CN114149943B - Sulfanilamide antibiotic degrading bacteria at low temperature and application thereof - Google Patents
Sulfanilamide antibiotic degrading bacteria at low temperature and application thereof Download PDFInfo
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- CN114149943B CN114149943B CN202111361246.8A CN202111361246A CN114149943B CN 114149943 B CN114149943 B CN 114149943B CN 202111361246 A CN202111361246 A CN 202111361246A CN 114149943 B CN114149943 B CN 114149943B
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
Abstract
The invention belongs to the technical field of biological treatment of antibiotic pollution, and particularly relates to sulfonamide antibiotic degrading bacteria at low temperature and application thereof. The degrading bacteria are gram negative Acinetobacter (Acinetobacter sp.) H-3 and are preserved in China general microbiological culture Collection center (CGMCC) No.22941 at the month 22 of 2021. The strain used in the invention has high efficiency for degrading sulfadiazine at low temperature, and provides scientific basis for degrading sulfadiazine in soil.
Description
Technical Field
The invention belongs to the technical field of biological treatment of antibiotic pollution, and particularly relates to sulfonamide antibiotic degrading bacteria at a low temperature (5-15 ℃) and application thereof.
Background
The antibiotic is a secondary metabolite produced by the life activities of microorganisms or animals and plants, and has good effect on preventing and treating diseases. Sulfadiazine is a kind of sulfa antibiotic, belongs to artificial synthetic antibiotic, and has the features of broad spectrum, stability, simple use, etc. and is used as animal antibiotic for preventing and treating animal diseases. With the development of animal husbandry and breeding industry, the use amount of sulfadiazine is greatly increased, and potential influence is caused on the ecological system and human health, so that the sulfadiazine becomes a novel environmental pollutant.
The antibiotics can not be completely absorbed by livestock and poultry when used in a large amount, and 30% -90% of the antibiotics taken by the livestock and poultry can be discharged out of the body along with the feces and enter the environment through the effects of rainwater runoff, soil infiltration and the like. The antibiotics are not easy to degrade after entering the environment, and can enter the animal and plant bodies after migration and transformation, so that the diffusion of the antibiotics is aggravated. When the environmental antibiotics exceed the self-purification capability of the environment, ecological balance is destroyed, and growth and development of animals, plants, microorganisms and the like are inhibited. The persistence of antibiotics can cause microorganisms to develop antibiotic resistance, causing contamination of antibiotic resistance genes. Antibiotics can influence biosafety through food chains and ecosystems, can generate reactions such as immunity decline, anaphylactic reaction, chronic poisoning and the like of animals and humans, and can greatly influence the health of the humans and the safety of the ecosystems.
The method for removing the sulfonamide antibiotics comprises a physical adsorption method, an electrochemical method, a photodegradation method, a biodegradation method and the like. The biodegradation is an environment-friendly, convenient, efficient, economical and sustainable degradation method, and the microbial degradation is particularly important in the biodegradation.
At present, the microbial degradation research of sulfadiazine is less, researchers screen and separate out strains with the capability of degrading sulfanilamide antibiotics, but most researches only aim at degrading at medium and high temperature, li Liancheng screens out the research that the degradation rate of FF bacteria 240h for degrading sulfamethoxazole is 22.4 percent, the degradation rate of NT16 bacteria 240h for degrading sulfamethazine is 28.6 percent, and the researches of degrading sulfadiazine at low temperature are rare. The sulfadiazine degradation can be inhibited at low temperature, so that screening of sulfadiazine high-efficiency degradation bacteria at low temperature is an important breakthrough.
Disclosure of Invention
The invention aims to break through the low temperature problem of degrading sulfadiazine and provides a degrading bacterium for degrading sulfa antibiotics at the temperature of 5-15 ℃ and application thereof.
In order to achieve the above purpose, the invention adopts the technical scheme that:
a strain of sulfa antibiotic degrading bacteria at low temperature is prepared from gram negative Acinetobacter sp.H-3, which is preserved in China general microbiological culture Collection center (CGMCC) No.22941 at 22 days 7 and 2021.
The application of the degrading microorganism in degrading sulfonamide antibiotics.
The degrading microorganism is applied to degrading the sulfonamide antibiotics at a low temperature.
A degradation bacteria preparation contains the degradation microorganism.
The preparation is one or more of culture solution, culture concentrated solution and culture separation solution containing degrading microorganism.
The culture medium is prepared by shaking culture of Acinetobacter (Acinetobacter sp.) H-3 in LB medium at 37deg.C and 160rpm for 24 hr.
A degrading bacterial preparation, the use of said preparation for degrading sulfonamide antibiotics.
The degrading microorganism is applied to degrading the sulfonamide antibiotics at a low temperature.
The application of the degrading microorganism in degrading sulfadiazine at the temperature of 5-20 ℃.
Compared with the prior art, the invention has the following technical effects:
the invention screens out a sulfadiazine degrading bacterium at low temperature, and the strain is identified as Acinetobacter sp (Acinetobacter sp.) and named as H-3 by 16S sequence analysis. The preservation number is CGMCC No.22941. The strain used in the invention has high efficiency for degrading sulfadiazine, and the strain can utilize sulfadiazine as the only carbon source for growth and propagation to degrade sulfadiazine. Under the laboratory condition, the 20d degradation rate of the strain can reach 63.5% under the conditions of 15 ℃ and 160rpm, and the 20d degradation rate can reach 52% under the conditions of 5 ℃ and 160rpm, so the invention provides a scientific basis for removing sulfadiazine in the environment.
Drawings
FIG. 1 is a colony morphology of strain H-3 according to an embodiment of the present invention.
FIG. 2 shows the quantitative detection results of sulfadiazine degradation by strain H-3 at 5℃in accordance with the present invention.
FIG. 3 shows the quantitative detection results of sulfadiazine degradation by strain H-3 at 15℃in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention is further provided in connection with the accompanying examples, and it should be noted that the embodiments described herein are for the purpose of illustration and explanation only, and are not limiting of the invention.
The drawings of the present invention are only for the purpose of combining the disclosure of the specification, and are not intended to limit the applicable limitations of the present invention, so that any structural modification, proportional changes, or size adjustment should fall within the scope of the disclosure without affecting the efficacy and achievement of the present invention.
The high performance liquid chromatography-tandem mass spectrometry determination method of the invention comprises the following steps: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantitatively analyzes the concentration of sulfadiazine. Atlantis T3 chromatographic column (3 μm, 2.1X105 mm), column temperature is 30deg.C, mobile phase is composed of 98% (v/v) ammonium acetate+0.1% formic acid water solution (A) and 2% (v/v) acetonitrile (B), sample injection speed is 0.4ml/min, and sample injection amount is 10 μl.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
The apparatus employed in the present invention is conventional in the art, unless otherwise specified.
The preparation method of the culture medium used in the following examples of the invention is as follows:
1. enrichment medium: NH (NH) 4 NO 3 1.0g，K 2 HPO 4 1.0g，KH 2 PO 4 1.0g，NaCl 0.5g，MgSO 4 ·7H 2 O 0.1g，FeCl 3 0.05g，CaCl 2 0.02g of trace element solution 10m L, distilled water l000m L and pH 7.0.
LB medium: peptone 10g, yeast extract 5g, naCl 10g, distilled water l000mL, pH 7.0.
3. Solid medium: LB medium, 2% agar.
4. Screening the culture medium: with the enrichment medium.
5. Trace element solution: feSO 4 0.1g，MnSO 4 0.1g，ZnSO 4 0.1g，Na 2 MoO 4 0.01g，CaCl 2 0.1g，MgSO 4 3g，CuSO 4 0.1g of distilled water l000m L, pH 7.0.
Example 1: isolation of Acinetobacter sp
Enriching, separating and purifying the soil of a livestock and poultry farm in a new Shenbei area of Shenyang city of Liaoning province; specifically, the method comprises the following steps:
step one: enrichment of strains
5g of soil is weighed and added into 250mL of enrichment medium, wherein the enrichment medium contains 20mg/L sulfadiazine, and the enrichment medium is cultivated for 7d in a shaking table at 15 ℃ and 160r/min in a dark place. And adding 5mL of enrichment culture solution into a new enrichment culture medium, continuing to culture according to the culture conditions, and then transferring again, wherein the concentration of sulfadiazine added into the enrichment culture medium is increased by 20mg/L after transferring again. Repeating the steps for transfer culture until the concentration of sulfadiazine in the enrichment medium reaches 100mg/L.
Step two: isolation and purification of strains
The enriched culture solution is treated by sterile water according to 10 -3 、10 -4 、10 -5 、10 -6 And (3) carrying out gradient dilution, respectively and uniformly coating the dilutions with different gradients on an LB solid culture medium, observing morphological characteristics of bacterial colonies, picking single bacterial colonies with different bacterial colony morphologies, further separating and purifying on the LB solid culture medium, and picking pure bacterial colonies on a flat plate for preservation by using a glycerol tube after activation.
Step three: screening of degradation strains
Inoculating each separated single strain into a screening culture medium containing 20mg/L sulfadiazine according to an inoculum size of 5%, shake culturing in a shaking table at 15 ℃ and 160rpm for 7d under dark condition, quantitatively detecting sulfadiazine residual concentration by using a high performance liquid chromatography-tandem mass spectrometry method, obtaining the degradation rate of sulfadiazine degrading bacteria, and designating the strain with the highest degradation rate as H-3.
High performance liquid chromatography-tandem mass spectrometry determination method
High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantitatively analyzes the concentration of sulfadiazine. Atlantis T3 chromatographic column (3 μm, 2.1X105 mm), column temperature is 30deg.C, mobile phase is composed of 98% (v/v) ammonium acetate+0.1% formic acid water solution (A) and 2% (v/v) acetonitrile (B), sample injection speed is 0.4ml/min, and sample injection amount is 10 μl.
The strain has a degradation effect on sulfadiazine at low temperature and has the following characteristics:
the purified strain H-3 was streaked on LB solid medium and cultured at 28℃for 1d, and the colony was small, round, smooth in surface, small in projections, and milky in surface, as shown in FIG. 1.
Example 2: identification of Strain 16S
(1) Genome extraction
Genomic DNA of strain H-3 was extracted according to the procedure described using the Tiangen Biochemical technologies Co., ltd. TIANamp Bacteria DNA Kit bacterial genomic DNA extraction kit.
(2) PCR amplification
The 16S universal primers are as follows: 27F: agagttttgatcctggcttag 1492R: TACGGCTACCTTGTTACGACTT, PCR has a length of about 1500 bp.
PCR reaction system:
PCR reaction procedure:
pre-denaturation at 95℃for 4min, deformation at 94℃for 30s, renaturation at 55℃for 30s, extension at 72℃for 40s, and 35 cycles of extension at 72℃for 7min,4℃until termination.
(3) Gel electrophoresis
Finally, the obtained PCR product was observed by electrophoresis on a 1% agarose gel under 80V for 30min. And (3) delivering the amplified PCR product to Huada gene technology Co.Ltd for sequencing, wherein the sequencing primer is identical with the amplification primer. The nucleotide sequence of the 16S gene of strain H-3 was obtained, and the nucleotide sequence of the 16S gene of strain H-3 was as follows.
Strain H-3 gene sequence table
The above sequence consists of 1401 bases (bp). The measured 16S gene sequences were subjected to BLAST search alignment on the National Center for Biological Information (NCBI) website to obtain strains with higher similarity, and analysis results show that the strain H-3 was identified as Acinetobacter.
The strain H-3 is Acinetobacter sp, which has been preserved in 2021, 7 and 22 days, and has a preservation number of CGMCC No.22941.
Example 3: verification of degradation effect of H-3 on sulfadiazine
H-3 was cultured in LB medium at 37℃and 160rpm for 24 hours with shaking, and inoculated into a selection medium containing sulfadiazine at 20mg/L in an inoculum size of 5%, and cultured in light-shielding shaking at 5℃and 15℃and 160rpm for 20 days, respectively, with three replicates per group, and the non-sterile culture was used as a blank. The obtained culture was passed through a 0.22um organic filter membrane, and the sulfadiazine residual concentration was quantitatively detected by using high performance liquid chromatography-tandem mass spectrometry.
The calculation formula is as follows:
the result shows that the degradation rate of the strain can reach 63.5% under the conditions of 15 ℃ and 160rpm for 20d, and the degradation rate of a blank control is 36.8%; the degradation rate of 20d can reach 52% under the conditions of 5 ℃ and 160rpm, and the degradation rate of blank control is 22.5%.
The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present invention and are not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention should be included in the scope of the present invention.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination of the various embodiments of the present disclosure may be made without departing from the spirit of the present disclosure, which should also be considered as the subject matter of the invention of the present disclosure.
Sequence listing
<110> Shenyang applied ecological institute of academy of sciences in China
<120> sulfanilamide antibiotics degrading bacteria at low temperature and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1401
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
caagtcgagc gggggaagga agcttgctac tggacctagc ggcggacggg tgagtaatac 60
ttaggaatct gcctattagt gggggacaac gtttcgaaag ggacgctaat accgcatacg 120
ccctacgggg gaaagcaggg gatcttcgga ccttgcgcta atagatgagc ctaagccgga 180
ttagctagtt ggtggggtaa aggcctacca aggcgacgat ctgtagcggg tctgagagga 240
tgatccgcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300
atattggaca atggggggaa ccctgatcca gccatgccgc gtgtgtgaag aaggcctttt 360
ggttgtaaag cactttaagc gaggaggagg ctcttctggt taatacctag aatgagtgga 420
cgttactcgc agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg 480
tgcgagcgtt aatcggattt actgggcgta aagcgtacgt aggcggcttt ttaagtcgga 540
tgtgaaatcc ctgagcttaa cttaggaatt gcattcgata ctgggaagct agagtatggg 600
agaggatggt agaattccag gtgtagcggt gaaatgcgta gagatctgga ggaataccga 660
tggcgaaggc agccatctgg cctaatactg acgctgaggt acgaaagcat ggggagcaaa 720
caggattaga tacccctggt agtccatgcc gtaaacgatg tctactagcc gttggggcct 780
ttgaggcttt agtggcgcag ctaacgcgat aagtagaccg cctggggagt acggtcgcaa 840
gactaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900
cgatgcaacg cgaagaacct tacctggcct tgacatacag agaactttcc agagatggat 960
tggtgccttc gggaactctg atacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttttcct tatttgccag cacttcgggt 1080
gggaacttta aggatactgc cagtgacaaa ctggaggaag gcggggacga cgtcaagtca 1140
tcatggccct tacggccagg gctacacacg tgctacaatg gtcggtacaa agggttgcta 1200
cctagcgata ggatgctaat ctcaaaaagc cgatcgtagt ccggattgga gtctgcaact 1260
cgactccatg aagtcggaat cgctagtaat cgcggatcag aatgccgcgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac catgggagtt tgttgcacca gaagtaggta 1380
gtctaaccgc aaggaggaaa c 1401
Claims (9)
1. A sulfadiazine degrading bacterium at low temperature is characterized in that: the degrading bacteria are gram negative Acinetobacter (Acinetobacter sp.) H-3 and are preserved in China general microbiological culture Collection center (CGMCC) No.22941 at the month 22 of 2021.
2. Use of a degrading bacterium according to claim 1, wherein: the application of the degrading bacteria in degrading sulfadiazine.
3. Use of a degrading bacterium according to claim 2, wherein: the degrading bacteria are applied to degrading sulfadiazine at low temperature.
4. A degrading bacteria preparation, which is characterized in that: the preparation comprises the degrading bacterium according to claim 1.
5. The degrading bacterial preparation according to claim 4, wherein: the preparation is a culture solution containing degradation bacteria.
6. The degrading bacterial preparation according to claim 5, wherein: the culture medium is obtained by shaking culture of Acinetobacter (Acinetobacter sp.) H-3 of claim 1 in LB medium at 37℃and 160rpm for 24 hours.
7. The use of the degrading bacterial preparation according to claim 4, wherein: the application of the preparation in degrading sulfadiazine.
8. The use according to claim 7, wherein: the degrading bacteria preparation is applied to degrading sulfadiazine at low temperature.
9. The use according to claim 8, wherein: the application of the degradation bacteria preparation in degrading sulfadiazine at the temperature of 5-20 ℃.
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