A method for producing succinic acid based on biochar fermentation

Abstract

The application discloses a method for producing succinic acid based on biochar fermentation, which comprises strain activation, seed culture and fermentation culture, wherein the strain is Escherichia coli BER208, and the preservation number is CCTCC NO: M2012351; the fermentation medium formula of the fermentation culture is as follows: 0-70 g / L biochar, 0.12 g / L betaine, 20-80 g / L glucose, 2.6 g / L (NH4)HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4.7H2O, 2.4 g / L FeCl3.6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2.6H2O, 0.15 g / L CuCl2.2H2O, 0.5 g / L ZnCl2.4H2O, 0.5 g / L NaMoO4.2H2O and 0.5 g / L MnCl2.4H2O, and the solvent is water. The succinic acid yield of the method reaches 78.90 g / L at most, the yield is 0.98 g / g, and the method can effectively reduce the content of by-products such as formic acid in the fermentation product. Compared with the succinic acid produced by the traditional chemical method, the natural succinic acid obtained by the biological method has the advantages of high efficiency and environmental protection, and has a wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a method for producing succinic acid through fermentation based on biochar. Background Technology

[0002] Succinic acid, also known as succinic acid, is a four-carbon platform compound with significant applications in the chemical, food, and agricultural industries. Firstly, succinic acid is used as an antibacterial agent, flavoring agent, and acidulant. In the food industry, sodium succinate can regulate and improve the quality of sauces and seasonings, and can also replace monosodium glutamate (MSG). Succinic acid has been used to synthesize many health-related products, including pharmaceuticals, antibiotics, vitamins, and amino acids. In the pharmaceutical field, succinic acid can also be used to synthesize antidotes, diuretics, sedatives, hemostatic agents, antibiotics, and vitamins A and B. Secondly, in the chemical industry, succinic acid is an excellent surfactant, detergent, foaming agent, and ion chelating agent used in the electroplating industry to prevent metal corrosion and pitting. Additionally, in agriculture, succinic acid can be used to synthesize low-toxicity, high-efficiency plant growth inhibitors to control plant growth, regulate nutrients, and increase drought, disease, and frost resistance, making it widely used in fruits, vegetables, and other cash crops. Meanwhile, succinic acid, as an important organic synthesis intermediate, is also a precursor used in the synthesis of a series of high-value-added specialty chemicals, such as 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, N-methylpyrrolidone, adipic acid, and other chemical products, as well as biopolymers such as polybutene succinate (PBS) and polyamides. Currently, the global production capacity of succinic acid reaches 100,000 to 120,000 tons per year, with bio-based succinic acid production at approximately 20,000 to 40,000 tons per year.

[0003] Traditional processes for producing succinic acid primarily involve catalytic hydrogenation: this method uses maleic anhydride as a raw material, producing succinic acid through hydrogenation and hydrolysis. While this method produces highly stable succinic acid, the hydrogenation catalyst is expensive and corrosive to pipelines, particularly to the intermediate product maleic anhydride, requiring high-quality equipment. Bio-fermentation, on the other hand, utilizes bacteria or other microorganisms to produce succinic acid and its derivatives from starch, sugar, or other microbial waste. This method has a longer process flow and more complex equipment, but it allows for the substitution of fossil fuels with biomass resources. Bio-fermentation synthesis of succinic acid uses renewable biomass as a raw material, offering advantages such as mild reaction conditions, high product purity, and a green and environmentally friendly process. Furthermore, bio-fermented succinic acid can completely replace the current petroleum-based electrolytic maleic anhydride process.

[0004] Biochar-mediated biofermentation for succinic acid production is a sustainable approach based on a reversible oxidation mechanism. It effectively addresses the problem of acid accumulation in organisms during fermentation, reduces the inhibitory effect of intermediate products in anaerobic fermentation, and promotes the fermentation process. Biochar's large specific surface area and porous structure provide more sites for microbial colonization, facilitating biofilm formation and serving as a carrier for cell growth. Furthermore, the minerals and metals released from biochar during fermentation can serve as nutrients for cell growth and solvent production.

[0005] Currently, researchers both domestically and internationally have conducted research on the synthesis of succinic acid via bio-fermentation, classifying the synthetic pathways into three categories:

[0006] 1) Succinic acid is synthesized via the reduced tricarboxylic acid pathway. The reduced tricarboxylic acid pathway is the main pathway for succinic acid synthesis by microorganisms under anaerobic conditions. This pathway first generates phosphoenolpyruvate through glycolysis. Then, phosphoenolpyruvate or pyruvate is converted into oxaloacetate by PEP carboxylase or pyruvate carboxylase. It is then further reduced in the cytoplasm by malate dehydrogenase, fumarate enzyme and fumarate reductase to generate succinic acid.

[0007] 2) The aerobic tricarboxylic acid cycle is the second pathway for succinate accumulation. Under aerobic conditions, oxaloacetate and acetyl-CoA are first catalyzed to citrate in the mitochondria, and then succinate is generated through multiple oxidation reactions.

[0008] 3) The glyoxylate cycle is another biosynthetic pathway used to synthesize succinate. The process is as follows: Acetyl-CoA and oxaloacetate condense under the action of citrate synthase to form citrate, which is then catalyzed by aconitase to form isocitrate. Subsequently, isocitrate lyase breaks down isocitrate into succinate and glyoxylate. The resulting glyoxylate combines with another molecule of acetyl-CoA under the action of malate synthase to form malate, which in turn generates oxaloacetate for the next round of the glyoxylate cycle.

[0009] However, regardless of the synthetic route, byproducts such as formic acid and acetic acid are inevitably produced. In practical applications, further separation and removal of these byproducts are required, which limits the use of bio-fermentation to synthesize succinic acid. Summary of the Invention

[0010] To address the aforementioned problems, this invention proposes a method for producing succinic acid based on biochar fermentation. This method not only rationally utilizes biomass resources and agricultural and forestry waste, but also applies the biochar prepared from it to the anaerobic fermentation of microorganisms to produce succinic acid, effectively protecting the ecological environment while creating economic benefits. Furthermore, the method of this invention can effectively reduce the content of byproducts such as formic acid in the fermentation products.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for producing succinic acid based on biochar fermentation includes strain activation, seed culture, and fermentation culture:

[0013] The bacterial strain was Escherichia coli BER208, with accession number CCTCCNO: M2012351;

[0014] The fermentation medium for the fermentation culture is formulated as follows: 5–70 g / L biochar, 0.12 g / L betaine, 20–80 g / L glucose, 2.6 g / L (NH4)HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, with water as the solvent.

[0015] Preferably, the fermentation conditions are: fermentation at 35-39℃ for 60-168 hours, pH 5.0-7.0, and rotation speed 0-200 rpm.

[0016] More preferably, the fermentation conditions are: fermentation at 37°C for 120 hours, pH 6.8, and rotation speed of 180 rpm.

[0017] Preferably, the seed culture process is as follows: the activated strain is transferred to the seed culture medium and activated at 37°C and 180 rpm for 12 hours.

[0018] Preferably, the seed culture medium is formulated with 0.12 g / L betaine, 6.54 g / L K₂HPO₄·3H₂O, 3.5 g / L KH₂PO₄, 3.5 g / L (NH₄)₂HPO₄, 0.25 g / L MgSO₄·7H₂O, 1.6 g / L FeCl₃·6H₂O, 0.05 g / L H₃BO₃, 0.2 g / L CoCl₂·6H₂O, 0.1 g / L CuCl₂·2H₂O, 0.2 g / L ZnCl₂·4H₂O, 0.2 g / L NaMoO₄·2H₂O, and 0.2 g / L ZnCl₂·4H₂O, with water as the solvent.

[0019] Preferably, the inoculation amount of the activated strain is 1 to 10% of the volume of the fermentation medium.

[0020] Preferably, the biochar is fruitwood biochar.

[0021] Preferably, the amount of glucose added to the fermentation medium is 50 g / L.

[0022] Preferably, during the fermentation culture, glucose is added when the glucose concentration is below 20 g / L.

[0023] Preferably, the amount of biochar added to the fermentation medium is 5-10 g / L.

[0024] Beneficial effects: Compared with the prior art, the technical advantages of the present invention are as follows:

[0025] (1) This invention utilizes biochar-mediated fermentation to apply biochar prepared from agricultural and forestry waste to anaerobic fermentation for high-yield succinic acid production, thereby creating economic benefits while making rational use of agricultural and forestry waste.

[0026] (2) This invention can produce succinic acid by adding biochar from agricultural and forestry waste, combining microbial fermentation with the characteristics of biochar. The fermentation broth after fermentation is relatively clear, which greatly reduces the cost of subsequent decolorization and separation. At the same time, the corresponding biochar can also be recycled.

[0027] (3) This invention utilizes the adsorption effect of biochar to use biochar as an immobilization carrier, thereby increasing the yield and production of succinic acid. Furthermore, the biochar is made from natural substrate components of renewable and sustainable raw materials, which has significant application value.

[0028] (4) This invention will enrich and develop the theory and practice of succinic acid biosynthesis, and realize the green and efficient synthesis of succinic acid; at the same time, it will provide new ideas and references for the synthesis of other organic acids, such as glutaric acid and malic acid by bio-fermentation, and has important value in both theory and practical application.

[0029] (5) The method of the present invention can effectively reduce the yield of by-products such as formic acid while increasing the yield of succinic acid. Attached Figure Description

[0030] Figure 1 This is a comparative graph showing the effects of different biochars on the final succinic acid yield and by-product yield.

[0031] Figure 2 This is a comparative graph showing the effects of different amounts of fruit tree biochar addition on the final succinic acid yield.

[0032] Figure 3 This is the result of scale-up culture in a 5L bioreactor of Example 3, which includes the fruitwood biochar group.

[0033] Figure 4 This is the result of scale-up culture in a 5L bioreactor of the group without biochar in Example 3. Detailed Implementation

[0034] Example 1

[0035] (1) Escherichia coli (Escherichiacoli) BER208, preservation number CCTCCNO: M2012351, was inoculated into seed culture medium at an inoculation rate of 2% v / v and cultured at 37℃ and 180rpm for 12h.

[0036] The seed culture medium formula is as follows: 0.12 g / L betaine, 6.54 g / L K2HPO4·3H2O, 3.5 g / L KH2PO4, 3.5 g / L (NH4)2HPO4, 0.25 g / L MgSO4·7H2O, 1.6 g / L FeCl3·6H2O, 0.05 g / L H3BO3, 0.2 g / L CoCl2·6H2O, 0.1 g / L CuCl2·2H2O, 0.2 g / L ZnCl2·4H2O, 0.2 g / L NaMoO4·2H2O, 0.2 g / L ZnCl2·4H2O, with water as the solvent, and the pH adjusted to 7.0.

[0037] (2) The inoculum was inoculated into a fermentation medium containing biochar at an inoculation rate of 10% v / v. The fermentation medium formula was: 50 g / L biochar, 0.12 g / L betaine, 2.6 g / L (NH4)HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, with water as the solvent.

[0038] Fermentation was conducted using coconut shell biochar, corn stalk biochar, bamboo biochar, reed biochar, rice husk biochar, and fruitwood biochar as experimental groups, as well as a blank control group without added biochar. Each group was tested in triplicate, and fermentation was carried out at 37℃ and 180rpm for 120h.

[0039] Biochar surface contains abundant functional groups such as aromatic C=C, COOH, phenolic C-OH, aromatic CH, and aliphatic CH, most of which are negatively charged and relatively dense. This enhances the polarity of biochar and its ability to exchange cations with external substances. During the cultivation process, the substrate consumption in the fermentation broth was measured at 0 h and 120 h of fermentation. When fruitwood biochar was added, the succinic acid yield was the highest, reaching 47.95 g / L, with a yield of 79.9%. Figure 1Compared to the control group without added biochar, the content of formic acid and acetic acid, byproducts, was reduced in the fermentation system, with formic acid content decreasing by 12.07% and acetic acid content decreasing by 26.61%.

[0040] Example 2

[0041] The method is the same as in Example 1, except that in the fermentation culture medium prepared in step (2), five groups of different fruit wood biochar addition amounts are set, with fruit wood biochar addition concentrations of 0 g / L, 16.7 g / L, 33.4 g / L, 50.0 g / L, and 66.7 g / L, for a total of 5 groups, with 3 replicates in each group.

[0042] During the cultivation process, the substrate consumption in the fermentation broth was measured at 0 h and 120 h of fermentation. The highest succinic acid yield (50.33 g / L) was observed when the amount of fruit wood biochar added was 16.7 g / L, with a yield of 85%. Figure 2 ).

[0043] The dosage of fruit wood biochar was further optimized, with 5 groups set at 0 g / L, 3.34 g / L, 8.33 g / L, 16.67 g / L, and 33.33 g / L, and 3 replicates in each group.

[0044] During the cultivation process, the substrate consumption in the fermentation broth was measured at 0 h and 120 h of fermentation. The highest succinic acid yield (55.51 g / L) was observed when the amount of fruit wood biochar added was 8.33 g / L, with a yield of 88.02%. Figure 2 Compared to the control group without biochar, succinic acid production increased by 31.45%, while the byproducts formic acid decreased by 14.16% and acetic acid decreased by 22.76%.

[0045] Example 3

[0046] The method was the same as in Example 1, except that the biochar used was fruitwood charcoal, and the addition amount was 8.33 g / L. The glucose and succinic acid concentrations in the fermentation broth were measured every 12 hours. When the glucose content in the fermentation broth decreased to 0–20 g / L, glucose was added to bring the concentration to 40–60 g / L. Results showed that when using a 5-L fermenter for scale-up cultivation, the experimental group with added biochar achieved a final succinic acid accumulation of 78.9 g / L, a yield of 96%, and a 20.59% reduction in formic acid and a 16.08% reduction in acetic acid accumulation. Figure 3 , 4 Comparative analysis shows that biochar can increase the yield of succinic acid, has a certain inhibitory effect on the yield of by-products formic acid and acetic acid, and the addition of biochar prolongs the survival time of the strain.

[0047] Biochar, as an immobilization carrier, can fix bacterial strains within its pores, providing some resistance to adverse environments in the later stages of fermentation. Figure 3 , 4 Analysis and comparison show that in the later stage of fermentation, the experimental group with added biochar still had a certain metabolic production capacity.

[0048] Compared to existing technologies, this invention combines biochar with the anaerobic fermentation of E. coli for high-yield succinic acid production, creating economic benefits while making rational use of agricultural and forestry waste. By combining microbial fermentation with the properties of biochar, the fermentation broth becomes clearer after fermentation, significantly reducing subsequent decolorization and separation costs. Furthermore, the current fermentation method is batch fermentation; a fed-batch fermentation method in the later stages can further greatly increase succinic acid yield.

Claims

1. A method for producing succinic acid based on biochar fermentation, comprising strain activation, seed culture, and fermentation culture, characterized in that: The bacterial strain is Escherichia coli (Escherichia coli) Escherichia coli BER208, accession number CCTCC NO: M2012351; The fermentation medium for the fermentation culture was formulated as follows: 5-10 g / L biochar, 0.12 g / L betaine, 20-80 g / L glucose, 2.6 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, and 0.5 g / L MnCl2·4H2O, with water as the solvent. The fermentation conditions were: 37 ℃ for 120 h, pH 6.8, and rotation speed of 180 rpm. The biochar mentioned is fruitwood biochar.

2. The method according to claim 1, characterized in that, The seed culture process is as follows: the activated strain is transferred to the seed culture medium and activated at 37 ℃ and 180 rpm for 12 h.

3. The method according to claim 2, characterized in that, The seed culture medium is formulated with 0.12 g / L betaine, 6.54 g / L K2HPO4•3H2O, 3.5 g / L KH2PO4, 3.5 g / L (NH4)2HPO4, 0.25 g / L MgSO4•7H2O, 1.6 g / L FeCl3•6H2O, 0.05 g / L H3BO3, 0.2 g / L CoCl2•6H2O, 0.1 g / L CuCl2•2H2O, 0.2 g / L ZnCl2•4H2O, 0.2 g / L NaMoO4•2H2O, and 0.2 g / L ZnCl2•4H2O, with water as the solvent.

4. The method according to claim 1, characterized in that, The amount of activated microbial strain inoculated is 1 to 10% of the volume of the fermentation medium.

5. The method according to claim 1, characterized in that, The amount of glucose added to the fermentation medium was 50 g / L.

6. The method according to claim 1, characterized in that, During the fermentation culture, glucose is added when the glucose concentration is below 20 g / L.

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