Thermophilic haloanaerobacter nounudulus and application of thermophilic haloanaerobacter nounudulus

Abstract

The invention belongs to the technical field of functional microorganism screening and application, and particularly relates to a thermophilic halogenobacter nounudi and application thereof. The halogenobacillusrupiensis is halogenobacillusrupiensis S2-5, the halogenobacillusrupiensis S2-5 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the halogenobacillusrupiensis S2-5 is CCTCC NO (China Center for Type Culture Collection): M 20241447. The strain S2-5 provided by the invention is screened out under high-temperature composting, and has the advantages of good thermal stability, no inhibition of metabolic activity by the temperature of high-temperature composting, and strong adaptability. The compost inoculant obtained by solid state fermentation of the anaerobic bacillus tubingensis S2-5 strain provided by the invention has the characteristics of being easy to preserve and transport, high in strain activity and the like. And the fertilizer prepared from the composting inoculant can shorten the composting period when the composting inoculant is applied to high-temperature composting treatment of organic solid wastes.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganism screening and application, and specifically relates to a thermophilic anaerobic Bacillus nubiquitus and an application thereof. Background Art

[0002] Straw and livestock manure are very valuable biomass resources in agricultural ecosystems. Their comprehensive utilization is of great significance to farmers' income increase, environmental protection, resource conservation and sustainable development of agricultural economy. If these resources are not used reasonably, they will pose a serious threat to human life, health and living space.

[0003] Composting is an important means of harmless treatment and resource utilization of organic solid waste. Composting can not only solve the environmental pollution problems caused by organic solid waste, but also convert organic solid waste into organic fertilizer, which is of great significance to maintaining and improving soil fertility and promoting sustainable agricultural development.

[0004] In the composting process, the prior art often uses compost inoculants containing microorganisms to decompose organic solid waste. However, the prior art has problems such as poor high temperature adaptability, poor metabolic capacity, and incomplete conversion of organic matter. At the same time, some of the currently available compost inoculants are liquid, which is not conducive to storage and transportation. Summary of the invention

[0005] The problem solved by the present invention is that the prior art has the problems of poor high temperature adaptability, poor metabolic capacity, incomplete degradation of organic matter, etc. At the same time, part of the currently available compost inoculants is liquid, which is not conducive to storage and transportation.

[0006] In view of the above technical problems, the present invention provides a thermophilic anaerobic Bacillus nubiquitus and its application.

[0007] Specifically, the present invention provides the following technical solutions: 1. A thermophilic Bacillus nubiquitosa, characterized in that the Bacillus nubiquitosa is: Bacillus nubiquitosa ( Anoxybacillus rupiensis )S2-5, which is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number: CCTCC NO: M 20241447.

[0008] 2. The anaerobic Bacillus nubifolia according to technical solution 1 is characterized in that the 16S rDNA gene sequence of the anaerobic Bacillus nubifolia is shown in SEQ ID NO.3.

[0009] 3. The anaerobic Bacillus of Nubihalogenis according to technical solution 1 or 2, characterized in that the anaerobic Bacillus of Nubihalogenis has the ability to survive under the condition of 40-80°C.

[0010] 4. The anaerobic Bacillus nubifolius according to any one of technical solutions 1 to 3, characterized in that the anaerobic Bacillus nubifolius has the ability to produce protease and / or amylase and / or cellulase.

[0011] 5. Use of the anaerobic Bacillus nubiquitus described in any one of technical solutions 1 to 4 in the preparation of a compost inoculant.

[0012] 6. A compost inoculant, characterized in that it contains the anaerobic Bacillus nubiquitus described in any one of technical solutions 1 to 4.

[0013] 7. The compost inoculant according to technical solution 6 is characterized in that the number of viable bacteria in the compost inoculant is 1×10 7 -1×10 10 CFU / g.

[0014] 8. The compost inoculant according to technical solution 6 or 7 is characterized in that it is prepared by a method comprising the following steps: inoculating the fermentation liquid of the anaerobic Bacillus nubiquitus into a solid fermentation medium for solid fermentation, drying and crushing to obtain the compost inoculant.

[0015] 9. The compost inoculant according to technical solution 8 is characterized in that the solid fermentation medium comprises one or more substances selected from the group consisting of wheat bran, corn flour, ammonium sulfate and potassium dihydrogen phosphate. Preferably, the solid fermentation medium is a combination of wheat bran, corn flour, ammonium sulfate and potassium dihydrogen phosphate; more preferably, the mixing weight ratio of wheat bran: corn flour: ammonium sulfate: potassium dihydrogen phosphate is 650-750: 250-350: 8-15: 0.5-0.9; most preferably, the mixing weight ratio of wheat bran: corn flour: ammonium sulfate: potassium dihydrogen phosphate is 680-720: 280-320: 9-11: 0.6-0.8.

[0016] 10. The compost inoculant according to technical solution 8 or 9 is characterized in that the water content of the solid fermentation medium is 50%-55% based on the weight of the solid fermentation medium.

[0017] 11. The compost inoculant according to any one of technical solutions 6 to 10, characterized in that the fermentation liquid of the anaerobic Bacillus nubigenes is prepared by a method comprising the following steps: inoculating the anaerobic Bacillus nubigenes into a liquid culture medium for culturing to obtain the fermentation liquid of the anaerobic Bacillus nubigenes, wherein the liquid culture medium comprises: 5-10 g / mL of tryptone, 3-7 g / mL of yeast extract powder and 3-7 g / mL of sodium chloride, and the balance is water.

[0018] 12. A fermentation method for preparing the compost inoculant according to any one of technical solutions 6 to 11, characterized in that it comprises the following steps: inoculating the fermentation liquid of the anaerobic Bacillus nubiquitus into a solid-state fermentation culture for solid-state fermentation, drying and crushing to obtain the compost inoculant.

[0019] 13. The fermentation preparation method according to technical solution 12 is characterized in that it comprises the following steps: (1) inoculating the anaerobic Bacillus nubigenes into a liquid culture medium for culturing to obtain a fermentation liquid of the anaerobic Bacillus nubigenes; (2) The fermentation liquid of the anaerobic Bacillus nubivorus obtained in step (1) is inoculated into a solid fermentation medium for solid fermentation, dried and crushed to obtain a compost inoculant.

[0020] 14. According to the fermentation preparation method of technical solution 12 or 13, the inoculation amount of the fermentation liquid of anaerobic Bacillus nubigenes per kg of solid fermentation medium is 3-10 mL, preferably, the inoculation amount of the fermentation liquid of anaerobic Bacillus nubigenes per kg of solid fermentation medium is 5-7 mL.

[0021] 15. According to the fermentation preparation method of technical solution 12 or 13, the solid-state fermentation temperature is 50-65°C, preferably, the solid-state fermentation temperature is 50-60°C.

[0022] 16. According to the fermentation preparation method of technical solution 12 or 13, the solid-state fermentation time is 24-60 hours, preferably, the solid-state fermentation time is 36-48 hours.

[0023] 17. The fermentation preparation method according to technical solution 12 or 13 is characterized in that the drying temperature is 50-55°C and the drying is performed to a moisture content of 5%-10%.

[0024] 18. The fermentation preparation method according to technical solution 12 or 13 is characterized in that the powder is sieved through a 80-100 mesh sieve after being crushed.

[0025] 19. The fermentation preparation method according to technical solution 13 is characterized in that in step (1), the inoculation amount is 3-10% by volume.

[0026] 20. According to the fermentation preparation method of technical solution 13, in step (1), the culture temperature is 50-60°C.

[0027] 21. Use of the compost inoculant according to any one of technical solutions 6 to 11 or the compost inoculant prepared by the fermentation preparation method according to any one of technical solutions 12 to 20 in the preparation of fertilizers.

[0028] 22. A fertilizer, characterized in that it contains the compost inoculant according to any one of technical solutions 6 to 11 or the compost inoculant prepared by the fermentation preparation method according to any one of technical solutions 12 to 20.

[0029] 23. The fertilizer according to technical solution 22 is characterized by comprising 1-2 parts of compost inoculant and 1000-2000 parts of organic matter.

[0030] 24. The fertilizer according to technical solution 22 or 23 is characterized in that it is prepared by a method comprising the following steps: mixing a compost inoculant and organic matter and fermenting the mixture to obtain the fertilizer.

[0031] 25. The fertilizer according to any one of technical solutions 22 to 24, characterized in that the carbon-nitrogen ratio of the organic material is 25-30, and / or the moisture content of the organic material is 45%-55%.

[0032] 26. The fertilizer according to any one of technical solutions 22 to 25, characterized in that the organic matter comprises one or more substances selected from the group consisting of rice bran, mushroom bran, cornmeal, straw, livestock and poultry excrement, garbage and sludge; preferably, the organic matter is a combination of mushroom bran, livestock and poultry excrement and straw, and more preferably, the mushroom bran is chestnut mushroom bran.

[0033] 27. The fertilizer according to technical solution 26 is characterized in that the weight ratio of chestnut mushroom chaff, livestock and poultry excrement and straw is (10-15): (5-10): (1-2).

[0034] 28. The fertilizer according to any one of technical solutions 24-27, characterized in that the fermentation time is greater than 0 day and less than or equal to 30 days, preferably, the fermentation time is 10-30 days, more preferably, the fermentation time is 20-30 days, and most preferably, the fermentation time is 25-30 days.

[0035] 29. The fertilizer according to any one of technical solutions 22 to 28, characterized in that the content of organic matter in the fertilizer is less than or equal to 65%, preferably, the content of organic matter in the fertilizer is 40%-65%.

[0036] Beneficial effects of the present invention: (1) The Nubihalogen anaerobic Bacillus S2-5 strain provided by the present invention is screened out under high-temperature composting, has good thermal stability, its metabolic activity is not inhibited by the high-temperature composting temperature, and has strong adaptability.

[0037] (2) The compost inoculant obtained by solid-state fermentation of the anaerobic Bacillus nubiquitus S2-5 strain provided by the present invention has the characteristics of easy preservation, easy transportation, high bacterial activity, etc. In addition, the fertilizer prepared by the compost inoculant can be applied to high-temperature composting of organic solid waste, thereby shortening the composting cycle.

[0038] Strain deposit information The present invention provides an anaerobic Bacillus nubivorus ( Anoxybacillus rupiensis )S2-5, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 1, 2024, with the deposit number CCTCC NO: M20241447, deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the colony morphology of anaerobic Bacillus S2-5 from Nubihalogen.

[0040] Figure 2 This is a Gram-stained microscopic image of the anaerobic Bacillus sp. S2-5 from Nubiqui.

[0041] Figure 3 This is a microscopic morphology of the anaerobic Bacillus of Nubiquitus S2-5 after spore staining.

[0042] Figure 4 This is the phylogenetic tree result constructed based on the 16S rDNA gene sequence of Nubihalogen Aanaerobic Bacillus S2-5.

[0043] Figure 5 This is a picture showing the transparent circle formed by the decomposition of protein by anaerobic Bacillus nubivorus S2-5 on a skimmed milk powder culture plate.

[0044] Figure 6 This is a picture showing the transparent circle formed by the decomposition of starch by anaerobic Bacillus S2-5 on the screening culture medium plate for amylase-producing strains.

[0045] Figure 7 This is a picture showing the transparent circle formed by the decomposition of cellulose by anaerobic Bacillus nubivorus S2-5 on a cellulose Congo red medium plate.

[0046] Figure 8Shown are the changes in fertilizer temperature of the experimental group and the control group during the composting period.

[0047] Fig. 9 Shown are the changes in organic matter content in fertilizers of the experimental and control groups during the composting period.

[0048] Fig.10 This is the effect of fertilizers in the experimental and control groups on seed germination index during composting. DETAILED DESCRIPTION

[0049] In order to better understand the above technical solution, the technical solution of the present invention is clearly and completely explained in conjunction with the specific implementation method below. It should be noted that the content in the specific implementation method is only a specific implementation and explanation of the technical solution of the present invention, and should not be understood as limiting the scope of protection of the present invention.

[0050] In some embodiments, the present invention provides a strain of Bacillus nubivorus ( Anoxybacillus rupiensis ) S2-5, which was isolated, purified and identified from the compost product of Angel Yeast (Chifeng) Co., Ltd. in Chifeng City, Inner Mongolia Autonomous Region. The colony texture of Nubi Halogen Anaerobic Bacillus S2-5 is round, with a raised center, moist, white, and regular edges; the microscopic morphology of the bacteria is rod-shaped and has spores. The 16S rDNA gene sequence of Nubi Halogen Anaerobic Bacillus S2-5 is shown in SEQ ID NO.1. The strain has the ability to survive under conditions of 40-80°C and can produce proteases, amylases and cellulases. At the same time, it can decompose substances such as cellulose and protein in organic solid waste at high temperatures, thereby accelerating the composting process of organic solid waste.

[0051] In some specific embodiments, when preparing NA solid culture medium in the present invention, the raw materials used include: 5-10 g / mL tryptone, 3-7 g / mL yeast extract powder, 3-7 g / mL sodium chloride and 10-20 g / mL agar powder, and the balance is water.

[0052] In some specific embodiments, when preparing the NB liquid culture medium in the present invention, the raw materials used include: 5-10 g / mL tryptone, 3-7 g / mL yeast extract powder and 3-7 g / mL sodium chloride, and the balance is water.

[0053] In some specific embodiments, when preparing the skimmed milk powder solid culture medium of the present invention, the raw materials used include: 5-10 g / mL of tryptone, 3-7 g / mL of yeast extract powder, 3-7 g / mL of sodium chloride, 10-20 g / mL of agar powder and 1-5 g / mL of skimmed milk powder, and the balance is water.

[0054] In some specific embodiments, when the present invention prepares a solid screening culture medium for amylase-producing strains, the raw materials used include: 5-10 g / mL tryptone, 3-7 g / mL yeast extract powder, 3-7 g / mL sodium chloride, 10-20 g / mL agar powder, and 5-15 g / mL soluble starch, with the balance being water.

[0055] In some specific embodiments, when the present invention prepares the cellulose Congo red solid culture medium, the raw materials used include: sodium nitrate 1-2g / mL, disodium hydrogen phosphate 1-2g / mL, potassium dihydrogen phosphate 0.5-1g / mL, magnesium sulfate 0.3-0.7g / mL, potassium chloride 0.3-0.7g / mL, yeast extract powder 0.3-0.7g / mL, acid hydrolyzed casein 0.3-0.7g / mL, Congo red 0.1-0.5g / mL, cellulose powder 3-7g / mL and agar powder 10-20g / mL, and the balance is water.

[0056] It should be noted that the tryptone and yeast extract are mainly present in the above-mentioned culture media as organic nitrogen sources to provide nitrogen elements necessary for the growth of microorganisms during fermentation. After being decomposed in the above-mentioned culture media, common organic nitrogen sources such as yeast extract, yeast extract, and peptone release amino acids and small molecule peptides, thereby becoming nitrogen sources required for the growth of microorganisms.

[0057] That is, when the present invention uses tryptone and yeast extract as common organic nitrogen sources to prepare the above-mentioned culture media, the present invention has no particular limitation on their sources, and their sources can be any commercially available or prepared by conventional methods. As long as tryptone is commercially available or prepared by conventional methods, and its total nitrogen content is greater than or equal to 10.0wt%, it can be used in the present invention. As long as yeast extract is commercially available or prepared by conventional methods, and its total nitrogen content is greater than or equal to 7.0wt% and its amino nitrogen content is greater than or equal to 3.0wt%, it can be used in the present invention.

[0058] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0059] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The sources of the experimental reagents and instruments used in the present invention are shown in Table 1 below.

[0060] Table 1. Reagent / Instrument Source Information The preparation of the culture medium involved in the embodiments of the present invention is specifically as follows: NA solid culture medium: Mix 10 g of tryptone, 5 g of yeast extract powder, 5 g of sodium chloride, 15 g of agar powder and distilled water and make up to 1000 mL. Adjust the pH to 7.5 ± 0.2 and sterilize at 121°C for 25 min to obtain NA solid culture medium.

[0061] Skim milk powder solid culture medium: Mix 10 g of tryptone, 5 g of yeast extract powder, 5 g of sodium chloride, 15 g of agar powder, 3 g of skim milk powder and distilled water and make up to 1000 mL. Adjust the pH to 7.5 ± 0.2 and sterilize at 121°C for 25 min to obtain a skim milk powder solid culture medium.

[0062] Solid screening medium for amylase-producing strains: Mix 10 g of tryptone, 5 g of yeast extract powder, 5 g of sodium chloride, 15 g of agar powder, 10 g of soluble starch and distilled water and make up to 1000 mL. After adjusting the pH to 7.5 ± 0.2, sterilize at 121° C. for 25 min to obtain a solid screening medium for amylase-producing strains.

[0063] Cellulose Congo red solid culture medium: Mix 1 g of sodium nitrate, 1.2 g of disodium hydrogen phosphate, 0.9 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of potassium chloride, 0.5 g of yeast extract powder, 0.5 g of acid hydrolyzed casein, 0.2 g of Congo red, 5 g of cellulose powder, 15 g of agar powder and distilled water and make up to 1000 mL. Adjust the pH to 7.0 ± 0.2 and sterilize at 121 °C for 25 min to obtain a cellulose Congo red solid culture medium.

[0064] NB liquid medium: Mix 10 g of tryptone, 3 g of yeast extract powder, 5 g of sodium chloride and distilled water and make up to 1000 mL. Adjust the pH value to 7.2 ± 0.2 and sterilize at 121°C for 25 min to obtain NB liquid medium.

[0065] Determination method of carbon-nitrogen ratio in application example: 1. Carbon-nitrogen ratio = organic carbon content / total nitrogen content, where the determination methods of organic carbon content and total nitrogen content are as follows: (1.1) Determination of organic carbon content: weigh 0.2g of the dried, crushed and sieved sample into a conical flask, add 10mL of 1 mol / L K2Cr2O7 solution, mix well, then slowly add 20mL of concentrated H2SO4, shake gently for 1min, let stand for 30min, transfer to a 250mL volumetric flask after cooling to room temperature, add water to make up the volume, add 3-4 drops of o-phenanthroline indicator, then titrate with 5mmol / L FeSO4 standard solution, the solution changes from green to dark green, then add FeSO4 drop by drop until the solution turns brick red as the sample group, and calculate the organic carbon content (g / kg) in the sample by (Formula 1). At the same time, the blank group is taken as the group without adding sample.

[0066] Organic carbon content (g / kg) = [C(V0-V) × 0.001 × 3.0 × 1.33 / m] × 1000 (Formula 1) Wherein, 1.33 represents the oxidation correction coefficient; C represents the concentration of the FeS04 standard solution; V0 represents the volume of FeS04 used for the blank group titration (mL); V represents the volume of FeS04 used for the sample group titration (mL); m represents the mass of the sample; 3.0 represents the molar mass of 1 / 4 carbon atom (g / mol); 0.001 represents the coefficient for converting mL to L; 1000 represents the coefficient for converting to content per kg.

[0067] (1.2) Determination of total nitrogen content: Weigh 0.3 g of the dried, crushed and sieved sample into a digestion tube, add 0.2 g of copper sulfate, 6 g of potassium sulfate and 20 mL of concentrated sulfuric acid, mix the solution evenly and then digest. Digest at 240°C for 30 min, then raise the temperature to 400°C and continue digestion. When the color of the solution changes from grayish white to light blue, continue digestion. After cooling to room temperature, distill the digestion solution using a Kjeldahl nitrogen analyzer, and titrate the final distillate with 0.01 mol / L hydrochloric acid standard solution. The endpoint is the critical point where the solution changes from blue-green to red, which is used as the sample group. At the same time, use the blank group without adding sample. Record the volume (mL) of the hydrochloric acid standard solution consumed and calculate the total nitrogen content in the sample by (Formula 2).

[0068] Total nitrogen content = [14 × C × (V1-V2)] / m (Formula 2) In the formula, C represents the molar concentration of the hydrochloric acid standard solution (mol / L); V1 represents the volume of the hydrochloric acid standard solution used for the sample group titration (mL); V2 represents the volume of the hydrochloric acid standard solution used for the blank group titration (mL); 0.014 represents the molar mass of the nitrogen atom (g / mol); and m represents the mass of the sample after drying (g).

[0069] Example 1: Isolation and screening of strains (1) 10 g of compost product (the compost product was purchased from Angel Yeast (Chifeng) Co., Ltd., Chifeng City, Inner Mongolia Autonomous Region) was placed in a conical flask containing 100 mL of sterile water and 10 glass beads. After shaking at 55°C and 180 rpm for 30 min, a gradient dilution was performed and 10 -5 , 10 -6 , 10 -7 The three dilutions were spread on NA solid culture medium plates and cultured at 55°C for 24 h, and single colonies of 36 bacterial strains were obtained on the NA solid culture medium plates.

[0070] (2) Screening of protease-producing strains: The single colonies of the 36 bacterial strains obtained in step (1) were inoculated into NB liquid culture medium and cultured at 180 r / min and 55°C for 24 h to obtain bacterial suspensions of the 36 bacterial strains. Then, the bacterial suspensions of the 36 bacterial strains were spotted onto skimmed milk powder culture plates, with 3 bacterial suspensions spotted on each plate and cultured at 55°C for 24 h. The strains that produced transparent circles were screened, and the colonies and transparent circle diameters of different strains were measured. The HC value (D / d) was calculated based on the transparent circle diameter (D) and colony diameter (d). Further, the bacteria with protease-producing ability were screened out in descending order based on the HC values ​​of the 36 bacterial strains, and 18 bacterial strains were obtained.

[0071] (3) Screening of amylase-producing strains: The 18 strains obtained in step (2) were selected and inoculated into NB liquid culture medium and cultured at 180 r / min and 55°C for 24 h to obtain a bacterial suspension of 18 strains. Then, the bacterial suspensions of the 18 strains were spotted onto solid screening culture medium plates for amylase-producing strains. Three strains of bacterial suspension were spotted onto each plate and cultured at 55°C for 24 h. Colonies that produced transparent circles were screened, and the colony and transparent circle diameters of different strains were measured. The HC value (D / d) was calculated based on the transparent circle diameter (D) and the colony diameter (d). Further, based on the HC values ​​of the 18 strains, bacteria with the ability to produce amylase were screened from large to small to obtain 8 strains.

[0072] (4) Screening of cellulase-producing bacteria: The eight strains obtained in step (3) were inoculated into NB liquid culture medium and cultured at 180 r / min and 55°C for 24 h to obtain bacterial suspensions of eight strains. Then, the bacterial suspensions of the eight strains were spotted onto cellulose Congo red solid culture medium plates, with three bacterial suspensions spotted on each plate and cultured at 55°C for 24 h. The strains that produced transparent circles were screened, and the colony and transparent circle diameters of different strains were measured. The HC value (D / d) was calculated based on the transparent circle diameter (D) and colony diameter (d). Further, based on the HC values ​​of the 36 strains, bacteria with the ability to produce cellulase were screened out in descending order, and two strains were obtained.

[0073] The HC values ​​of the two bacterial strains obtained in the above steps (1), (2), (3) and (4) are comprehensively ranked, and the strain with the highest comprehensive ranking is selected as the target strain.

[0074] The target strain was selected and inoculated in NB liquid culture medium and cultured at 180r / min and 55°C for 24h to obtain a bacterial suspension of the target strain. The bacterial suspension of the target strain was inoculated on a NA solid culture medium plate and cultured at 55°C until a single colony grew to observe the colony morphology of the target strain; at the same time, a single colony was selected and cultured in NB liquid culture medium at 180r / min and 55°C for 24h, and 10 μL was aspirated on a glass slide and Gram staining was performed using a Gram staining kit, and 10 μL was aspirated on a glass slide and spores were stained using malachite green and safflower aqueous solution, and the microscopic morphology of the target strain was observed by magnifying 100 times under an optical microscope.

[0075] The colony morphology of the target strain is round, with a raised center, moist, white, and regular edges; the microscopic morphology of the target strain is rod-shaped and has spores. The genome of the strain is extracted, and the genome of the target strain is extracted, using 27F (5'-AGAGTTTGATCCTGGCTCAG-3', i.e., SEQ ID NO.1) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', i.e., SEQ ID NO.2) as primers. The PCR program is: denaturation at 94°C for 30s, annealing at 55°C for 20s, and then extension at 72°C for 30s, for a total of 30-35 cycles. After the last cycle, the reaction tube is incubated at 72°C for 5min to ensure sufficient extension. It is used to amplify the 16S rDNA gene sequence. After 1% gel electrophoresis detection and sequencing, the measured 16S rDNA sequence is compared with the sequence on GenBank by Blast analysis. Sequence similarity greater than 99% indicates the same species. The measured 16S rDNA gene sequence of the target strain is shown in SEQ ID NO.3.

[0076] SEQ ID NO.3: Based on the 16S rDNA gene sequence shown in SEQ ID NO.3, MEGA7.0 was used to compare the differences in the 16S rDNA gene sequence to infer the evolutionary path of the organism and to construct a phylogenetic tree. The constructed phylogenetic tree is shown in Figure 4 As shown, from Figure 4 The phylogenetic tree showed that the strain was most closely related to the anaerobic Bacillus nubifolia. At the same time, further morphological analysis and molecular identification showed that the strain was anaerobic Bacillus nubifolia S2-5 ( Anoxybacillus rupiensis S2-5). It has been deposited in China Center for Type Culture Collection (CCTCC) on July 1, 2024, with the deposit number: CCTCC NO: M 20241447. Figure 1 Shown is the colony morphology of Anaerobic Bacillus nubifolia S2-5. Figure 2 Shown is a Gram-stained microscopic image of the anaerobic Bacillus nubifolia S2-5; Figure 3 Shown is the microscopic morphology of the anaerobic Bacillus nubifolia S2-5 after spore staining; Figure 5 The figure shows the transparent circle formed by the decomposition of protein by the anaerobic Bacillus nubidiolus S2-5 on the skim milk powder medium plate; Figure 6 The figure shows the result of the transparent circle formed by the decomposition of starch by the anaerobic Bacillus nubidiolus S2-5 on the screening medium plate of the amylase-producing strain; Figure 7 Shown is the result of the transparent circle formed by the decomposition of cellulose by anaerobic Bacillus S2-5 on the cellulose Congo red medium plate.

[0077] Example 2: Preparation of compost inoculant (1) Streak the strain stored in the glycerol tube onto NA medium and incubate at 55°C for 24 h for activation.

[0078] (2) After activation, the strain was transferred to NB liquid culture medium in a triangular flask and cultured at 180 r / min and 55°C for 24 h. The number of viable bacteria was 1.87×10 8 CFU / mL of seed solution.

[0079] (3) The seed solution obtained in step (2) was transferred into a fermenter containing NB liquid culture medium at an inoculum volume of 3%, and cultured at 55°C for 24 hours to obtain a viable bacterial count of 2.39×10 8 The fermentation tank has a volume of 1 L and a liquid volume of 500 mL.

[0080] (4) The fermentation broth in the fermentation tank obtained in step (3) is divided into 16 groups, namely, Examples 1 to 16, as shown in Table 2, and the fermentation broths of Examples 1 to 16 are inoculated into their respective solid-state fermentation cultures according to the inoculation amount, fermentation time and fermentation temperature in Table 2, and solid-state fermentation is performed to obtain fermented products of Examples 1 to 16, respectively, wherein the inoculation amount (mL / kg) in Table 2 is expressed as the amount of mL of inoculated fermentation broth per kg of solid-state fermentation medium. The fermented product is dried at 55°C to a moisture content of 5%-10%, and then crushed and sieved through an 80-mesh sieve to obtain a compost inoculant. Wherein, solid-state fermentation medium: wheat bran, corn flour, ammonium sulfate, and potassium dihydrogen phosphate are mixed in a weight ratio of 700:300:10:0.7, and the moisture content of the solid-state fermentation medium is controlled to be 55%, to obtain a solid-state fermentation medium.

[0081] The inoculum size, fermentation time, fermentation temperature of solid-state fermentation, and the number of effective live bacteria in the compost inoculant are shown in Table 2 below.

[0082] Table 2. Inoculum size, fermentation time, fermentation temperature, and effective viable bacteria count in compost inoculant for solid-state fermentation As shown in Table 2, the number of effective live bacteria in the fermentation product obtained in Example 2.14 was the highest. Therefore, the combination of an inoculum size of 5 (mL / kg), a fermentation time of 48 h, and a fermentation temperature of 50 °C was the optimal solid-state fermentation condition. The live bacteria content in the compost inoculant obtained under the optimal solid-state fermentation condition was 5×10 9 Therefore, in the following application examples, the present invention selects the compost inoculant prepared in Example 2.14 to further verify the application of the compost inoculant in composting.

[0083] Application example: Fertilizer preparation (1) The chestnut mushroom bran (the culture medium residue after the harvest of chestnut mushroom cultivation, the main component of which is a mixture of sawdust and wheat bran) was crushed and passed through a 60-mesh sieve as a compost carrier. The chestnut mushroom bran, livestock and poultry manure and straw were mixed in a weight ratio of chestnut mushroom bran: livestock and poultry manure: straw = 11:8:1 to obtain a mixture, and the initial carbon-nitrogen ratio of the mixture was adjusted to 26.3, and the initial moisture content of the material was adjusted to 60%. The composting reactor was a 66×60×93 cm mobile composting box, and the loading amount was 60 kg / box.

[0084] (2) Experimental group: Add the compost inoculant obtained in Example 2.14, and add 0.2% (w / w) of the compost inoculant to the weight of the mixture obtained in step (1) to carry out composting. The control group was used as the control group. During the composting period, the compost was turned every 6-8 days to supply oxygen and mix the materials. On the 0th, 5th, 10th, 15th, 20th, 25th and 30th days of composting, samples were taken from the top, middle, bottom, left and right of the compost bin and mixed to obtain fertilizer.

[0085] (3) The fertilizer obtained in step (2) is tested for organic matter content, such as Figure 8 As shown; and seed germination index test, such as Fig. 9 As shown. Among them, the method for detecting organic matter content: the method in Appendix C of NY / T 525-2021 is used for detection; the method for detecting germination index: the method in Appendix F of NY / T 525-2021 is used for detection.

[0086] (4) During the composting period, the temperature is measured regularly in the morning and afternoon every day, and the average value is taken as the temperature of the compost pile on that day. Figure 7 As shown. Wherein, temperature measurement: temperature measurement is performed using a thermometer.

[0087] like Figure 8 As shown, Figure 8 The results show that the maximum temperature of the experimental group and the control group during the composting period is 70.8°C when the inoculant of the present invention is added, while the control group without the compost inoculant is 64.1°C. Since the compost temperature is an important indicator of the metabolic intensity of microorganisms, the higher the temperature, the more active the strain. This shows that the compost inoculant provided by the present invention increases the maximum temperature of the composting high temperature period, promotes the degradation of organic matter, and improves the composting efficiency.

[0088] like Fig. 9 As shown, Fig. 9 The results show that the organic matter content of the experimental group and the control group was 63.77% after the experiment, and the organic matter content of the control group was 65.6%. The results show that the compost inoculant provided by the present invention is beneficial to promote the degradation of organic matter.

[0089] like Fig.10 As shown, Fig.10 The effect of fertilizers in the experimental group and the control group on the seed germination index during the composting period. It should be noted that the seed germination index is one of the indicators for evaluating the maturity of fertilizers, and the seed germination index also comprehensively reflects the toxicity of fertilizers to plants. Fig.10It can be seen that the germination index of the seeds in the experimental group is 92.5%, and the germination index of the seeds in the control group is 82.2%. Fig.10 It can also be seen that the seed germination index of the experimental group basically reached a stable state at 25 days, which proves that the fertilizer obtained by the experimental group has reached a mature state, while the seed germination index of the control group is still rising, which proves that the fertilizer obtained by the control group has not reached a mature state. It can be seen that the compost inoculant provided by the present invention has a promoting effect on compost maturity.

[0090] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A thermophilic anaerobic Bacillus nubiquitus, characterized in that The anaerobic bacillus of Nubihalogen is: anaerobic bacillus of Nubihalogen ( Anoxybacillus rupiensis )S2-5, which is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number: CCTCC NO: M 20241447.

2. The anaerobic Bacillus nubiquitus according to claim 1, characterized in that The 16S rDNA gene sequence of the anaerobic Bacillus nubiquitus is shown in SEQ ID NO.

3.

3. The anaerobic Bacillus nubiquitus according to claim 1, characterized in that The Nubihalogen anaerobic Bacillus has the ability to survive under the condition of 40-80°C.

4. The anaerobic Bacillus nubiquitus according to claim 1, characterized in that The anaerobic Bacillus nubivorus has the ability to produce protease.

5. The anaerobic Bacillus nubiquitus according to claim 1, characterized in that The anaerobic Bacillus nubifolia has the ability to produce amylase.

6. The anaerobic Bacillus nubiquitus according to claim 1, characterized in that The anaerobic Bacillus nubivorus has the ability to produce cellulase.

7. Use of the anaerobic Bacillus nubiquitus according to any one of claims 1 to 6 in preparing a compost inoculant.

8. A compost inoculant, characterized in that The invention comprises the anaerobic Bacillus nubiquitus of any one of claims 1 to 6.

9. The compost inoculant according to claim 8, characterized in that The viable bacteria count of the compost inoculant is 1×10 7 -1×10 10 CFU / g.

10. The compost inoculant according to claim 8, characterized in that The compost inoculant is prepared by a method comprising the following steps: inoculating the fermentation liquid of the anaerobic bacillus nubiquitus into a solid fermentation medium for solid fermentation, drying and crushing, and obtaining a compost inoculant.

11. The compost inoculant according to claim 10, characterized in that The solid-state fermentation medium comprises one or more substances selected from the group consisting of wheat bran, corn flour, ammonium sulfate and potassium dihydrogen phosphate.

12. The compost inoculant according to claim 11, characterized in that The solid-state fermentation medium is a combination of wheat bran, corn flour, ammonium sulfate and potassium dihydrogen phosphate.

13. The compost inoculant according to claim 12, characterized in that The mixing weight ratio of wheat bran: corn flour: ammonium sulfate: potassium dihydrogen phosphate is 650-750: 250-350: 8-15: 0.5-0.

9.

14. The compost inoculant according to claim 13, characterized in that The mixing weight ratio of wheat bran: corn flour: ammonium sulfate: potassium dihydrogen phosphate is 680-720: 280-320: 9-11: 0.6-0.

8.

15. The compost inoculant according to claim 10, characterized in that The water content of the solid-state fermentation medium is 50%-55% based on the weight of the solid-state fermentation medium.

16. The compost inoculant according to claim 10, characterized in that The fermentation liquid of the anaerobic Bacillus nubigenes is prepared by a method comprising the following steps: inoculating the anaerobic Bacillus nubigenes into a liquid culture medium for culturing to obtain the fermentation liquid of the anaerobic Bacillus nubigenes, wherein the liquid culture medium comprises: 5-10 g / mL of tryptone, 3-7 g / mL of yeast extract powder and 3-7 g / mL of sodium chloride, and the balance is water.

17. A fermentation method for preparing the compost inoculant according to any one of claims 8 to 16, characterized in that: The method comprises the following steps: inoculating the fermentation liquid of the anaerobic bacillus nubifolia into a solid-state fermentation culture for solid-state fermentation, drying and crushing, and obtaining a compost inoculant.

18. The fermentation preparation method according to claim 17, characterized in that: The steps include: (1) inoculating the anaerobic Bacillus nubigenes into a liquid culture medium for culturing to obtain a fermentation liquid of the anaerobic Bacillus nubigenes; (2) The fermentation liquid of the anaerobic Bacillus nubivorus obtained in step (1) is inoculated into a solid fermentation medium for solid fermentation, dried and crushed to obtain a compost inoculant.

19. The fermentation preparation method according to claim 18, characterized in that: The inoculation amount of the fermentation liquid of anaerobic Bacillus nubivorus in each kg of solid fermentation medium is 3-10 mL.

20. The fermentation preparation method according to claim 19, characterized in that: The inoculation amount of the fermentation liquid of anaerobic Bacillus nubiquitus in each kg of solid fermentation medium is 5-7 mL.

21. The fermentation preparation method according to claim 17, characterized in that: The solid state fermentation temperature is 50-65℃.

22. The fermentation preparation method according to claim 21, characterized in that: The solid state fermentation temperature is 50-60℃.

23. The fermentation preparation method according to claim 17, characterized in that: The solid-state fermentation time is 24-60h.

24. The fermentation preparation method according to claim 23, characterized in that: The solid-state fermentation time is 36-48h.

25. The fermentation preparation method according to claim 17, characterized in that: The drying temperature is 50-55℃, and the moisture content is 5%-10%.

26. The fermentation preparation method according to claim 17, characterized in that: After crushing, pass through 80-100 mesh sieve.

27. The fermentation preparation method according to claim 18, characterized in that: In step (1), the inoculation amount is 3-10% by volume.

28. The fermentation preparation method according to claim 18, characterized in that: In step (1), the culture temperature is 50-60°C.

29. Use of the compost inoculant according to any one of claims 8 to 16 in preparing fertilizer.

30. A fertilizer, characterized in that Contains the compost inoculant according to any one of claims 8 to 16.

31. The fertilizer according to claim 30, characterized in that 1-2 parts of compost inoculant and 1000-2000 parts of organic matter.

32. The fertilizer according to claim 31, characterized in that The fertilizer is prepared by a method comprising the following steps: mixing a compost inoculant and organic matter and fermenting the mixture to obtain fertilizer.

33. The fertilizer according to claim 31, characterized in that The carbon-nitrogen ratio of organic materials is 25-30.

34. The fertilizer according to claim 31, characterized in that The moisture content of organic materials is 45%-55%.

35. The fertilizer according to claim 31, characterized in that The organic matter includes one or more substances selected from the group consisting of rice bran, mushroom bran, cornmeal, straw, livestock and poultry excrement, garbage and sludge.

36. The fertilizer according to claim 35, characterized in that The organic matter is a combination of fungus dregs, livestock and poultry excrement and straw.

37. The fertilizer according to claim 36, characterized in that The mushroom bran is chestnut mushroom bran.

38. The fertilizer according to claim 37, characterized in that The weight mixing ratio of chestnut mushroom bran, livestock and poultry manure and straw is (10-15): (5-10): (1-2).

39. The fertilizer according to claim 32, characterized in that The fermentation time is greater than 0 days and less than or equal to 30 days.

40. The fertilizer according to claim 39, characterized in that The fermentation time is 10-30 days.

41. The fertilizer according to claim 40, characterized in that The fermentation time is 20-30 days.

42. The fertilizer according to claim 41, characterized in that The fermentation time is 25-30 days.

43. The fertilizer according to any one of claims 30 to 42, characterized in that The content of organic matter in the fertilizer is less than or equal to 65%.

44. The fertilizer according to claim 43, characterized in that The content of organic matter in the fertilizer is 40%-65%.

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