Multi-enzyme combination for precise characterization of compost fermentation progress and applications

Abstract

The application provides a multi-enzyme combination and application for accurately characterizing compost fermentation process, and belongs to the technical field of environmental biotechnology and solid waste resource utilization, and the multi-enzyme combination comprises alpha-glucosidase, alpha-galactosidase, endo-1, 3 glucanase, endo-cellulase and endo-chitosanase. The multi-enzyme combination is determined by screening the compost systems of different raw materials such as wheat straw, corn straw, chicken manure, alfalfa, sludge and the like under different C / N ratios. The multi-enzyme combination can accurately characterize four stages of composting, i.e. temperature rising, high temperature, temperature falling and maturity, and the enzyme activity can be determined by various detection methods such as fluorescence colorimetry and visible light colorimetry, so that the real-time monitoring and state evaluation of the composting process can be realized, technical support for the process regulation and quality release of composting is provided, and the application prospect is wide.

Description

Technical Field

[0001] This application relates to the fields of environmental biotechnology and solid waste resource utilization technology, and in particular to multi-enzyme combinations and applications for accurately characterizing the composting fermentation process. Background Technology

[0002] Accurately determining the fermentation process and status during composting is crucial for ensuring compost quality and optimizing the process. Composting typically involves a heating phase, a high-temperature phase, a cooling phase, and a maturation phase. Failure to accurately identify the fermentation process can easily lead to incomplete maturation or over-fermentation, affecting resource utilization and the safety of the final product.

[0003] Currently, determining the composting fermentation process mainly relies on traditional methods, including seed germination index (GI value), physicochemical indicators, microbial community analysis, and single enzyme activity detection. Among these, seed germination index testing has a long cycle (approximately 2-7 days), is greatly affected by seed sensitivity and environmental factors, has poor repeatability, and impacts composting efficiency and product quality. Physicochemical indicators, such as temperature and moisture content, are easily affected by environmental factors and lag behind microbial metabolic activity, failing to reflect the fermentation process in real time and easily leading to misjudgments of adequate levels but incomplete composting. Microbial community analysis can reveal microbial changes, but its detection process is complex, costly, and time-consuming, making it unsuitable for rapid industrial screening. Single enzyme activity detection cannot comprehensively reflect the material transformation characteristics during the composting process, limiting its accuracy and comprehensiveness.

[0004] How to obtain a precise and rapid testing system that can cover the entire composting cycle is an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a multi-enzyme combination and its application for accurately characterizing the composting fermentation process, in order to solve the problem of how to obtain a precise and rapid detection system that can cover the entire composting cycle.

[0006] The technical solution adopted in this application to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, embodiments of this application provide a multi-enzyme combination for accurately characterizing the composting fermentation process, the multi-enzyme combination comprising: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0008] Secondly, embodiments of this application provide a method for determining the composting fermentation process based on a multi-enzyme combination, including:

[0009] S01. Provide compost samples;

[0010] S02. Detect the activity of each enzyme in the multi-enzyme combination in the compost sample;

[0011] S03. Determine the fermentation stage of the compost sample by comparing the activity of each enzyme with the preset threshold of the enzyme corresponding to each fermentation stage.

[0012] S04, Output the results of the fermentation process determination of the compost sample;

[0013] The multi-enzyme combination includes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0014] In some embodiments, the compost sample includes one or more of chicken manure compost, sludge compost, and alfalfa compost; and / or

[0015] Methods for detecting enzyme activity include visible light colorimetry, fluorescence colorimetry, or DNS colorimetry.

[0016] In some embodiments,

[0017] The unit of activity for each enzyme is µmol·L⁻¹·h⁻¹·g⁻¹.

[0018] In some embodiments, the fermentation stage includes an initial stage, a heating stage, a high-temperature stage, a cooling stage, and a maturation stage.

[0019] In some embodiments, the initial composting center temperature is 0°C to 35°C; and / or

[0020] During the heating phase, the core temperature of the compost is 35℃~55℃; and / or

[0021] The core temperature of the composting process during the high-temperature phase is 55℃~75℃; and / or

[0022] During the cooling phase, the core temperature of the compost is 35℃~55℃; and / or

[0023] The core temperature of compost during the decomposition stage is 0℃~35℃.

[0024] In some embodiments, the initial threshold for α-glucosidase is 0.24 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 1.72 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.60 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.26 µmol·L⁻¹. -1 ·h -1 ·g-1 The threshold for endochitosanase is 0.72 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or

[0025] During the heating phase, the threshold for α-glucosidase was 0.28 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 0.50 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.21 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.31 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.43 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or

[0026] During the high-temperature phase, the threshold for α-glucosidase is 0.56 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.12 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.49 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.37 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or

[0027] During the cooling phase, the threshold for α-glucosidase was 0.16 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.13 µmol·L⁻¹. -1 ·h -1 ·g -1 The thresholds for α-galactosidase, endo-1,3-glucanase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹.-1 ·h -1 ·g -1 ; and / or

[0028] During the composting stage, the thresholds for α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 .

[0029] In some embodiments, determining the fermentation stage of a compost sample includes the following steps:

[0030] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than their respective initial stage thresholds, the compost sample is judged to be in the initial stage.

[0031] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than the threshold of their respective heating stages, the compost sample is judged to be in the heating stage.

[0032] When the activity of α-galactosidase is less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If so, the compost sample is considered to be in a high-temperature stage;

[0033] When the enzyme activities of α-galactosidase, endo-1,3-glucanase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the temperature is high, the compost sample is considered to be in the cooling phase.

[0034] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the compost sample is in the mature stage, it is determined that the sample is in the composting stage.

[0035] Thirdly, embodiments of this application provide a composting fermentation process detection system based on a multi-enzyme combination, comprising:

[0036] The enzyme activity detection module is used to detect the activity of each enzyme in a multi-enzyme combination.

[0037] The determination module is used to determine the fermentation stage of the compost sample based on the preset threshold of the enzymes corresponding to each fermentation stage.

[0038] The multi-enzyme combination includes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0039] Fourthly, embodiments of this application provide applications of multi-enzyme combinations in monitoring compost fermentation processes, controlling composting processes, or releasing compost quality. The multi-enzyme combination includes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0040] The multi-enzyme combination in this application was determined through screening composting systems using different raw materials such as wheat straw, corn straw, chicken manure, and alfalfa at different C / N ratios (30, 20, and 9). This multi-enzyme combination can accurately characterize the four stages of composting: heating, high temperature, cooling, and maturation. Enzyme activity can be measured using various detection methods such as fluorescence colorimetry and visible light colorimetry, thereby enabling real-time monitoring and status assessment of the composting process. It provides technical support for composting process control and quality release, and has broad application prospects. Attached Figure Description

[0041] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this application and are not intended to limit this application, wherein:

[0042] Figure 1 These are the results of the six composting stage divisions and seed germination index (GI) in the training set (J1, J2, J3, J4, M1, M2) of this application.

[0043] Figure 2 These are heatmaps of the relative activities of five enzymes in six sets of compost in the training set of this application;

[0044] Figure 3 ROC curves for single enzymes and multi-enzyme combinations to determine compost maturity status based on GI thresholds;

[0045] Figure 4 The results of composting stage division and seed germination index (GI) for the experimental set (MJ).

[0046] Figure 5 The results of the composting stage segmentation of the validation set (WN) and the seed germination index (GI). Detailed Implementation

[0047] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Furthermore, it should be understood that the specific embodiments described herein are only for illustration and explanation of this application and are not intended to limit this application.

[0048] In this application, "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0049] In a first aspect, embodiments of this application provide a multi-enzyme combination for accurately characterizing the composting fermentation process, including: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0050] This application screened out five enzymes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase. The enzyme activity of these enzymes gradually disappears during the composting fermentation process. Combining the five enzymes can jointly determine the stage of composting.

[0051] This application utilizes a multi-enzyme combination to achieve comprehensive monitoring of the composting fermentation process, accurately identify key stages, verify the stability of the degradation process, and ensure the final maturity quality. This combination effectively overcomes the limitations of traditional single indicators that cannot comprehensively characterize the degree of composting fermentation, providing reliable technical support for real-time monitoring, process control, and quality release of the composting process.

[0052] α-Glucosidase is a core functional enzyme in the degradation of carbohydrates in compost. It can specifically catalyze the hydrolysis of α-1,4-glycosidic bonds in starch oligosaccharides to generate glucose that can be directly utilized by microorganisms. This helps to promote the conversion of organic matter into humic precursors and facilitates the composting process.

[0053] α-Galactosidase can specifically degrade oligosaccharides containing α-1,6-glycosidic bonds, such as raffinose and stachyose, in composting materials, releasing usable monosaccharides to promote microbial metabolism and accelerate composting initiation.

[0054] Endoglucanase is a core enzyme in composting that degrades polysaccharides containing β-1,3-glycosidic bonds. It belongs to the glycosidic hydrolase family and specifically catalyzes the hydrolysis of β-1,3-glycosidic bonds in substrates such as fungal cell walls and algal polysaccharides, breaking down large polysaccharides into small oligosaccharides.

[0055] Endocellulases belong to the glycoside hydrolase family and specifically and randomly hydrolyze the β-1,4-glycosidic bonds within the cellulose macromolecule, breaking it down into smaller fragments such as cellobiose and cellooligosaccharides. These products can be further broken down into glucose by β-glucosidases, providing carbon and energy sources for composting microorganisms, driving the high-temperature sterilization process, and promoting the conversion of organic matter into humus.

[0056] Endochondrinase is the core functional enzyme for the degradation of chitinous substances in compost. It belongs to the glycoside hydrolase family and specifically catalyzes the hydrolysis of β-1,4-glycosidic bonds in chitosan molecules, randomly cleaving large chitosan molecules into smaller molecule products such as chitosan oligosaccharides and glucosamine.

[0057] The α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase selected in this application correspond to key substrate transformation pathways during composting, including the release of readily degradable oligosaccharides, supply of available glucose, hydrolysis of plant structural polysaccharides (cellulose), degradation of microbial cell wall polysaccharides (β-1,3-glucan), and transformation of chitosan residues. The dominant substrate at different stages of composting gradually shifts from soluble sugars to structural polysaccharides, and then to microbial residues and humification intermediates. These five enzymes complement each other in terms of substrate profile and target organisms, exhibiting a combination of phased enhancement and phased decay / exit during fermentation, thus achieving continuous coverage and key node identification during the heating, high-temperature, cooling, and maturation stages. Threshold determination is based on temperature nodes and GI reference standards, employing a CART decision tree combined with cross-validation and bootstrap confidence interval assessment to improve the robustness and transferability of the decision rules. Compared to GI assays that require more than 48 hours, this invention uses rapid enzyme activity detection methods such as colorimetric / fluorescent assays and can acquire multidimensional information in parallel, with a total detection cycle of approximately 6 hours. This enables rapid and objective process monitoring and quality release in industrial settings.

[0058] Secondly, embodiments of this application provide a method for determining the composting fermentation process based on a multi-enzyme combination, including:

[0059] S01. Provide compost samples;

[0060] S02. Detect the activity of each enzyme in the multi-enzyme combination in the compost sample;

[0061] S03. Based on the comparison between the activity of each enzyme and the preset threshold of the corresponding enzyme in each fermentation stage, determine the fermentation stage of the compost sample.

[0062] S04, Output the results of the fermentation process determination of the compost sample;

[0063] The multi-enzyme combination includes α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0064] In S01:

[0065] In some embodiments, the compost sample includes one or more of chicken manure compost, sludge compost, and alfalfa compost.

[0066] For example, the volume of chicken manure compost is 110L, the volume of sludge compost is 150L, ​​and the volume of alfalfa compost is 110L.

[0067] For example, chicken manure composting uses straw as a carbon source and chicken manure as a nitrogen source, and is a conventional composting process; sludge composting uses sludge as a nitrogen source and straw as a carbon source, and is a conventional composting process; alfalfa composting uses straw as a carbon source and alfalfa as a nitrogen source, and is a conventional composting process.

[0068] In some embodiments, methods for detecting enzyme activity include visible light colorimetry, fluorescence colorimetry, or DNS colorimetry.

[0069] It is understandable that the results of different methods for detecting enzyme activity need to be converted to µmol·L⁻¹·h⁻¹·g⁻¹ units before use.

[0070] In S02:

[0071] In some embodiments, the activity of each enzyme is measured in µmol·L⁻¹·h⁻¹·g⁻¹.

[0072] For example, the activity of each enzyme is defined as the increase in the concentration of the corresponding product of each enzyme generated in the catalytic reaction system per hour, based on 1g of compost sample on a dry weight, under conditions of pH 6.0-7.0 and reaction temperature of 37-50°C.

[0073] It can be understood that the reaction system is a standardized enzymatic reaction system constructed in vitro, using endogenous functional enzymes in compost samples as enzyme sources, adding reaction substrates corresponding to the target enzyme, and controlling pH and reaction temperature. The catalytic activity of the target enzyme in the sample is characterized by the increase in the concentration of characteristic products generated within a limited time.

[0074] In S03:

[0075] In some embodiments, the fermentation stage includes an initial stage, a heating stage, a high-temperature stage, a cooling stage, and a maturation stage.

[0076] Furthermore, the initial temperature ranges from 0℃ to 35℃, for example, it can be 0℃, 2℃, 5℃, 8℃, 10℃, 12℃, 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 34℃, 35℃, etc.

[0077] The temperature during the heating phase is 35℃~55℃, for example, it can be 35℃, 38℃, 40℃, 42℃, 44℃, 45℃, 46℃, 48℃, 50℃, 52℃, 54℃, 55℃, etc.

[0078] The high-temperature stage is 55℃~75℃, for example, it can be 55℃, 56℃, 58℃, 60℃, 62℃, 64℃, 65℃, 68℃, 70℃, 72℃, 74℃, 75℃, etc.

[0079] The temperature during the cooling phase is 35℃~55℃, for example, it can be 35℃, 38℃, 40℃, 42℃, 44℃, 45℃, 46℃, 48℃, 50℃, 52℃, 54℃, 55℃, etc.

[0080] The temperature during the composting stage is 0℃~35℃, for example, it can be 0℃, 2℃, 5℃, 8℃, 10℃, 12℃, 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 34℃, 35℃, etc.

[0081] It is understandable that the temperature at each stage refers to the temperature at the center of the reactor core.

[0082] Furthermore, in the initial stage, the threshold for α-glucosidase is 0.24 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 1.72 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.60 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.26 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.72 µmol·L⁻¹. -1 ·h -1 ·g -1 ;

[0083] During the heating phase, the threshold for α-glucosidase was 0.28 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 0.50 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.21 µmol·L⁻¹. -1 ·h -1 ·g-1 The threshold for endonuclease is 0.31 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.43 µmol·L⁻¹. -1 ·h -1 ·g -1 ;

[0084] During the high-temperature phase, the threshold for α-glucosidase is 0.56 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.12 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.49 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.37 µmol·L⁻¹. -1 ·h -1 ·g -1 ;

[0085] During the cooling phase, the threshold for α-glucosidase was 0.16 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.13 µmol·L⁻¹. -1 ·h -1 ·g -1 The thresholds for α-galactosidase, endo-1,3-glucanase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 ;

[0086] During the composting stage, the thresholds for α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 .

[0087] Furthermore, determining the fermentation stage of the compost sample includes the following steps:

[0088] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than their respective initial stage thresholds, the compost sample is judged to be in the initial stage.

[0089] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than the threshold of their respective heating stages, the compost sample is judged to be in the heating stage.

[0090] When the activity of α-galactosidase is less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If so, the compost sample is considered to be in a high-temperature stage;

[0091] When the enzyme activities of α-galactosidase, endo-1,3-glucanase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the temperature is high, the compost sample is considered to be in the cooling phase.

[0092] When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the compost sample is in the mature stage, it is determined that the sample is in the composting stage.

[0093] It is understandable that the enzyme activity threshold in the initial stage is used to characterize the basic enzymatic characteristics of the composting system in the fermentation start-up stage, serving as a starting reference for full-cycle monitoring.

[0094] Thirdly, embodiments of this application provide a composting fermentation process detection system based on a multi-enzyme combination, comprising:

[0095] The enzyme activity detection module is used to detect the activity of each enzyme in a multi-enzyme combination.

[0096] The determination module is used to determine the fermentation stage of the compost sample based on the preset threshold of the enzymes corresponding to each fermentation stage.

[0097] The multi-enzyme combination includes α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0098] Fourthly, embodiments of this application provide the application of a multi-enzyme combination in monitoring the compost fermentation process, the multi-enzyme combination including α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase and endo-chitosanase.

[0099] Fifthly, embodiments of this application provide the application of multi-enzyme combinations in composting process regulation, wherein the multi-enzyme combination includes α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0100] Sixthly, embodiments of this application provide the application of a multi-enzyme combination in compost quality release, the multi-enzyme combination comprising α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

[0101] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0102] Experimental Example 1

[0103] Candidate enzyme system screening and identification

[0104] Based on the organic matter degradation mechanism of composting—in the early stage of composting, the decomposition is mainly of easily degradable substances such as proteins and soluble sugars, while in the middle and late stages, it shifts to the hydrolysis of difficult-to-degrade macromolecules such as cellulose and hemicellulose. The dynamics of enzyme activity are highly synchronized with the degradation law of the substrate.

[0105] Based on literature review and mechanistic analysis, several candidate enzymes potentially involved in the degradation of compost organic matter were preliminarily screened, including glycosidases, hydrolases, and some esterases. Furthermore, preliminary experiments were conducted to evaluate the activity signal intensity, repeatability, and stage-discriminating ability of these candidate enzymes in compost samples at different fermentation stages. Enzymes with weak activity signals, poor repeatability, or insufficient stage indication were eliminated, resulting in a five-enzyme candidate system composed of α-glucosidase, α-galactosidase, endonuclease, endonuclease-1,3-glucanase, and endonuclease-chitosanase. This enzyme system exhibits complementarity in substrate coverage and stage response characteristics, enabling it to characterize the entire process of transformation from easily degradable organic matter to structured organic matter and microbial residues during compost fermentation, providing a foundation for subsequent stage determination and maturity evaluation.

[0106] Experimental Example 2

[0107] The composting training sets include: ① 110L wheat straw × chicken manure: C / N=30 (J1); ② 110L wheat straw × chicken manure: C / N=20 (J2); ③ 110L corn straw × chicken manure: C / N=30 (J3); ④ 110L corn straw × chicken manure: C / N=20 (J4); ⑤ 110L wheat straw × alfalfa: C / N=30 (M1); ⑥ 110L wheat straw × alfalfa: C / N=20 (M2).

[0108] According to industry standards, a core temperature ≥55℃ is considered the threshold for entering the high-temperature phase, <55℃ for entering the cooling phase, and a GI ≥80% for entering the maturation phase. In the six compost piles of training set, each pile exhibited a typical temperature succession process of "heating-high temperature-cooling-maturation." Figure 1 (a) The dynamic changes in the compost center temperature and ambient temperature during aerobic composting are shown, which can be divided into four stages: the initial stage (day 0), where the compost center temperature is basically the same as the ambient temperature, with no obvious trend of spontaneous temperature rise; the heating stage (days 1-3), where the compost center temperature rapidly breaks away from the influence of the ambient temperature and continues to rise above 55°C; the high-temperature stage (days 4-12), where the compost center temperature is stably maintained above 55°C, significantly higher than the ambient temperature, with microbial metabolic heat production dominating the high-temperature sustained effect; and the cooling stage (days 13-35), where the compost center temperature continues to decrease, gradually falling back to a level close to the ambient temperature, and finally entering the maturity stage. The temperature change trends of each treatment group are consistent, verifying the rationality of using the relative change of compost center temperature and ambient temperature as the basis for defining the fermentation stage. Based on the temperature succession, to avoid the subjectivity of judging the maturity stage solely by temperature, this application introduces GI as the maturity criterion. The maturity period is defined as GI ≥ 80%. The GI of each pile sample gradually increases over time and reaches the threshold on day 35 (…). Figure 1 (b)).

[0109] Figure 2 Figures (a)–(f) demonstrate the high consistency in the overall temporal pattern of the six compost piles in the training set during the composting process: High enzyme activity was prevalent in all piles during the early composting stage (approximately 0–3 days), with α-galactosidase exhibiting the most significant early peak in all six piles, indicating rapid utilization of readily degradable sugar substrates and the initiation of microbial metabolism. Subsequently, during the transition from heating to high temperatures (approximately 3–12 days), other hydrolysis-related enzymes (such as endonucleases, endochondralases, and endo-1,3-glucanases) showed moderate-intensity continuous activity bands or localized enhancements in different piles, reflecting the gradual participation of structural polysaccharide substrates and the maintenance of decomposition intensity. In the mid-to-late stages (approximately 12–20 days later), the overall activity of the five enzymes significantly decreased and tended to low levels. Furthermore, in the later stages (approximately 20–35 days), all piles generally maintained a low activity background, indicating a reduction in degradable substrates, a decrease in reaction rate, and the entry into a relatively stable phase.

[0110] Figure 2 In the diagram, (a) is J1, (b) is J2, (c) is M1, (d) is M2, (e) is J3, and (f) is J4.

[0111] It is understood that although the composting experiments in the training set of this application were carried out under different ambient temperature conditions (such as indoor ambient temperature of 15~25℃ and seasonal temperature difference of 5~35℃), the fermentation stage succession of each composting system was still determined by the enzyme activity threshold of the multi-enzyme combination as the core criterion, and temperature was only used as an auxiliary reference for stage division.

[0112] Experimental Example 3

[0113] To verify the applicability and stability of the five selected core enzymes as indicators for judging the fermentation stages of composting, and to eliminate the random influence of single measurements or trends in individual samples, this application repeatedly measured samples collected from the same compost pile at different stages of fermentation (initial, heating, high temperature, cooling, and maturation) to obtain enzyme activity data for the five enzymes at each stage. Based on the above repeated measurement data, the Friedman nonparametric repeated measures test was used to analyze the significance of the differences in enzyme activity at different fermentation stages to determine whether the changes in enzyme activity were statistically significant. Simultaneously, Kendall's W effect size was calculated to quantify the consistency of stage effects, thereby evaluating the stability and reproducibility of enzyme activity evolution with fermentation stages. As shown in Table 1, the activity changes of the five enzymes at different stages of composting fermentation were all highly significant. Kendall's W showed that the stage effects were moderate to highly consistent, indicating that these enzyme activity changes were not random fluctuations, but could stably and synergistically respond to the succession of composting fermentation stages. This provides reliable statistical support for determining the composting fermentation stage and maturity state based on multi-enzyme combinations.

[0114]

[0115] Table 1: 1. Friedman's nonparametric repeated measures test was used to statistically analyze the enzyme activity data of the same pile at different stages of composting fermentation (initial, heating, high temperature, cooling and maturation);

[0116] 2. Kendall's W is the uniformity effect size of the stage effect, used to characterize the stability of enzyme activity changes with fermentation stage;

[0117] 3. Significance markers: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0118] To further validate the effectiveness of the statistically screened enzymes in determining compost maturity, ROC curves were constructed, with GI≥80% used as the external true label for compost maturity determination. The discriminative performance of single enzymes and multi-enzyme combinations was compared. Figure 3The results showed that the overall discriminative ability of a single enzyme as an indicator was limited, with generally low AUC values ​​(0.16–0.28), indicating that a single indicator was insufficient to accurately characterize the composting process. After modeling with multiple enzymes, the discriminative ability was significantly improved: the AUC of the five-enzyme combined model reached 0.85. This indicates that different types of enzymes are complementary in the maturation process, and the integration of multiple indicators can more comprehensively reflect the characteristics of organic matter degradation and humification. Compared with single enzymes, the ROC curve of the combined model was closer to the upper left corner, and both sensitivity and specificity were significantly improved, verifying the scientific validity and stability of the "multi-enzyme combined-composting maturation stage determination" method.

[0119] Test Example 4

[0120] Determination of the threshold for determining the degree of decay

[0121] The relative temperature change characteristics of the compost pile center compared to the ambient temperature and the GI value are used as the basis for dividing the composting fermentation stages. Combined with temperature change trends, the composting process is divided into an initial stage, a heating stage, a high-temperature stage, and a cooling stage. The composting process is further divided into a maturation stage based on the GI value. Specifically, this application uses a compost center temperature ≥55℃ as the criterion for entering the high-temperature stage, and a compost center temperature <55℃ as the criterion for entering the cooling stage.

[0122] Initial stage: The temperature at the center of the compost pile is 0℃~35℃, for example, 10℃~35℃, 15℃~25℃, etc. This stage is the start-up and adaptation stage of composting, and the temperature has not yet shown a sustained upward trend.

[0123] Heating phase: The temperature at the core of the reactor continues to rise from the initial temperature, ranging from 35°C to 55°C, for example, from 25°C to 45°C. When the temperature first reaches or exceeds 55°C, it enters the high-temperature phase.

[0124] High-temperature stage: The core temperature of the reactor body is ≥55℃, preferably 55℃~75℃, for example 55℃~65℃ (higher temperatures can be achieved under some operating conditions).

[0125] When the temperature at the center of the reactor core drops from ≥55℃ to below 55℃, it enters the cooling stage. Cooling stage: The temperature at the center of the reactor core meets the condition of <55℃ and shows a continuous downward trend, with a temperature range of 35℃ to 55℃, for example, from 45℃ to 35℃.

[0126] This application uses a temperature center ≥55℃ as the standard for entering the high-temperature stage, a temperature center <55℃ as the standard for entering the cooling stage, and a GI ≥80% as the standard for entering the maturity stage. The CART decision tree algorithm was used, optimized through 5-fold cross-validation, and combined with a 1000-step bootstrapping method to calculate 95% confidence intervals, to determine the activity thresholds of five key enzymes. See Table 2.

[0127]

[0128] Using threshold values, the fermentation stages of compost can be accurately determined. When the enzyme activities of all five enzymes are greater than the threshold for the heating stage, the compost is considered to be in the heating stage; when the enzyme activity of α-galactosidase is less than 0.1 µmol·L⁻¹, the fermentation stage is determined to be complete. -1 ·h -1 ·g -1 This indicates that the compost is in a high-temperature stage; when the enzyme activities of α-galactosidase, endo-1,3-glucanase, and endo-chitosanase are less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 This indicates that the compost is in the cooling stage; when the enzyme activity values ​​of all five enzymes are less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 The compost can be judged to be in the mature stage. When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase and endo-chitosanase are all greater than the threshold of their respective initial stages, the compost sample is judged to be in the initial stage. Example

[0129] To verify the extrapolation ability of the multi-enzyme combination for judging the compost fermentation stage and to avoid bias caused by repeated use in the training set, this application sets up an experimental set as an external test set. The experimental set consists of two independent piles (denoted as M and J), with a pile size of 110 L × 2; specifically, the experimental set and the training set are different batches. The experimental set includes: ① 110L wheat straw × chicken manure: C / N = 30 (J); ② 110L wheat straw × alfalfa: C / N = 30 (M).

[0130] The purpose of the experimental set is solely to verify the applicability of the threshold on independent data without changing the feature processing methods and discrimination rules determined in the training set phase.

[0131] To ensure that external validation has a reproducible control benchmark, the "stage division" mentioned in this application refers to the compost fermentation stages divided using the relative change characteristics of the compost center temperature and ambient temperature as auxiliary references and the seed germination index (GI≥80%) as the core reference for maturity. This stage division serves as an external control benchmark for the multi-enzyme combination threshold determination rule, used to verify the accuracy of the multi-enzyme combination determination results. Stage division labels are thus obtained for each sampling time point in the experimental set, serving as a consistency control for subsequent multi-enzyme combination discrimination results.

[0132] The results of the composting stage division of the experimental set (MJ) are shown in the figure. Figure 4 ,from Figure 4(a) It can be seen that MJ compost is in a warming phase for 1-3 days, a high-temperature phase for 4-12 days, and a cooling phase for 13-40 days. In addition, Figure 4 Figure (a) shows that the temperature at the center of the composting center follows a pattern over time: "initial temperature equal to ambient temperature for 0 days - rapid increase (1-3 days) - sustained high temperature (4-12 days) - gradual decrease (13-40 days)," which clearly defines each fermentation stage, and the temperature change trends of groups M and J are consistent. Figure 4 (b) It can be seen that the GI value of MJ reaches 80% in 30 days.

[0133] The results, based on the threshold determination rules, are shown in Table 3: day 3 is in the heating phase, days 6, 9, and 12 are in the high-temperature phase, days 15, 20, and 25 are in the cooling phase, and days 30 and 40 are in the maturation phase. Table 3 shows that the discrimination between the two piles (M and J) in the experimental set is consistent with... Figure 4 The phase divisions remained consistent across all sampling time points.

[0134]

[0135] The multi-enzyme combination threshold determination rule established in this application can stably reproduce the division of the experimental stage in the experimental set, verifying the transferability and applicability of the rule under different pile conditions and different sampling days. Example

[0136] Compost validation set: ① 150L sludge × wheat straw: C / N = 9 (WN)

[0137] The validation set consisted of a 150L sludge composting system as an independent external sample to test the transferability and robustness of the multi-enzyme combination threshold rules established in the training set under different substrate and process scale conditions. Validation set samples were collected on preset sampling days (0, 3, 6, 9, 14, 19, 23, and 35 days), and the activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase were measured. During the validation phase, the CART threshold / rule table established in the training phase was directly used to output the composting stage determination results, and consistency with the stage division labels was evaluated to verify the external generalization ability of the method.

[0138] The results of the validation set (WN) composting stage division are shown below. Figure 5 , Figure 5 (a)WN compost is in a warming phase for 1-3 days, a high-temperature phase for 4-14 days, and a cooling phase for 15-35 days. In addition, Figure 5In (a), the temperature of sludge composting over time showed a pattern of "initially at the same level as the ambient temperature (day 0) - rapidly rising (days 1-3) - sustained high temperature (days 4-14) - gradually declining (days 15-35)," which clearly distinguishes each fermentation stage. (b) The GI value of WN compost reached 80% at 35 days.

[0139] The threshold rules for the training set were used to determine the stages of the 150 L sludge composting validation set point by point. The results are shown in Table 4 (threshold determination record table). The threshold rules determined the initial stage at 0 days, the heating stage at 3 days, the high-temperature stage from 6 to 14 days, the cooling stage from 19 to 23 days, and the maturation stage at 35 days. The overall succession sequence was consistent with the stage division. The above results show that, without adjusting the threshold parameters, this multi-enzyme combination threshold rule can stably reproduce the experimental stage division in 150 L sludge composting independent samples and under open-air conditions with greater environmental temperature fluctuations, demonstrating excellent external applicability, reproducibility, and resistance to environmental interference.

[0140]

[0141] This application establishes three independent datasets for research: a training set, an experimental set, and a validation set. These three datasets do not overlap in terms of compost pile source, sampling samples, or time points, and each dataset has a clearly defined function and purpose. Specifically: The training set contains 6 compost piles (J1, J2, J3, J4, M1, M2), primarily used to mine the statistical patterns of multi-enzyme combination activity changes at different fermentation stages of composting. Based on these patterns, the corresponding multi-enzyme activity thresholds for each fermentation stage are determined, providing a core basis for judging the composting fermentation process. The experimental set contains 2 compost piles (M, J), primarily used to externally test the multi-enzyme combination discrimination rules obtained in the training set under independent compost pile conditions, verifying the actual judgment effect of the rules in independent compost pile scenarios. The validation set consists of compost piles of different systems (WN), primarily used to test the generalization ability of the multi-enzyme combination discrimination rules under cross-raw material system conditions, verifying the adaptability and applicability of the rules in different composting raw material systems. The training set is used only for mining statistical patterns of multi-enzyme activity and determining the threshold for judgment, while the experimental and validation sets are used only for performance evaluation and practical usability verification of multi-enzyme combination discrimination rules.

[0142] The ambient temperature in this application is 0℃~35℃, for example, it can be 0℃, 5℃, 10℃, 12℃, 15℃, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, etc.

[0143] Figure 1 The ambient temperature is 18℃~28℃; Figure 4The ambient temperature ranges from 26.6℃ to 33.7℃. Figure 5 The ambient temperature is 5℃~17℃.

[0144] The composting in the training, experimental, and validation sets of this application all employs the intermittent aeration method.

[0145] In the experimental examples and embodiments of this application, the substrates for endonucleases and endochondrinases were AZCL-Barley β-Glucan (Cat. No. I-AZBGL, purity ≥98%) and AZCL-Chitosan (Cat. No. I-AZCHAN, purity ≥98%) from Megazyme, respectively. The substrate for endochondrinase was 4-nitrophenyl-β-phycotriosylglycoside (product number EN02422) from Biosynth. The substrates for α-glucosidase and α-galactosidase were 4-nitrophenyl-α-D-glucopyranoside (product number ABC-Y1031228, purity ≥99%) and 4-nitrophenyl-α-D-galactopyranoside (product number N503600, purity ≥99%) from Beijing Bailingwei Technology Co., Ltd. The dissolution procedures strictly followed the reagent usage specifications provided by the manufacturers.

[0146] Enzyme activity detection and conversion: all were performed in accordance with the manufacturer's standard testing procedures.

[0147] According to the NY / T 525-2021 standard, radish seeds were cultured in compost extract for 48 hours, and the seed germination index (GI value) at different stages of composting was measured.

[0148] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this application. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this application. Such modifications, improvements, and corrections are suggested in this application, and therefore remain within the spirit and scope of the exemplary embodiments of this application.

Claims

1. A method for determining the composting fermentation process based on multi-enzyme combinations, characterized in that: include: Provide compost samples; The activity of each enzyme in the multi-enzyme combination was detected in the compost sample; The fermentation stage of the compost sample is determined by comparing the activity of each enzyme with the preset threshold of the enzyme corresponding to each fermentation stage. Output the results of the fermentation process determination for the compost sample; The compost samples include one or more of chicken manure compost, sludge compost, and alfalfa compost. The multi-enzyme combination includes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

2. The method for determining the composting fermentation process based on multi-enzyme combinations according to claim 1, characterized in that: Methods for detecting enzyme activity include visible light colorimetry, fluorescence colorimetry, or DNS colorimetry.

3. The method for determining the composting fermentation process based on multi-enzyme combinations according to claim 1, characterized in that: The unit of activity for each enzyme is µmol·L⁻¹·h⁻¹·g⁻¹.

4. The method for determining the composting fermentation process based on multi-enzyme combinations according to claim 1, characterized in that: The fermentation process includes the initial stage, the heating stage, the high-temperature stage, the cooling stage, and the maturation stage.

5. The method for determining the composting fermentation process based on multi-enzyme combinations according to claim 4, characterized in that: The initial composting core temperature is 0℃~35℃; and / or During the heating phase, the core temperature of the compost is 35℃~55℃; and / or The core temperature of the composting process during the high-temperature phase is 55℃~75℃; and / or During the cooling phase, the core temperature of the compost is 35℃~55℃; and / or The core temperature of compost during the decomposition stage is 0℃~35℃.

6. The method for determining the composting fermentation process based on multi-enzyme combinations according to claim 4, characterized in that: Initially, the threshold for α-glucosidase was 0.24 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 1.72 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.60 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.26 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.72 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or During the heating phase, the threshold for α-glucosidase was 0.28 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is 0.50 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.21 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.31 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.43 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or During the high-temperature phase, the threshold for α-glucosidase is 0.56 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for α-galactosidase is less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endo-1,3-glucanase is 0.12 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.49 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endochitosanase is 0.37 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or During the cooling phase, the threshold for α-glucosidase was 0.16 µmol·L⁻¹. -1 ·h -1 ·g -1 The threshold for endonuclease is 0.13 µmol·L⁻¹. -1 ·h -1 ·g -1 The thresholds for α-galactosidase, endo-1,3-glucanase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 ; and / or During the composting stage, the thresholds for α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase were all less than 0.1 µmol·L⁻¹. -1 ·h -1 ·g -1 .

7. The method for determining the composting fermentation process based on multi-enzyme combination according to claim 1, characterized in that: Determining the fermentation stage of a compost sample includes the following steps: When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than their respective initial stage thresholds, the compost sample is judged to be in the initial stage. When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all greater than the threshold of their respective heating stages, the compost sample is judged to be in the heating stage. When the activity of α-galactosidase is less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If so, the compost sample is considered to be in a high-temperature stage; When the enzyme activities of α-galactosidase, endo-1,3-glucanase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the temperature is high, the compost sample is considered to be in the cooling phase. When the enzyme activities of α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase are all less than 0.1 µmol·L⁻¹ -1 ·h -1 ·g -1 If the compost sample is in the mature stage, it is determined that the sample is in the composting stage.

8. A compost fermentation process detection system based on multi-enzyme combinations, characterized in that, include: The enzyme activity detection module is used to detect the activity of each enzyme in a multi-enzyme combination. The determination module is used to determine the fermentation stage of the compost sample based on the preset threshold of the enzymes corresponding to each fermentation stage. The compost samples include one or more of chicken manure compost, sludge compost, and alfalfa compost. The multi-enzyme combination includes: α-glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

9. Based on the application of multi-enzyme combinations in monitoring compost fermentation processes, controlling composting processes, or releasing compost quality, the multi-enzyme combination includes: α-Glucosidase, α-galactosidase, endo-1,3-glucanase, endo-cellulase, and endo-chitosanase.

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