Method for degrading nicotine

Abstract

The invention provides a method for degrading nicotine, which comprises the following step of: adding sphingobacterium siyangense strain into a nicotine waste material for degrading. The method comprises the following steps of: adding the sphingobacterium siyangense strain into the nicotine waste material; the method for degrading the nicotine can efficiently and quickly treat the nicotine, can degrade organic matters in wastewater while efficiently degrading the nicotine, plays a dual role, and has a relatively high industrial value.

Description

Technical Field

[0001] This invention relates to the field of bacterial screening and culture technology, and more particularly to a method for degrading nicotine. Background Technology

[0002] Nicotine, commonly known as tobacco alkaloid, is an alkaloid found in common tobacco and a key component of tobacco. Its main function lies in its physiological potency, which is directly proportional to its content. Nicotine intake stimulates the central nervous system; moderate intake can quickly boost alertness and reduce fatigue and discomfort. However, excessive nicotine intake can cause dizziness and vomiting in smokers, and in severe cases, death. Therefore, high nicotine content in tobacco seriously harms smokers' health, and reducing nicotine levels can help reduce smoking-related diseases.

[0003] While some methods exist for reducing nicotine, such as agricultural measures like controlling nitrogen application during tobacco growth or chemical extraction, these techniques reduce other components that contribute to cigarette smoke, resulting in high costs, low efficiency, and significant subsequent pollution. Microbial degradation of nicotine is considered a highly efficient, safe, environmentally friendly, and low-cost method. Research on microbial degradation of nicotine has been reported since the last century, with researchers screening various fungi and bacteria capable of degrading nicotine, primarily including *Arthrobacter*, *Pseudomonas*, *Bacillus*, *Cellulomonas*, *Rhodococcus*, and *Agrobacterium*. *Arthrobacter* and *Pseudomonas* are the dominant species for nicotine degradation. These microorganisms have shown potential in nicotine degradation and have become a focus of research. However, although these strains have a certain degradation ability under laboratory conditions, the efficiency of nicotine degradation in practical applications is still unsatisfactory. Many strains perform poorly in the presence of high concentrations of nicotine and cannot even maintain stable degradation function.

[0004] Furthermore, existing nicotine-degrading strains generally suffer from poor tolerance to high concentrations of nicotine. Most known strains exhibit growth inhibition or even lose their degradation activity in environments with high nicotine concentrations. This problem significantly limits the effectiveness of these strains in large-scale applications, especially in industrial or environmental protection fields that require the treatment of high-concentration nicotine waste. Therefore, the practical application potential of existing degrading strains in nicotine pollution control is somewhat constrained.

[0005] Another limiting factor is that existing nicotine-degrading strains typically exhibit slow degradation rates, failing to meet the requirements of industrial-scale applications. This is particularly true when dealing with complex environments containing significant amounts of nicotine, such as tobacco waste and tobacco product residues, where the degradation efficiency of current technologies is severely inadequate. The degradation process of these strains often requires considerable time, making them highly inefficient for treating large-scale pollution sources and unsuitable for the efficient and rapid remediation of nicotine pollution. Therefore, there is an urgent need to find new, superior strains capable of efficiently degrading nicotine. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a method for degrading nicotine, thereby obtaining an efficient and rapid method for treating nicotine in waste.

[0007] In a first aspect, the present invention provides a method for degrading nicotine, comprising adding Sphingobacterium siyangense strain to nicotine waste for degradation.

[0008] In one embodiment of the present invention, the *Sphingobacterium siyangensestrain* is fermented in a fermentation medium containing nicotine waste to degrade nicotine.

[0009] In one embodiment of the present invention, the Sphingosine Bacillus includes live Sphingosine Bacillus cells, inactivated Sphingosine Bacillus, Sphingosine Bacillus lysates, and / or Sphingosine Bacillus culture supernatant.

[0010] In one embodiment of the present invention, the *Sphingomonas paucimobilis* includes strains isolated from natural environments, such as contaminated soils and agricultural soils, freshwater and seawater (e.g., lakes, rivers, groundwater), air, and extreme environments, such as high-salt or high-temperature areas. It also includes commercial strains, such as *Sphingomonas paucimobilis* ATCC 29837 and *Sphingomonas echinoides* DSM 19304, as well as genetically engineered and modified strains.

[0011] In one embodiment of the present invention, the waste includes, but is not limited to, tobacco waste: residues from the tobacco production process, such as tobacco leaves, stems, and fragments. Scrap materials from tobacco products: such as discarded parts of cigarettes and cigars. Tobacco processing wastewater: wastewater generated during tobacco processing, which may contain high concentrations of nicotine and other chemicals. E-cigarette liquids and waste: used e-cigarette liquid bottles and unused expired e-cigarette liquids, discarded e-cigarette devices and components. Cigarette ash and butts: cigarette butts and ash contain residual nicotine and other harmful substances. Pesticides and fertilizers: when nicotine is used as a natural pesticide, the related waste may contain nicotine. Laboratory and research waste: laboratory waste generated during tobacco-related research, including waste of nicotine extracts and related chemical reagents.

[0012] In one embodiment of the present invention, the amount of *Sphingosine monocytogenes* added is 10. 4 ~10 7 cfu / mL.

[0013] In one embodiment of the present invention, the fermentation temperature is 4-42℃, the pH is 6.0-8.5, and the time is 16h-24h. Preferably, the temperature is 30℃, the pH is 7, and the time is 16h.

[0014] In one embodiment of the present invention, the fermentation medium comprises inorganic salts, including but not limited to potassium salts, magnesium salts, calcium salts, iron salts, sodium salts, manganese salts, and ammonium salts. Specifically, the inorganic salts include dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, ferric sulfate, and sodium tungstate. In a preferred embodiment of the present invention, the fermentation medium comprises: 15.2 g / L dipotassium hydrogen phosphate trihydrate, 5.1 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride dihydrate, 0.1 g / L manganese sulfate monohydrate, 0.03 g / L ferric sulfate heptahydrate, and 0.3 g / L sodium tungstate dihydrate. In a preferred embodiment of the present invention, the fermentation medium comprises: 15.2 g / L dipotassium hydrogen phosphate trihydrate, 5.1 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride dihydrate, 0.1 g / L manganese sulfate monohydrate, 0.03 g / L ferric sulfate heptahydrate, 0.3 g / L sodium tungstate dihydrate, and 1.8% agar powder by mass.

[0015] In one embodiment of the present invention, the mass percentage concentration of nicotine in the fermentation culture medium is approximately 10%-30%.

[0016] In a second aspect, the present invention provides the application of Sphingobacterium siyangense strain in the degradation of nicotine in waste.

[0017] In one embodiment of the present invention, the *Sphingobacterium siyangense* strain has the characteristic of degrading nicotine and organic matter in waste, and can grow in a nicotine-containing culture medium.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method for degrading nicotine in this invention is a highly efficient and rapid method for treating nicotine. Furthermore, while efficiently degrading nicotine, it can simultaneously degrade organic matter in wastewater, thus achieving a dual function and possessing high industrial value. Attached Figure Description

[0020] Figure 1 The results of the screening of semi-synthetic culture medium in Example 1;

[0021] Figure 2 The figure shows the nicotine degradation curve in Example 3. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0023] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0024] Reagents used in the embodiments of this invention:

[0025] Bam HⅠ enzyme, HindⅢ enzyme, SacⅠ enzyme, and Taq DNA polymerase were all purchased from Takara; V-gene gel purification and recovery kit was purchased from V-GENE; λ-HindⅢ and DL2000 DNA Marker were purchased from Takara.

[0026] The culture medium used in the embodiments of this invention:

[0027] Initial screening culture medium: 10% wastewater from nicotine chemical synthesis (by amount of nicotine), 1% nicotine, 0.5% yeast extract, with distilled water as the solvent. After mixing, autoclave and cool to obtain the culture medium.

[0028] Slant culture medium: Add 300 mL of water to a 1 L container and heat to 50 °C. Add 20 g of peptone, 5 g of glucose, and 6 g of agar. Stir the system (300 rpm) and incubate for 10 min until the agar is completely dissolved. Cool the system to 25 °C, adjust the pH to 8.5 with 1% sodium hydroxide, filter through filter paper, dispense into glass bottles, and seal with cotton plugs for later use.

[0029] Secondary screening culture medium: 10% wastewater from nicotine chemical synthesis (based on the amount of nicotine), 0.1% yeast extract, with distilled water as the solvent, mixed, then autoclaved and cooled to obtain the medium.

[0030] Example 1: Screening of Nicotine-Degrading Strains

[0031] 1. Initial screening of neonicotinoid-degrading bacteria

[0032] Weigh 10g of soil from a tobacco-growing area (soil from Honghe County, Yunnan Province, where tobacco has been grown for over 10 years, provided by Wuhan Qirui Pharmaceutical Co., Ltd.), add physiological saline, shake, and let stand. Take the supernatant bacterial suspension and perform a 10-fold serial dilution (dilution gradient 10). -1 10 -2 10 -3 10 -4 10 -5 Different concentrations of diluted solutions were spread onto the primary screening medium and incubated at 30°C for 2 days.

[0033] 2. Streak plating purification of nicotine-degrading primary screening strains

[0034] Single colonies with good growth and typical characteristics were picked from the above primary screening medium using an inoculation loop and purified by streak plating on the same primary screening medium. The purified strains were then inoculated onto slant agar and stored at 4°C. A total of 7 strains were obtained through the above isolation and purification process.

[0035] 3. Secondary screening of nicotine-degrading strains

[0036] The seven isolated strains were inoculated into 30 mL of secondary screening medium at a volume ratio of 5%, and cultured on a shaker at 30°C for 16 hours at 150 rpm / min. Samples were taken periodically to detect the residual nicotine content in the fermentation broth; a decrease in the content indicated that nicotine had been degraded. The above screening process identified strain number 4 as a microbial strain capable of rapidly degrading nicotine.

[0037] The above seven strains degraded most of the nicotine in just two days on the primary screening medium (containing 1% nicotine). In the secondary screening medium containing 0.1% yeast extract and using wastewater as a solvent, strain #4 grew normally and degraded the nicotine in the wastewater. The screening results for the semi-synthetic medium are as follows: Figure 1 .

[0038] 4. Domestication of Nicotine-Degrading Strains

[0039] Using wastewater from nicotine chemical synthesis as the solute, with a nicotine content of 10%, and a starting yeast extract concentration of 0.5%, strain No. 4, selected through screening, was cultured to obtain a strain capable of directly growing and degrading nicotine in wastewater with a 10% nicotine content. Starting with this strain tolerant to 10% nicotine wastewater, the wastewater concentration was gradually increased while the yeast extract content was decreased, ultimately yielding a strain capable of growing and degrading nicotine in wastewater with a 30% nicotine content. The obtained strain was purified using the streak plate method to obtain the purified target strain.

[0040] Example 2 Identification of Nicotine-Degrading Strains

[0041] 1. Nicotine liquid culture medium

[0042] 15.2g dipotassium hydrogen phosphate trihydrate, 5.1g potassium dihydrogen phosphate, 0.3g magnesium sulfate heptahydrate, and 10mL of inorganic salt mixture (0.03g calcium chloride dihydrate, 0.01g manganese sulfate monohydrate, 0.003g ferric sulfate heptahydrate, and 0.03g sodium tungstate dihydrate, dissolved in 200mL 0.1mol / L hydrochloric acid solution) were added to 1000mL distilled water and the pH was adjusted to 7. The mixture was then extinguished at 120℃ for 25min, and 10% nicotine was added. Nicotine solid culture medium was prepared by adding 1.8% agar powder to liquid culture medium.

[0043] 2. Strain identification

[0044] The bacterial strain No. 4 obtained in Example 1 was cultured on nicotine solid medium at 30°C for 24 hours, and the colony morphology was observed. Common observation methods were used, and the data are as follows: The colonies were transparent pale yellow, with a smooth, raised surface, and a diameter of approximately 9.9 mm. Under a microscope, they appeared as short rods, without spores, and were Gram-negative bacteria. Methyl red and Vopper staining were positive, as were indole, nitrate reduction, hydrogen peroxide, starch hydrolysis, and gelatin liquefaction tests. The citrate test was negative.

[0045] 3. Homology analysis of 16S rRNA gene sequences

[0046] The target strain No. 4 was cultured in nicotine liquid medium at 30°C for 16 h. 5 mL of fermentation broth was centrifuged for 2 min in a high-speed centrifuge, the supernatant was discarded, and genomic DNA was extracted using an EF6 column as a template for PCR amplification of 16S rRNA.

[0047] The forward primer is 5'-AGTTTGATCCTGGCTCAG-3'.

[0048] Reverse primer: 5'-CGGTTACCTTGTTACGACTT-3';

[0049] Finally, the PCR amplified fragments were detected by agarose gel electrophoresis; the target bands were recovered from the gel and their DNA sequences were determined. The 16S rRNA sequence of the strain purified and isolated in this invention is shown in SEQ ID NO:1. Sequence homology was analyzed using BLAST software, and a neighbor-joining molecular phylogenetic tree was constructed using Mega software. The strain purified and isolated in this invention showed the highest homology with *Sphingobacterium siyangense* strain. Therefore, the strain purified and isolated in this invention is identified as *Sphingobacterium siyangense* strain.

[0050] Example 3: Effect of Sphingosine Bacillus on Nicotine Degradation under Different Temperature and pH Conditions

[0051] Sphingosporobacter sphingosine monocytogenes strain 4 was selected and cultured for 24 hours to the logarithmic growth phase to prepare a live bacterial suspension. The live bacterial suspension was inoculated into nicotine liquid medium. Different temperature (4℃, 20℃, 30℃, 42℃) and pH (6.0, 7.0, 8.5) conditions were set. Under different conditions, the inoculum size was 1×10⁻⁶. 6 The concentration of cfu / mL was controlled, and the fermentation time was 16 hours. Samples were collected every 4 hours, and the nicotine degradation effect was detected by high performance liquid chromatography (HPLC) and compared with the control group without inoculated strains.

[0052] The liquid chromatography method is as follows:

[0053] Instruments: High-performance liquid chromatograph, electronic balance

[0054] Reagents and reference standards: acetonitrile (chromatographic grade), methanol (chromatographic grade), acetic acid (chromatographic grade), ammonia, and purified water.

[0055] The chromatographic conditions were as follows: Column: YMC-Triart C18 ExRs 4.6mm*220, 1.5μm; Mobile phase A: 2ml acetic acid to 1000ml purified water, pH adjusted to 8.5 with ammonia; Mobile phase B: acetonitrile:methanol = 1:2; Diluent / injection wash: methanol; Detection wavelength: 254nm; Column temperature: 30℃; Flow rate: 1.0ml / min; Injection volume: 10μl.

[0056] Gradient procedure:

[0057] Time (min) Mobile phase A (%, V / V) Mobile phase B (%, V / V) 0 100 0 2 100 0 2.1 80 20 12 20 80 15 30 70 15.1 100 0 20 100 0

[0058] The experimental results are as follows:

[0059] At a constant pH, nicotine degradation was most efficient at 30°C, achieving a degradation rate of 95%, significantly higher than the degradation rates at 4°C and 42°C (58% and 72%, respectively). At a constant temperature, the highest degradation efficiency (90%) was observed at pH 7. The degradation rate was 75% at pH 6.0, but decreased to 67% at pH 8.5.

[0060] This experiment shows that *Sphingomonas* exhibits optimal nicotine degradation capacity at 30℃ and pH 7. Therefore, temperature and pH significantly affect the degradation effect, and selecting suitable fermentation conditions can greatly improve degradation efficiency.

[0061] Example 4: Detection of Nicotine Degradation Effect

[0062] The target bacterial strain No. 4 obtained in Example 1 was inoculated into nicotine liquid culture medium at an inoculation concentration of 1×10⁴. 7 Incubate at CFU / mL, pH 7, and 30℃ for 36 h. Centrifuge for 2 min, collect the supernatant, and filter through a 0.22 μm microporous membrane. Then, determine the nicotine content using high-performance liquid chromatography (HPLC) and plot the nicotine degradation curve.

[0063] The experimental results of the Sphingobacterium siyangense strain are as follows: Figure 2 As shown. From Figure 2 The data shows that after 4 hours, the residual nicotine in the system was less than 50 ppm after degradation, and after 8 hours, the residual nicotine remained almost at 0%, showing a stable trend. This indicates that after the initial decrease, nicotine basically no longer remains.

[0064] Example 5: Effect of different waste types on the degradation of nicotine by Sphingosine Bacillus

[0065] Sphingosporobacter spp. strain 4 was selected and cultured for 24 hours to the logarithmic growth phase to prepare a live bacterial suspension. Different types of tobacco waste (tobacco scraps, cigarette butts, cigarette ash, tobacco processing wastewater, and e-cigarette waste liquid) were added to the fermentation medium and inoculated with Sphingosporobacter spp. for fermentation. The effects of different wastes on the degradation of nicotine by the strain were observed. The inoculum size of Sphingosporobacter spp. was 1 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, fermentation temperature 30℃, pH 7, and fermentation time 16 hours. Samples were collected every 4 hours, and the nicotine degradation rate was detected by HPLC. The conversion products of nicotine during the degradation process were also analyzed.

[0066] The experimental results are as follows: Nicotine degradation rates were 85% and 88% for tobacco scraps and tobacco processing wastewater, respectively. Nicotine degradation rates were lower in cigarette butts and ash, at 70% and 60%, respectively. The highest nicotine degradation rate was observed in e-cigarette waste liquid, reaching 95%. This demonstrates that *Sphingomyelinatoria* exhibits varying degradation capabilities for different types of tobacco waste. The degradation effect is particularly significant when treating waste containing liquids (such as tobacco processing wastewater and e-cigarette waste liquid). Liquid wastes may facilitate more effective contact and degradation of nicotine by bacteria due to their high water solubility and low solids content.

[0067] Example 6: Detection of the effect of degrading organic matter in wastewater

[0068] The target bacterial strain No. 4 obtained in Example 1 was inoculated into a nicotine liquid culture medium containing 10% nicotine chemical synthesis wastewater (by amount of nicotine), with an inoculation concentration of 10%. 7 CFU / mL, cultured at 30℃ for 16 h, timed from the end of inoculation, samples were taken at 0 h and 16 h. The samples obtained at each time point were processed as follows: First, 10 ml of the mixture was placed in a 10 ml centrifuge tube, then centrifuged at 3000 r / min for 5 min, the supernatant was taken, filtered through a 0.22 μm microporous membrane, and the organic matter concentration of the supernatant was detected using a total organic carbon detector (SHIMADZU, Japan).

[0069] The results showed that the concentration of organic matter was 950 mg / L at 0 hours and 200 mg / L at 16 hours. This demonstrates that the strain of the present invention has excellent ability to degrade organic matter.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for degrading nicotine in waste, characterized in that: Sphingobacterium siyangense strain was added to nicotine waste for degradation.

2. The method for degrading nicotine in waste as described in claim 1, characterized in that: The *Sphingobacterium siyangense* strain was fermented in a fermentation medium containing nicotine waste to degrade nicotine.

3. The method for degrading nicotine in waste as described in claim 2, characterized in that: The amount of *Sphingosine monocytogenes* added was 10. 4 ~10 7 cfu / mL.

4. The method for degrading nicotine in waste as described in claim 2, characterized in that: The Sphingosine Bacillus includes live Sphingosine Bacillus cells, inactivated Sphingosine Bacillus, Sphingosine Bacillus lysates, and / or culture supernatant of live Sphingosine Bacillus.

5. The method for degrading nicotine in waste as described in claim 2, characterized in that: The waste includes, but is not limited to, tobacco waste, tobacco product scraps, tobacco processing wastewater, e-cigarette liquids and waste, cigarette ash and butts, pesticides and fertilizers, and laboratory and research waste.

6. The method for degrading nicotine in waste as described in claim 2, characterized in that: The fermentation temperature is 4-42℃, pH = 6.0-8.5, and the time is 16h-24h. Preferably, the temperature is 30℃, pH = 7, and the time is 16h.

7. The method for degrading nicotine in waste as described in claim 2, characterized in that: The fermentation medium includes inorganic salts, including dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, manganese sulfate monohydrate, ferric sulfate heptahydrate, and sodium tungstate dihydrate. Preferably, the fermentation medium comprises: 15.2 g / L dipotassium hydrogen phosphate trihydrate, 5.1 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride dihydrate, 0.1 g / L manganese sulfate monohydrate, 0.03 g / L ferric sulfate heptahydrate, and 0.3 g / L sodium tungstate dihydrate. Preferably, the fermentation medium comprises: 15.2 g / L dipotassium hydrogen phosphate trihydrate, 5.1 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride dihydrate, 0.1 g / L manganese sulfate monohydrate, 0.03 g / L ferric sulfate heptahydrate, 0.3 g / L sodium tungstate dihydrate, and 1.8% agar powder by mass. Preferably, the nicotine concentration in the fermentation medium is about 10%-30% by mass.

8. Application of a strain of Sphingobacterium siyangense in the degradation of nicotine in waste.

9. The application as described in claim 8, characterized in that: The *Sphingobacterium siyangense* strain possesses the ability to degrade nicotine. Preferably, the *Sphingobacterium siyangense* strain has the function of degrading organic matter in waste; Preferably, the *Sphingobacterium siyangense* strain can be grown in a nicotine-containing culture medium.

10. Application of primer pairs in screening nicotine-degrading bacteria, wherein the primer pairs include a forward primer: 5'-AGTTTGATCCTGGCTCAG-3'; and a reverse primer: 5'-CGGTTACCTTGTTACGACTT-3'.

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