Method for enhancing synergistic production of lactic acid and / or acetic acid by microorganisms

Abstract

The invention belongs to the technical field of agricultural waste recycling and bioelectrochemistry, and particularly relates to a method for enhancing synergistic production of lactic acid and / or acetic acid by microorganisms, which comprises the following steps: by taking agricultural waste as a fermentation substrate, adding microbial flora and quorum sensing signal molecules, and carrying out anaerobic fermentation to produce acid under the condition of an electric field; the microbial flora comprises one or more of clostridium cellulolyticum, lactobacillus acidophilus, Moorella thermoaceticum and sulfur reduction geobacter; and the quorum sensing signal molecule comprises one or more of AI-2, C < 8 >-HSL and 3-oxo-C12-HSL. A quorum sensing signal molecule is coupled with an electric field to form a micro-voltage environment, the metabolic pathway of microorganisms can be regulated and controlled, the yield of lactic acid and acetic acid is increased, generation of by-products is reduced, and when the volume of a fermentation substrate is 2.4 L, the total yield of lactic acid and acetic acid reaches 17.65-19.38 g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural waste resource utilization and bioelectrochemistry, and particularly relates to a method for enhancing the synergistic production of lactic acid and / or acetic acid by microorganisms. Background Art

[0002] Approximately 65% ​​of agricultural waste generated annually worldwide is lignocellulosic (35%-50% cellulose, 15%-20% lignin). Anaerobic technology can be used to recycle a significant amount of this waste into energy. Anaerobic fermentation hydrolyzes waste into short-chain fatty acids. Traditional anaerobic fermentation processes utilize multiple bacterial strains to decompose organic matter, but these processes result in a complex product spectrum, high levels of impurities, and a typically low lactic acid content of less than 15%, resulting in low yields.

[0003] In the prior art, although specific culture media and optimized culture conditions have been used to enrich acid-producing bacteria, these methods usually require complex equipment and high operating costs, and in the strain screening process, they can often only be optimized for a specific type of acid-producing bacteria, and cannot effectively cover a variety of acid-producing functional bacteria. For example, the enrichment of lactic acid bacteria is often carried out in a low pH and high sugar environment, while the enrichment of certain acetic acid-producing bacteria requires high oxygen and low sugar conditions. Such targeted culture conditions limit the diversity and adaptability of the strains. In addition, traditional enrichment methods also have problems of strain degeneration and contamination by foreign bacteria, which affect the quality and stability of the final product. Therefore, there is an urgent need to develop a method that can improve the quantity and quality of acid production. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for enhancing the cooperative production of lactic acid and / or acetic acid by microorganisms. Quorum sensing signal molecules are coupled with an electric field to form a microvoltage environment. The changes in the electric field can drive the directional movement of signal molecules, thereby precisely controlling the behavior of bacteria, increasing the production of lactic acid and acetic acid, reducing the generation of by-products, and improving the overall fermentation efficiency.

[0005] The present invention provides a method for enhancing the cooperative production of lactic acid and / or acetic acid by microorganisms, comprising the following steps: Using agricultural waste as fermentation substrate, adding microbial flora and quorum sensing signal molecules, and passing an electric field, anaerobic fermentation is carried out to produce acid; The microbial flora includes: one or more of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens; The quorum sensing signal molecules include: one or more of AI-2, C8-HSL and 3-oxo-C12-HSL.

[0006] As a preferred embodiment, the added volume of the microbial flora is 14.5% to 22.5% of the fermentation substrate.

[0007] As a preferred embodiment, the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Mooresella thermoacetica and Geobacter sulfurreducens is 0-56:0-26:9-66:9-36.

[0008] As a preferred embodiment, the anaerobic fermentation includes a start-up period, an acid production peak period and a stable period; During the startup phase, the added volume of the microbial flora is 8% to 12% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 54 to 56:24 to 26:9 to 11:9 to 11; During the peak period of acid production, the added volume of the microbial flora is 3% to 5% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 0:24-26:49-51:9-11; During the stable period, the added volume of the microbial flora is 3.5% to 5.5% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 0:0:64-66:34-36.

[0009] As a preferred solution, during the start-up phase, the quorum sensing signal molecule AI-2 is added, with the final concentration of AI-2 being 47-53 nM; At the peak of acid production, the quorum sensing signal molecule C8-HSL was added, with the final concentration of C8-HSL being 13-18 nM; During the stationary phase, AI-2 and 3-oxo-C12-HSL were added, with the final concentration of AI-2 being 47-53 nM and the final concentration of 3-oxo-C12-HSL being 13-18 nM.

[0010] As a preferred solution, the voltage of the electric field is 0.3-0.6V; an intermittent power-on mode is adopted.

[0011] As a preferred solution, the intermittent power-on mode includes: power-on for 5 to 15 minutes and an interval of 20 to 40 minutes.

[0012] As a preferred solution, the temperature of the anaerobic fermentation is 35-55° C.; the initial pH of the anaerobic fermentation is 4.0-6.0.

[0013] As a preferred embodiment, the carbon-nitrogen ratio of the agricultural waste is 20-30:1.

[0014] As a preferred embodiment, the agricultural waste includes one or more of straw, kitchen waste and excrement; and the total solid mass fraction of the agricultural waste is 5% to 15%.

[0015] Beneficial Effects: The present invention provides a method for enhancing the synergistic production of lactic acid and / or acetic acid by microorganisms, comprising the following steps: using agricultural waste as a fermentation substrate, adding a microbial consortium and a quorum sensing signal molecule, and applying an electric field to conduct anaerobic fermentation to produce acid; the microbial consortium includes one or more of Clostridium cellulolyticum, Lactobacillus acidophilus, Moore's thermoacetica, and Geobacter sulfurreducens; and the quorum sensing signal molecule includes one or more of AI-2, C8-HSL, and 3-oxo-C12-HSL. The present invention uses agricultural waste as a fermentation substrate, adds a microbial consortium to the fermentation process, simultaneously adds a quorum sensing signal molecule, and applies an electric field, thereby coupling the quorum sensing signal molecule with the electric field to accelerate substrate hydrolysis, enhance electron transfer, and optimize metabolic pathways. The present invention activates the endogenous regulatory network of the microbial consortium through intervention with exogenous quorum sensing signal molecules, thereby enhancing the acid tolerance and metabolic efficiency of the strain, inhibiting the accumulation of unfavorable intermediates, enhancing electron transfer between microorganisms, promoting metabolite conversion, and increasing the yield and selectivity of lactic acid and acetic acid. The results of the examples show that when the volume of the fermentation substrate is 2.4 L, the total yield of lactic acid and acetic acid reaches 17.65-19.38 g / L, a 73%-90% increase compared to the control group without the addition of quorum sensing signal molecules and electric field. This shows that the present invention optimizes the anaerobic fermentation process through electric field stimulation, increasing the yield of lactic acid and acetic acid, reducing the formation of byproducts, and improving overall fermentation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart of the anaerobic fermentation acid production method; Figure 2 Diagram of the fermentation device for anaerobic fermentation to produce acid; Figure 3 This is a graph showing the acid production after 30 days of anaerobic fermentation with different treatments. DETAILED DESCRIPTION

[0017] The present invention provides a method for enhancing the cooperative production of lactic acid and / or acetic acid by microorganisms, comprising the following steps: Using agricultural waste as fermentation substrate, adding microbial flora and quorum sensing signal molecules, and passing an electric field, anaerobic fermentation is carried out to produce acid; The microbial flora includes: one or more of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens; The quorum sensing signal molecules include: one or more of AI-2, C8-HSL and 3-oxo-C12-HSL.

[0018] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0019] The present invention uses agricultural waste as a fermentation substrate. The carbon-nitrogen ratio of the agricultural waste can be any value within the range of 20-30:1, for example, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. This carbon-nitrogen ratio can simultaneously meet the carbon and nitrogen requirements of both lactic acid bacteria and acetic acid bacteria, maintaining their high metabolic activity and being within a range that facilitates fermentation and acid production. The agricultural waste of the present invention can include one or more of straw, restaurant waste, and manure. Straw is a cellulose-rich raw material, manure is a high-nitrogen raw material, and restaurant waste is a perishable and easily acidified raw material. The total solids mass fraction of the agricultural waste of the present invention can be any value within the range of 5%-15%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. The restaurant waste of the present invention is a raw material that easily becomes rancid, so it is preferable to use agricultural waste with a low solids content to buffer acidification. Straw, manure, and food waste play different roles in anaerobic fermentation, creating a synergistic effect. Food waste serves as a carbon source, providing a primary energy source for microorganisms, continuously degrading them to produce precursors such as acetic acid and hydrogen. Manure carries a complete anaerobic microbial community (hydrolytic bacteria, acidogenic bacteria, and methanogenic archaea), rapidly initiating fermentation and maintaining bacterial diversity. The high soluble organic matter in food waste stimulates hydrolytic bacteria to secrete extracellular enzymes, accelerating the degradation of straw cellulose.

[0020] The amounts of straw, food waste, and manure used in the present invention can be calculated based on the carbon-nitrogen ratio and total solids mass fraction of the agricultural waste. For example, the measured carbon content of straw is 45.36% and the nitrogen content is 1.34%; the carbon content of manure is 29.5% and the nitrogen content is 1.63%; and the carbon content of food waste is 49.06% and the nitrogen content is 5.16. If you need to prepare 2.4L of agricultural waste with a total solid mass fraction of 5% and a carbon-nitrogen ratio of 25:1, first calculate the required total solid mass, that is, 2.4L×1000×5%=120g; the mass ratio of straw to manure is 7:3, assuming the mass of straw is x, the mass of manure is 3 / 7x, and the mass of food waste is y; the formula is x+3 / 7x+y=120g, (45.36x+29.5×3 / 7x+49.06y) / (1.34x+1.63×3 / 7x+5.16y)=25 / 1; the calculation results are x=79.12, y=6.97, that is, the solid mass of straw is 79.12g, the solid mass of food waste is 6.97g, and the solid mass of manure is 33.91g. The calculated mass is the dry matter mass, and the result is divided by the TS (obtained by detection) of straw, food waste, and manure to obtain the wet weight of the final feed, and the amount of straw, food waste, and manure added in the initial stage of fermentation is obtained.

[0021] In the present invention, microbial flora can be added to the fermentation substrate, and the microbial flora includes one or more of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens. In a specific embodiment, the collection center number of Clostridium cellulolyticum is ATCC 35319 (supplier: Ningbo Testo Biotechnology Co., Ltd., strain information can be found in the literature: Desvaux, M. (2005) Clostridium cellulolyticum: Model Organism of Mesophilic Cellulolytic Clostridia. FEMS Microbiology Reviews, 29, 741-764.), the collection center number of Lactobacillus acidophilus is ATCC 4356 (supplier: Shanghai Xuanya Biotechnology Co., Ltd.); the collection center number of Mooresella thermoacetica is ATCC 35608 (brand: Mingzhoubio, number: B89580); the collection center number of Geobacter sulfurreducens is DSM 12127 (supplier: BioVector NTCC Plasmid Vector Bacteria Cell Protein Antibody Gene Collection Center). The Clostridium cellulolyticum described in the present invention can accelerate fiber decomposition and secrete a highly active cellulase complex to hydrolyze cellulose into glucose, providing a substrate for subsequent acid production. The Lactobacillus acidophilus is resistant to acidic environments and stably produces lactic acid. The thermoacetic acid bacteria is an acetic acid-producing bacterium that is resistant to high temperatures and is suitable for high-temperature fermentation systems, improving the efficiency of acetic acid production. The Geobacter sulfurreducens is an electro-resistant bacterium that can enhance electron transfer. By constructing specific Clostridium cellulolyticum (hydrolytic bacteria), Lactobacillus acidophilus (lactic acid bacteria), Moorella thermoacetica (acetic acid bacteria), and Geobacter sulfurreducens (electro-resistant bacteria), the present invention optimizes the fermentation process. Through the division of labor and coordination of metabolic pathways, efficient substrate utilization, targeted product synthesis, and improved system stability are achieved during the anaerobic fermentation of agricultural waste, thereby increasing the yield and selectivity of lactic acid and acetic acid.

[0022] The added volume of the microbial flora of the present invention can be any value within the range of 14.5% to 22.5% of the fermentation substrate, for example, 14.5%, 15.5%, 16.5%, 17.5%, 18.5%, 19.5%, 20.5%, 21.5% or 22.5%; the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens can be any value within the range of 0-56:0-26:9-66:9-36, for example, 0-55:0-25:10-65:10-35.

[0023] The quorum sensing signal molecules of the present invention include one or more of AI-2, C8-HSL and 3-oxo-C12-HSL. The quorum sensing signal molecules of the present invention can accelerate the microbial perception of population density, activate QS regulatory genes in advance, and induce the synthesis of extracellular polysaccharides (EPS). AI-2 can shorten the "adaptation period" of the microbial community, enter the exponential growth phase in advance, and accelerate the acid production process. C8-HSL is an exclusive quorum sensing signal for Gram-negative bacteria (such as acetic acid bacteria and propionic acid bacteria). It directly activates acid-related genes (such as acetate kinase genes) by binding to LuxR receptor proteins. ackA , lactate dehydrogenase gene ldh ), specifically enhancing the lactate and acetate synthesis pathways while inhibiting unrelated metabolic pathways (such as methanogenesis). AI-2 ensures efficient substrate conversion, while 3-oxo-C12-HSL focuses on improving acid production, forming a "division of labor and collaboration" network that produces a synergistic effect.

[0024] In a specific embodiment, the anaerobic fermentation described in the present invention includes a startup period, a peak acid production period, and a stable period; the startup period is when the fermentation begins, the peak acid production period is 3 to 7 days after the start of fermentation, and the stable period is after the 20th day after the start of fermentation. At different stages of the anaerobic fermentation described in the present invention, the added mass and mass ratio of the microbial flora are adjusted according to the specific situation; the addition of quorum sensing signals at different stages plays different roles. The present invention regulates metabolic pathways by intervening in the behavior of microbial communities. Based on the functional characteristics of the flora at different fermentation stages, the types, concentrations, and addition nodes of signal molecules are dynamically adjusted to activate the synergistic metabolic network of the acid-producing flora and inhibit the activity of the competitive flora, thereby significantly improving the selective synthesis efficiency of target volatile fatty acids (such as acetic acid and butyric acid).

[0025] During the startup phase, the volume of the microbial consortium added can be anywhere within the range of 8% to 12% of the fermentation substrate, for example, 8%, 9%, 10%, 11%, or 12%. The volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens can be anywhere within the range of 54-56:24-26:9-11:9-11; for example, 55:25:10:10. During the startup phase, Clostridium cellulolyticum predominates and is added at a high concentration. High-density inoculation in the early stages of fermentation can rapidly initiate hydrolysis. Later, as fermentation primarily produces soluble sugars, the activity of cellulose-degrading bacteria naturally decreases, eliminating the need for further addition. Furthermore, during the early stages of fermentation, the activity of Lactobacillus acidophilus (lactic acid bacteria) and Moorella thermoacetica (acetic acid bacteria) is low, and Geobacter sulfurreducens (electrically resistant bacteria) has not yet been activated, resulting in a low inoculation percentage.

[0026] During the startup phase, quorum sensing signaling molecules can be added simultaneously. These include AI-2 (autoinducer molecule-2). The final concentration of AI-2 can be anywhere between 47 and 53 nM, such as 47, 48, 49, 50, 51, 52, or 53 nM (the total volume of the final concentration is based on the volume of agricultural waste during the initial fermentation). Adding AI-2 at this time can promote the secretion of extracellular enzymes by cellulose-degrading bacteria. Initially, adding AI-2 can shorten the microbial community's "adaptation period," allowing for earlier entry into the exponential growth phase and accelerating acid production.

[0027] During peak acid production, supplemental microbial flora can be added. The volume of the supplemental microbial flora can range from 3% to 5% of the fermentation substrate, for example, 3%, 3.5%, 4%, 4.5%, or 5% (the volume of the fermentation substrate is based on the volume of agricultural waste during the initial fermentation). The volume ratio of Clostridium cellulilyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens can range from 0:24 to 26:49 to 51:9 to 11, for example, 0:25:50:10. During peak acid production, lactic acid bacteria and acetic acid bacteria predominate, converting soluble sugars into lactic and acetic acids. Clostridium cellulilyticum breaks down the substrate into soluble sugars, weakening its function and requiring no supplementation. During peak acid production, when sugars are abundant, supplemental Lactobacillus acidophilus can accelerate conversion. Supplemental Moorella thermoacetica can fully utilize lactic acid or CO2 to maintain acetic acid synthesis. Adding electrotolerant bacteria can enhance electron transfer, preventing acid accumulation from leading to system collapse.

[0028] During the acid production phase, C8-HSL (octanoyl homoserine lactone) is added simultaneously. This addition can be done in two stages, for example, on the third and seventh days after fermentation. The final concentration of C8-HSL can be anywhere between 13 and 18 nM, such as 13, 14, 15, 16, 17, or 18 nM (the total volume of the final concentration is based on the volume of agricultural waste during the initial fermentation). C8-HSL can induce biofilm formation in lactic acid bacteria, enhance acid tolerance, and activate the Wood-Ljungdahl pathway in acetic acid bacteria, thereby dynamically regulating carbon source allocation. This preferentially converts substrates (glucose and xylose) into target organic acids, reducing the accumulation of byproducts such as propionic and butyric acids.

[0029] During the stabilization phase, additional microbial flora is added. The volume of the additional microbial flora can range from 3.5% to 5.5% of the fermentation substrate, for example, 3.5%, 4%, 4.5%, 5%, or 5.5% (the volume of the fermentation substrate is based on the volume of the agricultural waste during the initial fermentation). The volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens can range from 0:0:64 to 66:34 to 36, for example, 0:0:65:35. During the stabilization phase, Moorella thermoacetica and electrotolerant bacteria predominate. Adding Moorella thermoacetica can selectively enhance acetic acid synthesis; adding electrotolerant bacteria can ensure electron transfer efficiency. Since the cellulose in the substrate is depleted, there is no need to add Clostridium cellulolyticum.

[0030] During the stabilization phase, quorum sensing signal molecules AI-2 and 3-oxo-C12-HSL are added simultaneously. The final concentration of AI-2 is within the range of 47 to 53 nM, for example, 47, 48, 49, 50, 51, 52, or 53 nM (the total volume of the final concentration is based on the volume of agricultural waste during the initial fermentation). The final concentration of 3-oxo-C12-HSL is within the range of 13 to 18 nM, for example, 13, 14, 15, 16, 17, or 18 nM (the total volume of the final concentration is based on the volume of agricultural waste during the initial fermentation). AI-2, as a universal signal molecule, coordinates cross-species collaboration (such as cellulose-degrading bacteria and acid-producing bacteria), promoting substrate breakdown and sugar supply. 3-oxo-C12-HSL can specifically regulate Gram-negative acid-producing bacteria (such as Acetobacter), enhancing their metabolic activity and stress resistance. AI-2 ensures efficient conversion of substrates, while 3-oxo-C12-HSL focuses on improving acid production efficiency, forming a "division of labor-collaboration" network, coordinating the metabolic division of labor among bacterial communities, and inhibiting the growth of miscellaneous bacteria (such as methanogens).

[0031] The final concentration of the quorum sensing signal molecule in the fermentation substrate described herein can be any value within the range of 10 to 55 nM, for example, 10, 15, 20, 25, 30, 35, 40, 45, or 55 nM (the total volume of the final concentration is based on the volume of the agricultural waste during the initial fermentation). The quorum sensing signal molecule described herein can be added in minimal amounts, resulting in low cost and ease of operation. The quorum sensing signal molecule described herein has a stock solution concentration of 0.1 to 1 μM when added. In specific embodiments, the amount of quorum sensing signal molecule added can be calculated according to the following formula: V1 = C2 × V2 / C1, where C1 is the stock solution concentration of the quorum sensing signal molecule, C2 is the target final concentration in the fermentation system, and V2 is the total volume of the fermentation system.

[0032] The initial pH of the anaerobic fermentation described herein is any value within the range of 4.0 to 6.0, such as 4.0, 4.5, 5.0, 5.5, or 6.0. The temperature of the anaerobic fermentation described herein can be any value within the range of 35 to 50°C, such as 35, 38, 40, 42, 45, 47, or 50°C; high temperatures facilitate the production of lactic acid and acetic acid during anaerobic fermentation. By regulating fermentation conditions, the present invention significantly improves the activity of lactic acid bacteria and the efficiency of product formation. The anaerobic fermentation process described herein is dynamic, and the hydraulic retention time (HRT) can be any value within the range of 9 to 11 days, such as 9, 10, and 11 days. Fermentation performed with continuous feedstock replenishment can further increase acid production.

[0033] The present invention can introduce an electric field into the above-mentioned fermentation substrate to carry out anaerobic fermentation. The voltage of the electric field of the present invention can be any value in the range of 0.3~0.6V, such as 0.3, 0.4, 0.5 or 0.6V; an intermittent power-on mode can be adopted. The intermittent power-on mode of the present invention may include: power-on at any value in the range of 5~15min, such as 5, 8, 10, 12 or 15min, and an interval of any value in the range of 20~40min, such as 20, 23, 25, 28, 30, 33, 35, 38 or 40min. Coupling the quorum sensing signal molecules with the electric field can achieve multimodal regulation. The concentration of the signal molecules can be adjusted in real time by utilizing the changes in the electric field, thereby accurately controlling the behavior of the bacteria; the electric field drives the signal molecules to move in a directional manner, shortens the diffusion time, enables low-concentration signals to quickly reach the threshold, and improves the response speed of quorum sensing. The present invention forms a microvoltage environment in anaerobic fermentation through low voltage, which is beneficial to promoting the regeneration of NADH in lactic acid bacteria, coordinating with acetogenic bacteria to accelerate CO2 reduction to produce acetic acid, and can regulate the metabolic pathways of microorganisms, inhibit the accumulation of unfavorable intermediates, enhance electron transfer between microorganisms, and promote the conversion of metabolites.

[0034] The present invention can be used in anaerobic fermentation devices (such as Figure 2 The anode can be a high specific surface area carbon felt (specific surface area ≥ 2000m² / g), and the cathode can be a stainless steel platinum-plated electrode (platinum loading 1~2mg / cm²); the electrodes are immersed in the fermentation liquid with a spacing of 5~8cm, and the effective area of ​​the electrodes is 0.5~1.0m 2 / m 3 The electric field can promote the colonization of Lactobacillus acidophilus (lactic acid bacteria) and Moore's thermoacetica (acetic acid bacteria) in the functional bacteria on the electrode surface, forming a dense biofilm and improving the resistance of the bacteria and the sustainability of metabolism.

[0035] To further illustrate the present invention, a method for enhancing the synergistic production of lactic acid and / or acetic acid by microorganisms provided by the present invention is described in detail below with reference to the following examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] The collection number of Clostridium cellulolyticum is ATCC 35319 (supplier: Ningbo Testo Biotechnology Co., Ltd.), the collection number of Lactobacillus acidophilus is ATCC 4356 (supplier: Shanghai Xuanya Biotechnology Co., Ltd.); the collection number of Mooresella thermoacetica is ATCC 35608 (brand: Mingzhoubio, number: B89580); the collection number of Geobacter sulfurreducens is DSM 12127 (supplier: BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Collection Center).

[0037] Example 1 1. Anaerobic fermentation was performed using straw, food waste, and manure as the fermentation substrate. The fermentation substrate volume was 2.4 L, the feed carbon-nitrogen ratio was adjusted to 25:1, and the total solids (TS) was 5%. The pH was controlled to 5.0, the fermentation temperature was 50°C, and the HRT (hydraulic retention time) was 10 days.

[0038] Calculation process for the amount of straw, kitchen waste and manure: The C content, N content and TS of straw, kitchen waste and manure were measured using an elemental analyzer. The results are shown in Table 1.

[0039] Table 1 C content, N content and TS of straw, kitchen waste and manure

[0040] As can be seen from Table 1, the mass ratio of straw to manure is 7:3. To simplify the calculation process, let the mass of straw be x, the mass of manure be 3 / 7x, and the mass of food waste be y.

[0041] The volume of the fermentation substrate is 2.4 L, and the TS (total solids mass fraction) is 5%. The total solids mass fraction is calculated to be 2.4 L × 1000 × 5% = 120 g. This gives the formula x + 3 / 7x + y = 120 g.

[0042] Adjusting the carbon-nitrogen ratio to 25:1 and combining the C and N contents of straw, kitchen waste, and manure in Table 1, we obtain the formula (45.36x+29.5×3 / 7x+49.06y) / (1.34x+1.63×3 / 7x+5.16y)=25 / 1.

[0043] Calculation shows x=79.12, y=6.97, that is, the solid mass of straw is 79.12g, the solid mass of kitchen waste is 6.97g, and the solid mass of feces is 33.91g.

[0044] The amount of straw added = solid mass of straw / TS straw = 79.12 / 0.327 = 241.96 g, that is, the initial amount of straw added is 241.96 g.

[0045] The amount of food waste added = solid mass of straw / TS food waste = 6.97 / 0.18 = 38.72g, that is, the initial amount of food waste added is 38.72g.

[0046] The amount of manure added = solid mass of manure / TS straw = 33.91 / 0.135 = 251.19g, that is, the initial amount of manure added is 251.19g.

[0047] In anaerobic fermentation equipment (such as Figure 2 The anode can be made of high specific surface area carbon felt (specific surface area ≥ 2000m 2 / g), the cathode can be a stainless steel platinum-plated electrode (platinum loading 2mg / cm 2 The electrodes are immersed in the fermentation liquid with a spacing of 6 cm and an effective area of ​​1.0 m 2 / m 3 .

[0048] 2. During the startup phase, i.e. when anaerobic fermentation begins, perform the following operations for anaerobic fermentation: Add functional microbial flora: Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens, with a total inoculum volume of 240 mL. The inoculum volume of Clostridium cellulolyticum is 132 mL, accounting for 55% of the total inoculum volume; the inoculum volume of Lactobacillus acidophilus is 60 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica is 24 mL, accounting for 10% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens is 24 mL, accounting for 10% of the total inoculum volume.

[0049] Adding signal molecules: adding 1.2 μL of signal molecule AI-2 solution at one time, the initial concentration of the signal molecule AI-2 solution is 100 mM, and the final concentration of AI-2 in the fermentation substrate is 50 nM.

[0050] Electric field conditions: A weak electric field was applied, with a constant DC voltage of 0.5 V (error ± 0.05 V). An intermittent power-on mode was used, with power on for 10 minutes and an interval of 30 minutes. The voltage conditions remained unchanged during the entire anaerobic fermentation process.

[0051] 3. During the peak period of acid production, i.e., the 3rd to 7th day of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 96 mL of microbial flora agent was added. Among them, the inoculum volume of Lactobacillus acidophilus was 24 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica was 48 mL, accounting for 50% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens was 24 mL, accounting for 25% of the total inoculum volume.

[0052] Adding signal molecules: C8-HSL was added twice on the 3rd and 7th days after the start of anaerobic fermentation, with 0.36 μL of 100 mM signal molecule C8-HSL solution added each time. The final concentration of C8-HSL in the fermentation substrate was 15 nM each time.

[0053] 4. Stabilization period, i.e. anaerobic fermentation After 20 days of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 108 mL of microbial flora agent was added, including 70.2 mL of Moorella thermoacetica, accounting for 65% of the total inoculation volume, and 37.8 mL of Geobacter sulfurreducens, accounting for 35% of the total inoculation volume.

[0054] Adding signal molecules: adding composite signal molecules AI-2 and 3-oxo-C12-HSL; adding 1.2 μL of 100 mM signal molecule AI-2 solution, the final concentration of AI-2 in the fermentation substrate is 50 nM; adding 0.36 μL of 100 mM signal molecule 3-oxo-C12-HSL solution, the final concentration of 3-oxo-C12-HSL in the fermentation substrate is 15 nM.

[0055] At different stages of anaerobic fermentation, the added mass and mass ratio of microbial flora are adjusted according to specific circumstances; adding quorum sensing signals at different stages plays different roles, such as Figure 1 shown.

[0056] Example 2 1. Anaerobic fermentation was performed using straw, food waste, and manure as the fermentation substrate. The fermentation substrate volume was 2.4 L, the feed carbon-nitrogen ratio was adjusted to 20:1, and the TS was 5%. The pH was controlled to 5.0, the fermentation temperature was 50°C, and the HRT was 10 days.

[0057] Calculation process for the amount of straw, kitchen waste and manure: The results of the carbon, nitrogen, and TS contents of straw, food waste, and manure are shown in Table 1. The straw to manure mass ratio was 7:3. To simplify the calculation process, the mass of straw was set as x, the mass of manure as 3 / 7x, and the mass of food waste as y.

[0058] The volume of the fermentation substrate is 2.4 L, and the TS (total solids mass fraction) is 5%. The total solids mass fraction is calculated to be 2.4 L × 1000 × 5% = 120 g. This gives the formula x + 3 / 7x + y = 120 g.

[0059] Adjust the carbon-nitrogen ratio to 20:1, and combine the C and N contents of straw, kitchen waste, and manure in Table 1 to obtain the formula (45.36x+29.5×3 / 7x+49.06y) / (1.34x+1.63×3 / 7x+5.16y)=20 / 1.

[0060] Calculation shows x=68.70, y=21.86, that is, the solid mass of straw is 79.12g, the solid mass of kitchen waste is 6.97g, and the solid mass of feces is 33.91g.

[0061] The amount of straw added = solid mass of straw / TS straw = 68.70 / 0.327 = 210.09 g, that is, the initial amount of straw added is 210.09 g.

[0062] The amount of food waste added = solid mass of straw / TS food waste = 21.86 / 0.18 = 121.44 g, that is, the initial amount of food waste added is 121.44 g.

[0063] The amount of manure added = solid mass of manure / TS straw = 29.44 / 0.135 = 218.07g, that is, the initial amount of manure added is 218.07g.

[0064] 2. During the startup phase, i.e. when anaerobic fermentation begins, perform the following operations for anaerobic fermentation: Add functional microbial flora: Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens, with a total inoculum volume of 240 mL. The inoculum volume of Clostridium cellulolyticum is 132 mL, accounting for 55% of the total inoculum volume; the inoculum volume of Lactobacillus acidophilus is 60 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica is 24 mL, accounting for 10% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens is 24 mL, accounting for 10% of the total inoculum volume.

[0065] Adding signal molecules: adding 1.2 μL of a signal molecule AI-2 solution with an initial concentration of 100 mM at one time, and the final concentration of AI-2 in the fermentation substrate is 50 nM.

[0066] Electric field conditions: No electric field is applied during the startup period.

[0067] 3. During the peak period of acid production, i.e., the 3rd to 7th day of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 96 mL of microbial flora agent was added. Among them, the inoculum volume of Lactobacillus acidophilus was 24 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica was 48 mL, accounting for 50% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens was 24 mL, accounting for 25% of the total inoculum volume.

[0068] Adding signal molecules: adding 0.48 μL of 100 mM signal molecule C8-HSL solution, the final concentration of C8-HSL in the fermentation substrate is 20 nM.

[0069] Electric field conditions: A weak electric field was applied, with a constant DC voltage of 0.5 V (error ± 0.05 V), and an intermittent power-on mode of 10 min on and 30 min off.

[0070] 4. Stabilization period, i.e. anaerobic fermentation After 20 days of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 108 mL of microbial flora agent was added, including 70.2 mL of Moorella thermoacetica, accounting for 65% of the total inoculation volume, and 37.8 mL of Geobacter sulfurreducens, accounting for 35% of the total inoculation volume.

[0071] Adding signal molecules: adding composite signal molecules AI-2 and 3-oxo-C12-HSL; adding 0.24 mL of 100 mM signal molecule AI-2 solution and 3-oxo-C12-HSL solution respectively, the final concentration of AI-2 and 3-oxo-C12-HSL in the fermentation substrate is 10 nM.

[0072] Electric field conditions: The constant DC voltage was adjusted to 0.3 V (error ±0.05 V), and the intermittent power-on mode was adopted, with power-on for 10 min and an interval of 30 min.

[0073] Comparative Example 1 The method of Example 1 is as follows, except that: 1. Anaerobic fermentation was performed using straw, food waste, and manure as the fermentation substrate. The fermentation substrate volume was 2.4 L, the feed carbon-nitrogen ratio was adjusted to 20:1, and the total solids (TS) was 5%. The pH was controlled to 5.0, the fermentation temperature was 50°C, and the HRT (hydraulic retention time) was 10 days.

[0074] In anaerobic fermentation equipment (such as Figure 2 The anode can be made of high specific surface area carbon felt (specific surface area ≥ 2000m² / g), and the cathode can be made of stainless steel platinum-plated electrode (platinum loading 2mg / cm 2 The electrodes are immersed in the fermentation liquid with a spacing of 6 cm and an effective area of ​​1.0 m 2 / m3 .

[0075] 2. During the startup phase, i.e. when anaerobic fermentation begins, perform the following operations for anaerobic fermentation: Add functional microbial flora: Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens, with a total inoculum volume of 240 mL. The inoculum volume of Clostridium cellulolyticum is 132 mL, accounting for 55% of the total inoculum volume; the inoculum volume of Lactobacillus acidophilus is 60 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica is 24 mL, accounting for 10% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens is 24 mL, accounting for 10% of the total inoculum volume.

[0076] 3. During the peak period of acid production, i.e. the 7th day of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 96 mL of microbial flora agent was added. Among them, the inoculum volume of Lactobacillus acidophilus was 24 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica was 48 mL, accounting for 50% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens was 24 mL, accounting for 25% of the total inoculum volume.

[0077] 4. Stabilization period, i.e. anaerobic fermentation After 20 days of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 108 mL of microbial flora agent was added, including 70.2 mL of Moorella thermoacetica, accounting for 65% of the total inoculation volume, and 37.8 mL of Geobacter sulfurreducens, accounting for 35% of the total inoculation volume.

[0078] That is, in the anaerobic fermentation process of Comparative Example 1, only microbial flora was added without adding signal molecules or introducing an electric field, serving as a control group.

[0079] Comparative Example 2 The method of Example 1 is as follows, except that: 1. Anaerobic fermentation was carried out using straw, kitchen waste, and feces as fermentation substrates. The volume of the fermentation substrate was 2.4 L, the carbon-nitrogen ratio of the feed was adjusted to 25:1, the TS was 5%, the pH was adjusted to 5.0, and the fermentation temperature was 50°C.

[0080] 2. During the startup phase, i.e. when anaerobic fermentation begins, perform the following operations for anaerobic fermentation: Add functional microbial flora: Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica, and Geobacter sulfurreducens, with a total inoculum volume of 240 mL. The inoculum volume of Clostridium cellulolyticum is 132 mL, accounting for 55% of the total inoculum volume; the inoculum volume of Lactobacillus acidophilus is 60 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica is 24 mL, accounting for 10% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens is 24 mL, accounting for 10% of the total inoculum volume.

[0081] Electric field conditions: Anaerobic fermentation was started by applying a weak electric field, with a constant DC voltage of 0.5 V (error ± 0.05 V), and an intermittent power-on mode, i.e., power on for 10 min / interval of 30 min.

[0082] 3. During the peak period of acid production, i.e. the 7th day of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 96 mL of microbial flora agent was added. Among them, the inoculum volume of Lactobacillus acidophilus was 24 mL, accounting for 25% of the total inoculum volume; the inoculum volume of Moorella thermoacetica was 48 mL, accounting for 50% of the total inoculum volume; and the inoculum volume of Geobacter sulfurreducens was 24 mL, accounting for 25% of the total inoculum volume.

[0083] Electric field conditions: constant DC voltage of 0.5 V (error ±0.05 V), intermittent power-on mode, i.e., power-on for 10 min / interval of 30 min.

[0084] 4. Stabilization period, i.e. anaerobic fermentation After 20 days of anaerobic fermentation, perform the following operations for anaerobic fermentation: Supplementation of functional microbial flora: 108 mL of microbial flora agent was added, including 70.2 mL of Moorella thermoacetica, accounting for 65% of the total inoculation volume, and 37.8 mL of Geobacter sulfurreducens, accounting for 35% of the total inoculation volume.

[0085] Electric field conditions: constant DC voltage of 0.3 V (error ±0.05 V), intermittent power-on mode, i.e., power-on for 10 min / interval of 30 min.

[0086] That is, in the anaerobic fermentation process of Comparative Example 2, only microbial flora were added and an electric field was introduced, but no signal molecules were added.

[0087] Test Example 1 Anaerobic fermentation was carried out using the methods of Examples 1 and 2 and Comparative Examples 1 and 2. The acid production was measured after 30 days of anaerobic fermentation. The results are shown in Table 1. Figure 3 and Table 2. The lactic acid yield was determined by high performance liquid chromatography, and the acetic acid, butyric acid, and propionic acid yields were determined by GC-MS gas chromatography.

[0088] Table 2 Acid production after 30 days of anaerobic fermentation under different treatments

[0089] from Figure 3 As shown in Table 2, the test results show that the total lactic acid and acetic acid yields in Comparative Example 1 (control group) reached 10.2 g / L. The total lactic acid and acetic acid yields in Example 1 reached 17.65 g / L, a 73% increase compared to the control group. The total lactic acid and acetic acid yields in Example 2 reached 19.38 g / L, a 90% increase compared to the control group. The total lactic acid and acetic acid yields in Comparative Example 2 were 16.52 g / L, which was an improvement compared to the control group but significantly different from the Examples. Overall, the Examples showed lower levels of propionic acid and butyric acid, demonstrating fewer byproducts and higher lactic acid and acetic acid yields.

[0090] It can be seen that by using straw, manure and kitchen waste as fermentation substrates, constructing functional microbial communities, adding different quorum sensing signal molecules, and passing a weak electric field into the fermentation tank, the quorum sensing signal molecules can be coupled with the electric field to accelerate substrate hydrolysis, strengthen electron transfer and optimize metabolic pathways, thereby significantly improving the efficiency of lactic acid and acetic acid production.

[0091] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for enhancing the synergistic production of lactic acid and / or acetic acid by microorganisms, characterized in that: The following steps are involved: Using agricultural waste as fermentation substrate, adding microbial flora and quorum sensing signal molecules, anaerobic fermentation to produce acid under electric field conditions; The microbial flora includes: one or more of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens; The quorum sensing signal molecules include: one or more of AI-2, C8-HSL and 3-oxo-C12-HSL.

2. The method according to claim 1, characterized in that The added volume of the microbial flora is 14.5% to 22.5% of the fermentation substrate.

3. The method according to claim 2, characterized in that The volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 0-56:0-26:9-66:9-36.

4. The method according to claim 3, characterized in that The anaerobic fermentation includes a start-up period, an acid production peak period and a stable period; During the startup phase, the added volume of the microbial flora is 8% to 12% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 54 to 56:24 to 26:9 to 11:9 to 11; During the peak period of acid production, the added volume of the microbial flora is 3% to 5% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 0:24-26:49-51:9-11; During the stable period, the added volume of the microbial flora is 3.5% to 5.5% of the fermentation substrate, and the volume ratio of Clostridium cellulolyticum, Lactobacillus acidophilus, Moorella thermoacetica and Geobacter sulfurreducens is 0:0:64-66:34-36.

5. The method according to claim 4, characterized in that During the start-up phase, the quorum sensing signal molecule AI-2 was added, with a final concentration of 47-53 nM; At the peak of acid production, the quorum sensing signal molecule C8-HSL was added, with the final concentration of C8-HSL being 13-18 nM; During the stationary phase, AI-2 and 3-oxo-C12-HSL were added, with the final concentration of AI-2 being 47-53 nM and the final concentration of 3-oxo-C12-HSL being 13-18 nM.

6. The method according to claim 1, characterized in that The voltage of the electric field is 0.3-0.6V; an intermittent power-on mode is adopted.

7. The method according to claim 6, characterized in that The intermittent power-on mode includes: power-on for 5 to 15 minutes, with an interval of 20 to 40 minutes.

8. The method according to claim 1, characterized in that The temperature of the anaerobic fermentation is 35-50° C.; the initial pH of the anaerobic fermentation is 4.0-6.

0.

9. The method according to claim 1, characterized in that The carbon-nitrogen ratio of the agricultural waste is 20-30:

1.

10. The method according to claim 1, characterized in that The agricultural waste includes: one or more of straw, kitchen waste and excrement; the total solid mass fraction of the agricultural waste is 5% to 15%.

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