g/L, acetic acid concentration 9g/L, and remaining sugar concentration is 3g/L, fermented liquid pH6.8.
Fermented liquid is after centrifugally operated, the removal rate of thalline is 98%, the removal rate 35% of albumen, the micro-filtration penetrating fluid of acquisition is carried out decolorization operations, be specially and temperature is controlled, 45 DEG C time, to pump into ion exchange column with 3BV/h flow, collect and spread liquid, when to wear liquid transmittance be 60 ~ 80% of sample introduction liquid to stream, stop sample introduction.Now, fermented liquid percent of decolourization reaches more than 80%, and resin is saturated to pigment absorption in fermented liquid, and stream is worn liquid colourity and reduced obviously, and transmittance reaches 97%.
By transmittance be 97% stream wear liquid and carry out ultrafiltration, ultrafiltration adopts tubular fibre film device, organic membrane selected by mould material, it is 6000 ~ 20000 dalton that film retains fractional dose, adopt the mode of cross-flow operation, service temperature is 25 DEG C, working pressure is 0.5MPa, after ultrafiltration, the removal rate of protein is 82%, now succinic acid concentration is 90g/L, carry out nanofiltration operation further, wherein the relative molecular weight that retains of nanofiltration membrane is 90, temperature is room temperature, working pressure is 2MPa, after nanofiltration, the rejection of succinic acid reaches 96%, the transmitance of acetic acid is 89%, the transmitance 87% of formic acid, the rejection of sulfate ion is 98%, the rejection of magnesium ion is 98%, the rejection of calcium ion is 99%, the rejection of protein is 100%.After two step conjunctiva separation unit operation, the yield of succinic acid is 90%, and purity is more than 99.5%, and sulphate content is lower than 0.001%.
By the transmittance of above-mentioned acquisition be 97% stream wear liquid and carry out calcium salt method lock out operation, by Ca (OH)
2serum (25 ~ 33%w/v) at room temperature slowly adds in the fermentation clear liquid after decolouring, and rotating speed 300r/min, controls to stir, and when the acidity value of reaction system rises to 12.5, collected by centrifugation calcium succinate solid, washes three times with water, add H
2sO
4(40 ~ 60%w/v), stir until system pH is down to 2.0 rapidly, centrifugation calcium sulfate, clear liquor is placed in 60 DEG C of thermostat containers to spend the night and is obtained succinic acid crystallization.
By succinic acid fermentation liquor after reference ion exchange resin desolventing technology, find that its pigment content greatly reduces, fermented liquid after decolouring adopts calcium salt method technique to purify, by the gypsum (calcium sulfate) obtained in this subsequent technique, calcium succinate precipitates, and adopt further calcium salt method to be separated the succinic acid sterling obtained after ion-exchange resin decolorization process to compare discovery, apparent colour is crystal white, with purity be 99.9% commercial succinic acid product color close.
Claims (5)
1. the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor, is characterized in that, said method comprising the steps of:
1) by centrifugal for succinic acid fermentation liquor removal of impurities, obtain not containing the succinic acid fermentation liquor of solid substance;
2) by positively charged ion and anionite-exchange resin series connection, and pre-treatment is carried out;
3) at 30 ~ 50 DEG C by step 1) in succinic acid fermentation liquor add step 2) in process after ion exchange resin, decolour, coloring matter and impurity absorption on ion exchange resin, obtain decolour after succinic acid fermentation liquor;
Described step 2) Intake Quantity of ion exchange resin is 60 ~ 90% of column volume when calculating after resin expands;
Described step 3) flow of succinic acid fermentation liquor is 1.5 ~ 4.5BV/h.
2. utilize the method that ion exchange resin decolours to succinic acid fermentation liquor according to claim 1, it is characterized in that, described ion exchange resin comprises: ZGA455, ZGD301, ZGA331, NKD301, NKD380, XUS40285, Reillex425, Reillex402, IRA-93, IRA-35, AG-3 or AG-1.
3. utilize the method that ion exchange resin decolours to succinic acid fermentation liquor according to claim 1, it is characterized in that, described step 2) pre-treatment of ion exchange resin is soaked by ion exchange resin, and successively with 4wt%HCl, water, 4wt%NaOH and water carry out processing to effluent liquid be neutrality.
4. utilize the method that ion exchange resin decolours to succinic acid fermentation liquor according to claim 1, it is characterized in that, described method comprises step 3) in ion exchange resin alkali lye carry out wash-out, again with deionized water rinsing to pH5.0 ~ 8.5, realize the step of the regeneration of resin.
5. the method utilizing ion exchange resin to decolour to succinic acid fermentation liquor according to claim 4, is characterized in that, described alkali lye is NaOH, KOH, Na
2cO
3or K
2cO
3, its concentration is 0.1 ~ 10M, and flow is 2 ~ 5BV/h.
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| CN102935298A (en) * | 2012-11-28 | 2013-02-20 | 湖北益泰药业有限公司 | Method for recovering refined oxalate mother liquor |
| CN106967758B (en) * | 2017-05-09 | 2020-10-16 | 中国科学院过程工程研究所 | Method for extracting succinic acid and propionic acid |
| CN107916281A (en) * | 2017-07-10 | 2018-04-17 | 广西多得乐生物科技有限公司 | A kind of method that gamma aminobutyric acid is isolated and purified from streptococcus acidi lactici fermented solution |
| CN110713439A (en) * | 2019-10-31 | 2020-01-21 | 濮阳市盛源能源科技股份有限公司 | Preparation method of cyclohexane-1, 2-dicarboxylate environment-friendly plasticizer |
| CN114605255A (en) * | 2022-03-09 | 2022-06-10 | 宁夏恒力生物新材料有限责任公司 | Method for decoloring long-carbon-chain dibasic acid fermentation liquor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665428A (en) * | 2009-10-09 | 2010-03-10 | 安徽丰原发酵技术工程研究有限公司 | Method for extracting succinic acid from succinic acid fermentation liquor |
| CN101811953A (en) * | 2009-02-23 | 2010-08-25 | 凯能高科技工程（上海）有限公司 | Method for extracting succinic acid from fermentation broth |
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| EP1669459B1 (en) * | 2003-09-30 | 2014-10-22 | Ajinomoto Co., Inc. | Method of purifying succinic acid from fermentation liquid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101811953A (en) * | 2009-02-23 | 2010-08-25 | 凯能高科技工程（上海）有限公司 | Method for extracting succinic acid from fermentation broth |
| CN101665428A (en) * | 2009-10-09 | 2010-03-10 | 安徽丰原发酵技术工程研究有限公司 | Method for extracting succinic acid from succinic acid fermentation liquor |
Non-Patent Citations (2)
| Title |
|---|
| 发酵丁二酸提取液的脱色工艺研究;孙超等;《食品与发酵工业》;20081231;第34卷(第4期);第1-6页 * |
| 发酵丁二酸的提取工艺研究;孙超;《硕士学位论文》;20081231;第2.2.4.1节 * |
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