CN103952447A - Method for producing succinic acid by virtue of fermentation under anaerobic conditions - Google Patents
Method for producing succinic acid by virtue of fermentation under anaerobic conditions Download PDFInfo
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- CN103952447A CN103952447A CN201410212929.0A CN201410212929A CN103952447A CN 103952447 A CN103952447 A CN 103952447A CN 201410212929 A CN201410212929 A CN 201410212929A CN 103952447 A CN103952447 A CN 103952447A
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- succinic acid
- semen maydis
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- hydrolyzed solution
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Abstract
The invention relates to a method for producing succinic acid through anaerobic fermentation by using a corn flour full hydrolysate as a fermentation culture medium. The method comprises the following steps: by using a glucose hydrolysate produced by using a corn flour double-enzymolysis method as an only carbon source, and using a corn sugar residue hydrolysate obtained by performing enzymolysis on filter residue-corn residue generated in the process that glucose is produced from corn flour by using neutral protease as an only nitrogen source, combining the two hydrolysates to form the corn flour full hydrolysate as the fermentation culture medium, and producing succinic acid by anaerobic fermentation of the fermentation culture medium without adding other nutrients. The method for producing succinic acid through fermentation, disclosed by the invention, can be used for replacing expensive yeast powder and peptone; under the same conditions, compared with a method for producing succinic acid by fermenting an LB culture medium, the yield of succinic acid disclosed by the invention is increased by 1.37%, and the conversion rate of succinic acid is increased by 21.65%. The method disclosed by the invention is more economic and cleaner, and has important significances and economic values on industrialization of succinic acid.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of utilize Semen Maydis powder all-hydrolytic liquid fermentation production of succinic acid under anaerobic condition method.
Background technology
Succinic acid claims again succsinic acid, is the intermediate product of tricarboxylic acid cycle, is extensively present in human body, animal, plant and microorganism.It is as outstanding C
4hardware and software platform compound, can be used for organic chemicals and poly butylene succinate (PBS) the class Biodegradable materials such as synthetic BDO, tetrahydrofuran (THF), is thought one of biorefinery product of following 12 kinds of most worthies by USDOE.
Biosynthesizing succinic acid, is to utilize bacterium, the various microorganisms such as fungi, taking glucose or other various hydrolyzed solutions as carbon source, through producing succinic acid by microbial fermentation.Utilize microbe fermentation method to transform renewable resources (glucose, wood sugar etc.), because raw material sources are extensive and cheap, pollute little, environmental friendliness, and can absorb during the fermentation fixation of C O
2, can effectively alleviate Greenhouse effect, therefore become the focus of Recent study.
Corn is one of most important farm crop in the world, and China is one of big country producing corn, has abundant corn resources, reports, the corn annual production of China exceedes 100,000,000 tons.In addition, it is reported, in corn, containing thalli growth and producing acid has the factor of promoter action, as vitamin H, V
b1deng.Semen Maydis powder is the important source material of making glucose, but after filtration process, has also produced a large amount of by products, Semen Maydis grit.Therefore, Semen Maydis powder cheap and that be easy to get and by product Semen Maydis grit are made full use of in the middle of fermentation succinic acid-producing, this will have great significance to fermentation production of succinic acid industrialization.
Intestinal bacteria are in the situation that oxygen lacks, and the main the way of production of succinic acid is glucose through Embden-Meyerhof-Parnaspathway through generating phosphoenolpyruvic acid, and and then the synthetic oxaloacetic acid of metabolism, oxysuccinic acid, fumaric acid, finally with the form accumulation of succinic acid.G. the people such as N. Vemuri report utilizes the method for intestinal bacteria two stage fermentation succinic acid-producings, and principle is to be that thalline obtains Rapid Accumulation in the aerobic stage, then transfers anaerobism to and makes thalline produce a large amount of succinic acid.But in two stage fermentation processes, need to consume the growth of a large amount of nutritive ingredients for thalline, cause succinic acid total yield to decline, simultaneously because thalline grow aerobically is had relatively high expectations to dissolved oxygen, stir and the required higher energy consumption such as cooling and cost drop into, therefore, a step anaerobically fermenting succinic acid-producing receives increasing concern.But under specificity anaerobic condition, recombination bacillus coli is produced succinic acid needs the compound nitrogen source such as yeast powder, peptone as fermentation culture based component, but price is too expensive, in addition, also have the problems such as strain growth is slow, and succinic acid final concentration and yield are lower unresolved.For this reason, find and a kind ofly under specificity anaerobic condition, utilize the method that fermention medium bacterial strain Fast Growth improves succinic acid final concentration and yield industrialization has breakthrough meaning for fermentation production of succinic acid.
Summary of the invention
The object of the present invention is to provide a kind of method that can make bacterial strain under anaerobic still can fermentative production obtain high density and high yield succinic acid as fermentation culture based component without compound nitrogen sources such as expensive yeast powder, peptones.
For achieving the above object, the present invention adopts technical scheme as follows:
A kind of anaerobically fermenting provided by the invention is produced the method for succinic acid, and the fermention medium that it is characterized in that described method is Semen Maydis powder all-hydrolytic liquid.
Preferably, described Semen Maydis powder all-hydrolytic liquid comprises glucose sugar hydrolyzed solution and the Semen Maydis grit hydrolyzed solution of Semen Maydis powder hydrolysis gained.
Preferably, described glucose hydrolysis liquid is that Semen Maydis powder is through double enzymolysis method hydrolysis gained; Semen Maydis grit after described Semen Maydis grit hydrolyzed solution is overanxious by the hydrolysis of above-mentioned Semen Maydis powder double-enzyme method through neutral protein hydrolysis.
Preferably, the concrete steps of described Semen Maydis powder double enzymolysis method are: Semen Maydis powder is mixed with the ratio of mass fraction of solids 38 % with pure water, and add calcium ion, its addition adds 0.000175g calcium ion according to every gram of raw material, at pH=6.5, under 95 DEG C of conditions, add the α-amylase of 7 U/ gram raw material, and insulation to liquefaction finishes; By liquefier gone out enzyme live after at pH=4.5, under 60 DEG C of conditions, add the saccharifying enzyme of 200 U/ gram raw material, and be incubated 48 h, the filtrate that gained saccharified liquid obtains is after filtration glucose hydrolysis liquid;
Preferably, the concrete steps of described Semen Maydis grit hydrolysis are: filter residue and pure water after saccharified liquid is filtered mix in the ratio of solid masses percentage ratio 10%, at pH=7.0, under 55 DEG C of conditions, add neutral protease, its addition is 0.3% of filter residue quality, after 24 h, filtering gained filtrate is primverose slag hydrolyzed solution, and with triumphant formula nitriding, hydrolyzed solution is carried out the mensuration of nitrogen content.
Preferably, described recombination bacillus coli is recombination bacillus coli AFP111(
escherichia coliaFP111).
Preferably, the method concrete steps of described recombination bacillus coli AFP111 succinic acid-producing are as follows:
(1) test tube is cultivated: by microbiotic and recombination bacillus coli
escherichia coliaFP111 is seeded in anaerobism in 5 ml liquid LB substratum and cultivates, and culture temperature is 37 DEG C, 200r/min, and incubation time is 12 h;
(2) seed culture: microbiotic and test tube are cultivated
escherichia coliaFP111 is inoculated in seed culture medium, 500ml triangular flask liquid amount 50 ml, and aerobic is cultivated, and culture temperature is 37 DEG C, 200r/min, incubation time is 6 h;
(3) anaerobism shake flask fermentation is produced succinic acid: microbiotic and seed culture fluid are inoculated in fermention medium, and 100ml anaerobism shaking flask liquid amount is 30ml, and inoculum size is 10 %, and magnesium basic carbonate is as pH adjusting agent, carbonating; Culture temperature is 37 DEG C, 200r/min, and incubation time is 48 h.
Preferably, in described step 1), the formula of LB substratum is peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L;
Described step 2) in seed culture based formulas be peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L;
Described step 3) hydrolyzed solution culture medium prescription is: containing the primverose slag hydrolyzed solution of 1.8 g/L nitrogen elements,
Glucose hydrolysis liquid 10 g/L～80 g/L, magnesium basic carbonate 16 g/L.
Preferably, in described step 3), glucose hydrolysis liquid is 20 g/L.
Preferably, in described step 3), the aerated time is 2 minutes; Described microbiotic is kantlex and paraxin, and the final concentration adding is all 100 μ g/mL.
Beneficial effect of the present invention is:
The present invention only utilizes Semen Maydis powder to replace expensive LB substratum (containing import peptone and yeast powder) as raw material production carbon source (glucose) and nitrogenous source as fermention medium, do not adding under other nutritive substances and specificity anaerobic condition, the output that makes bacterial strain anaerobically fermenting produce succinic acid is compared LB substratum succinic acid output and yield with yield are slightly high.And Semen Maydis grit is fully utilized as industrial by product, this method is not only economical, saves, and also energy-saving and emission-reduction, reduce environmental pollution.As taking recombination bacillus coli as fermentation strain, in 5 L anaerobic fermentation tanks, two kinds of hydrolyzed solutions that utilize Semen Maydis powder to obtain through enzymolysis, 120 h that ferment consume the glucose of 50 g/L, the output of producing succinic acid is 42.29 g/L, and succinic acid transformation efficiency is 84.58%, compared with LB substratum fermentation production of succinic acid, the output of succinic acid has improved 1.37%, and transformation efficiency has improved 21.65%.
Brief description of the drawings
Fig. 1 is the contrast of recombination bacillus coli different initial sugar concentration fermentation succinic acid under pure anaerobic condition.
Fig. 2 is that recombination bacillus coli is at pure anaerobic condition bottom fermentation 120 h experimental result graphic representations.
Embodiment
According to following examples, can better understand the present invention.Concrete material proportion described in case study on implementation, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment of the present invention bacterial strain used is recombination bacillus coli AFP111(
escherichia coliaFP111 [F+
λ-rpoS396 (Am) rph-1△ (
pflAB:: Cam)
ldhA:: Kan 95
ptsG]) derive from open source literature (Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by
escherichia coli, Appl Environ Microbiol. Jan 2001; 67 (1): 148 – 154).Be so kind as to give by David P. Clark professor (Southern Illinois University), the inventor ensured in 20 years, to provide this biomaterial to the public from the application's day.But Semen Maydis powder all-hydrolytic liquid of the present invention is not only for intestinal bacteria Escherichia coliAFP111 as fermention medium, be also suitable for for other bacterial strain.
embodiment 1 the present embodiment explanation recombination bacillus coli Escherichia c
olithe technique of AFP111 fermentation production of succinic acid in anaerobism shaking flask
Culture medium prescription described in the present embodiment:
The formula of test tube, seed culture medium comprises: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermentative medium formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 20 g/L, magnesium basic carbonate 16 g/L.
Concrete steps are as follows:
(1) test tube is cultivated: by kantlex and paraxin together with colon bacillus
escherichia coliaFP111 is inoculated in test-tube culture medium, under anaerobic, and 37 DEG C of culture temperature, 200 r/min, incubation time 12 h.；
(2) seed culture: the AFP111 of kantlex and paraxin and test tube cultivation is inoculated in seed culture medium to 500 ml triangular flask liquid amount 50 ml, 37 DEG C of culture temperature, 200 r/min, incubation time 6 h;
(3) anaerobism shake flask fermentation is produced succinic acid: kantlex and paraxin and seed liquor are inoculated in fermention medium, inoculum size 10%(v/v), 100 ml serum bottle liquid amount 30 ml, carbonating 2 min, cultivating the output that detects succinic acid after 48 h is 16.39 g/L, succinic acid transformation efficiency is 81.95 %, refers to following table.
| ? | OD 600 | Consumption sugar (g/L) | Succinic acid (g/L) | Yield (%) |
| Hydrolyzed solution substratum | 4.31 | 20.00 | 16.39 | 81.95 |
| LB substratum | 6.00 | 20.00 | 14.32 | 71.6 |
embodiment 2 the present embodiment explanation colon bacillus
escherichia coliaFP111 is the contrast of 10 g/L, 20 g/L, 40 g/L, 60 g/L, 80 g/L condition bottom fermentations production succinic acid at glucose hydrolysis liquid addition
Culture medium prescription described in the present embodiment:
The formula of test tube, seed culture medium comprises: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermention medium (1) formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 10 g/L, magnesium basic carbonate 16 g/L.
Fermention medium (2) formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 20 g/L, magnesium basic carbonate 16 g/L.
Fermention medium (3) formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 40 g/L, magnesium basic carbonate 16 g/L.
Fermention medium (4) formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 60 g/L, magnesium basic carbonate 16 g/L.
Fermention medium (5) formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 80 g/L, magnesium basic carbonate 16 g/L.
Concrete steps are as follows:
(1) test tube is cultivated: by kantlex and paraxin together with colon bacillus
escherichia coliaFP111 is inoculated in test-tube culture medium, under anaerobic, and 37 DEG C of culture temperature, 200 r/min, incubation time 12 h.；
(2) seed culture: the AFP111 of kantlex and paraxin and test tube cultivation is inoculated in seed culture medium to 500 ml triangular flask liquid amount 50 ml, 37 DEG C of culture temperature, 200 r/min, incubation time 6 h;
(3) anaerobism shake flask fermentation is produced succinic acid: kantlex and paraxin and seed liquor are inoculated into respectively to fermention medium (1) in (5), inoculum size 10%(v/v), 100 ml serum bottle liquid amount 30 ml, carbonating 2 min, fermentation culture 48 h, fermentation results is as shown in Figure 1, when initial sugar concentration is at 20 g/L time, the output of succinic acid is at 16.09 g/L, and succinic acid transformation efficiency is 80.45%, be in different initial sugar concentration fermentation results succinic acid output and transformation efficiency the highest.
embodiment 3
The present embodiment explanation colon bacillus
escherichia coliaFP111 produces the technique of succinic acid in 5 L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment:
The formula of test tube, seed culture medium comprises: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermentative medium formula is: containing the Semen Maydis grit hydrolyzed solution of 1.8 g/L nitrogen elements, glucose hydrolysis liquid 20 g/L, magnesium basic carbonate 16 g/L.
Concrete steps:
(1) test tube is cultivated: by colon bacillus
escherichia coliaFP111 is inoculated in test-tube culture medium together with kantlex and paraxin, under anaerobic, and 37 DEG C of culture temperature, 200 r/min, incubation time 12 h;
(2) seed culture: the AFP111 of kantlex and paraxin and test tube cultivation is inoculated in seed culture medium to 500 ml triangular flask liquid amount 50 ml, 37 DEG C of culture temperature, 200 r/min, incubation time 6 h;
(3) anaerobism shake flask fermentation is produced succinic acid: kantlex and paraxin and seed liquor are inoculated in fermention medium to inoculum size 10%(v/v), 5 L fermentor tank liquid amount 3 L, CO
2flow velocity is per minute 0.2 L, 200 r/min, and 37 ° of C cultivate 120 h.
Fermentation results as shown in Figure 2, fermentation culture 120 h post consumptions the glucose of 50 g/L, the output of succinic acid is 42.29 g/L, succinic acid transformation efficiency is 84.58%; Compare the fermentation results of LB substratum, output and the yield of Semen Maydis powder all-hydrolytic liquid fermentation succinic acid have improved 1.37% and 21.65%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. anaerobically fermenting is produced a method for succinic acid, and the fermention medium that it is characterized in that described method is Semen Maydis powder all-hydrolytic liquid.
2. the method for succinic acid-producing according to claim 1, is characterized in that described Semen Maydis powder all-hydrolytic liquid comprises glucose sugar hydrolyzed solution and the Semen Maydis grit hydrolyzed solution of Semen Maydis powder hydrolysis gained.
3. the method for succinic acid-producing according to claim 2, is characterized in that: described glucose hydrolysis liquid is that Semen Maydis powder is through double enzymolysis method hydrolysis gained;
Semen Maydis grit after described Semen Maydis grit hydrolyzed solution is overanxious by above-mentioned Semen Maydis powder double-enzyme method hydrolysis is hydrolyzed gained through neutral protein.
4. the method for succinic acid-producing according to claim 3, the concrete steps that it is characterized in that described Semen Maydis powder double enzymolysis method are: Semen Maydis powder is mixed with the ratio of mass fraction of solids 38 % with pure water, and add calcium ion, its addition adds 0.000175g calcium ion according to every gram of raw material, at pH=6.5, under 95 DEG C of conditions, add the α-amylase of 7 U/ gram raw material, and insulation to liquefaction finishes; By liquefier gone out enzyme live after at pH=4.5, under 60 DEG C of conditions, add the saccharifying enzyme of 200 U/ gram raw material, and be incubated 48 h, the filtrate that gained saccharified liquid obtains is after filtration maize glucose hydrolyzed solution.
5. the method for succinic acid-producing according to claim 3, the concrete steps that it is characterized in that described Semen Maydis grit hydrolysis are: filter residue and pure water after saccharified liquid is filtered mix in the ratio of solid masses percentage ratio 10%, at pH=7.0, under 55 DEG C of conditions, add neutral protease, its addition is 0.3% of filter residue quality, after 24 h, filtering gained filtrate is Semen Maydis grit hydrolyzed solution, and total nitrogen content in hydrolyzed solution is measured with triumphant formula nitriding.
6. according to the method for the succinic acid-producing described in claim 1 claim, it is characterized in that described fermentation strain bacterium is recombination bacillus coli AFP111(
escherichia coliaFP111).
7. the method for succinic acid-producing according to claim 6, is characterized in that, described method concrete steps are as follows:
(1) test tube is cultivated: microbiotic and recombination bacillus coli AFP111 are seeded in to anaerobism cultivation in 5 ml liquid LB substratum, and culture temperature is 37 DEG C, 200r/min, and incubation time is 12 h;
(2) seed culture: the recombination bacillus coli AFP111 of microbiotic and test tube cultivation is inoculated in seed culture medium, 500ml triangular flask liquid amount 50 ml, culture temperature is 37 DEG C, 200r/min, incubation time is 6 h;
(3) anaerobism shake flask fermentation is produced succinic acid: microbiotic and seed culture fluid are inoculated in fermention medium, and 100ml anaerobism shaking flask liquid amount is 30ml, and inoculum size is 10 %, and magnesium basic carbonate is as pH adjusting agent, carbonating; Culture temperature is 37 DEG C, 200r/min, and incubation time is 48 h.
8. the method for succinic acid-producing according to claim 7, is characterized in that, in described step 1), the formula of LB substratum is peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L;
Described step 2) in seed culture based formulas be peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L;
Described step 3) hydrolyzed solution culture medium prescription is: containing the primverose slag hydrolyzed solution of 1.8 g/L nitrogen elements,
Glucose hydrolysis liquid 10 g/L～80 g/L, magnesium basic carbonate 16 g/L.
9. the method for succinic acid-producing according to claim 8, is characterized in that, in described step 3), glucose hydrolysis liquid is 20 g/L.
10. the method for succinic acid-producing according to claim 7, is characterized in that, in described step 3), the aerated time is 2 minutes;
Described microbiotic is kantlex and paraxin, and the final concentration adding is all 100 μ g/mL.
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| CN104846021A (en) * | 2015-06-02 | 2015-08-19 | 广西科学院 | Method for producing succinic acid through fermentation by utilizing lemna minor |
| CN105420296A (en) * | 2015-11-12 | 2016-03-23 | 武汉三江航天固德生物科技有限公司 | Method for producing succinic acid through fermentation method |
| CN105483167A (en) * | 2016-01-22 | 2016-04-13 | 南京工业大学 | Method for fermented production of succinic acid on the basis of electrochemical system for regulating intracellular reducing power regeneration |
| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
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Cited By (6)
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| CN105483167A (en) * | 2016-01-22 | 2016-04-13 | 南京工业大学 | Method for fermented production of succinic acid on the basis of electrochemical system for regulating intracellular reducing power regeneration |
| CN105483167B (en) * | 2016-01-22 | 2018-11-23 | 南京工业大学 | A method of based on reducing power regeneration fermentation succinic acid-producing in electro-chemical systems regulating cell |
| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
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