CN101475967A - Method for preparing butanoic acid from mixed fermentation ligno-cellulose - Google Patents
Method for preparing butanoic acid from mixed fermentation ligno-cellulose Download PDFInfo
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- CN101475967A CN101475967A CNA200910076954XA CN200910076954A CN101475967A CN 101475967 A CN101475967 A CN 101475967A CN A200910076954X A CNA200910076954X A CN A200910076954XA CN 200910076954 A CN200910076954 A CN 200910076954A CN 101475967 A CN101475967 A CN 101475967A
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Abstract
A method for preparing butanoic acid using bacteria mixing fermentation lignocellulose adopts the thermal fiber clostridium and thermophile butanoic clostridium, and takes the clostridium culture medium as medium, to formulate the seed culture medium; takes the preprocessed plant straw as raw material, adding the nitrogen source and inorganic salt to formulate a fermentation medium. The method includes main steps as follows: A, inoculating the thermal fiber clostridium and thermophile butanoic clostridium on a solid culture medium to perform anaerobic cultivation; B, respectively inoculating the thermal fiber clostridium and thermophile butanoic clostridium cultured by the step A onto a liquid seed culture medium; C, adding the thermal fiber clostridium seed culture medium in the step B in the fermentation medium for perform anaerobic liquid state fermentation; D. adding the thermophile butanoic clostridium seed culture medium in the step B in the fermentation tank, mixing evenly to perform high temperature bacteria mixing anaerobic fermentation to prepare the butanoic acid.
Description
Technical field
The present invention relates to a kind of method of butyric acid biological fermentation process preparation, relate in particular to a kind of method of preparing butanoic acid from mixed fermentation ligno-cellulose.
Background technology
Butyric acid is widely used in food, chemical industry and pharmaceutical industry.Butyric ester can be used as foodstuff additive increases food using, and the high purity butyric acid is used for dairy products industry increases flavor of dairy products.Beta-hydroxy-butanoic acid can be used for producing degradable poly-beta-hydroxy-butanoic acid.Butyric acid is extensively approved in the effect of treatment hemopathy, cancer and gastrointestinal tract disease.
Butyric acid mainly is that raw material is produced by the chemical industry method with the oil.But along with people improve year by year to the requirement of food, drug safety and green chemical, chemical industry method synthetic butyric acid has been difficult to enter food and medicine trade, and this makes the biological fermentation process synthetic butyric acid begin to come into one's own.
In the butyro-flow process of fermentative Production, expense of raw materials has occupied most of cost.Therefore, use wide material sources, cheap lignocellulose to carry out butyro-fermentative production as raw material and can reduce production costs significantly, and better commercial promise is arranged.
With the lignocellulose is that raw material carries out among the butyro-preparation technology, has several 'bottleneck' restrictions this The Application of Technology:
One, the commercialization cellulase costs an arm and a leg, and has reduced the price competitiveness of product.
Its two, for hexose and pentose, bacterial classification only can utilize wherein a kind of; The synchronous utilization that perhaps can only successively utilize and can't realize.Cause wastage of material, and prolonged the fermentation duration, increased product cost.
Summary of the invention
The cost of cellulase is eliminated or reduced significantly to the method that the purpose of this invention is to provide preparing butanoic acid from mixed fermentation ligno-cellulose under a kind of hot conditions, and realize the synchronous fermentation of pentose and hexose, finishes butyro-high temperature bacteria mixing fermentative preparation process.
Bacterial classification that the present invention adopts is respectively Clostridium thermocellum and thermophilic clostridium butylicum, and based on the clostridium substratum, the preparation seed culture medium; With the pre-treatment straw is raw material, adds nitrogenous source and inorganic salt, the preparation fermention medium, and its key step is as follows:
A, Clostridium thermocellum and thermophilic clostridium butylicum are inoculated in solid medium, carry out anaerobism and cultivate;
B, Clostridium thermocellum and thermophilic clostridium butylicum that steps A is cultivated are inoculated in liquid seed culture medium respectively, carry out anaerobism and cultivate;
C, the Clostridium thermocellum seed culture medium among the step B is added fermention medium, carry out anaerobism liquid state fermentation;
D, the thermophilic butyrate spindle bacillus seed substratum among the step B is added in the fermentor tank, mix, carry out high temperature bacteria mixing anaerobic fermentation, the preparation butyric acid.
It is raw material that the present invention adopts lignocellulose, significantly reduces raw materials cost; Utilize Clostridium thermocellum production of cellulose enzyme, the sky high cost of avoiding the commercial enzyme preparation to bring; Utilize thermophilic clostridium butylicum to cooperate, reduce the inhibition of intermediate product, and realize the synchronous fermentation of pentose and hexose cellulase activity with Clostridium thermocellum.The present invention uses the agriculture waste stalk to carry out the preparation of butyro-biological fermentation process as raw material, and is all significant to the research of the regeneration of resource and pollution-free new forms of energy.
Embodiment
Specifically, the method for preparing butanoic acid from mixed fermentation ligno-cellulose is as follows under a kind of hot conditions provided by the invention:
1) bacterial classification of Cai Yonging:
Clostridium thermocellum (Clostridium thermocellum)
Thermophilic clostridium butylicum (Clostridium thermobutyricum)
2) adopt liquid clostridium substratum, prepare seed culture medium on this basis.Its substratum is formulated as:
In the 1L substratum, add: 1.3g (NH
4)
2SO
4, 2.60gMgCl
2.6H
2O, 1.43gKH
2PO
4, 7.2gK
2HPO
4.3H
2O, 0.13g CaCl
2.2H
2O, 6g Sodium Glycerophosphate, 11mgFeCl
2.7H
2O, 5g yeast extract, 10g carbohydrate (Clostridium thermocellum-crystalline cellulose, thermophilic clostridium butylicum-glucose), 1mg resazurin, 0.1g halfcystine.PH is adjusted to 6.5-7.2, feeds N
2: H
2: CO
2=80:5:15 sterilized 20 minutes for 121 ℃ to keep the substratum anaerobic state.The substratum preparation, inoculation, sampling is all operated at the anaerobism workstation.
3) obtaining liq fermention medium
Stalk pre-treatment product 60g/l adds micro-nitrogenous source and inorganic salt and other material.PH is adjusted to 6.8-7.2.
4) Clostridium thermocellum and thermophilic clostridium butylicum are inoculated in solid medium, carry out anaerobism and cultivate; 55-60 ℃ of Clostridium thermocellum culture temperature, incubation time 120-168 hour, 55 ℃ of thermophilic clostridium butylicum culture temperature, incubation time 24 hours.
5) the two strain bacterium that step 4 is cultivated are inoculated in the 20-50ml liquid seed culture medium respectively, carry out anaerobism and cultivate; 55-60 ℃ of Clostridium thermocellum culture temperature, incubation time 120-168 hour, 55 ℃ of thermophilic clostridium butylicum culture temperature, incubation time 24 hours.
6) fermention medium is packed in the fermentor tank into 121 ℃ of sterilizations 20 minutes.Clostridium thermocellum seed culture medium in the step 2 is added in the fermentor tank, carry out anaerobism liquid state fermentation, behind the certain interval of time, thermophilic butyrate spindle bacillus seed substratum in the step 2 is added in the fermentor tank, the add-on of the seed liquor of two strain bacterium is the 5-10% of total add-on, two bacterial classification additional proportions are 1-3:1-3,55 ℃ of leavening temperatures, time 3-7 days.
7) adopt the HPLC method to measure butyric acid content.
Below in conjunction with embodiment the bright method of we is done further explanation.
Embodiment 1
1, preparation seed culture medium
Obtaining liq seed culture medium on the clostridium medium base, specifically composed as follows:
In the 1L substratum, add: 1.3g (NH
4)
2SO
4, 2.60gMgCl
2.6H
2O, 1.43gKH
2PO
4, 7.2gK
2HPO
4.3H
2O, 0.13g CaCl
2.2H
2O, 6g Sodium Glycerophosphate, 11mg FeCl
2.7H
2O, 5g yeast extract, 10g carbohydrate (Clostridium thermocellum-crystalline cellulose, thermophilic clostridium butylicum-glucose), 1mg resazurin, 0.1g halfcystine.PH is adjusted to 7.0, feeds N
2: H
2: CO
2=80:5:15 sterilized 20 minutes for 121 ℃ to keep the substratum anaerobic state.The substratum preparation, inoculation, sampling is all operated at the anaerobism workstation.
2, preparation fermention medium, stalk 60g after the acid treatment, urea 5g, KH
2PO
41g, K
2HPO
41g, water 1000ml, pH7.2.
3, zymotechnique
(1) Clostridium thermocellum and thermophilic clostridium butylicum are inoculated in the solid slant culture base respectively, carry out anaerobism and cultivate; 60 ℃ of Clostridium thermocellum culture temperature, incubation time 168 hours; 55 ℃ of thermophilic clostridium butylicum culture temperature, incubation time 24 hours.
(2) Clostridium thermocellum and thermophilic clostridium butylicum are inoculated in the liquid seed culture medium of 50ml and 20ml respectively, carry out anaerobism and cultivate; 60 ℃ of Clostridium thermocellum culture temperature, incubation time 120 hours; 55 ℃ of thermophilic clostridium butylicum culture temperature, incubation time 24 hours.
(3) fermention medium 1000ml is packed in the fermentor tank into 121 ℃ of sterilizations 20 minutes.The Clostridium thermocellum seed culture medium is added in the fermentor tank, and inoculum size 5% is carried out anaerobism liquid state fermentation, after 24 hours, thermophilic butyrate spindle bacillus seed substratum is added in the fermentor tank, and inoculum size is 5%, 55 ℃ of leavening temperatures, 3 days time.Sampling in each 6 hours is once measured butyric acid density.
(4) adopt the HPLC method to measure butyric acid content, with 0.05M H
2SO
4Be moving phase, sample introduction speed 0.6ml/ minute, sample size 15 μ l measure butyric acid density.Continuously fermented 72 hours, the butyric acid final concentration is 16.4g/l.
Embodiment 2
The seed culture medium preparation is with embodiment 1.
The fermention medium preparation, the quick-fried stalk 60g of ammonia, urea 5g, KH
2PO
41g, K
2HPO
41g, water 1000ml, pH7.2.
Fermention medium 1000ml is packed in the fermentor tank into 121 ℃ of sterilizations 20 minutes.The Clostridium thermocellum seed culture medium is added in the fermentor tank, and inoculum size 10% is carried out anaerobism liquid state fermentation, after 24 hours, thermophilic butyrate spindle bacillus seed substratum is added in the fermentor tank, and inoculum size is 10%, 55 ℃ of leavening temperatures, fermentation time 3 days.Sampling in each 6 hours is once measured butyric acid density.
Detect the method such as the embodiment 1 of butyric acid density.Continuously fermented 72 hours, the butyric acid final concentration is 15.8g/l.
Claims (5)
1, a kind of method of preparing butanoic acid from mixed fermentation ligno-cellulose adopts Clostridium thermocellum and thermophilic clostridium butylicum, and based on the clostridium substratum, the preparation seed culture medium; With the pre-treatment straw is raw material, adds nitrogenous source and inorganic salt, the preparation fermention medium; Its key step is as follows:
A, Clostridium thermocellum and thermophilic clostridium butylicum are inoculated in solid medium, carry out anaerobism and cultivate;
B, Clostridium thermocellum and thermophilic clostridium butylicum that steps A is cultivated are inoculated in liquid seed culture medium respectively, carry out anaerobism and cultivate;
C, the Clostridium thermocellum seed culture medium among the step B is added fermention medium, carry out anaerobism liquid state fermentation;
D, the thermophilic butyrate spindle bacillus seed substratum among the step B is added in the fermentor tank, mix, carry out high temperature bacteria mixing anaerobic fermentation, the preparation butyric acid.
2, the method for preparing butanoic acid from mixed fermentation ligno-cellulose according to claim 1, wherein, the anaerobism culture condition is in the steps A: 50-65 ℃ of Clostridium thermocellum culture temperature, incubation time 120-168 hour; 50-60 ℃ of thermophilic clostridium butylicum culture temperature, incubation time 12-24 hour.
3, the method for preparing butanoic acid from mixed fermentation ligno-cellulose according to claim 1, wherein, the volume of the liquid seed culture medium of inoculating among the step B is 20-50ml; The anaerobism culture condition is: culture temperature 50-60 ℃, and Clostridium thermocellum incubation time 120-168 hour, thermophilic clostridium butylicum incubation time 12-24 hour.
4, the method for preparing butanoic acid from mixed fermentation ligno-cellulose according to claim 1, wherein, the add-on of the seed liquor of Clostridium thermocellum and thermophilic clostridium butylicum is the 5-10% of total add-on among the step C, and two bacterial classification additional proportions are 1-3:1-3, leavening temperature 50-60 ℃, time 3-7 days.
5, the method for preparing butanoic acid from mixed fermentation ligno-cellulose according to claim 1 wherein, after step D ferments, adopts the HPLC method to measure butyric acid content.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643868A (en) * | 2012-04-27 | 2012-08-22 | 浙江大学 | Method for preparing butanol |
| CN103614315A (en) * | 2013-10-14 | 2014-03-05 | 湖北省农业科学院农产品加工与核农技术研究所 | Spent mushroom substrate high-temperature decomposition composite bacterial agent and preparation method thereof |
| CN104630292A (en) * | 2015-02-09 | 2015-05-20 | 哈尔滨工业大学宜兴环保研究院 | Method for preparing butyric acid by fermenting lignocellulose by using mixed flora |
| TWI638891B (en) | 2014-09-05 | 2018-10-21 | 鼎唐能源科技股份有限公司 | Microorganism co-culture system and uses of the same |
| CN110241053A (en) * | 2019-07-17 | 2019-09-17 | 厦门昶科生物工程有限公司 | A kind of method of mixing fermentation culture clostridium butyricum |
| CN112481316A (en) * | 2020-06-23 | 2021-03-12 | 哈尔滨工业大学 | Cathode electric fermentation method for preparing butyric acid by fermenting straw through enhanced anaerobic mixed flora |
| CN115521949A (en) * | 2022-11-10 | 2022-12-27 | 邯郸市生态环境局永年区分局 | Method for producing butyric acid by fermenting clostridium cellulovorum and method for measuring fermentable sugar content of wheat bran |
-
2009
- 2009-01-14 CN CNA200910076954XA patent/CN101475967A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643868A (en) * | 2012-04-27 | 2012-08-22 | 浙江大学 | Method for preparing butanol |
| CN102643868B (en) * | 2012-04-27 | 2013-11-20 | 浙江大学 | Method for preparing butanol |
| CN103614315A (en) * | 2013-10-14 | 2014-03-05 | 湖北省农业科学院农产品加工与核农技术研究所 | Spent mushroom substrate high-temperature decomposition composite bacterial agent and preparation method thereof |
| CN103614315B (en) * | 2013-10-14 | 2016-03-30 | 湖北省农业科学院农产品加工与核农技术研究所 | A kind of bacterium chaff pyrolytic decomposition composite fungus agent and preparation method thereof |
| TWI638891B (en) | 2014-09-05 | 2018-10-21 | 鼎唐能源科技股份有限公司 | Microorganism co-culture system and uses of the same |
| CN104630292A (en) * | 2015-02-09 | 2015-05-20 | 哈尔滨工业大学宜兴环保研究院 | Method for preparing butyric acid by fermenting lignocellulose by using mixed flora |
| CN110241053A (en) * | 2019-07-17 | 2019-09-17 | 厦门昶科生物工程有限公司 | A kind of method of mixing fermentation culture clostridium butyricum |
| CN112481316A (en) * | 2020-06-23 | 2021-03-12 | 哈尔滨工业大学 | Cathode electric fermentation method for preparing butyric acid by fermenting straw through enhanced anaerobic mixed flora |
| CN112481316B (en) * | 2020-06-23 | 2023-03-10 | 哈尔滨工业大学 | Cathode electric fermentation method for preparing butyric acid by fermenting straw through enhanced anaerobic mixed flora |
| CN115521949A (en) * | 2022-11-10 | 2022-12-27 | 邯郸市生态环境局永年区分局 | Method for producing butyric acid by fermenting clostridium cellulovorum and method for measuring fermentable sugar content of wheat bran |
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| CB03 | Change of inventor or designer information |
Inventor after: Ma Yujiu Inventor after: Zhang Chunhui Inventor after: Zhang Yuedong Inventor after: Yang Hua Inventor after: Yang Fangxiao Inventor before: Zhang Chunhui Inventor before: Ma Yujiu Inventor before: Yang Fangxiao Inventor before: Zhang Yuedong Inventor before: Yang Hua |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG CHUNHUI MA YUJIU YANG FANGXIAO ZHANG YUEDONG YANG HUA TO: MA YUJIU ZHANG CHUNHUI ZHANG YUEDONG YANG HUA YANG FANGXIAO |
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Open date: 20090708 |