CN112626133A - CO (carbon monoxide)2Method for directionally producing succinic acid through biotransformation - Google Patents

CO (carbon monoxide)2Method for directionally producing succinic acid through biotransformation Download PDF

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CN112626133A
CN112626133A CN202011553748.6A CN202011553748A CN112626133A CN 112626133 A CN112626133 A CN 112626133A CN 202011553748 A CN202011553748 A CN 202011553748A CN 112626133 A CN112626133 A CN 112626133A
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succinic acid
acid
nitrate
biological fermentation
sodium
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CN112626133B (en
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王雯
赵晴
张燚
杨紫怡
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Beijing University of Chemical Technology
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Abstract

The invention discloses CO2The method for producing the succinic acid directionally by biotransformation comprises the following steps: preparing culture medium suitable for growth of pure succinic acid-producing strain, inoculating pure succinic acid-producing strain, and culturing to obtain product with index increasing periodSuccinic acid pure bacteria; treating a precursor of the active metal catalyst, and grinding to obtain solid catalyst particles; adding a substrate, an acid neutralizer and solid catalyst particles into a biological fermentation culture medium to obtain a mixed biological fermentation culture medium; inoculating pure succinic acid-producing bacteria to a mixed biological fermentation culture medium, and mixing uniformly under anaerobic condition to obtain converted CO2A biological fermentation system for producing succinic acid; introducing sufficient CO into the reactor2The production of succinic acid is carried out under anaerobic conditions. In the invention, the substrate, the acid neutralizer and the catalyst are coupled with pure bacteria fermentation to form an optimized biological fermentation system, and the optimized system can accelerate CO2Can stably and directionally produce high-concentration succinic acid.

Description

CO (carbon monoxide)2Method for directionally producing succinic acid through biotransformation
Technical Field
The invention relates to a method for producing succinic acid. More particularly, it relates to a CO2A method for directionally producing succinic acid by biotransformation.
Background
At present, the increasing population and the ever-expanding industrialization lead to a drastic increase in global energy consumption, resulting in a large amount of CO2And discharged to cause serious problems such as global temperature rise and glacier melting. Therefore, renewable energy sources and CO reduction are sought2The problems of emission and the like are needed to be solved. Compared with the conventional physical and chemical technology, the method for capturing and sealing carbon by the anaerobic microbial fermentation technology is an eco-friendly means, and effectively reduces CO in the atmosphere2While can react with CO2And performing resource utilization, and converting the obtained product into high value-added chemicals or fuels.
Succinic Acid (SA) is an important tetracarbon compound and has great potential not only for use as a precursor for various fine chemicals including 1, 4-butanediol, tetrahydrofuran, gamma-butyrolactone, tetrahydrofuran, N-methyl-2-pyrrolidone, succinimide, succinate, etc., but also in the manufacture of polybutylene succinate (PBS), surfactants and detergents, perfumes and flavors, herbicides and fungicides, and food additives. Due to its widespread industrial application, global demand for SA has grown from 3-5 million tons/year in 2014 to over 70 million tons/year in estimated market size in 2020. Various chemical techniques for the production of succinic acid have been developed, including paraffin oxidation, catalytic hydrogenation and electrolytic reduction of maleic acid or maleic anhydride. However, these methods consume non-renewable petrochemical resources and cause serious environmental problems. In recent years, the fermentation production of succinic acid from biomass, which is a renewable natural resource, has received increasing attention. Renewable biomass is used as a substrate and is directionally converted into succinic acid under mild fermentation conditions, so that higher conversion rate and fermentation efficiency can be realized. Compared with the petrochemical synthesis process, the production of the succinic acid by the biological fermentation has more cost benefit and environmental protection, and can delay the exhaustion of non-biological resources and other environmental problems. Currently, many fungi and bacteria that produce SA have been selected, including Saccharomyces cerevisiae, Yarrowia lipolytica, Byssochlamysnivea, Paecilomyces varioti, Actinobacillus succinogenes, Anerobacteriosis succinogenes, Mannheimia succinogenes, Escherichia coli, Corynebacterium glutamicum, Basfia succinogenes, and the like.
Research shows that in the process of producing succinic acid by biological fermentation, a substrate is converted into a main intermediate enolpyruvic acid (PEP) through enzymatic hydrolysis and glycolysis pathways, and CO is concentrated in high concentration2(1mol CO21mol of glucose) to gradually convert PEP to finally synthesize succinic acid; or at low concentrations of CO2(0.1mol CO21mol glucose) is converted to pyruvate by the reverse tricarboxylic acid (TCA) cycle, ultimately forming monoacids and monoalcohols (acetic acid, formic acid, lactic acid and ethanol), the key factor determining these 2 pathways is CO2The degree of availability of (c). But CO2The solubility in water is not high (1.13g/L,37 ℃), the gas-liquid mass transfer rate is slow, a large amount of byproducts (lactic acid, formic acid and acetic acid) are easy to accumulate in the biological fermentation process, the yield of the succinic acid is low, and CO is generated2Poor directional transformation effect and the like, which makes the realization of the succinic acid produced by biological fermentation to meet the requirements of industrial production difficult.
In view of the foregoing, it would be desirable to provide an optimized CO2The method for directionally producing the succinic acid by biotransformation improves the yield and the productivity of the succinic acid produced by the method.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an optimized CO2A method for directionally producing succinic acid by biotransformation. The method combines specific substrate species, acid neutralizer and catalyst with pure bacteria fermentation to form a specific biological fermentation system which can convert CO into CO2Directed conversion to high concentrationAnd accelerating CO under the reinforcement of specific substrate types, acid neutralizers and catalysts2The metabolic rate of the biological conversion into the succinic acid is increased and the selectivity of the biological conversion into the succinic acid is improved. The fermentation system can maintain stable and efficient operation for 2-200h, and the catalyst is not easy to inactivate in a reaction system and cause death of pure strains.
In order to solve the technical problems, the invention adopts the following technical scheme:
CO (carbon monoxide)2The method for producing the succinic acid directionally by biotransformation comprises the following steps:
s1, preparing a culture medium suitable for growth of the succinic acid-producing pure strain, inoculating the succinic acid-producing pure strain, and culturing to obtain the succinic acid-producing pure strain with the index increment period;
s2, processing the precursor of the active metal catalyst, and grinding to obtain solid catalyst particles;
s3, adding a substrate, an acid neutralizer and solid catalyst particles into the biological fermentation medium to obtain a mixed biological fermentation medium;
s4, inoculating the succinic acid-producing pure bacteria to a mixed biological fermentation culture medium, and mixing uniformly under an anaerobic condition to obtain converted CO2A biological fermentation system for producing succinic acid;
s5, introducing sufficient CO into a shaking bed reactor containing a biological fermentation system2The production of succinic acid is carried out under anaerobic conditions.
In the present invention, the term "exponential growth period" means that when a microorganism is cultured in a closed system (batch culture), according to the change of the growth rate and the specific growth rate of the microorganism, the specific growth rate of the microorganism reaches a maximum after the microorganism grows for a certain period, which is called exponential growth period, and if no factor inhibiting or limiting the growth of the microorganism exists in the exponential growth period, the microorganism grows at a constant maximum specific growth rate, and the number of cells increases exponentially.
In the present invention, the term "catalyst" means a catalyst capable of promoting CO2The solid particles that are biologically converted to succinic acid are a mixture and not a compound.
In the present invention, the term "biological fermentation system" means that the whole of a pure strain and a mixed biological fermentation medium in a reactor exists as "fermentation conditions", and is a mixture of a pure strain and a mixed biological fermentation medium in the form of the present invention.
Preferably, in step S1, the formula of the culture medium is that the following substances are contained in each liter of solution: 0.75g of monopotassium phosphate, 0.75g of dipotassium phosphate, 0.45g of 2-ethanesulfonic acid, 5g of yeast extract, 2.5g of corn steep liquor, 0.006g of sodium selenite, 1.008g of sodium chloride, 0.008g of sodium tungstate, 0.5g of sodium hydroxide, 1.5g of ferrous chloride tetrahydrate, 0.19g of cobalt chloride hexahydrate, 0.1g of manganese chloride tetrahydrate, 0.07g of zinc chloride, 0.006g of boric acid, 0.036g of sodium molybdate, 0.024g of nickel chloride hexahydrate, 0.002g of copper chloride dihydrate, 0.5g of magnesium chloride hexahydrate, 0.3g of ammonium chloride, 0.3g of potassium chloride, 0.015g of calcium chloride dihydrate, 10mg of azurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L of cysteine and 0.077g of DL-dithiol (+) -.
Preferably, in step S1, before inoculating the succinic acid-producing pure strain for culturing, the culture medium is steam-sterilized at 90-130 deg.C and 0.1-0.25MPa for 20-30 min.
Preferably, in step S1, the pH of the medium is 6.0-7.5.
Preferably, in step S1, the temperature of the culture medium is 30-45 ℃.
According to some embodiments of the present invention, in step S1, the succinic acid-producing pure strain may be selected from pure bacteria known to be capable of producing succinic acid; the components of the culture medium are basic nutrient solution and other components which can meet the requirements of growth of pure strains and production of succinic acid.
Preferably, in step S1, the succinic acid-producing pure strain is selected from strains sold by German Collection of microorganisms and American type culture Collection. Generally including bacteria, fungi and genetically engineered bacteria: succinogens FZ53, succinogens 130Z, succinogens njj 113, succinogens CGMCC1593, succinicoproducens ATCC53488, succinicoproducens ATCC29305, m. succinicoproducens MBEL55E, b. succinicoproducens JF4016, c. glutamiccum R, m. succinicoproducens PALKG, m. succinicoproducens LPK7(pMS3-fdh2 meq), e.coli AFP111, e.coli as1600a, e.coli beb, e.coli e2, e.coli hx, e.zea jji1208, e.coli afc 111, e.coli asgiva, pcc, e.coli bejc. glucicumicin, g. coli c 3, g. glucicumicin, g. coli c 3-g. gavacu, g. glu, g. coli c 31-g. coli, g. coli c 31, g. coli.
Preferably, in step S1, the amount v/v of the inoculated succinic acid-producing pure strain is 5-15%, and the time for reaching the exponential growth period is generally 6-36 h.
As a further improvement of the technical solution, in step S2, the processing includes the following steps:
s2-1, drying a precursor of the active metal catalyst at 80-200 ℃ to remove water to obtain a solid A;
s2-2, dissolving the auxiliary salt into the solvent, and stirring to uniformly dissolve the auxiliary salt to obtain an auxiliary salt solution B;
s2-3, dipping the solution B on the solid A powder, stirring, and drying to evaporate water to obtain a solid C;
s2-4, reducing the solid C into iron carbide, oxide or a mixture thereof under the condition of reducing gas, tabletting to 20-40 meshes, and activating at 200-600 ℃ to obtain a solid D;
s2-5, grinding the solid D to obtain solid catalyst particles.
Preferably, in step S2-1, the precursor of the active metal catalyst is selected from Fe2O3、Fe3O4、Co3O4、Al2O3、MoO3、SiO2One or more of (a).
Preferably, in step S2-2, the auxiliary salt comprises one or more of the following: trisodium citrate, tripotassium citrate, trilithium citrate, sodium nitrate, potassium nitrate, lithium nitrate, rubidium nitrate, magnesium nitrate, copper nitrate, zinc sulfate, zirconium sulfate, gallium sulfate, manganese acetate, zinc acetate, potassium permanganate, sodium permanganate, zirconium nitrate, ruthenium chloride, platinum nitrate, chloroplatinic acid, palladium nitrate, tungsten nitrate, gallium nitrate, manganese nitrate, sodium sulfate, potassium sulfate, lithium sulfate, rubidium sulfate, magnesium sulfate, copper sulfate; preferably, the promoter salt of the catalyst comprises one or more of the following: trisodium citrate, tripotassium citrate, trilithium citrate, sodium nitrate, potassium nitrate, lithium nitrate, rubidium nitrate, magnesium nitrate, copper nitrate, zinc sulfate, zirconium sulfate, gallium sulfate, manganese acetate, zinc acetate, potassium permanganate, sodium permanganate, zirconium nitrate, ruthenium chloride, platinum nitrate, chloroplatinic acid, and palladium nitrate.
Preferably, in step S2-2, the solvent includes one or more of methanol, ethanol, propanol, acetone, hexane, cyclohexane, cyclohexanone, diethyl ether, propylene oxide, water, and ethylene glycol.
Preferably, in step S2-3, the drying temperature is 6-200 ℃ and the drying time is 2-20 h.
Preferably, in step S2-4, the reducing gas is selected from one or more of the following: h2、CO、CO2、CH4Synthetic gas (H)2/CO)。
Preferably, in step S2-4, the gas velocity of the reducing gas is 20-100 mL/min.
Preferably, in step S2-4, the reduction time is 6-20 h.
Preferably, in step S2-5, the solid catalyst particles obtained by grinding have a particle size of 20-500 meshes.
As a further improvement of the technical scheme, in step S3, the method for preparing the mixed biological fermentation medium specifically comprises the following steps:
s3-1, preparing biological fermentation culture medium
The formula of the prepared biological fermentation culture medium is that each liter of solution contains the following substances: 10g yeast extract, 3g dipotassium hydrogen phosphate, 0.2g magnesium chloride, 0.2g calcium chloride, 1g sodium chloride;
s3-2, adding the substrate into a biological fermentation culture medium;
s3-3, adding an acid neutralizer into the fermentation medium, and regulating and controlling the pH value to be 6.8-7.2 in the fermentation process;
s3-4, adding the solid catalyst particles into the biological fermentation culture medium to obtain the mixed biological fermentation culture medium.
Preferably, in step S3-2, the substrate is selected from one or more of the following: glucose, xylose, sucrose, fructose, sorbitol, lactose, cane molasses, oil palm leaf juice, rapeseed straws, sulfite waste liquid, raw bean pods, cellulose waste, duckweed, industrial hemp, cane sugar residues, cassava bagasse residues, rapeseed meal and fresh cassava roots.
Preferably, in step S3-3, the acid neutralizer is selected from one or more of the following: potassium carbonate, magnesium carbonate, sodium carbonate, calcium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate. Preferably, the acid neutralizing agent of the fermentation medium comprises one or more of the following: magnesium carbonate, sodium carbonate, calcium carbonate, sodium bicarbonate, sodium hydroxide, magnesium hydroxide.
Preferably, in step S3-3, the concentration of the acid neutralizer is 5-100 g/L.
Preferably, in step S3-4, the concentration of the solid catalyst particles in the biological fermentation medium is 2000-100000 mg/L.
As a further improvement of the technical scheme, in the step S4, the mixing temperature is 30-45 ℃.
Preferably, in step S4, mixing is performed in a swing bed reactor with an oscillation rate of 150 + -50 rpm.
As a further improvement of the technical scheme, in step S5, the swing bed reactor is a fully mixed anaerobic reactor or a semi-mixed slurry bed reactor, and the oscillation rate of the swing bed reactor is 150 +/-50 rpm.
Preferably, in step S5, the succinic acid is produced at a temperature of 30-45 ℃ and a pH of 6.0-7.5.
Preferably, in step S5, the volume of the biological fermentation medium in the biological fermentation system is 2-5.
In step S5 of the present invention, the following reaction may occur during the production of succinic acid:
C6H12O6+ATP→G6P+ADP
G6P+H2O+2NADP+→RL5P+CO2+2NADPH
RL5P→X5P
RL5P→R5P
X5P+R5P→S7P+GAP
S7P+GAP→E4P+F6P
X5P+E4P→F6P+GAP
ATP→ADP+Pi
G6P→F6P
F6P+ATP→FBP+ADP
FBP→2GAP
GAP+ADP+NAD+→PEP+ATP+NADH+H2O
PEP+CO2+ADP→OAA+ATP
OAA+NADH→MAL+NAD+
MAL→FUM+H2O
FUM+NADH→Succinate+NAD+
PEP+ADP→PYR+ATP
PYR+CoA+NAD+→ACA+CO2+NADH
ACA+Pi→+CoA+Ac-P
Ac-P+ADP→Acetate+ATP
PYR+NADH→Lactate+NAD+
PYR+CoA→Formate+ACA
ACA+2NADH→Ethanol+CoA+2NAD+
in the above formula, the abbreviation and formal names correspond to the following:
G6P: glucose-6-phosphate;
ATP is adenosine triphosphate;
ADP is adenosine diphosphate;
NADP+nicotinamide adenine dinucleotide phosphate;
NADPH, reduced nicotinamide adenine dinucleotide phosphate;
RL 5P: ribulose-5-phosphate;
X5P: xylulose 5-phosphate;
R5P: 5-phosphoribosyl;
S7P Heptaphosphoheptate;
E4P erythrose 4-phosphate;
F6P fructose-6-phosphate;
FBP is fructose;
GAP: glyceraldehyde-3-phosphate;
PEP is phosphoenolpyruvate;
OAA oxaloacetate;
MAL: malic acid;
FUM: fumaric acid;
4, sodium salt: a succinate salt;
PYR: pyruvic acid;
CoA: coenzyme A;
ACA is acetyl coenzyme A;
Ac-P: acetyl phosphate ester;
acetate: acetic acid;
lactate: lactic acid;
formate: formic acid;
ethanol: and (3) ethanol.
Any range recited herein is intended to include the endpoints and any number between the endpoints and any subrange subsumed therein or defined therein.
The starting materials of the present invention are commercially available, unless otherwise specified, and the equipment used in the present invention may be any equipment conventionally used in the art or may be any equipment known in the art.
Compared with the prior art, the invention has the following beneficial effects：
1) At present, the technology for producing succinic acid by chemical catalysis generally needs high-temperature and high-pressure conditions, meanwhile, raw materials are not renewable and expensive, and CO is easily discharged in the production process2Causing environmental problems. The biological fermentation system has mild reaction conditions and simple equipment structure, and can effectively utilize and convert greenhouse gas CO2Produce chemical succinic acid with high added value and can be efficiently and stably transportedLine of
2) Compared with the current technology for producing succinic acid by biological fermentation, the method has the advantages that the yield of succinic acid is low and the selectivity is poor, so that the main speed-limiting step for producing the succinic acid by the biological fermentation industry is realized. In the mixed biological fermentation system designed by the invention, the optimization condition is controlled within a specified range, so that CO is generated2The oriented biological conversion is carried out to obtain the succinic acid with high yield and high selectivity.
3) The catalyst is used as a biological fermentation optimization condition, and can strengthen the way of producing the succinic acid by pure strains through glucose metabolism. The catalyst can promote the conversion of glucose, accelerate the conversion of glucose into oxaloacetic acid or fumaric acid and other intermediates, and simultaneously improve the CO content of the high-purity strain2The utilization rate of. In addition, the pure bacteria can be adsorbed on the surface of the catalyst, so that the propagation density of the pure bacteria is improved, attachment points are provided for the pure bacteria, and the stability of a specific biological fermentation system is improved.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
CO (carbon monoxide)2The method for directionally producing the succinic acid by biotransformation comprises the following specific steps:
1) 5mL of pure strain A. succinogenes 130Z solution was removed and inoculated into 100mL of growth medium sterilized by autoclaving at 121 ℃ for 20 min. The formula of the growth medium is as follows: 0.75g of monopotassium phosphate, 0.75g of dipotassium phosphate, 0.45g of 2-ethanesulfonic acid, 5g of yeast extract, 2.5g of corn steep liquor, 0.006g of sodium selenite, 1.008g of sodium chloride, 0.008g of sodium tungstate, 0.5g of sodium hydroxide, 1.5g of ferrous chloride tetrahydrate, 0.19g of cobalt chloride hexahydrate, 0.1g of manganese chloride tetrahydrate, 0.07g of zinc chloride, 0.006g of boric acid, 0.036g of sodium molybdate, 0.024g of nickel chloride hexahydrate, 0.002g of copper chloride dihydrate, 0.5g of magnesium chloride hexahydrate, 0.3g of ammonium chloride, 0.3g of potassium chloride, 0.015g of calcium chloride dihydrate, 10mg of azurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L of cysteine and 0.077g of DL-dithiol (+) -;
2) controlling the pH value of a pure strain growth culture medium to be 6.8, controlling the culture temperature to be 37 ℃, and culturing for 12h to reach the exponential growth period of A. succinogenes 130Z, so as to obtain the succinic acid-producing pure strain with the exponential growth period;
3) weighing an auxiliary agent chloroplatinic acid, dissolving the auxiliary agent chloroplatinic acid in ethylene glycol, and stirring to uniformly dissolve the auxiliary agent chloroplatinic acid;
4) weighing metal Fe2O3Powder, dripping the solution obtained in the step 1) into Fe2O3Dripping while stirring to fully mix;
5) after the material prepared in the step 4) is formed into a paste shape, sealing the paste by using a preservative film, and vacuumizing the paste for 1 hour in a glass drier;
6) transferring the material prepared in the step 5) to a drying box, and drying for 12h at 150 ℃ in the air atmosphere;
7) tabletting, grinding and screening the dried catalyst in the step 6), and utilizing H at 400 DEG C2Reducing the synthesis gas with the volume ratio of/CO of 1:1 for 12 hours to obtain 1 percent Pt/Fe2O3An inorganic solid catalyst;
8) adding 40000mg/L magnesium carbonate as pH neutralizer and 60000mg/L glucose as substrate into biological fermentation culture medium sterilized by high pressure steam at 121 deg.C for 20min, and introducing sufficient CO2And 5% of succinic acid A.succinogenes 130Z pure strain in exponential growth phase is inoculated; the formula of the biological fermentation culture medium is that each liter of solution contains the following substances: 10g yeast extract, 3g dipotassium hydrogen phosphate, 0.2g magnesium chloride, 0.2g calcium chloride, 1g sodium chloride; the pH value is 6.8-7.2;
9) 1% of Pt/Fe prepared in the step 7)2O3Grinding the inorganic solid catalyst to 40 meshes, and adding the ground inorganic solid catalyst into the fermentation medium prepared in the step 8) to form a mixed fermentation culture system;
10) reacting the mixed biological fermentation culture system in a shaking bed reactor, and reacting CO2And performing biotransformation to produce succinic acid.
In this embodiment, chloroplatinic acid and Fe used in steps 3) and 4) are2O3Quality of (1)The quantity ratio is 0.03: 1;
in step 7), the inorganic solid catalyst obtained after the reduction reaction contains Fe2O3、Fe3C、Fe5C2、Fe2C and Pt auxiliary agents;
in step 9), 1% Pt/Fe2O3The concentration of the inorganic solid catalyst in the reactor is 10000 mg/L;
in the step 10), the culture condition is that the temperature is 37 ℃ and the initial pH value is 7.2;
in the step 10), the oscillation rate of the shaking bed reactor is 150 rpm.
This example fermentation System combines CO2The results of the analysis of the liquid substance biologically converted to succinic acid are shown in table 1 below:
table 1: analysis result of liquid substance in reactor
Time h Glucose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 59961.61 0.00 0.00 0.00 0.00
4 52378.32 4866.56 372.88 58.15 441.78
6 47698.52 9891.5 958.22 138.91 778.41
12 26802.9 15450.18 1447.42 250.91 917.92
18 19675.05 23188.42 1888.15 340.65 1110.57
24 11772.14 32250.43 2621.89 407.54 1412.91
36 6101.85 41269.32 3033.50 430 1830.47
48 0.00 53871.89 3405.93 447.6 2301.9
In this example, the catalyst added to the mixed biofermentation system was 1% Pt/Fe2O3The reaction completely consumes 59961.61mg/L glucose within 48h, and the reaction rate is faster. In the fermentation process, the main product is succinic acid, and other byproducts are monoacids including lactic acid, formic acid and lactic acid. The results in the table show that the final yield of succinic acid is 53871.89mg/L, the succinic acid accounts for 90.13% of the total carbon of the product, and compared with the existing biological fermentation system (the selectivity of succinic acid: 45-60%), the mixed biological fermentation system can directionally produce the main product succinic acid with high yield, and the production rate and the selectivity are higher.
Example 2
CO (carbon monoxide)2The method for directionally producing the succinic acid by biotransformation comprises the following specific steps:
1) 5mL of pure strain A. succinogenes 130Z solution was removed and inoculated into 100mL of growth medium sterilized by autoclaving at 121 ℃ for 20 min. The formula of the growth medium is as follows: 0.75g of monopotassium phosphate, 0.75g of dipotassium phosphate, 0.45g of 2-ethanesulfonic acid, 5g of yeast extract, 2.5g of corn steep liquor, 0.006g of sodium selenite, 1.008g of sodium chloride, 0.008g of sodium tungstate, 0.5g of sodium hydroxide, 1.5g of ferrous chloride tetrahydrate, 0.19g of cobalt chloride hexahydrate, 0.1g of manganese chloride tetrahydrate, 0.07g of zinc chloride, 0.006g of boric acid, 0.036g of sodium molybdate, 0.024g of nickel chloride hexahydrate, 0.002g of copper chloride dihydrate, 0.5g of magnesium chloride hexahydrate, 0.3g of ammonium chloride, 0.3g of potassium chloride, 0.015g of calcium chloride dihydrate, 10mg of azurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L of cysteine and 0.077g of DL-dithiol (+) -;
2) controlling the pH value of a pure strain growth culture medium to be 6.8, controlling the culture temperature to be 37 ℃, and culturing for 12h to reach the exponential growth period of A. succinogenes 130Z, so as to obtain the succinic acid-producing pure strain with the exponential growth period;
3) weighing an auxiliary agent chloroplatinic acid, dissolving the auxiliary agent chloroplatinic acid in ethylene glycol, and stirring to uniformly dissolve the auxiliary agent chloroplatinic acid;
4) weighing metal Fe2O3Powder, dripping the solution obtained in the step 1) into Fe2O3Dripping while stirring to fully mix;
5) after the material prepared in the step 4) is formed into a paste shape, sealing the paste by using a preservative film, and vacuumizing the paste for 1 hour in a glass drier;
6) transferring the material prepared in the step 5) to a drying box, and drying for 12h at 150 ℃ in the air atmosphere;
7) tabletting, grinding and screening the dried catalyst in the step 6), and utilizing H at 400 DEG C2Reducing the synthesis gas with the volume ratio of/CO of 1:1 for 12 hours to obtain 1 percent Pt/Fe2O3An inorganic solid catalyst;
8) adding 40000mg/L sodium carbonate as pH neutralizer and 60000mg/L glucose as substrate into biological fermentation culture medium sterilized by high pressure steam at 121 deg.C for 20min, and introducing sufficient CO2And 5% of succinic acid A.succinogenes 130Z pure strain in exponential growth phase is inoculated; the formula of the biological fermentation culture medium is that each liter of solution contains the following substances: 10g of yeast extract, 3g of dipotassium hydrogen phosphate, 0.2g of magnesium chloride, 0.2g of calcium chloride,1g of sodium chloride; the pH value is 6.8-7.2;
9) 1% of Pt/Fe prepared in the step 7)2O3Grinding the inorganic solid catalyst to 40 meshes, and adding the ground inorganic solid catalyst into the fermentation medium prepared in the step 8) to form a mixed fermentation culture system;
10) reacting the mixed biological fermentation culture system in a shaking bed reactor, and reacting CO2And performing biotransformation to produce succinic acid.
In this embodiment, chloroplatinic acid and Fe used in steps 3) and 4) are2O3The mass ratio of (A) to (B) is 0.03: 1;
in step 7), the inorganic solid catalyst obtained after the reduction reaction contains Fe2O3、Fe3C、Fe5C2、Fe2C and Pt auxiliary agents;
in step 9), 1% Pt/Fe2O3The concentration of the inorganic solid catalyst in the reactor is 10000 mg/L;
in the step 10), the culture condition is that the temperature is 37 ℃ and the initial pH value is 7.2;
in the step 10), the oscillation rate of the shaking bed reactor is 150 rpm.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 2 below:
table 2: analysis result of liquid substance in reactor
Figure BDA0002857900960000101
Figure BDA0002857900960000111
In this example, the acid neutralizer added to the mixed biological fermentation system was sodium carbonate, and 59761.45mg/L of glucose was completely consumed in 60 hours of the reaction. During the fermentation, the yield of the main product succinic acid was 46871.89mg/L, and the selectivity in the total product was 73.65%.
Example 3
Example 2 was repeated except that in step 6), the acid neutralizing agent in the mixed biological fermentation system was calcium carbonate.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 3 below:
table 3: analysis result of liquid substance in reactor
Time h Glucose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 59982.09 0.00 0.00 0.00 0.00
4 52855.52 1521.68 234.65 358.15 481.81
6 482541.34 13478.27 271.73 1216.12 1487.28
12 343885.07 22902.49 865.19 2651.67 5290.05
18 264550.41 23453.86 4598.52 3705.1 6040.21
24 188753.7 35024.81 4821.61 4236.74 6028.78
36 9983.71 38323.20 5692.77 5200.2 6720.54
48 10547.9 39776.54 7254.56 6313.41 7631.26
60 6547.8 40578.43 8902.56 7954.8 8457.9
72 0.00 42861.49 9595.93 8547.6 9851.9
In this example, the acid neutralizing agent added to the mixed biological fermentation system was calcium carbonate, and 59982.09mg/L of glucose was completely consumed in 72 hours of the reaction. During the fermentation, the yield of the main product succinic acid is 42861.49mg/L, and the selectivity in the total product is 63.53%.
Example 4
Example 1 was repeated, except that in step 6), the concentration of magnesium carbonate as an acid neutralizer added to the mixed biological fermentation system was 10000 mg/L.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 4 below:
table 4: analysis result of liquid substance in reactor
Time h Glucose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 59695.4 0.00 0.00 0.00 0.00
4 50308.58 2215 543.42 158.15 433.51
6 42844.9 5441.78 1469.98 472.84 1011.82
12 39315.25 12597.77 2482.51 550.91 1545.5
18 28768.66 24682.93 3817.38 583.74 1790.84
24 15458.15 38780.42 5328.43 603.11 2958.28
36 8790.96 42065.54 6479 771.84 3924.75
48 199.63 44353.81 7095.25 865.88 4290.46
60 0.00 46971.89 8695.93 947.6 4351.9
In this example, the concentration of magnesium carbonate, which is an acid neutralizing agent added to the mixed biological fermentation system, was 10000mg/L, and 59695.4mg/L of glucose was completely consumed within 60 hours of the reaction. The yield of the main product succinic acid during fermentation was 46971.89mg/L, and the selectivity in the total product was 77.76%.
Example 5
Example 1 was repeated, except that in step 6), the concentration of magnesium carbonate as an acid neutralizing agent added to the mixed biological fermentation system was 60000 mg/L.
This example fermentation System combines CO2The results of the analysis of the liquid substance biologically converted to succinic acid are shown in table 5 below:
table 5: analysis result of liquid substance in reactor
Figure BDA0002857900960000121
Figure BDA0002857900960000131
In this example, the concentration of magnesium carbonate, which is an acid neutralizing agent added to the mixed biological fermentation system, was 60000mg/L, and 59955.4mg/L of glucose was completely consumed within 60 hours of the reaction. During the fermentation, the yield of the main product succinic acid is 49871.89mg/L, and the selectivity in the total product is 83.99%.
Example 6
Example 1 was repeated, with the difference that, in step 6), the substrate added to the mixed biofermentation system was fructose 60000 mg/L.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 6 below:
table 6: analysis result of liquid substance in reactor
Time h Fructose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 60055.40 0.00 0.00 0.00 0.00
4 56354.58 2367 443.65 358.25 573.56
6 52284.95 5421.78 1165.25 702.24 807.89
12 49115.25 8575.82 1574.55 1057.54 912.55
18 42758.56 10689.72 2918.98 1359.77 1147.85
24 39488.17 15756.89 4358.63 1423.74 1588.28
36 34490.56 19455.94 6459.24 1721.84 2974.75
48 27999.83 24475.81 7055.25 2485.58 39020.46
60 22478.35 29851.89 8665.93 2957.6 4356.9
72 16475.2 31048.7 9145.7 3578.4 5871.4
84 12785.8 33578.9 10874.6 3874.5 6547.2
96 5524.5 36458.2 11478.2 4027.8 7984.3
108 0.00 38671.89 12695.93 4287.3 8351.9
In the example, the substrate added in the mixed biological fermentation system is fructose, and 60055.40mg/L of fructose is completely consumed in 108 h. During the fermentation, the yield of the main product succinic acid is 38671.89mg/L, and the selectivity in the total product is 62.23%.
Example 7
Example 1 was repeated, with the difference that in step 6), the substrate added to the mixed biofermentation system was 60000mg/L xylose.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted to succinic acid are shown in table 7 below:
table 7: analysis result of liquid substance in reactor
Time h Xylose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 59994.7 0.00 0.00 0.00 0.00
4 55348.58 3255 443.22 0.00 538.51
6 49884.9 5478.78 1469.98 0.00 911.89
12 37314.15 9597.98 2582.51 0.00 1447.5
18 30758.46 12684.93 3811.88 0.00 2790.85
24 25454.25 18730.52 4588.43 0.00 3955.28
36 18790.96 25071.54 5769 0.00 5424.78
48 12579.62 31347.61 6415.25 0.00 3890.46
60 8742.3 36951.87 7895.43 0.00 7351.9
72 4286.1 40451.3 86712.3 0.00 80745.2
84 0.00 41691.89 8999.93 0.00 8964.9
In this example, the substrate added to the mixed biofermentation system was xylose, and the reaction was completed consuming 59994.7mg/L of xylose in 84 h. The yield of the main product succinic acid during fermentation was 41691.89mg/L, and the selectivity in the total product was 70.24%.
Example 8
Example 1 was repeated, with the difference that in step 2), the metal of the catalyst added to the mixed biofermentation system was Al2O3And (3) powder.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 8 below:
table 8: analysis result of liquid substance in reactor
Figure BDA0002857900960000141
Figure BDA0002857900960000151
In this example, the metal used in the catalyst added to the mixed biological fermentation system was Al2O3Powder, reaction completely consumed 59997.13mg/L glucose within 48 h. During the fermentation, the yield of the main product succinic acid was 48671.89mg/L, and the selectivity in the total product was 76.21%.
Example 9
Example 1 was repeated, with the difference that in step 1), the promoter of the catalyst added to the mixed biofermentation system was palladium nitrate.
This example fermentation System combines CO2The results of the analysis of the liquid substance bioconverted into succinic acid are shown in table 9 below:
table 9: analysis result of liquid substance in reactor
Time h Glucose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 58997.9 0.00 0.00 0.00 0.00
4 50308.58 5215 443.42 258.18 233.51
6 42844.9 10441.28 969.58 402.44 811.84
12 39315.25 20547.72 1447.51 530.51 1045.7
18 28768.66 28622.95 2816.35 683.77 1490.74
24 15458.15 35740.45 3328.53 803.15 1658.58
36 8790.96 45075.54 4459 1171.87 2024.25
48 0.00 50691.89 5695.93 1287.3 2357.9
In the embodiment, the assistant used by the catalyst added in the mixed biological fermentation system is palladium nitrate, and 58997.9mg/L of glucose is completely consumed in 48 hours of reaction. During the fermentation, the yield of the main product succinic acid is 46971.89mg/L, and the selectivity in the total product is 85.30%.
Comparative example 1
Example 1 was repeated, with the difference that no acid neutralizer was added to the biological fermentation system in step 6).
The comparative example biological fermentation system uses CO2The results of the analysis of the liquid substance bioconverted to succinic acid are shown in table 10 below:
table 10: analysis result of liquid substance in reactor
Time h Glucose mg/L Succinic acid mg/L Lactic acid mg/L Formic acid mg/L Acetic acid mg/L
0 59982.09 0.00 0.00 0.00 0.00
6 51468.24 37.65 325.99 858.15 410.16
12 43854.52 3222.61 1628.2 1844.13 1984.21
18 38291.34 13824.06 10385.69 2977.31 2068.18
24 26786.07 15114.53 12262.28 4119.62 3674.99
36 16530.41 14899.97 13121.46 5077.05 4719.78
48 12325.56 16219.71 14933.69 6199.46 6384.28
60 8703.4 23896.88 16473.27 7033.18 7281.97
72 0.00 29671.89 18355.93 8647 8351.9
In this comparative example, 59982.09mg/L of glucose was consumed up to 72 hours without addition of the acid neutralizing agent. Compared to examples 1,4, 5, the final main product succinic acid concentration was not high (29671.89mg/L) with lower selectivity in the total product: 48.26 percent.
Comparative example 2
Example 1 was repeated, with the difference that, in step 7), no prepared solid catalyst was added to the biological fermentation system.
The comparative example biological fermentation system uses CO2The results of the analysis of the liquid substance biologically converted to succinic acid are shown in table 1 below:
table 11: analysis result of liquid substance in reactor
Figure BDA0002857900960000161
Figure BDA0002857900960000171
In the comparative example, 59997.43mg/L of glucose was consumed in 116h without adding a solid catalyst in the biological fermentation system. Compared with examples 8 and 9, the concentration of the final main product succinic acid is greatly reduced (34671.89mg/L), and the selectivity of the final main product succinic acid in the total product (58.85%) is also lower than 60%.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.

Claims (10)

1. CO (carbon monoxide)2The method for directionally producing the succinic acid by biotransformation is characterized by comprising the following steps of:
s1, preparing a culture medium suitable for growth of the succinic acid-producing pure strain, inoculating the succinic acid-producing pure strain, and culturing to obtain the succinic acid-producing pure strain with the index increment period;
s2, processing the precursor of the active metal catalyst, and grinding to obtain solid catalyst particles;
s3, adding a substrate, an acid neutralizer and solid catalyst particles into the biological fermentation medium to obtain a mixed biological fermentation medium;
s4, inoculating the succinic acid-producing pure bacteria to a mixed biological fermentation culture medium, and mixing uniformly under an anaerobic condition to obtain converted CO2A biological fermentation system for producing succinic acid;
s5, introducing sufficient CO into a shaking bed reactor containing a biological fermentation system2The production of succinic acid is carried out under anaerobic conditions.
2. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S1, the formula of the culture medium is that each liter of solution contains the following substances: 0.75g of monopotassium phosphate, 0.75g of dipotassium phosphate, 0.45g of 2-ethanesulfonic acid, 5g of yeast extract, 2.5g of corn steep liquor, 0.006g of sodium selenite, 1.008g of sodium chloride, 0.008g of sodium tungstate, 0.5g of hydrogenSodium oxide, 1.5g ferrous chloride tetrahydrate, 0.19g cobalt chloride hexahydrate, 0.1g manganese chloride tetrahydrate, 0.07g zinc chloride, 0.006g boric acid, 0.036g sodium molybdate, 0.024g nickel chloride hexahydrate, 0.002g copper chloride dihydrate, 0.5g magnesium chloride hexahydrate, 0.3 ammonium chloride, 0.3g potassium chloride, 0.015g calcium chloride dihydrate, 10mg resazurin (oxygen indicator), 0.015g sodium sulfide, 0.024g L (+) -cysteine and 0.077g DL-dithiothreitol.
3. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S1, before inoculating the succinic acid-producing pure strain for culture, steam sterilizing the culture medium at 90-130 deg.C and 0.1-0.25MPa for 20-30 min.
4. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S1, the pH of the culture medium is 6.0-7.5;
preferably, in step S1, the temperature of the culture medium is 30-45 ℃.
5. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S1, the succinic acid-producing pure strain is selected from strains sold by German culture collection center and American type culture collection center; including bacteria, fungi and genetically engineered bacteria: succinogens FZ53, succinogens 130Z, succinogens NJ113, succinogens CGMCC1593, succinicoproducens ATCC53488, succinicoproducens ATCC29305, M.succinicoproducens MBEL55E, B.succinicoproducens JF4016, C.glutamiccum R, M.succinicoproducens PALKG, M.succinicoproducens LPK7(pMS3-fdh2 meq), E.coli P111, E.coli AS1600a, E.coli BEP, E.coli E2, E.coli HX024, E.coli JJV 1208, E.coli JV 1208, E.coli C1, BO C1. coli C1-G23, BO C1. coli C23, BO.sucuci C1. coli C1. glutamicumC 1. G, G23, C.sucuci C1. coli C1PGC010037942；
Preferably, in step S1, the amount v/v of the inoculated succinic acid-producing pure strain is 5-15%, and the time for reaching the exponential growth period is generally 6-36 h.
6. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S2, the process includes the steps of:
s2-1, drying a precursor of the active metal catalyst at 80-200 ℃ to remove water to obtain a solid A;
s2-2, dissolving the auxiliary salt into the solvent, and stirring to uniformly dissolve the auxiliary salt to obtain an auxiliary salt solution B;
s2-3, dipping the solution B on the solid A powder, stirring, and drying to evaporate water to obtain a solid C;
s2-4, reducing the solid C into iron carbide, oxide or a mixture thereof under the condition of reducing gas, tabletting to 20-40 meshes, and activating at 200-600 ℃ to obtain a solid D;
s2-5, grinding the solid D to obtain solid catalyst particles;
preferably, in step S2-1, the precursor of the active metal catalyst is selected from Fe2O3、Fe3O4、Co3O4、Al2O3、MoO3、SiO2One or more of (a);
preferably, in step S2-2, the auxiliary salt comprises one or more of the following: trisodium citrate, tripotassium citrate, trilithium citrate, sodium nitrate, potassium nitrate, lithium nitrate, rubidium nitrate, magnesium nitrate, copper nitrate, zinc sulfate, zirconium sulfate, gallium sulfate, manganese acetate, zinc acetate, potassium permanganate, sodium permanganate, zirconium nitrate, ruthenium chloride, platinum nitrate, chloroplatinic acid, palladium nitrate, tungsten nitrate, gallium nitrate, manganese nitrate, sodium sulfate, potassium sulfate, lithium sulfate, rubidium sulfate, magnesium sulfate, copper sulfate; preferably, the promoter salt of the catalyst comprises one or more of the following: trisodium citrate, tripotassium citrate, trilithium citrate, sodium nitrate, potassium nitrate, lithium nitrate, rubidium nitrate, magnesium nitrate, copper nitrate, zinc sulfate, zirconium sulfate, gallium sulfate, manganese acetate, zinc acetate, potassium permanganate, sodium permanganate, zirconium nitrate, ruthenium chloride, platinum nitrate, chloroplatinic acid, palladium nitrate;
preferably, in step S2-2, the solvent includes one or more of methanol, ethanol, propanol, acetone, hexane, cyclohexane, cyclohexanone, diethyl ether, propylene oxide, water, ethylene glycol;
preferably, in the step S2-3, the drying temperature is 6-200 ℃, and the drying time is 2-20 h;
preferably, in step S2-4, the reducing gas is selected from one or more of the following: h2、CO、CO2、CH4And a synthesis gas;
preferably, in the step S2-4, the gas velocity of the reducing gas is 20-100 mL/min;
preferably, in the step S2-4, the reduction time is 6-20 h;
preferably, in step S2-5, the solid catalyst particles obtained by grinding have a particle size of 20-500 meshes.
7. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S3, the method for preparing the mixed biological fermentation medium specifically includes the following steps:
s3-1, preparing biological fermentation culture medium
The formula of the prepared biological fermentation culture medium is that each liter of solution contains the following substances: 10g yeast extract, 3g dipotassium hydrogen phosphate, 0.2g magnesium chloride, 0.2g calcium chloride, 1g sodium chloride;
s3-2, adding the substrate into a biological fermentation culture medium;
s3-3, adding an acid neutralizer into the fermentation medium, and regulating and controlling the pH value to be 6.8-7.2 in the fermentation process;
s3-4, adding the solid catalyst particles into the biological fermentation culture medium to obtain the mixed biological fermentation culture medium.
8. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S3-2, the substrate is selected from one or more of the following: glucose, xylose, sucrose, fructose, sorbitol, lactose, cane molasses, oil palm leaf juice, rapeseed straws, sulfite waste liquid, raw bean pods, cellulose waste, duckweed, industrial hemp, cane sugar residues, cassava bagasse residues, rapeseed meal and fresh cassava roots;
preferably, in step S3-3, the acid neutralizer is selected from one or more of the following: potassium carbonate, magnesium carbonate, sodium carbonate, calcium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate. Preferably, the acid neutralizing agent of the fermentation medium comprises one or more of the following: magnesium carbonate, sodium carbonate, calcium carbonate, sodium bicarbonate, sodium hydroxide, magnesium hydroxide;
preferably, in the step S3-3, the concentration of the acid neutralizer is 5-100 g/L;
preferably, in step S3-4, the concentration of the solid catalyst particles in the biological fermentation medium is 2000-100000 mg/L.
9. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S4, the mixing temperature is 30-45 ℃;
preferably, in step S4, mixing is performed in a swing bed reactor with an oscillation rate of 150 + -50 rpm.
10. CO according to claim 12The method for directionally producing the succinic acid by biotransformation is characterized in that: in step S5, the swing bed reactor is a fully-mixed anaerobic reactor or a semi-mixed slurry bed reactor, and the oscillation rate of the swing bed reactor is 150 +/-50 rpm;
preferably, in step S5, the production temperature of the succinic acid is 30-45 ℃, and the pH is 6.0-7.5;
preferably, in step S5, the volume of the biological fermentation medium in the biological fermentation system is 2-5.
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CA2654656A1 (en) * 2006-06-30 2008-01-03 Biogasol Ipr Aps Production of fermentation products in biofilm reactors using microorganisms immobilised on sterilised granular sludge
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