A strain of Weissella coagulans and its application in degrading industrial production wastewater

Abstract

The present invention discloses a Weizmannia coagulans and its application in degrading industrial production wastewater, belonging to the technical field of biodegradation. The Weizmannia coagulans can adapt to the environment of industrial production wastewater, grow and reproduce well, and has a good degradation and removal effect on the COD components in the wastewater, with the highest degradation rate reaching 100%. Therefore, the strain of the present invention can adapt to the harsh environment containing industrial production wastewater, can be applied to treat industrial production wastewater, and is beneficial to the sustainable development of the ecological environment.

Description

Technical Field

[0001] The present invention relates to a strain of Weissella coagulans and its application in degrading industrial production wastewater, belonging to the technical field of biodegradation. Background Art

[0002] Industrial production wastewater refers to the wastewater, sewage and waste liquid generated during industrial production. With the rapid development of industry, the types and quantities of wastewater have increased rapidly, and the pollution of water bodies has become increasingly widespread and serious, threatening human health and safety. Therefore, for environmental protection, the treatment of industrial production wastewater is more important than the treatment of urban sewage. Industrial production wastewater often contains a large amount of COD, that is, the chemical oxygen demand in industrial wastewater, which reflects the degree of pollution by reducing substances in water. The COD index is also one of the comprehensive indexes of the relative content of organic matter. COD can cause the inability of general aquatic organisms to survive. After their deaths, their bodies cannot be fully oxidized, and they will be incompletely oxidized by some anaerobic bacteria, resulting in the production of many organic poisons. For example, the sulfur element in the body will be converted into hydrogen sulfide, and the nitrogen element will become methylamine, etc. These are substances that are not only extremely smelly but also have strong biological toxicity. After entering natural water bodies, they will disrupt the water balance and cause the death of almost all organisms except microorganisms. They not only harm aquatic organisms such as fish in water bodies, but also can enter the human body through the enrichment of the food chain, causing chronic poisoning.

[0003] Currently, the main treatment methods for industrial production wastewater in China include adsorption method, filtration method, flocculation precipitation method, and oxidation-reduction method. The adsorption method for treating printing and dyeing wastewater has a relatively low cost, but the efficiency of treating wastewater is low and the problem of adsorbent regeneration has not been solved. The filtration method can treat printing and dyeing wastewater well, but the operation difficulty is relatively large. The flocculation precipitation method requires a large amount of flocculation precipitants, resulting in a relatively high cost. The oxidation-reduction method has a relatively high efficiency in treating printing and dyeing wastewater, but it will cause secondary pollution.

[0004] Currently, related to the microbial degradation of industrial production wastewater are: through the adsorption mechanism of Bacillus subtilis on cationic dyes, a composite material of Bacillus subtilis is prepared to degrade cationic dyes. However, the current experiments on the biodegradation of industrial production wastewater have the disadvantages of low biodegradation efficiency and high cost. In "Modification Conditions of Immobilized Carrier of Bacillus subtilis and Treatment of Simulated Sewage", immobilized Bacillus licheniformis is used to treat industrial production wastewater containing COD. After 24 hours of treatment, the maximum COD degradation rate reaches 23.5%, which is difficult to meet the requirements of industrial production. Therefore, it is still necessary to further improve the degradation efficiency, optimize the degradation scheme, and screen out highly efficient strains that can adapt to industrial production. Summary of the Invention

[0005] [Technical Problem] Currently, in the field of degrading industrial production wastewater, the biological method still has problems such as large equipment floor area, high cost, low degradation efficiency, and inability to be widely applied in industrial production.

[0006] [Technical Solution] In view of the existing problems, the present invention extracts a strain of Weizmannia coagulans from the sludge of Changzhou Sewage Treatment Plant, and this strain can be used to degrade COD in industrial production wastewater, providing an effective biological treatment method for treating industrial production wastewater.

[0007] The present invention provides a strain of Weizmannia coagulans, which was deposited at the China Center for Type Culture Collection on September 26, 2022, with the deposit number CGMCC No. 25803 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a microbial preparation containing the above-mentioned Weizmannia coagulans.

[0009] In one embodiment, the viable count of Weizmannia coagulans in the microbial preparation is ≥2×10 9 CFU / ml.

[0010] In one embodiment, the microbial preparation further contains a freeze-drying protectant.

[0011] The present invention also provides a method for preparing the microbial preparation, which ferments the Weizmannia coagulans in a culture medium.

[0012] In one embodiment, the fermentation is carried out at pH 7.0 - 10.0, 100 - 180 r / min, 20 - 40 °C for at least 22 h.

[0013] The present invention also provides the application of the Weizmannia coagulans or the above-mentioned microbial preparation in degrading COD in industrial wastewater.

[0014] The present invention also provides a method for degrading COD in industrial wastewater, which adds the Weizmannia coagulans or the microbial preparation into industrial wastewater.

[0015] In one embodiment, the COD value of the industrial wastewater is ≥205.4 mg / L.

[0016] In one embodiment, the bacterial liquid is added to the wastewater at a final concentration of (4×10 8 ~2×10 9 ) CFU / mL, and the degradation is carried out at pH 7.0 - 10.0, 100 - 180 r / min, 20 - 40 °C for at least 6 h.

[0017] The present invention also provides the application of the aforesaid Weizmannia coagulans, or the aforesaid microbial preparation, or the method for preparing the microbial preparation in the preparation of a sewage treatment agent.

[0018] Beneficial effects

[0019] A strain of Weizmannia coagulans was isolated from the sludge of a sewage treatment plant in Changzhou. The Weizmannia coagulans can grow normally under alkaline conditions with a pH of 7.0 - 10.0, and can degrade the COD in industrial production wastewater. For wastewater with a COD content of 205.4 - 1002 mg / L, the COD degradation rate reaches over 99%; for wastewater with a COD content of 17481.5 mg / L, the COD degradation rate reaches 63.82%. The degradation process is simple and has low requirements for the environment and process.

[0020] Biological material preservation

[0021] A strain of Weizmannia coagulans, taxonomically named Weizmannia coagulans, was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 26, 2022. The preservation number is CGMCC No. 25803, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0022] Figure 1 It is a diagram of the colony morphology and cell morphology of Weizmannia coagulans; A is the colony morphology, and B is the cell morphology diagram.

[0023] Figure 2 It is a diagram of the change of high - concentration COD wastewater treated by Weizmannia coagulans. Specific embodiments

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

[0025] LB liquid medium: Yeast powder 5 g·L -1 、Tryptone 10 g·L -1 、Sodium chloride 10 g·L -1 、1000 mL distilled water.

[0026] LB solid medium: Yeast powder 5 g·L -1 、Tryptone 10 g·L -1 、Sodium chloride 10 g·L -1 、1000 mL distilled water, agar 20 g.

[0027] pH adjustment of LB liquid medium and LB solid medium: As needed, adjust the pH of LB liquid medium or LB solid medium to 7, 9, 10, 11, 13, 14 using 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0028] Calculation of COD degradation rate of industrial production wastewater in some embodiments of the present invention:

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

[0030] COD degradation rate of industrial production wastewater = (total amount of COD at the inlet - total amount of COD at the outlet) / total amount of COD at the inlet. Use a multi-parameter water quality detector to measure the COD content of industrial production wastewater.

[0031] Technical terms:

[0032] Sludge: The term "sludge" is taken from Changzhou Wastewater Treatment Plant, with a density of 0.027 g·mL -1 , and a pH of 7.66.

[0033] Industrial production wastewater: Industrial production wastewater from Changzhou Wastewater Treatment Plant, which contains a large amount of organic and inorganic pollutants, such as dyes, heavy metals, salts, and oils, etc., with a pH of 8.7, and has the characteristics of complex composition, strong alkalinity, high salinity, large chromaticity, and containing toxic and harmful substances.

[0034] The present invention will be further described below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto.

[0035] Example 1: Screening of strains

[0036] (1) Take 10 mL of sludge with a density of 0.027 g·mL -1 from Changzhou Wastewater Treatment Plant and put it into 90 mL of LB liquid medium, and culture it in an environment of 140 - 180 rpm and 30 - 35 °C for 4 - 6 d;

[0037] (2) Take the bacterial liquid in step (1) and inoculate it into a new 100 mL LB liquid medium at an inoculation amount of 5 - 10% by volume, and culture it in an environment of 140 - 180 rpm and 30 - 35 °C for 4 - 6 d;

[0038] (3) Take the bacterial liquid in step (2) and inoculate it into a new 100 mL LB liquid medium at an inoculation amount of 5 - 10% by volume, and culture it in an environment of 140 - 180 rpm and 30 - 35 °C for 4 - 6 d;

[0039] (4) Take 5 μL of the bacterial liquid from step (3) and add it to the sterilized LB liquid medium. Culture it in a shaker at 140 - 180 rpm and 30 - 40 °C for 4 - 6 days. Then, use a pipette tip to aspirate 100 μL of the liquid and spread it on the alkaline LB solid medium.

[0040] (5) Place the alkaline LB solid medium coated with the bacterial liquid in an incubator at 37 °C for 1 - 2 days. Observe the colony morphology. Pick a small amount of bacteria from each colony and inoculate them into the alkaline LB liquid medium respectively. After culturing for 4 - 6 days, spread the bacterial liquid on the alkaline LB solid medium.

[0041] (6) Repeat step (5) multiple times until a single strain is obtained in each alkaline LB solid medium.

[0042] (7) Pick a single strain from the medium with a single strain grown and inoculate it into a new 100 mL alkaline LB liquid medium. Culture it in an environment of 140 - 180 rpm and 30 - 35 °C for 4 - 6 days, and measure the OD 600 value. When the OD value reaches 0.8, the required bacterial liquid is obtained.

[0043] Example 2: Identification of the strain

[0044] (1) Strain preservation: Preservation on a slant. It was preserved in the China General Microbiological Culture Collection Center on September 26, 2022. The preservation number is CGMCC No. 25803, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] (2) Strain identification: The strain was identified as Weizmannia coagulans by Sangon Biotech (Shanghai) Co., Ltd. through 16s RNA.

[0046] Example 3: Cultivation of the strain at different pH values and pH tolerance

[0047] (1) Inoculate the strain obtained in Example 1 into 100 mL of LB liquid medium at an inoculation volume ratio of 2%. Culture it under the conditions of 30 °C and 100 r / min.

[0048] (2) Continuously shake and culture for 48 h in LB liquid media with initial pH values of 7.0, 9.0, 10.0, 11.0, 13.0, and 14.0 respectively.

[0049] (3) Measure the OD 600 value in the media with different initial pH values at intervals of 6 h.

[0050] As shown in Table 3, Weizmannia coagulans enters the logarithmic growth phase earlier under the condition of an initial pH of 7. Therefore, its more suitable initial pH is 7. And the pH tolerance range of Weizmannia coagulans is 7.0 - 10.0.

[0051] Table 1 OD of Weizmannia coagulans at different initial pH values 600 Value

[0052]

[0053] Example 4: Culturing the strain at different rotation speeds

[0054] (1) Inoculate the strain obtained in Example 1 into 100 mL of LB liquid medium at an inoculation volume ratio of 2%, and culture it at 30 °C and a pH of 7.

[0055] (2) Continuously shake and culture in LB liquid medium at shaker rotation speeds of 0 r / min, 100 r / min, and 140 r / min for 48 h.

[0056] (3) Measure the OD in the culture medium at different shaker rotation speeds every 6 h 600 Value.

[0057] As shown in Table 2, Weizmannia coagulans enters the logarithmic growth phase of microorganisms earliest under the condition of a rotation speed of 180 r / min. Therefore, its optimal shaker rotation speed is 180 r / min, and Weizmannia coagulans can grow normally within the rotation speed range of 100 - 180 r / min.

[0058] Table 2 OD of Weizmannia coagulans at different rotation speeds 600 Value

[0059]

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

[0061] (1) Inoculate the strain obtained in Example 1 into 100 mL of LB liquid medium at an inoculation volume ratio of 2%, and culture it at a pH of 7 and a rotation speed of 180 r / min.

[0062] (2) Continuously shake and culture in LB liquid medium at temperatures of 20 °C, 30 °C, 40 °C, 50 °C, and 60 °C for 48 h.

[0063] (3) Measure the OD in the culture medium at different temperatures every 6 h600 Value

[0064] As shown in Table 3, Weizmannia coagulans enters the logarithmic growth phase earlier at 40°C, so its suitable temperature range is 40°C relatively speaking. And the temperature tolerance range of Weizmannia coagulans is 20°C - 40°C.

[0065] Table 3 OD of Weizmannia coagulans at different temperatures 600 Value

[0066]

[0067] Example 6: Application of Weizmannia coagulans in Degrading High-Concentration Industrial Production Wastewater

[0068] (1) Strain culture: The Weizmannia coagulans obtained in Example 1 was inoculated into 100 mL of LB liquid medium at an inoculation amount of 2% by volume, and shaken culture was carried out at 30°C, pH 7, and a rotation speed of 100 r / min. After culturing for 48 h, the cell concentration in the medium reached OD 600 of about 1.0, 2×10 9 CFU / ml;

[0069] (2) Wastewater treatment: The Weizmannia coagulans cell suspension after culturing for 48 h in step (1) was taken respectively, and the cell suspension was added to 100 ml of wastewater at final concentrations of 4×10 8 CFU / mL, 8×10 8 CFU / mL, 1.2×10 9 CFU / mL, 1.6×10 9 CFU / mL and 2×10 9 CFU / mL.

[0070] It was treated at 40°C and a rotation speed of 180 r / min for 6 - 72 h. The residual COD content in the wastewater was detected every 12 h, and the COD degradation rate of the strain for industrial production wastewater was calculated. At this time, the reaction parameters for treating COD were the optimal growth conditions of the strain. The total amount of COD at the wastewater inlet was 17481.5 mg / L.

[0071] (3) Result determination: The change in COD content before and after the sewage was measured, the degradation rate was calculated, and the concentration of the cell suspension with the best degradation effect was selected.

[0072] As can be seen from Table 4 and Figure 2 it is known that the dosage of Weizmannia coagulans prepared in step (1) is 1.2×109 When the concentration is CFU / mL, the efficiency of degrading high-concentration industrial production wastewater can reach the maximum, which is 63.82%.

[0073] Table 4 Treatment of high-concentration COD wastewater by Weizmannia coagulans for 72 h (total amount at the wastewater inlet is 17481.5 mg / L)

[0074]

[0075] Example 7: Application of Weizmannia coagulans in degrading low-concentration industrial production wastewater

[0076] The specific implementation method is as in Example 6, with the difference that the total amount at the industrial wastewater inlet is changed, and the total amounts at the wastewater inlet are 205.4 mg / L, 321.9 mg / L, and 1002 mg / L respectively.

[0077] As can be seen from Table 5, when the dosage of the Weizmannia coagulans bacteria prepared in step (1) is 2×10 9 CFU / mL, the efficiency of degrading low-concentration industrial production wastewater (total amount at the wastewater inlet is 205.4 mg / L) can reach the maximum. After degrading for 12 h, the COD degradation rate can reach 100%.

[0078] Table 5 Treatment of low-concentration COD wastewater by Weizmannia coagulans (total amount at the wastewater inlet is 205.4 mg / L)

[0079]

[0080] As can be seen from Table 6, when the dosage of the Weizmannia coagulans bacteria prepared in step (1) is 2×10 9 CFU / mL, the efficiency of degrading low-concentration industrial production wastewater (total amount at the wastewater inlet is 321.9 mg / L) can reach the maximum. After degrading for 12 h, the COD degradation rate can reach 99.14%.

[0081] Table 6 Treatment of low-concentration COD wastewater by Weizmannia coagulans (total amount at the wastewater inlet is 321.9 mg / L)

[0082]

[0083] As can be seen from Table 7, when the dosage of the Weizmannia coagulans bacteria prepared in step (1) is 2×10 9When the CFU / mL is [value], the efficiency of degrading low-concentration industrial wastewater (with a total amount of 1002 mg / L at the wastewater inlet) can reach the maximum. After 12 hours of degradation, the COD degradation rate can reach 100%.

[0084] Table 7 Treatment of low-concentration COD wastewater by Weizmannia coagulans (with a total amount of 1002 mg / L at the wastewater inlet)

[0085]

[0086] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Weizmannia coagulans, characterized in that, The described Weissella coagulans was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 26, 2022, with the deposit number CGMCC No. 25803.

2. A microbial preparation containing the Weizmannia coagulans described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable count of Weizmannia coagulans in the microbial preparation ≥ 2×10 9 CFU / ml.

4. The microbial preparation according to claim 3, characterized in that, The described 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, Ferment the Weissella coagulans described in claim 1 in a culture medium.

6. The method according to claim 5, characterized in that, At pH 7.0 - 10.0, 100 - 180 r / min, 20 - 40 °C, ferment for at least 22 h.

7. The application of the Weizmannia coagulans described in claim 1 or the microbial preparation according to any one of claims 2 to 4 in degrading the COD of industrial wastewater.

8. A method for degrading COD in industrial wastewater, characterized in that, Add the Weissella coagulans described in claim 1 or the microbial preparation described in any one of claims 2 to 4 into industrial wastewater.

9. The method according to claim 8, characterized in that, Add the bacterial cells to the industrial wastewater at a final concentration of 4×10 8 ~2×10 9 CFU / mL, and degrade for at least 6 h at pH 7.0 - 10.0, 100 - 180 r / min, and 20 - 40 °C.

10. The application of the Weizmannia coagulans described in claim 1, or the microbial preparation according to any one of claims 2 to 4, or the method according to claim 5 or 6 in the preparation of sewage treatment agents.

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