Bacillus aerius strain, bacterial agent and application thereof in tobacco stalk composting

Abstract

This invention discloses a thermoresistant strain of *Bacillus aeruginosa*, its inoculant, and its application in tobacco straw composting. *Bacillus aeruginosa* (… Aeribacillus sp.) Classification naming Aeribacillus sp. YT-6 was deposited at the China Center for Type Culture Collection on April 29, 2026, with the accession number CCTCC NO: M 2026836. This invention, *Bacillus aeruginosa* YT-6, can degrade nicotine in wastewater or tobacco straw compost at high temperatures, and simultaneously produces nicotinic acid and nicotinamide, achieving simultaneous degradation of pollutants and generation of active substances.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a thermoresistant airborne Bacillus strain, its inoculant, and its application in tobacco straw composting. Background Technology

[0002] Nicotine, commonly known as tobacco, is a highly toxic heterocyclic compound with a chemical structure consisting of a pyridine ring and a hydrogenated pyrrole ring. It is the main alkaloid found in tobacco and its processing waste, exhibiting strong environmental persistence and high biotoxicity, posing a potential threat to the ecological environment and human health. Therefore, nicotine degradation has become a crucial link in the green development and waste management of the tobacco industry.

[0003] Currently, nicotine degradation is mainly achieved through various methods, including physical, chemical, and microbial methods. Physical methods primarily include distillation, incineration, and solvent extraction. While these methods can effectively remove nicotine, they suffer from drawbacks such as high processing difficulty, high energy consumption, and complex operation. Chemical methods, such as Fenton oxidation, ozone catalytic oxidation, electrochemical oxidation, and photocatalytic oxidation, face challenges in practical applications, including high costs and numerous byproducts, limiting their large-scale application. Microbial degradation technology has become a hot research area in nicotine degradation due to its high efficiency and environmental friendliness. Compared to physical and chemical methods, microbial degradation technology offers high specificity, minimal impact on other components, low cost, and environmental friendliness. Furthermore, the metabolites produced during the microbial degradation process can be reused. Among the discovered nicotine-degrading microorganisms, those targeting the genus *Pseudomonas* (…) are particularly promising. Pseudomonas ) and Arthrobacter spp. ( Arthrobacter The study of nicotine-degrading strains is the most in-depth, and their degradation capabilities and characteristics are relatively clear. However, most of the nicotine-degrading strains currently screened and studied are room-temperature bacteria, which face problems such as poor adaptability to high-temperature environments, insufficient stability of degradation efficiency, and high costs for large-scale application. Therefore, there is an urgent need to screen for novel, high-efficiency nicotine-degrading strains that are resistant to high temperatures. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a heat-resistant strain of *Aeribacillus* sp. YT-6, along with its inoculant and its application in tobacco straw composting. *Aeribacillus* sp. YT-6 can degrade nicotine in wastewater or tobacco straw compost at high temperatures, and simultaneously produces nicotinic acid, achieving simultaneous degradation of pollutants and generation of active substances.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A thermostable strain of *Aeribacillus* sp., classified as *Aeribacillus sp.* YT-6, was deposited at the China Center for Type Culture Collection (CCTCC) on April 29, 2026, with accession number CCTCC NO: M 2026836.

[0006] A bacterial agent produced using Bacillus aerogenes.

[0007] Application of Bacillus aerogenes or its agents in the treatment of tobacco straw compost or wastewater at high temperatures, where the concentration of nicotine in the tobacco straw compost or wastewater is 30-1000 mg / L and the temperature is 40-60 ℃.

[0008] Application of Bacillus aerobicans or its agents in the production of nicotinic acid and nicotinamide at high temperatures. Bacillus aerobicans or its agents produce nicotinic acid and nicotinamide during the degradation of nicotine in tobacco straw compost or wastewater at temperatures of 40-60 ℃.

[0009] A method for treating tobacco straw compost or wastewater using microbial agents involves adding 2% to 10% by volume of microbial agents to the tobacco straw compost or wastewater for degradation treatment.

[0010] A method for preparing a microbial agent includes the following specific steps: Colonies of Bacillus aerogenes YT-6 were inoculated into LB liquid medium and cultured with shaking to obtain the bacterial agent.

[0011] Preferably, the composition of LB liquid culture medium is: 5 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, pH 7.0, culture temperature of 40-55 ℃, and shaking speed of 150-200 rpm.

[0012] The advantages of this invention are: the airborne Bacillus of this invention can degrade nicotine in wastewater or tobacco straw compost at high temperatures, and produces nicotinic acid and nicotinamide while degrading nicotine. Nicotinic acid and nicotinamide can be reused by microorganisms to enter the cell and participate in the metabolic and growth process, further improving the degradation rate of pollutants by microorganisms. Attached Figure Description

[0013] Figure 1 This is a colony morphology diagram of Bacillus aerogenes in this invention; Figure 2 This image shows the effect of various strains metabolizing nicotinic acid and nicotinamide in the initial screening of nicotinic acid production. Figure 3 This is a phylogenetic tree description of Bacillus aeruginosa YT-6 in this invention; Figure 4 The graph shows the degradation effect of Bacillus aeruginosa YT-6 on different concentrations of nicotine. Figure 5 Graphs showing the effect of Bacillus aerobicans YT-6 in degrading different concentrations of nicotine to produce nicotinic acid and nicotinamide; Figure 6 The graph shows the degradation effect of Bacillus aeruginosa YT-6 on nicotine at different temperatures; Figure 7 Graphs showing the effect of Bacillus aerobicans YT6 degrading nicotine to produce nicotinic acid and nicotinamide at different temperatures; Figure 8 This is a graph showing the temperature changes during the actual composting process. Figure 9 The graph shows the degradation effect of strain YT-6 on nicotine in actual compost. Figure 10 This is a diagram showing the effect of strain YT-6 on the production of nicotinic acid in actual compost. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0015] The culture medium formulations used in the following examples are as follows: LB liquid medium: peptone 5 g / L, yeast extract 10 g / L, NaCl 5 g / L.

[0016] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, agar powder 15 g / L, sterilized at pH 7.0 and temperature 121℃ for 30 min.

[0017] Inorganic salt culture medium: Na2HPO4·12H2O 9g / L, KH2PO4 1.5g / L, MgSO4·7H2O 0.2g / L, trace elements 1mL / L.

[0018] Screening medium: Na2HPO4·12H2O 9g / L, KH2PO4 1.5g / L, MgSO4·7H2O 0.2g / L, trace elements 1mL / L, agar powder 15g / L, nicotine 100mg / L.

[0019] Trace elements: H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, ZnSO4·7H2O 0.1 g / L, MgSO4·5H2O 0.03 g / L, (NH4)6Mo7O 24 ·4H2O 0.03 g / L, NiCl2·6H2O 0.02 g / L, CuSO4·5H2O 0.01g / L.

[0020] Example 1: Isolation and Identification of Strains Take 10 g of the mixed soil and compost samples respectively, and add them to an Erlenmeyer flask containing 90 mL of sterile water. Make two replicates for each sample, and shake on a shaker at 180 rpm and 55℃ for 1 h to ensure thorough mixing with the sterile water, obtaining a bacterial suspension. Transfer 2 mL of the bacterial suspension to 50 mL of inorganic salt liquid medium containing 10 mg / L nicotine and continue culturing until the liquid medium becomes turbid. Then, transfer another 2 mL of the bacterial suspension to fresh inorganic salt liquid medium containing 20 mg / L nicotine and continue culturing. The nicotine concentration in the medium is then increased in the following steps: 30 mg / L, 50 mg / L, and 80 mg / L. Continue to transfer and culture the turbid bacterial suspension until the nicotine concentration reaches 100 mg / L. Dilute 100 μL of the final bacterial suspension and spread it onto a solid medium containing 100 mg / L nicotine inorganic salts (dilution factor 10⁻¹⁰). 6 The culture medium was inverted and incubated at 55°C for 3 days until single colonies grew on the medium. Single colonies of different strains were selected and streaked onto LB solid medium until a single strain was obtained. A total of 35 nicotine metabolizing strains were obtained after initial screening.

[0021] (2) Secondary screening of strains The target strains obtained from the initial screening were inoculated into LB liquid medium and cultured at 55℃ for 24 h to obtain seed culture. The culture was centrifuged at 8000 rpm and 4℃ for 5 min, the LB supernatant was removed, and elution was performed with an appropriate amount of PBS. After three elutions, the PBS was removed, and the bacterial concentration was adjusted using inorganic salt liquid medium until the OD value was finally adjusted to 1. 10% of the inoculum was added to inorganic salt liquid medium containing 100 mg / L nicotine and cultured at 55℃ and 180 rpm for 3 days. A non-biological control group without inoculated strains was established. After 3 days, samples were taken to determine the nicotinic acid and nicotinamide content. The ability of the 35 nicotine-metabolizing strains to produce nicotinic acid and nicotinamide was preliminarily determined. Figure 2 Strains B3, which can produce both nicotinic acid and nicotinamide, were selected for identification.

[0022] (3) Identification of strains The species of bacterial strain B3 was identified using 16S rDNA sequencing, and its sequence is shown in SEQ ID NO: 1. Homology analysis was performed between the sequencing results and sequences in the NCBI database, and a phylogenetic tree was constructed. (See attached image.) Figure 3 The results showed that the strain belonged to *Aeribacillus sp.*, and was classified as *Aeribacillus sp.* YT-6. It is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026836, deposit date April 29, 2026, at Wuhan University, Wuhan, China.

[0023] Example 2: A method for preparing a microbial agent, comprising the following specific steps: Colonies of Bacillus aeruginosa YT-6 were inoculated into 100 mL of LB liquid medium and cultured at 180 rpm and 55℃ for 24 h with shaking to obtain a highly efficient nicotine-degrading bacterial agent.

[0024] Example 3: The degradation effect of Bacillus aeruginosa YT-6 on nicotine and its performance in producing nicotinic acid and nicotinamide. OD at a 10% inoculation rate 600 YT-6 strain with a concentration of 0.5 was inoculated into inorganic salt media containing 100 mg / L, 500 mg / L, and 1000 mg / L nicotine, respectively, at pH 7.0, and cultured with shaking at 180 rpm and 55℃ for 14 days. Simultaneously, samples were taken on days 1, 2, 3, 7, and 14 to determine the concentrations of nicotine, nicotinic acid, and nicotinamide. The experimental results are shown below. Figure 4 and Figure 5 .

[0025] like Figure 4 and Figure 5 As shown, after inoculation with 10% strain YT-6, the degradation rate of nicotine at concentrations of 100 mg / L, 500 mg / L, and 1000 mg / L reached 99.8% after 14 days. Simultaneously, with increasing nicotine concentration and culture time, the amounts of nicotinic acid and nicotinamide produced generally showed an increasing trend, with the total production reaching a maximum of 293.09 μg / L. This indicates that strain YT-6 has the potential to treat nicotine while simultaneously producing nicotinic acid and nicotinamide, demonstrating excellent application prospects.

[0026] Example 4: Effect of temperature on the degradation effect of Bacillus airborne bacteria YT-6 Bacillus airborne bacteria YT-6 were inoculated at a rate of 10% (v / v) (OD). 600 =0.5) Inoculated into an inorganic salt culture medium containing 500 mg / L nicotine, and placed in constant temperature shakers at 40℃, 45℃, 50℃, 55℃ and 60℃, respectively, and cultured at 180 rpm for 14 days. After the culture, samples were taken to determine the degradation rate of nicotine and the cumulative yield of metabolites nicotinic acid and nicotinamide under each temperature condition. The results are shown in the figure. Figure 6 and Figure 7 .

[0027] Depend on Figure 6 and Figure 7It was found that *Bacillus aerobicans* YT-6 exhibited highly efficient degradation capabilities for nicotine within the temperature range of 40-60℃. At 50-55℃, the nicotine degradation rate reached 98.1%-99.8%, demonstrating excellent high-temperature degradation performance. Even at 60℃, the degradation rate remained at 85.6%, indicating good heat resistance. Simultaneously, the production of nicotinic acid and nicotinamide was positively correlated with the nicotine degradation rate, with the total product yield reaching a maximum of 173.6 μg / L at 55℃, further validating the stable and high-yield capacity of the strain's metabolites. Considering both degradation efficiency and product yield, 55℃ was the optimal degradation temperature. These results indicate that *Bacillus aerobicans* YT-6 can adapt to high-temperature environments and simultaneously generate high-value-added nicotinic acid and nicotinamide while efficiently removing nicotine contamination.

[0028] Example 5: Degradation of Nicotine by Bacillus aeruginosa YT-6 in Actual Composting Pig manure, corn stalks, and tobacco stalks were composted at a mass ratio of 7:2:1, with an initial nicotine concentration of 30 mg / L. 5% (v / v) of *Bacillus aeruginosa* YT-6 inoculant (prepared according to Example 2) was added to the material. Samples were taken on days 0, 5, 13, 21, 28, and 49 to determine the residual concentration of nicotine and the cumulative concentrations of the metabolites nicotinic acid and nicotinamide in the compost. The group with added inoculant was the experimental group, and the group without added inoculant was the control group. The temperature changes during the composting process are shown in [Figure showing...]. Figure 8 The changes in nicotine and nicotinic acid concentrations are shown in the following figures. Figure 9 and Figure 10 .

[0029] like Figure 8 As shown, the composting temperature ranged from approximately 30-70℃, classifying it as high-temperature composting, which corresponds to the nicotine degradation temperature range of Bacillus airborne pathogen YT-6. Figure 9 and Figure 10As shown, the Bacillus aerobicans YT-6 inoculant demonstrated highly efficient nicotine degradation capabilities in actual composting. In the early stages of composting, the nicotine concentration in the experimental group with added Bacillus aerobicans YT-6 was higher than that in the control group. This is because the addition of the inoculant accelerated nicotine degradation, weakened its inhibition of the original microbial activity in the compost material, accelerated the decomposition of the material, and resulted in a large release of nicotine. Compared to the control group without added inoculant, the experimental group, due to the addition of exogenous nicotine-degrading inoculant, accelerated the original nicotine degradation during the composting process, further reducing toxicity. After 21 days of treatment, the residual nicotine concentration in the experimental group was 0.55 μg / g, almost undetectable, while the nicotine concentration in the control group was 6.85 μg / g. Simultaneously, with the extension of composting time, the concentration of the target metabolite nicotinic acid showed a trend of first increasing and then decreasing, reaching its highest level of 20.05 μg / g at 5 days, while the nicotinic acid concentration in the control group was 16.33 μg / g. Nicotinamide was not detected during the actual composting process, which may be because the small amount of nicotinamide was directly utilized by other microorganisms in the compost pile. This result indicates that even during high-temperature composting with complex compositions, *Bacillus aerobicans* YT-6 maintains high metabolic activity, not only efficiently degrading nicotine but also increasing nicotinic acid production, thus achieving resource utilization of pollutants and demonstrating significant application potential.

[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A thermoresistant airborne Bacillus strain, characterized in that, The Bacillus aerogenes is classified as... Aeribacillus sp. YT-6 was deposited at the China Center for Type Culture Collection on April 29, 2026, with the accession number CCTCC NO: M 2026836.

2. A bacterial agent produced using Bacillus aeruginosa as described in claim 1.

3. The application of the *Bacillus aeruginosa* of claim 1 or the microbial agent of claim 2 in tobacco straw composting, characterized in that, This is used to degrade nicotine in tobacco straw compost at high temperatures, wherein the concentration of nicotine in the tobacco straw compost is 30-1000 mg / L, and the temperature is 40-60 ℃.

4. The application of the *Bacillus aeruginosa* of claim 1 or the bacterial agent of claim 2 in the generation of nicotinic acid and nicotinamide at high temperature, characterized in that... The *Bacillus aeruginosa* of claim 1 or the microbial agent of claim 2 generates nicotinic acid and nicotinamide during the degradation of nicotine in tobacco straw compost at a temperature of 40-60 °C.

5. A method for treating tobacco straw compost using the microbial agent according to claim 2, characterized in that, Add 2% to 10% by volume of microbial agent to tobacco straw compost for degradation treatment.

6. A method for preparing the microbial agent according to claim 2, characterized in that, The specific steps include the following: Colonies of Bacillus aerogenes YT-6 were inoculated into LB liquid medium and cultured with shaking to obtain the bacterial agent.

7. The method for preparing the microbial agent according to claim 6, characterized in that, The LB liquid culture medium consists of: 5 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, pH 7.0, culture temperature of 40-55 ℃, and shaking speed of 150-200 rpm.

Images