Bacillus aryabhattai, bacterial agent and application thereof in treatment of chemical wastewater

Abstract

The application discloses a bacillus aryabhattai strain and a bacterial agent thereof and application of the bacillus aryabhattai strain in chemical wastewater treatment. The bacillus aryabhattai (Bacillus aryabhattai) is named JG-33, and was preserved in the China General Microbiological Culture Collection Center on March 17, 2025. The preservation number of the strain is CGMCC No. 33838. The bacillus aryabhattai of the application can degrade o-methylbenzoic acid and o-trifluoromethylbenzoic acid pollutants in chemical wastewater, and has the advantages of acid resistance, low-temperature resistance, excellent degradation effect and good stress resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Bacillus aryabhattai strain, a bacterial agent thereof and application of the bacterial agent in chemical wastewater treatment. BACKGROUND

[0002] O-methylbenzoic acid and o-trifluoromethylbenzoic acid are key intermediates indispensable for the synthesis of amide fungicides. These fungicides have a core structure of amide bond, and have the advantages of high efficiency, low toxicity and strong specificity for specific pathogenic fungi, and thus play an important role in the comprehensive prevention and control system of modern agricultural fungal diseases. With the continuous growth of agricultural demand, the production and use scale of such intermediates and downstream fungicides are increasing, however, the problems of residue and accumulation in the environment are also highlighted, which constitutes a potential ecological risk.

[0003] O-methylbenzoic acid and o-trifluoromethylbenzoic acid belong to substituted benzoic acids. Due to the introduction of methyl or strong electron-withdrawing trifluoromethyl on the benzene ring, the stability of the molecular structure is significantly enhanced, resulting in much lower biodegradability than unsubstituted benzoic acid, and easy to persist and accumulate in soil and water environment. Not only does it pose a threat to the ecological environment, but also has a clear harmfulness to human health. At present, the treatment technologies for such high-stability organic acid pollutants include advanced oxidation method or physical adsorption method, which generally have limitations such as high cost, complex operation, easy to produce secondary pollution or only achieve the transfer of pollutants rather than complete degradation, therefore, it is particularly urgent to develop a kind of efficient, complete and environmentally friendly biodegradation technology. SUMMARY

[0004] To solve the problems of the prior art, the present application aims to provide a Bacillus aryabhattai strain, a bacterial agent thereof and application of the bacterial agent in chemical wastewater treatment, which can degrade o-methylbenzoic acid and o-trifluoromethylbenzoic acid pollutants in chemical wastewater, and has excellent acid and low-temperature resistance and degradation effect.

[0005] To achieve the above-mentioned goal, the present application adopts the following technical solution:

[0006] A Bacillus aryabhattai strain, the Bacillus aryabhattai (Bacillus aryabhattai) is named JG-33, and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 17, 2025, the address of the preservation is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the strain is CGMCC No. 33838.

[0007] A bacterial agent produced by using the Bacillus aryabhattai.

[0008] The application of Bacillus aryabhattai or a bacterial agent in degrading amide bactericide intermediates in chemical industrial wastewater, the amide bactericide intermediates are o-methylbenzoic acid or o-trifluoromethylbenzoic acid, the concentration of the amide bactericide intermediates in the chemical industrial wastewater is 100-1000 mg / L, the pH of the chemical industrial wastewater is 4-7, and the temperature is 6-35 DEG C.

[0009] A method for treating chemical industrial wastewater by using a bacterial agent, wherein the bacterial agent is added in a volume ratio of 2-8% in the chemical industrial wastewater for degradation treatment.

[0010] A preparation method of a bacterial agent, comprising the following specific steps:

[0011] S1, inoculating the test tube seed of Bacillus aryabhattai JG-33 into LB culture medium and oscillating and culturing until the logarithmic phase;

[0012] S2, inoculating the cultured bacterial seed into a seed tank and culturing until the logarithmic growth phase, namely seed liquid;

[0013] S3, inoculating the seed liquid into a production tank for fermentation culture to obtain the bacterial agent.

[0014] Preferably, in the foregoing step S1, the formula of the LB culture medium is as follows: NaCl 10.00 g / L, peptone 10.00 g / L, yeast powder 5.00 g / L, o-methylbenzoic acid 0.1 g / L, and pH 7.0.

[0015] Preferably, in the foregoing step S2, the formula of the culture medium used in the seed tank is as follows: o-methylbenzoic acid 0.1 g / L, glucose 8 g / L, (NH4)2SO4 1 g / L, K2HPO4 2 g / L, MgSO4 0.5 g / L, NaCl 1 g / L, CaCO3 0.5 g / L, and yeast paste 2 g / L, and pH 7.2-7.5.

[0016] Preferably, in the foregoing step S3, the formula of the culture medium used in the production tank is the same as that of the culture medium used in the seed tank; and in the culture processes of steps S2 and S3, the inoculation amount is 8%-12%, the aeration amount of sterile air is 1:0.6-1.2, the stirring speed is 180-240 r / min, the culture temperature is 30-35 DEG C, and the culture time is 48-60 h.

[0017] The Bacillus aryabhattai and the preparation method thereof disclosed in the application can degrade o-methylbenzoic acid and o-trifluoromethylbenzoic acid pollutants in chemical industrial wastewater, are acid and low-temperature resistant, have excellent degradation effect, and have good stress resistance. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a colony morphology diagram of the Bacillus aryabhattai bacterial body in the application.

[0019] Figure 2 Strain JG-33 degradation effect diagram of o-methylbenzoic acid pollutants;

[0020] Figure 3 Strain JG-33 degradation effect diagram of o-trifluoromethylbenzoic acid pollutants;

[0021] Figure 4 Strain JG-33 degradation effect diagram at different degradation temperatures;

[0022] Figure 5 Strain JG-33 degradation effect diagram at different degradation pHs;

[0023] Figure 6 Strain JG-33 degradation effect diagram at different initial concentrations of o-methylbenzoic acid;

[0024] Figure 7 Strain JG-33 degradation effect diagram of o-methylbenzoic acid and o-trifluoromethylbenzoic acid in actual chemical wastewater;

[0025] Figure 8 Strain JG-33 degradation effect diagram of COD in actual chemical wastewater. DETAILED DESCRIPTION

[0026] The application will be specifically introduced below in combination with the drawings and specific examples.

[0027] Example 1, isolation and identification of strain:

[0028] Take 3.0 mL of activated sludge from a wastewater treatment tank of a pesticide enterprise, and add it into 100 mL of inorganic salt culture medium containing o-methylbenzoic acid (the concentration of o-methylbenzoic acid is 100 mg / L), and the formula is: NaCl 5.0 g, (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, and deionized water is added to 1000 mL, pH 7.0~7.2, sterilized at 121 ℃ for 20 min, and cultured under 10 ℃ condition, and every 10 days, inoculate 3% into fresh inorganic salt culture medium, and continuously inoculate for 5 times.

[0029] Take 1.0 mL of the above obtained enriched bacteria solution, add it into 9.0 mL of sterile water, and prepare 10 -1 of enriched solution, and then take 1.0 mL of the prepared 10 -1 of enriched solution, add it into 9.0 mL of sterile water, and fully mix to prepare 10 -2The enrichment liquid was diluted by gradient dilution, and so on. 0.1 mL of each gradient dilution liquid was coated on the inorganic salt solid culture medium containing o-methylbenzoic acid with a concentration of 100 mg / L (formula as above) and cultured at 30°C for 10 days. After 10 days, single colonies were picked from the above inorganic salt solid culture medium and cultured in 3.0 mL of LB liquid medium for 24 hours. The formula of the LB liquid medium was as follows: o-methylbenzoic acid 100 mg / L, NaCl 10.00 g / L, peptone 10.00 g / L, yeast powder 5.00 g / L, and pH 7.0. The supernatant was removed after centrifugation at 8000 r / min for 2 min, 3.0 mL of sterile water was added and shaken, and the bacteria were resuspended by adding 3.0 mL of sterile water after centrifugation at 8000 r / min for 2 min. The bacteria were resuspended by adding 3.0 mL of sterile water after washing twice with sterile water. 1.0 mL of the bacterial liquid was added to 100 mL of inorganic salt liquid medium containing o-methylbenzoic acid with a concentration of 100 mg / L (formula: 100 mg of o-methylbenzoic acid, 1.50 g of K2HPO4, 0.50 g of KH2PO4, 0.20 g of MgSO4·7H2O, 1.00 g of NaCl, 1.00 g of (NH4)2SO4, 20.00 g of agar per liter, and pH 7.0), and cultured at 160 r / min and 30°C for 7 days. The degradation effect was measured by liquid chromatography. A strain with high degradation efficiency was selected and preserved, i.e., Bacillus aryabhattai JG-33, which had a colony morphology on LB solid culture medium as shown in Figure 1

[0030] The main physiological characteristics of Bacillus aryabhattai are as follows: the cells are gram-negative and rod-shaped, with a size of 0.5-0.7 x 1.5-1.8 μm. The colonies are yellow, irregular, dry, tough, wrinkled, and some are round, white, opaque, raised in the middle, and smooth at the edge. The growth temperature range is 4-37°C, the respiratory type is aerobic or microaerophilic growth, the catalase is positive, and the oxidase is negative. The 16S rRNA gene sequence of Bacillus aryabhattai is shown in SEQ ID No. 1.

[0031] The above strain was preserved in the China General Microbiological Culture Collection Center on March 17, 2025, and was named Bacillus aryabhattai with a strain name of JG-33. The address of the preservation center is No. 1, Beichen West Road, Beijing City, Chaoyang District, No. 3, and the preservation number of the strain is CGMCC No. 33838.

[0032] Example 2, a preparation method of a bacterial agent, comprising the following specific steps:

[0033] ​S1. Inoculate the test tube culture of Bacillus argentea JG-33 into LB medium. The formula of LB medium is: NaCl 10.00 g / L, peptone 10.00 g / L, yeast extract 5.00 g / L, o-methylbenzoic acid 0.1 g / L, pH 7.0, and culture with shaking until the logarithmic phase.

[0034] S2. Inoculate the cultured inoculum into a seed tank. The culture medium used in the seed tank has the following formula: o-toluic acid 0.1 g / L, glucose 8 g / L, (NH4)2SO4 1 g / L, K2HPO4 2 g / L, MgSO4 0.5 g / L, NaCl 1 g / L, CaCO3 0.5 g / L, yeast extract 2 g / L, pH 7.2~7.5. Cultivate until the logarithmic growth phase, which is the seed culture.

[0035] S3. Introduce the seed liquid into the production tank for fermentation culture to obtain the microbial agent.

[0036] The culture medium used in the production tank is the same as that used in the seed tank; during the cultivation process in steps S2 and S3, the inoculum size is 8-12%, the sterile air ventilation rate is 1:0.6-1.2, the stirring speed is 180-240 r / min, the cultivation temperature is 30-35 ℃, and the cultivation time is 48-60 h.

[0037] Example 3: Degradation effect of strain JG-33 on o-methylbenzoic acid pollutant:

[0038] An inorganic salt liquid culture medium containing o-toluic acid (initial concentration 100 mg / L) was prepared. The culture medium formula was: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration 1 g / L, pH 7.0. The medium was autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. JG-33 seed culture was added to the inorganic salt liquid culture medium at a 5% inoculum (v / v), and cultured at 35 ℃ and 180 r / min with shaking for 144 h. The o-toluic acid content was measured every 24 h. The results are as follows: Figure 2 As shown.

[0039] Depend on Figure 2 It can be seen that after 144 h of culture, strain JG-33 has a degradation efficiency of over 90% for o-methylbenzoic acid, demonstrating excellent degradation performance.

[0040] Example 4: Degradation effect of strain JG-33 on o-trifluoromethylbenzoic acid pollutant:

[0041] An inorganic salt liquid culture medium containing o-trifluoromethylbenzoic acid (initial concentration 100 mg / L) was prepared. The culture medium formula was: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration 1 g / L, pH 7.0. The medium was autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. JG-33 seed culture was added at a 5% inoculum (v / v) to the culture medium containing each contaminant. The medium was incubated at 35 ℃ and 180 r / min with shaking for 144 h. The o-trifluoromethylbenzoic acid concentration was measured every 24 h. The results are as follows: Figure 3 As shown.

[0042] Depend on Figure 3 It can be seen that after 144 h of culture, strain JG-33 has a degradation efficiency of over 90% for o-trifluoromethylbenzoic acid, demonstrating excellent degradation performance.

[0043] Example 5: Effect of degradation temperature on the degradation effect of strain JG-33:

[0044] Multiple groups of inorganic salt liquid culture media containing o-toluic acid (initial concentration 100 mg / L) were prepared at temperatures of 3, 6, 10, 15, 20, 30, and 35 ℃. The culture medium formula was: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration 1 g / L, pH 7.0. The media were autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. JG-33 seed culture was added to the inorganic salt liquid culture medium at a 5% inoculum (v / v) and cultured with shaking at 180 r / min for 144 h. The o-toluic acid content was then determined, and the results are as follows: Figure 4 As shown.

[0045] Depend on Figure 4 It can be seen that the degradation rate gradually increases with the increase of degradation temperature. Under the degradation temperature of 6 ℃, the degradation rate of o-methylbenzoic acid can still reach more than 76%, indicating that strain JG-33 has excellent low temperature resistance.

[0046] Example 6: Effect of degradation pH on the degradation effect of strain JG-33:

[0047] Multiple groups of inorganic salt liquid culture media containing o-toluic acid (initial concentration 100 mg / L) were prepared, with pH values ​​set to 2, 3, 4, 5, 6, and 7, respectively. The culture medium formula was: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration 1 g / L, pH 7.0. The media were autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. JG-33 seed culture was added to the inorganic salt liquid culture medium at a 5% inoculum (v / v), and cultured at 35 ℃ and 180 r / min for 144 h with shaking. The o-toluic acid content was then determined, and the results are as follows: Figure 5 As shown.

[0048] Depend on Figure 5 It can be seen that under degradation conditions of pH 4-7, the degradation rate of o-methylbenzoic acid can reach more than 72%, indicating that strain JG-33 has excellent acid resistance.

[0049] Example 7: Effect of initial concentration of o-methylbenzoic acid on degradation efficiency of strain JG-33:

[0050] Inorganic salt liquid culture media were prepared with initial concentrations of o-methylbenzoic acid of 100, 400, 700, 1000, 1300, and 1600 mg / L, respectively. The culture medium formula was: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration 1 g / L, pH 7.0. The media were autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. JG-33 seed culture was added to the inorganic salt liquid culture medium at a 5% inoculum (v / v), and cultured at 35 ℃ and 180 r / min for 144 h with shaking. The o-methylbenzoic acid content was then determined. The results are as follows: Figure 6 As shown.

[0051] Depend on Figure 6 It can be seen that when the initial concentration of o-toluic acid is 100-1000 mg / L, strain JG-33 has an excellent degradation effect on o-toluic acid. When the initial concentration exceeds 1000 mg / L, the degradation rate decreases significantly, indicating that excessive o-toluic acid has a certain inhibitory effect on the degradation process.

[0052] Example 8: Degradation test of strain JG-33 in actual chemical wastewater:

[0053] The wastewater source was a wastewater treatment pond of a pharmaceutical company, with a treatment capacity of 10 L. A bacterial agent prepared from strain JG-33 was added to the wastewater at a volume ratio of 5%. The original wastewater had a pH of approximately 6.0, a temperature of 25 ℃, and initial concentrations of o-toluic acid and o-trifluoromethylbenzoic acid of 100 mg / L and 50 mg / L, respectively. Aeration was performed to maintain dissolved oxygen levels above 2 mg / L. The concentrations of pollutants and COD in the wastewater were measured every 12 hours. Figure 7 and Figure 8 As shown.

[0054] Depend on Figure 7 and Figure 8 It can be seen that after 5 days of adding the bacterial agent, the degradation rate of the target pollutants and COD in the wastewater reached more than 90%. The above experimental data show that strain JG-33 has excellent treatment effect on actual chemical wastewater.

[0055] 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 strain of Bacillus argentis, characterized in that, The Bacillus argentis ( Bacillus aryabhattai The strain was named JG-33 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 17, 2025, with the accession number CGMCC No. 33838.

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

3. The application of *Bacillus auricula-judae* as described in claim 1 or the bacterial agent as described in claim 2 in the degradation of amide-based bactericide intermediates in chemical wastewater, characterized in that... The intermediate of the amide bactericide is o-methylbenzoic acid or o-trifluoromethylbenzoic acid. The concentration of the intermediate of the amide bactericide in the chemical wastewater is 100~1000 mg / L, the pH of the chemical wastewater is 4~7, and the temperature is 6~35 ℃.

4. A method for treating chemical wastewater using the microbial agent according to claim 2, characterized in that, Add 2% to 8% by volume of bacterial agent to chemical wastewater for degradation treatment.

5. A method for preparing the microbial agent according to claim 2, characterized in that, The specific steps include the following: S1. Inoculate the test tube culture of Bacillus oryzae JG-33 into LB medium and culture with shaking until the logarithmic phase; S2. Inoculate the cultured inoculum into the seed tank and culture it to the logarithmic growth phase, i.e., the seed solution; S3. Introduce the seed liquid into the production tank for fermentation culture to obtain the microbial agent.

6. The method for preparing the microbial agent according to claim 5, characterized in that, In step S1, the LB medium formula is: NaCl 10.00 g / L, peptone 10.00 g / L, yeast extract 5.00 g / L, o-methylbenzoic acid 0.1 g / L, pH 7.

0.

7. The method for preparing the microbial agent according to claim 5, characterized in that, In step S2, the culture medium used in the seed tank has the following formulation: 0.1 g / L o-methylbenzoic acid, 8 g / L glucose, 1 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.5 g / L MgSO4, 1 g / L NaCl, 0.5 g / L CaCO3, 2 g / L yeast extract, and pH 7.2~7.

5.

8. The method for preparing the microbial agent according to claim 7, characterized in that, In step S3, the culture medium formula used in the production tank is the same as that in the seed tank; during the cultivation process in steps S2 and S3, the inoculation amount is 8%~12%, the sterile air ventilation rate is 1:0.6~1.2, the stirring speed is 180~240 r / min, the cultivation temperature is 30~35 ℃, and the cultivation time is 48~60h.

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