Method for identifying VFA degradation dominant metabolic pathway in AD based on energy efficiency ratio

Abstract

The invention relates to the technical field of biological fermentation, in particular to a method for identifying a dominant metabolic pathway of VFA degradation in AD based on an energy efficiency ratio. The method comprises the following steps: determining boundary conditions of a VFA metabolic pathway; and acquiring data in the operation process of the AD reactor. According to the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio, process parameters such as gas components, VFA concentration, pH value, bicarbonate ion concentration and the like in AD (anaerobic digestion) are regularly detected, a thermodynamic function in a reaction system is corrected according to a metabolic chemical equation of VFA in combination with phase state, temperature and substrate and product concentration factors, and the dominant metabolic pathway of VFA degradation in AD is identified. Calculating a delta G value corresponding to each metabolic pathway; the electron transfer mole number (Ne) involved in each metabolic reaction is calculated, and delta G is normalized into a form (delta G / Ne) of electron transfer per mole; preliminary identification of the dominant metabolic pathway can be realized through the physicochemical parameters easy to obtain in the AD system, and the method has the remarkable advantages of high efficiency, convenience and high universality.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and particularly to a method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio. Background Art

[0002] Under the severe background of climate change and resource shortage, the resource utilization of solid waste has become an important path and starting point for promoting green development and circular development. Anaerobic digestion (AD), as a mature and widely used core technology, through the synergistic action of microorganisms, converts complex organic substances into biogas and humified products, with the dual effects of energy recovery and environmental governance. As the core metabolic step in the AD process, the metabolic dynamics of volatile fatty acids (VFA) directly affect the operational stability and energy recovery efficiency of AD. Clarifying the metabolic mechanism of VFA and the changes in the dominant metabolic pathways in response to process disturbances can not only contribute to the dynamic regulation of the AD system but also provide a scientific basis for the operation optimization and instability warning of the AD system.

[0003] Currently, the identification of the dominant metabolic pathway of VFA mainly relies on microbiome technologies such as 16S rRNA sequencing and metagenomic analysis. Although these methods can provide high-resolution microbial community and functional information, due to high costs, complex operations, and long analysis cycles, it is difficult to meet the requirements of rapid diagnosis and real-time monitoring in engineering practice. How to achieve the rapid identification of the VFA metabolic pathway based on a generally applicable theoretical basis and without the cumbersome and time-consuming microbiome sequencing technology has become a scientific and engineering technical bottleneck that urgently needs to be solved.

[0004] Gibbs free energy (ΔG) is a key parameter describing the thermodynamic feasibility and directionality of biochemical reactions, which can intuitively reflect the microbial energy utilization efficiency and metabolic activity to a certain extent. However, existing studies mainly focus on the calculation of ΔG under ideal conditions (such as standard state) to explore the potential possibility of reaction occurrence, and the thermodynamic correction for the actual reaction conditions in the AD process is relatively limited. The failure to fully combine the unique reaction characteristics of AD (such as heterogeneous conditions, local environmental changes, etc.) for dynamic correction of thermodynamic functions has limited the guiding significance of thermodynamic analysis in practical engineering applications. Secondly, traditional ΔG calculations often ignore the changes in energy efficiency during the electron transfer process, and electron transfer is the core driving force of microbial metabolism in the anaerobic digestion process.

[0005] Therefore, by normalizing ΔG to the scale of per mole of electron transfer (ΔG / Ne), the energy efficiency differences of different metabolic pathways and their corresponding thermodynamic properties can be more accurately quantified. This analysis method based on ΔG / N e can not only profoundly reveal the internal laws of microbial metabolic activities, but also capture the dynamic conversion characteristics of the VFA metabolic pathway under different environmental stress conditions (such as overload). Especially when energy limitation or stress increases, this method can provide a quantitative diagnosis basis for identifying the operating state of AD, and is expected to provide theoretical support and practical guidance for optimizing the performance of the AD system in efficient resource utilization and long-term stable operation.

[0006] For this reason, a method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio is designed to provide another technical solution to the above technical problems. Summary of the Invention

[0007] Based on this, it is necessary to provide a method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio to solve the technical problems raised in the above background technology.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio, the steps are as follows:

[0010] Determine the boundary conditions of the VFA metabolic pathway;

[0011] Obtain the data during the operation of the AD reactor;

[0012] Correct the Gibbs free energy under non-standard state conditions;

[0013] Convert to ΔG per unit electron transfer.

[0014] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD provided by the present invention, determining the boundary conditions of the VFA metabolic pathway, the steps are as follows:

[0015] Calculate valeric acid, the expression is as follows:

[0016] CH3CH2CH2CH2COO - +2H2O→CH3COO - +CH3CH2COO - +2H2+H + ;

[0017] Calculate butyric acid, the expression is as follows:

[0018] CH3CH2CH2COO - + 2H2O → 2CH3COO - + 2H2 + H + ;

[0019] The calculation of propionic acid is as follows:

[0020]

[0021] The calculation of acetic acid oxidation is as follows:

[0022]

[0023] The calculation of acetic acid cleavage is as follows:

[0024]

[0025] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention, the data during the operation of the AD reactor is obtained as follows:

[0026] During the operation of the AD reactor, the relevant indicators of the gas phase and liquid phase are regularly detected;

[0027] According to Dalton's law of partial pressures, the partial pressures of CH4, CO2, and H2 are determined, and the concentrations of CH4, CO2, and H2 in the liquid phase are calculated based on Henry's law;

[0028] In the AD system containing the CO2 - HCO3- - CO3 2 - buffer system and VFAs, the concentration of HCO3- is calculated.

[0029] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention, during the operation of the AD reactor, the gas composition, pH, bicarbonate alkalinity, and VFA content are regularly detected.

[0030] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention, according to Henry's law, the expression is as follows:

[0031] C = H × P;

[0032] Wherein, C is the concentration of CH4, CO2, and H2 in the liquid phase, mol / L; H is the Henry solubility, mol / L / atm; P is the partial pressure of CH4, CO2, and H2 in the gas phase, atm;

[0033] In an AD system containing a CO2-HCO3--CO32- buffer system and VFAs, the concentration of HCO3- is calculated, and the expression is as follows:

[0034]

[0035] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention, the Gibbs free energy under non-standard state conditions is corrected, and the steps are as follows:

[0036] ① Calculate the standard Gibbs free energy of the current reaction;

[0037] ② When the reaction temperature is at a non-standard state temperature, use the Gibbs-Helmholtz equation to correct the standard Gibbs free energy at the actual temperature;

[0038] ③ Use the Nernst and van't Hoff equations to calculate ΔG of the relevant reaction at the current actual temperature and concentration.

[0039] As a preferred embodiment of the method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention, convert to ΔG per unit electron transfer, and the steps are as follows:

[0040] Based on the metabolic equation of VFA, calculate the number of electron transfers involved in each reaction, and convert ΔG into the form of per unit electron transfer, and the steps are as follows:

[0041]

[0042] It can be undoubtedly seen that through the above technical solutions of the present application, the technical problems to be solved by the present application can surely be solved.

[0043] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects:

[0044] The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio provided by the present invention regularly detects process parameters such as gas components (such as methane, hydrogen, and carbon dioxide concentrations), VFA (butyric acid, acetic acid, propionic acid, valeric acid) concentrations, pH value, and bicarbonate ion concentration in AD (anaerobic digestion). On this basis, according to the metabolic chemical equation of VFA, combined with factors such as phase state, temperature, and substrate and product concentrations, the thermodynamic functions in the reaction system are corrected, and the ΔG value corresponding to each metabolic pathway is calculated; subsequently, by calculating the number of moles of electron transfer (N e ) involved in each metabolic reaction, ΔG is normalized to the form of per mole of electron transfer (ΔG / N e ); for a single type of VFA degradation pathway, ΔG / N eThe smaller the value, the more significant the thermodynamic advantage of this pathway, that is, this pathway is the dominant metabolic reaction of this type of VFA (volatile fatty acid) in the current reactor.

[0045] 2. Compared with the existing methods relying on metagenomics technology, the present invention does not require expensive equipment, complex experimental operations and time-consuming data analysis, and can achieve the preliminary identification of the dominant metabolic pathway only through the easily accessible physical and chemical parameters in the AD system, having the remarkable advantages of high efficiency, convenience and strong universality. Brief Description of the Drawings

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0047] Figure 1 Schematic diagram of the response change curve of the ratio of Gibbs free energy to the number of electron transfers (△G / N e ) of the present invention in response to different organic loads;

[0048] Figure 2 Schematic diagram of the overview of the relative abundances of the dominant microorganisms and methanogenesis pathways with significant differences in the load groups (G1, G3 and G5) of the present invention. Detailed Embodiments

[0049] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further details the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0050] In order to enable those skilled in the art to better understand the solution of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings.

[0051] It should be noted that, without conflict, the embodiments in the present invention and the features and technical solutions in the embodiments can be combined with each other.

[0052] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0053] Example 1

[0054] Method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio.

[0055] 1) Determine the boundary conditions of the VFA (volatile fatty acid) metabolic pathway

[0056] Based on the VFA metabolic pathway, clarify the chemical reactions involved. Common VFA metabolic reactions include:

[0057] ① Valeric acid (Equation (1)):

[0058] CH3CH2CH2CH2COO - +2H2O→CH3COO - +CH3CH2COO - +2H2+H + (1)

[0060] ② Butyric acid (Equation (2)):

[0061] CH3CH2CH2COO - +2H2O→2CH3COO - +2H2+H + (2)

[0062] ③ Propionic acid (Equation (3)):

[0063]

[0064] ④ Acetic acid (oxidation) (Equation (4)):

[0065]

[0066] ⑤ Acetic acid (lysis) (Equation (5)):

[0067]

[0068] 2) Obtain the physical and chemical data during the operation of the AD reactor

[0069] During the operation of the AD reactor, regularly detect the relevant indicators of the gas phase and liquid phase, including: gas components (CH4, CO2, and H2), pH, bicarbonate alkalinity (BA), and VFA content (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, mol / L).

[0070] Among them, determine the partial pressures of CH4, CO2, and H2 according to Dalton's law of partial pressures, and then calculate the concentrations of CH4, CO2, and H2 in the liquid phase according to Henry's law (Equation (6)).

[0071] C = H×P (6)

[0072] C is the concentration of CH4, CO2, and H2 in the liquid phase, in mol / L; H is the Henry solubility, in mol / L / atm; P is the partial pressure of CH4, CO2, and H2 in the gas phase, in atm.

[0073] In an AD system containing a CO2-HCO3--CO3 2 - buffer system and VFAs, the concentration of HCO3- is calculated using Equation (7).

[0074]

[0075] 3) Correct the Gibbs free energy (ΔG) under non-standard state conditions

[0076] ΔG is the actual value observable during a chemical reaction and varies with the operating conditions and the concentrations of reactants and products. It is a variable. The steps to correct the Gibbs free energy under non-standard state conditions are as follows:

[0077] ① Calculate the standard Gibbs free energy (ΔG Θ ) of the current reaction. Under the conditions of a temperature of 25°C, pH = 7, a solute concentration of 1 mol / L, and an atmospheric pressure of 1 atm, Equation (8) is used to calculate ΔG of Equations (1-5) Θ :

[0078]

[0079] where is the standard Gibbs free energy of formation of the substance in the liquid phase, in kJ / mol.

[0080] ② Temperature correction. When the reaction temperature is not the standard state temperature (25°C), the Gibbs-Helmholtz equation (Equation (9)) is used to correct the standard Gibbs free energy at the actual temperature:

[0081]

[0082] where is the standard Gibbs free energy after temperature correction; Tref is the reference temperature, 298.15 K; Tact is the actual operating temperature of the AD system, in K; is the standard reaction heat, in kJ / mol.

[0083] ③ Concentration correction. Since the actual concentrations of substances during the reaction are usually not 1 mol / L, for a general biochemical reaction aA + bB = cC + dD, the Nernst and van't Hoff equation (Equation (10)) is used to calculate ΔG of the relevant reaction at the current actual temperature and concentration:

[0084]

[0085] Among them, ΔG is the actual Gibbs free energy under the current reaction; R is the gas constant, 8.314 J / (mol / L); T is the absolute temperature, K; A, B, C, and D are the concentrations of substances in the liquid phase in the AD system, mol / L;

[0086] 4) Convert ΔG to ΔG per unit electron transfer (ΔG / N e )

[0087] Based on the metabolic equation of VFA, calculate the number of electron transfers (N e ) involved in each reaction, and then convert ΔG to the form of per unit electron transfer (Equation (11)):

[0088]

[0089] Example 2

[0090] Refer to Figure 1 - Figure 2 , and on the basis of Example 1, disclose an application method.

[0091] Apply the content in Example 1 to: the change of the VFA-dominated metabolic pathway induced by overload

[0092] 1) Parameter setting: A total of 18 glass serum bottles with a total volume of 1 L and an effective volume of 800 ml were used as anaerobic reactors in the batch experiment. The initial volume ratios of the substrate (9.02%, 18.04%, 27.07%, 36.09%, 45.11%, and 54.13%) were used as variables to induce overload, and the experimental groups were labeled as G1, G2, G3, G4, G5, and G6 respectively.

[0093] 2) Detection means

[0094] During the experiment, the gas production, gas components (CH4, CO2, and H2), pH, BA, VFA (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) concentration and other common process parameters that can reflect the performance of the reactor were detected every two days. Combining with the metabolic chemical equation of VFA, according to the actual conditions such as the phase state, temperature, and substrate and product concentrations of the reaction system, the thermodynamic functions of Equation (1-5) were corrected and calculated to obtain the corresponding ΔG values of each metabolic pathway. Subsequently, by calculating the N e involved in each metabolic reaction, ΔG was normalized to the form of ΔG / N e . For simplicity of analysis, only the data of ΔG / N e at the initial stage (the fifth day) and the end stage (the thirtieth day) are listed. In addition, to verify the accuracy of the above method, microbial samples were collected at the initial stage (the 5th day) and the end stage (the 30th day) of digestion respectively for metagenomic analysis (AppendixFigure 2 , where a: potential dominant syntrophic bacteria; b: dominant methanogens; c: relative abundance of the methanogenic pathway identified based on the KEGG metabolic pathway).

[0095] 3) Identification effect

[0096] The case results showed that similar ΔG / N was observed from G1 to G6 e trends, but acetate degradation was a significant exception, showing its uniqueness in the energy dynamic distribution. Notably, with the increase in organic loading, the ΔG / N of acetate degradation via the syntrophic acetate oxidation (SAO) pathway and propionate degradation e first reached positive values, indicating a significant reduction in its thermodynamic feasibility, which may become a key limiting factor affecting process stability.

[0097] For acetate, the two main pathways of acetate degradation are acetoclastic methanogenesis (AM) and SAO. Through the AM pathway, acetate can be directly converted into methane, which is characterized by a e lower ΔG / N and higher energy utilization efficiency, and is usually dominant under neutral pH and stable conditions. However, when the organic loading increases, the dynamic change of energy distribution gradually makes SAO (-7.29 to -0.59 KJ / mol) more competitive than AM (-7.29 to -11.55 KJ / mol). The change in the ΔG / N value of acetate degradation e reveals the basic trade-off in microbial energy utilization, reflecting the ability of syntrophic acetate oxidation bacteria (SAOB) to adapt to environmental changes by maintaining thermodynamic feasibility under stress conditions of reduced energy supply and increased energy demand. Previous studies have shown that the shift of the metabolic pathway from AM to SAO can not only alleviate acid accumulation under stress conditions but also enhance the system's tolerance to high loads, thus enhancing the resilience and stability of the AD process.

[0098] Secondly, with the increase in organic loading, the analysis results of ΔG / N e were highly consistent with the results of microbial annotation. From a thermodynamic perspective, ΔG / N eThe increase in value indicates that the energy efficiency of acetic acid degradation through the AM pathway gradually decreases, while the thermodynamic feasibility of the SAO pathway relatively increases. This is consistent with the change in the microbial community structure: the results of microbial annotation show that during the process of load increase, the abundances of SAOB and hydrogenotrophic methanogens related to SAO increase significantly, and at the same time, their corresponding metabolic pathways (such as SAO and hydrogenotrophic methanogenesis pathways) are also significantly enriched. It is worth noting that the upregulation of SAOB further confirms its ability to adapt to environmental changes under stress conditions. By oxidizing acetic acid to CO2 and H2, it provides substrates for hydrogenotrophic methanogens, thus forming an efficient syntrophic cooperation relationship.

[0099] Meanwhile, the ΔG / N for propionate degradation e value is significantly higher than that of other intermediate product degradation pathways, highlighting its high energy consumption characteristics and profound impact on the AD metabolic balance. The high ΔG / N for propionate oxidation e value indicates its low spontaneity and requires a large amount of energy input to complete the conversion from propionate to acetic acid and H2. In addition, due to the specific growth rate of propionate-oxidizing bacteria (POB) (0.06 d-1) being much lower than that of acetic acid (0.3 d-1) and butyric acid (0.15 d-1), its competitive ability is weak, especially when other microorganisms can utilize thermodynamically more favorable intermediate products. This high energy consumption and low growth rate form a significant metabolic bottleneck, directly leading to a significant slowdown in the propionate degradation rate and limiting its dominant position under high load conditions. This phenomenon is also supported by the results of microbial annotation: as the organic load increases, the relative abundance of POB gradually decreases, and the expression level of its metabolic pathway also decreases. This indicates that under high load stress conditions, the adaptability of POB is weak and insufficient to effectively cope with the increase in energy demand and thermodynamically unfavorable conditions, thus further exacerbating the negative impact of propionate accumulation on the system metabolic balance.

[0100] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments to better explain the principle and practical application of the present invention, so that those skilled in the art in the relevant technical field can understand and utilize the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio, characterized in that The steps are as follows: Determine the boundary conditions of the VFA metabolic pathway; Obtain the data during the operation of the AD reactor; Correct the Gibbs free energy under non-standard state conditions; Convert to ΔG per unit electron transfer.

2. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 1, wherein, To determine the boundary conditions of the VFA metabolic pathway, the steps are as follows: Calculate valeric acid, and the expression is as follows: CH3CH2CH2CH2COO - +2H2O→CH3COO - +CH3CH2COO - +2H2+H + ; Calculate butyric acid, and the expression is as follows: CH3CH2CH2COO - +2H2O→2CH3COO - +2H2+H + ; Calculate propionic acid, and the expression is as follows: CH3CH2COO - + 3H2O → CH3COO - + 3H2 + HCO3 - + H + ; Calculate oxidized acetic acid, and the expression is as follows: CH3COO - + 4H2O → 2HCO3 - + 4H2 + H + ; Calculate cleaved acetic acid, and the expression is as follows: CH3COO - +H2O → CH4+HCO3 - 。 3. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 1, wherein To obtain the data during the operation of the AD reactor, the steps are as follows: During the operation of the AD reactor, regularly detect the relevant indicators of the gas phase and liquid phase; Determine the partial pressures of CH4, CO2, and H2 according to Dalton's law of partial pressures, and calculate the concentrations of CH4, CO2, and H2 in the liquid phase according to Henry's law; In an AD system containing a CO2-HCO3--CO3 2 - buffer system and VFAs, calculate the concentration of HCO3-.

4. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 3, wherein During the operation of the AD reactor, regularly detect the gas composition, pH, bicarbonate alkalinity, and VFA content.

5. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 3, wherein According to Henry's law, the expression is as follows: C = H × P; Among them, C is the concentration of CH4, CO2, and H2 in the liquid phase, mol / L; H is the Henry solubility, mol / L / atm; P is the partial pressure of CH4, CO2, and H2 in the gas phase, atm; In the AD system containing the CO2-HCO3--CO32- buffer system and VFAs, calculate the concentration of HCO3-, and the expression is as follows:

6. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 1, wherein To correct the Gibbs free energy under non-standard state conditions, the steps are as follows: ① Calculate the standard Gibbs free energy of the current reaction; ② When the reaction temperature is at a non-standard state temperature, use the Gibbs-Helmholtz equation to correct the standard Gibbs free energy at the actual temperature; ③ Use the Nernst and van't Hoff equations to calculate ΔG of the relevant reaction at the current actual temperature and concentration.

7. The method for identifying the dominant metabolic pathway of VFA degradation in AD based on the energy efficiency ratio according to claim 1, wherein To convert to ΔG per unit electron transfer, the steps are as follows: Based on the metabolic equation of VFA, calculate the number of electron transfers involved in each reaction, and convert ΔG into the form of per unit electron transfer, and the steps are as follows:

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