CN104152497A - Method for producing succinic acid by multistage continuous fermentation - Google Patents
Method for producing succinic acid by multistage continuous fermentation Download PDFInfo
- Publication number
- CN104152497A CN104152497A CN201410459744.XA CN201410459744A CN104152497A CN 104152497 A CN104152497 A CN 104152497A CN 201410459744 A CN201410459744 A CN 201410459744A CN 104152497 A CN104152497 A CN 104152497A
- Authority
- CN
- China
- Prior art keywords
- tank
- residual sugar
- sugar
- fermentor
- grades
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 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 43
- 230000004151 fermentation Effects 0.000 title claims abstract description 43
- 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
- 239000001963 growth medium Substances 0.000 claims abstract description 18
- 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
- 239000002609 medium Substances 0.000 claims description 23
- 235000015097 nutrients Nutrition 0.000 claims description 20
- 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
- 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
- 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
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 claims description 3
- 235000014380 magnesium carbonate Nutrition 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
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 239000013589 supplement Substances 0.000 abstract 4
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 description 10
- 239000007791 liquid phase Substances 0.000 description 7
- 230000004941 influx Effects 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 238000002360 preparation method Methods 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
- 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
- 150000001875 compounds Chemical class 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
Landscapes
- 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: firstly, preparing a seed culture medium, an initial fermentation culture medium, a carbon source feed tank culture solution and a culture medium feed tank culture solution; 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, has 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 Siping City platform compound, succinic acid is important intermediate product and 1, the precursor substance of the professional chemical preparationss 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 market total value 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 aspect the Application and preparation of biodegradable plastic.Succinic acid and BDO are carried out to 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 industrial application is produced succinic acid at present.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 petrochemical industry resource, ecocrisis is increasingly sharpened, and increasing investigator has both at home and abroad been placed on sight on Biological preparation succinic acid.Compared with traditional chemical method, biological process has obvious advantage.Biological process fermentation succinic acid has low, the good 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 anaerobions 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 of 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 solving: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, initial fermention medium, 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, initial fermention medium, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and serum bottle;
The 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;
The 4th step, multistage continuously fermenting, by after the fermentor tank series connection after sterilizing, in one grade fermemtation tank, access initial fermention medium, pass into gas, be 3-10% access fermented bacterium according to inoculum size volume ratio again, initial fermention medium flows into second order fermentation tank from one grade fermemtation tank, three grade fermemtation tank, flow to successively in last step fermentor tank, by regulating air flow, the initial sugar concentration of one grade fermemtation tank, the benefit sugar speed of fermentor tanks at different levels, fermented liquid flows out speed and fermented liquid flows into the sugared concentration in rate-controlling fermentor tanks at different levels, complete the fermentation of succinic acid by controlling cellular biomass and cell reflux rate in fermentor tanks 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, in the time that fermentor tank quantity is 3, in this three grade fermemtation tank tanks at different levels, sugared concentration is: one-level: 90%-80% residual sugar; Secondary: 60%-45% residual sugar; Three grades: 15%-0% residual sugar;
In the time that fermentor tank quantity is 4, in this level Four fermentor tank tanks 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;
In the time that fermentor tank quantity is 5, in this Pyatyi fermentor tank tanks 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;
In the time that 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;
In the time that 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, described gas is that volume ratio is the CO of 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 tanks at different levels is 10-40 ml/h, it is 40-160 ml/h that fermented liquid flows out speed, and it is 10-150 ml/h that fermented liquid flows into speed, 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 by adding one or more in sodium carbonate, salt of wormwood, magnesiumcarbonate, sodium hydroxide, potassium hydroxide or magnesium hydroxide to regulate.
beneficial effect
The present invention utilizes the method for the multistage production succinic acid that continuously ferments simple, easy to operate, and compared with existing fermentation technique, the output of succinic acid is high, is applicable to industrialization operation.
Brief description of the drawings
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, as three fermentor tanks of connecting are three grades and continuously ferment, 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 is produced a method for succinic acid, comprises the following steps:
The first step, joins seed culture medium, initial fermention medium, carbon source feed supplement tank nutrient solution and substratum feed supplement tank nutrient solution, fills a prescription as follows:
Seed culture medium (gL
-1): glucose 10 (divide and 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.
Initial fermention medium (gL
-1): glucose 40 (divide and 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 sterilizing 15 minutes at 121 DEG C of the serum bottle of getting five 1L fermentor tanks, carbon source feed supplement tank, substratum feed supplement tank, seed culture medium, initial fermention medium, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and 100ml, for subsequent use;
The 3rd step, gets the serum bottle after sterilizing, and the seed culture medium of access 20ml, passes into CO
2, then accessing Actinobacillus succinogenes NJ113,37 DEG C of rotating speeds of shaking table are under 180 rpm, to cultivate 10 hours, as fermented bacterium;
The 4th step by after the fermentor tank series connection after five sterilizings, accesses the initial fermention medium of 600ml in one grade fermemtation tank, passes into the CO that volume ratio is 1:1
2and H
2gas mixture, utilizing gas-holder control air flow is 0.02 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 20-30ml/h, it is 10-20 ml/h that glucose is mended sugared speed, it is 40-50 ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 50g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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, leavening temperature: 37 DEG C, control cell reflux rate 10 ml/h, after fermentation 120 h, get tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
embodiment 2
The first step to the three steps are with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is 200g/L sucrose solution.
The 4th step by after the fermentor tank series connection after five sterilizings, accesses the initial fermention medium of 600ml in one grade fermemtation tank, passes into the CO that volume ratio is 1:1
2and H
2gas mixture, utilizing gas-holder control air flow is 0.01L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 120-150 ml/h, it is 30 ml/h that sucrose is mended sugared speed, it is 150-160 ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 150g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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, cell reflux rate 50 ml/h, after fermentation 120 h, get tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
Embodiment 3
The first step to the three steps are with embodiment 2.
The 4th step, by after the fermentor tank series connection after five sterilizings, in one grade fermemtation tank, access the initial fermention medium of 600ml, utilize the pure N2 of gas-holder control, air flow is 0.04 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 90-110 ml/h, it is 20 ml/h that sucrose is mended sugared speed, it is 110-140ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 80g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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, cell reflux rate 40 ml/h, after fermentation 120 h, get tunning and carry out efficient liquid phase chromatographic analysis, and measure the amount of tunning.
embodiment 4
The first step to the three steps are with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is starch saccharificating liquid.
The 4th step by after the fermentor tank series connection after five sterilizings, accesses the initial fermention medium of 600ml in one grade fermemtation tank, passes into pure N
2, utilizing gas-holder control air flow is 0.01 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 50-70 ml/h, it is 30-40 ml/h that starch saccharificating liquid is mended sugared speed, it is 80-110 ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 50g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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 fermentation 120 h, measure the amount of tunning.
embodiment 5
The first step to the three steps are with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is molasses.
The 4th step by after the fermentor tank series connection after five sterilizings, accesses the initial fermention medium of 600ml in one grade fermemtation tank, passes into pure CO
2, utilizing gas-holder control air flow is 0.02 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 110-130 ml/h, it is 30-40 ml/h that molasses are mended sugared speed, it is 140-160 ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 50g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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, cell reflux rate 50 ml/h ferment after 120 h, measure the amount of tunning.
embodiment 6
The first step to the three steps are with embodiment 1, and wherein carbon source feed supplement tank nutrient solution is ligno-cellulose hydrolysate.
The 4th step by after the fermentor tank series connection after five sterilizings, accesses the initial fermention medium of 600ml in one grade fermemtation tank, passes into pure N
2, utilizing gas-holder control air flow is 0.04 L/(minL), be 10%(v/v according to inoculum size) access fermented bacterium, initial fermention medium flows into secondary from one grade fermemtation tank, three grades until Pyatyi, utilize carbon source feed supplement tank and substratum feed supplement tank controlled fermentation liquid rate of influx 120-130 ml/h, it is 30-40 ml/h that ligno-cellulose hydrolysate is mended sugared speed, it is 150-160ml/h that fermented liquid flows out speed, making initial sugar concentration in one grade fermemtation tank is 50g/L, in Continuous Fermentation Processes, in fermentor tanks at different levels, the initial sugared concentration of sugared relative concentration fermentation 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, cell reflux rate 50 ml/h, after fermentation 120 h, measure 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, the mode that adopts single-stage to continuously ferment, 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, the mode that adopts single-stage to continuously ferment, and 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.
Tunning of the present invention is carried out after liquid-phase chromatographic analysis, with the high-efficient liquid phase chromatogram contrast 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 is ferment the to obtain amount of product of different embodiments of the invention.
Ferment the to obtain amount of product of table 1 different embodiments of the invention
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, initial fermention medium, 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, initial fermention medium, carbon source feed supplement tank nutrient solution, substratum feed supplement tank nutrient solution and serum bottle;
The 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;
The 4th step, multistage continuously fermenting, by after the fermentor tank series connection after sterilizing, in one grade fermemtation tank, access initial fermention medium, pass into gas, be 3-10% access fermented bacterium according to inoculum size volume ratio again, initial fermention medium flows into second order fermentation tank from one grade fermemtation tank, three grade fermemtation tank, flow to successively in last step fermentor tank, by regulating air flow, the initial sugar concentration of one grade fermemtation tank, the benefit sugar speed of fermentor tanks at different levels, fermented liquid flows out speed and fermented liquid flows into the sugared concentration in rate-controlling fermentor tanks at different levels, complete the fermentation of succinic acid by controlling cellular biomass and cell reflux rate in fermentor tanks 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:
In the time that fermentor tank quantity is 3, in this three grade fermemtation tank tanks at different levels, sugared concentration is: one-level: 90%-80% residual sugar; Secondary: 60%-45% residual sugar; Three grades: 15%-0% residual sugar;
In the time that fermentor tank quantity is 4, in this level Four fermentor tank tanks 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;
In the time that fermentor tank quantity is 5, in this Pyatyi fermentor tank tanks 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;
In the time that 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;
In the time that 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, described gas is that volume ratio is the CO of 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 tanks at different levels is 10-40 ml/h, it is 40-160 ml/h that fermented liquid flows out speed, it is 10-150 ml/h that fermented liquid flows into speed, 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, pH is by adding one or more in sodium carbonate, salt of wormwood, magnesiumcarbonate, sodium hydroxide, potassium hydroxide or magnesium hydroxide to regulate.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410459744.XA CN104152497A (en) | 2014-09-11 | 2014-09-11 | Method for producing succinic acid by multistage continuous fermentation |
| CN201410609027.0A CN104372037A (en) | 2014-09-11 | 2014-11-04 | Method for producing succinic acid by multistage continuous fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410459744.XA CN104152497A (en) | 2014-09-11 | 2014-09-11 | Method for producing succinic acid by multistage continuous fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104152497A true CN104152497A (en) | 2014-11-19 |
Family
ID=51878123
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410459744.XA Withdrawn CN104152497A (en) | 2014-09-11 | 2014-09-11 | Method for producing succinic acid by multistage continuous fermentation |
| CN201410609027.0A Pending CN104372037A (en) | 2014-09-11 | 2014-11-04 | Method for producing succinic acid by multistage continuous fermentation |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410609027.0A Pending CN104372037A (en) | 2014-09-11 | 2014-11-04 | Method for producing succinic acid by multistage continuous fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN104152497A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106011216A (en) * | 2016-07-28 | 2016-10-12 | 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 |
| CN108130296A (en) * | 2018-01-17 | 2018-06-08 | 厦门昶科生物工程有限公司 | The method and the preparation method of clostridium butyricum probiotics that a kind of clostridium butyricum high density is continuously fermented |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282248B (en) * | 2016-08-08 | 2017-10-24 | 山东福洋生物科技有限公司 | A kind of production method of continuously fermenting 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302546B (en) * | 2008-06-06 | 2011-04-13 | 江南大学 | Method for producing amber acid by continuous fermentation or semi-continuous fermentation |
| CN101363033A (en) * | 2008-09-04 | 2009-02-11 | 江西省德兴市百勤异Vc钠有限公司 | 2-keto-D-gluconic acid continuous fermentation process |
| CN101712970B (en) * | 2009-12-29 | 2012-09-12 | 南京工业大学 | Method for preparing succinic acid by fermentation |
-
2014
- 2014-09-11 CN CN201410459744.XA patent/CN104152497A/en not_active Withdrawn
- 2014-11-04 CN CN201410609027.0A patent/CN104372037A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106011216A (en) * | 2016-07-28 | 2016-10-12 | 南京工业大学 | Method for producing 1, 5-pentanediamine by microorganism combined culture |
| 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 |
| CN108130296A (en) * | 2018-01-17 | 2018-06-08 | 厦门昶科生物工程有限公司 | The method and the preparation method of clostridium butyricum probiotics that a kind of clostridium butyricum high density is continuously fermented |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104372037A (en) | 2015-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104152497A (en) | Method for producing succinic acid by multistage continuous fermentation | |
| CN101302546B (en) | Method for producing amber acid by continuous fermentation or semi-continuous fermentation | |
| CN103602623A (en) | Bacterium strain with high yield of L-alanine and L-alanine biological fermentation production method | |
| CN101709263B (en) | Chemostat continuous culture device | |
| CN107746862A (en) | Utilize the method for clostridium tyrobutyricum fermented maize skin hydrolyzate production butyric acid | |
| CN106011216A (en) | Method for producing 1, 5-pentanediamine by microorganism combined culture | |
| CN101538592B (en) | Method for producing DHA by Crypthecodinium cohnii industrial fermentation | |
| CN104212851B (en) | method for producing L-phenylalanine by multistage continuous fermentation | |
| CN110029134B (en) | Process for producing and extracting glutamic acid | |
| CN103952447A (en) | Method for producing succinic acid by fermentation under anaerobic condition | |
| CN106399387A (en) | Nitrogen defect control method for production of ethanol through fermentation of synthesis gas with mixed bacteria | |
| CN103667373A (en) | Method for generating propionic acid by microbial fermentation and co-generating succinic acid and salt thereof | |
| CN112626134B (en) | Method for preparing malic acid by using citric acid fermentation tail gas | |
| CN105713932B (en) | A kind of method that microbial fermentation prepares 2,3- butanediol | |
| CN108456698A (en) | A kind of fermentation method for producing of 1,3- propylene glycol and lactic acid coproduction based on clostridium butyricum | |
| CN103966298A (en) | Recycling method of tetracycline fungi residue | |
| CN103320367A (en) | Screening and application of high-yield succinic acid escherichia coli by anaerobic utilization of synthetic culture medium | |
| CN104561139A (en) | Method for increasing final cell density of microorganisms and shortening culture time | |
| CN103382491B (en) | Preparation method of citric acid | |
| CN102321683B (en) | Process for preparing fumaric acid fermentation liquid by fermentation method and for separating and extracting pure fumaric acid from fumaric acid fermentation liquid | |
| CN106148426B (en) | It is a kind of containing molybdenum, zinc, nickel food gas anaerobe fermentation produce alcohol cultivating system and cultural method | |
| CN105274018A (en) | Thermo-sensitive corynebacterium glutamicum, cultivation medium, colloidal particles, preparation method and application | |
| CN111876446B (en) | Process for preparing L-malic acid by fermenting aspergillus oryzae | |
| CN101555504B (en) | Method for producing D-ribose by utilizing fermentation of bacillus pumilus transketolase variant | |
| CN116144516B (en) | Saccharomyces cerevisiae for producing succinic acid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C04 | Withdrawal of patent application after publication (patent law 2001) | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20141119 |