CN105331641A - Method for preparing succinic acid by using water hyacinth as fermentation raw material - Google Patents
Method for preparing succinic acid by using water hyacinth as fermentation raw material Download PDFInfo
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- CN105331641A CN105331641A CN201510831802.1A CN201510831802A CN105331641A CN 105331641 A CN105331641 A CN 105331641A CN 201510831802 A CN201510831802 A CN 201510831802A CN 105331641 A CN105331641 A CN 105331641A
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Abstract
The invention relates to a method for preparing succinic acid by using water hyacinth as a fermentation raw material. The method comprises the following steps: (1) pretreatment of water hyacinth; (2) one-stage cultivation: putting culture into a seed nutrient solution for cultivation, wherein the cultivation time is 14 h; (3) two-stage cultivation: inoculating a seed culture medium after the one-stage cultivation, wherein the inoculum size is 4%, and the culture medium is obtained after the cultivation time reaches 7 h; (4) three-stage cultivation: inoculating the culture medium into a fermentation culture medium, and feeding a sodium hydroxide solution with the molar concentration of 1 mol/L in the fermentation process to maintain the pH value of a fermentation system to be between 5.5 and 6.5, wherein the introduction amount of carbon dioxide in a fermentation tank is 0.5-5 L/min, the temperature is 37-39 DEG C, the fermentation time is 48 h, the sodium concentration reaches 1.5% to 2% when the fermentation is finished, and the succinic acid is obtained after the fermentation is finished. The requirement of reducing the pollution to the environment is reached while the production cost is low.
Description
Technical field
This patent relates to a kind of Herba Eichhorniae that uses and prepares the method for succinic acid as the carbon source of biological fermentation, belongs to technical field of bioengineering.
Background technology
Herba Eichhorniae, has another name called Herba Eichhorniae, water lettuce, phoenix eyes Nelumbo, China in 20th century the '30s introduce from South America as feed.The many growths of Herba Eichhorniae are in the water of eutrophication, and the speed of growth is generally acknowledged the fastest in the world, and general spring end starts growth, and time amount reproduction at the beginning of summer and autumn, when the speed of growth is the fastest, complete stool biomass dry-matter can reach 170 tons of per hectares.
Pollute due to environment in recent years and more and more seriously add the fecundity that Herba Eichhorniae is vigorous, in new environment, lack natural enemy, Herba Eichhorniae spreads unchecked in a lot of area of China simultaneously, and blocking river course, is paved with the water surface, causes serious harm.
In addition, Herba Eichhorniae covers with and covers whole lake surface, and the other plant in water can not grow very well, and the eubiosis in water will be broken.By the Herba Eichhorniae after pulling out, pile up like a mountain in a large number, causes very large harm further again to environment.
And on the other hand, containing very abundant fiber, protein, amino acid, crude fat and various trace element in Herba Eichhorniae, therefore, if utilize fermentable Herba Eichhorniae to prepare corresponding product, again can decreasing pollution, formed gradually and use Herba Eichhorniae to dispose of sewage, periodic collection is as the biological cycle system of the raw material of fermentable, and this has important practical significance to enterprise and society.
Both domestic and external focusing mostly on as the research emphasis of fermentation raw material for Herba Eichhorniae cleans reproducible alcohol fuel at the saccharomycetic microbe conversion of use at present.But because the added value of alcohol fuel is low, the economic benefit of Herba Eichhorniae maximally can not be improved; In addition, the wood sugar that the yeast for the preparation of ethanol commercial at present can not utilize hydrolysis of hemicellulose to produce mostly, this again reduces utilising efficiency undoubtedly for the Herba Eichhorniae that hemicellulose level is relatively high.
Succinic acid (succinicacid) has another name called succsinic acid, is extensively present in organism, is a kind of important binary organic carboxyl acid, is also the important meta-bolites of microorganism tricarboxylic acid cycle glycolytic pathway.Succinic acid has a wide range of applications in a lot of field, can be used for synthesized degradable plastics, the tranquillizer of field of medicaments, the foodstuff additive of foodstuffs industry, tensio-active agent etc.Therefore, the production method that is efficient, low cost of succinic acid also becomes the focus of Recent study, and wherein adopting the method for biological fermentation to prepare succinic acid is the most important thing.
The Main way reducing biological fermentation process succinic acid preparation cost uses cheap carbon source, how effectively to utilize natural Biological resources, can meet the normal life requirement of people, can reduce again the pollution of environment is a significantly problem.Undoubtedly, comprehensive utilization of waterhyacinth will have important practical significance to enterprise and society.
Summary of the invention
The object of the invention is to overcome above deficiency, providing a kind of Herba Eichhorniae that uses to prepare the method for succinic acid as fermentation raw material.
Object of the present invention realizes like this:
Use Herba Eichhorniae to prepare a method for succinic acid as fermentation raw material, its preparation method is as follows:
(1) pre-treatment of Herba Eichhorniae of the present invention:
1. first Herba Eichhorniae raw material is carried out centrifuge dripping, obtain dry Herba Eichhorniae; By Herba Eichhorniae volumetric molar concentration be 2%-3% aqueous sodium hydroxide solution soak 2 hours under 120 degree of conditions, the mass ratio of Herba Eichhorniae and aqueous sodium hydroxide solution is 1:10;
2. soak after terminating, leached by solid, use washed with de-ionized water, rinse to neutral, add citric acid or other organic acids adjustment pH value of solution to 4.0 ~ 4.5, temperature adjusts to 38 ~ 40 degree, and now the solid-to-liquid ratio of solution is 1:10, counts in mass ratio;
3. Novi is believed that cellulase Carezyme 4500L joins in the solution 2. processed through step (1) to be hydrolyzed, time is 48 hours, the dosage of Novi letter cellulase Carezyme 4500L is 10% of Mierocrystalline cellulose and hemicellulose level in Herba Eichhorniae solids, and quality is 2.6g; By solid filtering in solution, and collect filtrate, this filtrate is cellulase hydrolysis liquid;
5. cellulase hydrolysis liquid is concentrated, be concentrated into total sugar content and reach 350g/L, obtain hydrolysis sugar liquid;
(2) fermention medium is configured
Adopt deionized water preparation fermention medium 100mL, the moiety of fermention medium is counted by mass concentration: peptone 5g/L, corn steep liquor 10g/L, total reducing sugars content 100g/L, potassium primary phosphate 2.5g/L, Sodium phosphate dibasic 2.5g/L, yeast extract paste
5g/L, sodium acetate 1g/L, with deionized water preparation, wherein total reducing sugars takes from described hydrolysis sugar liquid.
(3) one-level is cultivated
Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 is put into seed culture fluid and cultivates, and incubation time is 14h.
(4) secondary is cultivated
One-level connects seed culture medium after cultivating, and inoculum size is 4%, and incubation time obtains nutrient solution after reaching 7h.
(5) third stage culture
Nutrient solution is accessed fermention medium, in fermentor tank, the intake of carbonic acid gas is 3L/min, in fermenting process, stream adds volumetric molar concentration is that the pH value of the sodium hydroxide solution maintenance fermentation system of 1mol/L is between 5.5 ~ 6.5, temperature is 37 ~ 39 DEG C, fermentation time is 48h, during fermentation ends, Na ion concentration reaches 1.5% ~ 2%, obtains succinic acid after fermentation ends.
Above-mentioned a kind of Herba Eichhorniae that uses prepares the method for succinic acid as fermentation raw material, and wherein, preferably, in described fermenting process, stream adds volumetric molar concentration is that to maintain the pH value of fermentation system be 6.0 ~ 6.2 for the sodium hydroxide solution of 1mol/L.
Above-mentioned a kind of Herba Eichhorniae that uses prepares the method for succinic acid as fermentation raw material, wherein, and the product after the Mierocrystalline cellulose that the carbon source that fermentation uses obtains through process for lotus skin powder, hydrolysis of hemicellulose.
Beneficial effect of the present invention is:
By the pre-treatment to Herba Eichhorniae, xylogen in Herba Eichhorniae is removed, adopt acidolysis and enzymolysis by hemicellulose and cellulose conversion the sugared source needed for fermentation succinic acid, this is for reducing raw materials cost of Biological preparation succinic acid and reducing Herba Eichhorniae and have great effect for the pollution of environment.The present invention adopts Herba Eichhorniae to produce succinic acid, and industrial step reasonable in design, the succinic acid impurity of output is low, and utilization ratio is high.While low production cost, accomplish the requirement reduced the pollution of environment.
Embodiment
Herba Eichhorniae used in embodiment produces to be longer than raises Chinese and Western Lai Qiaozhen pool near a river in Zhenjiang, and main project comprises fiber (comprising hemicellulose) content, protein content, ash oontent, water-content.
The mensuration of protein content, adopts micro-Kjeldahl, with reference to GB/T5009.5-1985.
The mensuration of fiber, is by dried quantitative Herba Eichhorniae is carried out oxygenolysis in the mixed solution of the vitriol oil and potassium bichromate, and uses the anti-method of dripping excessive potassium permanganate of Sulfothiorine.
The mensuration of moisture, adopts direct drying method, with reference to GB/T-14769-93.
The mensuration of ash content, with reference to GB5009.4-85
According to the detection of above method, Herba Eichhorniae (doing) is containing fiber and hemicellulose 25.56%, and protein content is 37.5%, ash content 14.2%, and other content (comprising xylogen) are 22.74%.
As can be seen from the above results, the material mainly Mierocrystalline cellulose as carbon source in Herba Eichhorniae, can be utilized.
Find in experiment, water hyacinth fibre element is comparatively fine and close with lignin structure, and pre-treatment needs use 2% ~ 3% diluted alkaline.
The TSB nutrient solution used in the embodiment of the present invention is pancreas peptone soybean broth.
Bacterial strain uses therefor Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 of the present invention, open in Chinese patent ZL200610038113.6, publication number CN1814747A, publication date on August 9th, 2006.
Below in conjunction with embodiment, the invention will be further described.
embodiment 1
Use Herba Eichhorniae to prepare a method for succinic acid as fermentation raw material, its preparation method is as follows:
(1) pre-treatment of Herba Eichhorniae of the present invention:
2. first get 100Kg Herba Eichhorniae, centrifuge dripping is carried out to Herba Eichhorniae raw material, then dry to substantially anhydrous, obtain dry Herba Eichhorniae 2.1kg; By Herba Eichhorniae volumetric molar concentration be 2.5% aqueous sodium hydroxide solution soak 2 hours under 120 degree of conditions, the mass ratio of Herba Eichhorniae and aqueous sodium hydroxide solution is 1:10;
2. soak after terminating, leached by solid, use washed with de-ionized water, rinse to neutral, add citric acid adjustment pH value of solution to 4.0 ~ 4.5, temperature adjusts to 38 ~ 40 degree, and now the solid-to-liquid ratio of solution is 1:10, counts in mass ratio;
3. Novi is believed that cellulase Carezyme 4500L joins in the solution 2. processed through step (1) to be hydrolyzed, time is 48 hours, and the dosage of Novi letter cellulase Carezyme 4500L is 10% of Mierocrystalline cellulose and hemicellulose level in Herba Eichhorniae solids; By solid filtering in solution, and collect filtrate, this filtrate is cellulase hydrolysis liquid;
5. cellulase hydrolysis liquid is concentrated, be concentrated into total sugar content and reach 350g/L, obtain hydrolysis sugar liquid;
(2) seed culture medium is configured
Preparation seed culture fluid 10mL, consist of peptone 5g/L, corn steep liquor 20g/L, glucose 20g/L, potassium primary phosphate 1.5g/L, Sodium phosphate dibasic 1.5g/L, yeast extract paste 5g/L, prepares with deionized water;
(3) fermention medium is configured
Adopt deionized water preparation fermention medium 100mL, the moiety of fermention medium is counted by mass concentration: peptone 5g/L, corn steep liquor 10g/L, total reducing sugars content 100g/L, potassium primary phosphate 2.5g/L, Sodium phosphate dibasic 2.5g/L, yeast extract paste
5g/L, sodium acetate 1g/L, with deionized water preparation, wherein total reducing sugars takes from described hydrolysis sugar liquid.
(4) one-level is cultivated
Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 is put into TSB nutrient solution cultivate, incubation time is 14h.
(5) secondary is cultivated
One-level connects seed culture medium after cultivating, and inoculum size is 4%, and incubation time obtains nutrient solution after reaching 7h.
(6) third stage culture
Nutrient solution is accessed fermention medium, in fermentor tank, the intake of carbonic acid gas is 3L/min, in fermenting process, stream adds volumetric molar concentration is that the pH value of the sodium hydroxide solution maintenance fermentation system of 1mol/L is between 6.0 ~ 6.1, temperature is 37 ~ 39 DEG C, fermentation time is 48h, during fermentation ends, Na ion concentration reaches 1.5%, obtains succinic acid after fermentation ends.
embodiment 2
Use Herba Eichhorniae to prepare a method for succinic acid as fermentation raw material, its preparation method is as follows:
(1) pre-treatment of Herba Eichhorniae of the present invention:
3. first get 100Kg Herba Eichhorniae, centrifuge dripping is carried out to Herba Eichhorniae raw material, then dry to substantially anhydrous, obtain dry Herba Eichhorniae 2.1kg; By Herba Eichhorniae volumetric molar concentration be 2.5% aqueous sodium hydroxide solution soak 2 hours under 120 degree of conditions, the mass ratio of Herba Eichhorniae and aqueous sodium hydroxide solution is 1:10;
2. soak after terminating, leached by solid, use washed with de-ionized water, rinse to neutral, add succinic acid adjustment pH value of solution to 4.0, temperature adjusts to 38 degree, and now the solid-to-liquid ratio of solution is 1:10, counts in mass ratio;
3. Novi is believed that cellulase Carezyme 4500L joins in the solution 2. processed through step (1) to be hydrolyzed, time is 48 hours, and the dosage of Novi letter cellulase Carezyme 4500L is 10% of Mierocrystalline cellulose and hemicellulose level in Herba Eichhorniae solids; By solid filtering in solution, and collect filtrate, this filtrate is cellulase hydrolysis liquid;
5. cellulase hydrolysis liquid is concentrated, be concentrated into total sugar content and reach 350g/L, obtain hydrolysis sugar liquid;
(2) seed culture medium is configured
Preparation seed culture fluid 10mL, consist of peptone 5g/L, corn steep liquor 20g/L, glucose 20g/L, potassium primary phosphate 1.5g/L, Sodium phosphate dibasic 1.5g/L, yeast extract paste 5g/L, prepares with deionized water;
(3) fermention medium is configured
Adopt deionized water preparation fermention medium 100mL, the moiety of fermention medium is counted by mass concentration: peptone 5g/L, corn steep liquor 10g/L, total reducing sugars content 100g/L, potassium primary phosphate 2.5g/L, Sodium phosphate dibasic 2.5g/L, yeast extract paste
5g/L, sodium acetate 1g/L, with deionized water preparation, wherein total reducing sugars takes from described hydrolysis sugar liquid.
(4) one-level is cultivated
Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 is put into TSB nutrient solution cultivate, incubation time is 14h.
(5) secondary is cultivated
One-level connects seed culture medium after cultivating, and inoculum size is 4%, and incubation time obtains nutrient solution after reaching 7h.
(6) third stage culture
Nutrient solution is accessed fermention medium, in fermentor tank, the intake of carbonic acid gas is 3L/min, in fermenting process, stream adds volumetric molar concentration is that the pH value of the sodium hydroxide solution maintenance fermentation system of 1mol/L is between 6.0, temperature is 37 DEG C, fermentation time is 48h, during fermentation ends, Na ion concentration reaches 1.5%, obtains succinic acid after fermentation ends.
embodiment 3
Use Herba Eichhorniae to prepare a method for succinic acid as fermentation raw material, its preparation method is as follows:
(1) pre-treatment of Herba Eichhorniae of the present invention:
4. first get 100Kg Herba Eichhorniae, centrifuge dripping is carried out to Herba Eichhorniae raw material, then dry to substantially anhydrous, obtain dry Herba Eichhorniae 2.1kg; By Herba Eichhorniae volumetric molar concentration be 2.5% aqueous sodium hydroxide solution soak 2 hours under 120 degree of conditions, the mass ratio of Herba Eichhorniae and aqueous sodium hydroxide solution is 1:10;
2. soak after terminating, leached by solid, use washed with de-ionized water, rinse to neutral, add acetic acid adjustment pH value of solution to 4.5, temperature adjusts to 40 degree, and now the solid-to-liquid ratio of solution is 1:10, counts in mass ratio;
3. Novi is believed that cellulase Carezyme 4500L joins in the solution 2. processed through step (1) to be hydrolyzed, time is 48 hours, and the dosage of Novi letter cellulase Carezyme 4500L is 10% of Mierocrystalline cellulose and hemicellulose level in Herba Eichhorniae solids; By solid filtering in solution, and collect filtrate, this filtrate is cellulase hydrolysis liquid;
5. cellulase hydrolysis liquid is concentrated, be concentrated into total sugar content and reach 350g/L, obtain hydrolysis sugar liquid;
(2) seed culture medium is configured
Preparation seed culture fluid 10mL, consist of peptone 5g/L, corn steep liquor 20g/L, glucose 20g/L, potassium primary phosphate 1.5g/L, Sodium phosphate dibasic 1.5g/L, yeast extract paste 5g/L, prepares with deionized water;
(3) fermention medium is configured
Adopt deionized water preparation fermention medium 100mL, the moiety of fermention medium is counted by mass concentration: peptone 5g/L, corn steep liquor 10g/L, total reducing sugars content 100g/L, potassium primary phosphate 2.5g/L, Sodium phosphate dibasic 2.5g/L, yeast extract paste
5g/L, sodium acetate 1g/L, with deionized water preparation, wherein total reducing sugars takes from described hydrolysis sugar liquid.
(4) one-level is cultivated
Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 is put into TSB nutrient solution cultivate, incubation time is 14h.
(5) secondary is cultivated
One-level connects seed culture medium after cultivating, and inoculum size is 4%, and incubation time obtains nutrient solution after reaching 7h.
(6) third stage culture
Nutrient solution is accessed fermention medium, in fermentor tank, the intake of carbonic acid gas is 3L/min, in fermenting process, stream adds volumetric molar concentration is that the pH value of the sodium hydroxide solution maintenance fermentation system of 1mol/L is between 6.1, temperature is 9 DEG C, fermentation time is 48h, during fermentation ends, Na ion concentration reaches 2%, obtains succinic acid after fermentation ends.
The succinic acid yield result that how obtained table 1 embodiment of the present invention 1-3 is:
| Succinic acid output | Unit | Numerical value |
| Embodiment 1 | g/L | 27 |
| Embodiment 2 | g/L | 24 |
| Embodiment 3 | g/L | 20 |
Above embodiment is only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (3)
1. use Herba Eichhorniae to prepare a method for succinic acid as fermentation raw material, its preparation method is as follows:
The pre-treatment of Herba Eichhorniae of the present invention:
First Herba Eichhorniae raw material is carried out centrifuge dripping, obtain dry Herba Eichhorniae; By Herba Eichhorniae volumetric molar concentration be 2%-3% aqueous sodium hydroxide solution soak 2 hours under 120 degree of conditions, the mass ratio of Herba Eichhorniae and aqueous sodium hydroxide solution is 1:10;
2. soak after terminating, leached by solid, use washed with de-ionized water, rinse to neutral, add citric acid or other organic acids adjustment pH value of solution to 4.0 ~ 4.5, temperature adjusts to 38 ~ 40 degree, and now the solid-to-liquid ratio of solution is 1:10, counts in mass ratio;
3. Novi is believed that cellulase Carezyme 4500L joins in the solution 2. processed through step (1) to be hydrolyzed, time is 48 hours, the dosage of Novi letter cellulase Carezyme 4500L is 10% of Mierocrystalline cellulose and hemicellulose level in Herba Eichhorniae solids, and quality is 2.6g; By solid filtering in solution, and collect filtrate, this filtrate is cellulase hydrolysis liquid;
5. cellulase hydrolysis liquid is concentrated, be concentrated into total sugar content and reach 350g/L, obtain hydrolysis sugar liquid;
(2) fermention medium is configured
Adopt deionized water preparation fermention medium 100mL, the moiety of fermention medium is counted by mass concentration: peptone 5g/L, corn steep liquor 10g/L, total reducing sugars content 100g/L, potassium primary phosphate 2.5g/L, Sodium phosphate dibasic 2.5g/L, yeast extract paste
5g/L, sodium acetate 1g/L, with deionized water preparation, wherein total reducing sugars takes from described hydrolysis sugar liquid;
(3) one-level is cultivated
Actinobacillus succinogenes (Actinobacillussuccinogenes) CGMCC1593 is put into seed culture fluid and cultivates, and incubation time is 14h;
(4) secondary is cultivated
One-level connects seed culture medium after cultivating, and inoculum size is 4%, and incubation time obtains nutrient solution after reaching 7h;
(5) third stage culture
Nutrient solution is accessed fermention medium, in fermentor tank, the intake of carbonic acid gas is 3L/min, in fermenting process, stream adds volumetric molar concentration is that the pH value of the sodium hydroxide solution maintenance fermentation system of 1mol/L is between 5.5 ~ 6.5, temperature is 37 ~ 39 DEG C, fermentation time is 48h, during fermentation ends, Na ion concentration reaches 1.5% ~ 2%, obtains succinic acid after fermentation ends.
2. a kind of Herba Eichhorniae that uses as claimed in claim 1 prepares the method for succinic acid as fermentation raw material, it is characterized in that, preferably, in described fermenting process, stream adds volumetric molar concentration is that to maintain the pH value of fermentation system be 6.0 ~ 6.2 for the sodium hydroxide solution of 1mol/L.
3. a kind of Herba Eichhorniae that uses as claimed in claim 1 prepares the method for succinic acid as fermentation raw material, it is characterized in that, the product after the Mierocrystalline cellulose that the carbon source that fermentation uses obtains through process for Herba Eichhorniae, hydrolysis of hemicellulose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106939286A (en) * | 2017-02-27 | 2017-07-11 | 华中农业大学 | A kind of method that phosphate solubilizing bacteria PA02 solid fungicides are prepared by culture matrix of plant biomass |
| CN112646843A (en) * | 2020-12-31 | 2021-04-13 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing lactic acid by using water hyacinth as raw material |
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| CN101215582A (en) * | 2007-12-28 | 2008-07-09 | 江南大学 | Method for producing succinic acid by fermenting straw raw material |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106939286A (en) * | 2017-02-27 | 2017-07-11 | 华中农业大学 | A kind of method that phosphate solubilizing bacteria PA02 solid fungicides are prepared by culture matrix of plant biomass |
| CN106939286B (en) * | 2017-02-27 | 2020-07-10 | 华中农业大学 | Method for preparing phosphate solubilizing bacterium PA02 solid microbial inoculum by taking plant biomass as culture medium |
| CN112646843A (en) * | 2020-12-31 | 2021-04-13 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing lactic acid by using water hyacinth as raw material |
| CN112646843B (en) * | 2020-12-31 | 2022-03-08 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing lactic acid by using water hyacinth as raw material |
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