Low-temperature-resistant lignin and cellulose-degrading bacterial strain and application thereof

Abstract

The present application belongs to the technical field of microorganisms and their applications, and in particular relates to a low-temperature-resistant composite microbial inoculum capable of degrading lignin and cellulose and application of the microbial inoculum in fecal fermentation. The microbial inoculum contains fusiform lysine bacillus LFS1 and bacillus tropicus BTS1. The microbial inoculum was preserved in the China General Microbiological Culture Collection Center on October 24, 2024, and the preservation numbers are GCMCC No: 32336 and No: 32338. The microbial inoculum can accelerate the fecal fermentation process under low-temperature conditions and is helpful to enrich the low-temperature-resistant strain resource library with lignin and cellulose degradation capability. The present application mixes two purified strains in different proportions to prepare a bacterial suspension and develops a series of high-efficiency composite microbial inoculums, which can effectively degrade lignin and cellulose under low-temperature conditions, promote the start-up temperature of composting, and improve the quality of composting. Compared with traditional methods such as artificial heating, the method has higher efficiency, lower cost, and is easier to operate.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and its application technology, specifically relating to a low-temperature resistant strain for degrading lignin and cellulose and its application. Background Technology

[0002] In recent years, China's livestock industry has developed rapidly, resulting in a large amount of livestock and poultry manure, which not only causes serious environmental pollution but also threatens the health of animals and humans. Aerobic composting technology, as an important way to utilize livestock and poultry manure resources, utilizes the metabolic activities of microorganisms to rapidly and effectively degrade organic matter in the manure, and uses high temperatures to kill pathogenic microorganisms and other harmful substances, ultimately forming safe and environmentally friendly organic fertilizer. However, traditional composting technology is inefficient in low-temperature environments, especially in northern China, where the long autumn and winter seasons and large diurnal temperature variations make it difficult for the compost pile to heat up quickly, leading to manure accumulation and environmental pollution.

[0003] Composting typically uses livestock and poultry manure, crop straw, and forestry waste as raw materials, with lignocellulose being the most difficult component to degrade. Adding exogenous lignin-degrading bacteria and highly efficient cellulose-producing bacteria can accelerate the degradation of lignin and cellulose, stimulate microbial reproduction, quickly initiate composting fermentation, and shorten the composting process. Although a large number of microorganisms capable of degrading lignin and cellulose exist in nature, their degradation effect is not ideal under low-temperature conditions. Traditional methods for solving the problems of starting and maintaining the temperature of low-temperature composting, such as injecting hot water and steam into the compost pile, laying heating devices or insulation materials, are not only energy-intensive but also ineffective. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide a low-temperature resistant strain for degrading lignin and cellulose and its applications.

[0005] To achieve the above objectives, the following technical solution is used:

[0006] A low-temperature resistant lignin and cellulose degradation strain, wherein the low-temperature resistant lignin and cellulose degradation strain is Bacillus BTS1 or / and Bacillus lysine LFS1; the Bacillus BTS1 (Bacillus sp.) was deposited at the China General Microbiological Culture Collection Center on October 24, 2024, with accession number CGMCC No: 32338;

[0007] The Lysinibacillus sp. was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 32336.

[0008] The strains are Bacillus BTS1 and Bacillus lysine LFS1; the two strains are mixed in a volume ratio of (1-3):(1-3).

[0009] Application of the aforementioned low-temperature resistant lignin and cellulose degrading strain, and the application of the strain in the degradation of lignin and / or cellulose.

[0010] Application of the strain in the degradation of lignin and / or cellulose at 10–15 °C.

[0011] Application of the aforementioned low-temperature resistant lignin and cellulose degrading strain, and application of the strain in low-temperature composting.

[0012] A low-temperature resistant, multifunctional bacterial agent containing the aforementioned bacterial strain.

[0013] The bacterial agent is a bacterial culture medium or bacterial suspension containing the bacterial strain.

[0014] The bacterial culture medium is prepared by culturing *Bacillus lysine-containing* LFS1 and *Bacillus thuringiensis* BTS1 separately in LB liquid medium at 10–15°C until the logarithmic growth phase. The culture medium is then centrifuged to collect the precipitate, which is then resuspended in sterile physiological saline to OD200. 600 The bacterial suspension has a concentration of 0.7–1.4.

[0015] A method of using the aforementioned microbial agent involves applying the agent to manure to be fermented, wherein the OD of the microbial agent... 600 When the concentration is 1.0, apply the inoculum to the manure to be fermented at an inoculum rate of 0.3 to 0.5 wt%.

[0016] A bio-organic fertilizer is obtained by adding the bacterial strain or the bacterial agent to manure and sewage for composting.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention isolates two cryogenically resistant bacterial strains from livestock and poultry manure, which are highly efficient at degrading lignin and cellulose. These strains were molecularly identified as LFS1 and BTS1. These bacteria can efficiently degrade lignin and cellulose under low-temperature conditions, contributing to a richer resource of cryogenically resistant bacterial strains capable of degrading both lignin and cellulose. Simultaneously, this bacterial agent can also promote compost initiation and temperature rise, improving compost quality. These strains exhibit strong adaptability, cryogenic tolerance, high efficiency, and are environmentally friendly, enabling the degradation of lignin and cellulose in a microbially adapted low-temperature environment, promoting rapid initiation of manure fermentation and improving the quality of fermentation products. Attached Figure Description

[0019] Figure 1 The images show the morphological characteristics of LFS1 and BTS1 strains provided in the embodiments of the present invention.

[0020] Figure 2 The figure shows the antagonistic experimental results of LFS1 and BTS1 strains provided in the embodiments of the present invention.

[0021] Figure 3 The graph shows the results of the lignin degradation ability of LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature (10℃) conditions.

[0022] Figure 4 The graph shows the results of the lignin degradation ability of LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature (15℃) conditions.

[0023] Figure 5 The graph shows the results of the lignin degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention under normal temperature (30℃).

[0024] Figure 6 The graph shows the cellulose degradation ability of LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature (10℃) conditions.

[0025] Figure 7 The graph shows the cellulose degradation ability of LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature (15℃) conditions.

[0026] Figure 8 The graph shows the cellulose degradation capabilities of the LFS1 and BTS1 strains provided in this embodiment of the invention at room temperature (30°C).

[0027] Figure 9 The temperature change diagram of LFS1 and BTS1 strains during cow manure composting at low temperature (15℃) provided in the embodiments of the present invention.

[0028] Figure 10 The image shows the seed germination index results of LFS1 and BTS1 strains provided in this embodiment of the invention under low temperature conditions (15℃). Detailed Implementation

[0029] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0030] Example 1

[0031] Isolation and identification of strains:

[0032] In this invention, the strains of *Bacillus fusiformis* LFS1 and *Bacillus tropicalis* BTS1 were obtained by screening livestock and poultry manure from a livestock and poultry farm in Shenyang City. The specific steps are as follows:

[0033] A. Screening for low-temperature resistant bacteria in livestock and poultry manure: The process of strain isolation and purification is as follows: Weigh 10g of fresh manure sample collected from a livestock and poultry farm and place it in a 250mL Erlenmeyer flask containing glass beads. Add 90mL of sterile water, shake thoroughly, and let stand for 2 hours. Take 1mL of the supernatant and dilute it to 10mL with sterile water to obtain 10... -1 Dilute the solution by 1:1. Mix well using a pipette, then take 1 mL and dilute with sterile water to 10 mL to make a 10:10 dilution. -2 Dilute the solution by 10 times, and dilute it to 10 using the method described above. -9 Times. Take 10. -1 10 -3 10 -5 10 -7 10 -9 Five different dilutions (100 μL each) were added to bacterial, fungal, and actinomycete media, respectively. The media were incubated upside down at 15°C for 1–7 days, and growth was observed. Single colonies were repeatedly streaked onto solid media based on colony morphology to isolate and purify the strains, ultimately obtaining single bacterial strains. For screening of psychrophilic strains, purified strains were inoculated onto solid media and cultured at 0°C, 4°C, 10°C, 15°C, and 30°C. After 7 days, strain growth was observed. Strains that could grow at 0°C with a maximum growth temperature not exceeding 20°C were preliminarily identified as psychrophiles. Strains that could grow normally at 0–5°C with a maximum growth temperature exceeding 20°C were preliminarily identified as psychrophilic bacteria, and their results were recorded.

[0034] B. Preliminary screening of low-temperature resistant lignin and cellulose degrading strains: The above-mentioned low-temperature resistant strains were streaked on lignin selection medium and incubated at 15℃. The growth of the strains was observed. Strains with good growth were inoculated onto aniline blue decolorizing medium and incubated at 15℃ for 48 hours. Strains with obvious decolorization zones were selected, indicating lignin-degrading strains, and their records were recorded. The above strains were then inoculated onto CMC-Na solid medium and placed in a 15℃ incubator. After single colonies grew, they were removed, stained with 1 mg / mL Congo red solution for 15 min, and then washed with 1 mol / L NaCl for 15 min. The ratio of the hydrolysis zone to the colony diameter was compared to preliminarily screen for low-temperature resistant strains with lignin and cellulose degradation capabilities, and the results were recorded.

[0035] C. Secondary Screening of Low-Temperature Resistant Lignin and Cellulose Degrading Strains: The low-temperature resistant lignin and cellulose degrading strains obtained from the initial screening were inoculated into 100mL Erlenmeyer flasks containing 40mL of lignin degradation medium and incubated at 15℃ for 7 days. Standard solutions of sodium lignin sulfonate with concentrations of 0, 5, 10, 20, 30, 40, 50, and 60 mg / L were prepared, and the absorbance values ​​corresponding to each concentration at 280nm were measured to construct a lignin standard absorption curve. Under aseptic conditions, 1mL of bacterial culture was transferred to a 1.5mL EP tube, centrifuged at 12000r / min for 5min, and the supernatant was collected and diluted 10-fold with distilled water. The absorbance was measured at a wavelength of 280nm. The lignin degradation rate of each strain was calculated using the lignin degradation rate calculation formula, and the strains with better lignin degradation effects were recorded.

[0036] Formula for calculating lignin degradation rate:

[0037]

[0038] Note: A1 represents the concentration of sodium lignosulfonate in the control (non-inoculated) sample.

[0039] A2 represents the concentration of sodium lignosulfonate in the sample to be tested.

[0040] Secondary screening of cellulose-degrading bacteria: The low-temperature resistant lignin- and cellulose-degrading strains obtained from the initial screening were inoculated into liquid fermentation medium and cultured for 7 days at 15℃ and 180 rpm. The cellulase activity was then measured. 2 mL of glucose standard solutions with concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mg / mL were prepared, 2 mL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min. The solutions were then rapidly cooled to room temperature, and distilled water was added to bring the volume to 20 mL. The absorbance values ​​corresponding to each concentration at 540 nm were measured, and a glucose standard curve was plotted. 4 mL of fermentation broth was transferred to a 5 mL centrifuge tube and centrifuged at 3000 rpm for 10 min. The supernatant was used as the crude enzyme solution. Four sterile and dried 10 mL centrifuge tubes were labeled, and 1.5 mL of citrate buffer was added to each tube, followed by 0.5 g of qualitative analysis filter paper. Add 0.5 mL of crude enzyme solution to each of the three centrifuge tubes (excluding the blank tube) and incubate at 50°C for 1 hour. After the incubation, add 0.5 mL of the enzyme solution to be tested to the blank tube, and then add 3 mL of DNS reagent to each of the four test tubes. Incubate at 100°C for 10 minutes, rinse with tap water and cool, then bring the volume to 10 mL. Zero the blank tube and measure its absorbance at 540 nm, taking the average value. Record the strains with high cellulose-degrading enzyme activity.

[0041] Formula for calculating cellulose-degrading enzyme activity:

[0042]

[0043] Note: 180 is the molecular weight of glucose.

[0044] Considering both the lignin degradation rate and cellulose-degrading enzyme activity of the strains, two highly efficient, low-temperature-resistant strains with both lignin and cellulose degradation capabilities were selected. The obtained strains were identified as *Bacillus fusiformis*, named LFS1, deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No: 32336; and *Bacillus sp.* BTS1, deposited on October 24, 2024, at the same center, with accession number CGMCC No: 32338.

[0045] The obtained strains underwent DNA extraction, PCR amplification, sequence sequencing, and sequence alignment. The 16S rDNA sequence of strain LFS1 showed over 99.93% homology with *Lysinibacillus fusiformis*, indicating the closest phylogenetic relationship, and was therefore identified as *Lysinibacillus fusiformis*, abbreviated as LFS1. The 16S rDNA sequence of strain BTS1 showed over 99.96% homology with *Bacillus tropicus*, indicating the closest phylogenetic relationship, and was also identified as *Bacillus tropicus*, abbreviated as BTS1. Morphology is shown in […]. Figure 1 .

[0046] Components of each culture medium used for screening:

[0047] Bacterial culture medium (LB): 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 20.0g agar, add distilled water to 1000mL, pH: 7.0, autoclave at 121℃ for 30min.

[0048] Fungal culture medium (PDA): 200g fresh potato, 20g glucose, 20g agar, add distilled water to 1000mL (fresh potatoes are peeled, cut into small pieces, boiled for half an hour, filtered through gauze, and then glucose and agar are added), autoclave at 115℃ for 30min.

[0049] Actinomycete culture medium (Gao's No. 1): 20g soluble starch, 1g KNO3, 0.5g NaCl, 0.5g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 0.01g FeSO4, 20g agar, add distilled water to 1000mL, pH: 7.2~7.4, autoclave at 121℃ for 30min.

[0050] Lignin screening medium: 1.0 g sodium lignin sulfonate, 1.0 g KH2PO4, 0.5 g NaCl, 0.5 g MgSO4·7H2O, 0.1 g CaCl2, add distilled water to 1000 mL, 20 g agar, autoclave at 121 °C for 30 min.

[0051] Primary screening medium (CMC-Na): 20g sodium carboxymethyl cellulose, 0.5g yeast extract, 2.5g Na2HPO4, 1.5g KH2PO4, 2.5g peptone, 15g agar, add distilled water to 1000mL, pH: 7.0~7.2, autoclave at 121℃ for 30min.

[0052] Aniline blue decolorizing medium: 10g yeast extract, 10g glucose, 0.1g aniline blue, 20g agar, add distilled water to 1000mL, autoclave at 115℃ for 30min.

[0053] Liquid fermentation medium: CMC-Na 10g, NH4SO4 2g, K2HPO4 1g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.1g, pH: 7.0~7.2, add distilled water to make up to 1000mL, sterilize at 121℃ for 30min.

[0054] Main reagent preparation:

[0055] Citrate buffer: Weigh 4.83g of citric acid monohydrate, dissolve it in 750mL of distilled water, add 7.94g of trisodium citrate while stirring, bring the volume to 1000mL with distilled water, and adjust the pH to 4.8.

[0056] CMC-Na solution: Accurately weigh 1g of CMC-Na, slowly add 80mL of citrate buffer solution with pH=4.8, heat to dissolve, cool and dilute to 90mL with buffer solution, adjust pH to 4.8 with HCl or NaOH, and bring the volume to 100mL with deionized water.

[0057] DNS reagent: Weigh 182.0g of potassium sodium tartrate, dissolve it in 500mL of distilled water, heat in a 45℃ water bath, and add 6.3g of 3,5-dinitrosalicylic acid, 21.0g of sodium hydroxide, 5.0g of phenol, and 5g of anhydrous sodium sulfite to the hot solution in sequence. Stir until the reagent is completely dissolved, cool to room temperature, and make up to 1000mL with distilled water. Transfer to a brown reagent bottle and store at room temperature for 7 days before use.

[0058] The 16S rDNA sequence is as follows:

[0059] LFS1 bacteria

[0060]

[0061] BTS1 bacteria

[0062]

[0063] Example 2

[0064] Antagonism Experiment: The two low-temperature resistant lignin and cellulose-degrading strains were streaked separately on LB solid medium, and the growth status of the strains at the cross-streaked points was observed. If the growth of the two strains at the cross-streaked points was relatively weak or nonexistent, it indicated an antagonistic effect between the two strains; if both strains grew well at the cross-streaked points, it indicated no antagonistic effect between the two strains, and mixed culture could be performed. The results of the antagonism experiment between the two strains are as follows: Figure 2 As shown, the colonies of both strains can grow continuously at the intersection of the cross lines, indicating that the two strains do not antagonize each other and will not inhibit each other's growth. They can be used together as backup strains for compound bacterial agents.

[0065] Preparation of microbial agents:

[0066] The single-strain inoculum was prepared by activating the two single strains LFS1 and BTS1 separately in sterile inorganic salt liquid medium. After activation, each strain was inoculated at a 5 wt% inoculum into 100 mL of liquid LB medium and cultured in a constant temperature shaker at 15°C and 180 rpm until the logarithmic growth phase. Then, the inoculum was washed with sterile physiological saline to prepare OD. 600 A bacterial suspension with a concentration of 1.0 is considered a single-strain inoculum.

[0067] The compound microbial agent is obtained by mixing the above-mentioned single microbial agents in a certain proportion.

[0068] Example 3

[0069] The degradation of lignin in a low-temperature environment (10℃) was tested using the above-obtained degrading microbial agent:

[0070] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0071]

[0072] The above five microbial agents were inoculated onto aniline blue decolorizing medium under sterile conditions, and then incubated upside down in a constant temperature incubator at 10℃ for 48 hours. The decolorization effect was then observed. The experimental results showed that all five microbial agents were tolerant of low temperatures and had a lignin-degrading effect. Among them, agents 2, 4, and 5 showed significant decolorization effects, with agent 5 exhibiting the best decolorization effect. (See results below.) Figure 3 .

[0073] Culture medium components:

[0074] Aniline blue decolorizing medium: 10g yeast extract, 10g glucose, 0.1g aniline blue, 20g agar, add distilled water to 1000mL, autoclave at 115℃ for 30min.

[0075] Example 4

[0076] The degradation of lignin in a low-temperature environment (15℃) was tested using the above-obtained degrading microbial agent:

[0077] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0078]

[0079] The above five microbial agents were inoculated onto aniline blue decolorizing medium under sterile conditions, inverted in a constant temperature incubator, and cultured at 15℃ for 48 hours. The decolorization effect was then observed. The experimental results showed that all five microbial agents were tolerant of low temperatures and had a lignin-degrading effect. Among them, agents 3, 4, and 5 showed significant decolorization effects, with agent 3 exhibiting the best decolorization effect. (See results below.) Figure 4 .

[0080] Culture medium components:

[0081] Aniline blue decolorizing medium: 10g yeast extract, 10g glucose, 0.1g aniline blue, 20g agar, add distilled water to 1000mL, autoclave at 115℃ for 30min.

[0082] Example 5

[0083] The degradation of lignin by the above-obtained degrading microbial agent in a normal temperature environment (30℃) was tested.

[0084] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0085]

[0086]

[0087] The above five microbial agents were inoculated onto aniline blue decolorizing medium under sterile conditions, inverted in a constant temperature incubator, and cultured at 30℃ for 48 hours. The decolorization effect was then observed. The experimental results showed that all five microbial agents had a lignin degradation effect, with agents 2, 4, and 5 showing significant decolorization effects, and agent 4 exhibiting the best decolorization effect. (See results below.) Figure 5 .

[0088] Culture medium components:

[0089] Aniline blue decolorizing medium: 10g yeast extract, 10g glucose, 0.1g aniline blue, 20g agar, add distilled water to 1000mL, autoclave at 115℃ for 30min.

[0090] Example 6

[0091] The degradation of cellulose in a low-temperature environment (10℃) was tested using the above-obtained degrading bacterial agent:

[0092] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0093]

[0094] The above five bacterial agents were aseptically inoculated onto CMC-Na solid medium, inverted in a constant temperature incubator, and cultured at 10℃ for 48 h. After staining with 1 mg / mL Congo red solution, the culture was immediately rinsed with sterile water, and then immersed in 1 mol / L NaCl for 30 min. The hydrolysis zone and colony diameter were observed.

[0095] Experimental results showed that all five microbial agents could tolerate low temperatures and degrade cellulose. Agents 3 and 4 exhibited obvious hydrolysis zones and showed better degradation effects. See results below. Figure 6 .

[0096] Culture medium components:

[0097] CMC-Na solid culture medium: 20g sodium carboxymethyl cellulose, 0.5g yeast extract, 2.5g Na2HPO4, 1.5g KH2PO4, 2.5g peptone, 15g agar, add distilled water to 1000mL, pH: 7.0~7.2, autoclave at 121℃ for 30min.

[0098] Example 7

[0099] The degradation of cellulose in a low-temperature environment (15℃) was tested using the above-obtained degrading bacterial agent:

[0100] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0101]

[0102] The above five bacterial agents were aseptically inoculated onto CMC-Na solid medium, inverted in a constant temperature incubator, and cultured at 15℃ for 48 h. After staining with 1 mg / mL Congo red solution, the culture was immediately rinsed with sterile water, and then immersed in 1 mol / L NaCl for 30 min. The hydrolysis zone and colony diameter were observed.

[0103] Experimental results showed that all five microbial agents could tolerate low temperatures and degrade cellulose. Agents 3 and 4 exhibited obvious hydrolysis zones and showed better degradation effects. See results below. Figure 7 .

[0104] Culture medium components:

[0105] CMC-Na solid culture medium: 20g sodium carboxymethyl cellulose, 0.5g yeast extract, 2.5g Na2HPO4, 1.5g KH2PO4, 2.5g peptone, 15g agar, add distilled water to 1000mL, pH: 7.0~7.2, autoclave at 121℃ for 30min.

[0106] Example 8

[0107] The degradation of lignin by the above-obtained degrading microbial agent in a normal temperature environment (30℃) was tested.

[0108] The two single-strain inoculants mentioned above are mixed in the following volume ratio to prepare a compound inoculant.

[0109]

[0110] The five bacterial agents were aseptically inoculated onto CMC-Na solid medium and incubated upside down in a constant temperature incubator at 30℃ for 48 h. After staining with 1 mg / mL Congo red solution, the culture was immediately rinsed with sterile water and then immersed in 1 mol / L NaCl for 30 min. The hydrolysis zone and colony diameter were observed. The experimental results showed that all five bacterial agents had a degrading effect on cellulose, with agents 2 and 5 showing obvious hydrolysis zones and better degradation effects. (See attached results). Figure 8 .

[0111] Culture medium components:

[0112] CMC-Na solid culture medium: 20g sodium carboxymethyl cellulose, 0.5g yeast extract, 2.5g Na2HPO4, 1.5g KH2PO4, 2.5g peptone, 15g agar, add distilled water to 1000mL, pH: 7.0~7.2, autoclave at 121℃ for 30min.

[0113] Example 9

[0114] The above experiment investigated the effect of the obtained degradation bacteria on the composting temperature of cow manure:

[0115] The cow manure used in the experiment was collected from a livestock farm in Shenyang City, Liaoning Province. The pH was 8.48, and the moisture content was 55.86%. The cow manure was mixed with straw at a mass ratio of 10:1 and composted in the open air. The compost pile weighed approximately 40 kg, and the composting period was 17 days. Two treatments were set up in the cow manure composting experiment. Samples from each treatment group were added to each treatment group during composting. The two treatment groups were as follows:

[0116] Treatment 1 (CK): Control treatment: Add 0.3% (w:w) sterile water.

[0117] Treatment 2 (T1~T5): Microbial agent treatment: The microbial agent described in Example 2 was added to each group. At the same time, the compound microbial agent was mixed in different proportions as described below to prepare a compound microbial agent, which was added to 5 different piles respectively (the inoculum amount of microbial agent was 0.3% (w:w) of cow manure). The microbial agent formula is shown in the table below:

[0118]

[0119]

[0120] During the cow manure composting process, the pile was turned over every two days. The pile temperature was measured daily from 9:00 to 10:00 and 16:00 to 17:00, and the average value was recorded. The ambient temperature was also recorded. Experimental results showed that all five microbial agents could promote composting initiation and heating, increase the peak compost temperature, and prolong the duration of high temperatures. The T3 treatment group had the highest peak temperature of 72℃, and the high-temperature period lasted for eight days. The peak temperatures of T2, T4, and T5 reached approximately 69℃, which was 6–8℃ higher than the control group (CK), and the high-temperature period was 1–2 days longer than the CK group. See the results below. Figure 9 .

[0121] Example 10

[0122] The above-mentioned experiment on the effect of the obtained degradation microbial agent on compost quality (seed germination index) was conducted:

[0123] The cow manure used in the experiment was collected from a livestock farm in Shenyang City, Liaoning Province. The pH was 8.28, and the moisture content was 54.70%. The cow manure was mixed with straw at a mass ratio of 10:1 and composted in the open air. The compost pile weighed approximately 40 kg, and the composting period was 16 days. Two treatments were set up in the cow manure composting experiment. Samples from each treatment group were added to each treatment group during composting. The two treatment groups were as follows:

[0124] Treatment 1 (CK): Control treatment: Add 0.3% (w:w) sterile water.

[0125] Treatment 2 (T1~T5): Microbial agent treatment: The microbial agents described in Example 2 were added to each group. Simultaneously, a compound microbial agent was prepared by mixing the two agents according to the different proportions described below, and added to five different compost piles (the inoculum amount of the microbial agent was 0.3% (w:w) of the cow manure). The microbial agent formulation is shown in the table below. After adding the microbial agent, the piles were thoroughly mixed to ensure even distribution. Subsequently, the piles were turned over every two days until the composting process was completed.

[0126]

[0127] After composting, take 10.00g of fresh sample and place it in a 200ml conical flask. After adjusting the sample moisture content, add water at a solid-liquid ratio (mass / volume) of 1:10. Tighten the cap and fix the flask vertically on a reciprocating horizontal shaker. Adjust the frequency to 100 times / min and the amplitude to not less than 40mm. Shake and extract at 25℃ for 1h. Take the supernatant and filter it on a filter device with filter paper pre-installed. Collect the filtered extract. Place one or two sheets of qualitative filter paper in a 9cm petri dish, and evenly place 10 plump cucumber seeds of similar size on them. Add 10mL of the sample extract, cover the culture, and incubate in a (25±2)℃ incubator in the dark for 48h. Count the number of germinated seeds, measure the root length of each seed with calipers, and calculate the germination index. Experimental results showed that all five microbial agents could promote the improvement of compost quality. Among them, T4 had the highest germination index (0.93693), while T3 and T5 had indices of 0.87348 and 0.859802, respectively, which were 0.26–0.29 higher than the control group. See the results below. Figure 10 .

[0128] In summary, the bacterial suspension provided by this invention has good potential for degrading lignin and cellulose. The free single bacteria screened by this invention can rapidly grow to the logarithmic phase within one day, exhibiting fast reproduction speed and strong adaptability. They can effectively degrade lignin and cellulose under low-temperature conditions, promote composting initiation and heating, improve compost quality, and are highly efficient, low-cost, and easy to operate, making them worthy of promotion.

Claims

1. A low-temperature resistant strain for lignin and cellulose degradation, characterized in that: The low-temperature resistant lignin and cellulose degrading strains are Bacillus BTS1 and Bacillus lysine LFS1; the two strains are mixed in a volume ratio of (1-3):(1-3); the Bacillus ( Bacillus sp. BTS1 was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 32338. The lysine-containing Bacillus ( Lysinibacillus sp. LFS1 was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 32336.

2. The application of the low-temperature resistant lignin and cellulose-degrading strain according to claim 1, characterized in that: Application of the strain in the degradation of lignin and / or cellulose.

3. The application of the low-temperature resistant lignin and cellulose-degrading strain according to claim 2, characterized in that: Application of the strain in the degradation of lignin and / or cellulose at 10–15 °C.

4. The application of the low-temperature resistant lignin and cellulose-degrading strain according to claim 1, characterized in that: Application of the strain in low-temperature composting.

5. A low-temperature resistant, multifunctional bacterial agent, characterized in that: The bacterial agent contains the strain described in claim 1.

6. The low-temperature resistant multifunctional bacterial agent according to claim 5, characterized in that: The bacterial agent is a bacterial culture medium or bacterial suspension containing the strain described in claim 1.

7. The low-temperature resistant multifunctional bacterial agent according to claim 6, characterized in that: The bacterial culture medium is prepared by culturing *Bacillus lysine-containing* LFS1 and *Bacillus thuringiensis* BTS1 separately in LB liquid medium at 10–15°C until the logarithmic growth phase. The culture medium is then centrifuged to collect the precipitate, which is then resuspended in sterile physiological saline to OD200. 600 The bacterial suspension has a concentration of 0.7–1.

4.

8. A method of using the microbial agent according to claim 5, characterized in that: The bacterial agent is applied to the manure to be fermented, and the OD of the bacterial agent... 600 When the concentration is 1.0, apply the inoculum to the manure to be fermented at an inoculum rate of 0.3 to 0.5 wt%.

9. A bio-organic fertilizer, characterized in that: The strain described in claim 1 or the microbial agent described in claim 5 is added to manure for composting to obtain bio-organic fertilizer.

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