CN1034579A - The method of microorganism fermentation n-paraffins production long-chain alpha alpha, omega-dicarboxylic acid - Google Patents
The method of microorganism fermentation n-paraffins production long-chain alpha alpha, omega-dicarboxylic acid Download PDFInfo
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- CN1034579A CN1034579A CN 87105445 CN87105445A CN1034579A CN 1034579 A CN1034579 A CN 1034579A CN 87105445 CN87105445 CN 87105445 CN 87105445 A CN87105445 A CN 87105445A CN 1034579 A CN1034579 A CN 1034579A
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Abstract
A kind of method of utilizing microorganism fermentation n-paraffins production long-chain alpha alpha, omega-dicarboxylic acid particularly relates to the method for high yield 16 carbon dicarboxylic acid.The kind mother liquor that one strain candida tropicalis UH-3-9 (Candida tropicalisUH-3-9) is cultivated inserts the pH value and contains in the mixed solution of the normal alkane of 10-18 carbon atom of 5-40% (V/V) and 95-60% (V/V) fermention medium for 5.5-9.0.Said mixture was kept under 24-34 ℃ 48-144 hour, then the α alpha, omega-dicarboxylic acid that obtains was separated purification.
Description
The present invention utilizes the method for microbial fermentation from normal alkane production long-chain alpha alpha, omega-dicarboxylic acid, the particularly method of high yield 16 carbon dicarboxylic acid.
Long chain dicarboxylic acid is a class important chemical material, can be used to make cold-resistant plasticizer, engineering plastics, resin, coating and spices.Particularly 16 carbon dicarboxylic acid are important source material of making exaltone, and the latter has pure Moschus fragrance, is a kind of senior musk odorant.
From the seventies, research and application that various countries utilize the microbiological oxidation normal alkane to produce long chain dicarboxylic acid have obtained breakthrough, and the eighties rises and entered the suitability for industrialized production stage.The interior tail of Japan in 1972 is very auxilliary to wait the people in the article of " agricultural biochemistry " magazine (Agr.Biol.Chem.Vol.36) " microorganism utilizes normal alkane to produce long chain dicarboxylic acid ", reported that they use cloaca candiyeast MR-12 bacterial strain in 1 liter fermentor tank, make the growth carbon source with sodium-acetate, adding 15%(V/V) n-hexadecane fermentation 72 hours, obtain 16 carbon dicarboxylic acid of 63.3 grams per liters, the transformation efficiency of n-hexadecane is 54%.Tail is again at " oil and microorganism " magazine (" oil と microorganism " NO.20 in 1978,1978) reported the result of expanding test: in 300 liter jars, acetic acid is as the growth carbon source, n-hexadecane was as fermenting carbon source fermentation 68 hours, obtain 54 grams per liters, 16 carbon dicarboxylic acid, n-hexadecane transformation efficiency 70%.This is the highest, the largest reports of present 16 carbon dicarboxylic acid output.1985, people such as village planted at " oil and microorganism " NO.33(" oil と microorganism " NO.33 in Japan) go up report and produce corresponding dicarboxylic acid respectively in 3 liters of jar top fermentations 94-113 hour normal alkane (add-on 30%V/V) from 9-18 carbon with the mutant strain M2030 of candida tropicalis.Wherein the output of 11-15 carbon dicarboxylic acid is respectively 62 grams per liters.82 grams per liters, 96 grams per liters, 100 grams per liters and 90 grams per liters, the normal alkane transformation efficiency is between 30-45%, and 16 carbon dicarboxylic acid output are lower, every liter less than 50 grams.
The purpose of this invention is to provide a kind of with the method, the particularly method of high yield 16 carbon dicarboxylic acid of candida tropicalis from 10-18 carbon normal alkane fermentative production long-chain alpha alpha, omega-dicarboxylic acid.
The used bacterial strain of the present invention is candida tropicalis UH-3-9 enhanced variant (Candida tropiealis UH-3-9), be to generate the stronger candida tropicalis (seeing " microorganism journal " 20(1) of long chain dicarboxylic acid ability: 88-93 with a strain two ends oxidation normal alkane, 1980) be starting strain, by nitroguanidine and repeatedly mutagenesis of ultraviolet ray, the strain omega oxidation ability that seed selection is come out is stronger, assimilation normal alkane ability and the more weak mutant strain of β-Yang Hua ability.The used bacterial strain of the present invention is deposited in Chinese microorganism strains preservation management committee common micro-organisms center, is numbered CGM CCNO.0129.
The female substratum of kind of the present invention has the wort of (1) 10 Bahrain to add the solid inclined-plane that 2% agar is made; The malt juice liquid medium of (2) 10 Bahrain; (3) the alkane liquid nutrient medium comprises KH
2PO
4The 6-8 grams per liter, sucrose 2-4 grams per liter, yeast extract paste 2-4 grams per liter, corn steep liquor 2-4 grams per liter, urea 2-4 grams per liter, heavy wax 40-50 milliliter/liter, the about 400ppm of defoamer (glycerin polyether), tap water preparation, pH value about 5.Cultivating kind of a female process is: get a transfering loop UH-3-9 yeast thalline, be coated on the wort solid inclined-plane (15 * 180 test tubes, every liquid amount 6-7ml is put into the inclined-plane) and cultivated two days in 28-30 ℃.Get an above-mentioned slant strains, insert the malt juice liquid medium or the 25ml/500ml triangular flask alkane substratum of 30ml/250ml triangular flask, on 200 rev/mins rotary shaker, cultivated 36-48 hour, as fermentation kind of mother in 28-30 ℃.
With enhanced variant candida tropicalis UH-3-9 production long-chain alpha alpha, omega-dicarboxylic acid of the present invention, particularly the concrete grammar of 16 carbon dicarboxylic acid is: kind of the female access pH value that will ferment is that 5.5-8.0 contains 5-40%(V/V), be preferably 10-25%(V/V) normal alkane and the 95-60%(V/V of a 10-18 carbon atom), be preferably 90-75%(V/V) and in the mixed solution of fermention medium.Consisting of of fermention medium: alkali metal phosphate 4-10 grams per liter, sodium-chlor 0.5-2.0 grams per liter, urea 0.5-2 grams per liter, vinylformic acid 0.5-2 milliliter/liter, tensio-active agent polysorbate60 0.1-2.0 grams per liter and other nutrition source.With above-mentioned mixed solution at 24-34 ℃, preferably kept 48-144 hour under 26-32 ℃, then the α alpha, omega-dicarboxylic acid that produces is separated purification, its specific practice is: the liquid heat after the fermentation ends adds NaOH to 80-90 ℃ pH value is transferred to 10-12, use whizzer centrifugal then, collect centrifugal clear liquid and remaining alkane, remove thalline.The centrifugal clear liquid that contains remaining alkane, 20 ℃ of following standing over night, alkane forms the white solid piece on the surface, reclaim and use again, a large amount of oarse-grained 16 carbon dicarboxylic acid sodium salt crystals are sunken to the bottom, inclining supernatant liquor, collects sodium salt crystal, and clear liquid concentrates to use with method handles collection 16 carbon dicarboxylic acid sodium salts.16 carbon dicarboxylic acid sodium salts are dissolved in the 80-90 ℃ of hot water, add dense HCl, transfer pH value, leave standstill crystallisation by cooling and spend the night to 2-3.Collecting the boiling hot quiet of white dicarboxylic acid knot Ч at last is baked to dried for 0 ℃.
Alkali metal phosphate not only as the phosphorus source but also as a kind of buffer reagent composition, behind the adding NaOH, is formed a buffer system in fermentation culture, keep the suitable pH value of fermented liquid.Its content changes with other composition in the substratum, and suitable phosphate content is the 6-8 grams per liter, preferably selects KH
2PO
4And NaHPO
4Urea content in substratum will suit, and as the only nitrogen source of used yeast bacterium, urea content is high, and is favourable to thalli growth, but excessive urea can reduce cytochrome P
450Capacity, influence the generation of α alpha, omega-dicarboxylic acid, therefore optimum urea content is the 0.8-1.2 grams per liter.Acrylic acid existence, yeast capable of blocking improve the output of α alpha, omega-dicarboxylic acid to the degraded of the β-Yang Hua of dicarboxylic acid, optimum concentration be the 0.5-1.2 milliliter/liter.The adding of low quantity of surfactant helps normal alkane
With the emulsification of water, shorten the adaptive phase, quicken thalli growth, improve dicarboxylic acid output.Tensio-active agent is a polysorbate60 preferably, and optimum content is the 0.3-0.7 grams per liter. other nutrition source comprises yeast extract paste, corn steep liquor, sucrose etc., and their content is generally the 0.5-3 grams per liter.
Fermention medium is prepared with tap water, transfers pH value to 6.8-7.3 with NaOH.Needn't add metallic elements such as Mg, Fe, Zn, Mn under normal conditions, the trace that contains in the tap water is just enough.Sterilized 30 minutes for 15 pounds.
Use method of the present invention, can obtain the higher 10-18 of output carbon α alpha, omega-dicarboxylic acid.16 carbon α alpha, omega-dicarboxylic acids particularly, with 16 liter fermentor tanks, the n-hexadecane input amount is 20%(V/V), fermented under optimum conditions 70 hours, and produced acid and be up to 77.5 grams per liters, and prolong fermentation time to 17 hour, produce the acid amount up to 123 grams per liters, transformation efficiency is 79%, obviously is better than the 16 carbon dicarboxylic acid output that Japan is produced with cloaca candida mutant strain MR-12 and candida tropicalis mutant strain M2030, and post-treating method is also simpler.
With vapor-phase chromatography normal alkane fermentative production hexadecane dicarboxylic acid is carried out qualitative and quantitative analysis, analysis condition is: 230 ℃ of column temperatures, N
2The 35ml/ branch, H
20.6kg/cm
2, Ar0.2Kg/cm
2, 280 ℃ of vaporizers.
Fig. 1,2,3 and table 1 can prove that the dicarboxylic acid (representing with DC) that n-Hexadecane fermentation produces is 16 carbon dicarboxylic acid (DC
16), purity is 97.7%.Fig. 4 shows that the purity of used n-hexadecane raw material is 99.7%.
Embodiment
(1) gets the UH-3-9 thalline of a transfering loop, be coated on 15 * 180 the Boiling tube wort solid inclined-plane, totally 6, cultivated 2 days for 28 ℃; (2) a big slant strains of 2 days of above-mentioned cultivation, insert in the 250ml triangular flask of dress 30ml malt extract medium, totally 6 bottles, 28 ℃, on 200 rev/mins rotary shaker, cultivated 2 days; (3) 2 bottles of the wort kind liquid of cultivating in (2), insert in the 3000ml triangular flask of a dress 50ml malt extract medium, totally three bottles, at 28 ℃, cultivated 43 hours on 120 rev/mins the rotary shaker, (4) two bottles of one of (about 1000ml) accesses of the kind liquid of cultivating in (3) are equipped with in 16 liters of automatic controlling tank of 10 liters of fermention mediums, wherein contain KH
2PO
480 grams.NaCl 10 grams, yeast extract paste 20 grams, corn steep liquor 10 grams, vinylformic acid 10ml, urea 10 grams (are dissolved in the 50ml water, separately sterilization, add before the inoculation), defoamer (glycerin polyether) 10 grams are dissolved in 7 liters of tap water, transfer pH value to 7.3 with NaOH, and the 2000ml n-hexadecane, 121 ℃ of sterilizations after 30 minutes, inoculation in the time of 29-30 ℃ is after (5) inoculation, at 29 ℃, stirring velocity 700-900 rev/min, air flow 1: 1-1: 1.5, tank pressure 10-15 pound/centimetre
2Condition under cultivation and fermentation 117 hours.In 24 hours, the breeding thalline was transferred a pH value to 7.0 by 24 hours with NaOH, transferred pH value to 7.5 again one time after in 40 hours, since 48 hours, pH value was controlled at 7.7 automatically, until fermentation ends.Recording 16 carbon dicarboxylic acid is 123.4 grams per liters, and transformation efficiency reaches 79%; (6) after the fermentation ends, heat temperature raising is to 80-90 ℃, add NaOH solution, transfer more than the pH value to 10, centrifugal while hot removal thalline (not having residual n-hexadecane basically), clear liquid is placed cool overnight, a large amount of macrobead 16 carbon dicarboxylic acid sodium salt crystals are separated out, pour out crystalline mother solution (16 carbon dicarboxylic acid also have 1.8%), sodium salt crystal is drained with washing from the beginning on a small quantity, after the crystalline mother solution evaporation concentration is a little volume, place crystallisation by cooling, collect 16 carbon dicarboxylic acid sodium salts, merge and wait to make with extra care.Per 220 grams, 16 carbon dicarboxylic acid sodium salts (water content is about 55%) are dissolved in (about 90 ℃) in 3 liters of hot water, heat HCl and transfer pH value to 2-3, generate white precipitate, place crystallisation by cooling and spend the night, and suction filtration is done, and 60 ℃ of oven dry, get white 16 carbon dicarboxylic acid, 91 grams.The separation and purification total recovery of 16 carbon dicarboxylic acid is about 80% in the fermented liquid.
Fig. 1. be the color atlas of standard dicarboxylic acid and tunning
Data are as follows:
Title time content MK peak area
1.2 0.3991 3537
1.72 0.2003 1775
2.12 8.9584 79397
2.63 8.399 ∨ 74438
3.3 16.2976 ∨ 144442
4.19 13.2711 ∨ 117619
5.37 1.2669 11229
6.94 51.2071 453836
Total amount 99.9999 886275
Fig. 2. be n-hexadecane fermentative production hexadecane dicarboxylic acid's purity check
Data are as follows:
Title time content MK peak area
1.71 0.0262 133
2.1 0.098 499
2.35 0.0634 323
2.62 0.1113 567
2.97 0.1165 593
3.3 0.6172 ∨ 3143
4.18 0.6772 3448
4.86 0.0564 287
5.35 0.5139 2617
6.91 97.7194 497647
Total amount 99.9999 509261
Fig. 3. dicarboxylic acid carbon number law figure
Illustrate: Fig. 3 is the chromatogram result according to Fig. 1, presses the appearance time of Fig. 1, carries out the carbon number law mapping.
Fig. 4. fermentation raw material n-hexadecane purity check
Data are as follows:
Title time content MK peak area
1.06 0.0005 38
1.48 0.0205 1547
1.84 0.0065 489
2.35 99.9724 7521908
Total amount 100 752398
Claims (7)
1, a kind of method of utilizing microbial fermentation production long-chain alpha alpha, omega-dicarboxylic acid is characterized in that:
(1) with a strain candida tropicalis UH-3-9 (Candida tropicalis UH-3-9) the common micro-organisms center C GMCC NO.0129 of China Committee for Culture Collection of Microorganisms) to insert pH value be that 5.5-9.0 contains in the mixed solution of the normal alkane of 10-18 carbon atom of 5-40% (V/V) and 95-60% (V/V) fermention medium for the kind mother liquor cultivated.Consisting of of fermention medium: alkali metal phosphate 4-12 grams per liter, sodium-chlor 0.5-2.0 grams per liter, urea 0.5-2.0 grams per liter, vinylformic acid 0.5-2 milliliter/liter, tensio-active agent leaf polysorbate60 0.1-2.0 grams per liter and other nutrition source.
(2) said mixture was kept under 24-34 ℃ 48-144 hour, then the α alpha, omega-dicarboxylic acid that produces was separated purification.
2, method according to claim 1, the content that it is characterized in that normal alkane in the mixed solution is 10-25%(V/V), fermention medium content 90-75%(V/V).
3, method according to claim 1 is characterized in that in the composition of fermention medium: alkali metal phosphate 6-8 grams per liter, and urea 0.8-1.2 grams per liter, vinylformic acid 0.5-1.2 milliliter/liter, tensio-active agent polysorbate60 0.3-0.7 grams per liter.
4, according to claim 1,2 method is characterized in that said normal alkane contains 14-16 carbon atom.
5, according to claim 1,2 method is characterized in that said normal alkane contains 16 carbon atoms.
6, according to claim 1,3 described methods is characterized in that preferably KH of alkali metal phosphate
2PO
4And Na
2HPO
4
7, method according to claim 1, the temperature of the mixed solution that it is characterized in that fermenting is controlled at 26-32 ℃, and pH value is 6.5-8.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 87105445 CN1017350B (en) | 1987-08-12 | 1987-08-12 | Production method of long chain alpha omega-dicarboxylic acid using microbe to ferment normal paraffin hydrocarbon |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 87105445 CN1017350B (en) | 1987-08-12 | 1987-08-12 | Production method of long chain alpha omega-dicarboxylic acid using microbe to ferment normal paraffin hydrocarbon |
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| Publication Number | Publication Date |
|---|---|
| CN1034579A true CN1034579A (en) | 1989-08-09 |
| CN1017350B CN1017350B (en) | 1992-07-08 |
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ID=4815321
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 87105445 Expired CN1017350B (en) | 1987-08-12 | 1987-08-12 | Production method of long chain alpha omega-dicarboxylic acid using microbe to ferment normal paraffin hydrocarbon |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1048754C (en) * | 1995-11-09 | 2000-01-26 | 中国科学院微生物研究所 | Process for producing long-chain alpha, omega-dicarboxylic acid by synchronous fermentation of microbe |
| CN1059903C (en) * | 1993-10-25 | 2000-12-27 | 朱守一 | Macrocyclic musk dilactone compound and its processing method |
| CN100360678C (en) * | 1999-09-30 | 2008-01-09 | 科金斯公司 | Improved fermentation process |
| CN101983242A (en) * | 2008-02-06 | 2011-03-02 | 拜康有限公司 | Fermentation media comprising urea-like nitrogen sources and its use for the production of secondary metabolits, enzymes and recombinant proteias |
| CN102115768B (en) * | 2009-12-30 | 2013-07-03 | 张艾琳 | Method for producing hexadecanedioic acid by synchronously fermenting n-hexadecane with microbe |
| WO2013131277A1 (en) * | 2012-03-09 | 2013-09-12 | Cathay R & D Center Co., Ltd | A new candida sake strain for producing long chain dicarboxylic acids |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698859B (en) * | 2009-09-11 | 2012-07-18 | 淄博广通化工有限责任公司 | Production method of n-hexadecyl dibasic acid |
-
1987
- 1987-08-12 CN CN 87105445 patent/CN1017350B/en not_active Expired
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059903C (en) * | 1993-10-25 | 2000-12-27 | 朱守一 | Macrocyclic musk dilactone compound and its processing method |
| CN1048754C (en) * | 1995-11-09 | 2000-01-26 | 中国科学院微生物研究所 | Process for producing long-chain alpha, omega-dicarboxylic acid by synchronous fermentation of microbe |
| CN100360678C (en) * | 1999-09-30 | 2008-01-09 | 科金斯公司 | Improved fermentation process |
| CN101983242A (en) * | 2008-02-06 | 2011-03-02 | 拜康有限公司 | Fermentation media comprising urea-like nitrogen sources and its use for the production of secondary metabolits, enzymes and recombinant proteias |
| CN101983242B (en) * | 2008-02-06 | 2015-12-16 | 拜康有限公司 | Fermention medium containing urea class nitrogenous source and the purposes for the production of recombinant protein thereof |
| CN102115768B (en) * | 2009-12-30 | 2013-07-03 | 张艾琳 | Method for producing hexadecanedioic acid by synchronously fermenting n-hexadecane with microbe |
| WO2013131277A1 (en) * | 2012-03-09 | 2013-09-12 | Cathay R & D Center Co., Ltd | A new candida sake strain for producing long chain dicarboxylic acids |
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| Publication number | Publication date |
|---|---|
| CN1017350B (en) | 1992-07-08 |
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