CN110885859A - Method for producing acetic acid by fermentation - Google Patents
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- CN110885859A CN110885859A CN201911237907.9A CN201911237907A CN110885859A CN 110885859 A CN110885859 A CN 110885859A CN 201911237907 A CN201911237907 A CN 201911237907A CN 110885859 A CN110885859 A CN 110885859A
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- 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
- C12P7/54—Acetic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a method for producing acetic acid by fermentation, which relates to the technical field of microbial fermentation and comprises three steps of activation culture, seed culture and fermentation culture, wherein acetic acid is produced by mixed culture of two types of acetic acid bacilli, namely acetic acid bacilli Shanghai brewing 1.01 and acetic acid bacilli AS 1.41. The yield of acetic acid is obviously improved by adjusting the inoculation ratio of the two acetobacter; the conversion rate of the ethanol is improved by adjusting the culture and the proportion; the fermentation time is effectively shortened by controlling the fermentation conditions, and the production cost is reduced. In addition, compared with the traditional chemical synthesis method for producing acetic acid, the method is more environment-friendly.
Description
The technical field is as follows:
the invention relates to the technical field of microbial fermentation, in particular to a method for producing acetic acid by fermentation.
Background art:
acetic acid is an important organic acid, is used as a basic organic chemical raw material, is mainly used as a solvent in the production process of rubber, plastics, dyes and the like, is also used as a basic chemical raw material for Producing Terephthalic Acid (PTA), Vinyl Acetate Monomer (VAM), acetate fibers, acetate and the like, and has wide application in the aspects of food, medicines, pesticide chemical fibers and the like.
Acetic acid can be prepared by two methods, namely chemical synthesis and biological fermentation, and the production methods of acetic acid in the world mainly comprise a methanol oxo-synthesis method, an acetaldehyde oxidation method and a butane liquid-phase oxidation method, wherein the capacity of a methanol oxo-synthesis method device accounts for about 75 percent of the total capacity. The acetic acid production process routes in China include an alcohol method, an ethylene method, a methanol method and the like, the methods are basically chemical synthesis methods, most of the methods adopt fossil resources as raw materials, the fossil resources are non-renewable and limited in reserves, and the development of the acetic acid industry is seriously influenced by the shortage and the rise of the price of petroleum. In addition, the synthesis method for producing acetic acid has the problems of serious pollution, more byproducts, difficult waste treatment and the like. Compared with the chemical synthesis method, the biological brewing method for producing the acetic acid is better in environmental protection and can save resources. However, the acetic acid produced by the biological fermentation method only accounts for 10% of the yield of the whole world, and the acetic acid or vinegar produced by the fermentation method is a process of oxidizing ethanol into acetic acid under the action of acetic acid bacteria. However, food safety regulations in many countries dictate that vinegar in foods must be prepared by biological processes, and thus biological fermentation processes are mainly used for the production of vinegar.
The acetic acid bacteria capable of producing acetic acid by fermentation are mainly Acetobacter (A), (B) and (C)Acetobaeter)It breathes aerobically and oxidizes ethanol (alcohol) to acetic acid under aerobic, neutral and acidic conditions. Ethanol and lactate are good carbon sources, and the metabolite CO is present during fermentation2And H2Acetic acid, lactic acid, propionic acid, butyric acid, acetone and the like. Since the metabolites required for acetic acid fermentation are mainly acetic acid, acetic acid fermentation is generally performed using acetobacter. The most widely used acetic acid bacteria in China are Huniang 1.01 (Acetobacter pasteurianus) and Acetobacter AS1.41, and the strains can be better applied to the production of the traditional brewed edible vinegar in China. However, in the production process of industrial acetic acid, the growth and metabolism of common acetic acid bacteria are inhibited by alcohol and acetic acid with higher concentration, and the acetic acid bacteria are single and easy to generate peroxidation, so that the conversion rate of the alcohol is lower, and the fermentation period of the acetic acid is longer. Therefore, the development of acetic acid production by fermentation has been greatly limited.
In order to solve the technical problems, the invention provides a method for producing acetic acid by fermentation, the content of the acetic acid and the conversion rate of ethanol produced by the method are obviously improved, and compared with the traditional chemical synthesis method, the method is more environment-friendly.
The invention content is as follows:
the invention aims to provide a method for producing acetic acid by fermentation.
In one aspect, the present invention provides a method for producing acetic acid by fermentation, said method comprising the steps of:
(1) activation culture: respectively activating and culturing Acetobacter Shanghai brewing 1.01 and Acetobacter AS 1.41;
(2) seed culture: respectively inoculating the activated bacillus aceticus Huniang 1.01 and the activated bacillus aceticus AS1.41 in a seed culture medium for seed culture to obtain a bacillus aceticus Huniang 1.01 seed solution and a bacillus aceticus AS1.41 seed solution;
the seed culture medium comprises: 3-8g/L of glucose, 2-6g/L of peptone, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 1-3% (v/v) of acetic acid and 1-3% (v/v) of absolute ethyl alcohol, wherein the pH value is 5.0-5.5;
(3) fermentation culture: mixing the seed solution of the bacillus aceticus Shanghai brewing 1.01 and the seed solution of the bacillus aceticus AS1.41 obtained in the step (2), inoculating the mixture into a fermentation culture medium for culturing, wherein the inoculation amount is 10-20%, and finishing the fermentation when the acidity is not increased any more;
the volume ratio of the seed solution of the acetobacter Shanghai brewing 1.01 to the seed solution of the acetobacter AS1.41 is 0.5-3: 1;
the fermentation medium comprises: 3-8g/L of glucose, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 2-6% (v/v) of absolute ethyl alcohol, 0.3% (v/v) of antifoaming agent and 5.0-5.5 of pH.
Preferably, the seed culture conditions are 28-32 ℃, 150-.
Further preferably, the seed culture conditions are 30 ℃ and 160r/min shake flask culture for 20 h.
Preferably, the fermentation culture conditions are that the temperature is 28-32 ℃, the stirring speed is 180-240r/min, and the ventilation quantity is 180-220L/h.
More preferably, the fermentation culture conditions are 30 ℃, the stirring speed is 220r/min, and the ventilation volume is 200L/h.
Preferably, the volume ratio of the seed solution of the bacillus aceticus Huniang 1.01 to the seed solution of the bacillus aceticus AS1.41 in the step (3) is 1: 1.
Preferably, said seed medium comprises: 5g/L glucose, 4g/L peptone, 3g/L yeast extract, 1g/L pantothenic acid, 2% (v/v) acetic acid, 2% (v/v) absolute ethyl alcohol, and pH of 5.0-5.5.
Preferably, the fermentation medium comprises: 5g/L glucose, 3g/L yeast extract, 1g/L pantothenic acid, 4% (v/v) absolute ethyl alcohol, 0.3% (v/v) antifoaming agent, and 5.0-5.5 pH.
Specifically, the method for producing the acetic acid by fermentation comprises the following steps:
(1) activation culture: respectively inoculating Acetobacter Shanghai brewing 1.01 and Acetobacter AS1.41 onto activation culture medium, and performing activation culture in incubator at 30 deg.C for 20 hr to obtain activated bacteria;
the activation medium is as follows: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L, NaCl 2.5.5 g/L yeast extract and 20g/L agar powder.
(2) Seed culture: inoculating a ring of well-grown activated bacteria Acetobacter Shanghai 1.01 and Acetobacter AS1.41 into a shake flask containing a seed culture medium for culturing respectively, wherein the liquid loading amount is 200mL/500mL, the temperature is 28-32 ℃, the shake flask culture is carried out for 18-22h at 150-620The value reaches 1.2, and a seed solution of the bacillus aceti Shanghai brewing 1.01 and a seed solution of the bacillus aceti AS1.41 are obtained;
the seed culture medium comprises: 3-8g/L of glucose, 2-6g/L of peptone, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 1-3% (v/v) of acetic acid and 1-3% (v/v) of absolute ethyl alcohol, wherein the pH value is 5.0-5.5;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a 10L fermentation tank containing a fermentation culture medium according to the inoculation amount of 10-20%, wherein the volume ratio of the seed solution of the acetobacter Shanghai brewing 1.01 to the seed solution of the acetobacter AS1.41 is 0.5-3:1, the liquid loading amount is 6L/10L, the culture temperature is 28-32 ℃, the stirring speed is 180-;
the fermentation medium comprises: 3-8g/L of glucose, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 2-6% (v/v) of absolute ethyl alcohol, 0.3% (v/v) of antifoaming agent and 5.0-5.5 of pH.
The v/v of the invention refers to volume ratio, including milliliter/milliliter, liter/liter and the like.
The invention has the beneficial effects that:
the invention provides a method for producing acetic acid by fermentation, which utilizes two acetobacter to produce acetic acid by mixed fermentation, the content of the produced acetic acid is higher, ethanol can be effectively utilized, and the fermentation time is shortened; in addition, the conversion rate of ethanol can be obviously improved by adding a proper amount of pantothenic acid into the culture medium. Compared with the traditional chemical synthesis method for producing acetic acid, the microbial fermentation method provided by the invention is more environment-friendly.
Detailed Description
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easily understood, the invention is further explained with the following embodiments, but the following embodiments are only the preferred embodiments of the invention, and not all embodiments. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and the bacterial species, drugs, reagents and the like used in the following examples are commercially available without otherwise specified.
Example 1
A method of producing acetic acid by fermentation, comprising the steps of:
(1) activation culture: respectively inoculating Acetobacter Shanghai brewing 1.01 and Acetobacter AS1.41 onto activation culture medium, and performing activation culture in incubator at 30 deg.C for 20 hr to obtain activated bacteria;
the activation medium is as follows: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L, NaCl 2.5.5 g/L yeast extract and 20g/L agar powder.
(2) Seed culture: inoculating a ring of well-grown activated bacteria, Acetobacter Shanghai 1.01 and Acetobacter AS1.41 into shake flask containing seed culture medium respectively, culturing at 28 deg.C and 150r/min with liquid loading of 200mL/500mL for 22 hr until thallus OD620The value reaches 1.2, and a seed solution of the bacillus aceti Shanghai brewing 1.01 and a seed solution of the bacillus aceti AS1.41 are obtained;
the seed culture medium comprises: 3g/L glucose, 2g/L peptone, 1g/L yeast extract, 0.5g/L pantothenic acid, 1% (v/v) acetic acid, 1% (v/v) absolute ethyl alcohol, and pH of 5.0-5.5;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a 10L fermentation tank containing a fermentation culture medium according to the inoculation amount of 10%, wherein the volume ratio of the seed solution of the acetobacter Shanghai 1.01 to the seed solution of the acetobacter AS1.41 is 0.5:1, the liquid loading amount is 6L/10L, the culture temperature is 28 ℃, the stirring speed is 180r/min, the ventilation amount is 180L/h, and the fermentation is finished when the acidity is not increased any more;
the fermentation medium comprises: 3g/L of glucose, 1g/L of yeast extract, 0.5g/L of pantothenic acid, 2% (v/v) of absolute ethyl alcohol, 0.3% (v/v) of antifoaming agent and 5.0-5.5 of pH.
Example 2
A method of producing acetic acid by fermentation, comprising the steps of:
(1) activation culture: respectively inoculating Acetobacter Shanghai brewing 1.01 and Acetobacter AS1.41 onto activation culture medium, and performing activation culture in incubator at 30 deg.C for 20 hr to obtain activated bacteria;
the activation medium is as follows: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L, NaCl 2.5.5 g/L yeast extract and 20g/L agar powder.
(2) Seed culture: inoculating a ring of well-grown activated bacteria, Acetobacter Shanghai 1.01 and Acetobacter AS1.41 into shake flask containing seed culture medium respectively, culturing at 30 deg.C and 160r/min for 20 hr until thallus OD620The value reaches 1.2, and a seed solution of the bacillus aceti Shanghai brewing 1.01 and a seed solution of the bacillus aceti AS1.41 are obtained;
the seed culture medium comprises: 5g/L glucose, 4g/L peptone, 3g/L yeast extract, 1g/L pantothenic acid, 2% (v/v) acetic acid, 2% (v/v) absolute ethyl alcohol, and pH of 5.0-5.5;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a 10L fermentation tank containing a fermentation medium according to the inoculation amount of 15%, wherein the volume ratio of the seed solution of the bacillus aceticus Shanghai brewing 1.01 to the seed solution of the bacillus aceticus AS1.41 is 1:1, the liquid loading amount is 6L/10L, the culture temperature is 30 ℃, the stirring speed is 220r/min, the ventilation amount is 200L/h, and the fermentation is finished when the acidity is not increased any more;
the fermentation medium comprises: 5g/L glucose, 3g/L yeast extract, 1g/L pantothenic acid, 4% (v/v) absolute ethyl alcohol, 0.3% (v/v) antifoaming agent, and 5.0-5.5 pH.
Example 3
A method of producing acetic acid by fermentation, comprising the steps of:
(1) activation culture: respectively inoculating Acetobacter Shanghai brewing 1.01 and Acetobacter AS1.41 onto activation culture medium, and performing activation culture in incubator at 30 deg.C for 20 hr to obtain activated bacteria;
the activation medium is as follows: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L, NaCl 2.5.5 g/L yeast extract and 20g/L agar powder.
(2) Seed culture: inoculating a ring of well-grown activated bacteria, Acetobacter Shanghai 1.01 and Acetobacter AS1.41 into shake flask containing seed culture medium respectively, culturing at 32 deg.C and 180r/min with liquid loading of 200mL/500mL for 18 hr until thallus OD620The value reaches 1.2, and a seed solution of the bacillus aceti Shanghai brewing 1.01 and a seed solution of the bacillus aceti AS1.41 are obtained;
the seed culture medium comprises: 8g/L glucose, 6g/L peptone, 5g/L yeast extract, 1.5g/L pantothenic acid, 3% (v/v) acetic acid, 3% (v/v) absolute ethyl alcohol, and pH of 5.0-5.5;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a 10L fermentation tank containing a fermentation medium according to the inoculation amount of 20%, wherein the volume ratio of the seed solution of the bacillus aceticus Shanghai brewing 1.01 to the seed solution of the bacillus aceticus AS1.41 is 3:1, the liquid loading amount is 6L/10L, the culture temperature is 32 ℃, the stirring speed is 240r/min, the ventilation amount is 220L/h, and the fermentation is finished when the acidity is not increased any more;
the fermentation medium comprises: 8g/L glucose, 5g/L yeast extract, 1.5g/L pantothenic acid, 6% (v/v) absolute ethyl alcohol, 0.3% (v/v) antifoaming agent, and 5.0-5.5 pH.
Comparative example 1
A method for producing acetic acid by fermentation, which is different from example 2 only in that the volume ratio of the seed liquid of Acetobacter Shanghai brewing 1.01 to the seed liquid of Acetobacter AS1.41 in step (3) is 4: 1.
Comparative example 2
A method for producing acetic acid by fermentation, which is different from example 2 only in that acetic acid is produced by fermentation using Acetobacter Huniang 1.01, an acetic acid bacterium.
Comparative example 3
A method for producing acetic acid by fermentation, which is different from example 2 only in that acetic acid is produced by fermentation using Acetobacter AS1.41, an acetic acid bacterium.
Comparative example 4
A process for the fermentative production of acetic acid differs from example 2 only in that pantothenic acid is not added to the seed medium and the fermentation medium.
Comparative example 5
A method for producing acetic acid by fermentation, which is different from example 2 only in that the inoculation amount in the fermentation culture of step (3) is 5%.
Comparative example 6
A method for producing acetic acid by fermentation only differs from the method in the embodiment 2 in that the fermentation culture conditions in the step (3) are that the culture temperature is 26 ℃, the stirring speed is 150r/min, and the ventilation quantity is 150L/h.
The method for producing acetic acid by fermentation provided by the invention has the following effects:
the contents of acetic acid and alcohol before and after the fermentation culture of examples 1 to 3 and comparative examples 1 to 5 were measured by high performance liquid chromatography, and the time taken for fermentation production was counted to calculate the acetic acid content and ethanol conversion rate produced during the fermentation culture.
Table 1: effect of different fermentation methods on the content of acetic acid produced by fermentation
As can be seen from Table 1, the yield of acetic acid produced by fermentation by the method provided by the invention is obviously improved, the yield of acetic acid is above 57g/L, and especially the effect of producing acetic acid by the method described in example 2 is best.
Table 2: effect of different fermentation methods on ethanol conversion
As can be seen from Table 2, the method provided by the invention for producing acetic acid by fermentation can effectively utilize ethanol, and can obviously improve the conversion rate of ethanol by adding a proper amount of pantothenic acid in a culture set.
Table 3: effect of different fermentation methods on fermentation time
As can be seen from Table 3, the method provided by the invention can be used for producing acetic acid by fermentation, the fermentation time is within 40, and the fermentation time can be shortened under the appropriate fermentation conditions, so that the cost is reduced.
In conclusion, the acetic acid produced by the method disclosed by the invention is higher in content, ethanol can be effectively utilized, the fermentation time is shortened, and the production cost is reduced; in addition, compared with the traditional chemical synthesis method for producing acetic acid, the method is more environment-friendly.
The foregoing is a preferred embodiment of the present invention, and is not intended to limit the invention in any way, so that any simple modification and equivalent changes made to the above embodiment without departing from the technical spirit of the present invention should be considered as the protection scope of the present invention.
Claims (6)
1. A method for producing acetic acid by fermentation, said method comprising the steps of:
(1) activation culture: respectively activating and culturing Acetobacter Shanghai brewing 1.01 and Acetobacter AS 1.41;
(2) seed culture: respectively inoculating the activated bacillus aceticus Huniang 1.01 and the activated bacillus aceticus AS1.41 in a seed culture medium for seed culture to obtain a bacillus aceticus Huniang 1.01 seed solution and a bacillus aceticus AS1.41 seed solution;
the seed culture medium comprises: 3-8g/L of glucose, 2-6g/L of peptone, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 1-3% (v/v) of acetic acid and 1-3% (v/v) of absolute ethyl alcohol, wherein the pH value is 5.0-5.5;
(3) fermentation culture: mixing the seed solution of the bacillus aceticus Shanghai brewing 1.01 and the seed solution of the bacillus aceticus AS1.41 obtained in the step (2), inoculating the mixture into a fermentation culture medium for culturing, wherein the inoculation amount is 10-20%, and finishing the fermentation when the acidity is not increased any more;
the volume ratio of the seed solution of the acetobacter Shanghai brewing 1.01 to the seed solution of the acetobacter AS1.41 is 0.5-3: 1;
the fermentation medium comprises: 3-8g/L of glucose, 1-5g/L of yeast extract, 0.5-1.5g/L of pantothenic acid, 2-6% (v/v) of absolute ethyl alcohol, 0.3% (v/v) of antifoaming agent and 5.0-5.5 of pH.
2. The method as claimed in claim 1, wherein the seed culture conditions are a temperature of 28-32 ℃ and a shake flask culture at 150-180r/min for 18-22 h.
3. The method as claimed in claim 1, wherein the fermentation culture conditions are a temperature of 28-32 ℃, a stirring speed of 180-.
4. The method of claim 1, wherein the volume ratio of the seed liquid of Bacillus aceticus Huniang 1.01 to the seed liquid of Bacillus aceticus AS1.41 in step (3) is 1: 1.
5. The method of claim 1, wherein said seed medium comprises: 5g/L glucose, 4g/L peptone, 3g/L yeast extract, 1g/L pantothenic acid, 2% (v/v) acetic acid, 2% (v/v) absolute ethyl alcohol, and pH of 5.0-5.5.
6. The method of claim 1, wherein the fermentation medium comprises: 5g/L glucose, 3g/L yeast extract, 1g/L pantothenic acid, 4% (v/v) absolute ethyl alcohol, 0.3% (v/v) antifoaming agent, and 5.0-5.5 pH.
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| CN111748491A (en) * | 2020-06-15 | 2020-10-09 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN112626137A (en) * | 2021-01-15 | 2021-04-09 | 驻马店华中正大有限公司 | Method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum |
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