CN108611285A - A kind of sulfa antibiotics degradation bacteria and its application - Google Patents
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Abstract
The invention discloses a kind of sulfa antibiotics degradation bacteria and its applications.The degradation bacteria is saccharomycete（Sakaguchia cladiensis）A5, the bacterial strain are preserved in Guangdong Province's Culture Collection, deposit number on January 26th, 2018：GDMCC NO：60319.The bacterial strain has good degradation effect to sulfa antibiotics such as sulfamerazine and Sulfamethoxazoles, the degradation that can be used for sulfa antibiotics pollution is administered, application may include the waste water and dregs containing sulfa antibiotics pollutant, water environment or soil etc., the harm that antibiotic generates ecological environment security can be reduced, and have many advantages, such as energy-saving and environmental protection, it has broad application prospects.
Description
Technical field
The invention belongs to environmental contaminants processing technology fields.More particularly, to a kind of sulfa antibiotics degradation bacteria
And its application.
Background technology
China be antibiotics production and use big country, have every year thousands of ton antibiotic medicines be used for livestock culture and
In personal medical treatment, wherein sulfa antibiotics are since the advantage of its broad spectrum activity and super quality and competitive price is as China's medicine and veterinary drug antibiosis
Larger one kind of usage amount in element.In fact, most of antibiotic can not be absorbed by body completely.The study found that up to
85%~90% antibiotic is entered through excreta in environment with drug original shape or metabolite form, is caused seriously to soil and water body etc.
Pollution.Mo Ce brightness seminar mainly for South China's antibiotic pollute influence to vegetable cultivation and agricultural food product safety into
Row research, it was demonstrated that antibiotic can be polluted by livestock excrement composting and bring the agricultural product such as vegetables into.It is aobvious from the data of literature survey
Show, sulfa antibiotics pollution condition is very severe in China's surface water, and antibiotic content is apparently higher than other countries in river water.
Detect that high concentration sulfa antibiotics remain in the major river basins in China.Zhujiang River dry season detects that sulfamethoxazole is dense
Degree is 1.4~157 ng/L, a concentration of 29.5~120 ng/L of sulfamerazine；The Liaohe River, Haihe basin detect concentration respectively
The sulfamethoxazole of up to 173 and 211 ng/L；A concentration of 14.9~623.3 ng/L of Huangpu River dry season sulfamerazine；Good fortune
It builds and also detects sulfamerazine in Jiulongjiang River, a concentration of 775.5 ng/L.
Municipal sewage plant is typically considered one of defence line that pollutant enters environment, but sewage disposal system now
System is not notable to the removal effect of antibiotic, and the water outlet still antibiotic residue containing higher concentration causes municipal sewage plant
Water outlet becomes one of the main source of antibiotic in environment.And in China's aquaculture, antibiotic as feed addictive more
It is directly to be devoted in water.Human and animal excreta is used as farm manure in use, wherein remaining antibiotic can also enter soil
In, form pollution of area source more rambunctious.Although antibiotic, which was once medically human health, makes tremendous contribution, however as
The abuse of antibiotic leads to drug-fast bacteria emergence and mass propagation, the resistant gene of generation is induced to cause to dive to ecological environment
Gene contamination.Currently, bacteria antibiotic drug resistance has become the focal issue for threatening human health and ecological safety.2000
A report in year WHO proposes that antibiotic resistance has become one of the key problem of 21 century challenge human health.
Degradation of Antibiotics includes the non-biodegradations such as photodissociation, hydrolysis and biodegradation, wherein microbial degradation in environment
It is the main approach of Degradation of Antibiotics in environment.The physics and chemistry processing method containing antibiotic residue sewage is had been carried out at present
A large amount of research and practice, including advanced oxidation processes, active carbon adsorption, lower temperature plasma technology and membrane technology etc.,
But of high cost, complex management needed for these physics and chemistry factures, it is other in addition to advanced oxidation processes can reach higher removal rate
Technical method removal effect is relatively low, and there are limitations to the remaining processing of antibiotic in solid state medium.Therefore, related anti-
The microbial degradation of raw element is increasingly becoming research hotspot.
Invention content
The technical problem to be solved by the present invention is to overcome existing sulfa antibiotics pollute treatment technology defect and deficiency,
A kind of microbial bacteria for capableing of efficient degradation sulfa antibiotics is provided.It is intended to promote microbial technique in environment sulfamido antibiosis
Application in plain pollution control solves environment sulfa antibiotics pollution problem.
The object of the present invention is to provide a kind of sulfa antibiotics degradation bacterias.
Another object of the present invention is to provide application of the degradation bacteria in terms of sulfa antibiotics pollutant process.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention the study found that saccharomycete (Sakaguchia cladiensis) to sulphurs such as sulfamerazine and Sulfamethoxazoles
Amine antibiotic has good degradation effect, and the degradation that can be used for sulfa antibiotics pollution is administered.
And the present invention is also screened from certain regional livestock and poultry farm polluted soil of Pearl River Delta, tames, is isolated and purified
To a saccharomycete A5 bacterial strains, Guangdong Province's Culture Collection, deposit number are preserved on January 26th, 2018：
GDMCC NO：60319.
Therefore, saccharomycete (Sakaguchia cladiensis) application in terms of degradation treatment sulfa antibiotics or
Application in improving sulfa antibiotics degradation removal rate, and administering the water environment polluted by sulfa antibiotics and soil
Application in terms of earth, should all be within protection scope of the present invention.
Preferably, the saccharomycete is above-mentioned saccharomycete A5 bacterial strains.
Preferably, the sulfa antibiotics are sulfamerazine, Sulfamethoxazole and/or sulphadiazine.
One kind containing saccharomycete (Sakaguchia cladiensis) sulfa antibiotics degradation bacterial agent, also Ying Ben
Within the protection domain of invention.Preferably, the saccharomycete is the saccharomycete A5 bacterial strains.
Preferably, the degradation bacterial agent is the bacteria suspension of saccharomycete A5 bacterial strains.
Preferably, it is to be prepared after saccharomycete A5 bacterial strain activation cultures.Specifically saccharomycete A5 bacterial strains are cultivated in LB
Activation is cultivated in base or yeast culture medium, and sediment is collected by centrifugation, dilutes and is prepared after washed.
It is highly preferred that the preparation method of the bacteria suspension is：The saccharomycete A5 that inclined-plane preserves is inoculated into liquid LB cultures
In base, in 25~30 DEG C, 150~160 r/min constant-temperature shaking cultures, 24~36 h, activated seed liquid is made；By inoculum concentration 1%
Seed liquor is inoculated into fresh culture by~10%, continues at 25 DEG C, 150 r/min expansion cultures, 24~36 h；Then 25 DEG C,
6000 r/min centrifuge 5~10min, abandon supernatant collection thalline, sterile water washing 2~3 times is finally configured to 1 with sterile water
The bacteria suspension of g/L.
Preferably, the formula of the LB culture mediums is：NaCl 5g/L, yeast extract 10g/L, tryptose 10g/L,
Solvent is water, and pH is 7.0~7.2.Autoclave sterilization before experiment.
The invention has the advantages that：
The present invention screens to obtain a Yeasts, has to sulfa antibiotics such as sulfamerazine and Sulfamethoxazoles good
Degradation effect, the degradation that can be used for sulfa antibiotics pollution are administered, and application may include polluting containing sulfa antibiotics
Waste water and dregs, water environment or soil of object etc. can reduce the harm that antibiotic generates ecological environment security
Moreover, the saccharomycete processing sulfa antibiotics using the present invention pollute, with the methods of physical absorption and chemical degradation phase
Than having many advantages, such as energy-saving and environmental protection, there is very wide application prospect.
Description of the drawings
Fig. 1 is the form pattern of the plated growth of saccharomycete A5.
Fig. 2 is the microscope imaging figure of saccharomycete A5.
Fig. 3 is the phylogenetic tree of saccharomycete A5.
Fig. 4 is the growth curve chart of saccharomycete A5.
Fig. 5 is degradation curves of the saccharomycete A5 to sulfamerazine.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
The separation and identification of 1 sulfa antibiotics degradation bacteria of embodiment
1, experiment material
Nutrient medium：5 g of sodium chloride, 3 g of beef extract, 10 g of albumen, distilled water 1000mL, pH are 7.0~7.2.Solid
Culture medium then adds agar powder（Item）15 g/L.Minimal medium：(NH4)2SO4 1g、MgSO4·7H2O 0.1g、KH2PO4 3g、
K2HPO4·3H2O 7g, sodium citrate 0.5g, distilled water 1000mL, pH are 7.0~7.2.
It is placed in high-pressure steam sterilizing pan before the culture medium experiment, 121 DEG C of sterilizing 30min.
2, the separation and identification of bacterial strain
（1）Sulfa antibiotics are resistant to the screening of bacterium, domestication：
4 stages of microbial acclimation point carry out.The contaminated soil-like of a certain livestock and poultry farm in Pearl River Delta is acquired, first sterile
Under operating condition, soil sample is added to 100 mL and contains sulfa antibiotics（10 mg/L）Nutrient medium in, be placed in 30 DEG C, 150
7 d of culture are protected from light in the constant-temperature table of r/min.Later, it is 30 to take the culture solution of 10% volume to be transferred to another antibiotic concentration
In the nutrient medium of mg/L, similarity condition continues to cultivate 7 d, carries out second stage domestication.Next, every 7 d is a period,
Continue culture domestication, the phase III tames antibiotic concentration and promoted to 50 mg/L；Fourth stage domestication antibiotic concentration is promoted to
100 mg/L；Domestication process changes except antibiotic concentration, and other condition of culture are constant.
（2）Sulfa antibiotics degradation bacteria isolating and purifying and screening：
Domestication terminates, and bacteria group culture liquid is pressed 10-1~10-6Gradient dilution takes 10 respectively-4、10-5、10-63 dilution gradients
Each 200 μ L coatings of culture solution are inoculated on solid nutrient medium, are inverted in 30 DEG C of 24~36 h of culture in incubator.To length
The bacterium colony gone out is numbered, and chooses further scribing line respectively and be separately cultured, and single bacterium colony is purified to until obtaining.
Later, respectively by the minimal medium of each single colony inoculation to the sole carbon source containing sulfa antibiotics into
Row degradation experiment investigates degradation capability of each bacterium to sulfa antibiotics.The sulfa antibiotics be sulfamerazine,
Sulfamethoxazole and sulphadiazine, preferably sulfamerazine.
The degradation experiment condition is：In every 20 mL minimal mediums, 1 mg/L of sulfa antibiotics concentration is thrown
1 g/L of bacterium amount is placed in 25 DEG C, culture is protected from light in the constant-temperature table of 150 r/min.
What the best growth performance of one plant of degradation capability of finally screening acquisition was stablized is used as aimed strain, number A5.
（3）Sulfa antibiotics degradation bacteria（Aimed strain A5）Identification
The preparation and observation of electron microscope sample：1 drop sterile water of drop, the inoculation cooled down with calcination among clean glass slide
After a small amount of bacterium A5 of ring picking is mixed well with water droplet on glass slide, a uniform thin layer is coated on glass slide；Naturally dry；
Ammonium oxalate crystal violet dyeing liquor is added dropwise on having dried fixed thalline, stands 1 ~ 2 min；Dye liquor is toppled over later, it is tilting
Glass slide washes away the extra dye liquor of residual with clear water, until the water of outflow is in colourless, naturally dry.It is walked by microscope work
Suddenly, it first finds suitable view in low power microscopic observation and further looks at somatic cells pattern under high power lens with oil mirror later.
Strain morphology and Physiology and biochemistry qualification result：The form pattern of the plated growth of saccharomycete A5 is as shown in Figure 1, micro-
Mirror image is as shown in Figure 2.Bacterium colony takes on a red color, is opaque, is round, the smooth moistening in surface, neat in edge；Bacterium cell under microscope
At single ellipsoid, cell is larger, and coloring is good.
The Molecular Identification of bacterium A5：The Molecular Identification commission Guangdong Microbes Inst of bacterium A5 carries out.It is extracted by DNA,
PCR amplification, sequence and alignment, the 26S rDNA sequencing sequences of bacterium A5 withSakaguchia cladiensisHave
Highest homology.And phylogenetic tree is constructed, as shown in Figure 3.
To sum up Morphological Identification and Molecular Identification are as a result, the bacterium A5 that the present invention screens is accredited as saccharomyceteSakaguchia cladiensis.And Guangdong Province's Culture Collection, deposit number are preserved on January 26th, 2018：GDMCC
NO：60319；Preservation address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100.
The measurement of the growth curve of 2 saccharomycete A5 of embodiment
1, saccharomycete A5 is inoculated into respectively in LB culture mediums and LB culture mediums containing sulfa antibiotics, is placed in 25 DEG C,
150 r/min shaking table constant temperature incubations, and respectively at 0.5,1,1.5,2,3,4,6,8,10,12,24,36,48,60,72,96,
120,144,168,192,216 h are sampled, and measure culture solutionOD 600, returned to zero with the LB liquid medium of sterilizing.
The LB culture mediums are：NaCl 5g/L, yeast extract 10g/L, tryptose 10g/L, water 1000mL, pH
It is 7.0~7.2.
The LB culture mediums containing sulfa antibiotics are：Sulfamerazine is added in above-mentioned LB culture mediums, makes
Its a concentration of 100mg/L.
2, the results are shown in Figure 4, and the saccharomycete A5 also can normally give birth in the cultivating system containing sulfa antibiotics
It is long.
Degradation experiments of the 3 saccharomycete A5 of embodiment to sulfamerazine
1, experimental method
（1）The preparation of bacteria suspension：
From conservation inclined-plane, 1 ~ 2 ring bacterium A5 is scraped, is linked into sterilized LB culture mediums, wrapping is placed on constant-temperature table culture
Case（25 DEG C, 150 r/min）, expand 24 ~ 36 h of culture.The bacterium A5 culture solutions activated are taken, are centrifuged（6000 r/min, 25
℃）, thalline is collected, with sterile water washing 2 times（6000 r/min, 25 DEG C）It is resuspended, is made certain density with sterile water later
Bacteria suspension.
（2）Degradations of the saccharomycete A5 to sulfamerazine：
A certain amount of sulfamerazine is added in autoclaved minimal medium as sole carbon source, make its a concentration of 1
mg/L；1 mL bacteria suspensions are accessed later, and it is 1 g/L to make throwing bacterium amount（Weight in wet base）；Design blank group simultaneously（It is not added with bacterium）Control.It is all
Sample wrapping is placed on 25 DEG C, and 150 r/min are protected from light culture, samples, measures molten respectively at 1,2,4,8,12,24,36,48,72h
The concentration of sulfamerazine is remained in liquid.
（3）Induce degradations of the saccharomycete A5 to sulfamerazine：
Sulfamerazine is added in the LB culture mediums that bacterium A5 expands culture, makes its a concentration of 100 mg/L, activation culture is to right
The number phase is prepared as described above bacteria suspension, and carries out degradation experiments of the bacterium A5 to sulfamerazine as stated above.
（4）The measurement of sulfamerazine：
Quantitative supernatant and extractant are taken after sample centrifugation（Containing 0.1% formic acid water：Acetonitrile：Methanol=10:3:1）By 1:1 is fully mixed
It closes, supernatant liquid filtering is taken after high speed centrifugation（0.22 um）, utilize high performance liquid chromatography（HPLC）It is dense to analyze sulfamerazine remnants
Degree.Analysis condition is：C18 reversed-phase columns, 5 μm, 4.6 × 250mm, UV detector, Detection wavelength 270nm, water（Containing 0.1% first
Acid）：Acetonitrile=30:70（V/V）For mobile phase, 1 mL/min of flow velocity, 20 μ L of sample size.
2, the results are shown in Figure 5, and bacterium A5 extends the degradation rate of sulfamerazine and increases at any time, it is dropped when 2 d
Solution rate just reaches 38.2 %.Bacterium A5 after induction significantly improves the degradation of sulfamerazine.
Study on degradation of the 4 saccharomycete A5 of embodiment to various concentration sulfamerazine
A certain amount of sulfamerazine is added in autoclaved minimal medium as sole carbon source, its concentration is made to distinguish
For 0.5,1,3 and 5 mg/L；1 mL bacteria suspensions are accessed later, and it is 1 g/L to make throwing bacterium amount（Weight in wet base）；Design blank group simultaneously（No
Add bacterium）Control.All samples wrapping is placed on 25 DEG C, and 150 r/min are protected from light culture, is sampled after reacting 2 d, measures in solution
Remain the concentration of sulfamerazine.Prepared by bacteria suspension and sulfamerazine method for measuring is as described in example 3 above.
The result shows that bacterium A5 all has degradation effect to the sulfamerazine of various concentration, wherein to the sulfanilamide (SN) of 1 mg/L
First pyrimidine degradation effect is best, takes second place to the sulfamerazine degradation effect of 0.5 mg/L, high concentration sulfamerazine（5 mg/
L）Degradation effect more take second place.
Study on degradation of the saccharomycete A5 of the different throwing bacterium amounts of embodiment 5 to sulfamerazine
A certain amount of sulfamerazine is added in autoclaved minimal medium as sole carbon source, make its a concentration of 1
mg/L；A certain amount of bacteria suspension is accessed later, and it is respectively 0.5,1,1.5 and 2 g/L to make throwing bacterium amount（Weight in wet base）；Design blank simultaneously
Group（It is not added with bacterium）Control.All samples wrapping is placed on 25 DEG C, and 150 r/min are protected from light culture, is sampled after reacting 2 d, measures molten
The concentration of sulfamerazine is remained in liquid.Prepared by bacteria suspension and sulfamerazine method for measuring is as described in example 3 above.
The experimental results showed that the best throwing bacterium amount of 1 mg/L sulfamerazines of degradation is 1 g/L, other concentration throwing bacterium amounts
Influence to sulfamerazine degradation is not notable.
Study on degradation of the 6 saccharomycete A5 of embodiment to sulfamerazine in different pH systems
A certain amount of sulfamerazine is added in autoclaved minimal medium as sole carbon source, make its a concentration of 1
mg/L；PH value of solution is adjusted, it is respectively acidity to make system pH（1~3）, faintly acid（5~6）, it is neutral（7）, alkalescent（9~10）And alkali
Property（12~14）.A certain amount of bacteria suspension is accessed later, and it is 1 g/L to make throwing bacterium amount（Weight in wet base）；Design blank group simultaneously（It is not added with bacterium）
It compares.All samples wrapping is placed on 25 DEG C, and 150 r/min are protected from light culture, is sampled after reacting 2 d, measures and is remained in solution
The concentration of sulfamerazine.Prepared by bacteria suspension and sulfamerazine method for measuring is as described in example 3 above.
The experimental results showed that being unfavorable for the removal of sulfamerazine under acid condition.In neutrality to alkaline pH range, to sulphur
The influence unobvious of amine first pyrimidine degradation.
Degradation treatments of the 7 saccharomycete A5 of embodiment to the waste water containing sulfa antibiotics
Containing there are many sulfa antibiotics（Sulfamerazine, Sulfamethoxazole and sulphadiazine）Water body in be added bacterium A5 bacterium
Suspension, throwing bacterium amount are 1 g/L（Weight in wet base）, design blank group（It is not added with bacterium）Control.25 DEG C are set, 150 r/min are protected from light 2 d
After sample, measure solution in residual sulfamerazine, Sulfamethoxazole and sulphadiazine concentration.Bacteria suspension prepares and sulfalene
Pyrimidine method for measuring is as described in example 3 above.
The experimental results showed that sulfa antibiotics all have good degradation effect in A5 pairs 3 of bacterium, wherein to sulfamerazine
Degradation removal effect it is best.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. one plant of sulfa antibiotics that can degrade saccharomycete (Sakaguchia cladiensis) A5 bacterial strains, feature exists
In the bacterial strain is preserved in Guangdong Province's Culture Collection, deposit number on January 26th, 2018：GDMCC NO：
60319。
2. saccharomycete (Sakaguchia cladiensis) application in terms of degradation treatment sulfa antibiotics or improving sulphur
Application in amine Degradation of Antibiotics removal rate.
3. saccharomycete (Sakaguchia cladiensis) in terms of administering the water environment polluted by sulfa antibiotics and soil
Application.
4. being applied according to described in Claims 2 or 3, which is characterized in that the saccharomycete is saccharomycete A5 bacterium described in claim 1
Strain.
5. being applied according to described in Claims 2 or 3, which is characterized in that the sulfa antibiotics are sulfamerazine, sulfalene
Oxazole and/or sulphadiazine.
6. a kind of sulfa antibiotics degradation bacterial agent, which is characterized in that containing saccharomycete (Sakaguchia cladiensis)。
7. degradation bacterial agent according to claim 6, which is characterized in that the saccharomycete is saccharomycete A5 described in claim 1
Bacterial strain.
8. degradation bacterial agent according to claim 7, which is characterized in that the degradation bacterial agent is that the bacterium of saccharomycete A5 bacterial strains is outstanding
Liquid.
9. degradation bacterial agent according to claim 7, which is characterized in that be prepared after saccharomycete A5 bacterial strain activation cultures.
10. degradation bacterial agent according to claim 8, which is characterized in that the preparation method of the bacteria suspension is：Inclined-plane is preserved
Saccharomycete A5 be inoculated into LB liquid medium or yeast culture medium, activated seed liquid is made in constant-temperature shaking culture；Then will
Seed liquor is inoculated into fresh culture, continues after expanding culture, and supernatant collection thalline is abandoned in centrifugation, sterile water be resuspended bacterium is outstanding
Liquid.
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| CN109280631A (en) * | 2018-10-24 | 2019-01-29 | 哈尔滨商业大学 | One plant of sulfamethazine degradation bacteria S-2 and its application |
| CN110331120A (en) * | 2019-08-28 | 2019-10-15 | 中国科学院烟台海岸带研究所 | The Vibrio alginolyticus of one plant of a variety of sulfa antibiotics that can degrade and its application |
| CN111018131A (en) * | 2019-12-16 | 2020-04-17 | 同济大学 | Method for degrading sulfamethoxazole |
| CN111528396A (en) * | 2020-05-08 | 2020-08-14 | 广东海洋大学 | Application of pichia guilliermondii in degradation of antibiotics |
| CN113215033A (en) * | 2021-04-30 | 2021-08-06 | 华南农业大学 | Sulfonamide antibiotic degrading bacteria and application thereof |
| CN114292764A (en) * | 2021-09-10 | 2022-04-08 | 暨南大学 | Achromobacter strain JD417 and application thereof |
| CN117645957A (en) * | 2023-12-04 | 2024-03-05 | 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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| CN111018131B (en) * | 2019-12-16 | 2022-04-26 | 同济大学 | Method for degrading sulfamethoxazole |
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| CN113215033A (en) * | 2021-04-30 | 2021-08-06 | 华南农业大学 | Sulfonamide antibiotic degrading bacteria and application thereof |
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| 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 |
| CN117645957A (en) * | 2023-12-04 | 2024-03-05 | 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 |
| 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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