Paracoccus denitrificans and application thereof

Abstract

The application specifically relates to a Paracoccus denitrificans strain and application thereof. A specific technical scheme is as follows: a Paracoccus denitrificans strain HY-1 is preserved in the Guangdong Microbial Culture Collection Center on December 12, 2022, and the preservation number is GDMCC NO: 62483. The application provides a new Paracoccus denitrificans strain which can rapidly reproduce under heterotrophic conditions, efficiently degrade ammonia nitrogen, and efficiently remove nitrate and nitrite. The Paracoccus denitrificans strain can be prepared into a microbial bacterial preparation and used in various places requiring deodorization, such as livestock and poultry farms, compost plants, sewage treatment plants and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of denitrifying paracoccus and its applications. Background Technology

[0002] Odor pollution refers to all gaseous substances that irritate the olfactory senses, cause unpleasantness, and damage the living environment. These substances are complex in composition, highly irritating, and pose a significant threat to public health. For the diverse range of odorous gases, existing deodorization methods mainly include physical, chemical, and biological methods, or a combination of these methods. Among these, biological methods have advantages such as low investment and operating costs, high treatment efficiency, ecological safety, no secondary pollution, and convenient equipment operation, and are gradually becoming the mainstream method for treating odor pollutants.

[0003] In biological methods, microorganisms are key and crucial. Most studies on biological deodorization focus on autotrophic microbial systems as the core microbial community, with few reports on applications centered on heterotrophic microorganisms. Heterotrophic microorganisms offer advantages over autotrophic microorganisms, including faster growth and metabolism, and higher pollutant removal efficiency. Among them, heterotrophic nitrifying-aerobic denitrifying (HN-AD) bacteria possess advantages such as rapid growth rate, high activity, a wide range of substrates for proliferation, and the ability to simultaneously degrade organic matter and perform biological denitrification. They can achieve simultaneous nitrification and denitrification in the same reactor under aerobic conditions, reducing secondary pollution from leachate while removing nitrogen. Furthermore, due to the alkali produced during denitrification, some HN-AD bacteria can neutralize the acidic liquid in the circulating solution, maintaining its pH. HN-AD bacteria can also remove organic matter from the circulating solution, achieving denitrification and carbon removal. Therefore, heterotrophic nitrifying-aerobic denitrifying bacteria have broad application prospects in deodorization and denitrification.

[0004] However, existing HN-AD bacteria have shortcomings such as unsatisfactory denitrification efficiency, limited types of nitrogen that can be degraded, and inability to efficiently degrade multiple forms of nitrogen. There is an urgent need to develop new HN-AD bacteria that can efficiently denitrify and deodorize. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of denitrifying paracoccus and its applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is as follows: a strain of Paracoccus denitrificans HY-1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with the accession number GDMCC NO: 62483.

[0007] Correspondingly, the 16S rDNA sequence of a strain of *Paracoccus denitrificans* is shown in SEQ ID No. 1.

[0008] Accordingly, the application of the denitrifying paracoccus in deodorization.

[0009] Accordingly, the application of the denitrifying paracoccus in denitrification.

[0010] Accordingly, a bacterial preparation containing the aforementioned denitrifying paracoccus.

[0011] Preferably, the bacterial preparation also includes *Pseudomonas stearothermiae*.

[0012] Preferably, the *Pseudomonas stearothermiae* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with accession number GDMCC NO: 62502. Its 16S rDNA sequence is shown in SEQ ID No. 2.

[0013] Preferably, the ratio of *Pseudomonas stearothermii* to *Paracoccus denitrificans* is 1:2-3, based on the viable bacterial count.

[0014] Accordingly, the application of the bacterial preparation in deodorization.

[0015] Accordingly, the application of the bacterial preparation in denitrification.

[0016] This invention offers the following advantages: It provides a novel denitrifying paracoccus strain that can rapidly multiply under heterotrophic conditions, efficiently degrade ammonia nitrogen, and efficiently remove nitrates and nitrites, exhibiting excellent deodorizing properties. This denitrifying paracoccus can be prepared as a microbial preparation for deodorization in odor-generating locations, including livestock farms, composting plants, and sewage treatment plants. When used alone or in combination with other microorganisms such as *Pseudomonas schistosomiasis*, this denitrifying paracoccus demonstrates good denitrification and deodorization capabilities even in relatively harsh environments (anaerobic environments, environments with multiple nitrogen sources, etc.). Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the colony morphology of *Paracoccus denitrificans* HY-1.

[0018] Figure 2 This is a schematic diagram of the colony morphology of Pseudomonas schistosomiasis YM-1.

[0019] Figure 3 This is a schematic diagram of the growth curve of Pseudomonas schistosomiasis YM-1.

[0020] Figure 4 This is a schematic diagram illustrating the ammonia nitrogen degradation process of Paracoccus denitrifyingis HY-1 under aerobic conditions.

[0021] Figure 5 This is a schematic diagram illustrating the nitrite degradation process of *Paracoccus denitrifyingans* HY-1 under aerobic conditions.

[0022] Figure 6This is a schematic diagram illustrating the nitrate degradation process of *Paracoccus denitrifyingans* HY-1 under aerobic conditions.

[0023] Figure 7 A schematic diagram showing the simultaneous degradation of three nitrogen sources by Paracoccus denitrifying HY-1 under aerobic conditions;

[0024] Figure 8 This is a schematic diagram illustrating the nitrate degradation process of *Paracoccus denitrifyingans* HY-1 under anaerobic conditions.

[0025] Figure 9 This is a schematic diagram illustrating the degradation of ammonia nitrogen and nitrate by Paracoccus denitrifyingis HY-1 under anaerobic conditions.

[0026] Figure 10 This is a schematic diagram illustrating the degradation of nitrates and nitrites by *Paracoccus denitrifyingans* HY-1 under anaerobic conditions.

[0027] Figure 11 This is a schematic diagram illustrating the degradation of three nitrogen sources by Paracoccus denitrifyingis HY-1 under anaerobic conditions. Detailed Implementation

[0028] This invention provides a novel strain of Paracoccus denitrificans HY-1, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 12, 2022, with accession number GDMCC NO: 62483. Its 16S rDNA sequence is shown in SEQ ID No. 1.

[0029] The denitrifying paracoccus is a facultative amphoteric microorganism that can rapidly reproduce under heterotrophic conditions, efficiently degrade ammonia nitrogen, and efficiently remove nitrates and nitrites, exhibiting excellent deodorization performance.

[0030] The denitrifying paracoccus can be prepared into a microbial preparation for use in various deodorization fields, such as livestock and poultry farms, composting plants, and sewage treatment plants.

[0031] A preferred method is to prepare a microbial preparation by combining the denitrifying paracoccus and Pseudomonas stutzeri, and then use it for deodorization. The preferred Pseudomonas stutzeri is Pseudomonas stutzeri YM-1, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 12, 2022, with accession number GDMCC NO: 62502. Its 16S rDNA sequence is shown in SEQ ID No. 2.

[0032] The preferred embodiment is that, based on the viable bacterial count, the ratio of *Pseudomonas stearothermiae* to *Paragonimococcus denitrificans* is 1:2-3.

[0033] Those skilled in the art can use conventional methods to deodorize using the denitrifying paracoccus or microbial preparations provided by the present invention, such as adding them to a bio-trickling filter tower for membrane formation and then deodorizing, or directly inoculating the microorganisms or microbial preparations into the liquid or solid to be deodorized for deodorization.

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0035] The culture media and solutions involved in the embodiments of the present invention are as follows:

[0036] 1. Activated sludge acclimation culture medium (g / L): KH2PO4 1.0g, K2HPO4 4.0g, MgCl2 0.5g, FeSO4 0.01g, glucose 5.0g, ammonium sulfate addition amounts are shown in Table 1, sodium succinate 5.0g; the ammonia nitrogen addition amounts are shown in the table below:

[0037] Table 1. Ammonium sulfate addition amount in activated sludge acclimatization culture medium (unit: g / L)

[0038] <![CDATA[(NH4)2SO4]]> 0.46 0.98 1.42 1.96 2.0

[0039] 2. LB medium (g / L): tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, pH=7.0. The LB medium is mainly used for large-scale culture.

[0040] 3. Heterotrophic nitrification medium (g / L): (NH4)2SO4 0.47g, sodium succinate 5.62g, Viss salt solution 50mL, C / N=10, pH=7.

[0041] 4. Nitrite medium (g / L): NaNO2 0.49g, sodium succinate 5.62g, Viss salt solution 50mL, C / N=10, pH=7.

[0042] 5. Nitrate medium (g / L): KNO3 0.72g, sodium succinate 5.62g, Viss salt solution 50mL, C / N=10, pH=7.

[0043] Unless otherwise specified, the nitrate medium is prepared as described above. If a carbon source is required in the nitrate medium, use another carbon source to replace sodium succinate, and adjust the amounts of carbon source and KNO3 accordingly to achieve a C / N ratio of 10. For example, if sucrose is used as the carbon source, add 1.5g of KNO3 and 4.94g of sucrose. If the nitrate concentration needs to be adjusted, adjust the amount of KNO3 accordingly. These adjustments are common knowledge in the field and will not be elaborated further.

[0044] 6. Combined nitrogen source culture medium (g / L): (NH4)2SO4 1.14g, NaNO2 1.47g, KNO3 2.16g, sodium succinate 16.86g, Vis salt solution 50mL, C / N=10, pH=7.

[0045] 7. Anaerobic denitrification medium (DM) (g / L): Sodium succinate 21.48g, potassium nitrate 3g, potassium dihydrogen phosphate 1.5g, disodium hydrogen phosphate heptahydrate 5.0g, trace element solution 2mL, magnesium sulfate heptahydrate 0.1g, deionized water 1000mL, adjust pH=7.0.

[0046] 8. Anaerobic combined nitrogen source culture medium (DM1) (g / L): Sodium succinate 21.48g, potassium nitrate 3g, ammonium sulfate 1.0g, potassium dihydrogen phosphate 1.5g, disodium hydrogen phosphate heptahydrate 5.0g.

[0047] 9. Anaerobic combined nitrogen source culture medium (DM2) (g / L): Sodium succinate 21.48g, potassium nitrate 3g, sodium nitrite 1.0g, potassium dihydrogen phosphate 1.5g, disodium hydrogen phosphate heptahydrate 5.0g.

[0048] 10. Anaerobic combined nitrogen source culture medium (DM3) (g / L): Sodium succinate 21.48g, potassium nitrate 3g, ammonium sulfate 1.0g, sodium nitrite 1.0g, potassium dihydrogen phosphate 1.5g, disodium hydrogen phosphate heptahydrate 5.0g.

[0049] 11. Trace element solution: EDTA 100mg, ZnSO4 4.4mg, CaCl2 11mg, MnCl2·4H2O 10.2mg, FeSO4·7H2O 10mg, (NH4)6MoO 24 ·4H2O 2.2mg, CuSO4·5H2O 3.2mg, CoCl2·6H2O 3.2mg, deionized water 1000mL.

[0050] 12. Vis salt solution (g / L): 5.0g dipotassium hydrogen phosphate, 2.5g magnesium sulfate heptahydrate, 2.5g sodium chloride, 0.05g ferrous sulfate heptahydrate, 0.05g manganese sulfate, 1000mL deionized water.

[0051] 13. Circulating culture medium (g / L): glucose 6g, sodium succinate 5.62g, KH2PO4 1.0g, K2HPO4 1.0g, MgCl2 0.4g, NaHCO3 0.4g.

[0052] If it is necessary to adjust the concentration of a certain component in the above culture media, the amount of the corresponding component can be adjusted accordingly, which will not be elaborated further below.

[0053] Example 1: Screening and Identification of Denitrifying Paracocci

[0054] 1. Acclimation and Screening. Activated sludge from the secondary sedimentation tank of Shuangliu Airport Wastewater Treatment Plant in Sichuan Province was collected and acclimated using a multi-carbon source aeration medium. The medium was gradually changed from low to high concentration of ammonium sulfate, with each concentration acclimated for 6 days, for a total of 30 days.

[0055] After acclimatization, the supernatant was collected and inoculated four times, each time for three days, into heterotrophic nitrification medium and aerobic denitrification medium (nitrate medium). Subsequently, serial dilutions were performed, starting from 10⁻⁶... -2 10 -3 10 -4 10 -5 10 -6 10 -7 At the desired dilution, 0.1 mL was spread onto LB medium and incubated at 30°C. Multiple colonies were obtained. Single colonies with good growth were selected and cultured individually on LB medium, repeated at least five times to obtain pure cultures. Preliminary efficacy experiments were conducted on the obtained strains; the results for some microorganisms are shown in Table 2.

[0056] Table 2 Experiment on the initial screening effect of strains

[0057]

[0058] 2. Identification

[0059] (1) Colony growth morphology of strain HY-1 on LB plates as follows Figure 1As shown, its colonies are round, milky white, smooth, and opaque. Gram staining is red, indicating it is a Gram-negative bacterium. SEM images of the strain show that strain HY-1 is a short rod-shaped bacterium, with individual cells measuring approximately 0.5 μm × (0.9–1.2) μm. 16S rDNA sequencing and comparison with NCBI revealed that this bacterium belongs to the genus *Paracoccus*, showing a 96% similarity to strain *Paracoccus denitrificans* NBRC-102528. It was identified as *Paracoccus denitrificans* HY-1 and deposited on December 12, 2022, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 62483.

[0060] The growth curve of strain HY-1 was plotted based on data obtained from the Bioscreen C instrument. The results showed that 0–24 h was the logarithmic growth phase of strain HY-1, 24–40 h was the stationary phase of strain growth, and the OD of the strain was observed after 40 h. 600 The value drops rapidly, entering the decline phase.

[0061] (2) The physiological and biochemical characteristics of strain YM-1 are shown in Table 3. In Table 3, "+" indicates positive and "-" indicates negative.

[0062] Table 3. Physiological and biochemical properties of Pseudomonas stearothermiae YM-1

[0063] Gram staining - VP - Bacterial shape spherical catalase + Oxidase - MR - Starch hydrolysis + gelatin - Nitrate reduction test + Good / Anaerobic Aerobic / Anaerobic glucose + maltose - L-arabinose - lactose - Xylose - grease +

[0064] The isolated and purified YM-1 strain was inoculated at a rate of 1% (v / v) into heterotrophic nitrification medium, nitrite medium, and nitrate medium HN-DA medium, respectively, and cultured in shakers at different temperatures (5℃, 10℃, 24℃, 27℃, 30℃, 33℃, 36℃, 40℃, 45℃, 50℃) for 12 h. The OD was measured. 600 The optimal growth temperature for strain YM-1 was determined to be 10–50℃, with 35℃ being the most suitable. Different initial pH values ​​(3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0, 11.0) were used to culture the strain at 30℃ for 24 h, and the OD values ​​were measured. 600 The optimal pH for this bacterium's growth was determined to be 7–8, within the range of 6–10.0. OD values ​​were measured at pH 6.5 and 35°C using different C / N ratios (1, 5, 10, 15, 20, 25, 30). 600 The tolerance range of this strain was determined to be C / N 1–25, with the optimal C / N ratio being 5.

[0065] When the purified YM-1 strain was shaken, the bacteria aggregated to form irregular small precipitates, such as... Figure 2 As shown. The whole genome of the purified bacterial strain was extracted using a rapid bacterial whole genome extraction kit. PCR amplification was performed using universal primers 27F and 1492R for bacterial 16S rDNA, followed by sequencing analysis. The sequencing results were identified as *Pseudomonas stutzeri* YM-1 by BLAST comparison with the NCBI database. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with accession number GDMCC NO: 62502.

[0066] The growth curves of *Pseudomonas stearothermii* YM-1 at different temperatures were studied using LB medium. The results are as follows: Figure 3 As shown in the figure. The results showed that *Pseudomonas stearothermia* YM-1 could grow rapidly in an organic carbon source heterotrophic medium with a temperature range of 20–40 °C. After culturing at 35 °C for 24 h, the OD of the bacterial culture was [data missing]. 600 The OD value was 0.589, indicating that the bacteria aggregated into irregular small precipitates during cultivation, resulting in a low OD value. In engineering applications, especially in membrane bioreactors, this strain can rapidly attach to cell membranes and grows quickly under heterotrophic conditions, shortening the start-up time of biofilm reactors and demonstrating significant potential for industrial application.

[0067] Example 2: Demonstration of the effect of denitrifying paracocci in deodorization

[0068] 1. Demonstration of nitrogen removal efficiency for various nitrogen forms under aerobic conditions.

[0069] (1) Demonstration of ammonia nitrogen removal effect.

[0070] The heterotrophic nitrification medium was sterilized at 115℃ for 30 min, and then inoculated with denitrifying paracoccus HY-1 bacterial suspension cultured for 24 h at an inoculation rate of 1% (v / v). The bottle mouth was sealed with an aerobic test tube stopper and placed in a shaker for incubation (35℃, 180 r / min).

[0071] A blank control group was also set up. The blank control group used an equal volume of sterile water instead of the *Paragonimus denitrifyingus* culture, with all other conditions remaining the same. The nitrogen source for cultivation was ammonia nitrogen, with an initial concentration of 212.06 mg / L. It should be noted that 200 mg / L is the design concentration, and 212.06 mg / L is the measured concentration; there is an experimentally acceptable error between the two. The same applies to subsequent differences in initial concentrations, which will not be elaborated further. Each group was set up in triplicate, and the ammonia nitrogen concentration in the culture medium of each group was measured every 2 hours. The results were averaged, as shown below. Figure 4 As shown. According to Figure 4After 12 hours of treatment, the ammonia nitrogen concentration in the culture medium decreased to 2.44 mg / L. At this point, HY-1 achieved a 99.8% removal rate of ammonia nitrogen, with a removal rate as high as 17.48 mg-N / L·h. This indicates that the strain has an extremely strong ammonia nitrogen degradation ability.

[0072] (2) Demonstration of nitrite removal effect.

[0073] The remaining conditions were the same as those in the ammonia nitrogen removal experiment of Experiment (1) in this embodiment, except that the culture medium was replaced with nitrite culture medium. The results are as follows: Figure 5 As shown in the figure, the initial nitrite concentration in the culture medium was 218.34 mg / L. After 10 h of treatment, the concentration in the culture medium decreased to 0 mg / L, with a removal rate as high as 21.83 mg-N / L·h.

[0074] (3) Demonstration of nitrate removal effect.

[0075] The remaining conditions are the same as those in the ammonia nitrogen removal experiment of Experiment (1) in this embodiment, except that the culture medium is replaced with nitrate culture medium. The results are as follows: Figure 6 As shown, the initial nitrate concentration in the culture medium was 213.76 mg / L. After 8 hours of culture, the concentration in the culture medium decreased to 13.83 mg / L, with a removal rate as high as 21.83 mg-N / L·h.

[0076] (4) Demonstration of the removal effect when multiple nitrogen pollutants are present at the same time.

[0077] The remaining conditions are the same as those in the ammonia nitrogen removal experiment of Experiment (1) in this embodiment, except that the culture medium is replaced with a combined nitrogen source culture medium (the initial design concentration of the three nitrogen sources is 100 mg / L, and the (NH4)2SO4 0.47 g, NaNO2 0.49 g, and KNO3 0.72 g in the culture medium is adjusted).

[0078] In the culture medium, NH4 + -N, NO2 - -N, NO3 - The initial measured concentrations of -N were 101.74 mg / L, 131.16 mg / L, and 134.43 mg / L, respectively. The results are as follows... Figure 7 As shown, after 8 hours of cultivation, the NH4+ in the culture medium... + The NO-N concentration decreased to 0 mg / L, and the removal rate was 12.72 mg-N / L·h. The NO2 in the culture medium... - The NO3- concentration decreased to 0 mg / L, with a removal rate as high as 32.79 mg-N / L·h, and the NO3- concentration in the culture medium decreased. -The -N concentration decreased to 24.32 mg / L within 2 hours, with a removal rate of 55.05 mg-N / L·h. When three forms of nitrogen pollutants were present simultaneously, the treatment environment became more complex, and the microbial treatment capacity relatively decreased, but HY-1 still maintained a strong degradation capacity. It should be noted that there is a certain degree of interconversion between different forms of nitrogen; therefore, nitrate (NO3)... - -N) rebounded somewhat after 2 hours.

[0079] 2. Demonstration of nitrogen removal efficiency under anaerobic conditions for various nitrogen forms.

[0080] (1) Demonstration of nitrate removal effect.

[0081] Sterilize the anaerobic denitrification medium at 115℃ for 30 min, then inoculate with HY-1 denitrifying paracoccus HY-1 culture at a 1% (v / v) inoculation rate. Seal the bottle mouth with an aerobic test tube stopper and place it in a shaker for incubation (35℃, 180 r / min).

[0082] A blank control group was also set up. The blank control group used an equal volume of sterile water instead of the *Paragonimus denitrifyingus* culture medium, with all other conditions remaining the same. Nitrate was used as the nitrogen source, with an initial designed concentration of 400 mg / L and an initial measured concentration of 453.74 mg / L. Each group was divided into three replicates, and the nitrate concentration in the culture medium was measured every 4 hours. The results were averaged. The results are as follows... Figure 8 As shown, after 28 hours of treatment, the nitrate nitrogen concentration in the culture medium decreased to 46.17 mg / L. At this point, HY-1 achieved a removal rate of 89.82% for ammonia nitrogen, with a removal rate as high as 14.56 mg-N / L·h. This indicates that the strain possesses extremely strong ammonia nitrogen degradation capabilities even under anaerobic conditions.

[0083] (2) Demonstration of the removal effect when ammonia nitrogen and nitrate coexist.

[0084] The remaining conditions were the same as those in Experiment (1) of this embodiment for nitrate removal, except that the culture medium was replaced with DM-1. The initial ammonia nitrogen concentration in the culture medium was 201.57 mg / L (design concentration was 200 mg / L), and the initial nitrate concentration was 455.46 mg / L (design concentration was 400 mg / L). The results are as follows: Figure 9 As shown, after 16 hours of treatment, the nitrate concentration in the culture medium decreased to 47.87 mg / L, the ammonia nitrogen concentration decreased to 127.03 mg / L, and the COD concentration decreased from 9850.5 mg / L to 6763.33 mg / L. Compared to aerobic denitrification, anaerobic denitrification requires less carbon source.

[0085] (3) Demonstration of the removal effect when nitrate and nitrite coexist.

[0086] The remaining conditions were the same as those in Experiment (1) of this embodiment for nitrate removal, except that the culture medium was replaced with DM-2. The initial nitrite concentration in the culture medium was 236.70 mg / L, and the initial nitrate concentration was 484.83 mg / L. The results are as follows: Figure 10 As shown, after 16 hours of treatment, the nitrate concentration in the culture medium decreased to 92.44 mg / L, and after 24 hours it decreased to 49.16 mg / L. After 24 hours, the nitrite concentration decreased to 0 mg / L.

[0087] (4) Demonstration of the removal effect when ammonia nitrogen, nitrite and nitrate coexist.

[0088] The remaining conditions were the same as those in Experiment (1) of this embodiment for nitrate removal, except that the culture medium was replaced with DM-3. The initial nitrite concentration in the culture medium was 257.98 mg / L, the initial ammonia nitrogen concentration was 202.77 mg / L, and the initial nitrate concentration was 412.23 mg / L. The results are as follows: Figure 11 As shown, after 24 hours of treatment, the nitrite concentration in the culture medium decreased to 33.89 mg / L, and after 32 hours it decreased to 0 mg / L. After 32 hours of treatment, the ammonia nitrogen concentration decreased to 149.29 mg / L, and the nitrate concentration decreased to 26.22 mg / L.

[0089] Example 3: Demonstration of the combined deodorization effect of Paracoccus denitrifyingis and Pseudomonas schrenckii

[0090] The heterotrophic nitrification medium was sterilized at 115℃ for 30 min, divided into two groups, and inoculated with *Pseudomonas stearothermii* YM-1 and *Paracoccus denitrificans* HY-1 at 1% (v / v) respectively. The bottle mouths were sealed with aerobic test tube stoppers, and the bottles were placed in shakers (35℃, 180 rpm) for approximately 24 h, incubating until their respective OD values ​​reached. 600 =1.4, yielding YM-1 and HY-1 bacterial cultures. Simultaneously, *Pseudomonas stearothermiae* and *Para dinitrate* from other sources were cultured to OD using the same method. 600 =1.4. *Pseudomonas schlegelii* and *Paragonimella denitrificans* bacterial suspensions from different sources were mixed separately according to the different volume ratios shown in Table 4 to obtain 11 groups of compound bacterial preparations. In Table 4, "\" indicates that the corresponding microorganism was not used. The inventors' research group did not only conduct the combination experiments shown in Table 4; for space considerations, only some representative combinations are presented.

[0091] Table 4. Formulas of compound microbial preparations for each group

[0092]

[0093]

[0094] The compound microbial agents from Table 4 were inoculated at a volume ratio of 1% (v / v) into a culture medium containing ammonia nitrogen, nitrate, and nitrite (using a combined nitrogen source medium; the initial design concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were all 300 mg / L). A blank control group was also set up for each group, using an equal volume of sterile water instead of the compound microbial agents, with all other conditions remaining the same. Each group was cultured at 35℃, 200 rpm, with air circulation (aerobic) for 12 h, and then under anaerobic conditions (air-free) for 24 h. Each treatment was repeated three times. The concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in each group were measured, and the average values ​​were taken. The results are shown in Table 5, in mg / L.

[0095] Table 5 shows the denitrification effect of each group of compound bacterial preparations.

[0096]

[0097] The results showed that the optimal ratio of HY-1:YM-1 = 3:1 (Group 5) was the most effective. Under aerobic conditions, when the treatment time was extended to 22 hours, the ammonia nitrogen concentration decreased to 0, the nitrite nitrogen concentration decreased to 15.2 mg / L (and further decreased to 0 at 24 hours), and the nitrate nitrogen concentration decreased to 37.35 mg / L. Under anaerobic conditions, when the treatment time was extended to 36 hours, the ammonia nitrogen concentration decreased to 101.22 mg / L, the nitrite nitrogen concentration decreased to 32.51 mg / L, and the nitrate nitrogen concentration decreased to 121.24 mg / L.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A bacterial preparation, characterized in that: The bacterial preparation includes denitrifying paracocci (… Paracoccus denitrificans HY-1, Paracoccus denitrificans, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with accession number GDMCC NO: 62483; The bacterial preparation also includes *Pseudomonas stearothermia* (… Pseudomonas stutzeri YM-1, the *Pseudomonas schlegelii* strain described therein was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with accession number GDMCC NO: 62502; Based on the viable count, the ratio of *Pseudomonas stearothermii* YM-1 to *Paracoccus denitrificans* HY-1 is 1:2-3.

2. The application of the bacterial preparation of claim 1 in deodorization.

3. The application of the bacterial preparation of claim 1 in denitrification.

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