A strain of Priesteria and its application in degradation of nitrate in acidic wastewater

Abstract

The invention discloses a strain of Priesteria and its application in degrading nitrate radicals in acidic wastewater, belonging to the field of biodegradation technology. The Priesteria can adapt to the environment of acidic wastewater, grow and reproduce well, and has a good degradation and removal effect on nitrate radical components in wastewater, with a degradation rate of up to 95.8%. Therefore, the strain of the invention can adapt to the harsh environment containing acidic wastewater, can be used to treat acidic wastewater, and is conducive to the sustainable development of the ecological environment.

Description

Technical Field

[0001] The invention relates to a strain of Priesteria and application thereof in degrading nitrate radicals in acidic wastewater, belonging to the technical field of biodegradation. Background Art

[0002] Acidic wastewater refers to wastewater containing certain acids and a pH value below 6. In addition to containing one or more acids, industrial acidic wastewater often contains harmful substances such as nitrates, heavy metal ions and their salts. If it is discharged directly into the environment without effective treatment, excessive nitrates will cause eutrophication of water bodies and deterioration of water quality, causing harm to animals, plants and even humans. Animals that ingest excessive nitrates will experience symptoms such as difficulty breathing, increased heart rate and breathing, physical incoordination, coma and death. Humans drinking high-nitrate well water will develop methemoglobinemia, with a mortality rate of 10%.

[0003] The existing methods for treating nitrates in industrial acidic wastewater include physical and chemical methods (such as ion exchange, reverse osmosis, nanofiltration and electrodialysis). These methods have high operating costs and are difficult to be applied on a large scale in industry. The biological treatment method has the advantages of low investment, good effect and low operating cost, and is most widely used in the treatment of urban wastewater and industrial wastewater. In the biological treatment process of sewage, microorganisms are the key factor. By providing sufficient nutrients, suitable temperature and pH value and other conditions, an environment conducive to the growth of microorganisms can be created, thereby increasing their metabolic activity and accelerating the degradation and inorganicization of organic matter. In this way, microorganisms can not only effectively purify sewage, but also quickly convert organic pollutants into harmless substances. In addition, the biological treatment method of sewage also helps to promote the purification process of sewage. Through the metabolic activities of microorganisms, organic pollutants are decomposed into carbon dioxide, water and other harmless substances, thereby achieving the purpose of purifying sewage. At the same time, microorganisms can also remove nutrients such as nitrogen and phosphorus in water to reduce the eutrophication of water bodies.

[0004] At present, microbial degradation of acidic wastewater is related to: using Pseudomonas, Bacillus, Proteus and some bacterial consortia to obtain higher nitrate removal efficiency under neutral or weakly alkaline conditions, but there are few reports of microorganisms that can degrade nitrates under acidic conditions, and it is even more difficult to obtain microorganisms that can degrade nitrates under acidic conditions containing a large amount of heavy metals and other harmful substances. Industrial acidic wastewater contains a large amount of heavy metals and other harmful substances. It is a huge challenge for microorganisms to survive in it and remove the nitrates in it. At present, no strains have been found that can survive in industrial acidic wastewater containing a large amount of harmful substances and can efficiently degrade nitrates. Summary of the invention

[0005] [Technical Issues] Currently, in the field of biological degradation of acidic wastewater, there is a lack of microorganisms that can survive in harmful acidic wastewater and degrade nitrates in acidic wastewater.

[0006] [Technical Solution] In view of the current problems, the present invention extracted a strain of Priesteria 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 Priesteria, which was deposited in the China Center for Type Culture Collection on October 25, 2023, with a deposit number of CGMCC No. 28758, 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 Priesteria.

[0009] In one embodiment, the microbial preparation contains the Priesteria according to claim 1 or its fermentation broth, or its immobilized cells, or its lyophilized powder, or its extract.

[0010] In one embodiment, the number of Priesteria cells in the microbial preparation is ≥ 2.0×10 9 CFU / mL.

[0011] In one embodiment, the microbial preparation further contains a lyoprotectant.

[0012] The invention also provides a product containing the Priesteria or the microbial preparation.

[0013] In one embodiment, the product includes but is not limited to a sewage treatment agent.

[0014] In one embodiment, the cell number of Priesteria spp. in the product is ≥ 2.0×10 9 CFU / mL.

[0015] The present invention also provides a method for preparing the microbial preparation, which comprises fermenting the Priesteria in a culture medium.

[0016] In one embodiment, the fermentation is carried out at pH 5.0-7.0, 100-180 r / min, 25-45° C., and for at least 22 hours.

[0017] The present invention also provides the use of the Priesteria or the microbial preparation in degrading nitrate in acidic wastewater.

[0018] The invention also provides a method for degrading nitrate in acidic wastewater, which comprises adding the Priesteria or the microbial preparation into the acidic wastewater.

[0019] In one embodiment, the nitrate value of the acidic wastewater is ≤23409 mg / L.

[0020] 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 3.0-7.0, 100-180 r / min, 25-45°C.

[0021] The present invention also provides the use of the Priesteria or the microbial preparation in treating acidic wastewater.

[0022] Beneficial Effects

[0023] The invention separates a strain of Priesteria from the sludge of a Changzhou sewage treatment plant, wherein the Priesteria can grow normally under an acidic condition of pH 5.0 to 7.0, and can degrade nitrate in acidic wastewater, and the nitrate degradation rate reaches 95.8% for wastewater with a nitrate content of 23409 mg / L. The degradation process is simple, and the requirements on the environment and process are low.

[0024] Biomaterial Deposit

[0025] A strain of Priestia, taxonomically named Priestia sp., was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2023, with the deposit number CGMCC No.28758, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the colony morphology and cell morphology of Priestia sp.; A is the colony morphology, and B is the cell morphology.

[0027] Figure 2 Diagram of changes in acidic wastewater treated with Prioccidioides. DETAILED DESCRIPTION

[0028] The culture medium involved in some embodiments of the present invention is as follows:

[0029] LB liquid medium: yeast powder 5g·L -1 , Tryptone 10 g·L -1 , sodium chloride 10g·L -1 , distilled water.

[0030] LB solid medium: yeast powder 5g L -1 , Tryptone 10 g·L -1 , sodium chloride 10g·L -1 Agar 20 g·L -1 , distilled water.

[0031] pH adjustment of LB liquid medium and LB solid medium: as needed, adjust the pH of LB liquid medium or LB solid medium to 1.0, 3.0, 5.0 and 7.0 with 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0032] In some embodiments of the present invention, the nitrate degradation rate of acidic wastewater is calculated as follows:

[0033] Calculate the degradation rate of nitrate in acidic wastewater:

[0034] 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 contents of nitrate and nitrite in the acidic wastewater were determined by ultraviolet spectrophotometer.

[0035] Technical terms:

[0036] Sludge: The term "sludge" is taken from the Changzhou sewage treatment plant, with a density of 0.027 g·mL -1 , pH 7.66.

[0037] 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.

[0038] 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.

[0039] The present invention is further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0040] Example 1: Screening and identification of strains

[0041] 1. Screening of strains

[0042] (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;

[0043] (2) taking the bacterial solution 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;

[0044] (3) taking the bacterial solution 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;

[0045] (4) Take 5 μL of the bacterial solution in step (3) and add it to the sterilized LB liquid medium. After culturing in a shaker at 140-180 r / min and 30-35° C. for 4-6 days, use a pipette to draw up 100 μL of the liquid and apply it to the acidic LB solid medium.

[0046] (5) Place the acidic LB solid medium coated with the bacterial solution in an incubator at 35°C for 1-2 days, observe the morphology of the colonies, pick a small amount of bacteria from each colony and inoculate it into the acidic LB liquid medium. After culturing for 4-6 days, coat the bacterial solution on the acidic LB solid medium;

[0047] (6) Repeat step (5) multiple times until a single bacterial strain is obtained in each acidic LB solid culture medium;

[0048] (7) Pick a single strain from the culture medium where a single strain grows and inoculate it into 100 mL of new acidic LB liquid culture medium and culture it at 140-180 rpm and 30-35°C for 4-6 days. Measure the OD of the bacterial solution. 600 When the value reaches 0.8, the desired bacterial solution is obtained.

[0049] 2. Identification of strains

[0050] (1) 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. 28758. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0051] (2) Identification of strains: The strain was identified as Priestia sp. by 16s RNA by Sangon Biotech (Shanghai) Co., Ltd. The colony morphology and cell morphology of the lysine Bacillus are as follows: Figure 1 As shown, the 16s RNA sequence is shown as SEQ ID NO.1.

[0052] Example 2: Culture of strains at different pH and pH tolerance

[0053] (1) The bacterial solution obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium with pH values ​​of 1.0, 3.0, 5.0, and 7.0 at a volume ratio of 2%, and cultured under continuous shaking at 35° C. and 180 r / min for 72 h;

[0054] (2) Measure OD in different culture systems 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.

[0055] As shown in Table 1, Priestia sp. enters the logarithmic growth phase earlier under the condition of an initial pH of 5.0, so its more suitable initial pH is 5.0, and the pH tolerance range of Priestia sp. is 5.0 to 7.0. In the pH range of 3.0 to 4.0, although the growth is restricted, the strain can maintain normal activity and physiological functions.

[0056] Table 1 OD of Priesteria at different initial pH 600 value

[0057]

[0058]

[0059] Example 3: Strain culture at different rotation speeds

[0060] (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 5.0, respectively, and the shaking culture was continuously carried out for 72 h.

[0061] (2) Measure the OD in the culture system 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 OD of the bacterial solution. 600 The values ​​are shown in Table 2.

[0062] As shown in Table 2, Priestia sp. enters the logarithmic growth phase of microorganisms at a rotation speed of 140 r / min, so its optimal shaking incubator speed is 140 r / min, and Priestia sp. can grow normally within the speed range of 100 to 180 r / min.

[0063] Table 2 OD of Priesteria at different speeds 600 value

[0064]

[0065] Example 4: Culture of strains at different temperatures and temperature tolerance

[0066] (1) The bacterial solution obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium at an inoculum volume ratio of 2%, and cultured at a pH of 5.0 and a rotation speed of 140 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.

[0067] (2) Measure the OD in the culture system at different temperatures 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 OD of the bacterial solution. 600 The values ​​are shown in Table 3.

[0068] As shown in Table 3, Priestia sp. enters the logarithmic growth phase earlier at 35°C, so its suitable temperature range is 35°C. And the temperature tolerance range of Priestia sp. is 25°C to 45°C.

[0069] Table 3 OD of Priesteria at different temperatures 600 value

[0070]

[0071] Example 5: Application of Priesteria in Degrading Acidic Wastewater

[0072] (1) Strain culture: The Priesteria bacterial solution 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 5.0, and a rotation speed of 140 r / min. After culturing for 18 h, the bacterial solution concentration in the culture medium reached OD 600 About 1.0, 2.0×10 9 CFU / mL, and the bacteria were collected by centrifugation.

[0073] (2) Wastewater treatment: Before the initial wastewater treatment, the wastewater was diluted with water so that the total amount of nitrate at the wastewater inlet was 23409 mg / L, the total amount of nitrite was 0 mg / L, and the pH was about 3.0. The Priesteria cells cultured for 18 hours in step (1) were respectively cultured at 2.0×10 8 CFU / mL, 6.0×10 8 CFU / mL, 1.0×10 9 CFU / mL, 1.4×10 9CFU / mL, 1.8×10 9 CFU / mL and 2.2×10 9 The final concentration of CFU / mL was added to 100mL of wastewater.

[0074] The treatment was carried out for 120 hours at a temperature of 35°C and a rotation speed of 140 r / min. The residual nitrate and nitrite content in the wastewater was detected every 48 hours, and the nitrate degradation rate of the strain to the acidic wastewater was calculated. The reaction parameters for treating nitrate at this time were the optimal growth conditions for the strain.

[0075] (3) Result determination: Determine the change of nitrate content before and after sewage treatment (such as Figure 2 The degradation rate was calculated and the concentration of the bacterial solution with the best degradation effect was selected.

[0076] As shown in Table 4, the dosage of Priesteria prepared in step (1) is 1.0×10 9 CFU / mL, after 120h of treatment, the efficiency of degrading nitrate in acidic wastewater can reach the maximum, which is 95.8%.

[0077] It can be seen from Table 5 that after degradation, the nitrate in the wastewater eventually generates nitrogen gas, and there is no accumulation of nitrite.

[0078] Table 4 Nitrate degradation of acidic wastewater treated by Priesteria (total amount at wastewater inlet is 23409 mg / L)

[0079]

[0080] Table 5 Changes of nitrite in acidic wastewater after Priestera treatment (total nitrite at wastewater inlet is 0 mg / L)

[0081]

[0082] Example 6: Preparation of Priesteria microbial preparation

[0083] Liquid microbial preparation: The Priesteria bacterial solution obtained in Example 1 was inoculated into 100 mL LB liquid culture 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 bacterial solution concentration in the culture medium reached OD 600 About 1.0, 2.0×10 9 CFU / mL.

[0084] 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.

[0085] Example 7: Preparation of a sewage treatment agent containing Priesteria

[0086] The solid microbial preparation obtained in Example 6 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.

[0087] 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 Priesteria Priestia sp. ), characterized in that, The Priesteria was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2023, with the deposit number CGMCC No.28758.

2. A microbial preparation, characterized in that: The microbial preparation contains the Priococcus aureus or its lyophilized powder as claimed in claim 1.

3. A product containing the Priesteria described in claim 1 or the microbial preparation described in claim 2.

4. The product according to claim 3, characterized in that The products include sewage treatment agents.

5. A method for preparing the microbial preparation according to claim 2, characterized in that: The method comprises fermenting the Priesteria described in claim 1 in a culture medium.

6. A method for degrading nitrate in acidic wastewater, characterized in that: The Priesteria bacteria described in claim 1 were added 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 h at pH 3.0, 140 r / min, and 35°C.

7. The use of Priesteria according to claim 1 in treating acidic wastewater, 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 h at pH 3.0, 140 r / min, and 35°C.

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