Bacillus subtilis and application thereof in aerobic composting deodorization and sulfur fixation

Abstract

The invention discloses a strain of bacillus subtilis and application thereof in aerobic composting deodorization and sulfur immobilization, the bacillus subtilis B.subtilis SHB-2 is preserved in China Center for Type Culture Collection on May 27, 2025, and the preservation number is CGMCC: No. 34689. The invention also discloses a method for preparing the bacillus subtilis SHB-2. The bacillus subtilis SHB-2 has the advantages that the bacillus subtilis SHB-2 is preserved in China Center for Type Culture Collection on May 27, 2025; the strain can rapidly grow under the condition of 60 DEG C. By means of the bacillus thermophilus, organic ammonia and sulfur elements in the aerobic composting process can be efficiently fixed, release of NH3 and H2S can be effectively reduced, the quality of organic fertilizer is improved, and the bacillus thermophilus has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of functional microbial application technology, specifically to a strain of Bacillus subtilis and its application in aerobic composting for deodorization and sulfur fixation. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Composting is a key technology that utilizes microbial decomposition to degrade and stabilize organic solid waste such as livestock and poultry manure into mature compost, thereby producing organic fertilizer. It is of great significance for realizing the resource utilization of organic waste, improving soil fertility, and protecting the environment. Among these methods, aerobic composting, due to its high efficiency in degradation and resource recovery, has become one of the mainstream methods for treating organic solid waste.

[0004] However, aerobic composting inevitably involves odor pollution, which has become a key factor restricting its widespread application and environmental benefits. Among the four typical stages of composting—heating, high temperature, cooling, and maturation—odor production is most pronounced during the high temperature stage. This is because the rapid decomposition of easily degradable organic matter in the early stages of composting leads to a sharp rise in temperature, accompanied by a large consumption of oxygen, easily creating localized micro-aerobic or even anoxic environments within the material. Under these conditions, microbial metabolic activities generate and release various odor-causing substances.

[0005] It is worth noting that ammonia (NH3) and hydrogen sulfide (H2S) are the most abundant and representative inorganic odor pollutants released during composting. Studies have shown that these two gases are mainly generated and released during the high-temperature stage: on the one hand, the mineralization and decomposition of nitrogen-containing organic matter is the main source of NH3; on the other hand, the anaerobic metabolism of sulfur-containing organic matter under localized hypoxic conditions leads to the generation of sulfur-containing compounds such as H2S, as well as a small amount of organic acids. Therefore, effectively controlling the release of odorous gases such as NH3 and H2S during the high-temperature stage is a core technical challenge that urgently needs to be addressed to improve the environmental friendliness of aerobic composting technology and reduce the risk of secondary pollution.

[0006] Biological deodorization technology mainly utilizes the physiological metabolic activities of microorganisms to degrade malodorous substances. Through adsorption and absorption, microorganisms oxidize malodorous substances into odorless and harmless final products, thus achieving deodorization. While the microorganisms currently selected show good removal effects on NH3 and H2S at low temperatures, their removal efficiency is poor at high temperatures. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a strain of Bacillus subtilis and its application in aerobic composting for deodorization and sulfur fixation.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a strain of Bacillus subtilis. B. subtilis SHB-2 was deposited at the China Center for Type Culture Collection (CGMCC) on May 27, 2025, with accession number CGMCC: No. 34689.

[0009] Secondly, the present invention provides the Bacillus subtilis. B. subtilis Application of SHB-2 in biological deodorization.

[0010] In some embodiments, the Bacillus subtilis B. subtilis Application of SHB-2 in aerobic composting for deodorization and sulfur fixation.

[0011] Thirdly, the present invention provides a biological deodorizing agent, comprising at least the aforementioned Bacillus subtilis. B. subtilis SHB-2.

[0012] In some embodiments, the biological deodorizing agent includes at least a porous carrier.

[0013] Preferably, the porous carrier is zeolite powder or attapulgite.

[0014] In some embodiments, the biological deodorizing agent further includes at least one other bacterium, enzyme, or its metabolite.

[0015] Preferably, the bacteria are selected from Bacillus, lactic acid bacteria, yeast, photosynthetic bacteria, bifidobacteria, actinomycetes, nitrifying bacteria, or Streptomyces flavus.

[0016] More preferably, the Bacillus is Bacillus subtilis, Bacillus megaterium, or Bacillus laterosporus; Alternatively, the lactic acid bacteria may be Bacillus acidophilus or plant lactic acid bacteria.

[0017] Preferably, the enzyme is selected from lysozyme, protease, or cellulase.

[0018] Thirdly, the present invention provides a method using the aforementioned Bacillus subtilis. B. subtilis SHB -2 The method for deodorization and sulfur fixation in aerobic composting includes the following steps: Bacillus subtilis cultured B. subtilis SHB -2 Mix thoroughly with composting materials, then carry out aerobic composting.

[0019] In some embodiments, the composting materials are mixed every 2-4 days during the composting process to ensure the composting trial can proceed.

[0020] In some embodiments, the aerobic composting time is 25-90 days.

[0021] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: The thermophilic Bacillus of the present invention can grow rapidly at 60°C. B. subtilis SHB-2 contains one sqr gene and one pdo gene. Its resting cells can completely consume 700 µM of exogenous H2S within 6 h. The H2S oxidation rate per unit biomass is: q =6.83 µmol·min -1 ·g -1 (Cell dry weight) B. subtilis During the oxidation of H2S by SHB-2, the main products are thiosulfate and intracellular thiosulfate, while the contents of extracellular thiosulfate, sulfite and sulfate do not change significantly, making it an effective application for the biological removal of H2S.

[0022] The Bacillus B. subtilis SHB-2 can be applied to aerobic composting of livestock and poultry manure. It not only significantly reduces the emission of odorous gases such as NH3 and H2S, but also significantly increases composting temperature, accelerates the composting process, and improves maturity and fertilizer efficiency. Compared with the control, inoculation... B. subtilis The thermophilic period of SHB-2 was extended to 22 days, H2S and NH3 emissions decreased by 31.52% and 26.28%, respectively, and sulfate and nitrate nitrogen contents increased to 15.18 mg / g and 2.60 mg / g, respectively. Inoculation B. subtilis SHB-2 significantly enhanced the driving role of key microbial components in sulfur and nitrogen transformation.

[0023] The Bacillus provided by this invention B. subtilis SHB-2 can be used as a deodorizing agent and an inoculant for composting organic fertilizers. It provides a new approach to reduce nitrogen and sulfur loss in aerobic composting of livestock and poultry manure and improve composting efficiency, and offers a new direction for the biological treatment of H2S. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 . B. subtilis SHB-2 colony morphology characteristics; Figure 2 . B. subtilis SHB-2 has a significant ability to oxidize exogenous H2S; Figure 3 . B. subtilisThe final product of SHB-2 oxidation of exogenous H2S; Figure 4 The temperature changes of the experimental and control groups during aerobic composting; Figure 5 The release of H2S in the experimental and control groups during aerobic composting; Figure 6 The release of NH3 in the experimental and control groups during aerobic composting; Figure 7 Changes in sulfate levels in the experimental and control groups during composting; Figure 8 Changes in water-soluble nitrate nitrogen (WSN) in the experimental and control groups during composting. Detailed Implementation

[0026] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example Obtaining Bacillus subtilis: The sample was isolated in May 2023 from the Jimo Hot Spring (120°38'52.548"E, 36°26'20.598"N) along the Qingdao Xiazhuang-Lingshanwei fault zone, using a mixture of hot spring mud and water. It was inoculated onto LB medium and enriched using temperature gradients of 30°C, 40°C, 50°C, 60°C, and 70°C.

[0029] The enrichment solution was diluted and spread on plates. The colonies were most prominent under 60°C incubation conditions. Single colonies were picked and streaked three times to isolate several pure bacteria. The fastest-growing strain, SHB-2, was selected as the target strain.

[0030] Colony morphology: The colony center is dense, the edges expand irregularly in a radial pattern, the surface is slightly rough, and it has a certain degree of adhesion and strong diffusion ability, showing good surface growth and migration characteristics.

[0031] The culture medium formula used was: Tryptone 10 g / L, Yeast extract 5 g / L, and Sodium chloride (NaCl) 10 g / L.

[0032] Will B. subtilisAfter inoculating the center of an LB plate and culturing for 10 hours, SHB-2 formed a colony with irregularly spreading radial edges, exhibiting the following morphological characteristics: Colony shape: dense in the center, with distinct feathery or serrated radial expansion at the edges, showing a typical "spiky" edge, which may be related to hyphal-like growth or motility.

[0033] Surface characteristics: The colonies are generally flat with a slight bulge in the middle. The surface is not smooth and has a certain roughness.

[0034] Color: Light gray with a slight yellow tinge.

[0035] Mobility: This outward-expanding radial morphology is usually associated with active migration of peripheral cells, and the strain may have some ability to swim or spread.

[0036] B. subtilis SHB-2 hydrogen sulfide oxidation rate test: Will B. subtilis SHB-2 cells were cultured overnight at 60℃ with shaking at 200 rpm for 12 hours. Cells were collected by centrifugation and washed once with pure water. The treated cells were then dissolved in PBS buffer at pH 8.0, and the bacterial turbidity was adjusted to OD. 600nm =2.

[0037] 10 mL of the adjusted cell suspension was added to a 50 mL anaerobic flask, followed by the addition of freshly prepared sodium hydrosulfide with a final concentration of 750 mM to initiate the reaction. The entire reaction was carried out at 60 °C with gentle shaking (100 ± 10 rpm). Samples were taken every 1 hour to determine the remaining concentration of hydrogen sulfide in the system.

[0038] After the reaction was complete, the cells were collected by centrifugation again, washed three times in pure water, and the supernatant was removed. The cells were then transferred to a vacuum freeze dryer and freeze-dried for at least 12 hours. The cell dry weight was then measured. The rate of oxidation of exogenous hydrogen sulfide per unit cell dry weight per unit time was calculated using the rate of change in hydrogen sulfide concentration and the cell dry weight.

[0039] Based on the above methods, B. subtilis SHB-2 exhibits a significantly high efficiency in oxidizing exogenous hydrogen sulfide, with an oxidation rate of [missing information]. q =6.83 µmol·min -1 ·g -1 (Cell dry weight), see Figure 2 .

[0040] Study on products of sulfide hydroxide oxidation: Under the same conditions as above, B. subtilisSHB-2 cells were cultured overnight at 60℃ with shaking at 200 rpm for 12 hours. Cells were collected by centrifugation and washed once with pure water. The treated cells were then dissolved in PBS buffer at pH 8.0, and the bacterial turbidity was adjusted to OD. 600nm =2. Add 10 mL of the adjusted cell suspension to a 50 mL anaerobic flask, and then add freshly prepared sodium hydrosulfide with a final concentration of 750 mM to initiate the reaction. The entire reaction condition is 60 ℃ with gentle shaking (100±10 r.pm). Samples are taken every 1 hour to determine the remaining concentration of hydrogen sulfide in the system, and the production of thiosulfate, sulfite, sulfate and thiosulfate are also tested.

[0041] The results showed that within seven hours B. subtilis The final products of SHB-2 oxidation of hydrogen sulfide are thioalkyl sulfur and thiosulfate (see...). Figure 3 ).

[0042] B. subtilis Evaluation of the deodorizing effect of SHB-2 during composting 1. Preparation of bacterial culture: Will B. subtilis Single colonies of SHB-2 were inoculated into test tubes containing 5 mL of LB medium and incubated at 60°C and 200 rpm for 20 hours. The bacterial culture was then transferred to Erlenmeyer flasks containing 2 L of fresh medium, and the initial OD was controlled. 600nm = 0.05. After 12 hours of incubation, the bacteria reached mid-log phase. The cells were collected by centrifugation at 4000 rpm for 10 minutes at 4°C, and washed with 100 mM pH 8.0 PBS buffer. The bacteria were resuspended in PBS buffer and the OD was adjusted. 600nm = 5. Then mix the bacterial suspension thoroughly with the compost material.

[0043] 2. Preparation of composting raw materials: The composting raw materials, fresh chicken manure and peanut shells, come from Qingdao Kanglilai Biotechnology Co., Ltd. The raw materials are thoroughly mixed, adjusting the C / N ratio to 30 and the moisture content to 60%. No additives will be added. B. subtilis Treatment with SHB-2 cells (with only 5% (v / w) PBS buffer) served as a control. Adding 5% (v / w) fresh bacterial suspension (OD200) was also included. 600nm = 5) as the experimental group.

[0044] 3. Composting process and sampling: 1 m 3The composting materials were placed in a greenhouse (1.8 m × 0.9 m × 0.9 m) for 35 days. During composting, samples were mixed every 3 days to ensure the aerobic composting experiment. Samples were taken every two days. The compost pile was divided diagonally into five sections, and samples were collected from the same location at a depth of 20-30 cm using a sterilized shovel containing 75% alcohol. These samples were then mixed into a single composite sample. Three samples were taken from each experimental and control group as replicates for each sampling.

[0045] 4. Determination of physicochemical indicators: Temperature changes in the experimental and control groups were recorded daily using a digital display thermometer (Pt100; Shanghai Qiekang Instrument Co., Ltd., China); the release of ammonia and hydrogen sulfide from the reactor pile was recorded daily using a portable gas detector (MS400; Erannetex, Shenzhen, China); and the determination of water-soluble ammonia nitrogen (WSA) and water-soluble nitrate nitrogen (WSN) was carried out in accordance with the national standard GB / T42485-2023.

[0046] 5. Odor control during the composting process: from Figure 4 The results showed that during the thermophilic phase of composting (>55 ℃), the highest temperature of the control group compost pile was 63.2 ℃, while the highest temperature of the experimental group compost pile was 67.0 ℃. The thermophilic phase (>55 ℃) in the experimental group was maintained for 13 days, after inoculation. B. subtilis The thermophilic period of the SHB-2 experimental group was prolonged to 20 days. The results indicate that inoculation... B. subtilis SHB-2 can significantly increase composting temperature and prolong the thermophilic period of composting.

[0047] from Figure 5 The results showed that H2S emissions were low in both the control and experimental groups at the initial stage of composting, then rose rapidly, peaking between 4 and 12 days. The peak H2S release values ​​for the control and experimental groups were 254.35 ppm and 184.35 ppm, respectively. Compared with the control group, the experimental group had lower H2S emissions, especially on day 12, with a removal rate of 31.52%. The results indicate that adding [the appropriate additive]... B. subtilis SHB-2 has a certain ability to remove H2S and has a good deodorizing effect.

[0048] from Figure 6The results showed that in the early stages of composting, with the rapid degradation of organic matter and the increase in composting temperature, both the control and experimental groups exhibited significant NH3 emissions. On day 16, when the temperature reached 63℃, the peak NH3 release values ​​for the control and experimental groups were 981.4 ppm and 903.05 ppm, respectively. NH3 release then decreased, reaching its lowest point at the end of composting. Compared to the control group, the experimental group released less NH3, particularly on day 4, with an NH3 removal rate of 26.28%.

[0049] The results above show that B. subtilis Inoculation with SHB-2 can not only significantly reduce the emission of NH3 and H2S odorous gases, but also significantly increase the composting temperature, accelerate the composting process, and improve the degree of decomposition and fertilizer efficiency.

[0050] 6. Nitrogen and sulfur conversion during composting: from Figure 7 The results showed that the sulfate content gradually increased during composting. The highest sulfate content was 9.06 mg / g in the control group and 12.18 mg / g in the experimental group. The higher sulfate content in the experimental group may be due to the higher sulfate content in the compost... B. subtilis The addition of SHB-2 promotes the sulfur oxidation process, converting H2S into sulfate and accumulating it. from Figure 8 The results show that water-soluble NO3 - The NO3- content is initially low, but begins to increase after the thermophilic phase as nitrification proceeds. From day 10 onwards, NO3- content... - The NO3- content increased rapidly, reaching its maximum at the end of composting. (Experimental group NO3 content...) - The peak value of -N was 1.60 mg / g, while the NO3 in the control group was... - The peak value of -N was 1.44 mg / g. Experimental group NO3 - The high -N content may be due to the high NH4 content in the compost. + -N content promotes the nitrification process, converting nitrogen into nitrate and accumulating it. Results indicate that inoculation... B. subtilis SHB-2 can enhance ammoniation during composting and reduce NH3 emissions.

[0051] The results above show that inoculation during the composting process is effective. B. subtilis SHB-2 can promote the conversion of sulfur and nitrogen between organic and inorganic processes, and enhance the driving role of key microorganisms in the sulfur and nitrogen cycle.

[0052] In summary, this invention provides theoretical and technical support for odor emission control, green composting, and environmental pollution control during the livestock and poultry manure composting process. Furthermore, B. subtilisThe discovery that SHB-2 can rapidly oxidize sulfides provides more possibilities and options for the biological treatment of H2S.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis B. subtilis SHB-2, characterized in that: It was deposited at the China Center for Type Culture Collection on May 27, 2025, with accession number CGMCC: No. 34689.

2. The Bacillus subtilis of claim 1 B. subtilis Application of SHB-2 in biological deodorization.

3. The Bacillus subtilis of claim 1 B. subtilis Application of SHB-2 in aerobic composting for deodorization and sulfur fixation.

4. A biological deodorizing agent, characterized in that: At least including the Bacillus subtilis as described in claim 1 B. subtilis SHB-2.

5. The biological deodorizing agent according to claim 4, characterized in that: The biological deodorizing agent includes at least a porous carrier; Preferably, the porous carrier is zeolite powder or attapulgite.

6. The biological deodorizing agent according to claim 4, characterized in that: The biological deodorizing agent also includes at least one other bacterium, enzyme, or its metabolite.

7. The biological deodorizing agent according to claim 6, characterized in that: The bacteria are selected from Bacillus, lactic acid bacteria, yeast, photosynthetic bacteria, bifidobacteria, actinomycetes, nitrifying bacteria, or Streptomyces flavus. Preferably, the Bacillus is Bacillus subtilis, Bacillus megaterium, or Bacillus laterosporus; Alternatively, the lactic acid bacteria may be Bacillus acidophilus or plant lactic acid bacteria; Preferably, the enzyme is selected from lysozyme, protease, or cellulase.

8. Using the Bacillus subtilis as described in claim 1 B. subtilis SHB -2 The method for deodorization and sulfur fixation in aerobic composting is characterized by: Includes the following steps: Bacillus subtilis cultured B. subtilis SHB -2 Mix thoroughly with composting materials, then carry out aerobic composting.

9. Bacillus subtilis according to claim 8 B. subtilis The method for deodorization and sulfur fixation in aerobic composting using SHB-2 is characterized by: During the composting process, the composting materials should be mixed every 2-4 days.

10. Bacillus subtilis according to claim 8 B. subtilis The method for deodorization and sulfur fixation in aerobic composting using SHB-2 is characterized by: Aerobic composting takes 25-90 days.

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