Sulfamethoxazole-degrading anaerobic bacterial strain sa-9 and application thereof

Abstract

The present application belongs to the field of environmental microbiology technology, in particular, the present application relates to a sulfamethoxazole anaerobic degradation strain SA-9 and its application. The present application is collected from the anaerobic sludge near the farm, after enrichment and domestication, a strain capable of efficiently and rapidly anaerobic degradation of sulfamethoxazole (SMX) is obtained after separation and purification, through morphological and molecular biological identification, the strain belongs to Cupidesulfovibrio genus, named Cupidesulfovibrio sp.SA-9. The strain can rapidly degrade SMX under anaerobic conditions, and the degradation half-life is 7.6 d. The strain of the present application can be used as a biological degradation bacteria for the bioremediation of SMX contaminated environment, equipment and facilities.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and in particular, this invention relates to a sulfamethoxazole anaerobic degrading strain SA-9 and its applications. Background Technology

[0002] Sulfamethoxazole [4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide, SMX] is a synthetic broad-spectrum antibiotic. Due to its chemical structure being similar to aminobenzoic acid, it competes with para-aminobenzoic acid, thus interfering with bacterial folic acid biosynthesis. It exhibits good antibacterial effects against most Gram-negative and Gram-positive bacteria. SMX can prevent and treat bacterial infections in livestock and humans, and is widely used in animal husbandry and the pharmaceutical industry.

[0003] SMX is not easily metabolized in humans and livestock, and most of it is excreted into the environment in the form of parent organisms or metabolites in feces, threatening ecological security. For example, SMX can significantly inhibit the normal photosynthesis of algae, reduce the diversity and richness of microbial communities, induce the transfer of resistance genes and enhance bacterial resistance, and more seriously, SMX can damage the human urinary, reproductive, hematopoietic, and renal systems. The main sources of SMX in the environment are wastewater and waste from medical treatment, industrial synthesis, and livestock and poultry farming. Among them, wastewater from SMX synthesis plants (28.4-1340 μg / L) and livestock and poultry manure from the livestock industry (140-18000 μg / L) have relatively high residual concentrations of SMX. The residual amount of SMX in the natural environment varies greatly in vertical space. For example, the residual concentrations of SMX in farmland soil, surface water, groundwater, and sediment are 0.9-671.5 μg / L, 0.036-4.3 μg / L, 0.009-1.1 μg / L, and 0.11-665 μg / L, respectively. More seriously, SMX residues have been detected in drinking water, reaching levels of 0.03-0.116 μg / L. It is evident that SMX can not only enter aerobic environments but also easily enter anaerobic environments, causing widespread and deep pollution and threatening ecological security and human health.

[0004] SMX can be eliminated in the environment through photolysis, chemical degradation, and microbial degradation, with microorganisms being the primary driver of SMX residue removal. Microbial remediation, with its advantages of simplicity, low cost, and high efficiency, is widely used for antibiotic removal in the environment. Currently, aerobic SMX-degrading strains such as *Achromobacter denitrificans* PR1, *Leucobacter* sp. GP, *Microbacterium* sp. BR1, and *Pseudomonas psychrophila* HA-4 have been isolated and screened; however, these are aerobic bacteria and not suitable for SMX removal in anaerobic environments. Due to SMX's high water solubility, it easily enters microaerobic or anaerobic environments such as livestock manure compost, wastewater, groundwater, and flooded soil during use. There are currently few reports on pure cultured anaerobic SMX-degrading strains; studies only exist on the anaerobic degradation of SMX by domesticated activated sludge, which hinders the propagation of anaerobic SMX-degrading strains through fermentation, increasing the cost of SMX remediation in anaerobic environments. Therefore, isolating and purifying SMX-degrading strains is of great significance for enriching SMX-degrading strain resources and reducing the remediation cost of SMX removal in anaerobic environments. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention obtained a strain capable of efficiently degrading sulfamethoxazole (SMX) under anaerobic conditions through extensive screening, thereby completing the present invention.

[0006] In a first aspect, the present invention provides a sulfamethoxazole (SMX) anaerobic degrading strain SA-9, which is classified as Cupidesulfovibrio sp.SA-9, and was deposited on October 31, 2024 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20242405, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The anaerobic degrading strain SA-9 of the present invention forms round colonies on MPM solid plates with a diameter of 0.5-3.0 mm, a moist, regular, and pale white surface; the bacterial cells are curved rod-shaped, flagellated, and 0.5-0.8 μm × 1.8-2.5 μm in size.

[0008] In a second aspect, the present invention provides a bacterial agent for degrading the antibiotic SMX, the bacterial agent containing Cupidesulfovibrio sp. SA-9. In some examples, the bacterial agent is a liquid bacterial agent, a solid bacterial agent, or a powder. Liquid bacterial agents include: emulsions, suspensions, and bacterial fermentation broths; in one embodiment, the bacterial agent is a solid bacterial agent prepared by immobilizing bacterial cells on a solid support. In one embodiment, the bacterial agent is a liquid bacterial agent prepared by resuspending bacterial cells in a buffer solution. Powders include lyophilized powders and wettable powders. The amount of Cupidesulfovibrio sp. SA-9 in the bacterial agent is 1.0 × 10⁻⁶. 4 –1.0×1.0 8 cfu / mL.

[0009] A third aspect of the invention provides the application of anaerobic strain SA-9 in the degradation of the antibiotic SMX. The application involves using anaerobic strain SA-9 to degrade SMX in the environment or facilities; preferably, the environment is SMX-contaminated livestock manure compost, livestock wastewater, groundwater, flooded soil, and wetlands. The facilities are wastewater treatment equipment or facilities; in one embodiment, the wastewater treatment facility is an anaerobic reactor. In one embodiment, the flooded soil is paddy field soil.

[0010] This invention utilizes anaerobic sludge collected from near a livestock farm. After enrichment, domestication, and purification, a strain capable of efficiently and rapidly degrading SMX anaerobically was obtained. Morphological and molecular biological identification confirmed that this strain belongs to the genus *Cupidesulfovibrio* and is named *Cupidesulfovibrio* sp. SA-9. This strain can rapidly degrade SMX under anaerobic conditions, with a degradation half-life of 7.6 days. This indicates that the anaerobic strain SA-9 can be used as a biodegrading bacterium for the bioremediation of SMX-contaminated environments.

[0011] Beneficial effects:

[0012] (1) This invention isolates and screens an anaerobic degrading strain SA-9. The test results show that the strain is a Gram-negative bacterium with flagella. Under strict anaerobic conditions, the half-life of SMX at a concentration of 100 μmol / L is 7.6 days, which shows that it has a high efficiency in anaerobic degradation of SMX. This enriches the seed resource bank of antibiotic anaerobic degradation and provides a basis for developing new antibiotic residue pollution remediation strategies.

[0013] (2) The anaerobic degrading strain SA-9 isolated by this invention can be applied in natural environments or wastewater treatment equipment. Attached Figure Description

[0014] Figure 1Colony and cell morphology of anaerobic degrading strain SA-9, where A represents the colony morphology of anaerobic degrading strain SA-9 and B represents the cell morphology.

[0015] Figure 2 Phylogenetic analysis of the 16S rRNA gene of anaerobic degrading strain SA-9;

[0016] Figure 3 The effects of various conditions on the degradation of SMX by the anaerobic degrading strain SA-9, where A is the effect of temperature on the degradation of SMX by the anaerobic degrading strain SA-9, B is the effect of pH on the degradation of SMX by the anaerobic degrading strain SA-9, C is the effect of electron acceptor on the degradation of SMX by the anaerobic degrading strain SA-9, and D is the effect of electron donor on the degradation of SMX by the anaerobic degrading strain SA-9.

[0017] Figure 4 Anaerobic strain SA-9 was used for the anaerobic remediation of SMX in flooded paddy soil. A and B were sterilized flooded paddy soil, while C and D were naturally flooded paddy soil. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0019] The culture medium formulations described in the following examples are as follows:

[0020] Anaerobic enrichment and acclimatization medium (MSM, g / L): K2HPO4·3H2O, 0.35g; KH2PO4, 0.27g; MgCl2·6H2O, 0.15g; NH4Cl, 0.53g; CaCl2·2H2O, 0.073g; Sodium pyruvate, 0.5g; FeSO4·7H2O, 0.5g (added before boiling and dispensing); Ascorbic acid, 0.1g (added before boiling and dispensing); Cysteine ​​hydrochloride, 0.2g (added before boiling and dispensing); Resazurin, 1.0mg; Trace element complex solution, 1.0mL; Vitamin complex solution, 1.0mL (added after sterilization); pH 7.0, diluted to 1.0L with pure water.

[0021] Anaerobic isolation and culture medium (MPM, g / L): NH4Cl, 1.0 g; NaCl, 1.0 g; K2HPO4·3H2O, 0.5 g; MgCl2·6H2O, 0.05 g; Na2SO4, 1.1 g; CaCl2·2H2O, 0.05 g; sodium lactate, 2.0 g; ascorbic acid, 0.10 g; cysteine ​​hydrochloride, 0.2 g; yeast extract, 1.0 g; resazurite, 1.0 mg; trace element complex solution, 1.0 mL; vitamin complex solution, 1.0 mL; pH adjusted to 7.2 with phosphate buffer, and purified water added to 1.0 L. Solid culture medium was supplemented with 1.7% agar.

[0022] Deoxygenation of the culture medium: Boil the prepared culture medium, and while it is still hot, dispense 30 mL of the medium into 50 mL serum bottles. Seal the bottles with rubber stoppers and secure them with aluminum caps. Purge with nitrogen for 30 minutes to remove oxygen, then autoclave at 115°C for 30 minutes. The sterilized culture medium should remain colorless. If it turns slightly red or red, it indicates that oxygen has not been completely removed.

[0023] SMX detection conditions: High performance liquid chromatography (1260 Infinity II, Agilent, USA); Agilent XDB-C 18 (5μm, 4.6×250mm); Mobile phase: acetonitrile: 0.1% formic acid water = 30:70 (V / V); Detection wavelength: 270nm; Flow rate: 1.0mL / min; Column temperature: 30℃; Injection volume: 20μL.

[0024] In this invention, "anaerobic degrading strain SA-9" and "Cupidesulfovibrio sp.SA-9" both refer to the strain with accession number CCTCC NO:M 20242405 screened in this invention. Those skilled in the art will understand that they have the same meaning in this invention.

[0025] Example 1: Isolation and Identification of Strain SA-9

[0026] (1) Enrichment, domestication and isolation of SMX anaerobic degrading bacteria

[0027] Anaerobic activated sludge collected from around the drainage outlets of livestock and poultry farms was inoculated into serum bottles containing 100 μM SMX and 30 mL MSM, and incubated at 37°C and 150 rpm for 7 days. The previous generation enrichment solution was transferred at a concentration of 5% to 30 mL of fresh MSM containing 100 μmol / L SMX. The SMX degradation rate in the bottle was measured to be above 90% after each transfer, and this process was repeated up to four generations. The fifth generation enrichment solution was then spread onto MPM solid medium containing 100 mg / L SMX using the dilution plating method in an anaerobic chamber. The plates were inverted and placed in an anaerobic jar, where they were incubated at 37°C for 7 days. After single colonies grew on the plates, they were streaked multiple times for purification, and their SMX degradation effect was verified.

[0028] Using SMX as the selection pressure, an SMX-degrading anaerobic bacterium, designated SA-9, was finally isolated and purified. It was classified and named Cupidesulfovibrio sp. SA-9, and deposited on October 31, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20242405, located at Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0029] (2) Morphological and molecular biological identification of anaerobic degrading strain SA-9

[0030] In an anaerobic chamber, anaerobic strain SA-9 was inoculated onto MPM solid plates and incubated at 37℃ for 7 days. Colony morphology was observed during this period. The colonies of anaerobic strain SA-9 on MPM solid plates were round, with a diameter of 0.5-3.0 mm, and had a moist, regular, and pale white surface. Figure 1 (A); the bacterial cells are curved rod-shaped, flagellated, and 0.5-0.8 μm × 1.8-2.5 μm in size. Figure 1 (B). This strain is Gram-negative and grows strictly anaerobicly; it can grow at temperatures ranging from 15 to 47°C, with an optimal growth temperature of 30 to 37°C; it can also grow at pH values ​​ranging from 5.0 to 9.0, with an optimal growth pH of 7.0 to 8.0.

[0031] Total DNA was extracted from anaerobic strain SA-9 using a high-salt method. The 16S rRNA gene sequence of strain SA-9 was amplified using the universal primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) as a template. The PCR amplification product was sequenced by a sequencing company. The 16S rRNA gene sequence of strain SA-9 obtained from the sequencing was compared with the 16S rRNA genes of reported type strains in the EzBioCloud database (www.ezbiocloud.net). Sequences with high homology to type strains were selected, and a phylogenetic tree of resistant strains was constructed using the neighbor-joining method with MEGA 11.0 software. Figure 2 As shown, the 16S rRNA gene sequence of strain SA-9 isolated in this invention is similar to that of Cupidesulfovibrio liaohensis XJ01. T The highest homology is 99%.

[0032] Therefore, based on the morphological and molecular biological identification of the anaerobic strain SA-9, it was identified as belonging to the genus *Cupidesulfovibrio* and named *Cupidesulfovibrio* sp. SA-9. The strain SA-9 isolated in this invention belongs to the genus *Cupidesulfovibrio*, enriching the seed resource bank of SMX-degrading bacteria. This strain was deposited on October 31, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20242405, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0033] Example 2: Study on the degradation characteristics of SMX by anaerobic strain SA-9

[0034] Add 1.0 mL of bacterial culture (OD500) of strain SA-9 to the deoxygenated MSM. 600 =0.6) and a certain amount of SMX were added to make a final concentration of 100 μM, and then the mixture was incubated in a shaker at 37℃ and 150 rpm for 14 days. The concentration of SMX was measured after 14 days. The control group did not contain strain SA-9. Each control group and treatment group had three replicates. Following the above procedure, the effects of different temperatures (15℃, 20℃, 25℃, 30℃, 37℃, 42℃ and 47℃), different pH (5.0, 6.0, 7.0, 8.0 and 9.0), different electron acceptors (Na2SO4, NaNO3 and FeCl3), and different electron donors (sodium formate, sodium acetate, sodium pyruvate, sodium lactate and sodium succinate) on the degradation of SMX by anaerobic strain SA-9 were studied.

[0035] Depend on Figure 3 It can be seen that the anaerobic strain SA-9 achieves a degradation rate of SMX greater than 50% at temperatures of 25-42℃ and greater than 70% at temperatures of 30-42℃, with the optimal temperature for SMX degradation being 30-37℃. Figure 3 (A). At pH 6.0-9.0, the anaerobic strain SA-9 exhibits a degradation rate of over 60% for SMX, with the optimal pH for SMX degradation being 7.0-8.0. Figure 3 (B); The effects of different electron acceptors on the degradation of SMX by anaerobic strain SA-9, as shown in Figure B). Figure 3 As shown in Figure C, without an electron acceptor, strain SA-9 cannot degrade SMX. With the addition of Na₂SO₄, NaNO₃, and FeCl₃ as electron acceptors, strain SA-9 can degrade SMX in all of them. The degradation rate of SMX is: Na₂SO₄ > NaNO₃ > FeCl₃, indicating that sulfate is the optimal electron acceptor for the anaerobic degradation of SMX by strain SA-9. The effects of different electron donors on the degradation of SMX by strain SA-9 are shown below. Figure 3 As shown in Figure D, compared with MSM without added electron acceptors, strain SA-9 with added sodium acetate and sodium succinate hardly degraded SMX; when sodium pyruvate and sodium lactate were added, strain SA-9 was able to rapidly degrade SMX, indicating that the optimal electron acceptors for anaerobic degradation of SMX by strain SA-9 are sodium pyruvate and sodium lactate.

[0036] The results above show that the optimal temperature range for anaerobic strain SA-9 to degrade SMX is 30-37℃, the optimal pH range is 7.0-8.0, the optimal electron acceptor is sulfate, and the optimal electron donors are sodium pyruvate and sodium lactate.

[0037] Example 3: Determination of the anaerobic degradation ability of anaerobic strain SA-9 on SMX

[0038] 1.0 mL of anaerobic degrading strain SA-9 bacterial culture (OD) 600 =0.6) was added to 30 mL of deoxygenated MSM at pH 7.0, followed by the addition of certain amounts of SMX to achieve final concentrations of 10 μM, 100 μM, 200 μM, and 400 μM. The mixture was then incubated at 37 °C and 150 rpm in a shaker. Smoke samples were taken at different time points to determine the SMX concentration, and the anaerobic degradation dynamics of SMX were fitted to calculate its half-life. The control group did not receive the anaerobic strain SA-9. Both the control and treatment groups were replicated three times.

[0039] The experimental results of anaerobic degrading strain SA-9 on SMX at different concentrations are shown in Table 1. The degradation dynamics of SMX by strain SA-9 conform to the first-order kinetic equation. With time as the horizontal axis and the residual concentration of SMX as the vertical axis, the first-order kinetic equation (C = COe^(-1 / 2)) is used to calculate the degradation dynamics.-kt The fitting equation is derived from the correlation coefficient R. 2 (0.9913-0.8552) indicates that the degradation dynamics of SMX by anaerobic strain SA-9 follows first-order kinetics. The degradation half-life (T0) of SMX by anaerobic strain SA-9 at concentrations of 10 μM, 100 μM, 200 μM, and 400 μM (T0) are shown below. 1 / 2 The degradation times (ln2 / k) were 6.0 days, 7.6 days, 11.6 days, and 30.8 days, respectively. The anaerobic strain SA-9 obtained in this invention exhibits high degradation efficiency for SMX and shows great potential for SMX pollution remediation.

[0040] Table 1. First-order kinetic parameters fitted by strain SA-9 for anaerobic degradation of different concentrations of SMX

[0041]

[0042] Example 4: Application of anaerobic strain SA-9 in SMX bioremediation of paddy field flooded soil pollution

[0043] Soil samples were collected from a flooded paddy field in Hefei City and stored in a sealed plastic bag in a cool place. The soil was diluted with MSM in a proportional ratio, filtered through gauze, and 60 mL was transferred to 100 mL vials. A certain amount of SMX was added to achieve concentrations of 10 μM and 100 μM, respectively. The vials were then sealed with butyl rubber stoppers and secured with aluminum caps. High-purity nitrogen was introduced for 30 minutes to remove dissolved oxygen. The flooded soil mixture was divided into two portions: one portion was autoclaved (sterilized soil), and the other portion was left unsterilized (natural soil). In an anaerobic chamber, a certain amount of SMX and 2.0 mL of bacterial culture of strain SA-9 (OD200) were added to each sample. 600 =0.6), the samples were placed in a shaker at 37℃ and 150rpm and the concentration of SMX in the bottles was measured at different time points. The control group was not added with bacterial solution. There were three replicates for each control group and treatment group. The experimental groups were set as follows: (1) sterilized soil + SMX (10μM / 100μM); (2) sterilized soil + SMX (10μM / 100μM) + strain SA-9; (3) natural soil + SMX (10μM / 100μM); (4) natural soil + SMX (10μM / 100μM) + strain SA-9.

[0044] The bioremediation effect of anaerobic degrading strain SA-9 on SMX in sterilized soil and natural soil is as follows: Figure 4As shown in the figure, in sterile flooded paddy soil, the decrease in SMX concentration in paddy soil without the addition of strain SA-9 was due to the adsorption of SMX by sterilized soil. The adsorption rates of 10 μM and 100 μM SMX by sterilized soil were less than 14% and 20%, respectively. The degradation half-lives of 10 μM and 100 μM SMX in sterile soil by strain SA-9 were 8.7 d and 9.6 d, respectively. In natural paddy soil without the addition of strain SA-9, the degradation half-lives of 10 μM and 100 μM SMX were 3.0 d and 2.6 d, respectively; while in natural paddy soil with the addition of strain SA-9, the degradation half-lives of 10 μM and 100 μM SMX were 1.5 d and 1.4 d, respectively. This indicates that the degradation efficiency of 10 μM and 100 μM SMX in flooded paddy soil by strain SA-9 was 2 times and 1.9 times that of the untreated strain. The above results indicate that the anaerobic strain SA-9 exhibits excellent remediation effects on SMX in flooded paddy field soil and has great application potential.

[0045] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A sulfamethoxazole-degrading anaerobic strain SA-9, characterized in that, The strain classification name is Cupidesulfovibrio sp.SA-9, preserved in China Center for Type Culture Collection on October 31, 2024, with a preservation number of CCTCC NO: M 20242405 and a preservation address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The sulfamethoxazole anaerobic degrading bacterial strain SA-9 according to claim 1, characterized by, The anaerobic degrading strain SA-9 forms round colonies on MPM solid plates, with a diameter of 0.5-3.0 mm. The surface is moist, regular, and pale white. The bacterial cells are curved rod-shaped, with flagella, and have a size of 0.5-0.8 μm × 1.8-2.5 μm.

3. A bacterial agent for degrading the antibiotic sulfamethoxazole, characterized in that, The bacterial agent contains the strain of claim 1 Cupidesulfovibrio sp. SA-9.

4. The bacterial agent of claim 3, characterized in that, The bacterial agent can be a liquid bacterial agent, a solid bacterial agent, or a powder.

5. The microbial agent according to claim 3, characterized in that, The number of sp. SA-9 in the bacterial agent was 1.0 × 10 Cupidesulfovibrio sp. SA-9 was 1.0 × 10 4 -1.0 × 1.0 8 cfu / mL.

6. The application of the sulfamethoxazole anaerobic degrading strain SA-9 according to claim 1, characterized in that, The anaerobic degrading strain SA-9 was used to degrade sulfamethoxazole in the environment or facilities.

7. The application according to claim 6, characterized in that, The environment refers to livestock and poultry manure compost, aquaculture wastewater, groundwater, flooded soil, and wetlands contaminated with sulfamethoxazole; the facility refers to a wastewater treatment facility.

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