Denitrifying paracoccus resistant to polypeptide antibiotics and use thereof

Abstract

The present invention relates to a method for treating wastewater in the presence of polymyxin B, the method comprising using aerobic denitrifying paracocci in the presence of a specific carbon source.

Description

Technical Field

[0001] This invention belongs to the field of wastewater biological treatment technology, specifically relating to a denitrifying paracoccus that can tolerate polypeptide antibiotics under aerobic conditions and can simultaneously remove nitrate nitrogen from water, and a method for optimizing its denitrification efficiency. Background Technology

[0002] Peptide antibiotics are a class of antibiotics with polypeptide structures that can directly act on bacterial cell membranes, increasing membrane permeability and leading to cell lysis and death. Due to their unique bactericidal mechanism, bacterial resistance to these antibiotics develops slowly. Therefore, peptide antibiotics are widely used in the clinical treatment of bacterial infections and as feed additives in livestock farming and aquaculture. Studies have shown that most antibiotics (30-90%) eventually accumulate in wastewater treatment plants through urban sewage systems. The presence of antibiotics affects the function and diversity of microorganisms in the environment and promotes the emergence of drug-resistant bacteria, endangering human health. Research has shown that peptide antibiotics have a strong inhibitory effect on anaerobic ammonia oxidation and nitrification processes.

[0003] Aerobic denitrification is a promising new wastewater treatment technology that relies on the ability of aerobic denitrifying bacteria to utilize both oxygen and nitrate as electron acceptors, enabling simultaneous nitrification and denitrification. Compared to traditional denitrification processes, aerobic denitrification offers advantages such as simpler processes, better nitrogen removal efficiency, and no need for additional acid or alkali addition, attracting widespread attention.

[0004] Given the broad application prospects of aerobic denitrification in wastewater denitrification and the widespread use of peptide antibiotics, reducing the inhibitory effect of peptide antibiotics on aerobic denitrifying bacteria will provide a reference for optimizing wastewater denitrification efficiency in practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the tolerance of aerobic denitrifying bacteria, such as Paracoccus denitrificans, to polypeptide antibiotics, such as polymyxin B, thereby optimizing the denitrification effect of wastewater and reducing the impact of polypeptide antibiotics on the wastewater denitrification process.

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

[0007] The *Paracoccus denitrificans* used in this invention is an aerobic denitrification model bacterium capable of removing nitrate nitrogen in an aerobic environment. In a specific embodiment, the *Paracoccus denitrificans* is *Paracoccus denitrificans* PD1222.

[0008] In a specific implementation scheme, the polypeptide antibiotic is polymyxin B (PMB).

[0009] The method for culturing denitrifying paracocci of the present invention involves inoculating denitrifying paracocci, such as denitrifying paracocci PD1222, into an aerobic denitrification medium and culturing it aerobically at 30°C and 180 rpm on a shaker.

[0010] In this field, aerobic denitrification media typically use glucose as a carbon source, and the elemental ratios of these media are usually adjusted according to individual experiments. Potassium nitrate and NO3 are commonly used as nitrogen sources in denitrification media. - The concentration range can be 20-500 mg / L, the carbon source concentration is determined according to the nitrogen source, and the C / N ratio can be 1-20. The remaining phosphates mainly provide potassium and sodium ions, and act as a pH buffer to keep the solution relatively stable.

[0011] The C / N ratio used in this invention is fixed at 5-10, preferably 5. Under the condition that other conditions remain unchanged, changing the type of carbon source can effectively improve the denitrification efficiency of Paracoccus denitrifying under polymyxin stress. The pH is 7.40 ± 0.10. The carbon source is selected from disaccharides, such as sucrose or trehalose.

[0012] In a specific implementation scheme, the composition of the aerobic denitrification culture medium of the present invention is as follows:

[0013] Sucrose or trehalose, KNO3 0.72-2.14 g / L, MgSO4 0.1-0.5 g / L, KH2PO4 1.5 g / L, Na2HPO4 7.9 g / L, trace element solution 1-2 mL / L, pH adjusted to 7.40 ± 0.10, the trace element solution composition is: Na2-EDTA 7.30 g / L, FeSO4·7H2O 2.50 g / L, MnCl2·4H2O 0.02 g / L, Na2MoO4·2H2O 0.242 g / L, CuCl2·2H2O 0.135 g / L, ZnCl2 0.34 g / L, wherein the C / N ratio is 5-10, preferably 5. Based on the C / N ratio and the concentration of KNO3, those skilled in the art can determine the concentration of the carbon source.

[0014] The formula for calculating carbon source concentration is: CON c-COD = CON NO3-N * k C / N

[0015] CON c-COD CON is the required carbon source concentration. NO3-N The concentration of the carbon source used, i.e., KNO3; k C / N This refers to the C / N ratio used.

[0016] In a specific implementation scheme, the composition of the aerobic denitrification culture medium of the present invention is as follows:

[0017] 4.75 g / L trehalose or 4.75 g / L sucrose, 1.44 g / L KNO3, 0.1 g / L MgSO4, 1.5 g / L KH2PO4, 7.9 g / L Na2HPO4, 1 mL / L trace element solution, adjusted to pH 7.40 ± 0.10. The trace element solution composition is: 7.30 g / L Na2-EDTA, 2.50 g / L FeSO4·7H2O, 0.02 g / L MnCl2·4H2O, 0.242 g / L Na2MoO4·2H2O, 0.135 g / L CuCl2·2H2O, 0.34 g / L ZnCl2.

[0018] The *Paracoccus denitrificans* described above is characterized by its ability to tolerate polymyxin B concentrations in the range of 0-0.75 mg / L. Preferably, the polymyxin B concentration is 0-0.5 mg / L.

[0019] The aforementioned *Paracoccus denitrificans* can perform aerobic denitrification using organic matter as a carbon source, thereby efficiently removing nitrate nitrogen. Preferably, this strain can still effectively remove nitrate nitrogen in the presence of polymyxin B, with a polymyxin B concentration ranging from 0 to 0.75 mg / L. More preferably, the polymyxin B concentration is 0 to 0.5 mg / L.

[0020] When trehalose and sucrose are used as carbon sources, the impact of PMB on the denitrification capacity of Paracoccus denitrifying can be minimized, for example, the total nitrogen removal rate can reach more than 80% at 48h.

[0021] The beneficial effects of this invention are:

[0022] (1) The denitrifying paracoccus used in this invention can tolerate high concentrations of polypeptide antibiotics and perform efficient aerobic denitrification.

[0023] (2) Optimizing the selection of carbon sources can minimize the impact of polypeptide antibiotics on the denitrification performance of Paracoccus denitrifying. Disaccharide carbon sources such as trehalose and sucrose are preferred. This provides a feasible optimization scheme for the actual treatment process of denitrification of antibiotic-containing wastewater. Attached Figure Description

[0024] Figure 1 The effect of different concentrations of polymyxin on the aerobic denitrification characteristics of Paracoccus denitrifying;

[0025] Figure 2 The growth characteristics of *Paracoccus denitrificans* in the presence of 0.50 mg / L polymyxin B in different carbon source systems.

[0026] Figure 3 The aerobic denitrification characteristics of Paracoccus denitrifying in the presence of 0.50 mg / L polymyxin B in different carbon source systems were investigated. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0028] The technical solutions of one or more embodiments of this specification will be described in detail below through specific examples.

[0029] The strains involved in the following examples are from: Paraacoccus denitrificans PD1222 T It was purchased from the American Type Culture Collection, accession number: ATCC 19367.

[0030] The culture media and reagents used in the following examples are:

[0031] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.20 ± 0.10.

[0032] Aerobic denitrification medium: glucose 5 g / L, KNO3 1.44 g / L, MgSO4 0.1 g / L, KH2PO4 1.5 g / L, Na2HPO4 7.9 g / L, trace element solution 1 mL / L, pH adjusted to 7.40 ± 0.10, autoclaved for 20 min. Trace element solution composition: Na2-EDTA 7.30 g / L, FeSO4·7H2O 2.50 g / L, MnCl2·4H2O 0.02 g / L, Na2MoO4·2H2O 0.242 g / L, CuCl2·2H2O 0.135 g / L, ZnCl2 0.34 g / L, sterilized by filtration through a 0.22 μm membrane.

[0033] Example 1. Effect of PMB on the aerobic denitrification characteristics of Paranitrogenous bacteria.

[0034] (1) Preparation of denitrifying paracoccus suspension

[0035] Add 5 μL of the frozen glycerol culture to LB liquid medium and incubate at 30°C and 180 rpm until the turbidity OD of the medium reaches a certain level. 600 The concentration is 1-1.5. After washing the bacterial solution twice with sterile water by centrifugation, the culture medium components are removed, and a bacterial suspension is prepared for use.

[0036] (2) Setting the PMB concentration gradient

[0037] The PMB addition concentration gradient is: 0, 0.25 mg / L, 0.50 mg / L, and 0.75 mg / L. The specific implementation method is as follows: Prepare a 1.00 g / L PMB stock solution with sterile water, filter to remove bacteria, and then dilute with sterile water to 0.75 g / L, 0.50 g / L, and 0.25 g / L respectively. When using, add it to the aerobic denitrification medium at a ratio of 1 / 1000.

[0038] (3) Determination of aerobic denitrification performance of Paracoccus denitrifying bacteria

[0039] The Paracoccus denitrifying bacterial suspension was inoculated at a rate of 1% into aerobic denitrification medium containing different concentrations of PMB and cultured in a shaker at 30°C and 180 rpm. Every 12 hours, 1 mL of bacterial suspension was taken in a clean bench, centrifuged at 4°C and 12,000 rpm for 2 minutes, and the supernatant was used to determine the remaining nitrate nitrogen and residual total inorganic nitrogen.

[0040] The results show that, Figure 1As the concentration of PMB increased, the nitrate removal rate of *Paracococcus denitrifyingus* decreased significantly. At 36 h, the nitrate nitrogen removal rates were 68.2%, 54.9%, and 34.7% under the conditions of 0, 0.25, and 0.50 mg / L PMB addition, respectively; at 60 h, the nitrate nitrogen removal rates were 97.4%, 85.7%, and 85.3%, respectively. When the PMB concentration was 0.75 mg / L, there was no significant change in nitrate content within the 60 h culture time range, indicating that *Paracococcus denitrifyingus* tolerates PMB at concentrations ranging from 0 to 0.75 mg / L, and that PMB significantly inhibits the aerobic denitrification capacity of *Paracococcus denitrifyingus*.

[0041] Example 2. Effects of different carbon sources on the growth characteristics of *Parasitic Paracoccus*.

[0042] A suspension of *Paragonimella denitrifyingis* was prepared according to the method described in Example 1, and 1% of the inoculum was inoculated into a carbon source-optimized denitrification medium containing 0.50 mg / L PMB. Seven carbon source optimization groups were selected: glucose, fructose, trehalose, sucrose, glycerol, sodium acetate, and sodium succinate. The dosage of each carbon source was calculated based on the same carbon-to-nitrogen ratio: glucose 5 g / L, fructose 5 g / L, trehalose 4.75 g / L, sucrose 4.75 g / L, glycerol 4.37 g / L, sodium succinate 3.68 g / L, and sodium acetate 6.8 g / L. The remaining components remained consistent with the aerobic denitrification medium. The turbidity (OD) of the medium was measured every 12 hours. 600 And a growth curve was plotted. The results are as follows: Figure 2 As shown, under the presence of polypeptide antibiotics, *Paragonimus denitrificans* exhibits the best growth in culture media with trehalose and sucrose as carbon sources, followed by sodium acetate and fructose. The lowest cell growth is observed when sodium succinate is used as the carbon source. This indicates that *Paragonimus denitrificans* shows a preference for carbon source utilization when tolerating polypeptide antibiotic stress.

[0043] Example 3. Effects of different carbon sources on the aerobic denitrification characteristics of Paracoccus denitrifyingus

[0044] A suspension of *Paragonimus westermani* was prepared according to the method described in Example 1. A 1% inoculum was inoculated into seven groups of carbon source-optimized denitrification media containing 0.50 mg / L PMB, and the total nitrogen removal rate was measured after 48 hours of incubation. Figure 3 As shown, consistent with the growth characteristics, *Paracococcus denitrifyingans* achieved total nitrogen removal rates of 87.5% and 81.5% respectively when trehalose and sucrose were used as carbon sources, almost indistinguishable from the control group. However, when glucose, sodium acetate, glycerol, and sodium succinate were used as carbon sources, the total nitrogen removal rate was significantly inhibited by peptide antibiotics. This indicates that the selection of carbon sources for denitrification should be cautious when peptide antibiotics are present, and trehalose or sucrose should be preferred.

Claims

1. A method for denitrifying wastewater containing polypeptide antibiotics, comprising culturing Paraacoccus denitrificans PD1222 (accession number ATCC 19367) in an aerobic denitrification medium, wherein the polypeptide antibiotic is polymyxin B, the concentration of polymyxin B is in the range of 0.25-0.75 mg / L, and the composition of the aerobic denitrification medium is as follows: Sucrose or trehalose, KNO3 0.72-2.14 g / L, MgSO4 0.1-0.5 g / L, KH2PO4 1.5 g / L, Na2HPO4 7.9 g / L, trace element solution 1-2 mL / L, pH adjusted to 7.40 ± 0.10, trace element solution composition: Na2-EDTA 7.30 g / L, FeSO4·7H2O 2.50 g / L, MnCl2·4H2O 0.02 g / L, Na2MoO4·2H2O 0.242 g / L, CuCl2·2H2O 0.135 g / L, ZnCl2 0.34 g / L, with a C / N ratio of 5-10.

2. The method for denitrification treatment of wastewater containing polypeptide antibiotics as described in claim 1, wherein the concentration of polymyxin B is 0.25-0.5 mg / L.

3. The method for denitrification treatment of wastewater containing polypeptide antibiotics as described in claim 1, wherein the concentration of polymyxin B is 0.5 mg / L.

4. The method for denitrification treatment of wastewater containing polypeptide antibiotics as described in claim 1, wherein the C / N ratio is 5.

5. The method for denitrifying wastewater containing polypeptide antibiotics according to claim 1, wherein the composition of the aerobic denitrification medium is as follows: 4.75 g / L trehalose or 4.75 g / L sucrose, 1.44 g / L KNO3, 0.1 g / L MgSO4, 1.5 g / L KH2PO4, 7.9 g / L Na2HPO4, 1 mL / L trace element solution, adjusted to pH 7.40 ± 0.

10. The trace element solution composition is: 7.30 g / L Na2-EDTA, 2.50 g / L FeSO4·7H2O, 0.02 g / L MnCl2·4H2O, 0.242 g / L Na2MoO4·2H2O, 0.135 g / L CuCl2·2H2O, 0.34 g / L ZnCl2.

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