Paracoccus denitrificans and fungicide thereof and application of paracoccus denitrificans in chemical wastewater treatment

Abstract

The invention discloses paracoccus denitrificans, a bacterial agent of the paracoccus denitrificans and application of the paracoccus denitrificans in chemical wastewater treatment, the paracoccus denitrificans is named as PH-21 and preserved in the China General Microbiological Culture Collection Center on March 17, 2025, and the preservation number of the paracoccus denitrificans is CGMCC No. 33832. The invention further discloses a preparation method of the paracoccus denitrificans, and the preparation method of the paracoccus denitrificans and the bacterial agent of the paracoccus denitrificans and the application of the paracoccus denitrificans and the bacterial agent of the paracoccus denitrificans and the application of the paracoccus denitrificans in chemical wastewater treatment. The paracoccus denitrificans PH-21 disclosed by the invention can be used for rapidly degrading 3-pyridine acetate hydrochloride and various organic nitrogen pollutants in wastewater under a high-salt condition (the NaCl concentration is 30g / L) and a low-temperature condition (the water temperature is 8 DEG C), and is excellent in degradation effect and strong in stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Paracoccus denitrifying, its inoculant, and its application in the treatment of chemical wastewater. Background Technology

[0002] 3-Pyridine acetate hydrochloride is an organic acid salt containing a pyridine ring. Its structure includes both a pyridine ring and a carboxyl group. It is chemically relatively stable and mainly used as an intermediate in the fine chemical industry, including as a raw material for pharmaceutical synthesis, a pesticide and dye intermediate, and a chemical research reagent. Pollution from 3-Pyridine acetate hydrochloride primarily originates from leaks or discharges during its production, storage, use, and disposal. It is readily soluble in water. If cleaning wastewater or reaction wastewater is discharged directly without treatment during production, or if leaks occur during storage, it can enter surface water or groundwater, leading to increased concentrations of organic pollutants in aquatic bodies. It has potential toxicity to aquatic organisms such as fish and algae, potentially inhibiting their growth and reproduction and disrupting the ecological balance of aquatic bodies. As an organic pollutant, it may accumulate in organisms through the food chain, indirectly affecting higher trophic level organisms. Existing biodegradation technologies for 3-Pyridine acetate hydrochloride, especially under high salinity and low temperature conditions, generally have low efficiency or have not been reported. Therefore, it is necessary to study a strain capable of degrading 3-Pyridine acetate hydrochloride. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a strain of Paracoccus denitrifying and its inoculant, as well as its application in the treatment of chemical wastewater. Under high-salt conditions (NaCl concentration of 30 g / L) and low-temperature conditions (water temperature of 8°C), it can rapidly degrade 3-acetic acid pyridine hydrochloride pollutants and various organic nitrogen pollutants in wastewater.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A strain of Paracoccus denitrificans, named PH-21, was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33832.

[0006] A microbial agent for the production of denitrifying paracocci.

[0007] Application of Paracoccus denitrifying or its inoculum in the degradation of 3-acetic acid pyridine hydrochloride in chemical wastewater, wherein the concentration of 3-acetic acid pyridine hydrochloride in chemical wastewater is 100-500 mg / L, the concentration of NaCl is 5-30 g / L, and the temperature is 8-30℃.

[0008] Application of Paracoccus denitrifying or its inoculum in the degradation of organic nitrogen pollutants in chemical wastewater, wherein the organic nitrogen pollutants are one or more of DMF, triethylamine, diethylamine, N-methylpyrrolidone or pyridine, the NaCl concentration in the chemical wastewater is 5-30 g / L, and the temperature is 8-30℃.

[0009] A method for treating chemical wastewater using the above-mentioned microbial agent involves adding 5% to 10% by volume of the microbial agent to the chemical wastewater for degradation treatment.

[0010] A method for preparing the above-mentioned microbial agent includes the following specific steps:

[0011] S1. Inoculate Paracoccus denitrifying PH-21 into LB medium and culture with shaking until the logarithmic phase;

[0012] S2. Inoculate the cultured inoculum into the seed tank and culture it to the logarithmic growth phase, i.e., the seed solution;

[0013] S3. Introduce the seed liquid into the production tank for fermentation culture to obtain the microbial agent.

[0014] Preferably, in step S1 above, the LB medium formula is: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, 3-acetylated pyridine hydrochloride 0.3g / L, pH 7.0.

[0015] Preferably, in step S2 above, the culture medium formula used in the seed tank is: 0.3 g / L pyridine 3-acetate hydrochloride, 8 g / L glucose, 1 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.5 g / L MgSO4, 1 g / L NaCl, 0.5 g / L CaCO3, 2 g / L yeast extract, pH 7.2-7.5.

[0016] Preferably, in step S3, the culture medium used in the production tank has the same formula as that used in the seed tank; during the cultivation process in steps S2 and S3, the inoculation amount is 8% to 12%, the sterile air ventilation rate is 1:0.6 to 1.2, the stirring speed is 180 to 240 r / min, the cultivation temperature is 30 to 35°C, and the cultivation time is 48 to 60 h.

[0017] The advantages of this invention are: This invention discloses a strain of Paracoccus denitrifying, PH-21, which can rapidly degrade 3-acetic acid pyridine hydrochloride pollutants and various organic nitrogen pollutants in wastewater under high salt conditions (NaCl concentration of 30 g / L) and low temperature conditions (water temperature of 8℃), with excellent degradation effect and good stress resistance. Attached Figure Description

[0018] Figure 1 This is a colony morphology diagram of Paracoccus denitrifying in this invention;

[0019] Figure 2 This is a graph showing the degradation rate of 3-acetylated pyridine hydrochloride contaminant by strain PH-21.

[0020] Figure 3 The effect of 3-acetic acid pyridine hydrochloride concentration on the degradation efficiency of strain PH-21;

[0021] Figure 4 The degradation effect of strain PH-21 on organic nitrogen pollutants

[0022] Figure 5 The effect of NaCl concentration on the degradation efficiency of strain PH-21;

[0023] Figure 6 The effect of temperature on the degradation efficiency of strain PH-21;

[0024] Figure 7 The image shows the effect of strain PH-21 in treating actual chemical wastewater. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] Example 1: Isolation and Identification of Strains:

[0027] 3.0 mL of activated sludge obtained from a wastewater treatment pond in a chemical industrial park in Jiangsu Province was added to 100 mL of nitrogen-free inorganic salt medium containing pyridine hydrochloride (pyridine concentration of 200 mg / L). The medium was formulated as follows: NaCl 5.0 g, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, and deionized water was added to bring the total volume to 1000 mL. The pH was adjusted to 7.0–7.2. The medium was sterilized at 121 °C for 20 min and cultured with shaking at 30 °C. Every 10 days, the medium was transferred to fresh inorganic salt medium at an inoculum rate of 3%, and this process was repeated 5 times.

[0028] Take 1.0 mL of the enriched bacterial solution obtained above and add it to 9.0 mL of sterile water to prepare 10... -1 The enrichment solution, then take 1.0 mL of the prepared 10 -1 The enrichment solution was added to 9.0 mL of sterile water and mixed thoroughly to prepare 10... -2The enrichment solution was serially diluted, and so on. 0.1 mL of each dilution was spread onto a 200 mg / L inorganic salt solid medium containing pyridine 3-acetate hydrochloride (formulation as above), and incubated at 30°C for 10 days. After 10 days, a single colony was picked from the inorganic salt solid medium and incubated for 24 hours in 3.0 mL of LB liquid medium containing pyridine. The LB liquid medium formulation was: 300 mg / L pyridine 3-acetate hydrochloride, 10.00 g / L NaCl, 10.00 g / L peptone, 5.00 g / L yeast extract, pH 7.0. The culture was centrifuged at 8000 rpm for 2 minutes, the supernatant was discarded, 3.0 mL of sterile water was added and mixed well, and the culture was centrifuged again at 8000 rpm for 2 minutes. After washing twice with sterile water using the same method, the bacteria were resuspended in 3.0 mL of sterile water. 1.0 mL of the bacterial culture was added to 100 mL of nitrogen-free inorganic salt liquid medium with a 3-acetylated pyridine hydrochloride concentration of 200 mg / L (formulation: 200 mg 3-acetylated pyridine hydrochloride, 1.50 g K₂HPO₄, 0.50 g KH₂PO₄, 0.20 g MgSO₄·7H₂O, 1.00 g NaCl, pH 7.0 per liter). The medium was incubated at 160 rpm and 30 °C for 96 h with shaking. The degradation efficiency was then measured by liquid chromatography. A strain with high degradation efficiency, namely *Paracoccus denitrifyingans* PH-21, was preserved. Its colony morphology on LB solid medium is shown in the figure below. Figure 1 As shown, subsequent experiments were conducted.

[0029] The main physiological characteristics of *Paracococcus denitrifyingus* are: spherical or short rod-shaped (coccal), approximately 0.5-0.9 μm in diameter and 0.9-1.2 μm in length; Gram-negative; positive for oxidases, catalases, and nitrate reduction. The 16S rRNA gene sequence of *Paracococcus denitrifyingus* is shown in SEQ ID No. 1.

[0030] The above-mentioned strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 17, 2025. Its classification name is Paracoccus denitrificans, the strain name is PH-21, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the accession number is CGMCC No. 33832.

[0031] Example 2: A method for preparing a microbial agent, comprising the following specific steps:

[0032] S1. Inoculate Paracoccus denitrifyingis PH-21 into LB medium. The LB medium formula is: NaCl 10.00 g / L, peptone 10.00 g / L, yeast extract 5.00 g / L, 3-acetylated pyridine hydrochloride 0.3 g / L, pH 7.0, and culture with shaking until the logarithmic phase.

[0033] S2. Inoculate the cultured inoculum into a seed tank. The culture medium used in the seed tank has the following formula: 0.3 g / L 3-acetic acid pyridine hydrochloride, 8 g / L glucose, 1 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.5 g / L MgSO4, 1 g / L NaCl, 0.5 g / L CaCO3, 2 g / L yeast extract, pH 7.2-7.5. Cultivate until the logarithmic growth phase, which is the seed culture.

[0034] S3. Introduce the seed liquid into the production tank for fermentation culture. The culture medium used in the production tank is the same as that used in the seed tank to obtain the microbial agent.

[0035] In steps S2 and S3, the inoculum size is 8%–12%, the sterile air ventilation ratio is 1:0.6–1.2, the stirring speed is 180–240 r / min, the culture temperature is 30–35℃, and the culture time is 48–60 h.

[0036] Example 3: Degradation effect of strain PH-21 on 3-acetylated pyridine hydrochloride pollutants:

[0037] An inorganic liquid culture medium containing pyridine 3-acetate hydrochloride with an initial concentration of 200 mg / L was prepared. The culture medium formula was: (NH4)2SO4 1 g / L, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl concentration 10 g / L, pH 5.0. The medium was autoclaved at 121℃ for 20 minutes and allowed to cool to room temperature. PH-21 seed culture was added to the culture medium at a 5% inoculum (v / v), and the medium was cultured at 30℃ and 180 rpm for 72 h with shaking. The contaminant content was measured every 8 h, and the degradation rate was calculated. Specific results are shown below. Figure 2 .

[0038] Depend on Figure 2 It can be seen that as the degradation time increases, the degradation rate of 3-acetylbiridine hydrochloride by strain PH-21 increases accordingly. At 72h, the degradation rate is 98%, which shows excellent degradation effect.

[0039] Example 4: Effect of 3-Acetylpyridine hydrochloride concentration on the degradation effect of strain PH-21:

[0040] Liquid culture media containing pyridine 3-acetate hydrochloride at different concentrations were prepared (initial concentrations of 100, 200, 300, 400, 500, and 600 mg / L). The culture medium formulation was: (NH4)2SO4 1 g / L, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl concentration 10 g / L, pH 5.0. The media were autoclaved at 121℃ for 20 minutes and allowed to cool to room temperature. PH-21 seed culture was added to the culture media containing each contaminant at a 5% inoculum (v / v). The media were then incubated at 30℃ and 180 rpm for 72 h with shaking. The contaminant content was measured, and the degradation rate was calculated. Specific results are shown below. Figure 3 .

[0041] Depend on Figure 3 It can be seen that the degradation rate of 3-acetylated pyridine hydrochloride by strain PH-21 decreases with the increase of the initial concentration of 3-acetylated pyridine hydrochloride, and the degradation rate can still reach 83% at a concentration of 500 mg / L.

[0042] Example 5: Degradation effect of strain PH-21 on organic nitrogen pollutants:

[0043] Liquid culture media containing DMF, triethylamine, diethylamine, N-methylpyrrolidone, and pyridine (initial concentration 100 mg / L for each) were prepared. The culture medium formula was: (NH4)2SO4 1 g / L, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl concentration 10 g / L, pH 5.0. The media were autoclaved at 121℃ for 20 minutes and allowed to cool to room temperature. PH-21 seed culture was added at a 5% inoculum (v / v) to the media containing each contaminant. The media were incubated at 30℃ and 180 rpm for 72 h with shaking. The concentration of each contaminant was measured, and the degradation rate was calculated. Specific results are shown in [link to results]. Figure 4 .

[0044] Depend on Figure 4 It can be seen that strain PH-21 has a degradation rate of over 95% for DMF, triethylamine, diethylamine, N-methylpyrrolidone and pyridine, demonstrating excellent degradation performance.

[0045] Example 6: Effect of NaCl concentration on the degradation effect of strain PH-21:

[0046] Liquid culture media containing pyridine acetate hydrochloride, DMF, triethylamine, diethylamine, N-methylpyrrolidone, and pyridine (initial concentration 100 mg / L) were prepared. The culture medium formula was: (NH4)2SO4 1 g / L, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, pH 5.0. The NaCl concentration in the culture medium was adjusted to 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L, respectively. The media were autoclaved at 121℃ for 20 minutes and allowed to cool to room temperature. The seed culture was inoculated into serum bottles at an inoculation volume of 5%. After incubation at 30℃ and 180 rpm for 72 h, the pollutant content was measured, and the degradation rate was calculated. Specific results are shown below. Figure 5 .

[0047] Depend on Figure 5 It can be seen that strain PH-21 has good tolerance to salt concentration. When the NaCl concentration is 30 g / L, the degradation efficiency of 3-acetic acid pyridine hydrochloride and organic nitrogen pollutants can still exceed 75%. This indicates that strain PH-21 has good tolerance to high salt conditions and is suitable for the treatment of high-salt chemical wastewater.

[0048] Example 7: Effect of temperature on the degradation efficiency of strain PH-21:

[0049] Liquid culture media containing pyridine acetate hydrochloride, DMF, triethylamine, diethylamine, N-methylpyrrolidone, and pyridine (initial concentration 100 mg / L for each) were prepared. The culture medium components were: (NH4)2SO4 1.0 g, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl concentration 10 g / L, pH 5.0. The media were autoclaved at 121℃ for 20 minutes and allowed to cool to room temperature. PH-21 seed culture was added at a 5% inoculum (v / v) to the media containing each contaminant. The media were then incubated on a shaker at 5℃, 8℃, 15℃, 25℃, and 30℃ at 180 rpm for 72 h. The contaminant concentration was then measured. Figure 6 As shown.

[0050] Depend on Figure 6 It can be seen that the degradation rate of 3-acetic acid pyridine hydrochloride and organic nitrogen pollutants by strain PH-21 increases with increasing temperature. At a degradation temperature of 8℃, the degradation rate can still reach more than 75%, indicating that strain PH-21 has good low-temperature resistance.

[0051] Example 8: Application of strain PH-21 in the degradation of actual chemical wastewater:

[0052] The wastewater source was a wastewater treatment pond of a chemical enterprise in Jiangsu Province, with a treatment capacity of 5L. A bacterial agent prepared by strain PH-21 was added to the wastewater at a 5% volume ratio. The original wastewater had a pH of 6.0, a NaCl concentration of 22 g / L, and a temperature of 30℃. The initial concentrations of 3-acetic acid pyridine hydrochloride, triethylamine, and N-methylpyrrolidone in the wastewater were 246 mg / L, 473 mg / L, and 360 mg / L, respectively. Aeration was performed to maintain dissolved oxygen levels above 2 mg / L. The concentrations of pollutants in the wastewater were measured every 12 hours. The results are as follows: Figure 7 As shown.

[0053] Depend on Figure 7 It was found that after 72 hours of bacterial inoculant addition, the degradation rates of 3-acetic acid pyridine hydrochloride, triethylamine, and N-methylpyrrolidone in the wastewater were 96%, 95%, and 93%, respectively. These experimental data indicate that strain PH-21 exhibits excellent degradation effects on 3-acetic acid pyridine hydrochloride and organic nitrogen pollutants in actual chemical wastewater, and has promising application prospects.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A strain of denitrifying paracoccus, characterized in that, The Paracoccus denitrificans strain was named PH-21 and was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33832.

2. A microbial agent produced using the denitrifying paracoccus as described in claim 1.

3. The application of the denitrifying paracoccus according to claim 1 or the bacterial agent according to claim 2 in the degradation of 3-acetic acid pyridine hydrochloride in chemical wastewater, characterized in that, The concentration of 3-acetic acid pyridine hydrochloride in the chemical wastewater is 100–500 mg / L, the concentration of NaCl is 5–30 g / L, and the temperature is 8–30 °C.

4. The application of the denitrifying paracoccus according to claim 1 or the bacterial agent according to claim 2 in the degradation of organic nitrogen pollutants in chemical wastewater, characterized in that, The organic nitrogen pollutants are one or more of DMF, triethylamine, diethylamine, N-methylpyrrolidone, or pyridine, and the NaCl concentration in the chemical wastewater is 5-30 g / L, and the temperature is 8-30℃.

5. A method for treating chemical wastewater using the microbial agent according to claim 2, characterized in that, Add 5% to 10% by volume of bacterial agent to chemical wastewater for degradation treatment.

6. A method for preparing the microbial agent according to claim 2, characterized in that, The specific steps include the following: S1. Inoculate Paracoccus denitrifying PH-21 into LB medium and culture with shaking until the logarithmic phase; S2. Inoculate the cultured inoculum into the seed tank and culture it to the logarithmic growth phase, i.e., the seed solution; S3. Introduce the seed liquid into the production tank for fermentation culture to obtain the microbial agent.

7. The method for preparing the microbial agent according to claim 6, characterized in that, In step S1, the LB medium is formulated as follows: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, pyridine 3-acetate hydrochloride 0.3g / L, pH 7.

0.

8. The method for preparing the microbial agent according to claim 6, characterized in that, In step S2, the culture medium used in the seed tank has the following formulation: 0.3 g / L pyridine 3-acetate hydrochloride, 8 g / L glucose, 1 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.5 g / L MgSO4, 1 g / L NaCl, 0.5 g / L CaCO3, 2 g / L yeast extract, and pH 7.2–7.

5.

9. The method for preparing the microbial agent according to claim 8, characterized in that, In step S3, the culture medium used in the production tank has the same formula as that used in the seed tank. During the cultivation process in steps S2 and S3, the inoculation amount is 8% to 12%, the ventilation rate of sterile air is 1:0.6 to 1.2, the stirring speed is 180 to 240 r / min, the cultivation temperature is 30 to 35℃, and the cultivation time is 48 to 60 h.

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