Sulfamethoxazole anaerobic degradation strain SA-9 and application thereof

Abstract

The invention belongs to the technical field of environmental microorganisms, and particularly relates to a sulfamethoxazole anaerobic degradation strain SA-9 and application thereof. According to the present invention, anaerobic sludge collected from the vicinity of a farm is subjected to enrichment domestication, separation and purification to obtain the bacterial strain capable of efficiently and rapidly performing anaerobic degradation on sulfamethoxazole (SMX), and the morphological and molecular biological identification results show that the bacterial strain belongs to Cupidesulfavibrio, and is named as Cupidesulfavibrio sp.SA-9; the strain can rapidly degrade SMX under an anaerobic condition, and the degradation half-life period is 7.6 d. The strain disclosed by the invention can be used as a biodegradation bacterium to be applied to bioremediation of SMX polluted environments, equipment and facilities.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and in particular, the invention relates to a sulfamethoxazole anaerobic degradation strain SA-9 and an application thereof. Background Art

[0002] Sulfamethoxazole [4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide, sulfamethoxazole, SMX] is a synthetic broad-spectrum antibiotic. Because its chemical structure is similar to aminobenzoic acid, it will compete with p-aminobenzoic acid, thereby interfering with the biosynthesis of bacterial folate, and has a good antibacterial effect on most Gram-negative and Gram-positive bacteria. SMX can prevent and treat bacterial infections in livestock and poultry and humans, and is widely used in animal husbandry and the pharmaceutical industry.

[0003] SMX is not easily metabolized in humans and livestock. Most of it will be discharged into the environment in the form of the parent substance or metabolites with feces, threatening ecological safety. For example, SMX can significantly inhibit the normal photosynthesis of algae, reduce the diversity and richness of microbial communities, induce resistance gene transfer and enhance bacterial resistance. More seriously, SMX can damage the human urinary system, reproductive system, hematopoietic system and kidneys. The main sources of SMX in the environment are wastewater and waste discharged from medical treatment, industrial synthesis, livestock and poultry breeding, etc. Among them, the residual concentration of SXM in the wastewater of SMX synthesis plants (28.4-1340μg / L) and livestock and poultry feces (140-18000μg / L) in the breeding industry is relatively high. The amount of SMX residues 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 0.9-671.5μg / L, 0.036-4.3μg / L, 0.009-1.1μg / L and 0.11-665μg / L, respectively. What is more serious is that SMX residues were detected in drinking water, reaching 0.03-0.116μg / L. This shows that SMX will not only enter the aerobic environment, but is more likely to enter the anaerobic environment, causing widespread and deep pollution, threatening ecological security and life and health.

[0004] SMX can be eliminated in the environment through photolysis, chemical degradation and microbial degradation, among which microorganisms are the main driving force for eliminating SMX residual pollution in the environment. Microbial remediation has the advantages of simple operation, low cost and high efficiency, and is widely used in the removal of antibiotics in the environment. At present, the isolated and screened SMX aerobic degradation strains include Achromobacterdenitrificans PR1, Leucobacter sp.GP, Microbacterium sp.BR1 and Pseudomonaspsychrophila HA-4, etc. However, they are aerobic bacteria and are not suitable for the removal of SMX in anaerobic environments. Due to the high water solubility of SMX, it is easy to enter micro-aerobic or anaerobic environments such as livestock manure compost or wastewater, groundwater, and flooded soil during use. At present, there are few reports on pure culture strains for anaerobic degradation of SMX, and only the anaerobic degradation of SMX by domesticated activated sludge has been studied, which is not conducive to the expansion of SMX anaerobic degradation strains through fermentation, and increases the cost of remediation of SMX in anaerobic environments. Therefore, isolating and purifying SMX anaerobic degrading strains is of great significance for enriching the SMX degrading strain resources and reducing the remediation cost of removing SMX in anaerobic environments. Summary of the invention

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

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

[0007] The colonies of the anaerobic degradation strain SA-9 of the invention are round in the MPM solid plate, with a diameter of 0.5-3.0 mm, a moist, regular and pale white surface, and a curved rod-shaped bacterial body with flagella, with a size of 0.5-0.8 μm×1.8-2.5 μm.

[0008] In the second aspect of the present invention, there is provided a bacterial agent for degrading the antibiotic SMX, wherein the bacterial agent contains 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, which is prepared by fixing the bacterial cells on a solid phase carrier. In one embodiment, the bacterial agent is a liquid bacterial agent, which is prepared by resuspending the bacterial cells in a buffer solution. Powders include freeze-dried powders and wettable powders. The amount of Cupidesulfovibrio sp.SA-9 in the bacterial agent is 1.0×10 4 –1.0×1.0 8 cfu / mL.

[0009] In the third aspect of the present invention, the present invention provides the application of anaerobic strain SA-9 in degrading the antibiotic SMX. The application is to use anaerobic strain SA-9 to degrade SMX in the environment or equipment facilities; preferably, the environment is livestock and poultry manure compost, aquaculture wastewater, groundwater, flooded soil and wetland contaminated with SMX. The equipment facilities are wastewater treatment equipment and wastewater treatment facilities; in one embodiment, the wastewater treatment facility is an anaerobic reaction tank. In one embodiment, the flooded soil is paddy field soil.

[0010] The present invention collects anaerobic sludge from the vicinity of a breeding farm, and after enrichment and domestication, separates and purifies, obtains a strain capable of efficiently and rapidly anaerobic degradation of SMX. After morphological and molecular biological identification, the strain belongs to the genus Cupidesulfovibrio and is named Cupidesulfovibrio sp.SA-9. The strain can rapidly degrade SMX under anaerobic conditions, and the degradation half-life is 7.6d. It shows that the anaerobic strain SA-9 can be used as a biodegradation bacterium for bioremediation of SMX-contaminated environments.

[0011] Beneficial effects:

[0012] (1) The present invention isolates and screens an anaerobic degradation strain SA-9, and through testing, it is found that the strain is a Gram-negative bacterium with flagella. Under strict anaerobic conditions, the half-life of the strain for degradation of SMX at a concentration of 100 μmol / L is 7.6 days. It has a high efficiency in anaerobic degradation of SMX, enriches the seed resource library of anaerobic degradation of antibiotics, and provides a basis for the development of new antibiotic residue pollution remediation strategies.

[0013] (2) The anaerobic degradation strain SA-9 isolated by the present invention can be applied in natural environments or wastewater treatment equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1, the colony and bacterial morphology of the anaerobic degradation strain SA-9, wherein A is the colony morphology of the anaerobic degradation strain SA-9 and B is the bacterial morphology;

[0015] Figure 2 , 16S rRNA gene phylogenetic analysis of the anaerobic degradation strain SA-9;

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

[0017] Figure 4 , anaerobic remediation of SMX in flooded paddy soil by anaerobic strain SA-9, where A and B are sterilized flooded paddy soils, and C and D are naturally flooded paddy soils. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] The culture medium formula described in the following examples is 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 aliquoting); ascorbic acid, 0.1g (added before boiling and aliquoting); cysteine ​​hydrochloride, 0.2g (added before boiling and aliquoting); resazurin, 1.0mg; trace element complex solution, 1.0mL; vitamin complex solution, 1.0mL (added after sterilization); pH 7.0, made up to 1.0L with pure water.

[0021] Anaerobic separation 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; resazurin, 1.0 mg; trace element complex solution, 1.0 mL; vitamin complex solution, 1.0 mL; pH was adjusted to 7.2 with phosphate buffer and pure water was added to 1.0 L. 1.7% agar was added to the solid culture medium.

[0022] Deoxygenation of culture medium: Boil the prepared culture medium, and while it is still hot, dispense 30 mL of the culture medium into 50 mL serum bottles, plug the bottles with rubber stoppers and secure them with aluminum caps, fill them with nitrogen for 30 minutes to deoxygenate, and sterilize them with high-pressure steam at 115°C for 30 minutes. The culture medium should remain colorless after sterilization. If the color changes to slightly red or red, it means that oxygen has not been completely removed.

[0023] Detection conditions of SMX: HPLC (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 the present invention, "anaerobic degradation strain SA-9" and "Cupidesulfovibrio sp. SA-9" both refer to the strain screened by the present invention with a deposit number of CCTCC NO: M 20242405. Those skilled in the art can understand that they have the same meaning in the present invention.

[0025] Example 1 Isolation and identification of strain SA-9

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

[0027] Take the anaerobic activated sludge collected from the drainage outlet of livestock and poultry farms, inoculate it in a serum bottle containing 100μM SMX and 30mL MSM, and place it in a shaker at 37℃ and 150rpm for 7 days. Take the enriched liquid of the previous generation and transfer it to a fresh 30mL MSM containing 100μmol / LSMX at a content of 5%. The degradation rate of SMX in the test bottle is above 90% each time, and transfer it to the 4th generation. Take the enriched liquid of the 5th generation, and spread the diluted enriched liquid on the MPM solid culture medium containing 100mg / L SMX in the anaerobic operation box by dilution coating method, put the plate upside down in the anaerobic tank, and culture it at 37℃ for 7 days. After a single colony grows on the plate, pick a single colony and purify it by streaking multiple times, and verify its degradation effect on SMX.

[0028] With SMX as the selection pressure, a strain of SMX anaerobic degrading bacteria was finally isolated and purified, numbered SA-9. It was classified and named Cupidesulfovibrio sp.SA-9, and was deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number CCTCC NO:M 20242405. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

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

[0030] In the anaerobic operation box, the anaerobic strain SA-9 was inoculated on the MPM solid plate and cultured at 37°C for 7 days. The colony morphology of the anaerobic strain SA-9 on the MPM solid plate was round, with a diameter of 0.5-3.0 mm, and a moist, regular, pale white surface ( Figure 1 Middle A); the bacteria are curved rod-shaped, have flagella, and are 0.5-0.8μm×1.8-2.5μm in size ( Figure 1 (B). The strain is Gram-negative and grows strictly anaerobic; it can grow at temperatures between 15-47°C, with an optimum temperature of 30-37°C; it can grow at pH between 5.0-9.0, with an optimum pH of 7.0-8.0.

[0031] The total DNA of the anaerobic strain SA-9 was extracted using the high salt method, and the bacterial identification universal primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) were used to amplify the 16S rRNA gene sequence of the strain SA-9 using the extracted total DNA of the strain SA-9 as a template. The product after PCR amplification was commissioned to a sequencing company for sequencing, and the 16S rRNA gene sequence of the strain SA-9 obtained by sequencing was analyzed for homology with the 16S rRNA gene of the reported model strain in the EzBioCloud database (www.ezbiocloud.net). The model strain-related sequences with higher homology were selected, and the phylogenetic tree of the resistant strains was constructed by the neighbor-joining method using the MEGA 11.0 software. Figure 2 As shown in FIG. 1 , the 16S rRNA gene sequence of the strain SA-9 isolated and obtained in the present 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, the anaerobic strain SA-9 was identified as the genus Cupidesulfovibrio and named Cupidesulfovibrio sp.SA-9. The strain SA-9 isolated by the present invention belongs to the genus Cupidesulfovibrio in terms of classification, enriching the seed resource library of SMX-degrading bacteria. The strain was deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number CCTCC NO:M 20242405, and the deposit address is: 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] To the deoxygenated MSM, 1.0 mL of strain SA-9 bacterial suspension (OD 600 =0.6) and a certain amount of SMX to a final concentration of 100 μM, then cultured in a shaker at 37°C and 150 rpm, and sampled and measured the concentration of SMX after 14 days. The control group did not add strain SA-9, and there were three replicates in each control group and treatment group. According to the above operation, the effects of different temperatures (15°C, 20°C, 25°C, 30°C, 37°, 42°C and 47°C), 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 degradation strain SA-9 were studied.

[0035] Depend on Figure 3 It can be seen that the degradation rate of SMX by anaerobic strain SA-9 is greater than 50% at a temperature of 25-42°C and greater than 70% at a temperature of 30-42°C. The optimum temperature for SMX degradation is 30-37°C ( Figure 3 At pH 6.0-9.0, the degradation rate of SMX by anaerobic strain SA-9 was greater than 60%, and the optimal pH for SMX degradation was 7.0-8.0 ( Figure 3 B); Effects of different electron acceptors on the degradation of SMX by anaerobic strain SA-9 Figure 3 As shown in Figure C, when no electron acceptor is added, strain SA-9 cannot degrade SMX. When Na2SO4, NaNO3 and FeCl3 are added as electron acceptors, strain SA-9 can degrade SMX. The degradation rate of SMX is: Na2SO4>NaNO3>FeCl3, indicating that sulfate is the best electron acceptor for anaerobic degradation of SMX by strain SA-9. Figure 3 As shown in D, compared with no electron acceptor added to MSM, strain SA-9 hardly degraded SMX when sodium acetate and sodium succinate were added; when sodium pyruvate and sodium lactate were added, strain SA-9 was able to quickly degrade SMX, indicating that the best electron acceptors for anaerobic degradation of SMX by strain SA-9 were sodium pyruvate and sodium lactate.

[0036] The above results show that the optimal temperature range for the anaerobic strain SA-9 to degrade SMX is 30-37°C, 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 SMX by anaerobic strain SA-9

[0038] 1.0 mL of anaerobic degradation strain SA-9 bacterial solution (OD 600 =0.6) was added to 30 mL of deoxygenated MSM at pH 7.0, and then a certain amount of SMX was added to make the final concentrations 10 μM, 100 μM, 200 μM and 400 μM, respectively, and then placed in a shaker at 37°C and 150 rpm for culture. The concentration of SMX was measured at different time points, and the anaerobic degradation dynamics of SMX was fitted to calculate its half-life. The control group did not add anaerobic strain SA-9, and the control group and the treatment group were repeated three times each.

[0039] The experimental results of the degradation of SMX at different concentrations by the anaerobic degradation strain SA-9 are shown in Table 1. The degradation dynamics of SMX by the strain SA-9 conforms to the law of the first-order kinetic equation. The time is taken as the horizontal axis and the residual concentration of SMX is taken as the vertical axis. The first-order kinetic equation (C = C0e-kt ) fitting, the correlation coefficient R of the fitting equation 2 (0.9913-0.8552) shows that the degradation dynamics of SMX by anaerobic strain SA-9 conforms to the law of first-order kinetics. The degradation half-life (T) of SMX by anaerobic strain SA-9 at concentrations of 10μM, 100μM, 200μM and 400μM 1 / 2 =ln2 / k) are 6.0d, 7.6d, 11.6d and 30.8d respectively. The anaerobic strain SA-9 screened and obtained by the present invention has a high degradation efficiency for SMX and has a good application potential for SMX pollution remediation.

[0040] Table 1 The first-order kinetic parameters of strain SA-9 for anaerobic degradation of SMX at different concentrations

[0041]

[0042] Example 4 Application of anaerobic strain SA-9 to SMX bioremediation of flooded paddy soil contaminated by water

[0043] Flooded soil from a rice field in Hefei was obtained, stored in a sealed plastic bag and placed in a cool place. The soil was diluted with MSM in equal proportions, filtered with gauze, and 60 mL was placed in a 100 mL vial, and a certain amount of SMX was added to make the concentrations reach 10 μM and 100 μM, respectively. Then, it was sealed with a butyl rubber stopper and fixed with an aluminum cap, and high-purity nitrogen was filled in for 30 minutes to remove all dissolved oxygen in the bottle. The flooded soil mixture was divided into two parts, one part was sterilized with high-pressure steam as sterilized soil, and the other part was not sterilized as natural soil. In the anaerobic operation box, a certain amount of SMX and 2.0 mL of strain SA-9 bacterial solution (OD 600 =0.6), the sample was placed in a shaker at 37°C and 150 rpm for incubation, and the concentration of SMX in the bottle was measured at different time points. The control group without bacterial solution was used as the control group, and the control group and the treatment group were repeated three times each. 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 degradation strain SA-9 on SMX in sterilized soil and natural soil Figure 4As shown. In the sterile paddy field flooded soil, the decrease in SMX concentration in the paddy field soil without the addition of strain SA-9 is due to the adsorption of SMX by the sterilized soil. The adsorption rate of 10μM and 100μM SMX by the sterilized soil is less than 14% and 20%, respectively; the degradation half-life of 10μM and 100μM SMX in the sterile soil by strain SA-9 is 8.7d and 9.6d, respectively. In the natural paddy field soil without the addition of strain SA-9, the degradation half-life of 10μM and 100μM SMX is 3.0d and 2.6d, respectively; in the natural paddy field soil with the addition of strain SA-9, the degradation half-life of 10μM and 100μM SMX is 1.5d and 1.4d, respectively, indicating that the degradation efficiency of 10μM and 100μM SMX in the flooded paddy field soil by strain SA-9 is 2 times and 1.9 times that of the unadded strain. The above results indicate that the anaerobic strain SA-9 exhibits a good remediation effect on SMX in flooded paddy soil and has great application potential.

[0045] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A sulfamethoxazole anaerobic degradation strain SA-9, characterized in that: The strains were classified as Cupidesulfovibrio sp.SA-9, deposited in China Center for Type Culture Collection on October 31, 2024, with the deposit number CCTCC NO: M 20242405, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The sulfamethoxazole anaerobic degradation strain SA-9 according to claim 1, characterized in that The colonies of the anaerobic degradation strain SA-9 on the MPM solid plate are round, with a diameter of 0.5-3.0 mm, a moist, regular, pale white surface; the bacteria are curved rods with flagella, and are 0.5-0.8 μm × 1.8-2.5 μm in size.

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

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is a liquid bacterial agent, a solid bacterial agent, or a powder.

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

6. The use of the sulfamethoxazole anaerobic degradation strain SA-9 according to claim 1, characterized in that: The anaerobic degradation strain SA-9 is used to degrade sulfamethoxazole in the environment or equipment facilities.

7. The use according to claim 6, characterized in that: The environment described is livestock and poultry manure compost, aquaculture wastewater, groundwater, flooded soil and wetlands contaminated by sulfamethoxazole; the equipment and facilities described are wastewater treatment equipment and wastewater treatment facilities.

Images