CN112626133B - CO (carbon monoxide) 2 Method for directionally producing succinic acid by bioconversion - Google Patents
CO (carbon monoxide) 2 Method for directionally producing succinic acid by bioconversion Download PDFInfo
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Abstract
The invention discloses a CO 2 The method for directionally producing the succinic acid by bioconversion comprises the following steps: preparing a culture medium suitable for growth of pure strain producing succinic acid, inoculating the pure strain producing succinic acid for culturing to obtain pure strain producing succinic acid in an exponential growth phase; treating the 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 medium to obtain a mixed biological fermentation medium; inoculating pure bacteria producing succinic acid into a mixed biological fermentation culture medium, and uniformly mixing under anaerobic condition to obtain converted CO 2 A bio-fermentation system for producing succinic acid; sufficient CO is introduced into the reactor 2 The production of succinic acid is performed under anaerobic conditions. The invention couples the substrate, the acid neutralizer and the catalyst with the pure bacteria fermentation to form an optimized biological fermentation system, and the optimized system can accelerate CO 2 Can stably and directionally produce high-concentration succinic acid.
Description
Technical Field
The invention relates to a method for producing succinic acid. And more particularly to a CO 2 A method for directionally producing succinic acid by bioconversion.
Background
Currently, the ever-increasing population and the ever-expanding industrialization lead to a dramatic increase in global energy consumption, with a concomitant rise in CO-production 2 Emissions, causing serious problems such as global temperature rise and glacier melting. Thus, renewable energy sources and CO reduction are sought 2 The problems of discharge and the like are to be solved. Compared with the conventional physicochemical technology, the carbon capture and sequestration by the anaerobic microorganism fermentation technology is an eco-friendly means, and is effectiveReducing CO in the atmosphere 2 Can be used for CO at the same time 2 And the method is used for recycling and converting the waste into high-added-value chemicals or fuels.
Succinic acid (abbreviated as SA) is an important four-carbon compound and can be used as a precursor of various fine chemicals, including 1, 4-butanediol, tetrahydrofuran, gamma-butyrolactone, tetrahydrofuran, N-methyl-2-pyrrolidone, succinimide, succinate and the like, and has great potential in the production of polybutylene succinate (abbreviated as PBS), surfactants and detergents, fragrances and perfumes, herbicides and fungicides, food additives and the like. Because of its wide industrial application, global demand for SA has grown from 3-5 ten thousand tons per year in 2014 to an estimated market size of over 70 ten thousand tons per year in 2020. Various chemical techniques for producing succinic acid have been developed, including paraffin oxidation, catalytic hydrogenation, and electrolytic reduction of maleic acid or anhydride. However, these methods consume non-renewable petrochemical resources and present serious environmental problems. In recent years, the fermentative production of succinic acid from renewable natural resources biomass has received increasing attention. The renewable biomass is used as a substrate, and is directionally converted into the succinic acid under mild fermentation conditions, so that higher conversion rate and fermentation efficiency can be realized. Compared with petrochemical synthesis technology, the bio-fermentation production of succinic acid is more cost-effective and more environment-friendly, and can delay the exhaustion of non-biological resources and other environmental problems. At present, many fungi and bacteria have been selected for SA production, including Saccharomyces cerevisiae, yarrowia lipolytica, byssochlamys nivea, paecilomyces varioti, actinobacillus succinogenes, anaerobiospirillum succiniciproducens, mannheimia succiniciproducens, escherichia coli, corynebacterium glutamicum, basfia succiniciproducens, etc.
It was found that during the production of succinic acid by biological fermentation, the substrate is converted into the main intermediate enolpyruvic acid (PEP) by enzymatic hydrolysis and glycolysis, and is converted into a high concentration of CO 2 (1mol CO 2 1mol glucose), gradually converting PEP into final synthetic succinic acid; or at low concentration of CO 2 (0.1mol CO 2 PEP is converted to pyruvic acid by reverse tricarboxylic acid (TCA) cycle under 1mol glucose conditions, and monoacids and monoalcohols (acetic acid, formic acid, lactic acid and ethanol) are finally formed, and the key factor determining these 2 pathways is CO 2 Is used for the availability of the product. But CO 2 The solubility in water is not high (1.13 g/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, so that the succinic acid yield is low, and CO is generated 2 Poor directional conversion effect, and the like, which makes the realization of the production of succinic acid by biological fermentation difficult to meet the requirements of industrial production.
In view of the foregoing, there is a need to provide an optimized CO 2 The method for directionally producing the succinic acid by bioconversion improves the yield and the productivity of the succinic acid.
Disclosure of Invention
The invention aims to provide a method for optimizing CO 2 A method for directionally producing succinic acid by bioconversion. The method couples a specific substrate species, acid neutralizer and catalyst with pure bacterial fermentation to form a specific biological fermentation system capable of converting CO 2 Oriented conversion to high concentration succinic acid and acceleration of CO under intensification of specific substrate species, acid neutralizers and catalysts 2 The metabolic rate of bioconversion to succinic acid and its selectivity are improved. The fermentation system can maintain stable and efficient operation for 2-200h, and the catalyst is not easy to deactivate in the reaction system and is not easy to cause death of pure strains.
In order to solve the technical problems, the invention adopts the following technical scheme:
CO (carbon monoxide) 2 The method for directionally producing the succinic acid by bioconversion comprises the following steps:
s1, preparing a culture medium suitable for growth of pure succinic acid-producing strains, inoculating the pure succinic acid-producing strains for culture to obtain pure succinic acid-producing strains in an exponential growth phase;
s2, treating a 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 pure bacteria producing succinic acid into a mixed biological fermentation medium, and uniformly mixing under anaerobic conditions to obtain converted CO 2 A bio-fermentation system for producing succinic acid;
s5, introducing enough CO into a shaking table reactor containing a biological fermentation system 2 The production of succinic acid is performed under anaerobic conditions.
In the present invention, the term "exponential growth phase" means that when a microorganism is cultured in a closed system (batch culture), the specific growth rate of the microorganism is maximized after a certain period of growth of the microorganism according to the growth rate and the change of the specific growth rate of the microorganism, and is referred to as an exponential growth phase, in which the microorganism maintains a constant maximum specific growth rate without the presence of factors inhibiting or limiting the growth of the microorganism, and the number of cells is exponentially increased.
In the present invention, the term "catalyst" means a catalyst capable of promoting CO 2 Solid particles that are bioconverted to succinic acid are a mixture rather than a compound.
In the present invention, the term "biological fermentation system" means that the whole of the pure strain, mixed biological fermentation medium in the reactor exists as "fermentation conditions", in the form of a mixture of pure bacteria, mixed biological fermentation medium in the present invention.
Preferably, in step S1, the culture medium is formulated to contain the following substances in each liter of solution: 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen 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 resazurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L (+) -cysteine and 0.077g of DL-dithiothreitol.
Preferably, in step S1, the medium is steam sterilized at 90-130℃and a pressure of 0.1-0.25MPa for 20-30min before inoculating the pure succinic acid-producing strain for cultivation.
Preferably, in step S1, the pH of the medium is between 6.0 and 7.5.
Preferably, in step S1, the temperature of the medium is 30-45 ℃.
According to certain embodiments of the present invention, in step S1, the pure strain producing succinic acid may be selected from pure bacteria known to be capable of producing succinic acid; the culture medium comprises basic nutrient solution and other components which can meet the growth of pure strains and the production of succinic acid.
Preferably, in step S1, the succinic acid-producing pure strain is selected from the group consisting of strains sold by the German collection of microorganisms and the American type culture Collection. Generally including bacteria, fungi and genetically engineered bacteria: succinogenes FZ53, A.succinogenes130Z,A.succinogenesNJ113,A.succinogenes CGMCC1593,A.succiniciproducens ATCC53488,A.succiniciproducens ATCC29305,M.succiniciproducens MBEL55E,B.succiniciproducens JF4016,C.glutamicum R,M.succiniciproducens PALKG,M.succiniciproducens LPK7 (pMS 3-fdh2 meq), E.coli AFP111, E.coliAS1600a, E.coliBE062, E.coliE2, E.coliHX024, E.coliJCL1208, E.coliJW1021, C.glutamicum BOL, C.glutamicum ELB-P, C.glutamicum NC-3-1,C.acetoacidophilum,S.elongatusPCC,Synechocystis sp.PCC 6803,P.kudriavzevii 13171,P.kudriavzevii 13723,Y.lipolytica Y-3314,Y.lipolytica PGC010037942.
Preferably, in the step S1, the amount v/v of the pure strain producing succinic acid is 5-15%, and the time for achieving the exponential growth phase is generally 6-36h.
As a further improvement of the technical solution, in step S2, the processing includes the steps of:
s2-1, drying and dehydrating a precursor of the active metal catalyst at 80-200 ℃ to obtain a solid A;
s2-2, dissolving the auxiliary agent salt into a solvent, and stirring to uniformly dissolve the auxiliary agent salt to obtain an auxiliary agent salt solution B;
s2-3, soaking the solution B on the solid A powder, stirring, 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 Fe 2 O 3 、Fe 3 O 4 、Co 3 O 4 、Al 2 O 3 、MoO 3 、SiO 2 One or more of the following.
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 comprises 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 2-20h.
Preferably, in step S2-4, whereThe reducing gas is selected from one or more of the following substances: h 2 、CO、CO 2 、CH 4 Synthesis gas (H) 2 /CO)。
Preferably, in the step S2-4, the gas speed of the reducing gas is 20-100mL/min.
Preferably, in step S2-4, the time for the reduction is 6-20h.
Preferably, in step S2-5, the solid catalyst obtained by grinding has a particle size of 20-500 mesh.
As a further improvement of the technical scheme, in step S3, the method for preparing the mixed biological fermentation medium specifically includes the following steps:
s3-1, preparing a biological fermentation culture medium
The formula of the prepared biological fermentation medium comprises the following substances in each liter of solution: 10g of yeast extract, 3g of dipotassium hydrogen phosphate, 0.2g of magnesium chloride, 0.2g of calcium chloride and 1g of sodium chloride;
s3-2, adding a substrate into a biological fermentation medium;
s3-3, adding an acid neutralizer into a fermentation culture medium, and regulating and controlling pH=6.8-7.2 in the fermentation process;
s3-4, adding the solid catalyst particles into the biological fermentation medium to obtain the mixed biological fermentation 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 straw, sulfite waste liquid, raw bean pods, cellulose waste, duckweed, industrial hemp, sucrose residues, cassava bagasse, rapeseed powder 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, and disodium hydrogen phosphate. Preferably, the acid neutralizer 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 5-100g/L.
Preferably, in step S3-4, the concentration of the solid catalyst particles in the biological fermentation medium is 2000-100000mg/L.
As a further improvement of the technical scheme, in the step S4, the mixing temperature is 30-45 ℃.
Preferably, in step S4, the mixing is carried out in a shaker reactor with an oscillation rate of 150.+ -.50 rpm.
As a further improvement of the technical scheme, in the step S5, the shaking table reactor is a full-mixed anaerobic reactor or a semi-mixed slurry bed reactor, and the shaking speed of the shaking table 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 mixed biofermentation medium in the biofermentation system is headspace volume = 2-5.
In step S5 of the present invention, the following reactions may occur during the production of succinic acid:
C 6 H 12 O 6 +ATP→G6P+ADP
G6P+H 2 O+2NADP + →RL5P+CO 2 +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+H 2 O
PEP+CO 2 +ADP→OAA+ATP
OAA+NADH→MAL+NAD +
MAL→FUM+H 2 O
FUM+NADH→Succinate+NAD +
PEP+ADP→PYR+ATP
PYR+CoA+NAD + →ACA+CO 2 +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 abbreviations and formal names correspond to the following:
G6P: glucose-6-phosphate;
ATP, adenine nucleoside triphosphate;
ADP, adenosine diphosphate;
NADP + nicotinamide adenine dinucleotide phosphate;
NADPH, reduced nicotinamide adenine dinucleotide phosphate;
RL5P: ribulose-5-phosphate;
X5P xylulose 5-phosphate;
R5P is 5-phosphoribosyl;
S7P is heptaphosphoric acid heptyl ester;
E4P.4-erythrose phosphate;
F6P is fructose-6 phosphoric acid;
FBP, fructose;
GAP: glyceraldehyde-3-phosphate;
PEP, phosphoenolpyruvic acid;
OAA oxaloacetate;
MAL: malic acid;
FUM: fumaric acid;
succinate: a succinate salt;
PYR: pyruvic acid;
CoA: coenzyme A;
ACA, acetyl CoA;
Ac-P: acetyl phosphate;
acetate: acetic acid;
lactate: lactic acid;
formate: formic acid;
ethanol: ethanol.
Any range recited in the invention includes any numerical value between the endpoints and any sub-range of any numerical value between the endpoints or any numerical value between the endpoints.
Unless otherwise indicated, all starting materials herein are commercially available, and the equipment used in the present invention may be conventional in the art or may be conventional 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 are high in price, and CO is easy to discharge in the production process 2 Causing environmental problems. The biological fermentation system has mild reaction condition and simple equipment structure, and can effectively utilize and convert greenhouse gas CO 2 The succinic acid with high added value chemical product is produced, and the succinic acid can be efficiently and stably operated
2) Compared with the existing technology for producing succinic acid by biological fermentation, the succinic acid yield is low and the selectivity is poor, so that the technology becomes the main speed limiting step for producing succinic acid in the biological fermentation industry. In the mixed biological fermentation system, the optimization condition is controlled in a specified range, so that CO 2 The directional biological transformation is to high yield and high selectivity succinic acid.
3) The catalyst is used as a biological fermentation optimization condition, and can strengthen the way of producing succinic acid by using glucose metabolism by pure strains. 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 of pure strains 2 Is used. In addition, pure bacteria can be adsorbed on the surface of the catalyst, so that the propagation density of the pure bacteria is improved, an attachment point is 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 present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1
CO (carbon monoxide) 2 The method for directionally producing the succinic acid by bioconversion comprises the following specific steps:
1) 5mL of pure strain A.succinogenes130Z solution was inoculated into 100mL of growth medium after autoclaving at 121℃for 20min for cultivation. The formula of the growth medium is as follows: 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen 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 resazurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L (+) -cysteine and 0.077g of DL-dithiothreitol;
2) Controlling the pH of a pure strain growth culture medium to be 6.8, culturing at 37 ℃ for 12 hours to reach an index growth period of A.succinogenes130Z, and obtaining pure succinic acid-producing bacteria in the index 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 Fe 2 O 3 Powder, dropping Fe into the solution obtained in the step 1) 2 O 3 In the process, stirring is carried out while dripping, so that the materials are fully mixed;
5) Sealing the materials prepared in the step 4) with a preservative film after the materials are in paste form, and vacuumizing in a glass dryer for 1h;
6) Transferring the material prepared in the step 5) to a drying oven, and drying for 12 hours in an air atmosphere at 150 ℃;
7) Tabletting, grinding and sieving the catalyst dried in the step 6), and then adding the catalyst into the catalyst obtained in the step 4At 00 ℃ utilize H 2 Reducing the synthetic gas with the volume ratio of/CO of 1:1 for 12 hours to obtain 1 percent Pt/Fe 2 O 3 An inorganic solid catalyst;
8) Adding 40000mg/L magnesium carbonate as pH neutralizer and 60000mg/L glucose as substrate into biological fermentation medium after high pressure steam sterilization at 121deg.C for 20min, and introducing enough CO 2 And 5% of pure strain producing succinic acid A.succinogenes130Z in exponential growth phase is accessed; the formula of the biological fermentation medium comprises the following substances in each liter of solution: 10g of yeast extract, 3g of dipotassium hydrogen phosphate, 0.2g of magnesium chloride, 0.2g of calcium chloride and 1g of sodium chloride; ph=6.8-7.2;
9) 1% Pt/Fe as prepared in step 7) 2 O 3 Grinding the inorganic solid catalyst to 40 meshes, and adding the inorganic solid catalyst into the fermentation culture medium prepared in the step 8) to form a mixed fermentation culture system;
10 Reaction of the mixed biological fermentation culture system in a shaking table reactor to obtain CO 2 And (5) bioconversion to produce succinic acid.
In this example, chloroplatinic acid and Fe used in steps 3) and 4) 2 O 3 The mass ratio of (2) is 0.03:1;
in the step 7), the inorganic solid catalyst obtained after the reduction reaction contains Fe 2 O 3 、Fe 3 C、Fe 5 C 2 、Fe 2 C and Pt auxiliary agent;
in step 9), 1% Pt/Fe 2 O 3 The concentration of the inorganic solid catalyst in the reactor is 10000mg/L;
in step 10), the culture conditions were 37℃and the initial pH was 7.2;
in step 10), the shaking speed of the shaker reactor was 150rpm.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 1 below:
table 1: analysis results of liquid substances 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 bio-fermentation system was 1% Pt/Fe 2 O 3 The reaction was carried out within 48h 59961.6The 1mg/L glucose consumption is complete 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 the succinic acid is up to 53871.89mg/L, the total carbon proportion of the succinic acid is 90.13 percent, and compared with the existing biological fermentation system (the selectivity of the succinic acid is 45-60 percent), the mixed biological fermentation system can directionally produce the main product succinic acid with high yield, and the production rate is faster and the selectivity is higher.
Example 2
CO (carbon monoxide) 2 The method for directionally producing the succinic acid by bioconversion comprises the following specific steps:
1) 5mL of pure strain A.succinogenes130Z solution was inoculated into 100mL of growth medium after autoclaving at 121℃for 20min for cultivation. The formula of the growth medium is as follows: 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen 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 resazurin (oxygen indicator), 0.015g of sodium sulfide, 0.024g L (+) -cysteine and 0.077g of DL-dithiothreitol;
2) Controlling the pH of a pure strain growth culture medium to be 6.8, culturing at 37 ℃ for 12 hours to reach an index growth period of A.succinogenes130Z, and obtaining pure succinic acid-producing bacteria in the index 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 Fe 2 O 3 Powder, dropping Fe into the solution obtained in the step 1) 2 O 3 In the process, stirring is carried out while dripping, so that the materials are fully mixed;
5) Sealing the materials prepared in the step 4) with a preservative film after the materials are in paste form, and vacuumizing in a glass dryer for 1h;
6) Transferring the material prepared in the step 5) to a drying oven, and drying for 12 hours in an air atmosphere at 150 ℃;
7) Tabletting, grinding and sieving the catalyst dried in the step 6), and using H at 400 DEG C 2 Reducing the synthetic gas with the volume ratio of/CO of 1:1 for 12 hours to obtain 1 percent Pt/Fe 2 O 3 An inorganic solid catalyst;
8) Adding 40000mg/L sodium carbonate as pH neutralizer and 60000mg/L glucose as substrate into biological fermentation medium after high pressure steam sterilization at 121deg.C for 20min, and introducing enough CO 2 And 5% of pure strain producing succinic acid A.succinogenes130Z in exponential growth phase is accessed; the formula of the biological fermentation medium comprises the following substances in each liter of solution: 10g of yeast extract, 3g of dipotassium hydrogen phosphate, 0.2g of magnesium chloride, 0.2g of calcium chloride and 1g of sodium chloride; ph=6.8-7.2;
9) 1% Pt/Fe as prepared in step 7) 2 O 3 Grinding the inorganic solid catalyst to 40 meshes, and adding the inorganic solid catalyst into the fermentation culture medium prepared in the step 8) to form a mixed fermentation culture system;
10 Reaction of the mixed biological fermentation culture system in a shaking table reactor to obtain CO 2 And (5) bioconversion to produce succinic acid.
In this example, chloroplatinic acid and Fe used in steps 3) and 4) 2 O 3 The mass ratio of (2) is 0.03:1;
in the step 7), the inorganic solid catalyst obtained after the reduction reaction contains Fe 2 O 3 、Fe 3 C、Fe 5 C 2 、Fe 2 C and Pt auxiliary agent;
in step 9), 1% Pt/Fe 2 O 3 The concentration of the inorganic solid catalyst in the reactor is 10000mg/L;
in step 10), the culture conditions were 37℃and the initial pH was 7.2;
in step 10), the shaking speed of the shaker reactor was 150rpm.
The fermentation system of this example converts CO 2 Analytical results of liquid substances bioconverted into succinic acid are shown in the following table2 is shown as follows:
table 2: analysis results of liquid substances in reactor
In this example, the acid neutralizer added to the mixed biological fermentation system was sodium carbonate, and the reaction consumed 59761.45mg/L glucose completely within 60 hours. During the fermentation, the yield of succinic acid as the main product was 46871.89mg/L, and the selectivity in the total product was 73.65%.
Example 3
Example 2 is repeated, with the difference that in step 6), the acid neutralizer in the mixed biological fermentation system is calcium carbonate.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 3 below:
table 3: analysis results of liquid substances 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 neutralizer added to the mixed biological fermentation system was calcium carbonate, and the reaction consumed 59982.09mg/L glucose completely within 72 hours. During the fermentation, the yield of succinic acid as the main product was 42861.49mg/L, and the selectivity in the total product was 63.53%.
Example 4
Example 1 was repeated, except that in step 6), the concentration of the acid neutralizer magnesium carbonate added to the mixed biological fermentation system was 10000mg/L.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 4 below:
table 4: analysis results of liquid substances 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 as an acid neutralizer added to the mixed biological fermentation system was 10000mg/L, and the reaction was completed within 60 hours with 59695.4mg/L glucose. During the fermentation, the yield of succinic acid as the main product 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 the acid neutralizer magnesium carbonate added to the mixed biological fermentation system was 60000mg/L.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 5 below:
table 5: analysis results of liquid substances in reactor
In this example, the concentration of magnesium carbonate as an acid neutralizer added to the mixed biological fermentation system was 60000mg/L, and the reaction was completed with 59955.4mg/L glucose consumption in 60 hours. During the fermentation, the yield of succinic acid as the main product was 49871.89mg/L, and the selectivity in the total product was 83.99%.
Example 6
Example 1 was repeated except that in step 6), the substrate added to the mixed biological fermentation system was 60000mg/L fructose.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 6 below:
table 6: analysis results of liquid substances 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 this example, the substrate added to the mixed biofermentation system was fructose and the reaction consumed 60055.40mg/L of fructose over 108 hours. During the fermentation, the yield of succinic acid as the main product was 38671.89mg/L, and the selectivity in the total product was 62.23%.
Example 7
Example 1 was repeated, except that in step 6), the substrate added to the mixed biological fermentation system was 60000mg/L xylose.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 7 below:
table 7: analysis results of liquid substances 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 biological fermentation system was xylose, and the reaction consumed 59994.7mg/L xylose completely within 84 hours. During the fermentation, the yield of succinic acid as the main product 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 Al 2 O 3 And (3) powder.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 8 below:
table 8: analysis results of liquid substances in reactor
In this example, the metal used for the catalyst added in the mixed bio-fermentation system is Al 2 O 3 The powder, the reaction consumed 59997.13mg/L glucose completely in 48 h. In the fermentation processIn the process, the yield of the main product succinic acid is 48671.89mg/L, and the selectivity in the total product is 76.21%.
Example 9
Example 1 was repeated, with the difference that in step 1), the auxiliary agent of the catalyst added to the mixed biological fermentation system was palladium nitrate.
The fermentation system of this example converts CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 9 below:
table 9: analysis results of liquid substances 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 auxiliary agent used by the catalyst added in the mixed biological fermentation system is palladium nitrate, and 58997.9mg/L glucose is completely consumed in 48 hours. During the fermentation, the yield of succinic acid as the main product was 46971.89mg/L, and the selectivity in the total product was 85.30%.
Comparative example 1
Example 1 was repeated, except that in step 6), no acid neutralizer was added to the biological fermentation system.
Comparative example biological fermentation System CO 2 The results of analysis of the liquid materials bioconverted to succinic acid are shown in table 10 below:
table 10: analysis results of liquid substances 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, the acid neutralizer was not added, and it took 72 hours to consume 59982.09mg/L of glucose. The final main product succinic acid concentration was not high (29671.89 mg/L) compared to examples 1,4, 5, with lower selectivity in the total product: 48.26%.
Comparative example 2
Example 1 was repeated, except that in step 7), no prepared solid catalyst was added to the biofermentation system.
Comparative example biological fermentation System CO 2 The results of analysis of the liquid material bioconverted to succinic acid are shown in table 1 below:
table 11: analysis results of liquid substances in reactor
In this comparative example, no solid catalyst was added to the biofermentation system, and the consumption of 59997.43mg/L glucose was completed over 116 hours. The final main product succinic acid concentration was greatly reduced (34671.89 mg/L) compared to examples 8, 9, with a selectivity in total product (58.85%) of less than 60%.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which come within the spirit of the invention are desired to be protected.
Claims (1)
1. CO (carbon monoxide) 2 The method for directionally producing the succinic acid by bioconversion is characterized by comprising the following steps of:
1) Transferring 5mL of pure strain A.succinogenes130Z solution, inoculating into 100mL of growth medium after high-pressure steam sterilization at 121 ℃ for 20min, and culturing;
the formula of the growth medium is as follows: 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen 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 resazurin (oxygen indicator), 0.015g of sodium sulfide, 0.024gL (+) -cysteine and 0.077 gDL-dithiothreitol;
2) Controlling the pH of a pure strain growth culture medium to be 6.8, culturing at 37 ℃ for 12 hours to achieve an index growth period of A.succinogenes130Z, and obtaining pure succinic acid-producing bacteria in the index 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 Fe2O3 powder, dripping the solution obtained in the step 1) into Fe2O3, and stirring while dripping to fully mix the solution;
5) Sealing the materials prepared in the step 4) with a preservative film after the materials are in paste form, and vacuumizing in a glass dryer for 1h;
6) Transferring the material prepared in the step 5) to a drying oven, and drying for 12 hours in an air atmosphere at 150 ℃;
7) Tabletting, grinding and screening the catalyst dried in the step 6), and reducing the catalyst for 12 hours at 400 ℃ by using synthesis gas with the H2/CO volume ratio of 1:1 to obtain a 1% Pt/Fe2O3 inorganic solid catalyst;
8) Adding 40000mg/L magnesium carbonate as a pH neutralizer and 60000mg/L glucose as a substrate into a biological fermentation culture medium after high-pressure steam sterilization at 121 ℃ for 20min, introducing enough CO2, and inoculating 5% of pure strain producing succinic acid A.succinogenes130Z in an exponential growth phase; the formula of the biological fermentation medium comprises the following substances in each liter of solution: 10g of yeast extract, 3g of dipotassium hydrogen phosphate, 0.2g of magnesium chloride, 0.2g of calcium chloride and 1g of sodium chloride; ph=6.8-7.2;
9) Grinding the 1% Pt/Fe2O3 inorganic solid catalyst prepared in the step 7) to 40 meshes, and adding the ground 1% Pt/Fe2O3 inorganic solid catalyst into the fermentation culture medium prepared in the step 8) to form a mixed fermentation culture system;
10 The mixed biological fermentation culture system reacts in a shaking table reactor, and CO2 is biologically converted to produce succinic acid;
the mass ratio of chloroplatinic acid to Fe2O3 used in the steps 3) and 4) is 0.03:1;
in the step 7), the inorganic solid catalyst obtained after the reduction reaction contains Fe2O3, fe3C, fe C2, fe2C and Pt auxiliary agent;
in the step 9), the concentration of the 1% Pt/Fe2O3 inorganic solid catalyst in the reactor is 10000mg/L;
in step 10), the culture conditions were at a temperature of 37℃and an initial pH of 7.2;
in step 10), the shaking speed of the shaker reactor was 150rpm.
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| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Succinic acid-producing strain and its screening method and uses |
| 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 |
| CN102851224A (en) * | 2012-01-19 | 2013-01-02 | 广西科学院 | Actinobacillus succinogenes strain and method for producing succinic acid by screening and fermentation of same |
| WO2013067850A1 (en) * | 2011-11-11 | 2013-05-16 | 南京工业大学 | Chemically defined culture medium for fermentation to produce succinic acid and application thereof |
| CN111909970A (en) * | 2020-08-10 | 2020-11-10 | 北京化工大学 | Method for producing medium-chain fatty acid by fermentation of exogenous medium reinforced anaerobic microorganisms |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Succinic acid-producing strain and its screening method and uses |
| 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 |
| WO2013067850A1 (en) * | 2011-11-11 | 2013-05-16 | 南京工业大学 | Chemically defined culture medium for fermentation to produce succinic acid and application thereof |
| CN102851224A (en) * | 2012-01-19 | 2013-01-02 | 广西科学院 | Actinobacillus succinogenes strain and method for producing succinic acid by screening and fermentation of same |
| CN111909970A (en) * | 2020-08-10 | 2020-11-10 | 北京化工大学 | Method for producing medium-chain fatty acid by fermentation of exogenous medium reinforced anaerobic microorganisms |
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