Brevibacterium autochromatum and method for decarburizing and denitrifying wastewater under anaerobic condition by using brevibacterium autochromatum

Abstract

The invention discloses a brevibacterium anodorum strain and a method for carbon and nitrogen removal of wastewater under anaerobic conditions, and belongs to the field of biological treatment of water body pollution. The brevibacterium pigmentum JLG5-316-4 is obtained through separation, the brevibacterium pigmentum has the capacity of efficiently degrading nitrogen and carbon in wastewater under the anaerobic condition, the pantoea agglomerans provided by the invention can have high denitrification capacity under the environments that the pH value is 7.5, the temperature is 30 DEG C, the illumination condition is 5000lux and the like, and it can be seen that the pantoea agglomerans can be used for efficiently degrading nitrogen and carbon in wastewater under the conditions that the pH value is 7.5, the temperature is 30 DEG C, the illumination condition is 5000lux and the like. The brevibacterium edulis provided by the invention can be used for improving the water quality in an anoxic environment.

Description

Technical Field

[0001] The invention relates to a strain of Bacillus brevis and a method for using the strain for removing carbon and nitrogen from wastewater under anaerobic conditions, and belongs to the field of biological treatment of water pollution. Background Art

[0002] Affected by human activities, the discharge of nitrogen-containing wastewater continues to grow, and nitrogen pollution in water bodies has become one of the most concerning environmental issues in the world. + -N), as the main substance of nitrogen pollution in water bodies, will stimulate the massive growth and reproduction of algae and other plankton in water. The death and decay of these short-lived organisms will lead to a decrease in the dissolved oxygen (DO) content in water, destroying the balance of aquatic ecosystems and causing eutrophication of water bodies.

[0003] The use of microbial methods to treat water pollution has the advantages of low investment, good results, low operating costs, clean and environmentally friendly. At present, a variety of microbial strains with the ability to simultaneously remove carbon and nitrogen pollutants in wastewater have been reported, which can be used to treat carbon and nitrogen pollutants in wastewater. For example, the "Isolation, Identification and Denitrification Performance Study of Two Nitrifying Bacteria" reported that the isolated plant nitrogen-fixing bacteria and Enterobacter can use organic carbon as a carbon source, and the removal rate of ammonia nitrogen and nitrite nitrogen is more than 85%. However, different types of microorganisms have different adaptability in different water environments. Different types of microorganisms need to be used to adapt to different water environments. The existing strain types still need to be expanded. For example, if the flow rate is slow and the dissolved oxygen in the water is insufficient, microbial agents adapted to the environment are needed. Therefore, it is necessary to continuously supplement different types of microorganisms, especially microorganisms suitable for anoxic environments, to meet the treatment needs of different water environments. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Bacillus brevis and a method for removing carbon and nitrogen from wastewater under anaerobic conditions, aiming to solve the technical problem that the prior art microbial strains with denitrification ability cannot adapt to different water quality environments, and thus cannot meet the denitrification and carbon removal engineering treatment requirements of actual water bodies, especially anoxic water bodies.

[0005] The first technical solution provided by the present invention is a strain of Brevibacterium pepigmentatum JLG5-316-4, which was deposited in the China Center for Type Culture Collection on November 29, 2024, with the deposit number: CCTCCNO: M 20242670.

[0006] The second technical solution provided by the present invention is a microbial preparation containing Bacillus brevis JLG5-316-4 described in the first technical solution.

[0007] In some embodiments, the content of Brevibacterium atrophaeum JLG5-316-4 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0008] The third technical solution provided by the present invention is a composition containing Brevibacterium atrophaeum JLG5-316-4 described in the first technical solution or the microbial preparation described in the second technical solution.

[0009] In some embodiments, the content of Brevibacterium atrophaeum JLG5-316-4 in the composition is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The fourth technical solution provided by the present invention is a wastewater treatment agent, which contains Brevibacterium atrophaeum JLG5-316-4 described in the first technical solution, the microbial preparation described in the second technical solution, or the composition described in the third technical solution.

[0011] In some embodiments, in the wastewater treatment agent, the content of Brevibacterium atrophaeum JLG5-316-4 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] The fifth technical solution provided by the present invention is the application of Brevibacterium atrophaeum JLG5-316-4 described in the first technical solution, the microbial preparation described in the second technical solution, the composition described in the third technical solution, or the wastewater treatment agent described in the fourth technical solution in improving the quality of wastewater.

[0013] In some embodiments, the wastewater is a water body with slow flow rate and dissolved oxygen concentration lower than 1 mg / L.

[0014] In some embodiments, the improvement of the wastewater quality includes at least one of the following effects:

[0015] (1) Reducing the chemical oxygen demand (COD) of the wastewater;

[0016] (2) Reducing the content of ammonia nitrogen (NH4 + -N) in the wastewater;

[0017] (3) Reducing the total nitrogen (TN) of the wastewater.

[0018] The sixth technical solution provided by the present invention is a method for treating wastewater, which is to introduce the Brevibacterium pigmentatum JLG5-316-4 described in the first technical solution, the microbial preparation described in the second technical solution, the composition described in the third technical solution, or the wastewater treatment agent described in the fourth technical solution into the wastewater for denitrification and carbon removal reactions.

[0019] In some embodiments, the reaction temperature is 28-32 °C, the reaction pH is 5-7.5, the reaction light condition is 1000-5000 lux, and the reaction time is not less than 24 h.

[0020] In some embodiments, the wastewater is a water body with slow flow rate and dissolved oxygen concentration lower than 1 mg / L.

[0021] In some embodiments, the carbon-nitrogen ratio of the wastewater is 7:1 to 10:1.

[0022] The technical effects of the present invention are as follows:

[0023] A strain of Brevibacterium pigmentatum JLG5-316-4 was isolated from a water sample of a certain lake in Changzhou. The Brevibacterium pigmentatum has the ability to efficiently degrade nitrogen and carbon in wastewater under anaerobic conditions. The Pantoea agglomerans provided by the present invention has a high denitrification ability in environments such as pH 7.5, temperature 30 °C, and light condition 5000 lux. It can be seen that the Brevibacterium pigmentatum provided by the present invention can improve water quality in anoxic environments.

[0024] Biological material preservation

[0025] Brevibacterium pigmentatum JLG5-316-4, classified and named Brevibacterium pigmentatum, was deposited at the China Center for Type Culture Collection (CCTCC) on November 29, 2024. The deposit address: Wuhan University, Wuhan, China; Deposit number: CCTCC NO: M 20242670. Description of the drawings

[0026] Figure 1 It is a growth plate diagram of Brevibacterium pigmentatum.

[0027] Figure 2 It is an evolutionary tree diagram of Brevibacterium pigmentatum. Specific embodiments

[0028] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] The culture media involved in the following examples are as follows:

[0030] Liquid medium (L -1 ): 3 g of sodium acetate, 1 g of ammonium chloride, 0.2 g of magnesium sulfate heptahydrate, 1.75 g of potassium dihydrogen phosphate, 2 g of sodium bicarbonate, 2 g of sodium chloride, 1 g of yeast powder.

[0031] Solid medium (L -1 ): 3 g of sodium acetate, 1 g of ammonium chloride, 0.2 g of magnesium sulfate heptahydrate, 1.75 g of potassium dihydrogen phosphate, 2 g of sodium bicarbonate, 2 g of sodium chloride, 1 g of yeast powder, 15 g of agar.

[0032] The measurement methods involved in the following examples are as follows:

[0033] 1. Detection of COD: Determined according to the method of HJ / T 399 - 2007.

[0034] 2. Detection of ammonia nitrogen: Determined according to the method of HJ 535 - 2009.

[0035] 3. Detection of nitrite nitrogen concentration: Determined according to the method of GB / T 7493 - 87.

[0036] 4. Detection of nitrate nitrogen concentration: Determined according to the method of HJ / T 346 - 2007.

[0037] 5. Degradation efficiency = (blank concentration - concentration after biological treatment) / blank concentration.

[0038] Example 1

[0039] 1. Screening of strains

[0040] (1) Take 10 mL of water sample from a certain lake in Changzhou and put it into 90 mL of liquid medium, and culture it in an anaerobic environment at 3000 lux of light and 30 °C for 240 h to obtain a preliminarily activated and enriched bacterial solution;

[0041] (2) Take the preliminarily activated bacterial solution in step (1) and inoculate it into a new 100 mL liquid medium at an inoculation amount of 10% by volume, and culture it in an anaerobic environment at 3000 lux of light and 30 °C for 240 h to obtain a re - activated and enriched bacterial solution; Repeat 2 - 3 times;

[0042] (3) Take 10 ml of the bacterial solution cultured in step (2), add glass beads and shake, then dilute it with sterile water to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 times, respectively absorb 200 μL of the bacterial solution and coat it on the solid medium, and culture it at 30 °C for 168 h;

[0043] (4) Pick a small amount of the single colony with better growth in step (3) and transfer it into sterile water. Take 200 μL of the bacterial solution and evenly spread it on the solid medium;

[0044] (5) Repeat step (4) multiple times until a single strain is obtained in each solid medium, as Figure 1 shown;

[0045] (6) Pick the single strain from the medium with the single strain grown and inoculate it into a new 100 mL liquid medium, and culture it in an anaerobic environment at 3000 lux of light and 30 °C for 240 h. Measure the OD600 value of the bacterial solution to reach stability (≈0.45). At this time, the bacterial solution concentration is 4×10 7 CFU / ml, and the required bacterial solution is obtained.

[0046] 2. Strain identification:

[0047] The strain was identified as Brevibacterium pigmentatum by Sangon Biotech (Shanghai) Co., Ltd. through 16S rRNA. The nucleotide sequence of 16S rRNA is shown in SEQ ID NO.1. The phylogenetic tree diagram is as Figure 2 shown.

[0048] Example 2: OD600 value and denitrification efficiency of the strain at different times

[0049] (1) Inoculate Brevibacterium pigmentatum JLG5-316-4 obtained in Example 1 into a 100 mL liquid medium with an initial ammonia nitrogen concentration of 140 mg / L and a COD concentration of 1120 mg / L at an inoculation amount of 1% by volume (4×10 6 CFU / ml), and culture it under anaerobic conditions at 3000 lux of light, 30 °C, and pH 7.5;

[0050] (2) Measure OD600 at 0, 24, 48, 96, 144, 192, and 240 h. During the implementation process, measure the concentrations of COD, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen at intervals of 48 h, and calculate the degradation efficiency.

[0051] As can be seen from Table 1, after 192 h, the OD value of the bacterial solution was basically stable above 0.4.

[0052] Table 1 OD of the bacteria at different culture times 600 value

[0053]

[0054] As can be seen from Table 2, when the bacterial liquid was cultured for 192 h, the removal rates of COD, ammonia nitrogen and TN could reach more than 30%.

[0055] Table 2 Ammonia nitrogen removal rate of bacteria at different culture times

[0056]

[0057] Note: TN is the sum of the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen.

[0058] Example 3: Denitrification and carbon removal performance of strains under different light conditions

[0059] Detect the denitrification and carbon removal performance of strains under different light conditions.

[0060] Inoculate the Brevibacterium pumilus JLG5-316-4 obtained in Example 1 into 100 mL of liquid medium at an inoculation amount of 1% by volume. The light conditions are set to 0, 230, 1000, 3000, and 5000 lux respectively, and culture for 240 h under anaerobic conditions at 30 °C and pH 7.5. Measure the concentrations of COD, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, as well as OD600, and calculate the degradation efficiency of the strains for COD, ammonia nitrogen and TN under different light conditions;

[0061] As shown in Table 3, Brevibacterium pumilus JLG5-316-4 has the highest COD degradation efficiency when the light condition is 5000 lux, reaching more than 45%; and this bacterium has a relatively high denitrification efficiency when the light is 1000 - 5000 lux, and the removal rates of ammonia nitrogen and TN can reach more than 30%.

[0062] Table 3 Degradation efficiency of Brevibacterium pumilus JLG5-316-4 for ammonia nitrogen and COD under different light conditions

[0063]

[0064] Example 4: Denitrification and carbon removal performance of strains under different carbon-nitrogen ratios

[0065] Detect the denitrification and carbon removal performance of strains under different carbon-nitrogen ratio conditions.

[0066] Inoculate the Brevibacterium pumilus JLG5-316-4 obtained in Example 1 into liquid media (100 mL) with different carbon-nitrogen ratios of 4:1, 7:1, 10:1, 15:1, and 20:1 at an inoculation amount of 1% by volume. After culturing for 240 h under the conditions of a temperature of 30 °C, a light of 5000 lux, and a pH of 7.5, measure the concentrations of COD, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, as well as OD600, and calculate the degradation efficiency of the strains for COD, ammonia nitrogen and TN under different carbon-nitrogen ratios.

[0067] As shown in Table 4, Brevibacterium epidermidis JLG5-316-4 had high removal efficiencies for COD, ammonia nitrogen, and TN under the condition of a carbon-nitrogen ratio of 7:1 to 10:1.

[0068] Table 4 Degradation efficiencies of Brevibacterium epidermidis JLG5-316-4 for ammonia nitrogen and COD at different carbon-nitrogen ratios

[0069]

[0070] Example 5: Denitrification and carbon removal performance of the strain at different pH values

[0071] Detect the denitrification and carbon removal performance of the strain under different pH conditions.

[0072] Inoculate Brevibacterium epidermidis JLG5-316-4 obtained in Example 1 into liquid medium (100 mL) with pH values of 3, 5, 7, 9, and 11 at an inoculation amount of 1% by volume. After culturing for 240 h at a temperature of 30 °C, a light intensity of 5000 lux, and a C / N ratio of 10, measure the concentrations of COD, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen, as well as OD600, and calculate the degradation efficiencies of the strain for COD, ammonia nitrogen, and TN under different pH conditions.

[0073] As shown in Table 5, Brevibacterium epidermidis JLG5-316-4 had high removal efficiencies for COD, ammonia nitrogen, and TN under the condition of pH from 5 to 7.5.

[0074] Table 5 Degradation efficiencies of Brevibacterium epidermidis JLG5-316-4 for ammonia nitrogen and COD at different pH values

[0075]

[0076] 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 Brevibacterium pigmentatum JLG5-316-4, characterized in that, It was deposited with the China Center for Type Culture Collection on November 29, 2024, and the deposit number is: CCTCC NO: M 20242670.

2. A microbial preparation containing the Brevibacterium rubrum JLG5-316-4 described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The content of Brevibacterium epidermidis JLG5-316-4 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A composition containing the Brevibacterium rubrum JLG5-316-4 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. A wastewater treatment agent, characterized in that, The wastewater treatment agent contains the Brevibacterium rubrum JLG5-316-4 described in claim 1, the microbial preparation described in claim 2 or 3, or the composition described in claim 4.

6. The application of the Brevibacterium rubrum JLG5-316-4 described in claim 1, the microbial preparation described in claim 2 or 3, the composition described in claim 4, or the wastewater treatment agent described in claim 5 in improving the quality of wastewater.

7. The application according to claim 6, characterized in that, The wastewater is a water body with a dissolved oxygen concentration of less than 1 mg / L.

8. The application according to claim 6, characterized in that The improvement of the wastewater quality includes at least one of the following effects: (1) Reducing the chemical oxygen demand (COD) of the wastewater; (2) Reduce the ammonia nitrogen content (NH4 + -N) in the wastewater; (3) Reducing the total nitrogen (TN) of the wastewater.

9. A method for wastewater treatment, characterized in that, The method is to introduce the Brevibacterium rubrum JLG5-316-4 described in claim 1, the microbial preparation described in claim 2 or 3, the composition described in claim 4, or the wastewater treatment agent described in claim 5 into the wastewater for denitrification and carbon removal reactions.

10. The method according to claim 9, wherein The temperature of the reaction is 28 - 32 °C, the pH of the reaction is 5 - 7.5, the light condition of the reaction is 1000 - 5000 lux, and the reaction time is not less than 24 h; The wastewater is a water body with a dissolved oxygen concentration of less than 1 mg / L, and the carbon-nitrogen ratio of the wastewater is 7:1 - 10:1.

Images