Low-temperature-resistant lignin and cellulose degradation strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms and application thereof, and particularly relates to a low-temperature-resistant complex microbial inoculant capable of degrading lignin and cellulose and application of the complex microbial inoculant in manure fermentation. The microbial inoculum contains lysinibacillus fusiformis LFS1 and bacillus tropicalis BTS1. The strain is preserved in the China General Microbiological Culture Collection Center on October 24, 2024, and the preservation number is GCMCCNO: 32336, No: 32338. The microbial agent disclosed by the invention can accelerate a manure fermentation process under a low-temperature condition, and is beneficial to enriching a low-temperature-resistant strain resource library with lignin and cellulose degradation capabilities. According to the invention, two purified strains are mixed according to different proportions to prepare a bacterial suspension, and a series of high-efficiency complex microbial inoculants are developed, so that lignin and cellulose can be effectively degraded in a low-temperature environment, starting temperature rise of compost is promoted, and the quality of the compost is improved. Compared with traditional methods such as manual heating, the method is higher in efficiency, lower in cost and easier to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and their applications, and specifically relates to a low-temperature resistant lignin and cellulose degrading strain and its application. Background Art

[0002] In recent years, China's animal husbandry has developed rapidly, and with it comes a large amount of livestock and poultry manure, which not only causes serious pollution to the environment, but also threatens the health of animals and humans. Aerobic composting technology is an important way to utilize livestock and poultry manure resources. It uses the metabolic activities of microorganisms to quickly and effectively degrade organic matter in manure, and kills pathogenic microorganisms and other harmful substances through high temperatures, ultimately forming safe and environmentally friendly organic fertilizer. However, traditional composting technology is inefficient in low temperature environments, especially in northern China, where the autumn and winter seasons are long and the temperature difference between day and night is large, making it difficult for the pile to heat up quickly, resulting in manure accumulation and environmental pollution.

[0003] Composting fermentation usually uses livestock and poultry manure, crop straw, forestry waste, etc. as raw materials, and lignocellulose is the most difficult component to degrade. Adding exogenous lignin-degrading bacteria, high-efficiency cellulose bacteria and other microbial agents can accelerate the degradation of lignin and cellulose, stimulate microbial reproduction, quickly start composting fermentation, and shorten the composting process. Although there are a large number of microorganisms that can degrade lignin and cellulose in nature, their degradation effect is not ideal under low temperature conditions. Traditional methods to solve the problems of starting and keeping warm in low-temperature composting, such as pouring hot water and steam into the pile, laying heating devices or insulation materials, etc., not only consume electricity and energy, but also have poor results. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a low-temperature resistant lignin and cellulose degrading strain and its application.

[0005] To achieve the above purpose, the following technical solutions are used:

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

[0007] The lysinibacillus sp. LFS1 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 24, 2024, with a deposit number of CGMCC No: 32336.

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

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

[0010] The strain is used in degrading lignin and / or cellulose at 10-15°C.

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

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

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

[0014] The bacterial agent culture solution is obtained by culturing the lysine Bacillus LFS1 and Bacillus BTS1 in LB liquid culture medium at 10-15°C to the logarithmic growth phase, and the culture solution is centrifuged to collect the precipitate, and the precipitate is resuspended in sterile saline to an OD 600 It is a bacterial suspension with a concentration of 0.7 to 1.4.

[0015] A method for using the bacterial agent, wherein the bacterial agent is applied to the feces to be fermented, and the OD of the bacterial agent is 600 =1.0, add 0.3-0.5wt% of inoculum into the manure to be fermented.

[0016] A biological organic fertilizer is obtained by adding the strain or the bacterial agent into feces and sewage for composting.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention screens and separates two low-temperature-resistant bacterial strains that can efficiently degrade lignin and cellulose from livestock and poultry feces. The two strains are identified as LFS1 and BTS1 by molecular identification. The bacteria can efficiently degrade lignin and cellulose under low temperature conditions, which is beneficial to enriching the resource library of low-temperature-resistant bacteria with the ability to degrade lignin and cellulose. At the same time, the bacterial agent can also promote the start-up temperature rise of composting and improve the quality of composting. The strain has strong adaptability, low temperature resistance, high efficiency, and is environmentally friendly and harmless. It can achieve the degradation of lignin and cellulose in a low-temperature environment adapted to microorganisms, promote the rapid start-up of feces fermentation, and improve the quality of fermentation products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphology of LFS1 and BTS1 strains provided in the embodiments of the present invention.

[0020] Figure 2 This is a graph showing the results of an antagonistic experiment between LFS1 and BTS1 strains provided in an embodiment of the present invention.

[0021] Figure 3 This is a graph showing the lignin degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature conditions (10°C).

[0022] Figure 4 This is a graph showing the lignin degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature conditions (15°C).

[0023] Figure 5 This is a graph showing the lignin degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention at room temperature (30°C).

[0024] Figure 6 This is a graph showing the ability of LFS1 and BTS1 strains provided in an embodiment of the present invention to degrade cellulose under low temperature conditions (10°C).

[0025] Figure 7 This is a graph showing the cellulose degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention under low temperature conditions (15°C).

[0026] Figure 8 This is a graph showing the cellulose degradation ability of the LFS1 and BTS1 strains provided in the embodiments of the present invention at room temperature (30°C).

[0027] Fig. 9 This is a temperature change diagram of the LFS1 and BTS1 strains provided in the embodiment of the present invention during cow dung composting in a low temperature environment (15°C).

[0028] Fig.10 This is a graph showing the seed germination index results of the LFS1 and BTS1 strains provided in an embodiment of the present invention under a low temperature environment (15°C). DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0030] Example 1

[0031] Isolation and identification of strains:

[0032] In the present invention, the lysinophilic Bacillus fusiformis LFS1 and tropical Bacillus fusiformis BTS1 strains are screened from livestock and poultry feces in a livestock and poultry farm in Shenyang, and the specific steps are as follows:

[0033] A. Screening of low-temperature resistant bacteria in livestock and poultry feces: Strain separation and purification process: Weigh 10g of fresh feces sample collected from livestock and poultry farms and place it in a 250mL conical flask with glass beads, add 90mL of sterile water, shake it thoroughly and let it stand for 2h. Take 1mL of supernatant and dilute it to 10mL with sterile water to make 10 -1 Use a pipette to mix well, then draw 1mL and dilute to 10mL with sterile water to make 10 times dilution. -2 Dilute to 10 times the dilution as above -9 times. Take 10 -1 , 10 -3 , 10 -5 , 10 -7 , 10 -9 100 μL of the 5 dilutions were added to bacterial culture medium, fungal culture medium, and actinomycete culture medium, respectively, and inverted and cultured in a 15°C constant temperature incubator for 1 to 7 days to observe their growth. According to the morphological characteristics of the colonies, single colonies were picked and repeatedly streaked on solid culture medium, so that the strains in the culture medium could be separated and purified, and finally a single strain was obtained. Screening of low-temperature resistant strains: The purified strains were inoculated on solid culture medium and cultured at different temperatures of 0°C, 4°C, 10°C, 15°C, and 30°C. After 7 days, the growth of the strains was observed. The strains that could grow at 0°C and whose maximum growth temperature was not higher than 20°C were preliminarily determined to be psychrophilic bacteria, and the strains that could grow normally at 0-5°C and whose maximum growth temperature was higher than 20°C were preliminarily determined to be psychrophilic bacteria, and recorded.

[0034] B. Preliminary screening of low-temperature resistant lignin and cellulose degradation strains: streak the above low-temperature resistant strains on the lignin screening medium, culture at a constant temperature of 15°C and observe the growth of the strains, select the strains with good growth and inoculate them on the aniline blue decolorization medium, culture at a constant temperature of 15°C for 48h, select the strains with obvious decolorization circles, that is, the lignin degradation strains, and record them. Inoculate the above strains on the CMC-Na solid culture medium and place it in a 15°C constant temperature incubator. After a single colony grows, take it out, stain it with 1mg / mL Congo red solution for 15min, and then soak it with 1mol / L NaCl for 15min. Compare the ratio of the hydrolysis circle to the community diameter, preliminarily screen out the low-temperature resistant strains with lignin and cellulose degradation capabilities, and record them.

[0035] C. Rescreening of cold-resistant lignin and cellulose degradation strains: Rescreening of lignin degradation bacteria: The cold-resistant lignin and cellulose degradation strains obtained in the above primary screening were inoculated in a 100mL conical flask containing 40mL lignin degradation medium and cultured at 15℃ for 7d. Prepare sodium lignin sulfonate standard solutions with concentrations of 0, 5, 10, 20, 30, 40, 50, and 60mg / L, measure the absorbance values ​​corresponding to each concentration at 280nm, and make a lignin standard absorption curve. Aseptically draw 1mL of bacterial solution into a 1.5mL EP tube, centrifuge at 12000r / min for 5min, take the supernatant, dilute it 10 times with distilled water, and measure its absorbance at a wavelength of 280nm. Calculate the degradation rate of lignin by each strain according to the lignin degradation rate calculation formula, and record the strains with better lignin degradation effect.

[0036] The calculation formula of lignin degradation rate is:

[0037]

[0038] Note: A1 represents the concentration of sodium lignin sulfonate in the control (no inoculation)

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

[0040] Rescreening of cellulose-degrading bacteria: The low-temperature resistant lignin and cellulose-degrading strains obtained in the initial screening were inoculated into the liquid fermentation medium, and the cellulase activity was determined after culturing in a constant temperature shaking incubator at 15°C and 180r / min for 7 days. Prepare 2mL of glucose standard solution with concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7mg / mL, add 2mL of DNS reagent and mix well, boil in a water bath for 5min, take out and quickly cool to room temperature, add distilled water to 20mL, measure the absorbance values ​​corresponding to each concentration at 540nm, and draw a glucose standard curve. Take 4mL of fermentation broth from the liquid fermentation medium, place it in a 5mL centrifuge tube, centrifuge it at 3000r / min for 10min, and use the supernatant as the crude enzyme solution. Take 4 sterilized and dried 10mL centrifuge tubes and number them, add 1.5mL of citric acid buffer to each tube, and then add 0.5g of qualitative analysis filter paper to each tube. Add 0.5mL of crude enzyme solution to the three centrifuge tubes outside the blank tube, and place them in a water bath at 50℃ for 1h. After the water bath, add 0.5mL of the enzyme solution to be tested to the blank tube, and then add 3mL of DNS reagent to the four test tubes, place them in a boiling water bath at 100℃ for 10min, rinse with tap water and cool, and dilute to 10mL. Zero the blank tube, measure its absorbance at a wavelength of 540nm, and take the average value. Record the strains with higher cellulose degrading enzyme activity.

[0041] Calculation formula for cellulose degradation enzyme activity:

[0042]

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

[0044] Taking into account the lignin degradation rate and cellulose degradation enzyme activity of the strains, two high-efficiency low-temperature resistant strains with both lignin and cellulose degradation capabilities were selected. After identification, the above-obtained strains were spindle-shaped lysine Bacillus, named LFS1, deposited in the General Microbiological Center of the China Microbiological Culture Collection on October 24, 2024, with the deposit numbers CGMCC No: 32336; Bacillus BTS1 (Bacillus sp.), deposited in the General Microbiological Center of the China Microbiological Culture Collection on October 24, 2024, with the deposit numbers CGMCC No: 32338.

[0045] The obtained strains were subjected to DNA extraction, PCR amplification, sequence sequencing and sequence comparison. The 16s rDNA sequence of strain LFS1 was more than 99.93% homologous to Lysinibacillus fusiformis, and the closest relationship was identified as Lysinibacillus fusiformis, referred to as LFS1; the 16s rDNA sequence of strain BTS1 was more than 99.96% homologous to Bacillus tropicus, and the closest relationship was identified as Bacillus tropicus, referred to as BTS1, and the morphology was shown in FIG. Figure 1 .

[0046] Components of each culture medium for screening:

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

[0048] Fungal culture medium (PDA): 200 g fresh potatoes, 20 g glucose, 20 g agar, add distilled water to 1000 mL (peel the fresh potatoes, cut into small pieces, boil for half an hour, filter with gauze, and then add glucose and agar), sterilize at 115°C for 30 min.

[0049] Actinomycete culture medium: (Gao's No. 1) soluble starch 20g, KNO3 1g, NaCl 0.5g, K2HPO4·3H2O0.5g, MgSO4·7H2O 0.5g, FeSO4 0.01g, agar 20g, add distilled water to 1000mL, pH: 7.2~7.4, autoclave at 121℃ for 30min.

[0050] Lignin screening medium: sodium lignin sulfonate 1.0 g, KH2PO4 1.0 g, NaCl 0.5 g, MgSO4·7H2O0.5 g, CaCl2 0.1 g, add distilled water to 1000 mL, agar 20 g, and autoclave at 121°C for 30 min.

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

[0052] Aniline blue decolorization medium: 10 g yeast extract, 10 g glucose, 0.1 g aniline blue, 20 g agar, add distilled water to 1000 mL, and sterilize at 115°C for 30 min.

[0053] Liquid fermentation medium: CMC-Na 10g, NH4SO4 2g, K2HPO4 1g, MgSO4·7H2O 0.5g, NaCl0.5g, 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 configuration:

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

[0056] CMC-Na solution: Accurately weigh 1 g of CMC-Na, slowly add 80 mL of citric acid buffer with pH = 4.8, heat to dissolve, dilute to 90 mL with buffer after cooling, adjust pH to 4.8 with HCl or NaOH, and make up to 100 mL with deionized water.

[0057] DNS reagent: Weigh 182.0 g of potassium sodium tartrate and dissolve it in 500 mL of distilled water. Heat in a water bath at 45°C. Add 6.3 g of 3,5-dinitrosalicylic acid, 21.0 g of sodium hydroxide, 5.0 g of phenol, and 5 g 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 1000 mL 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:

[0059] LFS1 bacteria

[0060]

[0061] BTS1 bacteria

[0062]

[0063] Example 2

[0064] Antagonism experiment: The two cold-resistant lignin and cellulose-degrading strains were streaked on LB solid culture medium and the growth of the strains at the intersection was observed. If the two strains grew relatively weakly or did not grow at the intersection, it indicated that there was antagonism between the two strains; if both strains could grow well at the intersection, it indicated that there was no antagonism between the two strains and mixed culture could be performed. The results of the antagonism experiment between the two strains are shown in Figure 2. Figure 2 As shown, the colonies of the two strains can grow continuously at the cross lines that intersect each other, indicating that the two strains have no antagonism to each other and will not inhibit each other's growth. They can be used in combination as backup strains for composite bacterial agents.

[0065] Preparation of inoculants:

[0066] The single bacterial inoculant was prepared by activating the two single strains LFS1 and BTS1 in a sterile inorganic salt liquid medium. After activation, they were inoculated into 100 mL of liquid LB medium at a 5 wt % inoculum and cultured in a constant temperature shaker at 15 ° C and 180 r / min until the logarithmic phase. The strains were then rinsed with sterile saline to obtain an OD 600 A bacterial suspension with a pH of 1.0 is a single-bacterial agent.

[0067] The composite bacterial agent is obtained by mixing the above-obtained single bacterial agents in proportion.

[0068] Example 3

[0069] The degradation test of lignin in low temperature environment (10°C) was carried out using the degradation bacteria obtained above:

[0070] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0071]

[0072] The above five bacterial agents were inoculated on aniline blue decolorization medium under a sterile environment, inverted in a constant temperature incubator, and cultured at 10℃ for 48h to observe the decolorization effect. The experimental results show that the five bacterial agents can tolerate low temperatures and have a degrading effect on lignin. Among them, bacterial agents 2, 4, and 5 have obvious decolorization effects, and bacterial agent 5 has the best decolorization effect. Figure 3 .

[0073] Medium ingredients:

[0074] Aniline blue decolorization medium: 10 g yeast extract, 10 g glucose, 0.1 g aniline blue, 20 g agar, add distilled water to 1000 mL, and sterilize at 115°C for 30 min.

[0075] Example 4

[0076] The degradation test of lignin in low temperature environment (15°C) was carried out using the degradation bacteria obtained above:

[0077] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0078]

[0079] The above five bacterial agents were inoculated on aniline blue decolorization medium under a sterile environment, inverted in a constant temperature incubator, and cultured at 15℃ for 48 hours to observe the decolorization effect. The experimental results showed that the five bacterial agents can tolerate low temperatures and have a degrading effect on lignin. Among them, bacterial agents 3, 4, and 5 have obvious decolorization effects, and bacterial agent 3 has the best decolorization effect. Figure 4 .

[0080] Medium ingredients:

[0081] Aniline blue decolorization medium: 10 g yeast extract, 10 g glucose, 0.1 g aniline blue, 20 g agar, add distilled water to 1000 mL, and sterilize at 115°C for 30 min.

[0082] Example 5

[0083] The degradation test of lignin in room temperature environment (30°C) was carried out using the degradation bacteria obtained above:

[0084] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0085]

[0086]

[0087] The above five bacterial agents were inoculated on aniline blue decolorization medium under a sterile environment, inverted in a constant temperature incubator, and cultured at 30℃ for 48h to observe the decolorization effect. The experimental results showed that all five bacterial agents had a degrading effect on lignin, among which bacterial agents 2, 4, and 5 had obvious decolorization effects, and bacterial agent 4 had the best decolorization effect. Figure 5 .

[0088] Medium ingredients:

[0089] Aniline blue decolorization medium: 10 g yeast extract, 10 g glucose, 0.1 g aniline blue, 20 g agar, add distilled water to 1000 mL, and sterilize at 115°C for 30 min.

[0090] Example 6

[0091] The degradation test of cellulose in a low temperature environment (10°C) was carried out using the above-obtained degradation bacteria agent:

[0092] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0093]

[0094] The five bacterial agents were inoculated on CMC-Na solid culture medium under a sterile environment, inverted in a constant temperature incubator, cultured at 10°C for 48 hours, stained with 1mg / mL Congo red solution, immediately rinsed with sterile water, and then soaked in 1mol / L NaCl for 30 minutes. The hydrolysis zone and community diameter were observed.

[0095] The experimental results show that all five bacterial agents can tolerate low temperatures and have a degrading effect on cellulose. Among them, bacterial agents 3 and 4 have obvious hydrolysis circles and have better degradation effects. Figure 6 .

[0096] Medium ingredients:

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

[0098] Example 7

[0099] The degradation test of cellulose in a low temperature environment (15°C) was carried out using the above-obtained degradation bacteria agent:

[0100] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0101]

[0102] The five bacterial agents were inoculated on CMC-Na solid culture medium under a sterile environment, inverted in a constant temperature incubator, cultured at 15°C for 48 hours, stained with 1 mg / mL Congo red solution, immediately rinsed with sterile water, and then soaked in 1 mol / L NaCl for 30 minutes. The hydrolysis zone and community diameter were observed.

[0103] The experimental results show that all five bacterial agents can tolerate low temperatures and have a degrading effect on cellulose. Among them, bacterial agents 3 and 4 have obvious hydrolysis circles and have better degradation effects. Figure 7 .

[0104] Medium ingredients:

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

[0106] Example 8

[0107] The degradation test of lignin in room temperature environment (30°C) was carried out using the degradation bacteria obtained above:

[0108] The above two single bacterial agents are mixed in the following volume ratio to prepare a composite bacterial agent.

[0109]

[0110] The above five bacterial agents were inoculated on CMC-Na solid culture medium under a sterile environment, inverted in a constant temperature incubator, cultured at 30°C for 48 hours, stained with 1mg / mL Congo red solution, immediately rinsed with sterile water, and then soaked in 1mol / L NaCl for 30 minutes. The hydrolysis circle and community diameter were observed. The experimental results showed that all five bacterial agents had a degrading effect on cellulose, among which bacterial agents 2 and 5 had obvious hydrolysis circles and better degradation effects. The results are shown in Figure 8 .

[0111] Medium ingredients:

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

[0113] Example 9

[0114] The above-obtained degradation bacteria agent has an effect on the temperature of cow dung composting:

[0115] The cow dung for the test was taken from a livestock and poultry farm in Shenyang, Liaoning Province. The pH was 8.48 and the moisture content was 55.86%. The cow dung and straw were mixed in a mass ratio of 10:1 and composted in the open air. The mass of the pile was about 40 kg and the composting cycle was 17 days. Two treatments were set up in the cow dung composting test. Samples of each treatment group were added 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): Bacterial agent treatment: The bacterial agent described in Example 2 was added to each group, and the composite bacterial agent was mixed in different proportions according to the following description to prepare a composite bacterial agent, which was added to 5 different piles (the inoculation amount of the bacterial agent accounted for 0.3% (w:w) of the cow dung). The bacterial agent formula is shown in the following table:

[0118]

[0119]

[0120] During the cow dung composting process, the compost was turned every 2 days, and the temperature of the compost was measured at 9:00-10:00 and 16:00-17:00 every day. The average value was taken as the temperature of the day, and the ambient temperature was recorded at the same time. The experimental results show that all five types of bacterial agents can promote the start of composting and increase the peak temperature of the compost and prolong the duration of high temperature. Among them, the T3 treatment group had the highest peak temperature of 72°C, and the high temperature period lasted for eight days. The peak temperatures of T2, T4, and T5 reached about 69°C, which was 6-8°C higher than that of the CK group, and the high temperature period was extended by 1-2 days compared with the CK group. Results are shown in Fig. 9 .

[0121] Example 10

[0122] The effect of the above-mentioned degradation bacteria on compost quality (seed germination index) was tested:

[0123] The cow dung for the test was taken from a livestock and poultry farm in Shenyang, Liaoning Province. The pH was 8.28 and the moisture content was 54.70%. The cow dung and straw were mixed in a mass ratio of 10:1 and composted in the open air. The mass of the pile was about 40 kg and the composting cycle was 16 days. Two treatments were set up in the cow dung composting test. Samples of each treatment group were added 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): Bacterial agent treatment: The bacterial agent described in Example 2 was added to each group, and the composite bacterial agent was mixed according to the following description in different proportions to prepare a composite bacterial agent, which was added to 5 different piles (the inoculation amount of the bacterial agent accounted for 0.3% (w:w) of the cow dung). The bacterial agent formula is shown in the following table. After adding the bacterial agent, it was fully turned and stirred to make it evenly distributed. Subsequently, the composting operation was performed every 2 days until the composting process was completed.

[0126]

[0127] After the composting is completed, take 10.00g of fresh sample and place it in a 200ml conical bottle. After converting the moisture content of the sample, add the corresponding mass of water according to the solid-liquid ratio (mass / volume) of 1:10. After tightening the bottle cap, fix it vertically on a reciprocating horizontal oscillator, adjust the frequency to 100 times / min, and the amplitude is not less than 40mm. Oscillate and extract at 25℃ for 1h. Take the supernatant and filter it on a filter device with filter paper pre-installed, and collect the filtered extract. Put 1 or 2 qualitative filter papers in a 9cm culture dish, evenly put 10 cucumber seeds of basically the same size and full on it, add 10mL of the sample extract, cover the culture cover, and culture in a light-proof incubator at (25±2)℃ for 48h. Count the number of germinated seeds, and use a vernier caliper to measure the taproot length one by one and calculate the germination index. The experimental results show that all five bacterial agents can promote the improvement of compost quality, among which T4 has the highest germination index of 0.93693, T3 and T5 are 0.87348 and 0.859802 respectively, which is 0.26-0.29 higher than that of CK group. Fig.10 .

[0128] In summary, the bacterial suspension provided by the present invention has good lignin and cellulose degradation potential. The free single bacteria screened by the present invention can grow rapidly to the logarithmic phase within one day, have a fast reproduction speed, strong adaptability, can effectively degrade lignin and cellulose in a low temperature environment, promote composting start-up temperature rise, improve compost quality, and have high efficiency, low cost, and easy operation, and are worthy of promotion.

Claims

1. A low temperature resistant lignin and cellulose degrading strain, characterized by: The low-temperature resistant lignin and cellulose degradation strain is Bacillus BTS1 or / and Bacillus lysinicus LFS1; the Bacillus BTS1 (Bacillus sp.) was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on October 24, 2024, with a deposit number of CGMCC No: 32338; The lysinibacillus sp. LFS1 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 24, 2024, with a deposit number of CGMCC No: 32336.

2. The low-temperature resistant lignin and cellulose degrading strain according to claim 1, characterized in that: The strains are Bacillus subtilis BTS1 and Bacillus lysinicola LFS1; the two strains are mixed in a volume ratio of (1-3): (1-3).

3. An application of the low-temperature resistant lignin and cellulose degrading strain according to claim 1, characterized in that: The strain is used in degrading lignin and / or cellulose.

4. The use of the low-temperature resistant lignin and cellulose degrading strain according to claim 3, characterized in that: The strain is used in degrading lignin and / or cellulose at 10-15°C.

5. An 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.

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

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

8. The low-temperature resistant multifunctional bacterial agent according to claim 7, characterized in that: The bacterial agent culture solution is obtained by culturing the lysine Bacillus LFS1 and Bacillus BTS1 in LB liquid culture medium at 10-15°C to the logarithmic growth phase, and the culture solution is centrifuged to collect the precipitate, and the precipitate is resuspended in sterile saline to an OD 600 It is a bacterial suspension with a concentration of 0.7 to 1.

4.

9. A method of using the bacterial agent according to claim 1, characterized in that: The bacterial agent is applied to the manure to be fermented, and the OD of the bacterial agent is 600 =1.0, add 0.3-0.5wt% of inoculum into the manure to be fermented.

10. A biological organic fertilizer, characterized in that: The strain described in claim 1 or the bacterial agent described in claim 6 is added to feces and sewage for composting to obtain biological organic fertilizer.

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