CN104372037A - Method for producing succinic acid by multistage continuous fermentation - Google Patents
Method for producing succinic acid by multistage continuous fermentation Download PDFInfo
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- CN104372037A CN104372037A CN201410609027.0A CN201410609027A CN104372037A CN 104372037 A CN104372037 A CN 104372037A CN 201410609027 A CN201410609027 A CN 201410609027A CN 104372037 A CN104372037 A CN 104372037A
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 238000000855 fermentation Methods 0.000 title claims abstract description 55
- 230000004151 fermentation Effects 0.000 title claims abstract description 55
- 239000001384 succinic acid Substances 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 22
- 235000015097 nutrients Nutrition 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 230000001954 sterilising effect Effects 0.000 claims abstract description 17
- 238000011218 seed culture Methods 0.000 claims abstract description 13
- 241000948980 Actinobacillus succinogenes Species 0.000 claims abstract description 8
- 210000002966 serum Anatomy 0.000 claims abstract description 8
- 239000006052 feed supplement Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 13
- 238000010992 reflux Methods 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 230000004941 influx Effects 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 8
- 230000008901 benefit Effects 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 4
- 239000000413 hydrolysate Substances 0.000 claims description 4
- 235000013379 molasses Nutrition 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 230000001276 controlling effect Effects 0.000 claims description 3
- 239000001095 magnesium carbonate Substances 0.000 claims description 3
- 235000014380 magnesium carbonate Nutrition 0.000 claims description 3
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 claims description 3
- 229960001708 magnesium carbonate Drugs 0.000 claims description 3
- 229910000021 magnesium carbonate Inorganic materials 0.000 claims description 3
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 claims description 3
- 239000000347 magnesium hydroxide Substances 0.000 claims description 3
- 229910001862 magnesium hydroxide Inorganic materials 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 3
- 235000015320 potassium carbonate Nutrition 0.000 claims description 3
- 239000002028 Biomass Substances 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims description 2
- 239000013589 supplement Substances 0.000 abstract 4
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 abstract 1
- 230000001502 supplementing effect Effects 0.000 abstract 1
- 239000000126 substance Substances 0.000 description 11
- 239000007791 liquid phase Substances 0.000 description 7
- 239000002253 acid Substances 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000004631 polybutylene succinate Substances 0.000 description 4
- 229920002961 polybutylene succinate Polymers 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 229920000704 biodegradable plastic Polymers 0.000 description 3
- 230000031018 biological processes and functions Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- 241000417230 Actinobacillus succinogenes 130Z Species 0.000 description 1
- 241000722955 Anaerobiospirillum Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 239000001361 adipic acid Substances 0.000 description 1
- 235000011037 adipic acid Nutrition 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- -1 poly butylene succinate Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A method for producing succinic acid by multistage continuous fermentation comprises the following steps: in the first step, a seed culture medium, an initial fermentation culture medium, a carbon source feed tank culture solution and a culture mediumCulture solution in a nutrient medium supplementing tank; secondly, sterilizing the series fermentation tank group, the carbon source supplement tank, the culture medium supplement tank, the seed culture medium, the initial fermentation culture medium, the culture solution of the carbon source supplement tank, the culture solution of the culture medium supplement tank and the serum bottle; thirdly, taking the sterilized serum bottle, inoculating the sterilized seed culture medium, and introducing CO2Then inoculating Actinobacillus succinogenes NJ113 to shake culture to be used as a fermentation strain; fourthly, multi-stage continuous fermentation. The production method is simple, and the succinic acid has high yield and purity, so the method is suitable for industrial production.
Description
Technical field
The invention belongs to succinic acid preparation field, be specifically related to a kind of multistage continuously fermenting and produce the method for succinic acid.
Background technology
Succinic acid, having another name called succsinic acid (succinate acid), is a kind of common natural organic acids, is one of mesostate of tricarboxylic acid cycle, is extensively present in animal and plant and microorganism.As a kind of important carbon four platform chemicals, succinic acid is important intermediate product and 1, the precursor substance of the speciality chemical goods such as 4-butyleneglycol (BDO), gamma-butyrolactone, tetrahydrofuran (THF), N-Methyl pyrrolidone (NMP), adipic acid, oxysuccinic acid and novel biodegradable plastic product poly butylene succinate (PBS), the total market of these chemical reaches 15,000,000,000 U.S. dollars.Up-to-date result of study also shows that succinic acid has boundless prospect in the Application and preparation of biodegradable plastic.Succinic acid and BDO are carried out polyreaction and can generate the novel biodegradable plastic product of a class---poly butylene succinate (PBS).
The synthetic method of succinic acid has chemical method and biological process, and the chemical method that is mainly of current industrial application produces succinic acid.Chemical method mainly contains shortening method, paraffin oxidation style, electrochemical synthesis.But chemical method mainly utilizes non-renewable petroleum resources as raw material, and cost is high, seriously polluted and cannot Sustainable development, seriously limit the development of succinic acid.In recent years, along with the exhaustion day by day of fossil resources, ecocrisis is increasingly sharpened, and sight has been placed on Biological preparation succinic acid by increasing investigator both at home and abroad.Compared with conventional chemical methods, biological process has obvious advantage.Biological process fermentation succinic acid has low, the excellent environmental benefit of cost and has fixing greenhouse gases CO
2etc. advantage.Succinic acid is the mesostate of tricarboxylic acid cycle, also be the reductibility terminal meta-bolites of numerous anaerobion and amphimicrobe, nearly all animals and plants are energy synthesizing succinic acid all, conventional microorganism has Anaerobiospirillum succinoproducens, Actinobacillus succinogenes, intestinal bacteria, Corynebacterium glutamicum, succsinic acid bacterium etc. is produced in mannheim.
Chinese patent CN101857888 A discloses a kind of method utilizing whey fermentation to produce succinic acid, adopts Actinobacillus succinogenes
actinobacillus succinogenessuccsinic acid is produced in NJ113 fermentation in the substratum of the whey containing 30 ~ 100g/L lactose, and production concentration is up to 47.51g/L, and yield is up to 67.9%.
Summary of the invention
the technical problem solved:for the deficiencies in the prior art, the object of the present invention is to provide a kind of multistage continuously fermenting to produce the method for succinic acid, preparation process is simple, and succinic acid output is high.
technical scheme:for solving prior art problem, the technical scheme that the present invention takes:
Multistage continuously fermenting produces a method for succinic acid, comprises the following steps:
The first step, joins seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution and substratum feed supplement tank nutrient solution;
Second step, carries out sterilising treatment to fermentor tank, carbon source feed supplement tank, substratum feed supplement tank, seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and serum bottle;
3rd step, gets the serum bottle after sterilizing, and the seed culture medium after access sterilizing, passes into CO
2after access again Actinobacillus succinogenes (
actinobacillus succinogenes) NJ113 shaking table cultivate after as fermented bacterium;
4th step, multistagely to continuously ferment, after the fermentor tank series connection after sterilizing, Preliminary fermentation substratum is accessed in one grade fermemtation tank, pass into gas, again according to inoculum size volume ratio be 3-10% access fermented bacterium, Preliminary fermentation substratum flows into second order fermentation tank from one grade fermemtation tank, three grade fermemtation tank, flow in last step fermentor tank successively, by regulating air flow, the initial sugar concentration of one grade fermemtation tank, the benefit sugar speed of fermentor tank at different levels, fermented liquid discharge rate and fermented liquid flow into the sugared concentration in rate-controlling fermentor tank at different levels, the fermentation of succinic acid is completed by controlling cellular biomass and cell reflux rate in fermentor tank at different levels, the omnidistance pH that ferments is 6.5-7.5, temperature is 35-40 DEG C and carries out 50-750h.
As preferably, when fermentor tank quantity is 3, in this three grade fermemtation tank tank at different levels, sugared concentration is: one-level: 90%-80% residual sugar; Secondary: 60%-45% residual sugar; Three grades: 15%-0% residual sugar;
When fermentor tank quantity is 4, in this level Four fermentor tank tank at different levels, sugared concentration is: one-level: 95%-85% residual sugar; Secondary: 70%-55% residual sugar; Three grades: 40%-25% residual sugar; Level Four: 15%-0% residual sugar;
When fermentor tank quantity is 5, in this Pyatyi fermentor tank tank at different levels, sugared concentration is: one-level: 95%-85% residual sugar; Secondary: 75%-60% residual sugar; Three grades: 55%-40% residual sugar; Level Four: 35%-20% residual sugar; Pyatyi: 15%-0% residual sugar;
When fermentor tank quantity is 6, in these six grades of fermentor tank tanks at different levels, sugared concentration is: one-level: 95%-90% residual sugar; Secondary: 80%-70% residual sugar; Three grades: 70%-60% residual sugar; Level Four: 50%-40% residual sugar; Pyatyi: 30%-20% residual sugar; Six grades: 15%-0% residual sugar;
When fermentor tank quantity is 7, in these seven grades of fermentor tank tanks at different levels, sugared concentration is: one-level: 95%-90% residual sugar; Secondary: 80%-70% residual sugar; Three grades: 70%-60% residual sugar; Level Four: 60%-45% residual sugar; Pyatyi: 45%-35% residual sugar; Six grades: 35%-15% residual sugar; Seven grades: 15%-0% residual sugar.
As preferably, the 4th step, the CO of described gas to be volume ratio be 1:1
2and H
2gas mixture, pure CO
2or pure N
2in one, air flow is 0.01-0.04 L/(minL).
As preferably, the 4th step, the initial sugar concentration of one grade fermemtation tank is 50-150 g/L, the benefit sugar speed of fermentor tank at different levels is 10-40 ml/h, fermented liquid discharge rate is 40-160 ml/h, and fermented liquid rate of influx is 10-150 ml/h, cell reflux rate 10-50 ml/h.
As preferably, carbon source feed supplement tank nutrient solution is a kind of in glucose solution, sucrose solution, starch saccharificating liquid, molasses or ligno-cellulose hydrolysate.
As preferably, the 4th step, pH is regulated by one or more adding in sodium carbonate, salt of wormwood, magnesiumcarbonate, sodium hydroxide, potassium hydroxide or magnesium hydroxide.
beneficial effect
The present invention utilizes the method for the multistage production succinic acid that continuously ferments simple, and easy to operate, compared with existing fermentation technique, the output of succinic acid is high, is applicable to industrial operation.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of succinic acid standard substance;
Fig. 2 is the high-efficient liquid phase chromatogram of the tunning of embodiment 1.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
Said multistage the continuously fermenting of the present invention is that number according to cascade fermentation tank is determined, continuously ferment as three fermentor tanks of connecting are three grades, wherein first fermentor tank is one grade fermemtation tank, and second fermentor tank is second order fermentation tank, and the 3rd fermentor tank is three grade fermemtation tank.
Embodiment 1
Multistage continuous fermentation apparatus produces a method for succinic acid, comprises the following steps:
The first step, join seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution and substratum feed supplement tank nutrient solution, fill a prescription as follows:
Seed culture medium (gL
-1): glucose 10 (point disappear), yeast extract paste 5, corn steep liquor 5, NaHCO
310, NaH
2pO
42H
2o 9.6, K
2hPO
43H
2o 15.5, pH 7.0.
Preliminary fermentation substratum (gL
-1): glucose 40 (point disappear), yeast extract paste 10, corn steep liquor 10, sodium acetate 1.36, KH
2pO
43, MgCl
26H
2o 0.2, CaCl
20.2, NaCl 1, Na
2hPO
412H
2o 0.31, NaH
2pO
42H
2o 1.6, pH 7.0.
Carbon source feed supplement tank nutrient solution (gL
-1): glucose 750.
Substratum feed supplement tank nutrient solution (gL
-1): yeast extract paste 30, corn steep liquor 30, sodium acetate 4.08, KH
2pO
49, MgCl
26H
2o 0.6, CaCl
20.6, NaCl 3, Na
2hPO
412H
2o 0.93, NaH
2pO
42H
2o 4.8, pH 7.0.
Second step, the serum bottle getting five 1L fermentor tanks, carbon source feed supplement tank, substratum feed supplement tank, seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and 100ml sterilizing 15 minutes at 121 DEG C, for subsequent use;
3rd step, gets the serum bottle after sterilizing, and the seed culture medium of access 20ml, passes into CO
2, then accessing Actinobacillus succinogenes NJ113, shaking table 37 DEG C of rotating speeds are cultivate 10 hours under 180 rpm, as fermented bacterium;
4th step, after the fermentor tank series connection after five sterilizings, accesses the Preliminary fermentation substratum of 600ml, passes into the CO that volume ratio is 1:1 in one grade fermemtation tank
2and H
2gas mixture, utilizing gas-holder to control air flow is 0.02 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 20-30ml/h, sugared speed mended by glucose is 10-20 ml/h, fermented liquid discharge rate is 40-50 ml/h, initial sugar concentration in one grade fermemtation tank is made to be 50g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use sodium hydroxide control pH is 6.5, and leavening temperature: 37 DEG C, controls cell reflux rate 10 ml/h, after 120 h that ferment, get tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
embodiment 2
The first step is to the 3rd step with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is 200g/L sucrose solution.
4th step, after the fermentor tank series connection after five sterilizings, accesses the Preliminary fermentation substratum of 600ml, passes into the CO that volume ratio is 1:1 in one grade fermemtation tank
2and H
2gas mixture, utilizing gas-holder to control air flow is 0.01L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 120-150 ml/h, sugared speed mended by sucrose is 30 ml/h, fermented liquid discharge rate is 150-160 ml/h, initial sugar concentration in one grade fermemtation tank is made to be 150g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use magnesium hydroxide control pH is 6.5, and cell reflux rate 50 ml/h, after 120 h that ferment, gets tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
Embodiment 3
The first step to the 3rd step with embodiment 2.
4th step, after the fermentor tank series connection after five sterilizings, the Preliminary fermentation substratum of 600ml is accessed in one grade fermemtation tank, gas-holder is utilized to control pure N2, air flow is 0.04 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 90-110 ml/h, sugared speed mended by sucrose is 20 ml/h, fermented liquid discharge rate is 110-140ml/h, initial sugar concentration in one grade fermemtation tank is made to be 80g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use sodium carbonate control pH is 6.5, and cell reflux rate 40 ml/h, after 120 h that ferment, gets tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
embodiment 4
The first step is to the 3rd step with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is starch saccharificating liquid.
4th step, after the fermentor tank series connection after five sterilizings, accesses the Preliminary fermentation substratum of 600ml, passes into pure N in one grade fermemtation tank
2, utilizing gas-holder to control air flow is 0.01 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 50-70 ml/h, it is 30-40 ml/h that starch saccharificating liquid mends sugared speed, fermented liquid discharge rate is 80-110 ml/h, initial sugar concentration in one grade fermemtation tank is made to be 50g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use salt of wormwood and magnesiumcarbonate mixture control pH are 6.5, cell reflux rate 30 ml/h, after 120 h that ferment, measure the amount of tunning.
embodiment 5
The first step is to the 3rd step with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is molasses.
4th step, after the fermentor tank series connection after five sterilizings, accesses the Preliminary fermentation substratum of 600ml, passes into pure CO in one grade fermemtation tank
2, utilizing gas-holder to control air flow is 0.02 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 110-130 ml/h, it is 30-40 ml/h that molasses mend sugared speed, fermented liquid discharge rate is 140-160 ml/h, initial sugar concentration in one grade fermemtation tank is made to be 50g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use potassium hydroxide control pH is 6.5, and cell reflux rate 50 ml/h ferments after 120 h, measures the amount of tunning.
embodiment 6
The first step is to the 3rd step with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is ligno-cellulose hydrolysate.
4th step, after the fermentor tank series connection after five sterilizings, accesses the Preliminary fermentation substratum of 600ml, passes into pure N in one grade fermemtation tank
2, utilizing gas-holder to control air flow is 0.04 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, Preliminary fermentation substratum flows into secondary from one grade fermemtation tank, three grades until Pyatyi, carbon source feed supplement tank and substratum feed supplement tank is utilized to control fermented liquid rate of influx 120-130 ml/h, it is 30-40 ml/h that ligno-cellulose hydrolysate mends sugared speed, fermented liquid discharge rate is 150-160ml/h, initial sugar concentration in one grade fermemtation tank is made to be 50g/L, in Continuous Fermentation Processes, in fermentor tank at different levels, sugared relative concentration fermentation Initial sugar concentration is followed successively by one-level: 95%-85% residual sugar, secondary: 75%-60% residual sugar, three grades: 55%-40% residual sugar, level Four: 35%-20% residual sugar, Pyatyi: 15%-0% residual sugar, omnidistance use sodium carbonate control pH is 6.5, and cell reflux rate 50 ml/h, after 120 h that ferment, measures the amount of tunning.
comparative example 1
With the succinic acid-producing that continuously ferments of current bibliographical information, with
a. succinogenes130Z is starting strain, adopts the mode that single-stage is continuously fermented, and controlling glucose sugar concentration is 20g/L, and thinning ratio is 0.2-1.2, and the maximum concentration of succinic acid only has 10.4g/L.
Comparative example 2
With
a. succiniciproducensaTCC No. 29305 is starting strain, and adopt the mode that single-stage is continuously fermented, control lactose concn is 20g/L, and thinning ratio is 0.03-0.14, and the maximum concentration of succinic acid only has 14.0g/L.
After tunning of the present invention is carried out liquid-phase chromatographic analysis, contrast with the high-efficient liquid phase chromatogram of succinic acid standard substance, determine that tunning of the present invention is succinic acid.Compared with prior art, the output of fermentation production of succinic acid of the present invention is high, and technique is simple, is applicable to large-scale production fermentation, and wherein table 1 to ferment to obtain the amount of product for different embodiments of the invention.
Table 1 different embodiments of the invention are fermented to obtain the amount of product
Claims (6)
1. multistage continuously fermenting produces a method for succinic acid, it is characterized in that, comprises the following steps:
The first step, joins seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution and substratum feed supplement tank nutrient solution;
Second step, carries out sterilising treatment to fermentor tank, carbon source feed supplement tank, substratum feed supplement tank, seed culture medium, Preliminary fermentation substratum, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and serum bottle;
3rd step, gets the serum bottle after sterilizing, and the seed culture medium after access sterilizing, passes into CO
2after access again Actinobacillus succinogenes (
actinobacillus succinogenes) NJ113 shaking table cultivate after as fermented bacterium;
4th step, multistagely to continuously ferment, after the fermentor tank series connection after sterilizing, Preliminary fermentation substratum is accessed in one grade fermemtation tank, pass into gas, again according to inoculum size volume ratio be 3-10% access fermented bacterium, Preliminary fermentation substratum flows into second order fermentation tank from one grade fermemtation tank, three grade fermemtation tank, flow in last step fermentor tank successively, by regulating air flow, the initial sugar concentration of one grade fermemtation tank, the benefit sugar speed of fermentor tank at different levels, fermented liquid discharge rate and fermented liquid flow into the sugared concentration in rate-controlling fermentor tank at different levels, the fermentation of succinic acid is completed by controlling cellular biomass and cell reflux rate in fermentor tank at different levels, the omnidistance pH that ferments is 6.5-7.5, temperature is 35-40 DEG C and carries out 50-750h.
2. multistage continuously fermenting produces the method for succinic acid according to claim 1, it is characterized in that:
When fermentor tank quantity is 3, in this three grade fermemtation tank tank at different levels, sugared concentration is: one-level: 90%-80% residual sugar; Secondary: 60%-45% residual sugar; Three grades: 15%-0% residual sugar;
When fermentor tank quantity is 4, in this level Four fermentor tank tank at different levels, sugared concentration is: one-level: 95%-85% residual sugar; Secondary: 70%-55% residual sugar; Three grades: 40%-25% residual sugar; Level Four: 15%-0% residual sugar;
When fermentor tank quantity is 5, in this Pyatyi fermentor tank tank at different levels, sugared concentration is: one-level: 95%-85% residual sugar; Secondary: 75%-60% residual sugar; Three grades: 55%-40% residual sugar; Level Four: 35%-20% residual sugar; Pyatyi: 15%-0% residual sugar;
When fermentor tank quantity is 6, in these six grades of fermentor tank tanks at different levels, sugared concentration is: one-level: 95%-90% residual sugar; Secondary: 80%-70% residual sugar; Three grades: 70%-60% residual sugar; Level Four: 50%-40% residual sugar; Pyatyi: 30%-20% residual sugar; Six grades: 15%-0% residual sugar;
When fermentor tank quantity is 7, in these seven grades of fermentor tank tanks at different levels, sugared concentration is: one-level: 95%-90% residual sugar; Secondary: 80%-70% residual sugar; Three grades: 70%-60% residual sugar; Level Four: 60%-45% residual sugar; Pyatyi: 45%-35% residual sugar; Six grades: 35%-15% residual sugar; Seven grades: 15%-0% residual sugar.
3. multistage continuously fermenting produces the method for succinic acid according to claim 1, it is characterized in that: the 4th step, the CO of described gas to be volume ratio be 1:1
2and H
2gas mixture, pure CO
2or pure N
2in one, air flow is 0.01-0.04L/(minL).
4. multistage continuously fermenting produces the method for succinic acid according to claim 1, it is characterized in that: the 4th step, the initial sugar concentration of one grade fermemtation tank is 50-150 g/L, the benefit sugar speed of fermentor tank at different levels is 10-40 ml/h, fermented liquid discharge rate is 40-160 ml/h, fermented liquid rate of influx is 10-150 ml/h, cell reflux rate 10-50 ml/h.
5. multistage continuously fermenting produces the method for succinic acid according to claim 1, it is characterized in that: carbon source feed supplement tank nutrient solution is a kind of in glucose solution, sucrose solution, starch saccharificating liquid, molasses or ligno-cellulose hydrolysate.
6. multistage continuously fermenting produces the method for succinic acid according to claim 1, it is characterized in that: the 4th step, and pH is regulated by one or more adding in sodium carbonate, salt of wormwood, magnesiumcarbonate, sodium hydroxide, potassium hydroxide or magnesium hydroxide.
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| CN106282248A (en) * | 2016-08-08 | 2017-01-04 | 山东福洋生物科技有限公司 | A kind of continuous fermentation production method of sodium gluconate |
| CN107858383A (en) * | 2017-12-21 | 2018-03-30 | 北京首钢朗泽新能源科技有限公司 | A kind of continous way fermentation method prepares the technique and device of alcohol |
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| CN104531782A (en) * | 2014-12-31 | 2015-04-22 | 南京工业大学 | Method for producing pyruvic acid by micro-aerobic fermentation of actinobacillus |
| CN106011216B (en) * | 2016-07-28 | 2019-12-27 | 南京工业大学 | Method for producing 1,5-pentanediamine by microorganism combined culture |
| CN106148444A (en) * | 2016-08-31 | 2016-11-23 | 南京工业大学 | Method for producing L-lysine by multistage continuous fermentation |
| CN108130296B (en) * | 2018-01-17 | 2020-09-01 | 厦门昶科生物工程有限公司 | High-density continuous fermentation method of clostridium butyricum and preparation method of clostridium butyricum microecological preparation |
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| CN101302546A (en) * | 2008-06-06 | 2008-11-12 | 江南大学 | Method for producing amber acid by continuous fermentation or semi-continuous fermentation |
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| CN107858383A (en) * | 2017-12-21 | 2018-03-30 | 北京首钢朗泽新能源科技有限公司 | A kind of continous way fermentation method prepares the technique and device of alcohol |
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