A thermostable denitrifying bacterium and its application in wastewater treatment

Abstract

This invention relates to the field of microbial wastewater treatment, specifically to a denitrifying bacterium and its application in wastewater treatment. Its classification name is Lysobacter sp. HBCW-DN-HT02, and its accession number is CCTCC NO: M20241308. This strain can grow rapidly under aerobic conditions using a variety of carbon and nitrogen sources. This bacterial agent is suitable for anaerobic denitrification of industrial wastewater in summer / high-temperature conditions.

Description

Technical Field

[0001] This invention relates to the field of microbial wastewater treatment, specifically to a heat-resistant denitrifying bacterium and its application in wastewater treatment. Background Technology

[0002] When nitrogen (such as ammonium salts, nitrates, and nitrites) from wastewater is discharged into water bodies, it promotes the excessive growth of algae and other aquatic plants, leading to eutrophication, algal blooms, and impacting the survival of aquatic organisms and disrupting the ecological balance. Meanwhile, my country has strict legal limits on the nitrogen content of wastewater discharge to protect the environment and public health.

[0003] Wastewater treatment plants typically employ biological nitrogen removal processes to treat nitrogen in wastewater, including aerobic nitrification and anaerobic denitrification. In aerobic nitrification, ammonia nitrogen in the wastewater is converted into nitrite and nitrate nitrogen by autotrophic bacteria under aerobic conditions. In anaerobic denitrification, denitrifying bacteria convert nitrate and nitrite nitrogen into nitrogen gas under anaerobic conditions, thus removing total nitrogen. Both nitrification and denitrification processes require suitable temperatures for microbial growth, generally 25-35℃. However, due to the influence of equipment operating temperatures, many industrial wastewater temperatures in summer often exceed 37℃, and reaction tank temperatures also exceed 35℃, sometimes even reaching 45℃. Under high-temperature conditions, the activity of microorganisms in activated sludge is greatly reduced or even killed, ultimately leading to substandard total nitrogen levels in the effluent. Therefore, companies often install cooling or temperature control equipment upstream of the biological treatment process to cool the wastewater before biological nitrogen removal. However, this would increase the company's operating costs. If high-performance, heat-resistant denitrifying bacteria could be used directly for denitrification, the stability of the biological treatment tank would be improved, and the company's wastewater treatment costs would be greatly reduced. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a thermoresistant denitrifying bacterium, Lysobacter sp., screened from landfill leachate, and its application in nitrogen-containing wastewater treatment.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A thermotolerant denitrifying bacterium, named HBCW-DN-HT02, belongs to the genus Lysobacter. This strain was deposited on June 19, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with the taxonomic name Lysobacter sp. HBCW-DN-HT02 and accession number CCTCC NO: M20241308.

[0007] The HBCW-DN-HT02 mentioned above can grow rapidly under aerobic conditions, thus enabling the large-scale production of microbial agents. Furthermore, this microbial agent is suitable for anoxic denitrification of various wastewaters, including industrial wastewater, under high-temperature conditions.

[0008] Compared with the prior art, the present invention has the following advantages and beneficial effects: There are currently no reported strains of Bacillus lysinensis that can withstand high temperatures of 45°C, while the Bacillus lysinensis HBCW-DN-HT02 involved in the present invention has a denitrification efficiency of over 30% in wastewater at 45°C for 24 hours, and removes a total nitrogen of 102 mg / L. Attached Figure Description

[0009] Figure 1 This is a scanning electron microscope image of the bacterial cell morphology of Bacillus lysinus HBCW-DN-HT02.

[0010] Figure 2 This describes the growth of Bacillus lysinensis HBCW-DN-HT02 in aerobic growth media with different nitrogen sources.

[0011] Figure 3 This describes the growth of Bacillus lysinensis HBCW-DN-HT02 in aerobic growth media with different carbon sources.

[0012] Figure 4 This describes the nitrate removal performance of Bacillus lysinus HBCW-DN-HT02 in anoxic conditions within a culture medium.

[0013] Figure 5 This describes the total nitrogen removal performance of Bacillus lysinus HBCW-DN-HT02 under anoxic conditions in automotive parts processing wastewater.

[0014] Figure 6 This describes the total nitrogen removal performance of Bacillus lysinus HBCW-DN-HT02 under anoxic conditions in food processing wastewater.

[0015] Figure 7 This describes the total nitrogen removal performance of Bacillus lysinus HBCW-DN-HT02 under anoxic conditions in industrial park wastewater. Detailed Implementation

[0016] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0017] The culture media used in the following examples were prepared as follows, and all culture media were sterilized by high temperature and autoclave (121°C, 20 min) before use:

[0018] Aerobic growth medium: FeSO4·7H2O 0.4g / L, MgSO4·7H2O 0.05g / L, K2HPO4 1g / L, NaHCO3 1.5g / L, CaCl2·2H2O 0.5g / L, NaCl 2g / L, nitrogen source (containing 200mg / L total nitrogen), carbon source (containing 1600mg / L COD), and trace element solution 1mL / L. The solid medium is the aerobic growth medium supplemented with 20g / L agar powder.

[0019] Anaerobic denitrification medium: FeSO4·7H2O 0.4g / L, MgSO4·7H2O 0.05g / L, K2HPO4 1 g / L, NaHCO3 1.5g / L, CaCl2·2H2O 0.5g / L, NaCl 2g / L, KNO3 1.4 g / L, sodium acetate 1.28g / L, trace element solution 1mL / L.

[0020] Trace element solution: EDTA 0.5 g / L, CuSO4·5H2O 0.075 g / L, ZnSO4·7H2O 0.3 g / L, CoCl2·6H2O 0.375 g / L, MnCl2·2H2O 0.3 g / L, H3BO4 0.014 g / L, NaMoO4·2H2O 0.22 g / L.

[0021] All water quality indicators tested were performed using national standard methods. Specifically, ammonia nitrogen was tested using Nessler's reagent spectrophotometric method (HJ 535-2009); nitrite nitrogen was tested using the spectrophotometric method (GB 7493-1987); nitrate nitrogen was tested using the ultraviolet spectrophotometric method (HZ / T346-2007); and total nitrogen was tested using the alkaline potassium persulfate digestion ultraviolet spectrophotometric method (HJ 636-2012).

[0022] Example 1: Screening and Identification of Microbial Strains

[0023] 10 mL of activated sludge from a landfill leachate biochemical treatment system in Shenzhen was placed in 100 mL of aerobic growth medium (nitrogen source: potassium nitrate 1.45 g / L, carbon source: sodium acetate 2.05 g / L). After continuous enrichment culture at 45°C for 3–5 days, three rounds of streak plating were performed. Single colonies with significant morphological differences were selected and placed in 5 mL of aerobic growth medium. After incubation at 45°C with shaking at 120 rpm for 2–3 days, the 16S rDNA gene was amplified by PCR, and molecular biological identification was performed by sequencing the 16S rDNA fragment. The primers used for PCR amplification were as follows:

[0024] Upstream primer 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO.1).

[0025] Downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO.2).

[0026] Sequencing of the PCR products was performed by Shanghai Sangon Biotech Co., Ltd., and the sequencing results are shown in SEQ ID NO.3. 16S rDNA sequence alignment revealed that the isolated strain belonged to the genus *Lysobacter*, and it was named HBCW-DN-HT02. This strain was deposited on June 19, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with the taxonomic name *Lysobacter sp.*HBCW-DN-HT02 and accession number CCTCC NO: M20241308.

[0027] Figure 1 The scanning electron microscope (SEM) sample preparation method was as follows: Single-colony bacterial cultures were centrifuged and fixed with 2.5% glutaraldehyde. Then, they were dehydrated using gradient solutions of 30%, 50%, 70%, 80%, and 90% ethanol-water solutions, respectively. Finally, the bacterial cells were lyophilized in tert-butanol. A small amount of the bacterial cells was sputter-coated with gold and observed under an electron microscope. Figure 1 The bacterial cells shown are rod-shaped and measure (0.7–2.0) μm × 0.3 μm.

[0028] Example 2: Growth of denitrifying bacteria in culture media with different carbon and nitrogen sources

[0029] Preparation of inoculum: Inoculate 1 mL of bacterial culture into an aerobic growth medium (nitrogen source: potassium nitrate 1.45 g / L, carbon source: sodium acetate 2.05 g / L) and incubate at 35℃ and 120 r / min for 2–3 days to obtain the inoculum.

[0030] Figure 2 The implementation method is as follows: Aerobic growth media (carbon source: sodium acetate 2.05 g / L) were prepared using ammonium chloride (0.77 g / L), sodium nitrite (0.99 g / L), and potassium nitrate (1.45 g / L) as nitrogen sources (each containing 200 mg / L of nitrogen). Bacterial suspensions were inoculated into the aerobic growth media at a 1 vol% inoculation ratio and cultured at 35℃ and 120 r / min. OD values ​​were measured every 24 hours. 600 .

[0031] Figure 3The implementation method is as follows: Prepare aerobic growth media (nitrogen source: potassium nitrate 1.45 g / L) using glucose, sodium acetate, and sodium citrate as carbon sources (COD concentration: 1600 mg / L each); inoculate bacterial suspensions into the aerobic growth media at a 1 vol% inoculation ratio, and culture at 35℃ and 120 r / min. Take samples every 24 hours to measure OD. 600 .

[0032] according to Figure 2 , Figure 3 The lysozyme HBCW-DN-HT02 can grow using different types of nitrogen sources (ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen) and different types of carbon sources (glucose, sodium acetate, and sodium citrate). When ammonia nitrogen is used as the sole nitrogen source and sodium acetate is used as the sole carbon source, the OD600 reaches 0.324 after 48 hours of culture; when nitrate nitrogen is used as the sole nitrogen source and sodium citrate is used as the sole carbon source, the OD600 reaches 1.049 after 84 hours of culture.

[0033] Example 3: Denitrification performance test of denitrifying bacteria at high temperature

[0034] The preparation of the inoculum solution is the same as in Example 2.

[0035] Figure 4 The implementation method is as follows: Inoculum solution is added to 100 mL of anaerobic denitrification medium at a ratio of 1 vol%. The culture conditions are anoxic, 45 °C, and 120 r / min. NO2 is sampled and detected every 24 h. - -N, NO3 - -N, TN.

[0036] according to Figure 4 Under hypoxic conditions at 45°C, total nitrogen decreased by 96 mg / L (43%) after 24 h of inoculation with Bacillus lysinus HBCW-DN-HT02, and by 195 mg / L (87%) after 48 h, indicating that Bacillus lysinus HBCW-DN-HT02 can utilize sodium acetate as a carbon source to remove total nitrogen through denitrification at 45°C.

[0037] Example 4: Denitrification effect of denitrifying bacteria in different wastewaters

[0038] The preparation method of the inoculum is the same as in Example 2.

[0039] Figure 5 - Figure 7The implementation method was as follows: Wastewater from different industries was collected, and potassium nitrate was used to supplement approximately 200 mg / L of nitrate nitrogen, while sodium acetate was used to supplement 1000 mg / L of carbon source. The wastewater was then subjected to high-temperature and high-pressure sterilization (121℃, 20 min). The blank control group consisted of sterilized wastewater without added bacterial solution. The experimental group was inoculated with 1 vol% Bacillus lysinensis HBCW-DN-HT02 inoculum and cultured at 45℃, 120 r / min, under anaerobic conditions. Samples were taken to detect the total nitrogen concentration.

[0040] according to Figure 5 - Figure 7 After culturing with Bacillus lysinensis HBCW-DN-HT02 for 20 hours, the total nitrogen in wastewater from automotive parts processing decreased by 89 mg / L (31%), in food processing wastewater by 53 mg / L (31%), and in industrial park wastewater by 102 mg / L (30%). These results indicate that at a high temperature of 45℃, Bacillus lysinensis HBCW-DN-HT02 can effectively remove total nitrogen through denitrification in wastewater from automotive parts processing, food processing, and industrial park wastewater, resulting in a decrease of approximately 30% in total nitrogen after 20 hours of cultivation.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A thermotolerant denitrifying bacterium is classified and named Bacillus lysinus (Bacillus). Lysobacter sp. HBCW-DN-HT02, accession number CCTCC NO: M20241308.

2. The heat-resistant denitrifying bacterium lysozyme as described in claim 1 ( Lysobacter sp. The application of HBCW-DN-HT02 in wastewater treatment, wherein the wastewater contains nitrate nitrogen.

3. The application according to claim 2, characterized in that, The wastewater is wastewater with a temperature of 45℃.

4. The application according to claim 3, characterized in that, The wastewater in question is either wastewater from automotive parts processing or food processing.

Images