CN113215033A

CN113215033A - Sulfonamide antibiotic degrading bacteria and application thereof

Info

Publication number: CN113215033A
Application number: CN202110478770.7A
Authority: CN
Inventors: 宋孟珂; 宋丹丹; 罗春玲
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-06
Anticipated expiration: 2041-04-30
Also published as: CN113215033B

Abstract

The invention discloses a sulfonamide antibiotic degrading bacterium and application thereof, belonging to the technical field of biology. The name of the bacterium is Paenarthrobacter nicotinovorans SMK-1, and the preservation number is as follows: GDMCC No: 61613. the strain can utilize sulfamethoxazole as a unique carbon source and can be degraded quickly, and the initial concentration of SMK-1 in antibiotics is 100 mg.L^‑1pH8 for 10 daysThen, the degradation rate can reach more than 73 percent; at pH6, an initial antibiotic concentration of 30 mg.L^‑1After the inorganic salt culture medium is cultured for 10 days, the degradation rate can reach more than 79 percent; the degradation effect of the bacterium was best overall at pH 8. The strain is a strain with strong antibiotic degradation and antibiotic tolerance, and has strong adaptability to sulfamethoxazole; has good application prospect in the bioremediation of sulfamethoxazole polluted environment.

Description

Sulfonamide antibiotic degrading bacteria and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a sulfonamide antibiotic degrading bacterium Paenarthrobacter nicotinovorans SMK-1 and application thereof.

Background

Antibiotics, which are chemical substances capable of inhibiting or killing microorganisms, are generally produced by microbial metabolism or synthesized artificially, and have significant effects in treating and preventing animal diseases and promoting animal growth and development, are widely used in livestock and poultry breeding industry worldwide (Chee-sanfordatal, 2001), including the commonly used tetracyclines and sulfonamides. The usage amount of the antibiotics in 2010 worldwide is reported to be 10-20 ten thousand tons (Tasho and Cho et al, 2016), and the usage amount of the antibiotics in 2013 in China is about 16.2 ten thousand tons (Zhang et al, 2015). Untreated or incompletely treated livestock manure and sewage are often discharged directly and continuously into the environment, resulting in the continuous accumulation and long-term maintenance of antibiotics at high levels, which are now a type of environmental pollutants that are of great concern.

The natural attenuation of toxic and harmful organic pollutants in the environment mainly depends on the metabolism of related microorganisms, the bioremediation technology has the advantages of low cost, good effect, no secondary pollution and the like, and the microbial degradation is one of the main approaches and main remediation measures for removing antibiotics. Various single or mixed flora capable of utilizing sulfonamides as carbon sources have been isolated, mainly from phyla proteobacteria, phyla actinomycetes and phyla firmicutes, and the most frequently occurring genera are Pseudomonas (Pseudomonas sp.), Bacillus (Microbacterium sp.), Rhodococcus (Rhodococcus sp.), Achromobacter (Achromobacter sp.), Bacillus Alcaligenes (Alcaligenes sp.), Ralstonia sp.), Shewanella (Shewanella sp.), Arthrobacter (Arthrobacter sp.), Paracoccus (Paracoccus sp.), coriobacter (kribbellella sp.), Bacillus (Bacillus sp.), Nocardioides sp.), Clostridium (Clostridium sp.), Clostridium (Clostridium sp., Clostridium 2018, Clostridium 2019, Bacillus 2012016).

In recent years, researchers at home and abroad prove the degradation capability and degradability of sulfamethoxazolePreliminary studies can be performed. SAs (sulfonamide antibiotics) pollutants serve as a carbon source in the microbial degradation process and provide essential nutrients for the growth and metabolism of microorganisms. The utilization process of the sulfonamide pollutants by the microorganisms has important influence on water environment and the like, and sulfonyl generates an electron-withdrawing effect by the high-polarity C-S bond, so that sulfonic acid combined with aromatic compounds is extremely firm, and the difficulty of degrading the sulfonamide pollutants by the microorganisms is greatly increased. Zhang Yu et al separated from activated sludge of urban sewage treatment plant to obtain a strain Bacillus cereus (Bacillus cereus) which can use sulfamethazine as sole carbon source, wherein the initial OD600 is 0.1 and the initial concentration of the sulfamethazine is 50 mg.L at the temperature of 30 ℃ and the pH is 8.0^-1Under the optimal condition, the degradation rate of the sulfamethazine within 36 hours can reach 100 percent (Zhang Jia yoget al, 2019). The scholars also used activated sludge from Harbin sewage plant as a sample and added the sample into a Minimum Mineral Salt Medium (MMSM) containing sulfamethoxazole, and the sample was cultured at 10 ℃ for 150 r.min^-1Under the conditions of (1), a strain of Pseudomonas psychrophila is selected, and inoculated to a strain containing 100 mg.L^-1MMSM of sulfamethoxazole at 10 deg.C and 150r min^-1The contaminants were degraded by 34.30% by incubation for 192h (Jiang et al, 2014). Gauthier (Gauthier et al, 2010) purchased Rhodococcus rhodochrous (Rhodococcus rhodochrous) and the like, and cultured in BHI medium once, then cultured in MMSM medium twice, and finally inoculated in a medium containing 30 mg.L^-1Sulfamethoxazole and 3 g.L^-1Adding glucose as carbon source, culturing at 26 deg.C and 150 r.min^-1The result of the degradation experiment in the incubator shows that the rhodococcus roseus can degrade 20 percent of sulfamethoxazole after being cultured for 36 days. At present, due to artificial activities of industrial and mining industry, agriculture and the like and high background value of soil environment and other factors, antibiotics in the environment are seriously polluted, and farmland soil antibiotics are continuously accumulated and kept at high content for a long time, so that the growth of animals and plants, the microbial community structure and the enzyme activity are influenced, and further the safety of agricultural products and the soil function are influenced (Kummerer, 2009; Zouet et al, 2011; Xuqiulou et al, 2014). Therefore, the temperature of the molten metal is controlled,the screened strain capable of effectively degrading the high-concentration antibiotic sulfamethoxazole has higher application value.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a sulfonamide antibiotic degrading bacterium Paenarthrobacter nicotinovorans SMK-1. The bacteria are high-efficiency antibiotic degrading bacteria capable of degrading high-concentration sulfamethoxazole, which are obtained by collecting activated sludge from an aeration tank of a Guangzhou new pond sewage treatment plant, carrying out long-term domestication and carrying out multiple screening, separation and purification.

The invention also aims to provide application of the sulfonamide antibiotic degrading bacteria.

The purpose of the invention is realized by the following technical scheme:

the invention provides a sulfonamide antibiotic degrading bacterium, which is named as Paenarthrobacter nicotinovorans SMK-1 and is obtained by collecting activated sludge from an aeration tank of a Guangzhou new pond sewage treatment plant, carrying out long-term domestication, and carrying out multiple screening, separation and purification.

The preservation information of Paenarthrobacter nicotinovorans SMK-1: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No: 61613, deposit address: the microbiological research institute of Guangdong province, No. 59 building, No. 5 building, Guangdong province, of the Fuli Zhonglu 100, Guangzhou city, the preservation date: 21/4/2021.

The mycological characteristics of the sulfonamide antibiotic-degrading bacteria Paenarthrobacter nicotinovorans SMK-1 are described as follows:

after the bacteria are activated and grow for 48 hours on a flat plate made of a solid nutrient medium under the aerobic condition at the temperature of 30 ℃, light yellow, round, smooth in surface, slightly convex upwards, opaque, neat-edged, spore-free and flagellum-free bacterial colonies with the diameter of 0.3-2.5 mm can be formed, and a bacterial colony graph is shown in figure 1.

Molecular biological characteristics: the DNA G + C content of the strain is 57.0%, and the 16s rRNA gene of the strain is amplified and sequenced to obtain the DNA sequence shown as SEQ ID NO: 1, and the result of constructing a phylogenetic tree by utilizing the sequence and adopting an adjacent phase connection method and a minimum evolution method shows that the bacterium has the highest homology with Paenarthrobacter nicotinovorans DSM 420, and the bacterium belongs to different strains of the same genus of the Paenarthrobacter on the aspect of molecular biology, so the sulfonamide antibiotic degrading bacterium SMK-1 is named as Paenarthrobacter nicotinovorans SMK-1.

The experimental result of applying Paenarthrobacter nicotinovorans SMK-1 to sulfamethoxazole degradation shows that: the Paenarthrobacter nicotinovorans SMK-1 can degrade antibiotics under the culture conditions of 30 ℃, pH 6-8 and an inorganic salt culture medium without adding NaCl, and the initial concentration of sulfamethoxazole is 30 mg.L^-1After the inorganic salt culture medium is cultured for 5 days, the degradation rate can reach more than 25 percent, and when the inorganic salt culture medium is cultured for 10 days, the degradation rate can reach more than 45 percent; at an initial sulfamethoxazole concentration of 100 mg.L^-1After the inorganic salt culture medium with the pH of 6-7 is cultured for 10 days, the degradation rate can reach more than 20 percent and is 100 mg.L^-1After the inorganic salt culture medium with the pH value of 8 is cultured for 10 days, the degradation rate can reach more than 73 percent; the Paenarthrobacter nicotinovorans SMK-1 is a bacterial strain with strong abilities of degrading sulfamethoxazole and resisting sulfamethoxazole, so that the bacterial strain can be expected to have better tolerance and degradability to sulfamethoxazole.

The invention also provides a biological agent which is prepared on the basis of the sulfonamide antibiotic degrading bacteria.

The biological agent is prepared by liquid culture of the sulfonamide antibiotic degrading bacteria, and preferably comprises the following steps: inoculating the sulfonamide antibiotic degrading bacteria into an LB liquid culture medium for culture to obtain the biological agent.

The culture is carried out at 25-40 ℃ and pH5.0-9.0 for 24-72 h.

Further, the culture is carried out for 24-72 h at 25-37 ℃ and pH of 5.0-9.0.

Furthermore, the culture is carried out for 24-72 h at 30-37 ℃ and pH of 5.0-9.0.

The invention also aims to provide the application of the Paenarthhromobacter nicotinovorans SMK-1 in degrading the sulfonamide antibiotics, and further the application of the Paenarthhromobacter nicotinovorans SMK-1 in degrading the sulfamethoxazole antibiotics.

The Paenarthrobacter nicotenovorans SMK-1 is a novel strain separated and identified in activated sludge, and has not been reported about the research of the Paenarthrobacter nicotenovorans on the degradation of antibiotics at home and abroad; the Paenarthrobacter nicotinovorans SMK-1 can be effectively degraded by taking high-concentration antibiotics as carbon sources, and the initial concentration of the antibiotics in the bacteria is 30 mg.L^-1After the inorganic salt culture medium with the pH of 6-8 is cultured for 5 days, the degradation rate can reach over 25 percent, and when the inorganic salt culture medium is cultured for 10 days, the degradation rate can reach over 45 percent; at an initial sulfamethoxazole concentration of 100 mg.L^-1After the inorganic salt culture medium with the pH of 6-7 is cultured for 10 days, the degradation rate can reach more than 20 percent and is 100 mg.L^-1After the inorganic salt culture medium with the pH value of 8 is cultured for 10 days, the degradation rate can reach more than 73 percent; the strain is a strain with strong antibiotic degradation and antibiotic tolerance, and has strong adaptability to sulfamethoxazole; has good application prospect in the bioremediation of sulfamethoxazole polluted environment.

A method for degrading sulfonamide antibiotics comprises applying the bacteria Paenarthrobacter niconovorans SMK-1 or the biological agent to an environment containing sulfonamide antibiotics to achieve the purpose of degrading the sulfonamide antibiotics.

The environment containing the sulfonamide antibiotics is water or soil containing the sulfonamide antibiotics.

The concentration of the sulfonamide antibiotics is 1-100 mg.L^-1。

The sulfonamide antibiotic is sulfamethoxazole.

In the environment, the temperature is 25-40 ℃, the initial pH is 5-9, and the environment is aerobic.

Furthermore, in the environment, the temperature is 25-37 ℃, the initial pH is 5-9, and the environment is aerobic.

Furthermore, in the environment, the temperature is 30-37 ℃, the initial pH is 6-8, and the environment is aerobic.

Compared with the prior art, the invention has the following advantages and effects:

the invention separates and obtains a degrading strain taking sulfamethoxazole as a carbon source, identifies the strain as different species of Paenarthrobacter and names the strain as Paenarthrobacter nicotinovanans SMK-1 according to the strain morphology, physiological characteristics, 16S rDNA gene sequencing analysis and phylogenetic analysis. The strain can utilize sulfamethoxazole as a unique carbon source and can quickly degrade antibiotic sulfamethoxazole, and the initial concentration of the strain SMK-1 in the antibiotic is 100 mg.L^-1After the inorganic salt culture medium with the pH of 8 is cultured for 10 days, the degradation rate can reach more than 73 percent; at pH6, the initial concentration of sulfamethoxazole is 30 mg.L^-1After the inorganic salt culture medium is cultured for 3 days, the degradation rate can reach more than 31 percent; at pH6, the initial concentration of sulfamethoxazole is 30 mg.L^-1After the inorganic salt culture medium is cultured for 10 days, the degradation rate can reach more than 79 percent; the degradation effect of the bacterium was best overall at pH 8. The strain is a strain with strong antibiotic degradation and antibiotic tolerance, and has strong adaptability to sulfamethoxazole; has good application prospect in the bioremediation of sulfamethoxazole polluted environment.

Drawings

FIG. 1 is a colony morphology of Paenarthrobacter nicotinovorans SMK-1 grown on solid nutrient medium.

FIG. 2 is a phylogenetic tree of Paenarthrobacter nicotinovorans SMK-1 and related bacteria based on 16s rRNA gene sequences, which is constructed by the least evolution method, and shows only the results of more than 90% of the auto-unfolding value, the scale bar 0.002 represents the substitution rate of each nucleotide, and SMK-1 represents Paenarthrobacter nicotinovorans SMK-1.

FIG. 3 is a graph showing the effect of different growth temperatures on Paenarthrobacter nicotinovorans SMK-1.

FIG. 4 is a graph showing the effect of different growth pH on Paenarthrobacter nicotinovorans SMK-1.

FIG. 5 is a graph showing the effect of different salinity tolerance on Paenarthrobacter nicotinovorans SMK-1.

FIG. 6 is a culture of Paenarthrobacter nicotinovorans SMK-1 in inorganic salts of pH7 containing different concentrations of antibioticsThe initial concentration of sulfamethoxazole in the growth curve in the medium is 1-100 mg.L^-1。

FIG. 7 shows the degradation of sulfamethoxazole after Paenarthrobacter nicotinovorans SMK-1 is cultured in inorganic salt culture media with different concentrations and different pH values for 7 days, wherein the initial concentration of sulfamethoxazole is 1-100 mg.L^-1The pH value is 4-9.

FIG. 8 shows the degradation of sulfamethoxazole in an inorganic salt medium containing sulfamethoxazole at different concentrations and pH6, where the initial concentration of sulfamethoxazole is 1-100 mg.L^-1。

FIG. 9 shows the degradation of sulfamethoxazole in an inorganic salt medium containing sulfamethoxazole at different concentrations and pH7, where the initial concentration of sulfamethoxazole is 1-100 mg.L^-1。

FIG. 10 shows the degradation of sulfamethoxazole in an inorganic salt medium containing sulfamethoxazole at different concentrations and pH8, where the initial concentration of sulfamethoxazole is 1-100 mg.L^-1。

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1

1 Material

1.1 sample sources

The bacteria are high-efficiency antibiotic degrading bacteria SMK-1 capable of degrading high-concentration sulfamethoxazole, which are obtained by collecting activated sludge from an aeration tank of a Guangzhou new pond sewage treatment plant, carrying out long-term domestication and carrying out multiple screening, separation and purification.

1.2 culture Medium

1.2.1 inorganic salt Medium

The inorganic salt culture medium is used for enrichment culture of microorganisms in a sample and sulfamethoxazole degradation experiments under the pure bacteria condition. The inorganic salt medium formulation is shown in Table 1 (without sulfamethoxazole).

TABLE 1 inorganic salt media formulation

Name of reagent	Concentration of
		NaCl	0.5g/L
NH₄Cl	1g/L
		Na₂HPO₄·12H₂O	8.5g/L
KH₂PO₄	3g/L
		MgSO₄·7H₂O	0.2g/L
CaCl₂	14.7mg/L
		CuSO₄	0.4mg/L
MnSO₄·H₂O	4.0mg/L
		ZnSO₄·7H₂O	4.0mg/L
H₃BO₃	5.0mg/L
		H₂MoO₄·2H₂O	1.6mg/L
FeCl₃·6H₂O	2.0mg/L

1.2.2 nutrient Medium

The nutrient medium (LB medium) is used for the culture of conventional microorganisms such as the separation, purification, preservation and activation of bacteria. The types and compositions of the liquid nutrient media used in this experiment are shown in Table 2. If a solid nutrient medium needs to be prepared in an experiment, only 1.5-2% of agar powder needs to be added on the basis of the original liquid nutrient medium formula. If the culture conditions of the strains are not specified, the pH of the culture medium is adjusted to 7.2.

TABLE 2 nutrient medium composition

Name of reagent	Concentration of
		Tryptone	10g/L
Yeast extract	5g/L
		NaCl	10g/L

2 method

2.1 acclimatization, screening and isolation of strains

Adding the collected activated sludge into an inorganic salt culture medium, taking the antibiotic sulfamethoxazole with the concentration of 100mg/L as a degradation substrate (namely, the inorganic salt culture medium contains the sulfamethoxazole), and placing the activated sludge in an incubator at 30 ℃ for shake culture in dark. Using an inorganic salt culture medium with sulfamethoxazole as a unique carbon source to carry out strain domestication, wherein 7d is a domestication period; 10% of the inoculum size was transferred to a fresh sulfamethoxazole-containing inorganic salt medium with the same culture system and the enrichment process was repeated three times.

And (3) coating and separating the fourth generation enrichment culture sample obtained by the method of dilution plate, and separating the sample by using nutrient medium. Culturing the coated sample at the original culture temperature for about 48 hours to form obvious single colonies on the surface of the culture medium, selecting a plurality of different single colonies according to the characteristics of the colony such as morphology size, color, transparency and the like, and carrying out streak purification and culture on a nutrient medium flat plate. If single colonies of different characteristics are still observed on the streaked plates, they are streaked again until only single colonies of the same characteristics are observed on the same plate. 1 strain SMK-1 with high-efficiency degradation performance on sulfamethoxazole is obtained by screening in the experiment. Selecting a single bacterial colony of the purified SMK-1 to be cultured in a corresponding liquid nutrient medium until the logarithmic phase, mixing the bacterial liquid and sterile glycerol, subpackaging the mixture into a sterile 2mL freezing storage tube (the concentration of the glycerol is 25 percent), and placing the freezing storage tube at the temperature of minus 80 ℃ for long-term storage.

2.2 morphological characteristics of Strain SMK-1

SMK-1 is a bacterium separated from activated sludge, and after being activated and growing for 48 hours on a flat plate made of a solid nutrient medium under the aerobic condition at the temperature of 30 ℃, a colony which is light yellow, round, smooth in surface, slightly convex upwards, opaque, neat in edge, free of spores and free of flagella and has the diameter of 0.3-2.5 mm can be formed, and the colony map is shown in figure 1.

2.3 characterization of the molecular biological Properties of the Strain SMK-1

The molecular biological characteristic identification mainly comprises the determination of DNA G + C content, the sequencing of the separated strain SMK-1 and the construction of a phylogenetic tree, and the experiments can provide scientific evidence for the location of bacteria in taxonomy.

2.3.1DNA G + C content determination

The determination of the G + C content of DNA was mainly carried out by high performance liquid chromatography, the detailed procedure being reported in Mesbah et al (1989). The DNA G + C content of SMK-1 was determined to be 57.0%.

2.3.2 sequencing and construction of phylogenetic Tree

Before sequencing and construction of phylogenetic trees, the DNA of bacteria needs to be extracted first (the bacterial genomic DNA rapid extraction kit used in experiments is from edley biotechnology limited, beijing). In order to study the taxonomy of bacteria, it is usually necessary to amplify a 16s rRNA gene, which is a DNA fragment of the component encoding rRNA in prokaryotes, and to construct phylogenetic trees, which are usually used for detecting and identifying bacteria because of its high degree of conservation, specificity and appropriate sequence length.

Polymerase Chain Reaction (PCR) is mainly used to amplify different gene segments, PCR requires different primers (27F and 1492R for forward and reverse primers; Baker et al, 2003), and PCR amplification system: 0.5 mu.L of forward primer 27F (10mmol/L), 0.5 mu.L of reverse primer 1492R (10mmol/L), 12.5 mu.L of Taq enzyme, 1 mu.L of DNA group template and 10.5 mu.L of deionized water. PCR amplification reaction conditions: denaturation at 95 ℃, annealing at 55 ℃, extension at 72 ℃, circulating for 30 times, extension at 72 ℃ for 5min, and storing at 4 ℃ after PCR reaction. Amplifying the needed gene, adding 1-1.5% agarose and nucleic acid coloring agent to prepare gel block, adding PCR product and DNA marker (marker) containing various length segments into the gel block, placing the gel block in an electrophoresis apparatus, filling TAE buffer solution into the electrophoresis apparatus, operating the electrophoresis apparatus for 20min under a certain voltage, taking out the gel block, and placing the gel block under an ultraviolet lamp of 300nm for observation to confirm the success of the PCR product amplification reaction. Then, the PCR product successfully amplified is sent to Huada gene science and technology limited company for sequencing, and the sequencing primer is the same as the amplification primer. An SMK-116s rRNA gene sequence with the length of 1410bp, which is obtained by carrying out gene sequencing on a PCR product, and the sequence of the gene sequence is shown as SEQ ID NO: 1 is shown.

The 16s rRNA gene sequence of the bacteria SMK-1 obtained by sequencing is uploaded to NCBI, and the website can compare the submitted sequence with the 16s rRNA gene sequence of a typical strain of a recognized species to obtain the similarity information among the sequences. According to the result analysis of sequence comparison, a corresponding typical strain can be selected as a model strain of the experimental isolation strain, and meanwhile, a 16s rRNA gene sequence of the model strain can be obtained, and phylogenetic analysis is constructed to prove that the model strain and the experimental isolation strain SMK-1 have difference, so that the isolated strain SMK-1 is identified. Construction of phylogenetic trees are constructed using the program MEGA 5.05 (Tamura et al, 2011), usually using the neighbor, minimum evolution and maximum reduction methods, where the most common is the neighbor and the bootstrap value is often set to repeat 1000 calculations (Felsenstein et al, 1985). A phylogenetic tree is made by using the 16s rRNA gene sequence of SMK-1 and the 16s rRNA gene sequence with higher similarity, so as to obtain the homology result between the 16s rRNA gene of SMK-1 and the 16s rRNA gene with higher similarity. The phylogenetic tree constructed by the minimum evolution method is shown in figure 2, and the result shows that the SMK-1 has the highest homology with Paenarthrobacter nicotenovorans DSM 420, and the strain is different strains belonging to the same genus of Paenarthrobacter on the aspect of molecular biology. Therefore, this strain SMK-1 was named as Paenarthrobacter nicotinovorans SMK-1.

2.4 growth conditions of Strain SMK-1

Measurement of growth temperature: preparing a liquid nutrient medium required by the growth of the strain SMK-1, and taking the prepared liquid nutrient medium into a sterilization pot for sterilization. Inoculating the activated strain SMK-1 into a culture medium (an experimental group), taking the culture medium without inoculating bacteria as a control (a control group), putting the culture medium into different temperatures for culturing, wherein the control group and the experimental group corresponding to each temperature are repeated three times, the growth condition of the bacteria needs to be observed every day, and when a result which is difficult to distinguish by naked eyes is met, measuring the light absorption value of the culture medium at the position of 600nm of wavelength lambda by using a visible-ultraviolet spectrophotometer to finally obtain the growth temperature and the optimal growth temperature range of the SMK-1. The test temperatures were as follows: 25 ℃, 30 ℃, 37 ℃ and 40 ℃. As shown in FIG. 3, in the liquid nutrient medium, the strain SMK-1 can grow at a temperature of 25-40 ℃, and the optimal growth temperature is 30 ℃ of the enrichment temperature of the strain.

Measurement of growth pH: preparing a liquid nutrient medium required by the growth of the strain SMK-1, and adjusting the pH of a culture solution by using a buffer system, wherein the pH is 4.0-5.0, and 0.1mol/L sodium citrate and 0.1mol/L citric acid are added; pH 6.0-8.0, 0.1mol/L NaOH and 0.1mol/L KH₂PO₄；pH 9.0～10.0，0.1mol/L NaHCO₃And 0.1mol/L Na₂CO₃(ii) a pH 11.0, 0.1mol/L NaOH and 0.05mol/L Na₂HPO₄(Zhang et al, 2009). Inoculating the strain SMK-1 into a liquid nutrient medium, repeating every pH value for three times, using a culture medium without inoculated bacteria as a control, putting the liquid nutrient medium into a new bacterium for culturing at the optimum growth temperature of 30 ℃, observing the growth condition of the bacteria every day, and measuring the light absorption value of the culture medium at the position of 600nm wavelength by using a visible-ultraviolet spectrophotometer when a result which is difficult to distinguish by naked eyes is met, thereby obtaining the growth pH value and the optimum growth pH range of the strain SMK-1. The pH tested was as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0. As shown in FIG. 4, the strain SMK-1 was able to grow at a pH of 5.0 to 9.0, and the optimum growth pH was 7.0.

Salt concentration tolerance: preparing a liquid nutrient medium required by the growth of the strain SMK-1, and adjusting the salt concentration of the medium. Inoculating activated strain SMK-1 into sterilized liquid nutrient medium, repeating the concentration of each salt for three times, using culture medium without inoculated strain as control, placing the liquid nutrient medium under the optimum growth condition (30 ℃, pH7.0) of SMK-1, culturing, observing the growth condition of bacteria every day, and when the bacteria are difficult to distinguish by naked eyes, measuring the light absorption value of the culture medium at the wavelength lambda of 600nm by using a visible-ultraviolet spectrophotometer, and preferably obtaining the salt concentration range which can be tolerated by new bacteria. The salt concentrations (mass fractions) tested were as follows: 0%, 1%, 3%, 5%, 7%, 10%. As shown in FIG. 5, the salt tolerance of the strain SMK-1 was weak, and the strain SMK-1 was able to grow at a salt concentration of 0% to 5%, and was the best at a salt concentration of 0%.

2.5 growth and degradation of Strain SMK-1 under different sulfamethoxazole concentrations

According to the above experimental results, the optimal growth conditions of the strain were determined to be at 30 ℃ and pH7.0, without adding NaCl. The growth and degradation experiments of the strain SMK-1 in different antibiotic concentrations and different pH values were carried out under these conditions (temperature 30 ℃ C., without addition of NaCl). The strain SMK-1 in logarithmic growth phase was inoculated at an inoculum size of 10% (v/v) to a medium containing an initial sulfamethoxazole concentration of 1, 10, 30, 50, 100 mg.L^-1The inorganic salt medium of (1), pH was as follows: 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, shaking and culturing, and performing parallel experiments for 3 times.

The sulfamethoxazole concentration is measured by adopting an ultraviolet spectrophotometry, namely, the collected bacterial liquid is centrifuged for 1min at 10000rpm, the bacterial liquid passes through a 0.22 mu m organic filter membrane, the wavelength is scanned to find the optimum wavelength, finally, the absorbance is measured at the wavelength of 255nm, the degradation rate is calculated, and all the experimental steps are protected from light as much as possible.

The cell concentration of the microorganisms is measured by a photoelectric turbidimetric method and is expressed by OD, namely the optical density value of ultraviolet light which penetrates through a measured bacterial liquid sample when the wavelength is 600 nm.

The growth curve of the strain SMK-1 is shown in FIG. 6. The results show that SMK-1 can be used in high concentration sulfamethoxazole conditionAnd (4) growing. The degradation condition of sulfamethoxazole by the strain SMK-1 is shown in figures 7-10, wherein figure 7 shows the degradation condition of sulfamethoxazole after 7 days, and figures 8-10 show the degradation condition of sulfamethoxazole by the strain under the conditions of pH6, pH7 and pH8 respectively. The initial concentration of the strain SMK-1 in the antibiotic is 30 mg.L measured and analyzed by an ultraviolet spectrophotometry method^-1After the inorganic salt culture medium with the pH of 6-8 is cultured for 5 days, the degradation rate can reach over 25 percent, and when the inorganic salt culture medium is cultured for 10 days, the degradation rate can reach over 45 percent; at an initial sulfamethoxazole concentration of 100 mg.L^-1After the inorganic salt culture medium with the pH of 6-7 is cultured for 10 days, the degradation rate can reach more than 20 percent and is 100 mg.L^-1After the inorganic salt culture medium with the pH value of 8 is cultured for 10 days, the degradation rate can reach more than 73 percent; the strain is a strain with strong antibiotic degradation and antibiotic tolerance, and has strong adaptability to sulfamethoxazole; has good application prospect in the bioremediation of sulfamethoxazole polluted environment.

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.

Sequence listing

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ggtctctggg ctgtaactga cgctgaggag cgaaagcatg gggagcgaac aggattagat 720

accctggtag tccatgccgt aaacgttggg cactaggtgt gggggacatt ccacgttttc 780

cgcgccgtag ctaacgcatt aagtgccccg cctggggagt acggccgcaa ggctaaaact 840

caaaggaatt gacgggggcc cgcacaagcg gcggagcatg cggattaatt cgatgcaacg 900

cgaagaacct taccaaggct tgacatgaac cggaaagacc tggaaacagg tgccccgctt 960

gcggtcggtt tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1020

agtcccgcaa cgagcgcaac cctcgttcta tgttgccagc ggttcggccg gggactcata 1080

ggagactgcc ggggtcaact cggaggaagg tggggacgac gtcaaatcat catgcccctt 1140

atgtcttggg cttcacgcat gctacaatgg ccggtacaaa gggttgcgat actgtgaggt 1200

ggagctaatc ccaaaaagcc ggtctcagtt cggattgggg tctgcaactc gaccccatga 1260

agtcggagtc gctagtaatc gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt 1320

gtacacaccg cccgtcaagt cacgaaagtt ggtaacaccc gaagccggtg gcctaaccct 1380

tgtgggggag ccgtcgaagg tgacggacta 1410

Claims

1. A sulfonamide antibiotic-degrading bacterium characterized by: the microbial strain preservation center is named as Paenarthrobacter nicotinovorans SMK-1, and is preserved in 21 days 4 and 21 months 2021 at the Guangdong province microbial strain preservation center of Guangdong province microbial research institute No. 59 building, No. 5 building, Guangdong province of Mieli Zhonglu 100 institute, Guangzhou, with the preservation number: GDMCC No: 61613.

2. a biological agent characterized by: the sulfonamide antibiotic-degrading bacterium according to claim 1.

3. The biological agent according to claim 2, characterized in that: prepared by liquid culture of the sulfonamide antibiotic-degrading bacteria of claim 1.

4. The biological agent according to claim 3, characterized in that: inoculating the sulfonamide antibiotic-degrading bacteria of claim 1 into LB liquid medium for culture to obtain the biological agent.

5. A biological agent according to claim 3 or 4, characterized in that:

the culture is carried out at 25-40 ℃ and pH5.0-9.0 for 24-72 h.

6. Use of the sulfonamide antibiotic-degrading bacterium of claim 1 or the biological agent of any one of claims 2 to 5 for degrading a sulfonamide antibiotic.

7. Use according to claim 6, characterized in that:

the sulfonamide antibiotic is sulfamethoxazole.

8. A method of degrading a sulfonamide antibiotic, comprising: the sulfonamide antibiotic degrading bacteria of claim 1 or the biological preparation of any one of claims 2 to 5 is applied to an environment containing sulfonamide antibiotics to realize the purpose of degrading the sulfonamide antibiotics.

9. The method of claim 8, wherein:

the environment containing the sulfonamide antibiotics is water or soil containing the sulfonamide antibiotics;

the concentration of the sulfonamide antibiotics is 1-100 mg.L^-1；

10. The method according to claim 8 or 9, characterized in that:

the sulfonamide antibiotic is sulfamethoxazole;

in the environment, the temperature is 25-37 ℃, the initial pH is 5-9, and the environment is aerobic.