CN107858305A - One plant of tetracycline efficient degradation bacterium and its application - Google Patents

One plant of tetracycline efficient degradation bacterium and its application Download PDF

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Publication number
CN107858305A
CN107858305A CN201711212339.8A CN201711212339A CN107858305A CN 107858305 A CN107858305 A CN 107858305A CN 201711212339 A CN201711212339 A CN 201711212339A CN 107858305 A CN107858305 A CN 107858305A
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China
Prior art keywords
tetracycline
efficient degradation
degradation bacterium
application
bacterium
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CN201711212339.8A
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CN107858305B (en
Inventor
康贻军
夏丹
印志峰
沈敏
赵庆新
张言周
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Yancheng Teachers University
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Yancheng Teachers University
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    • C12N1/205
    • C12R2001/22
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/30Organic compounds
    • C02F2101/38Organic compounds containing nitrogen
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/34Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32
    • C02F2103/343Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the pharmaceutical industry, e.g. containing antibiotics

Abstract

The invention discloses one plant of tetracycline efficient degradation bacterium and its application, present invention screening from the organic fertilizer with chicken manure etc. for raw material fermentation obtains one plant of tetracycline efficient degradation bacterium, pass through physio-biochemical characteristics and 16SrDNA sequence analyses, the bacterial strain is accredited as Klebsiella, and is named as TR5.Under the efficiency laboratory level of strains for degrading tetracycline used in the present invention, by response surface experimental design, prediction can reach more than 90%.Data than being reported in current most of documents improve 20% or so.The present invention also improves capacity of resisting impact load and biomass of the tetracycline efficient degradation bacterium in water body using immobilized microorganism technology, is handled for related aquaculture system and pharmaceutical factory's tetracycline antibiotics waste water control aspect provides green, the biological approach of non-environmental-pollution.

Description

One plant of tetracycline efficient degradation bacterium and its application
Technical field
The present invention relates to a kind of bacterial strain of tetracycline efficient degradation bacterium Klebsiella, more particularly to a kind of micro- Application of the biological fixnig embedding techniques to water body purification of aquaculture and pharmaceutical factory's tetracycline antibiotics waste water treatment.
Background technology
In recent years, the environment tetracycline antibiotics caused by medical treatment and a large amount of uses of livestock and poultry breeding industry is dirty Dye problem is on the rise, it has also become one of focus studied both at home and abroad.Studies have reported that claim the antibiotic in environment except that can make Into outside chemical contamination, it is also possible to the generation of resistant microorganism and resistant gene in meeting induced environment, and aggravate antibiotic resistance biography The risk broadcast.Some resistant microorganisms may directly or indirectly participate in the approach such as food chain and enter human body, cause human body Antibiotic Resistance increase, so as to bring danger to a certain degree to coerce to mankind's publilc health.
Tetracycline is one of main species of tetracycline antibiotics, and existing many documents and patent report cross phase at present The situation and method of tetracycline degraded are closed, is mainly urged including physico-chemical process, During High-Temperature Composting method, microbial degradation method, photoelectricity Change etc..Wherein, microbial degradation method because its condition is single, be easily controlled, selectivity compared with it is strong, application cost is low and will not trigger Secondary pollution, it is current both economical effective method.But in the prior art, the report to microbial degradation method and few, has Document report degradeds of one Achromobacter xylosoxidans to tetracycline, degradation rate 63.9%, also document report one Degraded of the bacillus subtilis to tetracycline, defect shortwave monad is mentioned in degradation rate 62.8%, also document (Brevundimonas diminuta), degraded of the human pallid bacillus (Ochrobactrum anthropi) to tetracycline, but Degradation efficiency is not high.Therefore the popularization and application of microbial degradation method be have impact on.
The content of the invention
It is an object of the invention to solve the deficiencies in the prior art, there is provided a kind of tetracycline efficient degradation bacterium, the bacterium Nearly 30% is improved than prior art to the degradation rate of tetracycline, can be applied to culture fishery water body purification and tetracycline Class antibiotic pharmacy sewage is administered.
Technical scheme
A kind of tetracycline efficient degradation bacterium, it is Friedlander's bacillus category (Klebsiella pneumoniae), hides In China Microbial Culture Preservation Commission's common micro-organisms center, deposit number is CGMCC No. 14393, preservation date For:On July 7th, 2017, preservation address are:BeiJing, China.
The present inventor is by the way that from being to screen to obtain in the organic fertilizer that raw material ferment with chicken manure etc., isolation medium uses The continuous enrichment culture of carbon-free culture medium using tetracycline as sole carbon source, flat board isolate and purify to obtain highly resistance to tetracycline Single bacterium colony, isolated single bacterium colony sterile working is added in the nutrient solution containing finite concentration tetracycline and cultivated, and set Blank control group is tested, and the concentration that tetracycline is remained in nutrient solution, each bacterium of com-parison and analysis are determined with HPLC after cultivating certain time Fall the degradation efficiency to tetracycline, further filter out tetracycline efficient degrading bacterial strain.The 16SrDNA gene orders of the bacterial strain See SEQ ID No1.By physio-biochemical characteristics and 16SrDNA sequence analyses, the bacterial strain is accredited as Klebsiella (Klebsiellapneumoniae), and it is named as TR5.
Application of the above-mentioned tetracycline efficient degradation bacterium in culture fishery water body purification.
Application of the above-mentioned tetracycline efficient degradation bacterium in the waste water control of tetracycline antibiotics pharmaceutical factory.
Beneficial effect:The present invention will be the industrialization of tetracycline efficient degrading bacteria by microbial immobilized processing method Using providing theoretical foundation, presence that the immobilized spherules of TR5 bacterial strains of the invention can be stable in water body, in water body Fourth Ring is known as good degradation effect, and can be recycled, and has a higher biological safety, and application cost is low and not Secondary pollution can be triggered, culture fishery water body purification is can be applied to and tetracycline antibiotics pharmacy sewage is administered.
Brief description of the drawings
Fig. 1 is TR5 evolution tree graph;
Fig. 2 is different carbon source to TR5 upgrowth situations and the influence figure of degradation efficiency;
Fig. 3 is different carbon source and influence of the different nitrogen sources compatibility to TR5 upgrowth situations and degradation efficiency;
Fig. 4 is temperature to TR5 upgrowth situations and the influence figure of degradation efficiency;
Fig. 5 is rotating speed to TR5 upgrowth situations and the influence figure of degradation efficiency
Fig. 6 is pH to TR5 upgrowth situations and the influence figure of degradation efficiency;
Fig. 7 is inoculum concentration to TR5 upgrowth situations and the influence figure of degradation efficiency.
Embodiment
Embodiment 1
Separation, purifying and the preservation of bacterial strain
Weigh the liquid for the organic manure 5g that raw material ferment to 50ml gradients containing various concentrations tetracycline with chicken manure etc. Culture medium, and using tetracycline as sole carbon source (culture medium prescription as:Contain 5g (NH4) in per 1L nutrient solutions2SO4、1g KH2PO4、0.5g MgSO4·7H2O、0.1g CaCl2, 0.1g NaCl), 7d is cultivated under 28 DEG C, 150rpm, according to same Condition of culture by continuous enrichment culture, obtain what can be grown under conditions of using high concentration tetracycline as sole carbon source Bacterial strain, the bacteria suspension of following absorption 0.2% are coated on the solid culture containing higher concentration tetracycline and using it as carbon source Base, typically single bacterium colony carries out isolation and purification culture to picking after cultivating 2~3d, the pure culture bacterial strain that final separation is obtained High concentration tetracycline nutrient solution culture is carried out, the initial concentration of tetracycline in nutrient solution, and the timing of culture one are measured using HPLC Between after in nutrient solution tetracycline residual concentration, obtain tetracycline degradation efficiency corresponding to different pure culture bacterial strains, analyze ratio Compared with data, tetracycline efficient degrading bacteria is obtained, the 16SrDNA gene orders of the bacterial strain are shown in SEQ ID No1, pass through Physiology and biochemistry Characteristic and 16SrDNA sequence analyses, the bacterial strain are accredited as Klebsiella (Klebsiellapneumoniae), and will It is named as TR5.The bacterial strain is preserved in China Microbial Culture Preservation Commission's common micro-organisms on July 7th, 2017 The heart, deposit number are CGMCC No. 14393.
Embodiment 2
The influence of growth and degrade tetracycline of different carbon source, nitrogen source, pH, temperature and the inoculum concentrations to TR5
Carbon source needed for experimental group using the carbohydrates as strain growth such as glucose, starch, lactose, with yeast extract, albumen Peptone, casein etc. are as the nitrogen source needed for strain growth, pH 3~11, temperature at 15 DEG C~40 DEG C, inoculum concentration is 2%~ Tested under the conditions of 10% laboratory cultures.
Fig. 1 is TR5 evolution tree graph.
Fig. 2 be carbon source to TR5 upgrowth situations and the influence figure of degradation efficiency, as seen from Figure 2, because sodium acetate is in Alkalescence, and the unstable easily decomposition of tetracycline under alkalescence condition, upgrowth situation of the TR5 bacterial strains under this laboratory condition is also bad, Finally determined from glucose as the optimum carbon source under the bacterial strain laboratory condition according to this figure.
Fig. 3 is different carbon source and different nitrogen sources compatibility to TR5 upgrowth situations and the influence figure of degradation efficiency;Can be with by Fig. 3 Find out, using glucose as optimum carbon source, while using yeast extract as nitrogen source in the case of, TR5 to tetracycline degraded effect Rate is optimal, therefore final choice yeast extract is as the optimum nitrogen source under the bacterial strain laboratory condition.
Fig. 4 be temperature to TR5 upgrowth situations and the influence figure of degradation efficiency, as seen from Figure 4, TR5 bacterial strains are in 25- Tetracycline efficiency of being degraded under the conditions of 30 DEG C is higher.
Fig. 5 be rotating speed to TR5 upgrowth situations and the influence figure of degradation efficiency, as seen from Figure 5, TR5 bacterial strains are in rotating speed For 180rpm when degradation efficiency it is optimal, according to growth curve can initial guess, the bacterium is more aerobic.
Fig. 6 be pH to TR5 upgrowth situations and the influence figure of degradation efficiency, as seen from Figure 6, TR5 is in pH7.0 or so It is higher to the degradation efficiency of tetracycline.
Fig. 7 be inoculum concentration to TR5 upgrowth situations and the influence figure of degradation efficiency, as seen from Figure 7, inoculum concentration is to four The degraded of ring element simultaneously has no significant effect.
To sum up, experimental result shows the bacterial strain using glucose as carbon source, and yeast extract is as nitrogen source, pH 7~9, Temperature degradation efficiency at 25 DEG C~30 DEG C is higher, and inoculum concentration influences little on degradation efficiency.
Embodiment 3
Degradation capability determination experiment
1st, application of the tetracycline efficient degradation bacterium (TR5) in culture fishery water body purification
The water in certain artificial farming fish pond is taken, after water body is carried out into preliminary purification processing, with HPLC to tetracycline in water body Content is measured, and is 200ppm, the moisture in fish pond is attached into blake bottle (every bottle of 20ml), and add 20 in every bottle of culture The beads of immobilization embedded TR5 bacterial strains cultivated, and a blank control group is set, cultivated after 5d to blake bottle The content of middle tetracycline is determined again, finds experimental group compared with control group, and Fourth Ring cellulose content has obvious reduction, Experimental group degradation efficiency is 86%.
2nd, application of the tetracycline efficient degradation bacterium in the waste water control of tetracycline antibiotics pharmaceutical factory
The tetracycline antibiotics pharmacy waste water for taking certain pharmaceutical factory to discharge, by waste water after preliminary purification is handled, reference State's calibration method is measured with HPLC to the content of tetracycline in water body, is 435ppm, by pharmacy waste water quantitative separating to training Bottle (every bottle of 20ml) is supported, and the bead for adding in every bottle of culture the immobilization embedded TR5 bacterial strains of 20 is cultivated, and One blank control group is set, the content of tetracycline in blake bottle determined again after cultivating 5d, discovery experimental group with it is right Compared according to group, Fourth Ring cellulose content has obvious reduction, and experimental group degradation efficiency is 88%.

Claims (3)

1. a kind of tetracycline efficient degradation bacterium, it is characterised in that the bacterium is Klebsiella, and it is micro- to be preserved in China Biological inoculum preservation committee common micro-organisms center, deposit number are CGMCC No.14393, and preservation date is:2017 7 The moon 7, preservation address are:BeiJing, China.
2. application of the tetracycline efficient degradation bacterium in culture fishery water body purification described in claim 1.
3. application of the tetracycline efficient degradation bacterium described in claim 1 in the waste water control of antibiotic pharmaceutical factory.
CN201711212339.8A 2017-11-28 2017-11-28 Bacterium for efficiently degrading tetracycline and application thereof Active CN107858305B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707559A (en) * 2018-05-15 2018-10-26 浙江省农业科学院 A kind of tetracycline antibiotics degradation arthrobacterium and its application
CN109337825A (en) * 2018-11-09 2019-02-15 北京农学院 One plant of Beijing Paecilomyces varioti bacterial strain LYZ7 and its application
CN110592067A (en) * 2019-09-20 2019-12-20 广东省农业科学院农业资源与环境研究所 In-situ microorganism repairing agent for tetracycline antibiotic contaminated soil, preparation method and application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105110486A (en) * 2015-09-01 2015-12-02 哈尔滨工业大学 Method for removing tetracycline from water by virtue of microbial flocculant
CN105130010A (en) * 2015-09-01 2015-12-09 哈尔滨工业大学 New application of Klebsiella sp.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105110486A (en) * 2015-09-01 2015-12-02 哈尔滨工业大学 Method for removing tetracycline from water by virtue of microbial flocculant
CN105130010A (en) * 2015-09-01 2015-12-09 哈尔滨工业大学 New application of Klebsiella sp.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707559A (en) * 2018-05-15 2018-10-26 浙江省农业科学院 A kind of tetracycline antibiotics degradation arthrobacterium and its application
CN109337825A (en) * 2018-11-09 2019-02-15 北京农学院 One plant of Beijing Paecilomyces varioti bacterial strain LYZ7 and its application
CN110592067A (en) * 2019-09-20 2019-12-20 广东省农业科学院农业资源与环境研究所 In-situ microorganism repairing agent for tetracycline antibiotic contaminated soil, preparation method and application

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