Aerobic denitrifying bacterium and application thereof

Abstract

The invention belongs to the technical field of environmental microorganisms, and particularly relates to an aerobic denitrifying bacterium and application thereof, the aerobic denitrifying bacterium is Raoultella sp. 752, is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 22, 2025, has the preservation number of CGMCC No.35338, and has the preservation number of CGMCC No.35338; the strain has the capability of synchronously degrading high-concentration AZM and nitrate, has the advantages of high adaptability, simplicity, feasibility and the like, and can be directly used for denitrification treatment of high-concentration AZM-containing wastewater in pharmacy, culture and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of environmental microorganisms, and particularly relates to an aerobic denitrifying bacterium and application thereof. BACKGROUND

[0002] The superimposed discharge of antibiotic residues and inorganic nitrogen pollution has become a prominent problem in water environment treatment. Azithromycin (AZM) is a macrolide antibacterial drug widely used in clinical and livestock breeding. Because of its strong resistance to biodegradation in the environment, AZM has been detected in sewage and surface water multiple times and may promote the spread of environmental drug resistance genes. Studies have shown that the concentration of AZM in animal breeding wastewater and municipal sewage is generally high, and about 60%-80% of the original drug can be discharged into water through urine and feces. At the same time, agricultural runoff and domestic sewage often contain high concentrations of inorganic nitrogen, which can easily cause eutrophication and degradation of aquatic ecosystems. However, under the condition of high concentration of AZM, the nitrification-denitrification process is easily inhibited, affecting the removal of nitrogen. SUMMARY

[0003] In order to solve the above technical problems, the application provides an aerobic denitrifying bacterium and application thereof.

[0004] The aerobic denitrifying bacterium strain provided by the application is Raoultella sp. 752, which was preserved in the China General Microbiological Culture Collection Center on July 22, 2025, and the preservation number is CGMCC No: 35338.

[0005] The aerobic denitrifying bacterium strain provided by the application can simultaneously degrade AZM and perform aerobic denitrification under the condition of high concentration of AZM, solving the problem in the prior art that the nitrification-denitrification process is easily inhibited under the condition of high concentration of AZM.

[0006] The aerobic denitrifying bacterium is applied to the preparation of a microbial agent for treating wastewater.

[0007] Preferably, the wastewater is azithromycin-containing wastewater.

[0008] Preferably, the microbial agent takes the aerobic denitrifying bacterium as an active ingredient.

[0009] Preferably, the microbial agent is a suspension or a fermentation broth.

[0010] Preferably, the preparation method of the microbial agent is as follows: the aerobic denitrifying bacterium is cultured in a BTB denitrification solid culture medium for 20h-30h to obtain a bacterial liquid, the bacterial liquid is centrifuged to collect a precipitate, the precipitate is washed and resuspended to make OD600 be 1.0, and the microbial agent is obtained.

[0011] Preferably, the microbial inoculant is added to the water body in an amount of 5-15% by volume.

[0012] Preferably, the nitrogen is inorganic nitrogen.

[0013] Based on the similarity comparison of 16S rRNA genes, the strain of the application has the highest similarity to Raoultella sp., which is 99.69%.

[0014] The application also provides domestication culture conditions, denitrification effects and applications of the above-mentioned strain.

[0015] The strain of the application is a kind of bacteria with aerobic denitrification, which can remove AZM and inorganic nitrogen.

[0016] The strain of the application can use sodium acetate, sodium succinate and glucose as carbon sources to perform aerobic denitrification reaction under aerobic conditions, and simultaneously degrade AZM, and has excellent dual tolerance and metabolic efficiency.

[0017] The strain 752 of the application can make nitrate nitrogen finally removed as nitrous oxide or nitrogen.

[0018] The strain of the application can simultaneously and efficiently degrade AZM and perform aerobic denitrification in a liquid culture medium with 20mg / L AZM. The optimal temperature for degradation of the strain is 25-37℃, the optimal pH is pH≈7.0, the optimal carbon-nitrogen ratio is C / N≥10, and the strain can normally grow under the concentration of azithromycin of 0-40mg / L.

[0019] Compared with the prior art, the application has the beneficial effects that: The application screens and isolates a Raoultella sp. strain with dual metabolic characteristics of azithromycin (AZM) degradation and aerobic denitrification from an antibiotic stress environment, and the acquisition of the strain provides a new functional resource for microbial remediation of complex contaminated water bodies.

[0020] The strain exhibits significant tolerance under high-concentration pollutant conditions, can stably grow in an environment with 40mg / L AZM and 200mg / L nitrate nitrogen coexisting, and can maintain a high level of aerobic denitrification efficiency. This shows that the strain not only has the survival ability under antibiotic stress, but also can efficiently complete nitrogen removal, and is suitable for treatment of multiple types of composite contaminated wastewater.

[0021] The strain of the application is inoculated into organic carbon wastewater with 40mg / L AZM and 200mg / L nitrate nitrogen, under the conditions of C / N=10 and temperature of 30℃, 49.65% of the nitrate nitrogen is removed within 72h, and 47.12% of the AZM is degraded. The strong AZM degradation capacity of the strain greatly improves its practicability in the antibiotic wastewater.

[0022] Unlike traditional multi-stage processes, the strain can simultaneously achieve antibiotic removal and nitrogen conversion in a single reactor, without the need for additional anaerobic zones or the addition of external carbon sources, significantly simplifying the operation process, reducing energy consumption and operation costs.

[0023] In summary, the Raoultella sp. provided by the present application has both AZM degradation and aerobic denitrification functions, has good drug resistance and nitrogen metabolism gene background, and can efficiently convert nitrate nitrogen into nitrogen and nitrous oxide. In typical complex pollution systems such as livestock breeding wastewater and pharmaceutical wastewater, the strain shows higher efficiency, more economy, and more green application prospects than existing denitrification processes, and provides a practical technical approach to solve the problem of coexistence of antibiotic and nitrogen pollution. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A phylogenetic tree of Raoultella sp. 752.

[0025] Figure 2 A concentration change curve of nitrous oxide of Raoultella sp. 752 under the stress of nitrate nitrogen and AZM.

[0026] Figure 3 A removal efficiency of nitrate nitrogen and AZM of Raoultella sp. 752 within 72h of culture.

[0027] Figure 4 A pseudo-first-order kinetics fitting curve of nitrate nitrogen degradation of Raoultella sp. 752 within 72h.

[0028] Figure 5 A pseudo-first-order kinetics fitting curve of AZM of Raoultella sp. 752 within 72h. DETAILED DESCRIPTION

[0029] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0030] In the following examples, the percentage content is mass percentage content unless otherwise specified.

[0031] The common chemicals used in the following examples were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. with analytical grade; the mobile phase chemicals such as formic acid and methanol were chromatographically pure and purchased from Merck KGaA, Germany; the azithromycin antibiotic purity was > 98% and purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.

[0032] Example 1 Domestication, isolation and identification of strains The following are the culture media with different functions in the experiment as follows: Denitrification liquid medium: KNO31.2 g / L, glucose 3.0 g / L, KH2PO40.15 g / L, MgSO4·7H2O 0.05 g / L, pH 7.0±0.2, trace element solution 1 mL / L. The pH was adjusted to 7.0±0.2 with 20% KOH solution, and high-pressure sterilization was carried out in a 121℃ high-pressure sterilization pot for 25 min. The trace element solution composition: EDTA 0.5 g / L, FeSO40.15 g / L, ZnSO40.01 g / L, MnCl20.02 g / L, H3BO30.3 g / L, CoCl20.01 g / L, CuCl20.001 g / L, NiCl20.001 g / L, NaMoO40.003 g / L.

[0033] BTB denitrification solid medium: 1.5 mL of 10% bromothymol blue (BTB) aqueous solution, 40 mg of CAP and 10 g of agar powder were added to 1 L of denitrification liquid medium. High-pressure sterilization was carried out in a 121℃ high-pressure sterilization pot for 25 min, and the temperature was cooled to about 40℃ when pouring the plate, and after solidification, it was inverted for standby.

[0034] The domestication and isolation method of the strain has the following steps: The active sludge of a pharmaceutical factory in Xi'an, Shaanxi Province was used as the inoculum, and the active sludge was centrifuged at high speed using a high-speed centrifuge at 9000 r / min, and 2 g of the lower bacterial body was inoculated into 200 ml of denitrification liquid medium.

[0035] AZM was added to the denitrification liquid medium inoculated with the bacterial body, and the concentration of AZM was gradually increased to 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, and 40 mg / L, a total of 8 stages, each stage for 7 days. During the domestication period, the concentration of nitrate nitrogen was monitored daily using a gas-phase molecular absorption spectrometer, and the concentration of AZM was monitored using HPLC-MS.

[0036] The bacterial liquid after domestication was gradiently diluted (10 -3 ~10 -6Afterwards, the bacterial liquid was evenly coated on the BTB denitrification solid medium, and cultured at 30°C for 3-5 days. The denitrification process caused the medium to change color due to the generation of N2O. Single colonies with a blue discoloration ring with a diameter of ≥1.5 cm around the colonies were selected and purified by plate streaking for 4 times.

[0037] The purified strain was inoculated into the denitrification liquid medium, and AZM was added to the denitrification liquid medium to make the concentration of AZM 40 mg / L. The residual concentrations of nitrate nitrogen and AZM were measured every 24 h. The bifunctional strain with the ability of aerobic denitrification and AZM degradation was selected, and the bifunctional strain with the ability of aerobic denitrification and AZM degradation was recorded as Raoultella sp 752.

[0038] The target strain was subjected to 16S rRNA gene PCR amplification. 27F (5'-AGAGTTTGATCMTGGCTCAG-3', recorded as SEQ ID NO. 1) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3', recorded as SEQ ID NO. 2) were used as primers for PCR amplification, wherein M represents base A or base C, Y represents base C or base T, 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles, 72°C final extension for 10 min. After sequencing, the strain classification was confirmed by NCBI BLAST comparison.

[0039] The sequencing results were subjected to BLAST comparison in the NCBI database. The results showed that the strain Raoultella sp 752 had a homology of more than 99% with Raoultella sp. Combined with morphological and physiological and biochemical characteristics, the strain Raoultella sp 752 was determined to be a bacterium of the genus Raoultella, and was classified and named as Raoultella sp.

[0040] Example 2 Concentration change of nitrous oxide of strain Raoultella sp 752 under the stress of nitrate nitrogen and AZM The strain Raoultella sp 752 with the ability of simultaneously degrading AZM and aerobic denitrification can completely convert nitrate nitrogen into nitrogen gas. The concentration change of the intermediate product nitrous oxide can be measured to determine the generation of nitrogen gas. The specific steps are as follows: 100 mL of denitrification liquid medium was added to a 300 mL brown serum bottle, and the serum bottle was placed in an autoclave for sterilization at 121°C for 20 min.

[0041] After cooling, the strain Raoultella sp 752 was inoculated into the denitrification liquid medium, sealed and placed in a constant temperature shaker at 30℃ and 160rpm for 96h, during which the nitrous oxide concentration in the headspace gas was determined every 24h using gas chromatography.

[0042] The results, as shown in Figure 2 Figure 1, showed that the nitrous oxide reached a peak of 155ppm at 48h, and then gradually decreased to 136ppm at 72h, indicating that the strain Raoultella sp 752 could further convert nitrous oxide into nitrogen, completing the whole denitrification process.

[0043] Example 3 Degrading ability of the strain Raoultella sp 752 under different environmental factors. The different environmental factors and degradation results are shown in Table 1.

[0044] Table 1 Different environmental factors and degradation results Note: "-" represents a non-optimal value, and "★" represents an optimal value.

[0045] The range of carbon sources The strain Raoultella sp 752 was cultured in denitrification liquid medium containing different carbon sources (glucose, sodium succinate, sodium acetate) at 30℃ and 150rpm for 48h, and AZM and nitrate nitrogen showed different removal efficiencies, but both had good growth rate and degradation effect, indicating that the carbon source adaptation range of the strain Raoultella sp 752 was very wide.

[0046] The range of temperature The strain Raoultella sp 752 could grow between 25-38℃ and still maintain good pollutant removal effect.

[0047] The range of pH The strain Raoultella sp 752 could grow, denitrify and degrade AZM under the conditions of pH 6-8, but the growth rate was the fastest under neutral conditions (pH≈7.0).

[0048] The range of AZM concentration The strain Raoultella sp 752 could grow normally and perform aerobic denitrification under the concentration range of 0-40mg / L of AZM, indicating that the strain AZM had high tolerance to AZM.

[0049] Carbon-nitrogen ratio The AZM and denitrification effects of the strain Raoultella sp 752 change with the change of the carbon-nitrogen ratio. Since the strain has a faster growth rate at a high concentration of the carbon-nitrogen ratio, the removal effects of the AZM and the nitrate nitrogen are optimal under the condition of C / N >= 10.

[0050] Example 4 Degradation ability of the strain Raoultella sp 752 to AZM and nitrate nitrogen within 72h The initial concentration of the AZM in the denitrification liquid culture medium is set to 40mg / L, and the initial concentration of the nitrate nitrogen is set to 200mg / L, and 200ml is taken in a 500ml conical flask.

[0051] The strain Raoultella sp 752 is picked from the BTB denitrification solid culture medium, diluted with physiological saline to OD600 = 1.0 to obtain a bacterial solution, and 10mL of the bacterial solution is inoculated into the above denitrification liquid culture medium, and the concentrations of the residual AZM and the nitrate nitrogen are measured every 24h.

[0052] As shown in Figure 3 , the strain Raoultella sp 752 shows the ability of simultaneously degrading AZM and aerobic denitrification. After 72h of inoculation culture, the degradation rates of the AZM and the nitrate nitrogen can reach 47.12% and 49.65%, respectively.

[0053] Among 0-48h, the concentration of the bacterial solution rapidly rises, and the AZM and the nitrate nitrogen are rapidly degraded, which is a rapid degradation stage.

[0054] As shown in Figure 4 , the degradation of the AZM within 72h conforms to pseudo-first-order kinetics, and the degradation rate constant k reaches 0.00870h -1 .

[0055] As shown in Figure 5 , the degradation of the nitrate nitrogen within 72h conforms to pseudo-first-order kinetics, and the degradation rate constant k reaches 0.00966 h -1 .

[0056] The present application obtains a strain Raoultella sp., which not only can efficiently degrade AZM, but also can remove nitrate nitrogen in a single system. The strain has both antibiotic resistance and aerobic denitrification ability, and has potential industrialization prospects for integrated wastewater treatment and compound pollution treatment.

[0057] It should be noted that when the present application claims involving numerical ranges, it should be understood that each numerical range has two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0058] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0059] It is apparent that those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. It is therefore intended that the present application embrace all such changes and modifications as fall within the scope of the claims and their equivalents.

Claims

1. An aerobic denitrifying bacterium, characterized in that, The aerobic denitrifying bacteria is Raoultellasp. 752, which was deposited on July 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35338.

2. The application of the aerobic denitrifying bacteria according to claim 1 in the preparation of microbial agents for wastewater treatment.

3. The application according to claim 2, characterized in that, The wastewater is wastewater containing azithromycin and / or nitrogen.

4. The application according to claim 3, characterized in that, The nitrogen mentioned is inorganic nitrogen.

5. The application according to claim 2, characterized in that, The microbial agent uses the aerobic denitrifying bacteria as its active ingredient.

6. The application according to claim 2, characterized in that, The microbial agent is a suspension or fermentation broth of the aerobic denitrifying bacteria.

7. The application according to claim 6, characterized in that, The suspension is prepared by culturing the aerobic denitrifying bacteria in a culture medium for 20-30 hours to obtain a bacterial solution, centrifuging to collect the precipitate, washing the precipitate and resuspending it to achieve an OD600 of 0.8-1.5, thus obtaining the suspension.

8. The application according to claim 6, characterized in that, The amount of the suspension added to the water body is 5% to 15% of the water volume.

Images