An indomicrobacterium capable of tolerating low and high temperatures simultaneously, a microbial agent and application thereof

Abstract

The application provides an indomicrobium KY183 strain capable of resisting low temperature and high temperature simultaneously, a microbial inoculum and application thereof, and relates to the technical field of microorganisms and application thereof.The KY183 strain is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 62698.From 1140 strains of microorganisms, 46 strains capable of degrading cellulose at low temperature (4 DEG C) and high temperature (40 DEG C) are screened out, and finally, a strain KY183 capable of strongly degrading cellulose at low temperature (4 DEG C), normal temperature (30 DEG C) and high temperature (40 DEG C) is selected.The strain not only has a wide temperature resistance range and strong cellulose degradation capacity, but also has the abilities of degrading protein, degrading starch, activating mineral potassium and silicon elements in soil, and promoting the rotting of straw, and has obvious comprehensive advantages.

Description

Technical Field

[0001] This invention relates to the field of microbiology and its application technology, and in particular to a type of Microbacterium indicum that can tolerate both low and high temperatures, a microbial agent, and its application. Background Technology

[0002] Crop straw refers to all agricultural byproducts and by-products generated during the initial processing of main crop products after harvesting, including straw from some legumes and non-legumes, such as wheat straw, corn straw, and soybean straw (Zang Qicong, 2016). Straw is an important source of nutrients for soil and a potential source of fertilizer, significantly increasing soil organic matter content. Berhane et al. found a significant positive correlation between the annual soil fixation rate of straw return treatment and the amount of straw returned. Li Feng et al. compared milkvetch straw return with a control group without fertilization, finding that milkvetch straw return effectively increased soil organic matter (3.98%–46.49%), available nitrogen (4.46%–35.88%), and particulate organic carbon (3.59%–35.77%) content. Straw resources are important renewable resources, rich in heat energy and essential nutrients for crop growth such as carbon, nitrogen, phosphorus, potassium, magnesium, calcium, and sulfur. They have wide-ranging applications; for example, magnesium significantly impacts corn growth, and combining straw return to the field with magnesium fertilizer application can effectively increase corn yield. Therefore, long-term straw return to the field combined with mineral fertilization can effectively increase crop yield and soil organic carbon reserves, significantly improving soil fertility and bringing substantial economic and ecological benefits to agriculture.

[0003] my country has abundant crop straw resources, with a wide variety of straw types. Nearly 20 major crop straws are included, with rice, wheat, and corn accounting for the largest share (77.2%), while other straw resources account for only about one-third (Shi Zuliang et al., 2019). Returning straw to the field is currently a widely used method for comprehensive utilization of crop straw resources in my country. This requires straw to be crushed and deeply tilled to thoroughly mix with the soil. This necessitates efficient straw crushing by farmers after or during harvest, requiring additional straw crushing machinery and increasing farmers' operating costs. Furthermore, while straw is rich in various nutrients, it also contains various pests and diseases. Directly returning straw to the field can increase the incidence of pests and diseases, posing a potential risk to food security. In recent years, regional, seasonal, and structural overproduction of straw has emerged, leading to widespread straw piling and centralized burning in the fields. This not only severely pollutes the environment but also wastes a significant amount of resources. Therefore, how to efficiently utilize straw resources is a major problem that urgently needs to be solved.

[0004] Straw is mainly composed of cellulose, hemicellulose, and lignin. These three components have complex chemical structures and are cross-linked together, exhibiting strong resistance to decomposition. The long cellulose chains are primarily maintained by hydrogen bonds. Because the basic unit is a homogeneous cellobiose without branches, the resulting long chains have a relatively regular molecular structure and exhibit high overall stability (Nishiyama Y et al., 2003). Under normal conditions, the long cellulose chains are stable and not easily degraded; acids, alkalis, and organic solvents have little impact on their structure (He Yun, 2004).

[0005] Lignocellulose is the most prevalent form of cellulose in the environment. In natural plant resources, cellulose often combines with hemicellulose and lignin to form lignocellulose in various forms (FREITAG M et al., 1992). The fibrillar structure extending from the long chains of cellulose can cross-link with lignin and hemicellulose in various forms, including covalent and non-covalent bonds, forming a complex structure after mixing and coiling (Dai Yunyun et al., 2016). This structure is not only very dense, but also has the coating of hemicellulose and the protection of lignin, which is more stable and harder to degrade, making lignocellulose extremely difficult to degrade (COWLINGEB et al., 1976). Most of the cellulose resources in nature exist in the form of lignocellulose, so the biggest obstacle to the utilization of this type of resource is its difficulty in degradation.

[0006] Currently, the utilization of straw mainly relies on chemical or biological treatment to achieve resource recovery. The cellulose in straw has an insoluble, rigid structure, insoluble in water, dilute acids, and dilute alkalis at room temperature (Chen Hongzhang, 2005). It decomposes slowly under natural conditions, and its insolubility and heterogeneity make it difficult to degrade (Luo Hui et al., 2008), thus hindering its use as industrial raw material and animal feed. Therefore, microbial decomposition of cellulose has become the core of cellulose biological treatment technology. However, low temperatures directly affect straw decomposition, leading to unsatisfactory results from straw biochemical decomposition agents. For example, in Northeast China, where soil temperature is low and soil is sandy, agricultural straw residues decompose slowly or even fail to degrade completely. Low temperatures inhibit microbial growth and metabolism, preventing the aerobic compost pile from heating up, making it difficult or even impossible to start aerobic composting of cellulosic biomass waste. Simultaneously, it slows down the degradation of lignocellulose, prolonging the composting cycle and increasing production costs. Therefore, screening for low-temperature-resistant, highly efficient cellulose-degrading bacteria to ensure the smooth initiation of aerobic composting of cellulosic biomass waste under low-temperature conditions is of great significance in solving this problem. However, since straw decomposition is essentially a composting process, and aerobic composting generally consists of a heating phase, a high-temperature phase, and a maturation phase, with the high-temperature phase often reaching above 40℃, screening for cellulose-degrading bacterial strains with a wide temperature tolerance range is even more crucial for aerobic composting treatment of cellulosic biomass waste in low-temperature environments.

[0007] Currently, there are few reports on low-temperature cellulase-producing bacteria. Mu Chunlei et al. (2013) isolated a fungus M11 from straw-returned soil that efficiently decomposes cellulose at a low temperature of 13℃, and identified it as *Penicillium oxalicum*. Zhang Dan et al. (2008) isolated two bacterial strains, B9 (Cytophage) and B21 (Cellulomonas), and determined that they have a significant degradation effect on cellulose at 10-15℃. Zhao Xu et al. (2017) screened a fungal strain D5 that can degrade carboxymethyl cellulose, corn straw cellulose, and produce high levels of cellulase at 15℃ using 30 samples collected from mountainous areas in Weiyuan County. It was preliminarily identified as *Penicillium* sp. by ITS rDNA sequence analysis. Qu Qingtao et al. (2020) discovered a *Pseudomonas* strain that can degrade cellulose well and grow stably at 4℃. The *Graminis* strain exhibits reduced cellulose-degrading activity at temperatures above 30°C. Regarding thermophilic cellulose-degrading strains, researchers have isolated strains including *Bacillus amyloliquefaciens*, *Aspergillus oryzae*, *Cladosporium*, *Bacillus subtilis*, *Bacillus thuringiensis*, *B. burgdorferi*, *Cladosporium*, *Anoxybacillus rupiensis*, *Geobacillus thermocatenulatus*, *Bacillus pumilus*, and *Bacillus stearophilus*. Therefore, while some cellulose-degrading strains have been studied and applied under both high and low temperature conditions, there are currently no reports of microbial strains capable of simultaneously tolerating both low and high temperatures.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a type of Indo-eucobacterium that can tolerate both low and high temperatures, a microbial agent, and its application, which solves the problem of the lack of cellulose-degrading bacteria that can tolerate both low and high temperatures in the prior art, and provides a new strain with excellent comprehensive performance and a wide temperature tolerance range.

[0010] In one aspect, the present invention provides an Exiguobacterium indicum KY183 strain that can tolerate both low and high temperatures. The KY183 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62698.

[0011] The 16S rDNA sequence of the *Exiguobacterium indicum* KY183 strain of this invention is shown in SEQ ID No. 1.

[0012] Existing cellulose-degrading bacteria cannot simultaneously achieve effective cellulose degradation at both low and high temperatures. This invention screened a multifunctional strain (KY183) from 1140 microbial strains, exhibiting strong thermostability and versatility. This strain can strongly degrade cellulose simultaneously at low temperatures (4℃), normal temperatures (30℃), and high temperatures (40℃); it also possesses the ability to decompose proteins and starches, activate potassium and silicon minerals in the soil, and promote the decomposition of straw.

[0013] In one aspect, the present invention provides a microbial inoculant comprising the *Exiguobacterium indicum* KY183 strain or the fermentation broth of the *Exiguobacterium indicum* KY183 strain.

[0014] The microbial inoculant of the present invention may also contain other excipients or additives. For example, it may contain peat moss, diatomaceous earth, and calcium carbonate.

[0015] In another aspect, the present invention provides the application of the *Exiguobacterium indicum* KY183 strain or the microbial agent, characterized in that the application includes at least one of the following aspects: (a) degrading cellulose; (b) degrading protein; (c) degrading starch; (d) activating trace elements in soil.

[0016] In one embodiment, the activated trace elements in the soil include activated potassium and / or silicon. Potassium or silicon may be activated from sources such as potassium feldspar, plagioclase, microcline, illite, vermiculite, montmorillonite, silicates, and / or mica.

[0017] The strains or agents of this invention can be used to degrade organic matter containing cellulose.

[0018] In another aspect, the present invention provides the application of the *Exiguobacterium indicum* KY183 strain or the microbial agent in promoting the degradation and / or composting of straw and / or kitchen waste. The strain or agent of the present invention can be prepared as a composting agent for degrading straw and kitchen waste.

[0019] In one embodiment, the straw includes straw from wheat, sorghum, corn, soybeans, and rice.

[0020] In another aspect, the present invention provides the application of the aforementioned Exiguobacterium indicum KY183 strain or the aforementioned microbial agent in the preparation of microbial organic fertilizer, plant growth modifier or soil conditioner.

[0021] The *Exiguobacterium indicum* KY183 strain or the microbial agent provided by this invention can also be used in organic material composting, including, for example, agricultural waste or industrial byproducts.

[0022] In another aspect, the present invention provides a method for promoting cellulose degradation under different temperature conditions, comprising mixing and fermenting the *Exiguobacterium indicum* KY183 strain or the microbial agent with a cellulose-containing substance.

[0023] In another aspect, the present invention provides a method for in-situ return of straw to the field, comprising fermenting and culturing the straw using the aforementioned Exiguobacterium indicum KY183 strain or the aforementioned microbial agent.

[0024] In one embodiment, the fermentation culture temperature is 4°C to 40°C. In one embodiment, the *Indian Microbe* KY183 undergoes a strain activation step prior to inoculation.

[0025] Preservation Information: Exiguobacterium indicum strain KY183 was deposited on August 12, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 62698. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, 510070, China. The strain was confirmed as viable by the collection center on August 12, 2022.

[0026] Beneficial effects:

[0027] The strain provided by this invention has the advantage of a wide temperature tolerance range, that is, it has a strong ability to degrade cellulose at both low temperatures of 4°C and high temperatures of 40°C; and it also has the ability to degrade proteins, starches, and activate potassium and silicon minerals in the soil, which has obvious advantages.

[0028] The *Microbacterium indicum* or its containing agents of the present invention have significant economic and application value in fermentation, agriculture, and industry. They can be used to improve the efficiency of straw return to the field, increase soil organic matter content, improve soil properties and fertility, enhance the resource utilization capacity of kitchen waste, reduce processing costs, and promote the rapid development of related industries. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 The results show the ability of some strains provided in this embodiment of the invention to degrade cellulose on CMC medium at different temperatures;

[0031] Figure 2 The morphology of strain KY183 provided in the embodiments of the present invention under a microscope (10×100);

[0032] Figure 3 The results show the ability of strains on CMC medium to degrade cellulose at different temperatures, as provided in the embodiments of the present invention.

[0033] Figure 4 Growth curves of different strains at 4°C provided for embodiments of the present invention;

[0034] Figure 5 Growth curves of different strains at 30°C provided for embodiments of the present invention;

[0035] Figure 6 Growth curves of different strains at 40°C provided for embodiments of the present invention;

[0036] Figure 7 The results of the determination of the composting ability of different strains on straw mixtures at 4°C are provided in the embodiments of the present invention.

[0037] Figure 8 The results of the determination of the composting ability of different strains on straw mixtures at 30°C are provided in the embodiments of the present invention.

[0038] Figure 9 The ability of strain KY183 provided in this embodiment of the invention to decompose proteins at 4°C, 30°C and 40°C was determined.

[0039] Figure 10 The starch-degrading ability of strain KY183 provided in the embodiments of the present invention at 4℃, 30℃ and 40℃;

[0040] Figure 11 The results of the ability test of strain KY183 to degrade silica minerals provided in the embodiments of the present invention;

[0041] Figure 12 The results of the determination of the potassium-solubilizing ability of strain KY183 provided in the embodiments of the present invention. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1. Screening of strains capable of degrading cellulose at 4°C with strong thermostability

[0044] 1. Screening for microorganisms capable of degrading cellulose under low-temperature conditions (4℃).

[0045] 1.1 Screening of cryogenic strains in microbial resource banks

[0046] Representative soil samples were collected from various provinces across the country, including sandy soil, clay, and black soil. The samples came from different regions such as farmland, pasture, and forest soil. 15-20g of sample was collected from each point, and the source (province, county), year and month of collection, and source of the soil (plant, sandy soil, or others) were marked. The samples were stored in a -80℃ freezer. A total of 169 soil samples from various provinces and cities across the country were collected. A rare microbial resource bank with a total of 1140 strains was constructed by screening functional strains from different soil samples.

[0047] 1.2 Screening of microbial strains capable of degrading cellulose at 4℃

[0048] Strains from the rare microbial resource bank were streaked onto R2A solid medium (0.50g yeast extract, 0.50g peptone, 0.50g tryptone, 0.50g glucose, 0.50g soluble starch, 0.30g dipotassium hydrogen phosphate, 0.30g sodium pyruvate, 0.05g magnesium sulfate heptahydrate, 15.0g agar powder, 1000mL water) and incubated at 30℃ for 24h. Single colonies were then picked and needled onto CMC solid medium (1.0g dipotassium hydrogen phosphate, 0.25g magnesium sulfate heptahydrate, 2.0g yeast extract, 10.0g agar powder, sodium carboxymethyl cellulose CMC). In a solution of 2.0 g of water and 1000 mL of water, 92068 (Bacillus subtilis) was used as a control and cultured at 4°C. After the low-temperature strains grew, they were fumigated with iodine solution. The strains that degraded CMC at 4°C were recorded, and the results were measured. The larger the transparent zone produced, the better the ability to degrade cellulose.

[0049] CMC solving capability = number of millimeters of CMC loop diameter solved + X;

[0050] Where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the CMC ring of the strain.

[0051] (Note: X is a weighting coefficient, corresponding to -1, 0, 1, and 2 based on the transparency of the hydrolysis zone of the bacterial strain. A number 2 represents a completely transparent hydrolysis zone; a number 1 represents a semi-transparent hydrolysis zone; a number 0 represents an opaque hydrolysis zone, but with traces of hydrolysis on the culture medium surface, barely visible to the naked eye, but after rinsing the colony with water, faint traces of hydrolysis are visible at the inoculation site; -1 represents no hydrolytic activity. This method has also been used to test the protein-degrading and starch-degrading activities of bacteria.)

[0052] 1.3. Screening for microbial strains that degrade cellulose at 30℃

[0053] Select the microbial strains from 1.2 that can degrade cellulose at 4℃, puncture them in sodium carboxymethyl cellulose medium, set Bacillus subtilis 92068 as a control, and incubate at 30℃ for 2 days. After the strains have grown, fumigate with iodine solution and measure the diameter of the degraded CMC rings in mm.

[0054] 1.4. Screening for microbial strains that degrade cellulose at 40℃

[0055] Select the microbial strains from 1.2 that can degrade cellulose at 4℃, puncture them into sodium carboxymethyl cellulose medium, set Bacillus subtilis 92068 as a control, and incubate at 40℃ for 2 days. After the strains have grown, fumigate with iodine solution and measure the diameter of the degraded CMC rings in mm.

[0056] 1.5. Duplicate Validation

[0057] To ensure the CMC degradation ability of the strain, the strain that can strongly degrade CMC at 4℃, 30℃ and 40℃ was inoculated again into CMC solid medium and cultured at 4℃, 30℃ and 40℃ respectively to verify whether the selected colonies have the function of degrading CMC at 4℃, 30℃ and 40℃ at the same time, and to exclude false positive colonies.

[0058] 1.6. Results

[0059] From Table 1 and Figure 1 It is evident that: ① strains with a strong ability to degrade cellulose at 30℃ do not necessarily have the ability to degrade cellulose at high temperatures (40℃); ② strains with a relatively poor ability to degrade cellulose at 30℃ can also have a strong ability to degrade cellulose at 4℃; ③ strains with a strong ability to degrade cellulose at 4℃ do not necessarily have a strong ability to degrade cellulose at both room temperature (30℃) and high temperature (40℃).

[0060] Table 1. Results of the ability of some strains to degrade cellulose on CMC medium at different temperatures.

[0061]

[0062] This invention screened 140 strains capable of low-temperature cellulose degradation from 1140 strains in a rare microbial strain resource bank, and further screened 46 strains capable of degrading cellulose at low temperature (4℃), as well as at room temperature (30℃) and high temperature (40℃). Finally, this invention comprehensively analyzed the strains with the ability to degrade cellulose at low temperature (4℃), room temperature (30℃), and high temperature (40℃), and selected a strain KY183 that can strongly degrade cellulose at low temperature (4℃), room temperature (30℃), and high temperature (40℃). The results of 16S rDNA sequence determination of this strain showed that it had a high homology of 99.59% with Exiguobacterium indicum. Based on the obtained 16S rDNA sequence of KY183, homologous sequences were searched in GenBank and compared with each other. Simultaneously, sequence alignment was performed with the 16S RNA database (Chun's Lab) recognized by the International Committee for Bacteriology, and combined with literature analysis to determine the taxonomic position of the target microorganism (Yoon, SH, Ha, SM, Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol. 67: 1613-1617). The results showed that the 1456-base sequence of this strain was highly homologous (99.59% similarity) to the strain *Exiguobacterium indicum*, confirming that KY183 is *Exiguobacterium indicum*.

[0063] 2.16S DNA determination and strain physiological morphology analysis:

[0064] 2.1 Determination of 16S DNA of KY183 strain

[0065] 2.1.1 Extraction of bacterial DNA by CTAB method

[0066] 1. Inoculate a single colony into 5 mL of R2A and incubate overnight at 30°C;

[0067] 2. Take 1 mL of seed culture medium and inoculate it into 100 mL of LR2A liquid, and incubate at 37℃ and 220 r / min for 16 hours;

[0068] 3. Centrifuge at 5000 r / min for 10 minutes and discard the supernatant.

[0069] 4. After centrifugation and washing with 10 mL TE, dissolve the bacterial cells with 10 mL TE, mix well, and store at -20℃ for later use.

[0070] 5. Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS, mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour.

[0071] 6. Add 740 μL of 5 mol / L NaCl, then add 512 μL of CTAB / NaCl, mix well, and incubate at 65°C for 10 minutes.

[0072] 7. Add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant;

[0073] 8. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant;

[0074] 9. Add 0.6 times the amount of isopropanol, mix well, centrifuge at 10000 r / min for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol by centrifugation.

[0075] 10. Dissolve the DNA in 1 mL TE buffer, add RNase A to a final concentration of 20 μg / mL, and store at 4°C.

[0076] 2.1.2 Amplification and Sequencing

[0077] PCR amplification of 16S rDNA was performed using universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 2) and 1492r (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 3). PCR reaction conditions were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for 35 cycles. The PCR products were subjected to 1.5% agarose gel electrophoresis, and after agarose gel electrophoresis, the PCR products were recovered, purified, and sequenced (Beijing Meiyimei Biotechnology Co., Ltd.). Homologous sequences were searched for in GenBank using BLAST based on the obtained 16S rDNA sequence, and homologous sequence analysis was performed for comparison.

[0078] 2.1.3 16S sequencing results of KY183 strain

[0079] The results of the 16SDNA sequence determination of strain KY183 (Sequence 1) and the comparison with the NCBI database showed that Exiguobacterium indicum had a high homology of 99.59%.

[0080] Sequence 1 (SEQ ID No. 1):

[0081]

[0082] 2.2 Observation of strain morphology

[0083] The selected strains were inoculated onto R2A plates and cultured at 30°C for 2 days. The size, shape, color, gloss, viscosity, raised shape, transparency, edge characteristics, and presence or absence of spores of the colonies were observed.

[0084] 2.2.1 Results of Observation of Strains' Morphology

[0085] After observing the growth of strain KY183 (Exiguobacterium indicum) on R2A medium for 2 days, the colony morphology showed that the colonies were round, orange-yellow and opaque, with a smooth and moist surface, regular and slightly raised edges, and no halo. Microscopic measurements showed that the bacterial diameter was approximately 2-5 μm.

[0086] 3. Cellulose degradation activity test of strain KY183 at different temperatures

[0087] 3.1 Determination of the cellulose degradation ability of strain KY183 at different temperatures

[0088] Strawberry strain KY183 was streaked onto R2A medium and incubated at 30°C for 24 hours. Single colonies were then picked and needled into three identical CMC solid media (1g dipotassium hydrogen phosphate, 0.25g magnesium sulfate heptahydrate, 2g yeast, 2g sodium carboxymethyl cellulose, agar, and 1000mL water). Low-temperature strains KY240 (Pseudomonas graminis, a patented strain for cellulose degradation at 4°C, patent number CN201911394095.9) and 92068 (Bacillus subtilis, a major component in commercially available composting agents) were used as controls. These strains were incubated at 4°C (4 days), 30°C, and 40°C (1 day), respectively. After the strains grew, they were fumigated with iodine solution, and the hydrolysis zone was measured. The larger the transparent zone, the better the ability to degrade cellulose.

[0089] CMC resolution capability = number of millimeters of CMC ring diameter + X; where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the CMC ring resolved by the strain.

[0090] (Note: X is a weighting coefficient, corresponding to -1, 0, 1, and 2 based on the transparency of the hydrolysis zone of the bacterial strain. A number 2 represents a completely transparent hydrolysis zone; a number 1 represents a semi-transparent hydrolysis zone; a number 0 represents an opaque hydrolysis zone, but with traces of hydrolysis on the culture medium surface, barely visible to the naked eye, but after rinsing the colony with water, faint traces of hydrolysis are visible at the inoculation site; -1 represents no hydrolytic activity. This method has also been used to test the protein-degrading, starch-degrading, and silicate-degrading activities of bacteria.)

[0091] 3.2 Results of the determination of the cellulose degradation ability of strain KY183 at different temperatures

[0092] Table 2. Cellulose degradation ability of strains on CMC medium at different temperatures

[0093]

[0094] From Table 2 and Figure 3 The results showed that strain KY183 had a significantly higher cellulose-degrading ability at 4℃ than the patented low-temperature cellulose-degrading strain KY240 and Bacillus subtilis strain 92068, while strain 92068 had no ability to degrade cellulose at 4℃. At 30℃, strain KY183 had a significantly higher cellulose-degrading ability than Bacillus subtilis 92068. At 40℃, the cellulose-degrading ability of strain KY183 was not much different from that of Bacillus subtilis 92068, while the low-temperature cellulose-degrading strain KY240 had no ability to degrade cellulose at both 30℃ and 40℃.

[0095] 4. Determination of the growth of strain KY183

[0096] 4.1 Determination of the growth of strain KY183 at 4℃ / 30℃ / 40℃

[0097] Bacterial suspensions of strains KY183, 92068, and KY240 were prepared, mixed thoroughly, and OD was quantified. 600nm =0.05 was inoculated into 100 mL of liquid LB medium (5 g yeast extract, 10 g peptone, 10 g sodium chloride, 1000 mL water) and cultured in shake flasks at 4℃ / 30℃ / 40℃ and 200 rpm. The OD of the strain was measured at (30℃ / 40℃: 0 h, 4 h, 8 h, 12 h, 24 h) / (4℃: 0 h, 24 h, 48 h, 72 h, 96 h). 600nm Values. A curve was plotted with time / h on the x-axis and effective colony count on the y-axis. Bacillus subtilis strain 92068 and the patented low-temperature cellulose degradation strain KY240 were set as controls.

[0098] 4.2 Results of growth determination of strain KY183 at 4℃ / 30℃

[0099] 4. Determination of the growth of strain KY183

[0100] 4.1. Determination of the growth of strain KY183 at 4℃ / 30℃ / 40℃

[0101] Bacterial suspensions of strains KY183, 92068, and KY240 were prepared, mixed thoroughly, and OD was quantified. 600nm=0.05 was inoculated into 100 mL of liquid LB medium (5 g yeast extract, 10 g peptone, 10 g sodium chloride, 1000 mL water) and cultured in shake flasks at 4℃ / 30℃ / 40℃ and 200 rpm. The OD of the strain was measured at (30℃ / 40℃: 0 h, 4 h, 8 h, 12 h, 24 h) / (4℃: 0 h, 24 h, 48 h, 72 h, 96 h). 600nm Values. A curve was plotted with time / h on the x-axis and effective colony count on the y-axis. Bacillus subtilis strain 92068 and the patented low-temperature cellulose degradation strain KY240 were set as controls.

[0102] 4.2 Results of growth determination of strain KY183 at 4℃ / 30℃

[0103] Table 3. OD values ​​of different strains grown in shake flasks at 4℃ 600nm value

[0104]

[0105] From Table 3 and Figure 4 The results show that strain 92068 hardly grows at 4℃, indicating that the growth of strain 92068 is inhibited at low temperatures; the patented low-temperature cellulose degradation strains KY240 and KY183 can both grow at 4℃.

[0106] Table 4. OD values ​​of different strains grown in shake flasks at 30℃ 600nm value

[0107]

[0108] From Table 4 and Figure 5 The results show that all three different strains can grow at 30℃, with strain KY183 growing the fastest, while the low-temperature cellulose degradation patented strain KY240 grows relatively slowly.

[0109] Table 5. OD values ​​of different strains grown in shake flasks at 40℃ 600nm value

[0110]

[0111] From Table 5 and Figure 6 The results showed that both strain KY183 and strain 92068 could grow at 40℃, with strain KY183 showing the fastest growth rate, while the growth of the patented low-temperature cellulose degradation strain KY240 was inhibited.

[0112] In summary, strain KY183 can grow under low temperature (4℃), normal temperature (30℃), and high temperature (40℃) conditions, and its growth rate is relatively fast. This indicates that strain KY183 is a strain with strong temperature adaptability and can be used as a target strain for screening in this invention that can degrade cellulose at low temperature of 4℃ and has strong temperature adaptability.

[0113] 5. Straw composting experiment

[0114] 5.1 Determination of the composting ability of strain KY183 on straw mixtures at 4℃ / 30℃

[0115] Using corn stalks, wheat stalks, and tomato stalks as substrates, 1g of corn stalks, 1g of tomato stalks, and 1g of wheat stalks were weighed into the same 50mL centrifuge tube, mixed thoroughly, and divided into 8 portions. A small amount of equal volume of liquid R2A was added to moisten each portion, followed by the addition of 1mL of water, KY183 bacterial solution, KY240 bacterial solution, and 92068 bacterial solution. The mixtures were shaken well and placed at 4℃ and 30℃ respectively for incubation and composting. According to the growth requirements, equal amounts of bacterial solution were applied as needed, and the composting status of the straws was observed weekly. KY240 bacterial solution and 92068 bacterial solution were set as controls, and water was set as a blank control.

[0116] 5.2 Results of the determination of the composting ability of strain KY183 on straw mixtures at 4℃ / 30℃

[0117] from Figure 7 It can be seen that the mixed straw decomposed by strain KY183 at low temperature (4℃) is more delicate and more moist, indicating that strain KY183 has a stronger degree and speed of decomposition of mixed straw at low temperature (4℃) than the control Bacillus subtilis strain 92068 and strain KY240.

[0118] from Figure 8 It can be seen that the mixed straw decomposed by strain KY183 at 30℃ is darker in color, and compared with strain KY240, the straw is more delicate and moist. This indicates that at room temperature (30℃), strain KY183 has a significantly stronger degree and speed of decomposition of mixed straw than the control Bacillus subtilis strain 92068 and strain KY240.

[0119] Example 2. Determination of the multifunctionality of strain KY183

[0120] In summary, strain KY183 exhibits stable growth at low temperatures and demonstrates strong cellulose degradation capabilities at 4℃, 30℃, and 40℃. It can also decompose straw at 4℃ and 30℃. Therefore, it can be used as a microbial inoculant for straw decomposition in practical applications. As a key component of decomposition inoculants with a wide temperature tolerance range, strain KY183 can be explored to determine if it can expand its application scenarios, such as the degradation and decomposition of household kitchen waste. Starch, dietary fiber, animal protein, and fat are the main organic components of municipal solid waste; therefore, expanding the application scenarios of this product mainly involves exploring whether strain KY183 has the ability to degrade starch, cellulose, and protein.

[0121] 6.1 Determination of the protein-degrading ability of strain KY183

[0122] 6.1.1 Determination of the protein-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0123] Strain KY183, cultured on R2A medium for 1 day, was needled into protein medium (0.50 g yeast extract, 0.50 g peptone, 0.50 g trypone, 0.50 g glucose, 0.50 g soluble starch, 0.30 g dipotassium hydrogen phosphate, 0.30 g sodium pyruvate, 0.05 g magnesium sulfate, 15 g agar, 5 g skim milk powder, 1000 mL water). Bacillus subtilis 92068 and KY240 were used as controls. The cultures were incubated at 4℃, 30℃, and 40℃, respectively. After the strains grew, the diameter of the protein-degrading zone was measured to determine the strains' ability to degrade proteins, in mm.

[0124] Protein decomposition capability = number of millimeters of protein ring diameter + X;

[0125] Where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the protein zone of the strain.

[0126] 6.1.2 Results of the determination of the protein-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0127] Table 6. Protein-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0128]

[0129] From Table 6 and Figure 9The results showed that strain KY183 had significantly higher protein-degrading ability than the control strain Bacillus subtilis 92068 at 4℃ and 30℃; at 40℃, strain KY183 had slightly better protein-degrading ability than the control strain Bacillus subtilis 92068; the patented low-temperature cellulose degradation strain KY240 had no protein-degrading ability at 4℃, 30℃, and 40℃.

[0130] 6.2 Determination of starch-dissolving ability of strain KY183

[0131] 6.2.1 Determination of starch-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0132] Strain strain KY183, which was cultured on R2A medium for 1 day, was needled into starch medium (10g peptone, 5g sodium chloride, 2g soluble starch, 20g agar, 1000mL water). Bacillus subtilis 92068 was set as a positive control and Pseudomonas KY240 as a negative control. The strains were cultured at 4℃, 30℃, and 40℃, respectively. After the strains grew, the diameter of the starch-degrading ring was measured to determine the strains' ability to degrade starch, in mm.

[0133] Starch-dissolving capacity = diameter of the starch-dissolving ring in millimeters + X;

[0134] Where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the starch ring of the strain.

[0135] 6.2.2 Results of starch-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0136] Table 7. Starch-degrading ability of strain KY183 at 4℃, 30℃ and 40℃

[0137]

[0138] From Table 7 and Figure 10 The results showed that strain KY183 had a significantly higher starch-degrading ability than the control strain KY240 at 4℃, while the control strain Bacillus subtilis 92068 had no starch-degrading ability at 4℃. At 30℃, strain KY183 had a slightly stronger starch-degrading ability than the control strain Bacillus subtilis 92068. At 40℃, strain KY183 also had starch-degrading ability, but its degradation ability was not as good as that of the control strain Bacillus subtilis 92068. The patented low-temperature cellulose degradation strain KY240 had no starch-degrading ability at both 30℃ and 40℃.

[0139] Example 3. Determination of the ability of strain KY183 to activate trace elements in soil.

[0140] Straw composting involves piling collected crop straw in layers 15-20cm thick, sprinkling diluted straw composting agent on each layer, then adding a thin layer of soil, and repeating this process until the pile is about 1.2m high. The pile is then covered with a layer of soil or wrapped with a thin film to prevent moisture loss. After composting, the straw is mainly used as a base fertilizer for the next crop, thus further research is needed to investigate whether strain KY183 has the ability to activate trace elements (potassium, silicon, phosphorus, etc.) in the soil, thereby improving the plant's ability to utilize these trace elements.

[0141] 7.1 Determination of the ability of strain KY183 to degrade silica minerals

[0142] 7.1.1 Determination of the ability of strain KY183 to degrade silica minerals

[0143] Strain KY183, grown on R2A medium for 1 day, was needled into silica-degrading medium (0.50 g yeast extract, 0.50 g peptone, 0.50 g tryptone, 0.50 g glucose, 0.50 g soluble starch, 0.30 g dipotassium hydrogen phosphate, 0.30 g sodium pyruvate, 0.05 g magnesium sulfate, 15 g agar, 5 g magnesium silicate, 1000 mL water). Bacillus subtilis 92068 was set as a control. The culture was carried out at 30℃. After the strain grew, the diameter of the silica-degrading zone was measured to determine the strain's ability to degrade silica minerals, in mm.

[0144] Silicon removal capability = Silicon removal ring diameter in millimeters + X;

[0145] Where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the silica ring of the strain.

[0146] 7.1.2 Results of the determination of the ability of strain KY183 to degrade silica minerals

[0147] Depend on Figure 11 The results showed that strain KY183 had the ability to degrade silicon minerals, and its degradation ability was significantly stronger than that of the control strain 92068, indicating that strain KY183 had the ability to activate trace elements (silicon) in the soil.

[0148] 7.2 Determination of potassium mineral degradation ability of strain KY183

[0149] 7.2.1 Determination of the potassium mineral degradation ability of strain KY183

[0150] Strain KY183, grown on R2A medium for 1 day, was needled into potassium-degrading medium (10.0 g glucose, 0.5 g yeast extract, 1.0 g ammonium sulfate, 2.0 g disodium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1.0 g calcium carbonate, 15.0 g agar powder, 1.0 g potassium feldspar, 1000 mL water). Bacillus subtilis 92068 was used as a control. The strain was cultured at 30 °C. After the strain grew, the diameter of the potassium-degrading zone was measured to determine the strain's ability to degrade potassium minerals, in mm.

[0151] Potassium-dissolving capacity = Potassium-dissolving zone diameter in millimeters + X;

[0152] Where X is a weighting coefficient, which is -1, 0, 1, or 2 depending on the transparency of the potassium-solubilizing zone of the strain.

[0153] 7.2.2 Results of the determination of potassium mineral degradation ability of strain KY183

[0154] Depend on Figure 12 The results showed that strain KY183 had the ability to degrade potassium minerals, and its degradation ability was significantly stronger than that of the control strain 92068, indicating that strain KY183 had the ability to activate trace elements (potassium) in the soil.

[0155] Conclusion: This invention screened a rare microbial strain, KY183, from 1140 strains in a rare microbial resource library constructed by screening functional strains from different soil samples. KY183 exhibits strong thermostability and can simultaneously degrade cellulose at low temperature (4℃), normal temperature (30℃), and high temperature (40℃). The 16SDNA sequence determination of this strain showed a high homology of 99.59% with *Exiguobacterium indicum*. Compared with the commercial strain *Bacillus subtilis* 92068 and the patented low-temperature cellulose degradation strain KY240, strain KY183 exhibits a stronger ability to degrade cellulose under low-temperature (4℃), normal-temperature (30℃), and high-temperature (40℃) conditions. Furthermore, strain KY183 can grow stably at these temperatures. Simultaneously, strain KY183 also demonstrates superior ability to degrade proteins and starches at low-temperature (4℃), normal-temperature (30℃), and high-temperature (40℃) conditions compared to the commercial strain *Bacillus subtilis* 92068 and the patented low-temperature cellulose degradation strain KY240. This provides a theoretical basis for expanding its application as a composted product. Therefore, it can be seen that KY183 is a functional microbial strain that can degrade cellulose at low temperature (4℃), has strong temperature adaptability, and is multifunctional. It can strongly degrade cellulose at low temperature (4℃), normal temperature (30℃) and high temperature (40℃), and also has the ability to decompose proteins, starch, activate potassium and silicon minerals in the soil, and promote the decomposition of straw.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Indomicrobacterium that can tolerate both low and high temperatures ( Exiguobacterium indicum KY183 strain, characterized in that, The KY183 strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCCNo: 62698; The application of the *Inonotus indicus* KY183 strain includes at least one of the following aspects: (a) Degradation of cellulose; (b) Degradation of proteins; (c) Activating trace elements in the soil; said activated trace elements in the soil include activated potassium and / or silicon.

2. A microbial inoculant, characterized in that, Fermentation broth containing the *Indiana microbacterium* KY183 strain as described in claim 1 or the *Indiana microbacterium* KY183 strain; The application of the microbial inoculant includes at least one of the following aspects: (a) Degradation of cellulose; (b) Degradation of proteins; (c) Activating trace elements in the soil; said activated trace elements in the soil include activated potassium and / or silicon.

3. The application of the *Microbacterium indicum* KY183 strain as described in claim 1 or the microbial agent as described in claim 2 in promoting the degradation and / or composting of straw and / or kitchen waste.

4. The application according to claim 3, characterized in that, The straw includes straw from wheat, sorghum, corn, soybeans, and rice.

5. The application of the Inonotus KY183 strain of claim 1 or the microbial agent of claim 2 in the preparation of microbial organic fertilizer, plant growth conditioner or soil conditioner.

6. A method for promoting cellulose degradation under different temperature conditions, characterized in that, This includes fermenting and culturing a mixture of the *Inonotus indicus* KY183 strain as described in claim 1 or the microbial agent as described in claim 2 with a cellulose-containing substance.

7. A method for in-situ straw return to the field, characterized in that, This includes fermenting and culturing the straw using the *Microbacterium indicum* KY183 strain according to claim 1 or the microbial agent according to claim 2.

8. The method according to claim 6 or 7, characterized in that: The fermentation culture temperature is 4℃~40℃.

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