High-adaptability low-carbon-nitrogen-ratio denitrifying stenotrophomonas and application thereof

Abstract

The invention discloses denitrifying stenotrophomonas with high adaptability and low carbon nitrogen ratio and application thereof, and relates to the technical field of environmental microorganisms, and the technical scheme of the denitrifying stenotrophomonas with high adaptability and low carbon nitrogen ratio is characterized in that the denitrifying bacterium with high adaptability and low carbon nitrogen ratio is stenotrophomonas R2 and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is October 10, 2024, and the preservation number is CCTCC NO: M 20242155. The invention provides a microbial strain, namely stenotrophomonas maltophilia R2, which is obtained by carrying out actual industrial sewage enrichment and domestication on activated sludge and finally separating and purifying the activated sludge, and has the advantages of low carbon nitrogen ratio and high adaptability, and a preparation method of the stenotrophomonas maltophilia R2 and a preparation method of the stenotrophomonas maltophilia R2 and a preparation method of the stenotrophomonas maltophilia R2. The strain can still keep an extremely high denitrification effect under the stress of toxic pollutants such as different dyes (50mg / L), heavy metals (50mg / L) and aniline (1g / L), can tolerate heavy metals including MnSO4, CoCl2, FeSO4, ZnSO4 and NiSO4, and can tolerate dyes including acid orange, acid red, direct blue 1, golden orange G and Congo red.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms, and more particularly to a strain of denitrifying oligotrophic bacteria with high adaptability and low carbon-nitrogen ratio and application thereof. Background Art

[0002] In recent years, improvements in human production and lifestyles have led to the overuse of nitrogen-containing compounds and the direct discharge of wastewater. Cases of nitrogen pollution are common worldwide, including red tides in the oceans, eutrophication of lakes and rivers, and the deterioration of drinking water sources. Nitrogen pollution has become a threat to aquatic life and human health. Therefore, efforts are needed to control and reduce nitrogen levels in water bodies. Biological denitrification methods, which offer advantages such as high efficiency, cost-effectiveness, and environmental protection, are often preferred. Biological denitrification generally involves nitrification driven by autotrophic microorganisms and denitrification driven by heterotrophic microorganisms. Most denitrification processes occur under anaerobic or anoxic conditions.

[0003] At present, many microbial strains with denitrification function have been screened out, such as Pseudomonas ( Pseudomonas sp.), Bacillus ( Bacillus sp.), Paracoccus denitrificans ( Paracoccus denitrificans ), Alcaligenes ( Alkaligenes sp.) etc. Stenotrophomonas sp.) is a common environmental microorganism. To date, research on this genus of microorganisms in environmental remediation has mainly focused on the degradation of pollutants such as saponins, chlorinated pesticides, pyridine, toluene, and cyanide, and the treatment of heavy metals such as hexavalent chromium reduction, low-valent sulfur oxidation, and zinc adsorption. There are only a few reports on denitrification research on this genus of microorganisms, such as patent CN109401997, which discloses a strain of Oligotrophomonas, its application, and microbial agent. This strain ( Stenotrophomonas sp. DB-2) can remove about 99.8% of nitrate (initial concentration 185 mg / L) within 6 days under anaerobic conditions with sodium acetate as the carbon source and a carbon-nitrogen ratio (COD / TN) of 11; Patent CN103074277 discloses a denitrifying bacterium and its application, the maltophilia ( Stenotrophomonas maltophilia ) CM-NRD3 can remove more than 95% of nitrate (initial concentration 140 mg / L) within 36-48 hours under anaerobic conditions with sodium acetate as the carbon source and a carbon-nitrogen ratio (COD / TN) of 11. This shows that current research on denitrification by Oligotrophomonas only focuses on ideal simulated nitrate or nitrite culture media, and the carbon-nitrogen ratio required for denitrification is relatively high. It is worth considering whether such microorganisms can still maintain excellent denitrification ability when faced with low carbon-nitrogen ratio water bodies with different toxic pollutants.

[0004] Therefore, a highly adaptable low carbon-nitrogen ratio denitrifying oligotrophic bacteria and its application are proposed to solve or alleviate the above problems. Summary of the Invention

[0005] The present invention aims to provide a microbial strain, Stenotrophomonas maltophilia R2, derived from activated sludge enriched with actual industrial wastewater, followed by final separation and purification, exhibiting a low carbon-to-nitrogen ratio and high adaptability, and its use in wastewater denitrification. Compared to currently discovered denitrifying strains, the Stenotrophomonas maltophilia R2 strain in the present invention exhibits a low carbon-to-nitrogen ratio for denitrification, strong adaptability, and high denitrification efficiency. Furthermore, the strain demonstrates excellent denitrification effects on actual textile and dyeing wastewater and mixed industrial wastewater, demonstrating broad application prospects in the field of microbial denitrification.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: 1. A highly adaptable low carbon-nitrogen ratio denitrifying bacterium, which is Oligotrophomonas ( Stenotrophomonas maltophilia )R2, deposited in China Center for Type Culture Collection (CCTCC), the deposit date is October 10, 2024, and the deposit number is CCTCC NO: M 20242155.

[0007] The 16SrDNA gene sequence of the Stenotrophomonas sp. of the present invention is shown in SEQ ID NO.1.

[0008] The colonies of the oligotrophomonas strain R2 on the LB plate are yellow and opaque, with a moist and smooth surface and regular edges.

[0009] 2. The present invention provides the application of highly adaptable low carbon-nitrogen ratio denitrifying bacteria for efficient nitrogen removal in different sewage.

[0010] Furthermore, the application comprises the following steps: Stenotrophomonas R2 was inoculated into LB liquid medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h. After the culture was completed, the bacterial liquid was centrifuged at 8000 × g for 5 min, the lower layer of bacteria was removed, and the cells were diluted with sterile water to an OD of 600 =1.0 as the seed solution of Stenotrophomonas R2; The seed liquid was added to the nitrate nitrogen and nitrite nitrogen denitrification simulated denitrification culture medium at a ratio of 5%, the temperature was adjusted to 30°C and the pH was 7 for static culture, the carbon-nitrogen ratio (COD / TN) of the culture system was set between 4:1-12:1 (preferably 8:1), the nitrate nitrogen concentration was between 140mg / L-980mg / L (preferably 140mg / L), and the nitrite nitrogen concentration was between 100mg / L-500mg / L (preferably 100mg / L), and its continuous denitrification ability was explored by regular feeding.

[0011] Furthermore, the R2 seed liquid was added to simulated toxic wastewater (heavy metal, dye and aniline wastewater), textile printing and dyeing wastewater and mixed industrial wastewater in a certain proportion and statically cultivated to explore its adaptability and actual enhanced denitrification capacity.

[0012] Furthermore, the LB liquid culture medium is composed of: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and the solvent is water.

[0013] Furthermore, the denitrification simulation culture medium is composed of: 1g / L NaH2PO4, 1g / L Na2HPO4, 0.05g / LMgSO4, appropriate amounts of trisodium citrate, potassium nitrate and sodium nitrite, and the solvent is water.

[0014] The present invention provides a microbial strain, Stenotrophomonas maltophilia R2, which is enriched and domesticated from activated sludge through actual industrial wastewater and finally separated and purified, and has a low carbon-nitrogen ratio and high adaptability. The strain has a removal rate of nitrate nitrogen and nitrite nitrogen higher than 98% under a carbon-nitrogen ratio of 4:1; it can tolerate 140-700 mg / L nitrate nitrogen and 100-300 mg / L nitrogen under a carbon-nitrogen ratio of 8:1. mg / L of nitrite-nitrogen, with nitrate-nitrogen removal rates exceeding 97%, and a maximum nitrate-nitrogen denitrification rate of 28.4 mg / L / h. Nitrite-nitrogen removal rates exceeding 98.9%, with a maximum nitrite-nitrogen denitrification rate of 12.5 mg / L / h. Under continuous feeding conditions, Oligotrophomonas R2 achieved nitrate-nitrogen removal rates exceeding 90%, with a maximum denitrification cycle of five times, and nitrite-nitrogen removal rates exceeding 98%, with at least eight denitrification cycles, demonstrating excellent denitrification stability. Furthermore, the strain maintained high denitrification efficiency despite exposure to toxic pollutants such as dyes (50 mg / L), heavy metals (50 mg / L), and aniline (1 g / L). It was tolerant to heavy metals including MnSO4, CoCl2, FeSO4, ZnSO4, and NiSO4, and dyes including Acid Orange, Acid Red, Direct Blue 1, Golden Orange G, and Congo Red. In addition, Oligotrophomonas R2 has a certain enhancing effect in the biochemical denitrification treatment of actual textile printing and dyeing wastewater and mixed industrial wastewater.

[0015] Biomaterial Deposit The present invention provides oligotrophic monocytogenes ( Stenotrophomonas maltophilia )R2, deposited in China Center for Type Culture Collection (CCTCC), the deposit date is October 10, 2024, and the deposit number is CCTCC NO: M20242155. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) R2 strain morphology; Figure 2 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) R2 phylogenetic tree; Figure 3 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Removal of nitrate nitrogen by R2 at different carbon-nitrogen ratios; Figure 4 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Removal of nitrite nitrogen by R2 at different carbon-nitrogen ratios; Figure 5 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Removal of nitrate nitrogen at different concentrations by R2; Figure 6 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Removal of nitrite nitrogen at different concentrations by R2; Figure 7 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Continuous removal effect of R2 on nitrate nitrogen; Figure 8 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Continuous removal effect of R2 on nitrite nitrogen; Figure 9 :Stereotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Denitrification effect of R2 under high concentration aniline stress. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0018] 1. A highly adaptable low carbon-nitrogen ratio denitrifying bacterium, which is Oligotrophomonas ( Stenotrophomonas maltophilia)R2, deposited in China Center for Type Culture Collection (CCTCC), the deposit date is October 10, 2024, and the deposit number is CCTCC NO: M 20242155.

[0019] The 16SrDNA gene sequence of the Stenotrophomonas sp. of the present invention is shown in SEQ ID NO.1.

[0020] The colonies of the oligotrophomonas strain R2 on the LB plate are yellow and opaque, with a moist and smooth surface and regular edges.

[0021] 2. The present invention provides the application of highly adaptable low carbon-nitrogen ratio denitrifying bacteria for efficient nitrogen removal in different sewage.

[0022] Furthermore, the application comprises the following steps: Stenotrophomonas R2 was inoculated into LB liquid medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h. After the culture was completed, the bacterial liquid was centrifuged at 8000 × g for 5 min, the lower layer of bacteria was removed, and the cells were diluted with sterile water to an OD of 600 =1.0 as the seed solution of Stenotrophomonas R2; The seed liquid was added to the nitrate nitrogen and nitrite nitrogen denitrification simulated denitrification culture medium at a ratio of 5%, the temperature was adjusted to 30°C and the pH was 7 for static culture, the carbon-nitrogen ratio (COD / TN) of the culture system was set between 4:1-12:1 (preferably 8:1), the nitrate nitrogen concentration was between 140mg / L-980mg / L (preferably 140mg / L), and the nitrite nitrogen concentration was between 100mg / L-500mg / L (preferably 100mg / L), and its continuous denitrification ability was explored by regular feeding.

[0023] Furthermore, the R2 seed liquid was added to simulated toxic wastewater (heavy metal, dye and aniline wastewater), textile printing and dyeing wastewater and mixed industrial wastewater in a certain proportion and statically cultivated to explore its adaptability and actual enhanced denitrification capacity.

[0024] Furthermore, the LB liquid culture medium is composed of: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and the solvent is water.

[0025] Furthermore, the denitrification simulation culture medium is composed of: 1g / L NaH2PO4, 1g / L Na2HPO4, 0.05g / LMgSO4, appropriate amounts of trisodium citrate, potassium nitrate and sodium nitrite, and the solvent is water.

[0026] The culture medium involved in the present invention is as follows: LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, solvent is water; Acclimation medium 1: 0.5 g / L KNO3, 0.5 g / L NaNO2, 1.8 g / L trisodium citrate, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, solvent is mixed industrial wastewater; Acclimation medium 2: 1 g / L KNO3, 1 g / L NaNO2, 3.6 g / L trisodium citrate, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, solvent is mixed industrial wastewater; Acclimation medium 3: 1.5 g / L KNO3, 1.5 g / L NaNO2, 5.4 g / L trisodium citrate, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, solvent is mixed industrial wastewater; Selection medium: 0.5 g / L KNO3, 0.5 g / L NaNO2, 1.8 g / L trisodium citrate, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, 1 mL BTB (1% concentration), 20 g / L agar, solvent is water; Denitrification liquid medium: 1g / L NaH2PO4, 1g / L Na2HPO4, 0.05g / L MgSO4, appropriate amounts of trisodium citrate, potassium nitrate and sodium nitrite, the solvent is water; The above industrial wastewater was taken from the effluent of the industrial wastewater biochemical pool of a sewage treatment plant in Shaoxing. The COD was about 110 mg / L, TN (mainly NO3-N) was about 20 mg / L, and the color was yellowish.

[0027] The present invention relates to an experimental index detection method: 1. Detection of nitrate nitrogen (NO3-N): UV spectrophotometry; 2. Detection of nitrite nitrogen (NO2-N): N-(1-naphthyl)-ethylenediamine spectrophotometry; Nitrite nitrogen or nitrate nitrogen removal rate (%) = (C0-Ct) / C0 C0 is the initial nitrite nitrogen or nitrate nitrogen concentration, and Ct is the nitrite nitrogen or nitrate nitrogen concentration after treatment for t hours.

[0028] Example 1: Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Screening and identification of R2 The present invention uses actual mixed industrial wastewater as a culture solvent, gradually domesticates activated sludge by continuously increasing the concentrations of nitrate nitrogen and nitrite nitrogen in the culture system, and separates and purifies denitrifying bacteria with high adaptability and low carbon-nitrogen ratio from the activated sludge. The specific implementation method is as follows: Bacteria source collection Activated sludge was collected from sewage stations and wastewater treatment plants of different textile printing and dyeing enterprises in Shaoxing, China, frozen with ice and transported to a refrigerator for storage at 4°C until use.

[0029] Activated sludge enrichment and acclimation Take 1 mL of different activated sludges and place them in 100 mL of acclimation medium 1. Incubate at 30°C for acclimation. Take samples regularly every day and use 1% diphenylamine reagent to qualitatively monitor the removal of nitrate nitrogen and nitrite nitrogen. When the water sample does not turn blue after adding diphenylamine reagent, transfer the lower layer of acclimation sludge to acclimation medium 2. Maintain the same culture conditions. After the culture system indicators are significantly reduced, transfer to acclimation medium 3 for further acclimation.

[0030] Strain screening Take the mixture after the last enrichment and acclimation, centrifuge at 8000xg for 5 minutes, discard the supernatant and retain the bacteria, and dilute the bacteria to 10 with sterile water. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Five gradient bacterial suspensions were spread onto BTB selective medium solid plates and placed in a 30°C constant temperature incubator. The blue colonies on the plates were picked and streaked several times until a single strain was present on the plates. The strains screened were inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 24 hours. After the culture was complete, the lower layer of bacteria was centrifuged and diluted to OD 600 =1.0, and inoculated into acclimation medium 1 at a 5% inoculation rate. After constant temperature culture at 30℃ for 24h, nitrate nitrogen and nitrite nitrogen were qualitatively monitored. It was finally found that only the water sample of the strain R2 treatment group did not turn blue after adding diphenylamine reagent. Therefore, this strain was preserved and used for subsequent research.

[0031] Strain identification like Figure 1 As shown, the colonies of strain R2 on the LB plate were yellow, opaque, with a smooth, moist surface and regular edges. Meanwhile, the streaked LB plate of strain R3 was sent to Hangzhou Qingke Biological Company for 16S rDNA sequencing. Using 16S bacterial universal primers 27F and 1492R, the full-length sequence of strain R2 was found to be 1485 bp. The specific 16S rDNA nucleotide sequence is shown in SEQ ID NO. 1.

[0032] SEQ ID NO.1 R2 strain sequence: The 16S rDNA sequence of strain R2 (SEQ ID NO.1) was then submitted to the NCBI database for sequence comparison analysis. The results showed that strain R2 was Stenotrophomonas maltophilia The homology is the highest, so the strain R2 is determined to be Stenotrophomonas maltophilia. Stenotrophomonas maltophilia R2 Phylogenetic tree constructed based on 16S rDNA sequence homology Figure 2 As shown. The Stenotrophomonas is Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) R2, deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, Deposit date: October 10, 2024, Deposit number: CCTCCNO: M 20242155.

[0033] Example 2: Denitrification study of Stenotrophomonas maltophilia R2 at different C / N ratios The R2 strain was inoculated into LB liquid medium and cultured on a shaker at 30°C and 150 rpm for 24 h. After the culture was complete, the bacterial liquid was centrifuged at 8000 x g for 5 min, the lower layer of cells was removed, and the cells were diluted with sterile water to an OD of 600 =1.0 as the seed solution of Oligotrophomonas R2. 5% of the seed solution of Oligotrophomonas R2 was added to 100 mL of denitrification liquid medium modified with nitrate nitrogen (140 mg / L) and nitrite nitrogen (100 mg / L), respectively. The carbon-nitrogen ratio (COD / TN) was set to 4:1, 6:1, 8:1, 10:1, and 12:1 by adding appropriate amounts of trisodium citrate. After incubation at 30°C for 24 hours, samples were taken to test the nitrate and nitrite nitrogen contents. The results are shown in Figure 2. Figure 3 and 4 As shown, Oligotrophomonas R2 can almost completely remove nitrite-nitrogen at a COD / TN ratio of 4:1, with a nitrite removal rate exceeding 99%, and virtually no nitrification and nitrate accumulation occurs. Furthermore, Oligotrophomonas R2 achieves a nitrate-nitrogen removal rate of 98.1% within 24 hours at a COD / TN ratio of 4:1. However, due to strain adaptability and the fact that the denitrification pathway is longer than the denitrification process, 53 mg / L of nitrite-nitrogen accumulates. This nitrite-nitrogen concentration gradually decreases as denitrification progresses, remaining below 10 mg / L after 48 hours (data not shown). Therefore, Oligotrophomonas R2 can maintain efficient denitrification even under low COD / TN conditions.

[0034] Example 3: Study on the tolerance of Stenotrophomonas maltophilia R2 to nitrate and nitrite The same oligotrophic monosaccharide R2 seed solution as in Example 2 was taken and added at a dosage of 5% to 100 mL of denitrification liquid culture medium modified with different concentrations of nitrate nitrogen (140 mg / L, 420 mg / L, 700 mg / L, and 980 mg / L) and different concentrations of nitrite nitrogen (100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L). The carbon-nitrogen ratio (COD / TN) of the culture system was controlled to be 8:1 by adding an appropriate amount of trisodium citrate. After static culture at 30°C for 24 hours, samples were taken to detect the nitrate and nitrite nitrogen contents. The results are as follows: Figure 5 and 6 As shown, the removal rate of nitrate nitrogen by Oligotrophomonas R2 for 140-700 mg / L was higher than 97%, the highest nitrate nitrogen denitrification rate reached 28.4 mg / L / h, and the maximum residual nitrite nitrogen in the culture system after 24 hours was only 42 mg / L. When the nitrate nitrogen concentration was increased to 980 mg / L, the strain could also remove 57.4% of nitrate nitrogen in 24 hours. In addition, Oligotrophomonas R2 could basically completely remove nitrite nitrogen from 100-300 mg / L within 24 hours, with a nitrite nitrogen removal rate higher than 98.9% and a highest nitrite nitrogen denitrification rate of 12.5 mg / L / h. Moreover, the strain did not reverse nitrification and accumulate nitrate nitrogen. This shows that Oligotrophomonas R2 has great potential to tolerate high concentrations of nitrate nitrogen and nitrite nitrogen.

[0035] Example 4: Study on the Continuity of Denitrification by Stenotrophomonas maltophilia R2 Take the same oligotrophic monosaccharide R2 seed solution as in Example 2, add the seed solution to 100 mL of nitrate nitrogen (140 mg / L) and nitrite nitrogen (100 mg / L) denitrification liquid culture medium at a dosage of 5%, add appropriate amount of citric acid to control the carbon-nitrogen ratio (COD / TN) to 8:1, and culture at 30°C. Samples are taken every 24 hours to detect the nitrate nitrogen and nitrite nitrogen content, and additional nitrate nitrogen (140 mg / L), nitrite nitrogen (100 mg / L) and trisodium citrate (controlling the carbon-nitrogen ratio to 8:1) are added to the corresponding culture system to explore the continuous denitrification ability of the strain. The results are as follows Figure 7 and 8 As shown, after five consecutive cultivations in nitrate-rich water, Oligotrophomonas R2 maintained a nitrate-nitrogen removal rate of over 90%, and no excessive nitrite-nitrogen accumulation occurred during the first four cultivations (nitrite-nitrogen concentration <4 mg / L). Furthermore, Oligotrophomonas R2 exhibited improved denitrification stability during continuous cultivation in nitrite-rich water, achieving a nitrite-nitrogen removal rate exceeding 98% over eight consecutive cultivations, with the final nitrite-nitrogen content being only 3.1 mg / L. Therefore, Oligotrophomonas R2 maintains excellent genetic stability in wastewater denitrification treatment.

[0036] Example 5: Study on denitrification of Stenotrophomonas maltophilia R2 under different heavy metal stresses In order to explore the effect of heavy metal ions on denitrification of Oligotrophomonas R2, the same Oligotrophomonas R2 seed solution as in Example 2 was taken and added at a dosage of 5% to 100 mL of nitrate nitrogen (140 mg / L) denitrification liquid culture medium modified with manganese sulfate (50 mg / L), nickel sulfate (50 mg / L), zinc sulfate (50 mg / L), ferrous sulfate (50 mg / L) and cobalt chloride (50 mg / L), respectively. An appropriate amount of trisodium citrate was added to control the carbon-nitrogen ratio (COD / TN) to 8:1. After static culture at 30°C for 24 hours, samples were taken to detect the nitrate nitrogen and nitrite nitrogen contents. The results are shown in Table 1 below. Oligotrophomonas R2 maintained efficient denitrification under the stress of manganese sulfate (50 mg / L), nickel sulfate (50 mg / L), or ferrous sulfate (50 mg / L), with nitrate nitrogen removal rates of 99.8%, 97.8%, and 98.7% within 24 hours, respectively, and final nitrite nitrogen contents below 1 mg / L. Furthermore, strain R2 was still able to carry out denitrification under the stress of zinc sulfate (50 mg / L) and cobalt chloride (50 mg / L), with nitrate nitrogen removal rates of 96.7% and 98.6% within 72 hours, respectively, and final nitrite nitrogen contents of 3.22 mg / L and 0.15 mg / L, respectively. This suggests that Oligotrophomonas R2 has great application prospects in the field of heavy metal wastewater denitrification.

[0037] Table 1: Denitrification effect of Oligotrophomonas R2 under different heavy metal stresses Example 6: Study on denitrification of Stenotrophomonas maltophilia R2 under different dye stresses To investigate the denitrification performance of Oligotrophomonas R2 under different dye conditions, the same Oligotrophomonas R2 seed solution as in Example 2 was added at a 5% dosage to 100 mL of a denitrification liquid culture medium containing nitrate (140 mg / L) and nitrite (100 mg / L) modified with Acid Red (50 mg / L), Congo Red (50 mg / L), Direct Blue 1 (50 mg / L), Golden Orange G (50 mg / L), and Acid Orange (50 mg / L), respectively. Trisodium citrate was added to control the carbon-nitrogen ratio (COD / TN) to 8:1. After incubation at 30°C for 24 hours, samples were collected to measure nitrate and nitrite content. The results, as shown in Table 2 below, show that Oligotrophomonas R2 demonstrated excellent denitrification capabilities under the stress of all five dyes. Clear denitrification bubbles appeared on the surface of the culture medium, and both nitrate and nitrite were almost completely removed from the culture system. Therefore, Oligotrophomonas R2 has the potential to be applied in the denitrification of actual dye wastewater.

[0038] Table 2: Denitrification effect of Oligotrophomonas R2 under different dye stress Example 7: Study on denitrification of Stenotrophomonas maltophilia R2 under high concentration aniline stress To investigate the effect of aniline on denitrification by oligotrophomonas R2, the same oligotrophomonas R2 seed solution as in Example 2 was added at a dosage of 5% to 100 mL of a denitrification liquid culture medium containing 140 mg / L of nitrate nitrogen modified with 1 g / L of aniline. An appropriate amount of trisodium citrate was added to control the carbon-nitrogen ratio (COD / TN) to 8:1. The culture was incubated at 30°C, and samples were taken every 24 hours to detect the nitrate and nitrite nitrogen contents. The results are shown in Figure 2. Figure 9 As shown, high concentrations of aniline (1 g / L) inhibited the denitrification efficiency of Oligotrophomonas R2 to a certain extent, prolonging the denitrification process. Within 24 hours, the nitrate-nitrogen removal rate reached 98.3%, while the nitrite-nitrogen accumulation reached 108.9 mg / L. As Oligotrophomonas R2 gradually adapted to aniline, the inhibitory effect gradually weakened. After 72 hours, the nitrite-nitrogen content in the culture system decreased to 13.65 mg / L, and after 96 hours, it decreased to 0.52 mg / L. Therefore, this strain has great potential for engineering applications in the denitrification of chemical wastewaters containing aniline, among other applications.

[0039] Example 8: Study on enhanced denitrification of textile printing and dyeing wastewater by Stenotrophomonas maltophilia R2 Aerobic effluent from a textile printing and dyeing enterprise in Shaoxing, Zhejiang (COD in the range of 300-400 mg / L, TN in the range of 30-40 mg / L, NO₃-N in the range of 30-40 mg / L, and NH₃-N and NO₂-N concentrations less than 1 mg / L) and anoxic tank sludge were sampled. Appropriate amounts of activated sludge and Oligotrophomonas R2 seed solution were added to the effluent according to the table below. After incubation at room temperature for 24 hours, samples were collected and analyzed for the relevant indicators. The results, shown in Table 3 below, show that compared with the control group, the experimental groups treated with sludge or seed solution showed significantly and uniform reductions in COD and TN levels. The optimal treatment group, treated with both sludge and seed solution, achieved a TN removal rate of only 0.9 mg / L after 24 hours of treatment, achieving a 97.5% TN removal efficiency. Therefore, Oligotrophomonas R2 can be applied to the enhanced denitrification treatment of textile printing and dyeing wastewater.

[0040] Table 3: Effect of enhanced application of Oligotrophomonas R2 on denitrification of textile printing and dyeing wastewater Example 9: Study on enhanced denitrification of mixed industrial wastewater by Stenotrophomonas maltophilia R2 The seed solution of Oligotrophomonas R2 was applied to mixed industrial wastewater. Biochemical effluent from a mixed industrial wastewater treatment plant in Shaoxing (COD approximately 60 mg / L, TN approximately 25 mg / L, NO₃-N approximately 20 mg / L, and NH₃-N and NO₂-N concentrations less than 3 mg / L) and activated sludge were used. Given the wastewater's poor biodegradability and low carbon-nitrogen ratio, 0.2 g / L trisodium citrate was added as a denitrification carbon source. The specific experimental groups and results are shown in Table 4. Oligotrophomonas R2 enhanced denitrification in the mixed industrial wastewater. The group treated with the seed solution alone achieved a nitrate-nitrogen removal rate of 96.2% and a TN concentration of 9.4 mg / L. The combined treatment of the seed solution and sludge achieved even greater results, with an effluent TN of 6.5 mg / L, both meeting the Class A discharge standard for industrial wastewater.

[0041] Table 4: Effect of enhanced application of Oligotrophomonas R2 on denitrification of mixed industrial wastewater The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A highly adaptable low carbon-nitrogen ratio denitrifying bacterium, which is Oligotrophomonas ( Stenotrophomonas maltophilia )R2, deposited in China Center for Type Culture Collection (CCTCC), the deposit date is October 10, 2024, and the deposit number is CCTCC NO: M 20242155.

2. The highly adaptable low carbon-nitrogen ratio denitrifying bacteria according to claim 1, characterized in that: The bacterial 16SrDNA gene sequence is shown in SEQ ID NO.

1.

3. The highly adaptable low carbon-nitrogen ratio denitrifying bacteria according to claim 1, characterized in that: The colonies of the strain R2 on the LB plate were yellow, opaque, with a moist and smooth surface and regular edges.

4. Use of the highly adaptable, low carbon-nitrogen ratio denitrifying bacteria according to claim 1 for efficient denitrification in different sewage.

5. The use of denitrifying bacteria for efficient nitrogen removal in different sewage according to claim 4, characterized in that: The application includes the following steps: Stenotrophomonas R2 was inoculated into LB liquid medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h. After the culture was completed, the bacterial liquid was centrifuged at 8000 × g for 5 min, the lower layer of bacteria was removed, and the cells were diluted with sterile water to an OD of 600 =1.0 as the seed solution of Stenotrophomonas R2; The seed liquid was added to the nitrate nitrogen and nitrite nitrogen denitrification simulated denitrification culture medium at a ratio of 5%, the temperature was adjusted to 30°C and the pH was 7 for static culture, the carbon-nitrogen ratio (COD / TN) of the culture system was set between 4:1-12:1 (preferably 8:1), the nitrate nitrogen concentration was between 140mg / L-980mg / L (preferably 140mg / L), and the nitrite nitrogen concentration was between 100mg / L-500mg / L (preferably 100mg / L), and its continuous denitrification ability was explored by regular feeding.

6. The use of denitrifying bacteria for efficient nitrogen removal in different sewage according to claim 5, characterized in that: The R2 seed liquid was added to simulated toxic wastewater (heavy metal, dye and aniline wastewater), textile printing and dyeing wastewater and mixed industrial wastewater in a certain proportion and statically cultivated to explore its adaptability and actual enhanced denitrification capacity.

7. The use of denitrifying bacteria for efficient nitrogen removal in different sewage according to claim 5, characterized in that: The LB liquid culture medium consists of 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract, and the solvent is water.

8. The use of denitrifying bacteria for efficient nitrogen removal in different sewage according to claim 5, characterized in that: The denitrification simulation culture medium is composed of: 1g / L NaH2PO4, 1g / L Na2HPO4, 0.05g / L MgSO4, appropriate amounts of trisodium citrate, potassium nitrate and sodium nitrite, and the solvent is water.

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