A strain of Bacillus subtilis JN-K1 and its application in sewage treatment

Abstract

The present invention provides a strain of Bacillus subtilis JN-K1, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee, referred to as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit number of CGMCC No. 25650, and a deposit date of September 5, 2022. The strain has strong degradation performance for indole and three different indole heterocyclic derivatives of 1-methyl indole, 2-methyl indole, and 3-methyl indole, and the degradation rates of indole in vermicelli processing wastewater and livestock and poultry wastewater are 85.7% and 86.4%, respectively.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Bacillus subtilis JN-K1 and application thereof in sewage treatment. Background Art

[0002] Indole, with the chemical formula C8H7N and CAS registration number 120-72-9, is a nitrogen heterocyclic aromatic compound formed by the parallel connection of a benzene ring and a pyrrole ring. Indole is an imine that has a jasmine aroma at low concentrations and a fecal odor at high concentrations, similar to its derivative 3-methylindole, but the odor of 3-methylindole (skatole) is stronger and more persistent at high concentrations. Indole and 3-methylindole coexist in the intestines and excrement of animals and are one of the main causes of odor.

[0003] In addition to being exposed to indole during the production and manufacturing process, humans actually always have a certain amount of indole in their bodies. The effect of indole is small when the concentration is low, but high concentrations of indole are significantly toxic to animals. In chronic toxicity experiments with high-dose indole (20-200 mg / kg), mice, rats, rabbits, and dogs were given oral and subcutaneous injections for three months, and it was found that their blood cells underwent abnormal changes, with symptoms of anemia, leukocytosis or decrease, and the possibility of leukemia was not ruled out. Oral toxicity experiments with high-dose indole (100 or 200 mg / kg) on ​​cattle showed that higher concentrations of indole could be detected in cattle plasma. In the later stages of the experiment, the subjects developed symptoms such as diarrhea, snowmelt reaction, and hemoglobinuric nephropathy, and eventually died. In terms of environmental pollution, with the rapid development of animal husbandry, livestock and poultry farms around the world produce a large amount of livestock manure and wastewater every year. Among them, my country's slaughterhouses produce 3 billion tons of wastewater every year. The concentration of indole and 3-methylindole in the wastewater is high, the odor is strong, and it has a certain degree of toxicity, which has a very adverse impact on the healthy development of animal husbandry and the ecological environment. Research on the treatment and resource utilization of livestock wastewater is imminent.

[0004] Indole is weakly alkaline and low in toxicity. Some researchers have tried to use ultrasound, ultraviolet light and other methods to degrade indole, but in practical applications, there are practical limitations such as processing costs. Conventional treatment often requires removal during anaerobic or anoxic denitrification, which increases the difficulty and cost of wastewater treatment. Currently reported aerobic strains have limited effects on the degradation rate of high-concentration indole, have a long culture cycle, or can only degrade low-concentration indole, which is still a certain distance from the requirements for efficient removal of indole. It is necessary to screen strains with higher efficiency for production. Summary of the invention

[0005] Technical Solution

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A Bacillus subtilis JN-K1, including the isolation of the strain, primary screening and rescreening, identification of the strain, analysis of the indole degradation characteristics of the Bacillus subtilis JN-K1 strain, and analysis of the indole degradation conditions of the Bacillus subtilis JN-K1 strain.

[0008] Preferably, the Bacillus subtilis JN-K1 is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, abbreviated as CGMCC, whose address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No. 25650, and the deposit date is September 5, 2022.

[0009] Preferably, the Bacillus subtilis active ingredient is Bacillus subtilis strain JN-K1.

[0010] Preferably, the Bacillus subtilis JN-K1 degrades indole and three indole-derived compounds, namely 1-methylindole, 2-methylindole and 3-methylindole, in sewage.

[0011] Preferably, the sewage is vermicelli processing sewage and livestock and poultry sewage.

[0012] Compared with the prior art, the present invention provides a strain of Bacillus subtilis JN-K1 and its application in sewage treatment, which has the following beneficial effects:

[0013] The Bacillus subtilis provided by the present invention ( Bacillus subtilis )JN-K1 has a significant treatment effect on high-concentration wastewater from vermicelli processing and livestock and poultry wastewater, and the indole degradation rates in vermicelli processing wastewater and livestock and poultry wastewater are 85.7% and 86.4% respectively. This strain can not only efficiently degrade indole, but also efficiently degrade three different types of indole derivatives, namely 1-methylindole, 2-methylindole, and 3-methylindole. In addition, this strain is a beneficial bacterium with no pathogenicity. The subtilis spore JN-1 bacterial agent prepared by treating sewage can also be used to prepare bacterial fertilizer or organic fertilizer, which has high added value and solves the defects and deficiencies in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A is a colony photo of Bacillus subtilis JN-K1 of the present invention, and 1B is a photo of the cell morphology of the strain under a microscope.

[0015] Figure 2 The figure shows the effect of pH value on the degradation of indole by the Bacillus subtilis JN-K1 of the present invention.

[0016] Figure 3 The figure shows the effect of temperature on the degradation of indole by the Bacillus subtilis JN-K1 of the present invention.

[0017] Figure 4 The figure shows the effect of indole concentration on the degradation of indole by the Bacillus subtilis JN-K1 of the present invention. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] See also Figure 1-4 , a strain of Bacillus subtilis JN-K1, including strain isolation, primary screening and rescreening, strain identification, analysis of indole degradation characteristics of Bacillus subtilis JN-K1 strain, and analysis of indole degradation conditions of Bacillus subtilis JN-K1 strain.

[0020] Furthermore, the Bacillus subtilis active ingredient is Bacillus subtilis strain JN-K1.

[0021] Furthermore, the Bacillus subtilis JN-K1 degrades indole and three indole-derived compounds, namely 1-methylindole, 2-methylindole and 3-methylindole, in sewage.

[0022] Furthermore, the sewage is vermicelli processing sewage and livestock and poultry sewage.

[0023] Furthermore, the method includes the collection of strains, the cultivation of strains, and the preparation of strain culture medium.

[0024] Furthermore, the identification of the strain includes isolating and purifying the strain, performing physiological and biochemical identification on the strain JN-1, performing molecular biological identification on the strain JN-1, and screening the strain.

[0025] Furthermore, the analysis of the indole degradation characteristics of the Bacillus subtilis JN-K1 strain includes degradation experiments and comparisons of the strain.

[0026] Furthermore, the Bacillus subtilis JN-K1 strain was used in application experiments to degrade indole in vermicelli processing wastewater and livestock and poultry wastewater.

[0027] The following will be further described by means of specific embodiments:

[0028] Example 1: Isolation, primary screening and secondary screening of strains

[0029] S1: 10g of vermicelli processing wastewater sludge collected from a village in Feicheng, Tai'an, Shandong (collector: Liu Qiang, collection time: 2022) was dispersed in 100ml of sterile physiological saline, and cultured at 37℃, 200rpm shaking for 2h as the original mixed bacterial solution for later use. The original mixed bacteria were diluted 10-fold in a gradient ratio of 10-5-10-7, and 200ul of bacterial suspensions with different dilutions were aspirated and spread on enriched solid culture media respectively. They were cultured at 37℃ for 2d to obtain 5 single colonies with different colony shapes, which were coded as bacteria No. 1, bacteria No. 2, bacteria No. 3, bacteria No. 4, and bacteria No. 5, respectively. The 5 strains were further purified and cultured.

[0030] S2: The purified strains obtained in step S1 were inoculated into enriched liquid culture medium and cultured at 37°C and 200rpm for 24h. 10% of the culture solution was added into a conical flask containing 200ml of vermicelli processing wastewater (with a strong odor). After sealing with a sealing film, the flask was placed at room temperature for 3 days. The degree of odor degradation of the strain was determined by sensory evaluation, and the degree of indole degradation was determined by liquid chromatography (HPLC) to determine the degradation level of the strain. Finally, a strain No. 1 with the highest degree of indole compound degradation was obtained and named JN-K1. The strain was purified, cultured and frozen for preservation.

[0031] The components and proportions of the above enriched liquid medium are: glucose 10g / L, tryptone 10g / L, yeast extract powder 5g / L, ammonium sulfate 2g / L, sodium chloride 0.5g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.25g / L, sterilized at 121°C for 20min before use. The enriched solid medium is the above liquid medium with 2g / L agar added.

[0032] Example 2: Identification of strains

[0033] The strain JN-K1, which is highly efficient indole degrading, was isolated and purified as described above. After being cultured in LB solid medium at 37°C for 24 hours, its colonies showed a flower-shaped, white and opaque shape with irregular edges and a rough surface. Figure 1 As shown in A), spore-forming, Gram-positive aerobic bacteria, cell morphology observed under a microscope ( Figure 1 As shown in B), the cell morphology and size are (0.6-0.7) μm × (2-3) μm.

[0034] S1: Physiological and biochemical characterization of strain JN-1 (Table 1)

[0035] Table 1:

[0036] Biochemical indicators result Biochemical indicators result Tryptophan decarboxylase - glucose + Lysine decarboxylase - maltose + Arginine decarboxylase - Trehalose - Tyrosine decarboxylase - fructose - Ornithine decarboxylase - Xylose + Catalase + Arabinose + Catalase + Mannitol + Nitrate reduction + Sorbitol + Gelatin liquefaction + Sodium Citrate + VP test + glycerin +

[0037] Note: “+”: positive reaction; “-”: negative reaction.

[0038] S2: Molecular biological identification of strain JN-1 was performed by amplifying 16S rDNA using universal bacterial primers:

[0039] Primer: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0040] 1492R:5'-TACGACTTAACCCCAATCGC-3'

[0041] Reaction system: 2×TaqPCR Master Mix 12.5μL, upstream and downstream primers 1μL each, DNA template 0.5μL, ddH2O 10μL; reaction program: 95℃ 5 min; 94℃ 1 min, 55-58℃ 1 min, 72℃ 90s, 30 cycles; 72℃ 10 min.

[0042] S3: The PCR product obtained in step S1 was subjected to agarose gel electrophoresis and then sent to Shanghai Bioengineering for sequencing. A BLAST sequence comparison was performed on the NCBI website. The results showed that it was consistent with the registered Bacillus subtilis ( Bacillus subtili s) sequence homology reached 99%, confirming that the strain was Bacillus subtilis. The strain obtained by screening was named Bacillus subtilis ( Bacillus subtilis )JN-K1.

[0043] Bacillus subtilis ( Bacillus subtilis )JN-K1 was deposited on September 5, 2022 in the General Microbiology Center of the China Microorganism Culture Collection Administration, referred to as CGMCC, with its address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25650.

[0044] Example 3: Analysis of indole degradation characteristics of Bacillus subtilis JN-K1 strain

[0045] Take the frozen Bacillus subtilis JN-K1 strain, inoculate it in a fresh LB liquid medium, and culture it in a shaking table at 37°C and 200rpm for 24h. Then inoculate it with a 2% inoculum (by volume ratio) in 200ml liquid medium containing 200mg / L indole (analytical pure) of a glucose-containing inorganic salt formula and an inorganic salt formula, and ferment it aerobically at 37°C and 150rpm for 3d. At the same time, inoculate it with an inoculum of 8% in the inorganic salt formula as a control group. The indole concentration and cell content in each fermentation broth were determined, and the results are shown in Table 2. It was verified by experiments that the indole degradation rate in the glucose-containing inorganic salt formula was 84.5%, and the indole degradation rate in the inorganic salt formula was only 2.0-3.0%. It shows that the indole degradation characteristic of the Bacillus subtilis JN-K1 provided by the present invention is glucose-indole co-metabolism degradation, and indole cannot be degraded as the sole carbon source. The indole metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS), and the detection results showed that all of them contained oxidized indole and isocyanate products.

[0046] Table 2:

[0047] Type of culture medium Formula containing glucose and inorganic salts (2% inoculation amount) Inorganic salt formula (2% inoculation amount) Inorganic salt formula (8% inoculation amount) Different initial cell contents (cfu / ml) 1.76×106 1.85×106 2.41×108 Cell content at the end of fermentation (cfu / ml) 2.56×1010 1.78×106 2.63×108 Initial indole concentration (mg / L) 200 200 200 Concentration at the end of fermentation (mg / L) 30 196 194 Indole degradation rate (w / w) 84.5% 2.0% 3%

[0048] The above configuration contains the formula of glucose inorganic salt: glucose 10g / L, ammonium sulfate 2g / L, sodium chloride 0.5g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.25g / L, PH7.0, sterilized at 121°C for 20min before use.

[0049] The above-mentioned inorganic salt formula is as follows: ammonium sulfate 2g / L, sodium chloride 0.5g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.25g / L, pH 7.0, sterilized at 121°C for 20min before use.

[0050] The above-mentioned method for detecting indole by liquid chromatography (HPLC) is as follows: 5 ml of fermentation liquid is taken into a test tube, 5 ml of ethyl acetate is added, and the mixture is shaken for 2 minutes and then allowed to stand. The suspension on the surface is aspirated with a pipette, and filtered with a 0.22 um organic filter membrane. After filtration, the sample is injected for analysis. HPLC detection conditions: the chromatographic column is a Hypersill BDSC18 column (250 mm×4.6 mm, 5µm), the injection volume is 20 ul, the flow rate is 1 ml / min, the mobile phase is a mixture of 60% methanol and 40% water, and the injection time is 20 min.

[0051] The above-mentioned method for detecting indole metabolites using gas chromatography-mass spectrometry (GC-MS) is as follows: 200 ml of fermentation liquid is taken into a conical flask, 5 ml of acetone is added for extraction, and the sample is injected and analyzed after extraction and filtration through a filter membrane. Detection conditions: carrier gas is high-purity helium (99.999%), carrier gas flow rate: 1 mL / min, sample volume: 2 μL, split ratio: 1:1, column temperature starts at 70 °C, increases to 280 °C at 10 °C / min, and maintains for 5 min. The chromatographic column is a capillary column HP-5MS (30 m×250 μm×0.25 μm). The mass spectrometry conditions are EI ionization source, ion source temperature 230 °C, quadrupole 150 °C, and interface temperature 280 °C.

[0052] Example 4: Analysis of the degradation of different types of indole by Bacillus subtilis JN-K1 strain

[0053] 1-Methylindole, 2-methylindole and 3-methylindole are all indole heterocyclic derivatives. The degradation analysis of different types of indole derivatives was carried out by Bacillus subtilis JN-K1 strain. The frozen Bacillus subtilis JN-K1 strain was inoculated into fresh LB liquid medium and cultured in a shaking incubator at 37°C and 200rpm for 24h. Then, the inoculation amount of 2% (by volume ratio) was inoculated into 200ml liquid medium containing 200mg / L of 1-methylindole, 2-methylindole and 3-methylindole, respectively, and aerobic fermentation was carried out at 37°C and 150rpm for 3d. The concentration of each indole was determined by liquid HPLC analysis, and the results are shown in Table 3. It can be seen from the results that the degradation rate of 1-methylindole, 2-methylindole and 3-methylindole was 81.5%, 80.5% and 82.5% respectively. The experimental results show that the Bacillus subtilis JN-K1 strain provided by the present invention has a highly efficient degradation effect on three different types of indoles.

[0054] Table 3:

[0055] Indole type Monomethylindole dimethylindole Trimethylindole Initial cell content (cfu / ml) <![CDATA[2.16×10 6 ]]> <![CDATA[2.35×10 6 ]]> <![CDATA[2.34×10 6 ]]> End cell content (cfu / ml) <![CDATA[3.26×10 10 ]]> <![CDATA[3.86×10 10 ]]> <![CDATA[3.35×10 10 ]]> Initial indole concentration (mg / L) 200 200 200 Concentration at the end of fermentation (mg / L) 37 39 35 Indole degradation rate (w / w) 81.50% 80.50% 82.50%

[0056] Example 5: Analysis of indole degradation conditions of Bacillus subtilis JN-K1 strain

[0057] S1: Take the frozen Bacillus subtilis JN-K1 strain, inoculate it into fresh LB liquid medium, and culture it in a shaking incubator at 37℃ and 200rpm for 24h. Then inoculate it with 2% inoculum (by volume ratio) into 200ml liquid medium containing glucose inorganic salt formula with pH of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The indole content in the medium is 200mg / L. Ferment it aerobically at 37℃ and 150rpm for 3d. The indole concentration is determined by liquid HPLC. Figure 2It can be seen that the Bacillus subtilis JN-K1 strain provided by the present invention can effectively degrade indole under the condition of pH 6.0-8.0, and the degradation rate is as high as 85.54%.

[0058] S2: Take the frozen Bacillus subtilis JN-K1 strain, inoculate it into fresh LB liquid medium, and culture it in a shaking incubator at 37℃ and 200rpm for 24h. Then inoculate it into 200ml liquid medium containing 200mg / L indole and glucose inorganic salt formula with 2% inoculum (by volume ratio). A total of 6 shaking bottles were placed in a shaking incubator at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, respectively, and cultured at 150rpm for 3d. The indole concentration was determined by liquid HPLC. Figure 3 It can be seen that the Bacillus subtilis JN-K1 strain provided by the present invention has the highest indole degradation rate at 35° C., with a degradation rate of 84.57%.

[0059] S3: Take the frozen Bacillus subtilis JN-K1 strain and inoculate it into fresh LB liquid medium. Incubate it in a shaking incubator at 37°C and 200 rpm for 24 hours. Then inoculate it into 200 ml liquid medium containing 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 500 mg / L indole at a volume ratio of 2%, respectively. Incubate it at 150 rpm for 3 days, and determine the indole concentration by liquid HPLC. Figure 4 It can be seen that the Bacillus subtilis JN-K1 strain provided by the present invention can effectively degrade indole with an initial concentration of less than 200 mg / L within 72 hours, and the degradation rate is more than 84%.

[0060] Example 6: Application of Bacillus subtilis JN-K1 strain to degrade indole in vermicelli processing wastewater

[0061] S1: The pretreatment method of rice noodle processing wastewater is to mix the rice noodle processing wastewater discharged from a village in Feicheng, Tai'an, Shandong with tap water in a ratio of 1:2, stir evenly, let it stand for 24 hours, and then take the upper layer of liquid as the rice noodle processing wastewater to be treated in the laboratory.

[0062] S2: Add 5% of sugarcane waste sugar residue as an additional carbon source to the above-mentioned vermicelli processing wastewater, adjust the pH to 7.0 with sodium hydroxide regulator, and sterilize at 121℃ for 20min. Take the frozen Bacillus subtilis JN-K1 strain, inoculate it in fresh LB liquid culture medium, and activate it by shaking culture at 37℃ and 200rpm for 24h. Under sterile conditions, 200ml vermicelli processing wastewater was inoculated with activated JN-K1 bacteria after centrifugation to remove the culture medium at a volume fraction of 2%, and the culture conditions were set to 35℃ and 150rpm. After 3d, samples were taken to detect the indole content in the vermicelli processing wastewater. In this experiment, conventional Bacillus subtilis 168 (kindly donated by Professor Wang Tengfei of Qilu University of Technology) was used as a control experiment. The experimental results showed that the initial indole concentration of vermicelli processing wastewater was 245mg / L, and the indole concentration was 35mg / L after 3d, and the indole removal rate was as high as 85.7%. The bacterial content of JN-K1 increased from 2.43×10 6 / ml and rapidly grew to 1.56×10 10 / ml. From the intuitive feeling, the fecal odor was significantly reduced. The degradation rate of indole by the control strain 168 was only 8.45%.

[0063] Example 7: Application of Bacillus subtilis JN-K1 strain in degrading indole in livestock and poultry wastewater

[0064] S1: The pretreatment method of livestock and poultry wastewater is to mix the cow dung wastewater collected from Jiabao Dairy in Changqing, Jinan, Shandong with tap water in a ratio of 1:4, stir evenly, let it stand for 24 hours, and then take the upper layer of liquid as the livestock and poultry wastewater to be treated in the laboratory.

[0065] S2: Add 5% of sugarcane waste residue as an additional carbon source to the above livestock and poultry wastewater, adjust the pH to 7.0 with sodium hydroxide regulator, and sterilize at 121℃ for 20min. Take the frozen Bacillus subtilis JN-K1 strain, inoculate it in fresh LB liquid culture medium, and activate it by shaking culture at 37℃ and 200rpm for 24h. Under sterile conditions, inoculate JN-K1 activated bacteria after centrifugation to remove the culture medium at a volume fraction of 2% into 200ml of livestock and poultry wastewater, set the culture conditions to 35℃ and 150rpm, and take samples after 3 days to detect the indole content in livestock and poultry wastewater. This experiment uses conventional Bacillus subtilis 168 (kindly donated by Professor Wang Tengfei of Qilu University of Technology) as a control experiment. The experimental results show that the initial indole concentration of livestock and poultry wastewater is 214mg / L, and the indole concentration is 29mg / L after 3d, and the indole removal rate is as high as 86.4%. The JN-1 bacterial content increased from the initial inoculation of 2.56×10 6 / ml and rapidly grew to 2.86×10 10 / ml. From an intuitive point of view, the fecal odor was significantly reduced, while the degradation rate of indole by the control strain 168 was only 9.12%.

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Bacillus subtilis JN-K1, characterized in that: The Bacillus subtilis JN-K1 strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 25650 and the deposit date September 5, 2022.

2. The use of Bacillus subtilis JN-K1 in sewage treatment according to claim 1, characterized in that: The Bacillus subtilis JN-K1 degrades indole and three indole-derived compound substances, namely, 1-methylindole, 2-methylindole and 3-methylindole, in sewage.

3. The use of Bacillus subtilis JN-K1 in sewage treatment according to claim 2, characterized in that: The sewage is vermicelli processing sewage and livestock and poultry sewage.

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