Lysinibacillus sp. And application thereof in acid wastewater treatment

Abstract

The invention discloses lysinibacillus sp. And an application thereof in acid wastewater treatment, and belongs to the technical field of biodegradation. The lysinibacillus sp. Can adapt to the environment of acid wastewater, is good in growth and reproduction, and has good degradation and removal effects on nitrate radicals in the wastewater, and the highest degradation rate can reach 80.6%; meanwhile, the lysinibacillus sp. Can neutralize the pH value of the acidic wastewater, so that the pH value is recovered to 6.43 or above. Therefore, the strain disclosed by the invention can adapt to a severe environment containing the acid wastewater, can be applied to treatment of the acid wastewater, and is beneficial to sustainable development of an ecological environment.

Description

Technical Field

[0001] The invention relates to a lysine bacillus and application thereof in treating acidic wastewater, belonging to the technical field of biodegradation. Background Art

[0002] Acidic wastewater refers to wastewater with a pH value below 7, which is mainly generated during industrial production and treatment. It mainly comes from the following industries and processes: pickling, degreasing, and arsenic removal processes in metallurgical processes produce a large amount of acidic wastewater, which contains a large amount of acidic substances and heavy metal ions; wastewater generated in chemical synthesis, solvent extraction, desulfurization, etc. in the chemical industry usually contains acidic substances and organic acids, such as sulfuric acid, hydrochloric acid, nitric acid, etc.; acidic solutions and corrosive agents used in circuit board manufacturing, semiconductor production, and other processes in the electronics manufacturing industry lead to the generation of acidic wastewater. When untreated acidic wastewater is discharged into the water body, the excessive nitrate in the acidic wastewater can promote the excessive growth of algae and other aquatic plants, trigger eutrophication, lead to hypoxia in the water body, affect the survival of fish and other aquatic organisms, and destroy the aquatic ecosystem. Excessive discharge of nitrate can indirectly affect the food chain. Through bioaccumulation, high concentrations of nitrate will accumulate in organisms at the top of the food chain, thereby affecting the health of the entire ecosystem. In view of the above hazards, it is crucial to degrade nitrate in acidic wastewater.

[0003] In view of this, the effective management and purification of acidic wastewater has become a core issue in environmental protection work, aiming to reduce damage to the ecological environment and ensure public health and safety. This requires enterprises to adopt advanced wastewater treatment technology to ensure that emission standards comply with environmental protection regulations, while promoting the recycling of resources and achieving green and sustainable development. At present, the commonly used acidic wastewater treatment and disposal methods can be divided into three categories: physical method, chemical method and biological method. The physical method mainly separates insoluble pollutants in wastewater through physical action, including precipitation, filtration, flotation and other methods. These methods are simple and safe, but there are limitations in the treatment and recovery. The chemical method uses chemical reactions to degrade pollutants in acidic wastewater to achieve a certain standard of effluent concentration, but there are many problems such as restrictions on wastewater concentration, high energy consumption, huge amount of waste residue, inability to directly reuse heavy metals, and the possibility of causing secondary pollution.

[0004] Biological treatment is a wastewater treatment method that uses microorganisms in the natural environment to oxidize and decompose organic matter and certain inorganic poisons (such as cyanide and sulfide) in wastewater and convert them into stable and harmless inorganic substances. The biological treatment method of sewage is an artificial enhancement technology based on the self-purification of the environment. Its significance lies in creating a good environment conducive to the growth and reproduction of microorganisms, enhancing the metabolic function of microorganisms, promoting the proliferation of microorganisms, accelerating the inorganicization of organic matter, and enhancing the purification process of sewage. This method has the advantages of low investment, good effect, and low operating costs. It is most widely used in the treatment of urban wastewater and industrial wastewater. Among them, for neutral or alkaline industrial wastewater, the prior art has found that individual strains can effectively degrade nitrates therein; however, for acidic industrial wastewater, no strains that can effectively degrade nitrates in wastewater and neutralize the pH of wastewater have been found, and no strains that can degrade nitrates in industrial wastewater and neutralize the pH of wastewater under acidic conditions containing a large amount of harmful substances such as heavy metals have been found. Industrial pickling wastewater contains a large amount of heavy metals and nitrates. Removing nitrate and neutralizing the pH of wastewater are of great significance for subsequent wastewater treatment. Therefore, it is urgent to screen out strains that can effectively remove nitrate from acidic wastewater and neutralize the pH of acidic wastewater. Summary of the invention

[0005] [Technical Issues] Currently, in the field of acidic wastewater degradation, the use of biological degradation methods has too high requirements on water quality and the environment. There is a lack of microbial species that can degrade nitrates in acidic wastewater and neutralize the pH of the wastewater, so it cannot be widely used in industrial production.

[0006] [Technical Solution] In view of the current problems, the present invention extracted a strain of lysine Bacillus from the sludge of the Changzhou sewage treatment plant. The strain can be used to degrade nitrate in acidic wastewater and increase the pH of acidic wastewater, providing an effective biological treatment method for treating acidic wastewater.

[0007] The present invention provides a strain of Bacillus lysinicus, which was deposited in the China Center for Type Culture Collection on October 25, 2023, with a deposit number of CGMCC No. 28761, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0009] In one embodiment, the microbial preparation contains 2.0×10- 9 CFU / mL.

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

[0011] The invention also provides a method for preparing the microbial preparation, which comprises fermenting the lysine bacillus in a culture medium.

[0012] In one embodiment, the fermentation is carried out at pH 5.0-7.0, 100-180 r / min, 15-45° C., and for at least 22 hours.

[0013] The present invention also provides application of the lysine bacillus or the microbial preparation in degrading nitrate in acidic wastewater.

[0014] The invention also provides a method for degrading nitrate in waste water, wherein the lysine bacillus or the microbial preparation is added into the waste water.

[0015] In one embodiment, the wastewater includes acidic wastewater; the acidic wastewater contains nitrates and heavy metals; and the nitrate content in the acidic wastewater is ≤23409 mg / L.

[0016] In one embodiment, the bacterial solution is (2.0×10 8 ~2.2×10 9 ) CFU / mL was added into the wastewater and degraded for at least 12 h at pH 3.37-7.0, 100-180 r / min, 15-45°C.

[0017] The present invention also provides a method for neutralizing acidic wastewater, wherein the lysine bacillus or the microbial preparation is added into the acidic wastewater for reaction.

[0018] In one embodiment, the pH of the acidic wastewater is 3.0 to 7.0; the bacterial cells are cultured at 2.0×10 8 CFU / mL~2.2×10 9 The final concentration of CFU / mL is added into the acidic wastewater and reacted at 100-180 r / min and 15-45°C for at least 12 hours.

[0019] The present invention also provides the use of the lysine Bacillus or the microbial preparation in preparing a sewage treatment agent.

[0020] Beneficial Effects

[0021] The present invention separates a lysine bacillus from the sludge of a Changzhou sewage treatment plant. The lysine bacillus can grow normally under an acidic condition of pH 5.0 to 7.0. Under strong acid conditions, the lysine bacillus can effectively neutralize the pH of acidic wastewater, and the pH of the wastewater is restored from 3 to 4 to above 6.43. In addition, the lysine bacillus can degrade nitrate in the wastewater, and the nitrate degradation rate reaches 80.6% for wastewater with a nitrate content of 23409 mg / L. The degradation process is simple, the requirements on the environment and the process are low, and the industrial application of treating acidic wastewater is conducive.

[0022] Biomaterial Deposit

[0023] A strain of Lysinibacillus, taxonomically named Lysinibacillus sp., was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2023, with the deposit number CGMCC No.28761, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology and cell morphology of Bacillus lysinicus; A is the colony morphology, and B is the cell morphology.

[0025] Figure 2 This is the change diagram of acidic wastewater treatment by Bacillus lysinicus. DETAILED DESCRIPTION

[0026] The culture medium involved in some embodiments of the present invention is as follows:

[0027] LB liquid medium: yeast powder 5g·L -1 , Tryptone 10 g·L -1 , sodium chloride 10g·L -1 , distilled water.

[0028] LB solid medium: yeast powder 5g L -1 , Tryptone 10 g·L -1 , sodium chloride 10g·L -1 , distilled water, agar 20g·L -1 .

[0029] pH adjustment of LB liquid medium and LB solid medium: as needed, adjust the pH of LB liquid medium or LB solid medium to 3.0, 4.0, 5.0, 6.0 and 7.0 with 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0030] In some embodiments of the present invention, the nitrate degradation rate of acidic wastewater is calculated as follows:

[0031] Calculate the degradation rate of nitrate in acidic wastewater:

[0032] Nitrate degradation rate of acidic wastewater = (total amount of nitrate at the inlet - total amount of nitrate at the outlet) / total amount of nitrate at the inlet. The nitrate and nitrite contents of acidic wastewater were determined by ultraviolet spectrophotometer.

[0033] Technical terms:

[0034] Sludge: The term "sludge" is taken from the Changzhou sewage treatment plant, with a density of 0.027 g·mL -1 , pH 7.66.

[0035] Acidic wastewater: Acid wastewater from a factory in Changzhou, which contains a large amount of organic and inorganic pollutants, such as heavy metals (Fe 3+ , Cu 2+ ), salts (nitrates), etc., pH <1, with complex ingredients, strong acidity, high chromaticity and containing toxic and harmful substances.

[0036] Nitrate degradation: The nitrate degradation process described in the present invention refers to the bacterial strain converting nitrate in the wastewater into nitrite, and further converting it into nitrogen gas.

[0037] The present invention is further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0038] Example 1: Screening and identification of strains

[0039] (1) The density obtained from Changzhou Wastewater Treatment Plant is 0.027 g mL -1 10 mL of sludge was added into 90 mL of LB liquid culture medium and cultured at 140-180 r / min and 30-35°C for 4-6 days;

[0040] (2) taking the bacterial solution obtained in step (1) and inoculating it into a new 100 mL LB liquid culture medium at a volume ratio of 5-10%, and culturing it at 140-180 r / min and 30-35° C. for 4-6 days;

[0041] (3) taking the bacterial solution obtained in step (2) and inoculating it into a new 100 mL LB liquid culture medium at a volume ratio of 5-10%, and culturing it at 140-180 r / min and 30-35° C. for 4-6 days;

[0042] (4) taking 5 μL of the bacterial solution obtained in step (3) and adding it to a sterilized LB liquid medium, culturing it in a shaker at 140-180 r / min and 30-35° C. for 4-6 days, then taking up 100 μL of the liquid with a pipette tip and applying it to an acidic LB solid medium at pH 5.0;

[0043] (5) The acidic LB solid medium with a pH of 5.0 coated with the bacterial solution was placed in an incubator at 35°C for 1 to 2 days, and the morphology of the colonies was observed. A small amount of bacteria was picked from each colony and inoculated into an acidic LB liquid medium with a pH of 5.0. After culturing for 4 to 6 days, the bacterial solution was coated on an acidic LB solid medium with a pH of 5.0;

[0044] (6) Repeat step (5) multiple times until a single bacterial strain is obtained in each acidic LB solid culture medium;

[0045] (7) Pick a single strain from the culture medium where a single strain grows and inoculate it into 100 mL of fresh acidic LB liquid culture medium with a pH of 5.0. Cultivate at 140-180 rpm and 30-35°C for 4-6 days and measure the OD of the bacterial solution. 600 When the value reaches 0.8, the desired bacterial solution is obtained.

[0046] (8) Culture deposit: The culture was deposited on slant surface at the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2023. The deposit number is CGMCC No. 28761. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] (9) Identification of strains: The strain was identified as Lysinibacillus sp. by 16s RNA by Sangon Biotech (Shanghai) Co., Ltd. The colony morphology and cell morphology of the Lysinibacillus sp. Figure 1 As shown, the 16sRNA sequence is shown as SEQ ID NO.1.

[0048] Example 2: Growth characteristics of strains

[0049] 1. Culture of strains at different pH and pH tolerance

[0050] (1) The strain obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium with an initial pH of 3.0, 4.0, 5.0, 6.0 and 7.0 at a volume ratio of 2%, and cultured at 35° C. and 180 r / min for 72 h;

[0051] (2) Measure the OD in different initial pH medium at 6 h intervals 600 Values, respectively, are based on OD at 0h 600 The blank value was used as the blank control, and the blank value was deducted to obtain the bacterial solution OD 600 The values ​​are shown in Table 1.

[0052] As shown in Table 1, Bacillus lysinin entered the logarithmic growth phase earlier under the condition of an initial pH of 6.0, so its more suitable initial pH is 6.0. The pH tolerance range of Bacillus lysininin is 5.0-7.0. In the pH range of 3.0-4.0, although the growth is restricted, the strain can maintain normal activity and physiological functions.

[0053] Table 1 OD of Bacillus lysinicus at different initial pH 600 value

[0054]

[0055] 2. Cultivation of strains at different speeds

[0056] (1) The bacterial solution obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium at a volume ratio of 2%, and the shaking speed was set to 0 r / min, 100 r / min, 140 r / min and 180 r / min at 35° C. and pH 6.0, respectively, and the shaking culture was continuously carried out for 72 h.

[0057] (2) Measure the OD of the culture medium at different shaking speeds every 6 hours 600 Values, respectively, are based on OD at 0h 600 The blank value was used as the blank control, and the blank value was deducted to obtain the bacterial solution OD 600 The values ​​are shown in Table 2.

[0058] As shown in Table 2, Bacillus lysinin entered the logarithmic growth phase of microorganisms at the earliest under the condition of a rotation speed of 180 r / min, so its optimal shaking incubator speed is 180 r / min, and Bacillus lysininin can grow normally within the speed range of 0 to 180 r / min.

[0059] Table 2 OD of Bacillus lysinicus at different rotation speeds 600 value

[0060]

[0061]

[0062] 3. Culture of strains at different temperatures and temperature tolerance

[0063] (1) The bacterial solution obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium at a volume ratio of 2%. Under the conditions of pH 6.0 and a rotation speed of 180 r / min, the culture temperatures were set to 15° C., 25° C., 35° C. and 45° C., respectively, and the culture was continuously shaken for 72 h.

[0064] (2) Measure the OD in the culture medium at different temperatures every 6 hours600 Values, respectively, are based on OD at 0h 600 The blank value was used as the blank control, and the blank value was deducted to obtain the bacterial solution OD 600 The values ​​are shown in Table 3.

[0065] As shown in Table 3, Bacillus lysinicus enters the logarithmic growth phase earlier at a temperature of 35°C, so its suitable temperature range is 35°C. And the temperature tolerance range of Bacillus lysinicus is 15°C to 45°C.

[0066] Table 3 OD of Bacillus lysinicus at different temperatures 600 value

[0067]

[0068] Example 3: Application of Lysinibacillus in Degrading Acidic Wastewater

[0069] (1) Strain culture:

[0070] The lysinibacillus liquid obtained in Example 1 was inoculated into 100 mL LB liquid culture medium at a volume ratio of 2%, and cultured at 35°C, pH 6.0, and a rotation speed of 180 r / min. After culturing for 18 h, the concentration of the liquid in the culture medium reached OD 600 About 1.0, 2.0×10 9 CFU / mL.

[0071] (2) Wastewater treatment:

[0072] Take the lysine Bacillus culture solution after culturing for 24 hours in step (1), collect the cells, and separate them into 2.0×10 8 CFU / mL, 6.0×10 8 CFU / mL, 1.0×10 9 CFU / mL, 1.4×10 9 CFU / mL, 1.8×10 9 CFU / mL and 2.2×10 9 The final concentration of CFU / mL was added to 100mL of wastewater. The wastewater was diluted before treatment. The total amount of nitrate at the inlet of the diluted wastewater was 23409mg / L, the total amount of nitrite was 0mg / L, and the pH was about 3.0.

[0073] The wastewater was treated under aerobic conditions at 35°C and 180 r / min for 72 hours, and the pH of the wastewater was tested every 24 hours. Then, the wastewater was treated under anaerobic conditions at 35°C and 180 r / min for 120 hours, and the residual nitrate and nitrite content in the wastewater was tested every 24 hours to calculate the nitrate degradation rate of the strain on the acidic wastewater.

[0074] (3) Result determination:

[0075] The change of nitrate content in sewage before and after treatment was determined, the degradation rate was calculated, and the concentration of bacterial solution for the best degradation effect was selected.

[0076] As shown in Table 4, the addition of Bacillus lysine into wastewater will increase the pH value of the wastewater. 9 CFU / mL and 2.2×10 9 CFU / mL, the effect was better, and the pH of acidic wastewater was restored to 6.43 and 7.32 respectively.

[0077] As shown in Table 5, the dosage of the lysine Bacillus prepared in step (1) is 1.8×10 9 CFU / mL, the efficiency of reducing acidic wastewater can reach the maximum, and the nitrate degradation rate in 120h is 80.6%.

[0078] During the experiment, gas generation was observed, and upon testing, the gas was found to be nitrogen. As shown in Table 6, no nitrite was accumulated during the process.

[0079] Table 4 pH changes during aerobic treatment of acidic wastewater by Bacillus lysininus (pH at wastewater inlet is 3.13)

[0080]

[0081] Table 5 Nitrate degradation of acidic wastewater treated by Bacillus lysininus (total amount at wastewater inlet is 23409 mg / L)

[0082]

[0083]

[0084] Table 6 Changes of nitrite in acidic wastewater treated by Bacillus lysininus (total nitrite at the wastewater inlet is 0 mg / L)

[0085]

[0086] Example 4: Preparation of Lysinibacillus microbial preparation

[0087] Liquid microbial preparation: The lysine Bacillus liquid obtained in Example 1 was inoculated into 100 mL LB liquid medium at a volume ratio of 2%, and cultured under shaking conditions of 25-45°C, pH 7.0, and rotation speed of 100-180 r / min. After culturing for 24 hours, the concentration of the liquid in the culture medium reached OD 600 About 1.0, 2.0×10 9CFU / mL.

[0088] Solid microbial preparation: A protective agent (such as skim milk) is used as a carrier, and the strain is added to prepare a bacterial suspension, which is dehydrated and freeze-dried using a vacuum freeze-drying machine to prepare a freeze-dried powder, which is then vacuum-sealed for storage. The number of viable bacteria in the solid microbial preparation is ≥2.0×10 9 CFU / g.

[0089] Example 5: Preparation of a sewage treatment agent containing lysine Bacillus

[0090] The solid microbial preparation obtained in Example 5 is mixed with a nitrifying bacteria agent, a denitrifying bacteria agent, a nitrifying and denitrifying composite bacteria agent, and an organic matter strong degradation composite biological bacteria agent to obtain a sewage treatment agent.

[0091] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lysinibacillus sp., characterized in that: The lysine Bacillus was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2023, with the deposit number CGMCC No.28761.

2. A microbial preparation containing the lysinic Bacillus according to claim 1.

3. The microbial preparation according to claim 2, characterized in that: The number of viable Bacillus lysine in the microbial preparation is ≥2.0×10 9 CFU / mL.

4. The microbial preparation according to claim 3, characterized in that: The microbial preparation further contains a lyoprotectant.

5. A method for degrading nitrate in wastewater, characterized in that: The lysinic Bacillus described in claim 1 or the microbial preparation described in any one of claims 2 to 4 is added into the wastewater.

6. The method according to claim 5, characterized in that The wastewater includes acidic wastewater; the acidic wastewater contains nitrates and heavy metals.

7. The method according to claim 5 or 6, characterized in that: The bacteria were plated at 2.0×10 8 CFU / mL~2.2×10 9 The final concentration of CFU / mL was added into acidic wastewater and degraded for at least 12 hours at pH 3.0-7.0, 100-180r / min, 15-45°C.

8. A method for neutralizing acidic wastewater, characterized in that: The lysinic Bacillus described in claim 1 or the microbial preparation described in any one of claims 2 to 4 is added into acidic wastewater for reaction.

9. The method according to claim 8, characterized in that The pH of the acidic wastewater is 3.0-7.0; the bacterial cells are cultured at 2.0×10 8 CFU / mL~2.2×10 9 The final concentration of CFU / mL is added into the acidic wastewater and reacted at 100-180 r / min and 15-45°C for at least 12 hours.

10. Use of the lysinibacillus according to claim 1 or the microbial preparation according to any one of claims 2 to 4 in the preparation of a sewage treatment agent.

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