CN107760753B - Method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolyzing sugar and clostridium acetobutylicum - Google Patents
Method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolyzing sugar and clostridium acetobutylicum Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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Abstract
The invention discloses a method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolysis sugar and clostridium acetobutylicum, which comprises the following steps: (1) inoculating activated high-temperature anaerobic bacteria of the pyrolysis sugar into a fermentation culture medium containing xylan for fermentation to obtain fermentation liquor; (2) and (2) inoculating the activated clostridium acetobutylicum into the fermentation liquor obtained in the step (1), and fermenting to produce butanol. When the high-temperature anaerobe M5 for the pyrolysis sugar is cultured for 60h, the clostridium acetobutylicum bacterial mud is added, the butanol yield is the highest and reaches 8.34g/L, and the highest butanol yield is obtained by co-culture when xylan is used as a unique carbon source at present. The method reduces the cost of industrial production of butanol, solves the problem that the pyrolysis sugar high-temperature anaerobic bacteria can utilize hemicellulose but the yield of butanol is low, and has important application value.
Description
Technical Field
The invention belongs to the field of microbial fermentation, relates to butanol fermentation, and particularly relates to a method for producing butanol by co-culture fermentation of high-temperature anaerobes for pyrolysis of sugar and clostridium acetobutylicum.
Background
As a fuel, the butanol has the advantages of high energy density, high stability to water, direct application to internal combustion engines, convenient transportation and the like, and has a wide development prospect as a fuel at present with increasingly severe energy crises. The butanol can be produced mainly by acetaldehyde condensation, propylene oxo synthesis, fermentation, and the like. With the rising price of petroleum and the accelerated exhaustion of resources, butanol production by fermentation method has received extensive attention and is gradually one of the research hotspots of biological energy.
The traditional fermentation method for producing butanol mainly takes grain or other starchy agricultural byproducts as raw materials, fermentation liquor is obtained through hydrolysis, then butanol is obtained under the action of fermentation bacteria, and most strains are solvent clostridium, such as clostridium beijerinckii, clostridium acetobutylicum and the like. Because the lignocellulose can not be used as a carbon source, the production cost is greatly increased, and the method is not beneficial to industrial production. The main components of the grain straws are cellulose and hemicellulose, wherein the main component of the hemicellulose is xylan, so that the production cost can be greatly reduced by using the xylan as a unique carbon source to produce butanol, and waste can be changed into valuable.
The thermophilic anaerobic bacillus is the only thermophilic wild strain which can directly utilize hemicellulose to produce biological butanol by integrated biological processing engineering, but the yield of the butanol is lower, and the reported yield is generally lower than 0.1 g/L. CN106995790A discloses a pyrogenous sugar high temperature anaerobic strain M5(Thermoanaerobacterium thermosaccharolyticum) capable of directly utilizing lignocellulose as a carbon source to produce butanol, wherein the yield of the butanol is up to 1.32g/L, but the yield of the butanol is still at a lower level. The solvent clostridium such as clostridium acetobutylicum is a traditional solvent-producing clostridium, is a gram-positive bacterium, is in a shape of a short rod with two round ends, has flagella, is movable, has high butanol yield which can generally reach about 10g/L, but can only use monosaccharide as a carbon source such as glucose, xylose and the like, has high cost and greatly influences the industrial production. At present, few reports are made on co-culture by using lignocellulose as a raw material.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problem that the production cost is too high by using grains or other starchy byproducts as raw materials in the traditional butanol fermentation method, the invention provides a method for producing butanol by co-culture fermentation of high-temperature anaerobes for pyrolyzing sugar and clostridium acetobutylicum.
The technical scheme is as follows: the invention relates to a method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolysis sugar and clostridium acetobutylicum, which comprises the following steps:
(1) inoculating activated high-temperature anaerobic bacteria of the pyrolysis sugar into a fermentation culture medium containing xylan for fermentation to obtain fermentation liquor;
(2) and (2) inoculating the activated clostridium acetobutylicum into the fermentation liquor obtained in the step (1), and fermenting to produce butanol.
And (2) when the enzyme activity of the xylanase in the fermentation liquor obtained in the step (1) reaches 0.3-0.8U/mL, inoculating the activated clostridium acetobutylicum into the fermentation liquor obtained in the step (1). At the moment, a certain amount of xylose exists in the fermentation liquor, the activity of xylanase and xylanase is highest, xylan can be continuously degraded into xylose, and clostridium acetobutylicum can produce butanol by using the degraded xylose. The inoculation time is too early, the enzyme activity of the xylanase is low, and the subsequent degradation of xylan is not utilized; when the inoculation time is too late, the culture time of the strain M5 is too long, the secretion amount of xylanase and xylanase is reduced, the enzyme activity of the previously secreted enzyme at 55 ℃ is reduced, so that the enzyme activity of an enzyme system is reduced, and the xylose obtained by partial degradation is also utilized by the strain M5, so that the carbon flow of clostridium acetobutylicum is reduced, and the yield of butanol is finally reduced. Therefore, the inoculation time of C.acetobutylicum is a very critical step in the co-cultivation of fermented D.acetobutylicum.
Preferably, when the enzyme activity of the xylanase in the fermentation liquor obtained in the step (1) reaches 0.5-0.6U/mL, the activated clostridium acetobutylicum is inoculated into the fermentation liquor obtained in the step (1) for producing butanol through fermentation.
The inoculation amount of the activated high-temperature anaerobic bacteria for the pyrolysis sugar in the step (1) is 1-10% of the volume of a fermentation culture medium; the fermentation medium containing xylan comprises the following formula: 0.5-2.0g/L NaCl, 0.5-2.0g/L K2HPO4，0.5-2.0g/L KH2PO41.0-5.0g/L yeast powder, 0.2-1.0g/L MgCl2·6H2O，0.1-0.6g/L NH4Cl，0.01-0.05g/L CaCl2·2H2O，0.5-2.0g/L FeCl2·4H2O, 0.1-0.5g/L KCl, 30-90g/L xylan and water as solvent, adjusting pH to 6.0-6.5, introducing nitrogen for 10-20min, and sterilizing at 121 deg.C for 15 min; the fermentation conditions are as follows: fermenting at 50-65 deg.C for 36-72h, and fermenting at pH 5.5-8.5 (adjusting pH every 12-24 h) at 0-120rpm, and standing or stirring for fermentation.
Preferably, the xylan concentration in the xylan-containing fermentation medium is 60 g/L.
Preferably, the fermentation conditions in step (1) are: the fermentation temperature is 55 ℃, the fermentation time is 60h, the fermentation pH is 7.5, and the rotating speed is 120 rpm.
The formula of the activation medium of the high-temperature anaerobic bacteria for the pyrolysis sugar is 0.5-2.0g/L NaCl and 0.5-2.0g/L K2HPO4，0.5-2.0g/L KH2PO41.0-5.0g/L yeast powder, 0.2-1.0g/L MgCl2·6H2O，0.1-0.6g/L NH4Cl，0.01-0.05g/L CaCl2·2H2O，0.5-2.0g/L FeCl2·4H2O, 0.1-0.5g/L KCl, 30-90g/L xylan and water as solvent, and adjusting the pH value to 6.0-6.5; the activation conditions were: inoculating the pyrogenous sugar high-temperature anaerobic bacteria into an activation culture medium with the inoculation amount of 5% v/v, activating at 50-65 ℃ and 0-120rpm for 48-96h, and adjusting the pH to 5.5-8.5 every 12 h.
Wherein, the strain number of the high-temperature anaerobe (Thermoanaerobacterium thermosaccharolyticum) is M5, the high-temperature anaerobe has been preserved in China center for type culture collection with the preservation date of 2017, 2 and 27 days, the preservation number is CCTCC NO: M2017072, and the preservation addresses are as follows: wuhan university, which is published in Chinese application CN 106995790A. The pyrogenous sugar thermophilic anaerobe M5 can degrade xylan through xylanase to obtain xylose, and obtain 3-P-glyceraldehyde under the action of xylose isomerase and xylulokinase, so as to enter tricarboxylic acid cycle to obtain pyruvic acid, and finally obtain products such as acetic acid, ethanol, butyric acid, butanol and the like through the catalysis of a series of enzymes. And the enzymes have strong temperature tolerance and have high value for industrial production. The strain M5 has all genes from xylose to butanol, does not produce acetone, is the only wild strain reported so far, which can directly utilize xylan to produce butanol and does not produce acetone, but has low butanol yield, and the highest butanol yield is only 1.32g/L after being optimized by a culture medium and the like and is still at a low level. In addition, the strain M5 can grow at 50-65 ℃, and secreted enzymes from xylan to butanol production have higher temperature stability. The Clostridium acetobutylicum is classified and named as Clostridium acetobutylicum (Clostridium acetobutylicum) with the strain number of ATCC 824.
The activated clostridium acetobutylicum of the step (2) is inoculated in the following way: centrifuging the activated acetone-butanol clostridium culture solution, and inoculating the precipitate obtained by centrifugation into the fermentation liquor obtained in the step (1), wherein the volume of the activated acetone-butanol clostridium culture solution is the same as that of the fermentation liquor obtained in the step (1); the fermentation conditions are as follows: the fermentation temperature is 35-39 ℃, the fermentation time is 60-168h, the fermentation pH is 4.5-7.0, and the rotation speed is 100-200 rpm.
Preferably, the fermentation conditions in step (2) are: the fermentation temperature is 37 ℃, the fermentation time is 120h, the fermentation pH is 5.5, and the rotation speed is 150 rpm.
The activating culture medium formula of the clostridium acetobutylicum is 0.5-2.0g/L NaCl and 0.5-2.0g/L K2HPO4，0.5-2.0g/L KH2PO41.0-5.0g/L yeast powder, 0.2-1.0g/L MgCl2·6H2O，0.1-0.6g/L NH4Cl，0.01-0.05g/L CaCl2·2H2O，0.5-2.0g/L FeCl2·4H2O, 0.1-0.5g/L KCl, 30-90g/L xylose and water as solvent; the activation conditions were as follows: inoculating Clostridium acetobutylicum with an inoculum size of 5% v/v into an activation medium, activating at 37 deg.C and 150rpm for 48-96h, and adjusting pH to 4.5-7.0 every 12h to obtain activated Clostridium acetobutylicum culture solution.
Has the advantages that: compared with the prior art, the invention has the following technical advantages:
(1) according to the invention, the high-temperature anaerobic bacteria M5 of the pyrolysis sugar capable of growing by using xylan as a unique carbon source is cultured firstly, the xylan is degraded into xylose under the condition of high-temperature anaerobism, but the butanol yield obtained by the production is low, so that the clostridium acetobutylicum with higher butanol yield but incapable of directly using hemicellulose is utilized for co-culture, so that the butanol yield is improved;
(2) when the high-temperature anaerobe M5 for the pyrolysis sugar is cultured for 60h, the clostridium acetobutylicum bacterial mud is added, the butanol yield is the highest and reaches 8.34g/L, and the highest butanol yield is obtained by co-culture when xylan is used as a unique carbon source at present. When M5 is cultured for 60h, the enzyme activity of xylanase in the fermentation liquid is highest, and the xylanase effectively degrades xylan to obtain 18g/L xylose; in a co-culture system, xylanase and xylanase still have high enzyme activity, xylan can be continuously degraded into xylose, clostridium acetobutylicum can produce butanol by using the degraded xylose, and synchronous saccharification and fermentation are realized;
(3) the method reduces the cost of industrial production of butanol, solves the problem that the pyrolysis sugar high-temperature anaerobic bacteria can utilize hemicellulose but the yield of butanol is low, and has important application value.
Drawings
FIG. 1 is a graph showing the variation of xylose concentration and xylanase activity in fermentation broth under optimized co-culture fermentation conditions;
FIG. 2 is a graph showing the change in product concentration in fermentation broth under optimized co-culture fermentation conditions.
Detailed Description
Example 1 production of butanol Using Clostridium acetobutylicum ATCC 824 with xylose as carbon Source
The strain Clostridium acetobutylicum ATCC 824 was picked from the plate and inoculated into 5mL of fermentation medium, cultured at 37 ℃ for 48 hours at 150rpm, then inoculated into a new fermentation medium at an inoculum size of 5% v/v, shake-cultured at 37 ℃ at 150rpm, pH was adjusted to 5.5 every 12 hours, and after 120 hours, the concentration of each product was measured by GC, and the final butanol yield was 8.97 g/L.
The formula of the fermentation medium is 1g/L NaCl and 0.75g/L K2HPO4，0.75g/L KH2PO43g/L yeast powder, 0.5g/L MgCl2·6H2O，0.3g/L NH4Cl，0.015g/L CaCl2·2H2O，1.5g/L FeCl2·4H2O, 0.3g/L KCl and 60g/L xylose, adjusting the pH value to 5.5,introducing nitrogen gas for 10-20min, and sterilizing at 121 deg.C for 15 min.
Example 2 Effect of different xylan concentrations on the Final Butanol yields
(1) Inoculating the pyrogenous sugar high-temperature anaerobic bacteria into an activation culture medium with the inoculation amount of 5% v/v, activating at 55 ℃ and 120rpm for 60h, and adjusting the pH to 7.5 every 12 h;
the formula of the activation medium is as follows: 1g/L NaCl, 0.75g/L K2HPO4，0.75g/L KH2PO43g/L yeast powder, 0.5g/L MgCl2·6H2O，0.3g/L NH4Cl，0.015g/L CaCl2·2H2O，1.5g/L FeCl2·4H2O, 0.3g/L KCl, 60g/L xylan and water as a solvent, and adjusting the pH value to 7.5;
(2) inoculating activated high-temperature anaerobic bacteria M5 of the pyrolysis sugar into a fermentation culture medium with the inoculation amount of 5% v/v, fermenting at 55 ℃ and 120rpm for 60h, and adjusting the pH to 7.5 every 12h to obtain fermentation liquor;
the formula of the fermentation medium is as follows: 1g/L NaCl, 0.75g/L K2HPO4，0.75g/L KH2PO43g/L yeast powder, 0.5g/L MgCl2·6H2O,0.3g/L NH4Cl,0.3g/L KCl,0.015g/L CaCl2·2H2O，1.5g/L FeCl2·4H2O, introducing nitrogen for 10-20min, and sterilizing at 121 ℃ for 15min, wherein the solvent is water and the pH value is 7.5; setting three groups of fermentation culture media, wherein the concentrations of xylan are respectively 30g/L, 60g/L and 90 g/L;
(3) inoculating clostridium acetobutylicum into an activation culture medium of clostridium acetobutylicum with an inoculation amount of 5% v/v, activating at 37 ℃ for 60 hours at 150rpm, and adjusting the pH to 5.5 every 12 hours;
the formula of the activation medium is as follows: 1g/L NaCl, 0.75g/L K2HPO4，0.75g/L KH2PO43g/L yeast powder, 0.5g/L MgCl2·6H2O，0.3g/L NH4Cl，0.015g/L CaCl2·2H2O，1.5g/L FeCl2·4H2O, 0.3g/L KCl, 60g/L xylose and water as a solvent, and adjusting the pH value to 5.5;
(4) inoculating the activated clostridium acetobutylicum into the fermentation liquor obtained in the step (2) at the volume ratio of 10% v/v, fermenting at the temperature of 37 ℃ and the rpm of 150 for 132 hours, and adjusting the pH to 5.5 every 12 hours.
During the culture process, samples are taken every 12h to determine the butanol yield. When the concentration of xylan is 60g/L, the highest concentration of butanol obtained by co-culture reaches 2.61 g/L; when the concentration of xylan is 30g/L, the concentration of butanol obtained by co-culture is only 1.53 g/L; the butanol concentration at 90g/L xylan concentration was also slightly lower than 60g/L xylan, 2.37 g/L.
Example 3 Effect of different inoculation methods of Clostridium acetobutylicum ATCC 824 on butanol production
The method is the same as the example 2, except that the concentration of xylan in the fermentation medium is 60g/L, the activated acetone butanol clostridium culture solution in the step (3) is placed into a 4 ℃ centrifuge for centrifugation, the rotating speed is 6000rpm and 10min, then the supernatant is poured off, and the bacterial sludge is added into the fermentation liquor in the step (2), wherein the volume of the activated acetone butanol clostridium culture solution is the same as that of the fermentation liquor; then continuing culturing at 37 ℃, rotating at 150rpm, adjusting the pH to 5.5 every 12h, and fermenting for 132 h;
samples were taken every 12h during the culture and their butanol production was determined. When co-cultured in the inoculation mode of example 2, the butanol yield was only 2.61 g/L; when co-cultured in the inoculation mode of the embodiment, the yield of butanol reaches 8.34 g/L; therefore, the inoculation method of the present embodiment is more preferable.
Example 4 Effect of different inoculation times for Clostridium acetobutylicum on Final Butanol production
The method is the same as example 3, except that 4 sets of experiments are set in the step (2), and the fermentation time is 36h, 60h, 84h and 108h respectively.
Samples were taken every 12h during the culture and their butanol production was determined. When the saccharide pyrolysis thermophilic anaerobe M5 is cultured for 60h, the yield of butanol is the highest and reaches 8.34g/L when the clostridium acetobutylicum bacterial sludge is added, which is also the highest yield of butanol obtained by co-culture when xylan is used as a unique carbon source at present, and the figure is shown in figure 1 and figure 2. In the system, xylanase secreted by the strain M5 effectively degrades xylan and obtains about 18g/L xylose, and then in a co-culture system, the xylanase and the xylanase still have high enzyme activity and can continuously degrade xylan into xylose, and clostridium acetobutylicum can produce butanol by using the degraded xylose. The inoculation time is too early, the xylanase activity is low, the subsequent degradation of xylan is not utilized, and the late inoculation can result in too long culture time of the strain M5, the secretion of xylanase and xylanase is reduced, the enzyme activity of the previously secreted enzyme at 55 ℃ is reduced, so that the enzyme activity of an enzyme system is reduced, and the xylose obtained by partial degradation is also utilized by the strain M5, so that the carbon flow of clostridium acetobutylicum is reduced, and the yield of butanol is finally reduced.
Claims (3)
1. A method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolysis of sugar and clostridium acetobutylicum is characterized by comprising the following steps:
(1) inoculating activated high-temperature anaerobic bacteria of the pyrolysis sugar into a fermentation culture medium containing xylan for fermentation to obtain fermentation liquor;
the fermentation conditions in the step (1) are as follows: the fermentation temperature is 55 ℃, the fermentation time is 60h, the fermentation pH is 7.5, and the rotating speed is 120 rpm;
(2) inoculating activated clostridium acetobutylicum into the fermentation liquor obtained in the step (1), and fermenting to produce butanol;
the fermentation conditions in the step (2) are as follows: the fermentation temperature is 37 ℃, the fermentation time is 120h, the fermentation pH is 5.5, and the rotation speed is 150 rpm; the activated clostridium acetobutylicum was inoculated in the following manner: centrifuging the activated acetone-butanol clostridium culture solution, and inoculating the precipitate obtained by centrifugation into the fermentation liquor obtained in the step (1), wherein the volume of the activated acetone-butanol clostridium culture solution is the same as that of the fermentation liquor obtained in the step (1);
when the enzyme activity of xylanase in the fermentation liquor obtained in the step (1) reaches 0.5-0.6U/mL, inoculating activated clostridium acetobutylicum into the fermentation liquor obtained in the step (1) for fermentation to produce butanol;
the sugar pyrolysis high-temperature anaerobic bacteria classified and named in the step (1) are sugar pyrolysis high-temperature anaerobic bacteria (Thermoanaerobacterium thermosaccharolyticum), the strain number is M5, the strain number is preserved in China center for type culture collection, the preservation date is 2017, 2 and 27 days, and the preservation number is CCTCC NO: M2017072; the Clostridium acetobutylicum of the step (2) is classified and named as Clostridium acetobutylicum (Clostridium acetobutylicum) with the strain number of ATCC 824.
2. The method of claim 1, wherein the activated thermophilic anaerobe of step (1) is inoculated in an amount of 1-10% by volume of the fermentation medium.
3. The method according to claim 1, wherein the xylan-containing fermentation medium of step (1) is formulated as: 0.5-2.0g/L NaCl, 0.5-2.0g/L K2HPO4，0.5-2.0g/L KH2PO41.0-5.0g/L yeast powder, 0.2-1.0g/L MgCl2·6H2O，0.1-0.6g/L NH4Cl，0.01-0.05g/L CaCl2·2H2O，0.5-2.0g/L FeCl2·4H2O, 0.1-0.5g/L KCl, 30-90g/L xylan and water as solvent, and regulating pH to 6.0-6.5.
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| CN111269869B (en) * | 2020-02-10 | 2021-08-17 | 南京工业大学 | Construction method of recombinant clostridium acetobutylicum and application of recombinant clostridium acetobutylicum in preparation of butanol through semi-fiber fermentation |
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| CN106995790A (en) * | 2017-06-02 | 2017-08-01 | 南京工业大学 | One plant utilizes bacterial strain and its application that xylan is that sole carbon source directly produces butanol |
| CN109055441A (en) * | 2018-08-30 | 2018-12-21 | 江南大学 | A method of butanol is produced using Pichia pastoris solid-state castoff high-efficiency fermenting |
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| CN106995790A (en) * | 2017-06-02 | 2017-08-01 | 南京工业大学 | One plant utilizes bacterial strain and its application that xylan is that sole carbon source directly produces butanol |
| CN109055441A (en) * | 2018-08-30 | 2018-12-21 | 江南大学 | A method of butanol is produced using Pichia pastoris solid-state castoff high-efficiency fermenting |
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