CN108611285B - Sulfonamide antibiotic degrading bacteria and application thereof - Google Patents
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Abstract
The invention discloses a sulfonamide antibiotic degrading bacterium and application thereof. The degrading bacteria are saccharomycetesSakaguchia cladiensis) A5, wherein the strain has been deposited in Guangdong province culture Collection in 2018, 1 month and 26 days, and the deposit number is: GDMCC NO: 60319. the strain has a good degradation effect on sulfadiazine, sulfamethoxazole and other sulfonamide antibiotics, can be used for degradation treatment of sulfa antibiotic pollution, can reduce harm of antibiotics to ecological environment safety by using application objects including waste water and waste residues containing sulfa antibiotic pollutants, water environment or soil and the like, has the advantages of energy conservation, environmental protection and the like, and has a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of environmental pollutant treatment. More particularly, relates to a sulfonamide antibiotic degrading bacterium and application thereof.
Background
China is a big country for producing and using antibiotics, thousands of tons of antibiotic medicines are used in livestock breeding and personal medical treatment every year, and the sulfonamide antibiotics become a class of antibiotics with larger use amount in Chinese medicines and veterinary medicines due to the advantages of broad spectrum, high quality and low price. In fact, most antibiotics are not completely absorbed by the body. Researches show that up to 85-90% of antibiotics enter the environment in the form of drug prototypes or metabolites through excrement, and cause serious pollution to soil, water and the like. The Moscome problem group mainly researches the influence of antibiotic pollution in south China on vegetable planting and agricultural food safety, and proves that the antibiotic pollution can be brought into agricultural products such as vegetables and the like through livestock and poultry manure compost. Data researched and researched by literatures show that the pollution condition of sulfonamide antibiotics in surface water of China is very severe, and the content of antibiotics in river water is obviously higher than that in other countries. High-concentration sulfonamide antibiotic residues are detected in all large river basins in China. The sulfamethoxazole concentration is 1.4-157 ng/L and the sulfamethazine concentration is 29.5-120 ng/L when detected in the dry water period of Zhujiang; sulfamethoxazole with the concentration as high as 173 ng/L and 211 ng/L is detected in the Liaohe river basin and the Haihe river basin respectively; the concentration of sulfamethazine in the dry season of Huangpu river is 14.9-623.3 ng/L; sulfamethazine is also detected in Fujian Jiulong river, and the concentration is 775.5 ng/L.
The municipal sewage treatment plant is generally considered as a defense line for pollutants entering the environment, but the antibiotic removal effect of the current sewage treatment system is not obvious, and the effluent still contains antibiotic residues with higher concentration, so that the effluent of the municipal sewage treatment plant becomes one of the main sources of antibiotics in the environment. In the aquaculture of China, antibiotics as feed additives are directly put into water. When human and animal excreta are used as farmyard manure, residual antibiotics also enter soil to form more difficult-to-control non-point source pollution. Although antibiotics have made great contribution to human health in medicine, drug-resistant bacteria continuously appear and multiply along with the abuse of antibiotics, and the induced resistance genes cause potential gene pollution to the ecological environment. Currently, bacterial antibiotic resistance has become a focus of threat to human health and ecological safety. A report by WHO in 2000 suggested that antibiotic resistance has become one of the key challenges for human health in the 21 st century.
The degradation of antibiotics in the environment comprises non-biodegradation such as photolysis and hydrolysis and biodegradation, wherein microbial degradation is the most main path for the degradation of antibiotics in the environment. At present, a great deal of research and practice is carried out on physicochemical treatment methods of the sewage containing antibiotic residues, including an advanced oxidation method, an activated carbon adsorption method, a low-temperature plasma technology, a membrane treatment technology and the like, but the physicochemical treatment methods have high cost and complex management, except that the advanced oxidation method can achieve higher removal rate, other technical methods have lower removal effect, and the treatment of the antibiotic residues in a solid medium has limitation. Therefore, microbial degradation of antibiotics is becoming a research focus.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing sulfonamide antibiotic pollution treatment technology and provide a microbial bacterium capable of efficiently degrading sulfonamide antibiotics. Aims to promote the application of microbial technology in the environmental sulfonamide antibiotic pollution treatment and solve the problem of environmental sulfonamide antibiotic pollution.
The invention aims to provide a sulfonamide antibiotic degrading bacterium.
The invention also aims to provide application of the degrading bacteria in the treatment of sulfonamide antibiotic pollutants.
The above purpose of the invention is realized by the following technical scheme:
the present invention discovers that the yeast (A), (B), (C)Sakaguchia cladiensis) Has good degradation effect on sulfanilamide antibiotics such as sulfamethazine, sulfamethoxazole and the like, and can be used for degradation treatment of sulfanilamide antibiotic pollution.
In addition, the invention also obtains a yeast A5 strain by screening, domesticating, separating and purifying polluted soil of a certain livestock and poultry farm in the Kyowa area, which is preserved in Guangdong province microbial strain preservation center in 2018, 1 month and 26 days, and the preservation number is as follows: GDMCC NO: 60319.
therefore, yeast (C)Sakaguchia cladiensis) The application in degrading and treating the sulfanilamide antibiotics or in improving the degrading and removing rate of the sulfanilamide antibiotics and the application in treating the water environment and soil polluted by the sulfanilamide antibiotics are both within the protection scope of the invention.
Preferably, the yeast is the yeast A5 strain.
Preferably, the sulfa antibiotic is sulfamethazine, sulfamethoxazole and/or sulfadiazine.
A health food containing yeast (Saccharomycetes)Sakaguchia cladiensis) The sulfonamide antibiotic degrading bacteria also fall within the protection scope of the invention. Preferably, the yeast is the yeast A5 strain.
Preferably, the degrading bacteria agent is a bacterial suspension of yeast A5 strain.
Preferably, the yeast A5 strain is prepared after activated culture. Specifically, the yeast A5 strain is cultured and activated in LB culture medium or yeast culture medium, and the precipitate is collected by centrifugation and is diluted after washing to prepare the yeast A5 strain.
More preferably, the preparation method of the bacterial suspension comprises the following steps: inoculating the yeast A5 preserved on the inclined surface into a liquid LB culture medium, and carrying out constant-temperature shaking culture at the temperature of 25-30 ℃ and the constant temperature of 150-160 r/min for 24-36 h to prepare an activated seed solution; inoculating the seed solution into a fresh culture medium according to the inoculation amount of 1-10%, and continuously carrying out amplification culture at 25 ℃ for 24-36 h at 150 r/min; and centrifuging at 25 ℃ and 6000 r/min for 5-10 min, removing supernatant, collecting thalli, washing with sterile water for 2-3 times, and finally preparing 1g/L bacterial suspension with sterile water.
Preferably, the formulation of the LB medium is: 5g/L of NaCl, 10g/L of yeast extract, 10g/L of tryptanthrin, water as a solvent and 7.0-7.2 of pH. High temperature and high pressure sterilization before experiment.
The invention has the following beneficial effects:
the saccharomycete obtained by screening has good degradation effect on sulfadiazine, sulfamethoxazole and other sulfonamide antibiotics, can be used for degradation treatment of sulfonamide antibiotic pollution, can be applied to objects including waste water and waste residues containing sulfonamide antibiotic pollutants, water environment or soil and the like, and can reduce harm of antibiotics to ecological environment safety
Moreover, the saccharomycete for treating the pollution of the sulfonamide antibiotics has the advantages of energy conservation, environmental protection and the like compared with methods such as physical adsorption, chemical degradation and the like, and has very wide application prospect.
Drawings
FIG. 1 is a morphological view of the plate growth of yeast A5.
FIG. 2 is a microscope image of yeast A5.
FIG. 3 is a phylogenetic tree of yeast A5.
FIG. 4 is a graph of the growth of yeast A5.
FIG. 5 is a graph showing the degradation profile of yeast A5 on sulfamethazine.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1 isolation and identification of sulfonamide antibiotic-degrading bacteria
1. Experimental Material
Nutrient medium: 5g of sodium chloride, 3 g of beef extract, 10g of egg white, 1000mL of distilled water and 7.0-7.2 of pH. Agar powder (strips) 15 g/L was added to the solid medium. Inorganic salt culture medium: (NH)4)2SO41g、MgSO4·7H2O 0.1g、KH2PO43g、K2HPO4·3H27g of O, 0.5g of sodium citrate, 1000mL of distilled water and 7.0-7.2 of pH.
The culture medium is placed in a high-pressure steam sterilization pot before the experiment, and is sterilized for 30min at 121 ℃.
2. Isolation and characterization of strains
(1) Screening and domesticating sulfonamide antibiotic resistant bacteria:
the microorganism acclimatization is carried out in 4 stages. Collecting a polluted soil sample of a certain livestock and poultry farm in the triangle of pearl, firstly adding the soil sample into 100 mL of a nutrient medium containing sulfonamide antibiotics (10 mg/L) under the aseptic operation condition, and placing the soil sample in a constant-temperature shaking table at 30 ℃ and 150 r/min for light-shielding culture for 7 d. Then, 10% of the culture medium was transferred to another nutrient medium with 30 mg/L antibiotic concentration, and the culture was continued for 7 days under the same conditions for the second-stage acclimation. Then, continuously culturing and domesticating every 7 d as a period, and increasing the concentration of domesticated antibiotics to 50 mg/L in the third stage; the concentration of the domesticated antibiotics in the fourth stage is increased to 100 mg/L; the acclimation process is not changed except the change of antibiotic concentration, and other culture conditions are not changed.
(2) And (3) separating, purifying and screening sulfonamide antibiotic degrading bacteria:
after the acclimatization, the culture solution of the flora is treated according to the formula of 10-1～10-6Diluting with gradient, respectively taking 10-4、10-5、10-6And (3) respectively coating 200 mu L of culture solution with dilution gradient on a solid nutrient medium, and inversely placing the culture medium in an incubator for 24-36 h at 30 ℃. The colonies that grew out were numbered and individually picked for further streaking and isolation until a single colony was obtained that was purified.
Then, respectively inoculating each single colony to an inorganic salt culture medium containing a unique carbon source of the sulfonamide antibiotics for degradation experiments, and investigating the degradation capability of each bacterium on the sulfonamide antibiotics. The sulfonamide antibiotics are sulfamethazine, sulfamethoxazole and sulfadiazine, and preferably sulfamethazine.
The degradation experimental conditions are as follows: in each 20 mL of inorganic salt culture medium, the concentration of the sulfonamide antibiotics is 1 mg/L, the bacterial feeding amount is 1g/L, and the culture is carried out in a constant-temperature shaking table at 25 ℃ and 150 r/min in a dark place.
Finally, a target strain with the best degradation capability and stable growth performance is obtained through screening, and the number of the target strain is A5.
(3) Identification of sulfonamide antibiotic-degrading bacteria (target Strain A5)
Preparation and observation of electron microscope samples: 1 drop of sterile water is dripped in the middle of a clean glass slide, a small amount of bacteria A5 is picked by an ignition cooled inoculating loop and is fully and uniformly mixed with water drops on the glass slide, and then the glass slide is coated with a uniform thin layer; naturally drying; dripping ammonium oxalate crystal violet staining solution on the dried and fixed thalli, and standing for 1-2 min; and then, pouring the dye solution, obliquely placing a glass slide, flushing residual redundant dye solution by using clear water until the effluent water is colorless, and naturally drying. According to the operation steps of a microscope, firstly observing under a low-power microscope, and further observing the morphology of the somatic cells under a high-power microscope by using an oil lens after finding a proper visual field.
The strain morphology and physiological and biochemical identification results are as follows: the morphological appearance of the plate growth of the yeast A5 is shown in figure 1, and the image of the microscope is shown in figure 2. The bacterial colony is red, opaque and round, the surface is smooth and wet, and the edge is neat; the bacterial cells under the microscope are in a single ellipsoid shape, the cells are larger, and the colorability is good.
Molecular characterization of bacterium a 5: molecular identification of bacterium A5 was carried out by the institute of microorganisms of Guangdong province. After DNA extraction, PCR amplification, sequence sequencing and sequence comparison, 26S rDNA sequencing sequence of bacterium A5 andSakaguchia cladiensiswith the highest homology. And a phylogenetic tree was constructed as shown in fig. 3.
In conclusion of the morphological identification and the molecular identification results, the bacterium A5 screened by the invention is identified as the yeastSakaguchia cladiensis. And has been preserved in Guangdong province microorganism culture collection center in 2018, 1 month and 26 months, with the preservation number: GDMCCNO: 60319, respectively; and (4) storage address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
Example 2 determination of growth curves of Yeast A5
1. Respectively inoculating yeast A5 into LB culture medium and LB culture medium containing sulfonamide antibiotics, culturing at 25 deg.C and 150 r/min with shaking table at constant temperature, respectively sampling at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, 192, and 216 h, and determining the culture solutionOD 600And (4) zeroing with a sterilized LB liquid culture medium.
The LB culture medium is as follows: 5g/L of NaCl, 10g/L of yeast extract, 10g/L of tryptanthrin, 1000mL of water and 7.0-7.2 of pH.
The LB culture medium containing sulfonamide antibiotics is: sulfamethazine was added to the LB medium to a concentration of 100 mg/L.
2. As shown in FIG. 4, the yeast A5 was able to grow normally in the culture system containing sulfonamide antibiotics.
Example 3 degradation of sulfamethazine by Yeast A5
1. Experimental methods
(1) Preparation of bacterial suspension:
scraping 1-2 cyclosporins A5 from a seed preservation slope, inoculating the scraped cyclosporins A5 into a sterilized LB culture medium, binding, placing the bound cyclosporins A5 in a constant temperature shaking table incubator (25 ℃, 150 r/min), and carrying out amplification culture for 24-36 h. Taking the activated bacterium A5 culture solution, centrifuging (6000 r/min, 25 ℃), collecting the thallus, washing with sterile water for 2 times (6000 r/min, 25 ℃), then resuspending with sterile water, and preparing into a bacterial suspension with a certain concentration.
(2) Degradation of sulfamethazine by yeast a 5:
adding a certain amount of sulfamethazine serving as a unique carbon source into an autoclaved inorganic salt culture medium to ensure that the concentration of the sulfamethazine is 1 mg/L; then inoculating 1 mL of bacterial suspension to ensure that the bacterial feeding amount is 1g/L (wet weight); blank (no added bacteria) controls were also designed. All samples are packed and then are placed at 25 ℃ and are cultured in the dark at 150 r/min, samples are taken for 1, 2, 4, 8, 12, 24, 36, 48 and 72 hours respectively, and the concentration of the residual sulfamethazine in the solution is determined.
(3) Inducing saccharomycete A5 to degrade sulfamethazine:
adding sulfamethazine into LB culture medium of the enlarged culture of the bacteria A5 to make the concentration of the sulfamethazine 100mg/L, activating and culturing to a logarithmic phase, preparing bacterial suspension according to the method, and performing a degradation experiment of the bacteria A5 on the sulfamethazine according to the method.
(4) Determination of sulfamethazine:
after the sample is centrifuged, a quantitative supernatant is taken and fully mixed with an extracting agent (containing 0.1% formic acid water: acetonitrile: methanol =10:3: 1) according to a ratio of 1:1, after the sample is centrifuged at a high speed, the supernatant is taken and filtered (0.22 um), and the residual concentration of the sulfamethazine is analyzed by using a High Performance Liquid Chromatography (HPLC). The analysis conditions were: c18 reversed phase column, 5 μm,4.6 × 250mm, ultraviolet detector, detection wavelength 270nm, water (containing 0.1% formic acid): acetonitrile =30:70 (V/V) as a mobile phase, flow rate 1 mL/min, and sample size 20 μ L.
2. As shown in FIG. 5, the degradation rate of sulfamethazine by the bacterium A5 increased with time, and reached 38.2% at the 2 d. The degradation of the induced bacterium A5 to the sulfamethazine is obviously improved.
Example 4 degradation study of yeast A5 on sulfamethazine at various concentrations
Adding a certain amount of sulfamethazine serving as a unique carbon source into an autoclaved inorganic salt culture medium, wherein the concentrations of the sulfamethazine are 0.5, 1, 3 and 5 mg/L respectively; then inoculating 1 mL of bacterial suspension to ensure that the bacterial feeding amount is 1g/L (wet weight); blank (no added bacteria) controls were also designed. All samples are packed and then are placed at 25 ℃ and 150 r/min for dark culture, and samples are taken after reaction for 2 d, and the concentration of the residual sulfamethazine in the solution is determined. The bacterial suspension preparation and the sulfamethazine assay were as described in example 3.
The result shows that the bacterium A5 has degradation effects on sulfadiazine with different concentrations, wherein the degradation effect on sulfadiazine with concentration of 1 mg/L is the best, the degradation effect on sulfadiazine with concentration of 0.5 mg/L is the second, and the degradation effect on sulfadiazine with concentration of high concentration (5 mg/L) is the second.
Example 5 degradation study of sulfamethazine by Yeast A5 at various dosages
Adding a certain amount of sulfamethazine serving as a unique carbon source into an autoclaved inorganic salt culture medium to ensure that the concentration of the sulfamethazine is 1 mg/L; then inoculating a certain amount of bacterial suspension, and enabling the bacterial adding amount to be 0.5, 1, 1.5 and 2 g/L (wet weight) respectively; blank (no added bacteria) controls were also designed. All samples are packed and then are placed at 25 ℃ and 150 r/min for dark culture, and samples are taken after reaction for 2 d, and the concentration of the residual sulfamethazine in the solution is determined. The bacterial suspension preparation and the sulfamethazine assay were as described in example 3.
Experimental results show that the optimal bacterium adding amount for degrading 1 mg/L sulfamethazine is 1g/L, and the bacterium adding amount at other concentrations has no significant influence on the degradation of the sulfamethazine.
Example 6 degradation study of Yeast A5 on sulfamethazine in different pH systems
Adding a certain amount of sulfamethazine serving as a unique carbon source into an autoclaved inorganic salt culture medium to ensure that the concentration of the sulfamethazine is 1 mg/L; and adjusting the pH value of the solution to make the system pH value respectively acidic (1-3), weakly acidic (5-6), neutral (7), weakly alkaline (9-10) and alkaline (12-14). Then inoculating a certain amount of bacterial suspension to ensure that the bacterial adding amount is 1g/L (wet weight); meanwhile, a blank group (without adding bacteria) is designed to be used as a control. All samples are packed and then are placed at 25 ℃ and 150 r/min for dark culture, and samples are taken after reaction for 2 d, and the concentration of the residual sulfamethazine in the solution is determined. The bacterial suspension preparation and the sulfamethazine assay were as described in example 3.
The experimental result shows that the removal of the sulfamethazine is not facilitated under the acidic condition. Within the neutral to alkaline pH range, the influence on the degradation of sulfamethazine is not obvious.
Example 7 degradation treatment of wastewater containing sulfonamide antibiotics by Yeast A5
Adding bacterium A5 bacterium suspension into water containing various sulfanilamide antibiotics (sulfamethazine, sulfamethoxazole and sulfadiazine), wherein the bacterium adding amount is 1g/L (wet weight), and designing blank group (without bacterium) control. And (3) placing the mixture at 25 ℃ and 150 r/min in a dark place for reaction for 2 d, then sampling, and determining the concentration of the residual sulfamethazine, sulfamethoxazole and sulfadiazine in the solution. The bacterial suspension preparation and the sulfamethazine assay were as described in example 3.
Experimental results show that the bacterium A5 has a good degradation effect on 3 sulfonamide antibiotics, wherein the degradation and removal effect on sulfamethazine is the best.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A yeast (Sakaguchia cladiensis) A5 strain capable of degrading sulfonamide antibiotics is characterized in that the strain is deposited in Guangdong province microorganism strain collection center in 2018, 1 month and 26 days, and the preservation number is as follows: GDMCC NO: 60319.
2. the use of the yeast (Sakaguchia cladiensis) a5 strain according to claim 1 for degrading and treating sulfonamides or for increasing the degradation removal rate of sulfonamides.
3. The use of the yeast (Sakaguchia cladiensis) a5 strain according to claim 1 for the remediation of water and soil contaminated with sulfonamides.
4. Use according to claim 2 or 3, characterized in that the sulfonamide antibiotic is sulfamethazine, sulfamethoxazole and/or sulfadiazine.
5. A sulfonamide antibiotic-degrading bacterial agent comprising the yeast (Sakaguchiacladiaensis) A5 strain according to claim 1.
6. The degrading bacterial agent of claim 5, wherein the degrading bacterial agent is a bacterial suspension of yeast A5 strain.
7. The degrading bacterial agent of claim 5, which is prepared by activated culture of yeast A5 strain.
8. The degrading bacterial agent of claim 6, wherein the preparation method of the bacterial suspension comprises the following steps: inoculating the yeast A5 preserved on the inclined plane into a liquid LB culture medium or a yeast culture medium, and performing constant-temperature shaking culture to obtain an activated seed solution; then inoculating the seed liquid into a fresh culture medium, continuing to enlarge culture, centrifuging, removing supernatant, collecting thalli, and resuspending sterile water to obtain a bacterial suspension.
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| CN109280631B (en) * | 2018-10-24 | 2021-07-09 | 哈尔滨商业大学 | Sulfadimidine degrading bacterium S-2 and application thereof |
| CN110331120B (en) * | 2019-08-28 | 2019-12-24 | 中国科学院烟台海岸带研究所 | Vibrio alginolyticus capable of degrading various sulfanilamide antibiotics and application thereof |
| CN111018131B (en) * | 2019-12-16 | 2022-04-26 | 同济大学 | Method for degrading sulfamethoxazole |
| CN111528396B (en) * | 2020-05-08 | 2022-09-02 | 广东海洋大学 | Application of candida guilliermondii in degradation of antibiotics |
| CN113215033B (en) * | 2021-04-30 | 2022-06-28 | 华南农业大学 | Sulfonamide antibiotic degrading bacteria and application thereof |
| CN114292764B (en) * | 2021-09-10 | 2024-02-20 | 厦门众仁合美生物科技有限公司 | Achromobacter strain JD417 and application thereof |
| CN117645957B (en) * | 2023-12-04 | 2024-04-30 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Pseudomonas stutzeri strain for degrading sulfonamide antibiotics and application thereof |
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