Application of Trichoderma composting agent in compost preparation and methods for compost preparation

Abstract

This invention discloses the application of Trichoderma composting agents in compost preparation and a method for compost preparation, belonging to the field of biocomposting technology. It provides the application of Trichoderma composting agents in compost preparation, wherein the Trichoderma composting agents include *Trichoderma longibrachiatum* T6, *Trichoderma viride* P6, and *Trichoderma longibrachiatum* Lu3. *Trichoderma longibrachiatum* T6 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 13183 and deposit date November 14, 2016. This invention utilizes Trichoderma composting agents and an aerobic composting method to greatly increase the activity of *Trichoderma* fungi in the compost. Regular aeration and constant temperature composting significantly accelerate the composting speed of straw and cow manure.

Description

Technical Field

[0001] This invention belongs to the field of biological composting technology, and particularly relates to the application of Trichoderma composting agent in compost preparation and the method for preparing compost. Background Technology

[0002] In my country, most crop straw is used as fuel, with only a small portion used for composting. Crop straw is an important type of biomass, and its resource utilization is a current hot topic in scientific research. Common agricultural waste includes livestock and poultry manure and crop straw. Livestock and poultry manure refers to solid waste generated during livestock farming, containing a large amount of macromolecular organic matter that is not fully utilized. For example, cow manure contains about 40% unutilized cellulose, and pig manure contains about 30% crude protein. Crop straw refers to the general term for the stems of mature crops, which are rich in cellulose and lignin. Currently, aerobic composting is a frequently used, widely applied, and relatively environmentally friendly treatment process for livestock and poultry manure and crop straw residues. The basic technical route of aerobic composting involves first quantitatively mixing animal manure, effective microbial communities, and corresponding conditioners according to a prescribed carbon-to-nitrogen ratio. Secondly, by controlling the material's moisture content, ambient temperature, pH, carbon-to-nitrogen ratio, and oxygen concentration, the target microorganisms within the research object can proliferate in large quantities. Finally, the biological characteristics of the target microorganisms are utilized to transform and decompose macromolecules that are difficult to absorb and utilize. Aerobic fermentation is an important method for straw reduction and resource utilization. Through aerobic fermentation, the lignocellulose in straw can be effectively degraded, while simultaneously generating humus. The products obtained from aerobic fermentation contain a large amount of nutrients and humus, which can be used as fertilizer for agricultural and forestry crops and as soil conditioners. Therefore, it is urgent to develop a stable and highly effective aerobic composting method. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes the application of Trichoderma composting agent in compost preparation and a method for compost preparation. The use of Trichoderma composting agent in aerobic composting greatly increases the activity of Trichoderma fungi in the compost. Regular aeration and constant temperature composting significantly accelerate the composting speed of straw and cow manure, and optimize the degradation rate of cellulose in cow manure and straw.

[0004] To achieve the above objectives, this invention provides the application of Trichoderma composting agents in the preparation of compost, wherein the Trichoderma composting agents include Trichoderma longibrachiatum T6, Trichoderma viride P6, and Trichoderma longibrachiatum Lu3; wherein Trichoderma longibrachiatum T6 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.13183 and deposit date of November 14, 2016.

[0005] Preferably, the method for preparing the Trichoderma composting agent is as follows:

[0006] Trichoderma longifolia T6, Trichoderma viride P6 and Trichoderma longifolia Lu3 were mixed in a volume ratio of 2:1:1 to obtain a seed mixture. The seed mixture was then inoculated into microcrystalline cellulose medium at a volume fraction of 2% and cultured at 26°C, light for 12 h / d, and 180 rpm for 3 days to obtain a Trichoderma composting agent.

[0007] More preferably, the effective viable count of *Trichoderma longifolia* T6 bacterial solution in the *Trichoderma* composting agent is 1 × 10⁻⁶. 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 cfu / mL.

[0008] Preferably, the compost is prepared from poultry and livestock manure and crop straw.

[0009] The present invention also provides a method for preparing compost using the aforementioned Trichoderma composting agent, comprising the following steps:

[0010] (1) Mix poultry and livestock manure with crop straw to obtain compost substrate;

[0011] (2) Inoculate the Trichoderma composting agent into the compost substrate obtained in step (1) and perform aerobic fermentation to obtain compost.

[0012] Preferably, the poultry and livestock manure and crop straw in step (1) are mixed at a C / N mass ratio of 27:1; and the initial moisture content of the compost substrate in step (1) is 50-60%.

[0013] Preferably, the poultry and livestock manure mentioned in step (1) is cow manure, and the crop straw mentioned in step (1) is corn straw.

[0014] Preferably, the inoculation amount of the Trichoderma composting agent in step (2) is calculated as 3 to 4% of the compost substrate volume.

[0015] Preferably, the temperature of the aerobic fermentation in step (2) is 20-35°C, the aeration time of the aerobic fermentation is 1-2 h / d, and the time of the aerobic fermentation is 18-22 d.

[0016] The present invention also provides the method for preparing compost to obtain mature compost.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] This invention proposes the application of Trichoderma composting agents in compost preparation and a method for compost preparation. Utilizing Trichoderma composting agents and employing an aerobic composting method significantly increases the activity of Trichoderma fungi in the compost. Regular aeration and constant-temperature composting greatly accelerate the composting speed of straw and cow manure, optimizing the degradation rate of cellulose in cow manure and straw. Aerobic fermentation is an important method for straw reduction and resource utilization. Through aerobic fermentation, the lignocellulose in straw can be effectively degraded, while simultaneously generating humus. The products obtained from aerobic fermentation contain a large amount of nutrients and humus, and can be used as fertilizer for agricultural and forestry crops and as a soil conditioner.

[0019] This invention reflects the degree of substrate composting by measuring the cellulose content in cow manure and straw compost substrate. The fermentation conditions for cow manure and straw composting were optimized. The cow manure and straw were mixed and loaded at a C / N ratio of 27:1. The optimal composting conditions were: Trichoderma inoculant amount of 3.9%, initial moisture content of 59.78%, and aeration time of 1.48 h / d. After 20 days of fermentation, the substrate cellulose content was 173.19 mg / g, representing a 29.81% increase in cellulose degradation rate compared to the initial conditions.

[0020] Biological preservation instructions for Trichoderma longifolia T6:

[0021] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0022] Accession number: CGMCC NO.13183;

[0023] Date of deposit: November 14, 2016;

[0024] Taxonomic name: Trichoderma longibrachiatum. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is the standard curve for glucose.

[0027] Figure 2 The effect of Trichoderma composting agent addition on the cellulose content in compost substrate is shown in the figure. a, b, c and d represent significant differences.

[0028] Figure 3 The effect of initial moisture content on cellulose content in compost substrate is shown in the figure. a, b, c, and d represent significant differences.

[0029] Figure 4 The effect of aeration time on the cellulose content in compost substrate is shown in the figure. a, b, c, d and e represent significant differences.

[0030] Figure 5 The effect of ambient temperature on the cellulose content in compost substrate is shown in the figure, where a, b, and c represent significant differences.

[0031] Figure 6 Contour plot showing the interaction between Trichoderma composting agent inoculum amount and initial moisture content;

[0032] Figure 7 Three-dimensional surface plot of the interaction between Trichoderma composting agent inoculum amount and initial moisture content;

[0033] Figure 8 Contour plot showing the interaction between Trichoderma composting agent inoculum amount and aeration time;

[0034] Figure 9 Three-dimensional surface plot of the interaction between Trichoderma composting agent inoculum amount and aeration time;

[0035] Figure 10 Contour plot showing the interaction between initial moisture content and aeration time;

[0036] Figure 11 This is a three-dimensional surface plot showing the interaction between initial water content and aeration time. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” and “contain” used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] Materials used in this invention: Microcrystalline cellulose culture medium: 10g microcrystalline cellulose, 0.3g urea, 0.75g peptone, 1.4g (NH4)2SO4, 2g KH2PO4, 0.06g MgSO4, 0.06g CaCl2, trace elements (CoCl2·6H2O 0.0037g, FeSO4·7H2O 0.005g, ZnSO4·7H2O 0.0014g, MnSO4·H2O 0.0016g), 2mL Tween 80, pH 6.

[0043] Trichoderma viride P6 and Trichoderma longibrachiatum Lu3 are disclosed in non-patent literature: Efficacy of Trichoderma longibrachiatum SC5 Fermentation Filtrate in Inhibiting the Sclerotinia sclerotiorum Growth and Development in Sunflower. Enchen Li 1, Na Zhu 1, Shuwu Zhang 1,2,*, Bingliang Xu 1,*, Lilong Liu 3,4 and Aiqin Zhang.

[0044] Example 1

[0045] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 (cfu / mL).

[0046] (1) Cow manure and corn stalks were mixed at a C / N mass ratio of 27:1 to obtain compost substrate. The initial moisture content of the compost substrate was adjusted to 59.78%.

[0047] (2) Inoculate the Trichoderma composting agent into the compost substrate. The inoculation amount is calculated as 3.19% of the compost substrate volume. Ferment aerobically for 20 days at 28℃ with an aeration time of 1.48h / d to obtain compost.

[0048] Example 2

[0049] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL.3 (cfu / mL).

[0050] (1) Cow manure and corn stalks were mixed at a C / N mass ratio of 27:1 to obtain compost substrate. The initial moisture content of the compost substrate was adjusted to 60%.

[0051] (2) Inoculate the Trichoderma composting agent into the compost substrate. The inoculation amount is calculated as 4% of the volume of the compost substrate. Ferment at 28℃ for 20 days with an aeration time of 1h / d to obtain composted compost.

[0052] Example 3

[0053] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 (cfu / mL).

[0054] (1) Cow manure and corn stalks were mixed at a C / N mass ratio of 27:1 to obtain compost substrate. The initial moisture content of the compost substrate was adjusted to 50%.

[0055] (2) Inoculate the Trichoderma composting agent into the compost substrate. The inoculation amount is calculated as 3% of the volume of the compost substrate. At 20℃, aerobic fermentation is carried out for 18 days with an aeration time of 1h / d to obtain compost.

[0056] Example 4

[0057] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 (cfu / mL).

[0058] (1) Cow manure and corn stalks were mixed at a C / N mass ratio of 27:1 to obtain compost substrate. The initial moisture content of the compost substrate was adjusted to 60%.

[0059] (2) Inoculate the Trichoderma composting agent into the compost substrate. The inoculation amount is calculated as 4% of the volume of the compost substrate. Ferment at 35℃ for 22 days with an aeration time of 2h / d to obtain composted compost.

[0060] Example 5

[0061] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 (cfu / mL).

[0062] (1) Cow manure and corn stalks were mixed at a C / N mass ratio of 27:1 to obtain compost substrate. The initial moisture content of the compost substrate was adjusted to 55%.

[0063] (2) Inoculate the Trichoderma composting agent into the compost substrate. The inoculation amount is calculated as 3.5% of the compost substrate volume. Ferment aerobically at 26℃ for 20 days with an aeration time of 1.5h / d to obtain compost.

[0064] Experimental Example

[0065] 1. Materials and Methods

[0066] 1.1 Test Materials

[0067] Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 were mixed at a volume ratio of 2:1:1 to obtain a seed culture solution. This seed culture solution was then inoculated into microcrystalline cellulose medium at a volume fraction of 2%, and cultured at 26°C, under a light intensity of 12 h / d and a 180 rpm incubation for 3 days to obtain a Trichoderma composting agent (the effective viable count of Trichoderma longifolia T6 was 1 × 10⁻⁶). 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 (cfu / mL).

[0068] Cow dung (collected from a dairy farm in Honggu District, Lanzhou City) and corn stalks.

[0069] 1.2 Test Methods

[0070] 1.2.2 Compost Preparation

[0071] The composting materials (Table 1) were prepared according to a C / N ratio of 27:1, with a substrate moisture content of 60%. Trichoderma composting agent was mixed with the composting substrate at 2% of the compost volume, and an equal amount of sterile water was added as a control. The initial composting conditions were 20℃ and the aeration time was 1 h / d.

[0072] Table 1 Properties of compost raw materials

[0073] Material Total nitrogen Total carbon C / N cow dung 2.17 39 15.4 corn stalks 0.84 37.74 46

[0074] 1.2.3 Determination of cellulose content in compost substrate

[0075] The cellulose content in the compost substrate was determined using the Solarbio cellulose content assay kit.

[0076] 1.2.3.1 Extraction of cellulose from the sample

[0077] Cellulose extraction was performed on the sample according to the instructions of the Solarbio Cellulose Content Detection Kit.

[0078] 1.2.3.2 Determination of the standard curve

[0079] Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 620 nm, and zero the instrument with distilled water. Dilute the 10 mg / mL standard solution with distilled water to prepare standard solutions of 0.09, 0.08, 0.07, 0.05, 0.025, 0.0125, and 0.00625 mg / mL for later use. Add 300 μL of the prepared standard solution, 70 μL of the working solution (included in the Leybold cellulose content assay kit), and 630 μL of concentrated sulfuric acid to 1.5 mL centrifuge tubes, with 300 μL of distilled water added as a control. Mix well, incubate in a 95°C water bath for 10 minutes (tightly capped to prevent moisture loss), remove and cool to room temperature, and measure the absorbance at 620 nm, using a blank tube as a control.

[0080] 1.2.3.3 Determination of cellulase in samples

[0081] Add 300 μL of the sample solution extracted in 1.2.3.1, 70 μL of working solution and 630 μL of concentrated sulfuric acid to a 1.5 mL centrifuge tube. The determination method is the same as in 1.2.3.2.

[0082] Cellulose (mg / g) = 22.52X ÷ W3, where X is the glucose content (mg / mL) and W3 is the mass (g) of cell wall material (CWM) weighed during cellulose extraction.

[0083] 1.3 Single-factor optimization of composting conditions on cellulose content

[0084] 1.3.1 Effect of Trichoderma composting agent dosage on cellulose content of cow manure straw

[0085] Five treatment groups were designed for the experiment. The inoculum amount of composting agent was 1%, 2%, 3%, 4% and 5% by volume, respectively. The other conditions were the same as in 1.2.2 of this chapter. Compost samples were collected on the 20th day of composting fermentation and their cellulose content was determined.

[0086] 1.3.2 Effect of initial moisture content on the cellulose content of cow manure straw

[0087] Composting was carried out according to the conditions in 1.2.2, with initial moisture contents set at 30%, 40%, 50%, 60%, and 70%, respectively. Compost samples were collected on the 20th day of composting fermentation, and their cellulose content was determined.

[0088] 1.3.3 Effect of different ventilation times on the cellulose content of cow manure straw

[0089] Composting was carried out according to the conditions in 1.2.2, with the time intervals set to 0.5 h / d, 1 h / d, 1.5 h / d, 2 h / d and 2.5 h / d. Compost samples were collected on the 20th day of composting fermentation, and their cellulose content was determined.

[0090] 1.3.4 Effect of different temperatures on the cellulose content of cow dung straw

[0091] Composting was carried out according to the conditions in 1.2.2. The temperature of the composting chamber was set to 20℃, 28℃ and 35℃ respectively. Compost samples were collected on the 20th day of composting and their cellulose content was determined.

[0092] 1.4 Response surface methodology for optimizing composting fermentation conditions

[0093] Response surface methodology was used for optimization using Design-Expert 10 software. The dosage of Trichoderma composting agent, temperature, and initial moisture content were set as independent variables, and cellulose content as the response value. A total of 17 experimental treatments were designed using the Box-Behnken response surface methodology to simulate the preparation process of the Trichoderma composting agent, establish the optimal fermentation conditions, and validate the results.

[0094] 2. Results and Analysis

[0095] 2.1 Determination of the standard curve and the cellulose content of the compost substrate under initial conditions

[0096] Composting was carried out according to the cultivation conditions in 1.2.2. Compost samples were collected after 20 days. Figure 1 The glucose standard curve shown indicates that its cellulose content is 246.73 mg / g.

[0097] 2.2 Single-factor optimization of composting conditions on cellulose content

[0098] 2.2.1 Effect of Trichoderma composting agent dosage on cellulose content of cow manure straw

[0099] The results are as follows Figure 2 As shown, the amount of Trichoderma composting agent added can significantly affect the degradation of cellulose. With increasing inoculum dosage, the degradation rate of cellulose in the compost gradually increases. When the inoculum dosage is 5%, the cellulose content in the compost substrate is 180.69 mg / g. However, when the inoculum dosage exceeds 4%, the synergistic effect on cellulose degradation is no longer significant. This may be because the growth space of the Trichoderma strains has reached saturation at 4%, so further increasing the inoculum dosage would only increase economic costs.

[0100] 2.2.2 Effect of initial moisture content on the cellulose content of cow dung straw

[0101] The results are as follows Figure 3 As shown, the initial moisture content of the substrate directly affects the degradation rate of cellulose. With increasing moisture content, the degradation of cellulose in the substrate gradually accelerates. When the initial moisture content is 60%, the cellulose content in the substrate is the lowest, at 192.2 mg / g, which is significantly different from other treatment groups. However, when the moisture content is greater than 60%, the degradation of cellulose in the substrate begins to slow down again.

[0102] 2.2.3 Effect of different ventilation times on the cellulose content of cow manure straw

[0103] The results are as follows Figure 4 As shown, the aeration time of composting has a significant impact on the cellulose content in the substrate. With the increase of aeration time, the cellulose degradation rate in the substrate first increases and then decreases. When the aeration time is 1 h / d, the cellulose content in the substrate is the lowest, indicating that the cellulose degradation rate is also the highest at this time.

[0104] 2.2.4 Effect of different temperatures on the cellulose content of cow dung straw

[0105] The results are as follows Figure 5 As shown, controlling the ambient temperature has a significant effect on the degradation of cellulose during composting. The optimal ambient temperature is 28℃. When the temperature is 35℃, the cellulose content is higher than that at 28℃. This may be because the high temperature in the early stage limits the reproduction of Trichoderma strains, thereby affecting the secretion of cellulase and resulting in a slower cellulose degradation rate. However, due to the difficulty in controlling the ambient temperature and the limited number of constant-temperature composting reactors, the requirements for response surface methodology cannot be met. Therefore, the influence of ambient temperature was eliminated in the response surface design, and composting conditions were optimized under a constant temperature of 20℃.

[0106] 2.3 Optimization of Conditional Response Surface for Straw and Cow Manure Composting

[0107] 2.3.1 Response surface design for significant influencing factors

[0108] Based on the single-factor experiment and Box-Benhnken design principle, response surface methodology was conducted using Design Expert 8.0 software. Initial moisture content, Trichoderma composting agent inoculum amount, and aeration time were selected as three factors as independent variables. A total of 17 center combinations with 3 factors and 3 levels were designed for response surface methodology. The cellulose content in the compost substrate was used as the response value. The center value experiment was repeated 5 times. The design and experimental analysis results are shown in Tables 2 and 3.

[0109] Table 2. Box-Benhnken Experimental Factor Level Design

[0110]

[0111] Table 3 Optimization Design of Composting Fermentation Conditions

[0112]

[0113]

[0114] 2.3.2 Analysis of Variance for Regression Model

[0115] Analysis of variance of the predictive regression model showed that the regression level was significant. The interactions of the inoculum size (A), initial moisture content (B), and aeration time (C) of the Trichoderma composting agent, as well as the interactions of the inoculum size (A) and initial moisture content (B), the inoculum size (A) and aeration time (C), and the initial moisture content (B) and aeration time (C) (C) had the most significant effects on the cellulase activity of the Trichoderma composting agent (P<0.0001). Meanwhile, the coefficient of determination of the regression model equation and the predicted coefficient of determination showed good correlation with each other, indicating that the predictive regression model equation had a good fit and could predict the optimal fermentation conditions and cellulose content under optimal composting conditions. Meanwhile, among the three significant factors of Trichoderma composting agent inoculation amount (A), initial moisture content (B), and aeration time (C), initial moisture content and Trichoderma composting agent inoculation amount had the most significant impact on compost activity, followed by aeration time. However, since the interaction terms of Trichoderma composting agent inoculation amount (A)-initial moisture content (B) and Trichoderma composting agent inoculation amount (A)-aeration time (C) were not significant (Table 4), and the sum of squares of terms AB and AC was small, they needed to be removed to obtain the best prediction equation and a second regression model fitting analysis was performed (Table 5).

[0116] Table 4. Analysis of Variance Table for Regression Model

[0117]

[0118]

[0119] Table 5. Quadratic Regression Fit Analysis of the Model

[0120] source sum of squares Degrees of freedom Mean Square F value p-value Model 1.262E+005 7 18030.98 892.38 <0.0001 A-Inoculant dosage 1490.31 1 1490.31 73.76 <0.0001 B - Initial moisture content 37248.12 1 37248.12 1843.46 <0.0001 C - Ventilation duration 492.82 1 492.82 24.39 0.0008 BC 221.27 1 221.27 10.95 0.0091 <![CDATA[A 2 ]]> 22313.89 1 22313.89 1104.34 <0.0001 <![CDATA[B 2 ]]> 20390.90 1 20390.90 1009.17 <0.0001 <![CDATA[C 2 ]]> 35099.95 1 35099.95 1737.14 <0.0001 residual 181.85 9 20.21 - - Missing item 128.05 5 25.61 1.90 0.2762 Pure error 53.80 4 13.45 - - Total deviation 1.264E+005 16 - - - Coefficient of determination 0.9986 - - - - Corrected determination coefficient 0.9974 - - - -

[0121] Based on the multiple regression fitting analysis, the regression equation model between the significant influencing factors of inoculum size (A), initial moisture content (B), and aeration time (C) and the total cellulase activity of the Trichoderma composting agent is as follows:

[0122] Y=181.62-13.65A-68.23B+7.85C-7.44BC+72.80A 2 +69.59B 2 +91.30C 2 In the equation, Y represents the predicted cellulose content in the composting substrate. Furthermore, based on a regression model, the optimal composting conditions and minimum cellulose content were predicted. The results showed that the optimal fermentation conditions were: Trichoderma inoculant dosage of 3.19%, initial moisture content of 59.78%, and aeration time of 1.48 h / d. After 20 days of composting, the predicted minimum cellulose content in the substrate was 164.07 mg / g.

[0123] 2.3.3 Analysis of the interaction of significant influencing factors

[0124] Response surface optimization (RSI) 3D surface plots and 2D contour plots visually reflect the relationship between experimental factors and response values, and comprehensively demonstrate the interaction between the two factors. The steepness of the 3D surface plot reflects the sensitivity of enzyme activity to changes in fermentation conditions; the steeper the slope, the more significant the impact on the experimental results. The RSI contour plot visually reflects the influence of each factor on the response value; the center point of the smallest ellipse in the contour lines is the lowest point of the response surface. Furthermore, the shape of the contour lines reflects the strength of the interaction effect; an ellipse indicates a significant interaction between the two factors, while a circle indicates the opposite. Analysis results from 2D contour plots and 3D surface plots show that B (initial moisture content) - C (aeration time) (e.g., ...) Figure 10 and Figure 11 The shape is nearly elliptical with a significant slope, indicating a substantial interaction between the two factors. The interaction between A (inoculant inoculum amount) and B (initial moisture content) and A (inoculant inoculum amount) and C (aeration time) (e.g.) Figure 6 , Figure 7 , Figure 8 and Figure 9 The three-dimensional graph has a small slope and weak interaction, so it has no significant effect on the rate of cellulose degradation.

[0125] 2.3.4 Validation of Optimal Fermentation Conditions via Response Surface Methodology

[0126] The results of the optimal fermentation conditions validation showed that the predicted optimal cellulose content in the compost substrate (164.07 mg / g) was only slightly different from the measured value of 173.19 mg / g, thus proving the reliability of the established prediction model. Before optimization of the compost fermentation conditions, the cellulose content in the compost substrate after 20 days was 246.73 mg / g. After response surface methodology optimization, the cellulose degradation rate increased by 29.81%.

[0127] 3. Conclusions and Discussion

[0128] Aerobic fermentation is an important method for reducing and recycling straw. Through aerobic fermentation, the lignocellulose in straw can be effectively degraded, while simultaneously generating humus. The products obtained from aerobic fermentation contain a large amount of nutrients and humus, which can be used as fertilizer for agricultural and forestry crops and as a soil conditioner. Therefore, the aerobic composting method used in this invention greatly increases the activity of Trichoderma fungi in the compost, and the application of regular aeration and a constant-temperature composting box maximizes the decomposition process of straw and cow manure.

[0129] This invention reflects the degree of substrate composting by measuring the cellulose content in cow manure and straw compost substrates. The fermentation conditions for cow manure and straw composting were optimized based on single-factor significance analysis and response surface methodology. Results showed that when cow manure and straw were mixed in a C / N ratio of 27:1, the cellulose content in the compost substrate after 20 days of fermentation, after optimization using response surface methodology, was 164.07 mg / g. The optimal composting conditions were: Trichoderma inoculant at 3.9%, initial moisture content at 59.78%, and aeration time at 1.48 h / d. After verification experiments, the cellulose content in the substrate after 20 days of fermentation was 173.19 mg / g, which was close to the predicted value, and the cellulose degradation rate increased by 29.81% compared to before optimization.

[0130] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of Trichoderma composting agent in compost preparation, characterized in that, The Trichoderma composting agent includes Trichoderma longibrachiatum T6, Trichoderma viride P6, and Trichoderma longibrachiatum Lu3; Trichoderma longibrachiatum T6 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.13183 and deposit date of November 14, 2016.

2. The application according to claim 1, characterized in that, The preparation method of the Trichoderma composting agent is as follows: Trichoderma longifolia T6, Trichoderma viride P6 and Trichoderma longifolia Lu3 were mixed in a volume ratio of 2:1:1 to obtain a seed mixture. The seed mixture was then inoculated into microcrystalline cellulose medium at a volume fraction of 2% and cultured at 26°C, light for 12 h / d, and 180 rpm for 3 days to obtain a Trichoderma composting agent.

3. The application according to claim 2, characterized in that, The effective viable count of *Trichoderma longifolia* T6 bacterial solution in the *Trichoderma* composting agent is 1×10⁻⁶. 5 The effective viable count of Trichoderma viride P6 culture was 5 × 10⁻⁶ cfu / mL. 3 The effective viable count of Trichoderma longifolia Lu3 culture was 5 × 10⁻⁶ cfu / mL. 3 cfu / mL.

4. The application according to claim 1, characterized in that, The compost is prepared using poultry and livestock manure and crop straw as raw materials.

5. A method for preparing compost using the Trichoderma composting agent as described in claim 1, characterized in that, Includes the following steps: (1) Mix poultry and livestock manure with crop straw to obtain compost substrate; (2) Inoculate the Trichoderma composting agent into the compost substrate obtained in step (1) and perform aerobic fermentation to obtain compost.

6. The method for preparing compost according to claim 5, characterized in that, The poultry and livestock manure and crop straw mentioned in step (1) are mixed at a C / N mass ratio of 27:1; the initial moisture content of the compost substrate mentioned in step (1) is 50-60%.

7. The method for preparing compost according to claim 5 or 6, characterized in that, The poultry and livestock manure mentioned in step (1) is cow manure, and the crop straw mentioned in step (1) is corn straw.

8. The method for preparing compost according to claim 5, characterized in that, The amount of Trichoderma composting agent in step (2) is calculated as 3-4% of the volume of the compost substrate.

9. The method for preparing compost according to claim 5, characterized in that, The temperature of the aerobic fermentation in step (2) is 20-35℃, the aeration time of the aerobic fermentation is 1-2h / d, and the time of the aerobic fermentation is 18-22d.

10. The compost prepared by the method for preparing compost as described in any one of claims 5 to 9.

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