Bacillus velezensis and application thereof in degradation of wastewater cod

Abstract

The application discloses a bacillus velezensis strain and application thereof in degrading COD of wastewater, and belongs to the technical field of biological degradation. The bacillus velezensis strain can adapt to the environment of PTA wastewater and agricultural breeding wastewater, and grows and reproduces well, and has a good degradation and removal effect on the COD component in the wastewater, the degradation rate of the COD in the PTA wastewater can reach 50.50%, and the degradation rate of the COD in the agricultural breeding wastewater can reach 96.55%. The strain can effectively degrade the COD in the wastewater, and is beneficial to sustainable development of an ecological environment.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus belye and its application in degrading COD in wastewater, belonging to the field of biodegradation technology. Background Technology

[0002] PTA wastewater is a type of wastewater with high concentrations and difficulty in degrading organic pollutants. Due to the large scale of modern industrial production, the volume of wastewater discharged is correspondingly large, and if not effectively treated, it can cause significant harm to the ecological environment and human health. PTA wastewater mainly includes benzoic acid, terephthalic acid, various phenols, and other organic compounds. This type of wastewater has the following characteristics: high COD concentration: Although the situation varies among different industrial plants, the COD concentration of PTA wastewater from most factories worldwide is between 10,000 and 55,000 mg / L, and even exceeds 100,000 mg / L; low pH value: This type of wastewater contains a large amount of acidic compounds, such as benzaldehyde and benzoic acid, so its pH value is generally low, mostly between 3 and 5. Currently, methods for treating PTA wastewater include physicochemical methods and biological treatment methods. Commonly used physicochemical methods include chemical oxidation, adsorption, membrane separation, and electrochemical treatment. Biological treatment methods can use microorganisms to degrade the difficult-to-degrade organic matter into harmless substances.

[0003] The main characteristics of agricultural and livestock wastewater are high organic matter concentration, high suspended solids content, deep color, and a large number of bacteria. The high COD concentration is also due to the presence of large amounts of animal feces. The solid residue in the wastewater is mainly organic matter. This type of wastewater often requires effective solid-liquid separation; otherwise, it will cause difficulties in subsequent treatment, increase the treatment load, and affect the treatment effect. Currently, the most commonly used technologies for treating livestock wastewater are physical and biological methods. Physical methods use solid-liquid separation technologies such as filtration, centrifugation, and sedimentation. Commonly used equipment includes screens, sedimentation tanks, and sieves. Biological methods include activated sludge processes, biological contact oxidation processes, SBR, A / O, and oxidation ditches, but these processes are complex.

[0004] Microbial degradation is an effective, environmentally friendly, sustainable, and simple method for treating PTA wastewater and agricultural / livestock wastewater. However, there are currently no Bacillus species capable of simultaneously degrading COD in both PTA and agricultural / livestock wastewater. Summary of the Invention

[0005] [Technical Issues] Currently, in the field of wastewater degradation, biological degradation still faces problems such as high cost and low degradation efficiency, which prevent it from being widely applied in industrial production.

[0006] [Technical Solution] In view of the existing problems, the present invention extracts a strain of Bacillus belye from the sludge of Changzhou sewage treatment plant. This strain can be used to degrade COD in PTA wastewater and agricultural and aquaculture wastewater, providing an effective biological treatment method for wastewater treatment.

[0007] This invention provides a strain of Bacillus belye, which was deposited at the China Center for Type Culture Collection on September 26, 2022, with accession number CGMCC No. 25800.

[0008] The present invention also provides a microbial preparation containing the aforementioned Bacillus belyssus.

[0009] In one embodiment, the microbial preparation contains ≥2 × 10⁶ live Bacillus belyceae. 9 CFU / mL.

[0010] In one embodiment, the microbial preparation further contains a lyophilization protectant.

[0011] The present invention also provides a method for preparing the microbial preparation, wherein the Bacillus belye is fermented in a culture medium.

[0012] In one embodiment, the fermentation is carried out at pH 1.0-7.0, 0-180 r / min, 20-60°C, for at least 22 hours.

[0013] The present invention also provides the application of the Bacillus berberis or the microbial preparations in the degradation of COD in wastewater, the wastewater including PTA wastewater and agricultural and aquaculture wastewater.

[0014] The present invention also provides a method for degrading COD in PTA wastewater, wherein the aforementioned Bacillus belye or the aforementioned microbial preparation is added to the wastewater, the wastewater including PTA wastewater and agricultural and aquaculture wastewater.

[0015] In one embodiment, the COD value of the PTA wastewater is 2798.5 mg / L, and the COD value of the agricultural and livestock wastewater is 265.5~389.2 mg / L.

[0016] In one embodiment, the bacterial solution is prepared at (4 × 10⁻⁶) / 2. 8 ~2×10 9 Add the final concentration of CFU / mL to the wastewater and degrade it for at least 6 hours at pH 1.0-7.0, 0-180 r / min, and 20-60℃.

[0017] The present invention also provides the application of the aforementioned Bacillus belyssus, or the aforementioned microbial preparation, or the method for preparing the aforementioned microbial preparation, in the preparation of wastewater treatment agents.

[0018] Beneficial effects

[0019] This invention isolates a plant from sludge at a wastewater treatment plant in Changzhou. Bacillus velezensis It can grow normally under acidic conditions of pH 3.0-7.0 and can effectively degrade COD in PTA wastewater and agricultural and livestock wastewater. When the *Bacillus belye* was used to degrade COD in PTA wastewater, the COD degradation rate reached 50.50% after 72 hours for wastewater with a COD content of 2798.5 mg / L; when used to degrade COD in agricultural and livestock wastewater, the COD degradation rate reached 96.55% after 48 hours for wastewater with a COD content of 265.5 mg / L, and 81.37% after 24 hours for wastewater with a COD content of 389.2 mg / L.

[0020] Preservation of biological materials

[0021] A strain of Bacillus belye, taxonomically named Bacillus belye. Bacillus velezensis It was deposited on September 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25800, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0022] Figure 1 Bacillus belesi Bacillus velezensis Colony morphology and cell morphology diagrams; A shows colony morphology, and B shows cell morphology.

[0023] Figure 2 A graph showing the changes in high-concentration COD wastewater treated with Bacillus belyssus. Detailed Implementation

[0024] The culture media involved in some embodiments of the present invention are as follows:

[0025] LB liquid medium: 5 g / L yeast extract -1 Tryptone 10g•L -1 10 g / L of sodium chloride -1 1000mL distilled water.

[0026] LB solid medium: 5 g / L yeast extract -1 Tryptone 10g•L -1 10 g / L of sodium chloride -1 1000mL distilled water, 20g agar.

[0027] pH adjustment of LB liquid and LB solid media: Adjust the pH of LB liquid or LB solid media to 1, 3, 5, 6, or 7 as needed, using 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0028] In some embodiments of the present invention, the wastewater COD degradation rate is calculated as follows:

[0029] Calculate the degradation rate of COD in wastewater:

[0030] Wastewater COD degradation rate = (Total COD at inlet - Total COD at outlet) / Total COD at inlet

[0031] The COD content of wastewater was measured using a multi-parameter water quality analyzer.

[0032] Technical terms:

[0033] Sludge: The term "sludge" is taken from the Changzhou Wastewater Treatment Plant, with a density of 0.027 g / mL. -1 The pH value is 7.66.

[0034] PTA wastewater: PTA wastewater from Changzhou Sewage Treatment Plant. This wastewater contains a large amount of organic and inorganic pollutants, such as benzoic acid, terephthalic acid, various phenols and other organic substances. It has a pH of 3.5 and is characterized by complex composition, strong acidity, high salinity, high color and the presence of toxic and harmful substances.

[0035] Agricultural and livestock wastewater: This wastewater comes from the Changzhou sewage treatment plant. It has a high concentration of organic matter, a lot of suspended solids, a dark color, and contains a large number of bacteria, with a pH of 4.0.

[0036] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0037] Example 1: Screening of strains

[0038] (1) Take a sample with a density of 0.027 g·mL from the Changzhou wastewater treatment plant. -1 10 mL of sludge was placed in 90 mL of LB liquid medium and cultured at 140-180 rpm and 30-35℃ for 4-6 days.

[0039] (2) Take the bacterial culture from step (1) and inoculate it into 100 mL of new LB liquid medium at a volume ratio of 5-10% and incubate it at 140-180 rpm and 30-35℃ for 4-6 days;

[0040] (3) Take the bacterial culture from step (2) and inoculate it into 100 mL of new LB liquid medium at a volume ratio of 5-10% and incubate it at 140-180 rpm and 30-35℃ for 4-6 days;

[0041] (4) Take 5 μL of the bacterial solution from step (3) and add it to the sterilized LB liquid medium. After culturing in a shaker at 140-180 rpm and 30-40℃ for 4-6 days, use a pipette tip to draw 100 μL of liquid and spread it on the LB solid medium.

[0042] (5) Place the LB solid medium coated with bacterial solution in an incubator at 37°C for 1-2 days, observe the morphology of the colonies, pick a small amount of bacteria from each colony and inoculate them into LB liquid medium. After culturing for 4-6 days, spread the bacterial solution onto LB solid medium.

[0043] (6) Repeat step (5) multiple times until a single strain is obtained in each LB solid medium;

[0044] (7) Pick a single bacterial strain from the culture medium that has grown a single bacterial strain and inoculate it into 100 mL of fresh LB liquid medium. Incubate at 140-180 rpm and 30-35°C for 4-6 days, and measure the OD of the bacterial solution. 600 The desired bacterial solution is obtained when the value reaches 0.8.

[0045] Example 2: Identification of the strain

[0046] (1) Culture preservation: slant culture, deposited on September 26, 2022 at the China General Microbiological Culture Collection Center, with accession number CGMCC No.25800, and the deposit address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0047] (2) Strain identification: The strain was identified as Bacillus belye by Sangon Biotech (Shanghai) Co., Ltd. using 16S RNA. Bacillus velezensis .

[0048] Example 3: Culture and pH tolerance of the strain at different pH levels

[0049] (1) The strain obtained in Example 1 was inoculated into 100 mL of LB liquid medium at a volume ratio of 2% and cultured at 30 °C and 100 r / min. The strain was cultured continuously with shaking for 48 h in acidic LB liquid medium with initial pH of 1.0, 3.0, 4.0, 5.0 and 7.0, respectively.

[0050] (2) OD in culture media with different initial pH values ​​was measured at 6-hour intervals. 600 value.

[0051] As shown in Table 3 Bacillus velezensis The bacteria enter the logarithmic growth phase earlier at an initial pH of 7, therefore, its optimal initial pH is 7. Furthermore, *Bacillus belye* has a pH tolerance range of 3.0-7.0.

[0052] Table 1 Bacillus velezensis OD of bacteria at different initial pH 600 value

[0053]

[0054] Example 4: Culture of strains at different rotation speeds

[0055] (1) The strain obtained in Example 1 was inoculated into 100 mL of LB liquid medium at a volume ratio of 2% and cultured at 30°C and pH 7. The culture was continuously shaken for 48 h in LB liquid medium at shaking speeds of 0 r / min, 100 r / min, 140 r / min and 180 r / min, respectively.

[0056] (2) OD in culture medium at different shaking speeds was measured at 6-hour intervals. 600 value.

[0057] As shown in Table 2 Bacillus velezensis The bacteria enter the logarithmic growth phase earlier at a shaking speed of 140 r / min, therefore its optimal shaking speed is 140 r / min. The strain can grow normally at a shaking speed of 100~180 r / min.

[0058] Table 2 Bacillus velezensis OD of bacteria at different rotation speeds 600 value

[0059]

[0060] Example 5: Culture and temperature tolerance of the strain at different temperatures

[0061] (1) The strain obtained in Example 1 was inoculated into 100 mL of LB liquid medium at a volume ratio of 2%, and cultured at pH 7 and a rotation speed of 140 r / min. The culture was continuously shaken for 48 h in LB liquid medium at temperatures of 20℃, 30℃, 40℃, 50℃ and 60℃ respectively.

[0062] (2) OD in culture media at different temperatures was measured at 6-hour intervals. 600 value.

[0063] As shown in Table 3 Bacillus velezensisThe bacteria enter the logarithmic growth phase earlier at 30℃, therefore its suitable temperature range is 30℃. Furthermore, *Bacillus belye* has a temperature tolerance range of 20℃-50℃.

[0064] Table 3 Bacillus velezensis OD of bacteria at different temperatures 600 value

[0065]

[0066] Example 6: Application of bacterial strains in degrading PTA wastewater

[0067] (1) Culture of strain: The strain obtained in Example 1 Bacillus velezensis The bacteria were inoculated into 100 mL LB liquid medium at a volume ratio of 2%, and cultured with shaking at 30℃, pH 7, and a rotation speed of 140 r / min for 48 h to achieve an OD concentration of 100%. 600 It is around 1.0, 2×10 9 CFU / mL;

[0068] (2) Wastewater treatment: Take the Bacillus belye, which has been cultured for 48 h in step (1), and separately add the bacterial solution at 4×10 8 CFU / mL, 8×10 8 CFU / mL, 1.2×10 9 CFU / mL, 1.6×10 9 CFU / mL, 2×10 9 A final concentration of CFU / mL was added to 100 ml of wastewater, and the mixture was treated at 30℃ and 140 r / min for 6–72 h. The residual COD content in the wastewater was measured every 12 h, and the COD degradation rate of the strain on PTA wastewater was calculated. The reaction parameters for COD treatment at this time represent the optimal growth conditions for the strain. The total COD at the wastewater inlet was 2798.5 mg / L.

[0069] (3) Results determination: The change in COD content before and after wastewater was measured, the degradation rate was calculated, and the concentration of bacterial solution under the best degradation effect was selected.

[0070] From Table 4 and Figure 2 It can be seen that the product prepared in step (1) Bacillus velezensis The dosage of bacteria is 2×10 9 The efficiency of PTA wastewater degradation reaches its maximum at CFU / mL, which is 50.50%.

[0071] Table 4 Bacillus velezensis Bacterial treatment of high-concentration COD wastewater (total amount at wastewater inlet: 2798.5 mg / L)

[0072]

[0073] Example 7: Application of bacterial strains in degrading agricultural and livestock wastewater

[0074] The specific implementation method is as described in Example 6, except that the PTA wastewater is replaced with agricultural and aquaculture wastewater, and the total COD at the inlet of the agricultural and aquaculture wastewater is 265.5 mg / L and 389.2 mg / L, respectively.

[0075] As shown in Table 5, the product prepared in step (1) Bacillus velezensis The dosage of bacteria was 1.6 × 10⁻⁶. 9 At a concentration of CFU / mL, the efficiency of degrading agricultural and livestock wastewater with a total COD of 265.5 mg / L at the wastewater inlet can reach its maximum. After 48 hours of degradation, the COD degradation rate can reach 96.55%.

[0076] Table 5 Bacillus velezensis Bacterial treatment of agricultural and livestock wastewater (total COD at wastewater inlet is 265.5 mg / L)

[0077]

[0078] As shown in Table 6, the product prepared in step (1) Bacillus velezensis The dosage of bacteria is 2×10 9 At a concentration of CFU / mL, the efficiency of degrading agricultural and livestock wastewater with a total COD of 389.2 mg / L at the wastewater inlet can reach its maximum. After 24 hours of degradation, the COD degradation rate can reach 81.37%.

[0079] Table 6 Bacillus velezensis Bacterial treatment of agricultural and livestock wastewater (total COD at wastewater inlet is 389.2 mg / L)

[0080]

[0081] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis ), characterized in that, The Bacillus belyssus was deposited on September 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25800.

2. A microbial preparation containing the Bacillus belye as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation contains ≥2×10⁶ live Bacillus vesiculus bacteria. 9 CFU / ml.

4. The microbial preparation according to claim 3, characterized in that, The microbial preparation also contains a freeze-drying protectant.

5. A method for preparing the microbial preparation according to any one of claims 2 to 4, characterized in that, The Bacillus berleis described in claim 1 is fermented in a culture medium.

6. The method according to claim 5, characterized in that, Fermentation should be carried out at pH 3.0-7.0, 100-140 r / min, and 20-50℃ for at least 22 h.

7. The application of *Bacillus belye* as described in claim 1 or the microbial preparation as described in any one of claims 2-4 in the degradation of COD in wastewater, characterized in that, The wastewater includes PTA wastewater and agricultural and aquaculture wastewater.

8. A method for degrading COD in wastewater, characterized in that, The Bacillus berberis of claim 1 or any of the microbial preparations of claims 2 to 4 are added to the wastewater, wherein the wastewater includes PTA wastewater and agricultural and aquaculture wastewater.

9. The method according to claim 8, characterized in that, Bacterial cells were 4 × 10 8 ~2×10 9 Add the final concentration of CFU / mL to the wastewater and degrade it for at least 6 hours at pH 3.0-7.0, 100-140 r / min, and 20-50℃.

10. The use of Bacillus belye as described in claim 1, or the microbial preparation as described in any one of claims 2 to 4, or the method as described in claim 5 or 6 in the preparation of a wastewater treatment agent.

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