A method for treating high-concentration inorganic nitrogen wastewater by using paracoccus mixed bacteria wg2 and wg1

Abstract

This invention relates to a method for treating high-concentration inorganic nitrogen wastewater using a mixed culture of *Paragonimus* strains wg2 and wg1, belonging to the field of resource and environmental microbiology. This invention utilizes the co-culture of strains wg2 and wg1 to treat simulated wastewater, primarily exploring the effect of the mixed strains wg2 and wg1 on NO2 under anoxic conditions. ‑ -N, NO3 ‑ The study investigated the nitrogen removal capacity of nitrite-nitrogen (N), the optimal ratio of mixed bacteria, and the nitrogen removal performance of the mixed bacteria in treating simulated wastewater. Results showed that *Paragonimus* strains wg2 and wg1 acted synergistically in a mixed culture system, effectively addressing both nitrite accumulation and efficient nitrate degradation, demonstrating high nitrogen removal capacity. This study provides important practical guidance for the development of biological nitrogen-removing agents that simultaneously remove nitrite and nitrate.

Description

Technical Field

[0001] This invention relates to a method for treating high-concentration inorganic nitrogen wastewater using a mixed strain of Paracoccus wg2 and wg1, belonging to the field of resource and environmental microbiology. Background Technology

[0002] With the expansion of global aquaculture, nitrogen pollution caused by aquaculture wastewater discharge is becoming increasingly serious. Abnormally high nitrogen concentrations in aquaculture wastewater lead to increased nitrite (NO2) levels. - -N) and nitrates (NO3) - The continuous accumulation of nitrogen (N-N) not only poses a direct threat to aquatic life but also carries potential risks to human health through the food chain. While traditional physicochemical denitrification technologies are effective in removing nitrogen pollutants, they suffer from high energy consumption, secondary pollution, and operational complexity. Biological denitrification technology, due to its environmental friendliness, low cost, and sustainability, is considered an ideal approach to solving nitrogen pollution in aquaculture wastewater.

[0003] Chinese patent document CN116854262A (application number 202310952899.6) discloses a method using Paracoccus wg1 T Methods for treating wastewater containing high concentrations of inorganic nitrogen, Paracoccus wg1 T It exhibits tolerance to high concentrations of ammonia nitrogen and nitrate nitrogen, and demonstrates excellent denitrification ability under high concentrations of inorganic nitrogen conditions. This is achieved using *Paracoccus paragallinarum* wg1. T Biological denitrification treatment of high-concentration inorganic nitrogen wastewater has great application potential. Although strains P. binzhouensis wg1 T It has a high efficiency in reducing nitrates, but nitrites will accumulate during the degradation of nitrates.

[0004] To address this deficiency, this invention proposes a strategy of co-culturing strains wg2 and wg1. The selected strain wg2 ​​is an activated sludge strain isolated from propylene oxide saponification wastewater. P. shandongensis When WG2 and WG1 are co-cultured, it can effectively reduce the degradation of NO3 by WG1. - The -N process produces nitrite. The two work synergistically to solve both the problem of nitrite accumulation and the efficient degradation of nitrate. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for treating high-concentration inorganic nitrogen wastewater using a mixed strain of Paracoccus bacteria wg2 and wg1.

[0006] The technical solution of the present invention is as follows:

[0007] Application of mixed strains of Paracoccus wg2 and Paracoccus wg1 in the treatment of high-concentration inorganic nitrogen wastewater.

[0008] According to a preferred embodiment of the present invention, the paracoccus ( Paracoccus sp. wg1 is deposited at the China Center for Type Culture Collection, accession number CCTCC AB 2019400; the paracoccus ( Paracoccus shandongensis sp. nov. wg2 is deposited at the China Center for Type Culture Collection, accession number CCTCC AB 2019401.

[0009] A compound microbial agent comprising Paracoccus wg2 and Paracoccus wg1.

[0010] According to a preferred embodiment of the present invention, the compound microbial agent is used for efficient nitrogen removal in high-concentration inorganic nitrogen wastewater.

[0011] A method for treating high-concentration inorganic nitrogen wastewater using a mixed culture of Paracoccus wg2 and Paracoccus wg1 includes the following steps:

[0012] (1) Preparation of wg2 seed culture: After activating paracoccus wg2, pick a single colony and inoculate it in LB liquid medium and culture for 24-26 h; centrifuge and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline; transfer to simultaneous nitrification and denitrification medium at a volume ratio of 15-20% and culture for 18-20 h; centrifuge again and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline to obtain wg2 ​​seed culture;

[0013] (2) Preparation of wg1 seed culture: After activating paracoccus wg1, pick a single colony and inoculate it into LB liquid medium and culture for 12-14 h; centrifuge and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline; transfer to simultaneous nitrification and denitrification medium at a volume ratio of 5-10% and culture for 12-14 h; centrifuge again and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline to obtain wg1 seed culture;

[0014] (3) Mix the wg2 seed liquid and wg1 seed liquid obtained in steps (1) and (2) in proportion, inoculate them into the wastewater, and culture them under the conditions of 30~32℃ and 200~220rpm to complete the treatment of the wastewater.

[0015] According to a preferred embodiment of the present invention, the culture conditions described in step (1) are: 30~32℃, 200~220 rpm.

[0016] According to a preferred embodiment of the present invention, the culture conditions described in step (2) are: 35~37℃, 200~220 rpm.

[0017] According to a preferred embodiment of the present invention, the components of the simultaneous nitrification and denitrification culture medium in steps (1) and (2) are: NaNO3 0.3 g / L, NH4Cl 0.2 g / L, NaNO2 0.2 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, water as solvent, and pH adjusted to 7.

[0018] More preferably, the trace element components are: EDTA-2Na 50 g / L, (NH4)6Mo7O 24 The following solvents were used: 1.1 g / L of 4H₂O, 1.6 g / L of CoCl₂·6H₂O, 5.1 g / L of MnCl₄·4H₂O, 1.6 g / L of CuSO₄·5H₂O, 5.5 g / L of CaCl₂, 5.0 g / L of FeSO₄·7H₂O, and 2.2 g / L of ZnSO₄·7H₂O. The solution was water, and the pH was adjusted to 7.

[0019] According to a preferred embodiment of the present invention, the mixing ratio in step (3) is: OD of Paracoccus wg2 and Paracoccus wg1. 600 The ratio is 10:1.

[0020] According to a preferred embodiment of the present invention, the nitrate concentration in the wastewater in step (3) is 10~20 mg / L and the nitrite concentration is 3~7 mg / L.

[0021] Beneficial effects:

[0022] This invention utilizes the co-culture of strains wg2 and wg1 to treat simulated wastewater, primarily investigating the effect of the co-culture of strains wg2 and wg1 on NO2 under anoxic conditions. - -N, NO3 - The study investigated the nitrogen removal capacity of *N*, the optimal ratio of mixed bacteria, and the nitrogen removal performance of the mixed bacteria in treating simulated wastewater. Results showed that *Paragonimus* strains wg2 and wg1 acted synergistically in the mixed culture system, effectively addressing both nitrite accumulation and nitrate degradation, demonstrating high nitrogen removal capacity. This study provides important practical guidance for the development of biological nitrogen-removing agents that simultaneously remove nitrite and nitrate. Attached Figure Description

[0023] Figure 1 NO2 in mixed nitrogen source medium for both wg2 and wg1 co-culture and single-cell culture - A bar chart showing the concentration changes of -N;

[0024] Figure 2 NO3 in mixed nitrogen source medium for both wg2 and wg1 co-culture and single-cell culture -A bar chart showing the concentration changes of -N;

[0025] Figure 3 NO2 in NNM medium for cultures of strains wg2 and wg1 in different mixing ratios - -N concentration variation dotted line graph;

[0026] Figure 4 NO3 in DM medium for different mixing ratios of strains wg2 and wg1 - -N concentration variation dotted line graph;

[0027] Figure 5 This is a bar chart showing the nitrogen removal performance of Experimental Group 1 in a simulated wastewater environment;

[0028] Figure 6 This is a bar chart showing the nitrogen removal performance of Experiment Group 2 under a simulated wastewater environment;

[0029] Figure 7 This is a bar chart showing the nitrogen removal performance of Experiment Group 3 under a simulated wastewater environment;

[0030] Figure 8 This is a bar chart showing the nitrogen removal performance of experimental group four in a simulated wastewater environment. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and medicines involved in the embodiments are all commercially available products.

[0032] Paracoccus involved in the examples ( Paracoccus sp.)wg1 was deposited at the China Center for Type Culture Collection (CCTCC) in 2019 via non-patented accession, with accession number CCTCC AB 2019400; Paracoccus ( Paracoccus shandongensis sp. nov.)wg2 was deposited at the China Center for Type Culture Collection in 2019 via non-patented strain preservation, with accession number CCTCC AB 2019401.

[0033] The culture medium formulations involved in the examples consist of:

[0034] LB liquid medium: NaCl 10.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, solvent is water.

[0035] Simultaneous nitrification-denitrification (SND) medium: NaNO3 0.3 g / L, NH4Cl 0.2 g / L, NaNO2 0.2 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, water as solvent, pH adjusted to 7.

[0036] NNM medium: NaNO2 0.2 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, solvent is water, pH adjusted to 7.

[0037] DM medium: NaNO3 0.3 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, solvent is water, pH adjusted to 7.

[0038] Mixed nitrogen source culture medium: NaNO3 0.15 g / L, NaNO2 0.12 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, solvent is water, pH adjusted to 7.

[0039] The above trace element components are: C 10 H 14 N2Na2O8·2H2O (EDTA-2Na) 50 g / L, (NH4)6Mo7O 24 The following ingredients were used: 1.1 g / L of 4H₂O, 1.6 g / L of CoCl₂·6H₂O, 5.1 g / L of MnCl₄·4H₂O, 1.6 g / L of CuSO₄·5H₂O, 5.5 g / L of CaCl₂, 5.0 g / L of FeSO₄·7H₂O, and 2.2 g / L of ZnSO₄·7H₂O. The solvent was water, and the pH was adjusted to 7.

[0040] The nitrate nitrogen (NO3) involved in the examples - Nitrite nitrogen (NO2) was detected by ultraviolet spectrophotometry. - -N) was detected by the naphthylethylenediamine hydrochloride spectrophotometric method.

[0041] Example 1

[0042] Investigating the effect of mixed bacteria wg2 and wg1 on NO2 under hypoxic conditions - -N, NO3 --N removal capability

[0043] Preparation of seed culture for strain wg2: The preserved strain was taken from a -80℃ ultra-low temperature freezer and activated twice by streak plating. Single colonies were picked and inoculated into 100 mL LB liquid medium and cultured at 30℃ and 200 rpm with shaking for 24 h. After centrifugation at 4℃ and 7000 rpm for 5 min, the supernatant was discarded, and the cells were washed three times with sterile water to remove residual nutrients in the LB liquid medium. The cells were then resuspended in an equal volume of physiological saline. The inoculum was transferred to 100 mL SND medium (300 mL Erlenmeyer flask) at a volume ratio of 15% and cultured at 30℃ and 200 rpm with shaking for 18 h. After centrifugation again at 4℃ and 7000 rpm for 5 min, the supernatant was discarded, and the cells were washed three times with sterile water. The cells were then resuspended in an equal volume of physiological saline to prepare the seed culture.

[0044] Preparation of seed culture for strain wg1: The preserved strain was taken from a -80℃ ultra-low temperature freezer and activated twice by streak plating. Single colonies were picked and inoculated into 100 mL LB liquid medium and cultured at 37℃ and 200 rpm with shaking for 12 h. After centrifugation at 4℃ and 7000 rpm for 5 min, the supernatant was discarded, and the cells were washed three times with sterile water to remove residual nutrients in the LB liquid medium. The cells were then resuspended in an equal volume of physiological saline. The inoculum was transferred to 100 mL SND medium (300 mL Erlenmeyer flask) at a volume ratio of 5% and cultured at 37℃ and 200 rpm for 12 h. After centrifugation at 4℃ and 7000 rpm for 5 min again, the supernatant was discarded, and the cells were washed three times with sterile water. The cells were then resuspended in an equal volume of physiological saline to prepare the seed culture.

[0045] Three experimental groups were set up. Experimental group 1: wg1 seed culture was inoculated into 100mL of mixed nitrogen source medium to construct OD. 600 For experimental group 2: A single-cell culture system of 0.1 was prepared, and the volume of the seed culture was recorded as V1; For experimental group 3: wg2 seed culture was inoculated into 100 mL of mixed nitrogen source medium to construct OD. 600 For the 1.0 single-cell culture system, the volume of the seed culture is recorded as V2; Experimental group three: V1 volume of wg1 seed culture and V2 volume of wg2 seed culture are mixed and inoculated into 100mL of mixed nitrogen source medium to construct a mixed co-culture system. At this time, the OD of wg2 in the medium is... 600 This can be considered as 1.0, the OD of wg1 in the culture medium 600 This can be considered as 0.1, the OD of the two bacteria in the culture medium. 600 The ratio is 10:1.

[0046] After inoculation, the mixed nitrogen source medium was transferred to a sealed headspace bottle and placed in a constant temperature shaker at 30℃ and 200 rpm for incubation. Bacterial culture samples were collected at 0, 6, 12, 18, and 24 h and centrifuged to obtain the supernatant. The NO2 content in the supernatant of the mixed nitrogen source medium bacterial culture samples was then measured. - -N and NO3 - -N content.

[0047] The results are as follows Figure 1 , Figure 2 As shown, under hypoxic conditions, experimental group one, inoculated with a single bacterium wg1, was able to completely degrade NO3. - -N but will accumulate NO2. - -N, leading to NO2 - The -N content increases due to the NO3- content of wg1. - -N has a higher reduction rate than NO2. - -N reduction rate, leading to NO2 - -N accumulates only when all NO3- is produced. - After -N is restored, NO2 - -N will then be reduced, and the content will gradually decrease; in experimental group two, which was inoculated with a single bacterium wg2, only NO2 in the mixed nitrogen source medium was degraded. - -N but does not degrade NO3 - -N; Experimental group three, inoculated with a mixture of wg2 and wg1 bacteria, NO3 in the mixed nitrogen source medium. - -N and NO2 - -N was completely degraded within 18 hours. These results demonstrate that strain wg2 ​​can specifically and efficiently degrade NO2. - While it contains NO3-N, it exhibits low activity in degrading NO3-N. Conversely, strain wg1 can specifically and efficiently degrade NO3-N. - -N, but in the degradation of NO3 - -N will also produce NO2. - The accumulation of -N, therefore, choosing a mixed strain of wg2 and wg1 is effective against NO2. - -N and NO3 - -N removal is most effective. Mixing strains wg2 and wg1 can compensate for the shortcomings of single-strain denitrification, thus degrading NO3-. - -N does not accumulate NO2 - -N.

[0048] Example 2

[0049] Investigating the nitrogen removal capacity of wg2 and wg1 at different mixing ratios

[0050] Experimental groups with different mixing ratios were set up, including seed cultures of activated strains wg2 and wg1 mixed according to OD ratios.600 The ratios of 5:1, 8:1, 10:1, 15:1, 25:1, and 30:1 were mixed thoroughly (the method for adjusting the mixing ratio was the same as in Example 1). The preparation of the seed culture of wg2 and wg1 was the same as in Example 1. The OD values ​​were set accordingly. 600 The mixed bacteria were inoculated separately into NNM and DM media, and then transferred to sealed headspace bottles for incubation at 30 °C and 200 rpm using a shaker. Samples were taken at 0, 6, 12, 18, 24, 30, and 36 h, centrifuged, and the supernatant was collected. The NO2 content in the supernatant of the NNM culture samples was then measured. - The concentration of -N and the NO3 in the supernatant of the bacterial culture sample from DM culture medium. - -N concentration.

[0051] The results are as follows Figure 3 , Figure 4 As shown, during the hypoxic denitrification process, when the mixing ratio of the seed culture of strains wg2 and wg1 was 10:1, the mixed bacteria were able to completely degrade NO2 in NNM medium within 12 hours. - -N completely degrades NO3 in DM medium within 24 hours. - -N, NO2 - -N and NO3 - The removal rate of -N was as high as 100%. The nitrogen removal efficiency under other ratio conditions was lower than this level, indicating that the mixing ratio of strain wg2 ​​to wg1 seed culture at 10:1 has better adaptability and nitrogen removal performance. Therefore, the optimal ratio for mixed bacterial nitrogen removal under the experimental conditions is a mixing ratio of strain wg2 ​​to wg1 seed culture of 10:1.

[0052] Example 3

[0053] Investigating the nitrogen removal capacity of a mixed bacterial culture of WG2 and WG1 under simulated wastewater conditions.

[0054] An experiment was conducted using water samples from aquaculture ponds as a simulation object. The NO2 content in this water sample... - -N concentration is close to 0 mg / L, while NO3 - -N concentration was close to 10~12 mg / L. To simulate potential nitrogen exceedance during aquaculture, NaNO3 and NaNO2 were added to the simulated water sample, reducing the NO3- concentration in the simulated water sample. - The -N concentration is approximately 15 mg / L, NO2 --N concentration was approximately 5 mg / L. Based on this simulated wastewater, four experimental groups were designed. In experimental group one, a mixed culture of wg2 and wg1 was added, with a mixing ratio of 10:1 between strains wg2 and wg1 in the simulated wastewater (the method for adjusting the mixing ratio was the same as in experimental group three of Example 1). In experimental group two, a single-strain wg2 ​​seed culture was added to construct an OD. 600 The experimental group was a single-strain culture system with a culture volume of 1.0; in experimental group three, a single-strain wg1 seed culture was added to construct an OD. 600 A single-strain culture system of 0.1 μL was used; experimental group four served as a blank control, without inoculation of any strains. The preparation of the wg2 and wg1 seed cultures was the same as in Example 1. After inoculation, the simulated wastewater was transferred to a sealed headspace bottle and placed in a constant-temperature shaker at 30 °C and 200 rpm for incubation. Bacterial culture samples were collected at 0, 6, 12, 18, 24, and 30 h, and centrifuged. The supernatant was used to detect NO2 in the samples. - -N, NO3 - -N concentration.

[0055] Figure 5 This describes the nitrogen removal in experimental group 1 under a simulated wastewater environment. The results show that after 6 hours of treatment, NO2... - -N and NO3 - -N concentration can reach a low level, and mixed bacteria have a highly efficient nitrogen removal ability; Figure 6 The results of experimental group 2 on nitrogen removal under simulated wastewater conditions showed that single bacterium wg2 effectively reduced NO2 levels. - -N has a better degradation effect, NO3 - -N was partially degraded, possibly because the water sample contained other microorganisms that played a role in its degradation. Figure 7 The experiment group 3 tested nitrogen removal in a simulated wastewater environment. The results showed that the single bacterium wg1 could degrade NO3 in a short time. - -N but it accumulates NO2. - -N, leading to NO2 - The NO-N content suddenly increased after 12 hours, due to the complete degradation of NO3 by strain wg1. - -N, begins to gradually degrade NO2 - -N, NO2 - -N content gradually decreases, with NO2 content decreasing 24 hours prior. - -N is completely degraded; Figure 8 This refers to the nitrogen removal performance of experimental group four in a simulated wastewater environment, specifically NO2. - -N and NO3 - The -N concentration decreased after 24 hours. This is because there are a small number of microorganisms in the aquaculture wastewater. They can reduce the concentration of nitrate and nitrite in the wastewater through processes such as denitrification, assimilation and nitrification-denitrification coupling, but the degradation rate is slow.

[0056] By simulating the wastewater treatment environment of a real aquaculture farm, it was demonstrated that adding mixed bacteria to the wastewater can rapidly and simultaneously remove NO2. - -N, NO3 - The presence of -N and a synergistic effect indicates that the mixed culture of Paracoccus wg2 and wg1 has a highly efficient nitrogen removal capacity, which has important practical guiding significance for the development of biological nitrogen-removing agents that simultaneously remove nitrite and nitrate.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for treating high concentration inorganic nitrogen wastewater using a mixed culture of Paracoccus sp. wg2 and Paracoccus sp. wg1, characterized in that, Includes the following steps: (1) Preparation of wg2 seed culture: After activating paracoccus wg2, pick a single colony and inoculate it in LB liquid medium and culture for 24-26 h; centrifuge and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline; transfer to simultaneous nitrification and denitrification medium at a volume ratio of 15-20% and culture for 18-20 h; centrifuge again and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline to obtain wg2 ​​seed culture; (2) Preparation of wg1 seed culture: After activating paracoccus wg1, pick a single colony and inoculate it into LB liquid medium and culture for 12-14 h; centrifuge and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline; transfer to simultaneous nitrification and denitrification medium at a volume ratio of 5-10% and culture for 12-14 h; centrifuge again and discard the supernatant, wash with sterile water and resuspend with an equal volume of physiological saline to obtain wg1 seed culture; (3) After mixing the wg2 seed liquid and wg1 seed liquid obtained in steps (1) and (2) in proportion, they are inoculated into the wastewater and cultured under shaking conditions at a temperature of 30~32℃ and a rotation speed of 200~220rpm to complete the treatment of the wastewater; The Paracoccus (P. Paracoccus sp. ) wg1 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC AB 2019400; the Paracoccus (P. Paracoccus shandongensis sp. nov. ) wg2 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC AB 2019401.

2. The method of claim 1, wherein, The culture conditions described in step (1) are: 30~32℃, 200~220 rpm.

3. The method of claim 1, wherein, The culture conditions described in step (2) are: 35~37℃, 200~220 rpm.

4. The method of claim 1, wherein, The components of the simultaneous nitrification and denitrification medium mentioned in steps (1) and (2) are: NaNO3 0.3 g / L, NH4Cl 0.2 g / L, NaNO2 0.2 g / L, Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, CH3COONa·7H2O 3.4 g / L, trace elements 2 mL / L, water as solvent, and pH adjusted to 7.

5. The method of claim 4, wherein, The trace element components are: EDTA-2Na 50 g / L, (NH4)6Mo7O 24 The following ingredients were added: 1.1 g / L of 4H₂O, 1.6 g / L of CoCl₂·6H₂O, 5.1 g / L of MnCl₄·4H₂O, 1.6 g / L of CuSO₄·5H₂O, 5.5 g / L of CaCl₂, 5.0 g / L of FeSO₄·7H₂O, and 2.2 g / L of ZnSO₄·7H₂O. The solvent was water, and the pH was adjusted to 7.

6. The method of claim 1, wherein, The mixing ratio described in step (3) is: OD of Paracoccus wg2 and Paracoccus wg1. 600 The ratio is 10:

1.

7. The method of claim 1, wherein, The nitrate concentration in the wastewater in step (3) is 10~20 mg / L and the nitrite concentration is 3~7 mg / L.

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