CN107058156A - Tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application - Google Patents
Tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application.The bacterial strain is acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB42C005, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on July 22nd, 2016, it is referred to as CGMCC, and preserving number is CGMCC No.12812.Bacterial strain JZB42C005 has extraordinary degradation effect to tetracycline antibiotics especially terramycin in soil or water body.For the terramycin that concentration in inorganic salts nutrient solution is 10mg/L, degradation rate reaches 67.8% after handling 5 days.
Description
Technical field
The present invention relates to the technical field of Biochemical method antibiotic pollution, and in particular to a kind of tetracycline antibiotics drop
Solve bacterial strain, the microbial inoculum containing the bacterial strain and its application.
Background technology
Antibiotic is found in water body and agricultural environment extensively, although the concentration of the antibiotic found is generally relatively low,
It is that they are constantly entering environment by different approach, them is become " false persistence ".Research shows, in soil
Antibiotic residue also can produce different degrees of influence even toxic action to plant, animal, microorganism.With China's industry
The pollution of antibiotic also constantly occurs in change, urbanization, the development of agricultural and livestock and poultry breeding industry, soil.Some areas farmland soil
The pollution of antibiotic is more serious in earth, meanwhile, large area soil antibiotic pollution risk has also shown.Into resisting in soil
Raw element class medicine easily induces a large amount of anti-medicine bacterium of generation, and may induce generation group resistance, by life including humans
State system health exerts far reaching influence.Because most antibiotic is difficult to degrade, also antibiotic is caused largely to be accumulated in soil, and
Food chain can be entered by plant absorption, health is influenceed.Therefore, caused by antibiotic environment and health problem in Chinese day
Benefit is prominent.
Tetracycline antibiotics, are mainly used in the treatment of humans and animals infectious bacteria.The Fourth Rings such as the terramycin in soil
Plain class antibiotic can by plant absorption, enrichment, and may in plant different parts transmit, eventually enter into food chain to
Health brings potential risk.There is terramycin class antibiotic in the soil of certain areas to pollute, (Zhang Huimin, chapter such as Zhang Huimin
Teracycline antibiotic residues ecologies and rural environment in bright Kui, Gu Guoping Northern Zhejiang areas feces of livestock and poultry and agricultural land soil
Report, 2008,24 (3)：The total amount for 69-73) finding tetracycline antibiotics in the topsoil of China's Northern Zhejiang area farmland is 80-
6006 μ gkg-1, its content already close to other pesticide organic pollutions in soil level, Hu etc. (Hu X.G.,
Zhou Q.X.,Luo Y.Occurrence and source analysis of typical veterinary
antibiotics in manure,soil,vegetables and groundwater from organic vegetable
bases,northern China.Environmental Pollution,2010,158:2992-2998) research finds Tianjin
The content of terramycin is up to 2683 μ g kg-1 in the soil of regional organic vegetable base.
At present, non-biodegradation is broadly divided into and biodegradable two kinds to the processing method that antibiotic pollutes.Abiotic drop
Solution method is generally physically or chemically means, and major way has photodissociation, hydrolysis and oxidative degradation etc., and its advantage is to be swift in response, go
Except rate is high.But in recent years both at home and abroad there are some researches show, applied to the chemical material in Degradation of Antibiotics to environment there is also
A certain degree of toxic side effect.Biodegradable major way is that microbial degradation, plant degradation and plant-microorganism are compound
Degraded.It is exactly microbial degradation that antibiotic, which enters topmost degradation pathway in environment,.Relevant tetracycline antibiotics microorganism
The report of degraded is simultaneously few.Such as research shows amino bacillus, Phanerochaete chrysosporium, human pallid bacillus and Bacillus cercus
There is preferable degradation to tetracycline antibiotics such as terramycin.But, the terramycin degradation bacteria strains having now been found that also compare
It is less, and antibiotic is degraded using microorganism realization, mechanism is more complicated, with very strong specific aim.At present not
See the report of the tetracycline antibiotics such as acinetobacter calcoaceticus degraded terramycin.
Therefore, carry out the tetracycline antibiotics contaminating microorganisms recovery technique researchs such as terramycin, screen different types of
The efficient degradation microbial strains of the tetracycline antibiotics such as terramycin are very necessary, can be attenuating water body, soil or work
Determination of oxytetracycline residues provides effective means in thing, to ensure that agricultural product security and farm environment safely provide technical support.
The content of the invention
For the defect of prior art, an object of the present invention is to provide a kind of tetracycline antibiotics degradation bacteria
Strain.
The second object of the present invention is to provide a kind of microbial inoculum containing above-mentioned bacterial strains.
The third object of the present invention is the application for providing above-mentioned bacterial strains or microbial inoculum.
To achieve these goals, the present invention uses following technical scheme：
The first aspect of the present invention is there is provided a kind of tetracycline antibiotics degradation bacteria strains, and it is acinetobacter calcoaceticus
(Acinetobacter calcoaceticus) JZB42C005, the bacterial strain is preserved in Chinese micro- life on July 22nd, 2016
Thing culture presevation administration committee common micro-organisms center, it is referred to as CGMCC, and preserving number is CGMCC No.12812, and it is protected
Hiding unit address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Above-mentioned bacterial strains are enriched, separate and purify in the activated sludge of sewage treatment plant of Pang Gezhuang of Beijing
Arrive.The bacterial strain has very strong degradation to tetracycline antibiotics particularly terramycin, with following feature：
(1) bacterial strain JZB42C005 after purification is seeded on LB solid mediums, electron microscopic observation after 28 DEG C of culture 48h
Strain morphology feature, its result is：Colonial morphology is circle, and smooth opaque, bacterium colony is of medium size；For shaft-like, size (0.9
~1.6) μ m (1.5~2.5) μm, no gemma, atrichia, referring to Fig. 5.
(2) logarithmic phase of the strain growth of purifying is taken to carry out the dyeing such as gram, pod membrane, physiological and biochemical property determines reference
《Common bacteria system identification handbook》, physio-biochemical characteristics are shown in Table 1.
The bacterial strain JZB42C005 of table 1 physio-biochemical characteristics
| Experiment | Feature |
| Gram's staining | - |
| Gelatin liquefaction | - |
| Glucose production acid | + |
| Catalase | + |
| Oxidizing ferment | - |
| Citrate is utilized | + |
| Malonate | + |
| Glutamic acid transferase | + |
| Nitrate reductase | - |
(3) PCR expands the 16S rDNA of above-mentioned bacterial strains, and size is 1.5kb, and sequencing result is shown with SEQ in sequence table
Nucleotide sequence shown in ID No.1, DNAMAN version 5.2.2 and BLAST softwares and the sequence in GenBank are used by it
Row carry out sequence analysis, find the homologous of the bacterium and acinetobacter calcoaceticus (Acinetobacter calcoaceticus)
Property highest, reaches 100%.
Reference of the present invention《Common bacteria system identification handbook》The feature of (east show pearl, Cai Miaoying) acinetobacter, and tie
The feature for stating (1)-(3) is closed, bacterial strain JZB42C005 of the present invention is accredited as acinetobacter calcoaceticus (Acinetobacter
calcoaceticus)。
Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB42C005 of the present invention can it is common solid/
Grown on fluid nutrient medium or LB solid-liquid body culture mediums, also can be in the basic inorganic salt liquid that terramycin concentration is 1-50mg/L
Cultivated in culture medium, growth temperature is 25-35 DEG C, pH is 6-7.5, optimum growth temperature is 30 DEG C, and pH is 7.
There is provided a kind of microbial inoculum containing above-mentioned bacterial strains for the second aspect of the present invention, it is preferable that calcium acetate is not in the microbial inoculum
Lever bacterium (Acinetobacter calcoaceticus) JZB42C005 concentration is 0.5-5 × 107CFU/mL。
The third aspect of the present invention is there is provided the preparation method of above-mentioned microbial inoculum, by acinetobacter calcoaceticus after purification
JZB42C005 is inoculated in common liq culture medium or LB fluid nutrient mediums, is centrifuged after culture and is collected sediment, Ran Houxi
The sediment is washed, is finally diluted using diluent and obtains the microbial inoculum, be i.e. thalline suspension.Preferably, the culture
Specially：24-48h is cultivated under the conditions of 25-35 DEG C, 180r/min；The centrifugal condition is：4-5 DEG C of temperature, rotating speed 10000-
12000rpm, centrifugation time is 5-12min；The diluent is 0.1mol/L Na2HPO4-KH2PO4Buffer solution.
Application of the fourth aspect of the present invention there is provided above-mentioned bacterial strains in terms of tetracycline antibiotics of degrading.
Application of the fifth aspect of the present invention there is provided above-mentioned microbial inoculum in terms of tetracycline antibiotics of degrading.
In the fourth aspect of the present invention and the 5th aspect, the tetracycline antibiotics are terramycin.
Beneficial effects of the present invention：Bacterial strain JZB42C005 is especially native mould to tetracycline antibiotics in soil or water body
Element has extraordinary degradation effect.For the terramycin that concentration in inorganic salts nutrient solution is 10mg/L, degradation rate after handling 5 days
Reach 67.8%.
Brief description of the drawings
Fig. 1 is that different degradation time condition bacterial strain JZB42C005 of the present invention are 10mg/L to concentration in inorganic salts nutrient solution
Terramycin degradation effect figure；
Fig. 2 is degradation effect figures of the bacterial strain JZB42C005 of the present invention to the terramycin of different initial concentrations；
Fig. 3 be under condition of different pH bacterial strain JZB42C005 of the present invention to soil that concentration in inorganic salts nutrient solution is 10mg/L
The degradation effect figure of mycin；
Fig. 4 is that bacterial strain JZB42C005 of the present invention is 10mg/L to concentration in inorganic salts nutrient solution under condition of different temperatures
The degradation effect figure of terramycin；
Fig. 5 is acinetobacter calcoaceticus of the present invention (Acinetobacter calcoaceticus) JZB42C005 Electronic Speculum
Scanned photograph.
Embodiment
In order that the object, technical solutions and advantages of the present invention are clearer, below by by the way of embodiment to this
Invention is described in detail, but these embodiments are only used for explaining the present invention, without limiting the present invention.
The various culture mediums used in the present invention are prepared using conventional method, the molecular biology behaviour being related in embodiment
Make such as unreceipted specific experimental condition and method, with reference to the chief editor such as SambrookJ, Science Press, 2002, molecular cloning is real
Test guide (third edition)；Or product description.
Culture medium that the present invention is used is formulated as follows：
1) enriched medium：Peptone 5g, beef extract 3g, sodium chloride 5g, distilled water 1000mL adjust pH to 7.0, high pressure is gone out
Bacterium 20min.
2) ordinary solid culture medium：Peptone 5g, beef extract 3g, sodium chloride 5g, agar 15g, distilled water are mended to 1000mL,
PH to 7.0, autoclaving 20min are adjusted, plate is then down flat.
3) common liq culture medium：Peptone 5g, beef extract 3g, sodium chloride 5g, distilled water are mended to 1000mL, adjust pH extremely
7.0, autoclaving 20min.
4) basic inorganic salt culture medium：1g NH4NO3, 0.5g MgSO4·7H2O, 0.5g (NH4)2SO4,0.5g KH2PO4,
0.5g NaCl, 1.5g K2HPO4, distilled water mended to 1L, adjusts pH to 7.0, autoclaving 20min.
5) isolation medium：1g NH4NO3, 0.5g MgSO4·7H2O, 0.5g (NH4)2SO4,0.5g KH2PO4, 0.5g
NaCl, 1.5g K2HPO4, 15g agar, distilled water mends to 1L, adjusts pH to 7.0, autoclaving 20min, be then down flat plate.
6) LB fluid nutrient mediums：Tryptone 10g, yeast extract 5g, NaCl10g, distilled water is mended to 1L, adjusts pH extremely
7.0, autoclaving 20min.
7) LB solid mediums：Tryptone 10g, yeast extract 5g, NaCl10g, 15g agar, distilled water is mended to 1L,
PH to 7.0, autoclaving 20min are adjusted, plate is then down flat.
0.1mol/L Na2HPO4-KH2PO4The compound method of buffer solution (pH=7.0)：
First, by 61mL 0.2mol/L Na2HPO4The aqueous solution and 39mL 0.2mol/L-KH2PO4The aqueous solution is mixed
Close uniform, adjust pH to 7.0.Then, obtained mixed liquor is diluted into 1 times of Na for obtaining 0.1mol/L2HPO4-KH2PO4Buffering
Liquid (pH=7.0).
Embodiment 1：Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB42C005 separation with
Purifying
3 parts of activated sludge sample for coming from sewage treatment plant of Pang Gezhuang of Beijing is fetched, mixes respectively, 10g is respectively taken, in nothing
Under bacterium operating condition, it is added separately in enriched mediums of the 100mL containing terramycin, concentration of the terramycin in enriched medium is
10mg/L, is then put into shaking table progress lucifuge culture 7d by the enriched medium for adding mud sample, condition is 30 DEG C,
180r/min；By 10% inoculum concentration, (" 10% inoculum concentration " refers to inoculation liquid and be vaccinated culture medium in the present embodiment afterwards
Volume ratio) be transferred to concentration containing terramycin be 20mg/L next group enriched medium in, continue cultivate 7d；Afterwards again
It is transferred to by 10% inoculum concentration in the enriched medium that concentration containing terramycin is 50mg/L, continues to cultivate 7d；Then press again
10% inoculum concentration is transferred in the basic inorganic salt culture medium that concentration containing terramycin is 50mg/L, continues to cultivate 7d；Press again
10% inoculum concentration is transferred in the basic inorganic salt culture medium that concentration containing terramycin is 100mg/L, continues to cultivate 7d；Then will
The basic inorganic salt nutrient solution of three parts of gained is diluted to 10-1-10-6Plinth inorganic salts nutrient solution (is diluted 10 by serial solution successively
Again, 100 times, 1000 times, 10000 times, 100000 times, 1000000 times), respectively take 1mL to be transferred to corresponding ordinary solid training respectively
Support on base flat board, spread plate is placed in 30 DEG C of constant incubators and cultivates 1d-3d, choose the bacterial strain of different shape feature, respectively
It is inoculated on the isolation medium that concentration containing terramycin is 100mg/L, carries out isolating and purifying for 3 times respectively using plate streak.
The preferable single bacterium colony strain number of growing way on flat board is chosen after purification, wherein one plant is named as JZB42C005, is preserved in general
In the slant tube of logical solid medium.
Embodiment 2：Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) JZB42C005 16SrDNA
Identification
1st, the acquisition of pcr template：It includes two methods, and one kind is to extract genomic DNA with as pcr template,
Another is that direct heat treatment bacterium solution is used as pcr template using the bacterium solution after by heat treatment.Below two methods are done with an introduction.
1) genomic DNA is extracted, it is specific as follows using lysozyme plus 10%SDS methods：
(a) bacterium after purification is inoculated in LB fluid nutrient mediums, trained on 180r/min shaking table under the conditions of 28 DEG C
24h is supported, fresh cultured bacterium solution 2ml, 12000rpm is then taken, 1-2min, supernatant discarding is centrifuged；
(b) 1mL, 1 × TE (pH8.0) buffer are added into sediment again and mixes washing, 12000rpm centrifuges 1-
2min, supernatant discarding；
(c) 400 μ L, 5 × TE (pH8.0) buffer are added into sediment again and mixes washing, 12000rpm centrifuges 1-
2min, supernatant discarding；
(d) 50 μ L, 20mg/ml lysozyme solns, 37 DEG C of water-bath 30-60min are added into sediment again；
(e) 50 μ L, 10wt%SDS, 20 μ L protease (20mg/ml), 55 DEG C of water-bath 60min are added；
(f) 500 μ L phenol/(volume ratio is 25 to chloroform/isoamyl alcohol are added:24:1) mix, 12000rpm, centrifuge 10min；
(g) supernatant is taken to be transferred in new centrifuge tube, (volume ratio is 24 with the isometric chloroform/isoamyl alcohol of supernatant for addition:
1), mix, 12000rpm, centrifuge 10min；
(h) take supernatant to be transferred in new centrifuge tube (1.5ml), add the absolute ethyl alcohol of 2 times of supernatant volumes, it is light to mix directly
To DNA precipitations, 12000rpm centrifuges 10min；
(i) abandoning supernatant, 70% ethanol washing, 12000rpm centrifuges 10min；
(j) abandoning supernatant, operating desk dries, transparent to DNA；
(k) 50 μ L aqua sterilisas are added, 0.5 μ L RNase are mixed, and -20 DEG C of refrigerators are preserved, standby.
2) directly heat treatment obtains pcr template, specific as follows：The strain after purification is inoculated in LB solid mediums
On, then 28 DEG C of culture 24h dip a small amount of thalline with sterile pipette tips, mix in 100 μ L ddH2In O, after boiling water treating 2min
12000rpm centrifuges 5min, and supernatant is PCR amplification templates.The present embodiment employs this method and obtains pcr template.
2、PCR
PCR primer uses 16S rDNA universal primers 27F (5 '-GAG AGT TTG ATC CTG GCT CAG-3 ', i.e. sequence
SEQ ID No.2 in list) and 1492R (5 '-ACG GAT ACC TTG TTA CGA CTT-3 ', i.e., SEQ ID in sequence table
No.3)。
The μ L of PCR reaction systems 25：2 × Taq PCR Mix 12.5 μ L, primer 2 7F 1 μ L, primer 1492R 1 μ L, ddH2O
8.5 μ L, the μ L of DNA profiling 2.
PCR programs：94℃5min；94 DEG C of 40s, 55 DEG C of 40s, 72 DEG C of 90s, 30 circulations；72℃10min.
PCR uses 1% agarose gel electrophoresis after terminating, and PCR primer is 1.5kb, and carrier T is connected to after purified recovery
Upper clone, extracts plasmid after blue and white screening, is passed to the sequencing of Sino-U.S. calm and peaceful biotechnology (Beijing) Co., Ltd, sequencing knot
Nucleotide sequence of the fruit display with SEQ ID No.1 in sequence table is soft with DNAMAN version 5.2.2 and BLAST by it
Part carries out sequence analysis with the sequence in GenBank, finds the bacterium and acinetobacter calcoaceticus (Acinetobacter
Calcoaceticus homology highest), reaches 100%.
Embodiment 3：Bacterial strain JZB42C005 is determined to the degradation effect of terramycin under different disposal time conditions
Bacterial strain JZB42C005 after purification is cultivated to logarithm period in LB fluid nutrient mediums, connect with 10% inoculum concentration
Enter in the basic inorganic salt culture medium for being 10mg/L containing terramycin, the shaken cultivation on 30 DEG C, 180r/min constant-temperature table, together
When set and do not connect the basic inorganic salt culture medium of bacterium and compare, each 3 repetitions of processing.Respectively 0,1,3,5,7,10 days when, inhale
Supernatant 2mL is taken, 0.2 μm of filter membrane is crossed, the residual quantity of terramycin in supernatant is then determined using liquid chromatogram UV-detector,
Wherein sample size is 20 μ L.
The specific detection method of the residual quantity of terramycin is as follows：
1) drafting of standard curve
Using quantified by external standard method.Terramycin standard liquid is diluted to 0.05 successively with acetonitrile, 0.1,0.2,0.5,1,2,
5th, 10mg/L concentration, then with hplc determination, each μ L of sample introduction 2, each processing is repeated 3 times, and takes the flat of its peak area
Average.Then using concentration as abscissa, peak area is that ordinate does standard curve.
2) the liquid chromatogram measuring condition of terramycin
Liquid chromatogram is the HPLC of Waters 2695, chromatographic column：Waters C18 (250mm × 4.6mm, 5 μm)；Flowing
Phase：Acetonitrile (A)：0.1% aqueous formic acid (B)=75%：25% (volume ratio), Detection wavelength 254nm；The μ L of sample size 2；Post
35 DEG C of temperature.
3) culture 0,1,3,5,7, after 10 days in nutrient solution oxytetracycline residues calculating：
By step 1) standard curve obtain oxytetracycline residues in different time nutrient solution.
The degradation rate of terramycin in different time nutrient solution can be obtained by residual quantity, as a result referring to Fig. 1, wherein degrading
Terramycin is residual in rate=(oxytetracycline residues in oxytetracycline residues-processing nutrient solution in control nutrient solution)/control nutrient solution
Allowance × 100%.
Bacterial strain JZB42C005 is shown in Fig. 1 to concentration in basic inorganic salt culture medium for the degradation curve of 10mg/L terramycin.From
As can be seen that bacterial strain JZB42C005 has certain degradation effect to the terramycin in basic inorganic salt culture medium in Fig. 1, with
The growth of time, the degradation rate increase of terramycin reaches highest degradation rate 67.8%, then declined at 5 days.
Embodiment 4：Degradation effects of the bacterial strain JZB42C005 to various concentrations terramycin
Bacterial strain JZB42C005 after purification is cultivated to logarithm period in LB fluid nutrient mediums, with 10% inoculum concentration point
Not access containing terramycin be 1,5,10,20, in 50mg/L basic inorganic salt culture medium, at 30 DEG C, 180r/min constant temperature shakes
Shaken cultivation 5 days on bed, are compared while setting and not connecing the basic inorganic salt culture medium of bacterium, each 3 repetitions of processing.Culture terminates
Afterwards, the residual quantity of liquid chromatographic detection terramycin, specific detection method be the same as Example 3.Bacterial strain is in the different initial concentrations of terramycin
Under degradation curve see Fig. 2.
Test result indicates that, inoculated and cultured is under the conditions of 5 days, and bacterial strain JZB42C005 is to the native mould of 1~10mg/L of initial concentration
The degradation rate of element also has higher degradation capability more than 65% to 20mg/L terramycin, degradation rate more than 50%,
Degradation rate to concentration 50mg/L terramycin is 38.5%.
Embodiment 5：Influences of the pH to bacterial strain JZB42C005 degraded terramycin
Bacterial strain JZB42C005 after purification is cultivated to logarithm period in LB fluid nutrient mediums, with 10% inoculum concentration point
Be not linked into the basic inorganic salt culture medium that the concentration containing terramycin with different pH is 10mg/L, pH is respectively 4,5,6,7,
8th, 9, shaken cultivation 5 days on 30 DEG C, 180r/min constant-temperature table are opposed while setting and not connecing the basic inorganic salt culture medium of bacterium
According to each 3 repetitions of processing.After culture terminates, the residual quantity of liquid chromatographic detection terramycin, specific detection method be the same as Example
3.Bacterial strain JZB42C005 is shown in Fig. 3 under different pH to the degradation curve of terramycin.
Test result indicates that, inoculated and cultured 5 days, under condition of different pH, bacterial strain JZB42C005 is to basic inorganic salt nutrient solution
The degradation rate of middle 10mg/L terramycin is different, when pH is 7, reaches highest to the degradation capability of 10mg/L terramycin, pH is 6
When, 50% also can reach to the degradation capability of 10mg/L terramycin.
Embodiment 6：Influence of the different temperatures to bacterial strain JZB42C005 degraded terramycin
Bacterial strain JZB42C005 after purification is cultivated to logarithm period in LB fluid nutrient mediums, connect with 10% inoculum concentration
Enter in the basic inorganic salt culture medium for being 10mg/L containing terramycin, in different temperatures 20,25,30,35,40,45,50 DEG C of conditions
Under, shaken cultivation 5 days on 180r/min constant-temperature table are compared, each while setting and not connecing the basic inorganic salt culture medium of bacterium
Handle 3 repetitions.After culture terminates, the residual quantity of liquid chromatographic detection terramycin, specific detection method be the same as Example 3.Bacterial strain
JZB42C005 is shown in Fig. 4 to the degradation curve of terramycin at different temperatures.
Test result indicates that, under condition of different temperatures, inoculated and cultured 5 days, bacterial strain JZB42C005 is to inorganic salts nutrient solution
The degradation rate of middle 10mg/L terramycin is different, and when temperature is 30 DEG C, highest is reached to the degradation capability of 10mg/L terramycin,
For more than 60%；When temperature is 25 DEG C or 35 DEG C, 55% or so can also be reached to the degradation capability of 10mg/L terramycin.
Embodiment 7：The preparation of microbial inoculum
Bacterial strain JZB42C005 after purification is inoculated in 20mLLB fluid nutrient mediums, under the conditions of 30 DEG C, 180r/min
Cultivate after 36h, thalline is collected with 4300r/min centrifugations 10min at 5 DEG C, then with 10mL 0.1mol/L Na2HPO4-
KH2PO4Buffer solution washing thalline 2 times, then thalline suspension, i.e. microbial inoculum are made into above-mentioned buffer solution 4mL, bacterium in the microbial inoculum
Strain JZB42C005 concentration is 1.0 × 107CFU·mL-1。
Embodiment 8
(1) Degrading experiment of terramycin in terramycin sewage is simulated
Terramycin is dissolved in 10mL methanol, this solution is then added into 500mL water, preparation makes the concentration of terramycin
Two kinds for 5mg/L and 10mg/L are simulated terramycin sewage.Degradation bacterial agent prepared by embodiment 7 is respectively added to above two
Simulate in terramycin sewage, concentration of the bacterial strain JZB42C005 in two kinds of sewage is 1.0 × 107CFU/mL sewage, and set not
Add the control of microbial inoculum, be placed in 30 DEG C of incubators, cultivate 1 under dark condition, 3,5,7, after 10 days, it is separately sampled to determine dirty
The residual quantity of terramycin in water, specific detection method be the same as Example 3, the results are shown in Table 2.It is mould that this degradation bacterial agent can effectively remove soil
Residual of the element in water, removal effect is preferable.When terramycin concentration is 5mg/L and 10mg/L, bacterial strain JZB42C005 degrades to it
Rate is respectively 75.3% and 58.6%.
Degradation rates of the bacterial strain JZB42C005 of table 2 to different initial concentration terramycin in sewage
(2) Degrading experiment of terramycin in the agricultural sludge soil of terramycin pollution is simulated
Beijing suburb field topsoil (0-20cm) for choosing non-dispenser is a certain amount of, and sludge of sewage treatment plant is a certain amount of,
According to sludge：The mass ratio of soil is 1:3 ratio mixing, is made agricultural sludge soil, then weighs the agricultural sludge soil
Every part of 280g, is placed in large surface ware, terramycin is added respectively, addition concentration is respectively 5mg/kg and 10mg/kg, by embodiment
7 degradation bacterial agents prepared are uniformly sprayed in soil surface, and concentration is 1.0 × 107CFU/g soil, and sprinkling clear water control is set, put
In 30 DEG C of incubators, cultivate 1 under dark condition, 3,5,7, after 10 days, take the residual quantity for determining terramycin in soil, as a result
3 are shown in Table, specific assay method is as follows：Terramycin in soil is extracted using phosphate buffer with acetonitrile mixed solution, SAX-
HLB solid phase extraction columns are purified, ESI(+)Ionization mode, multiple-reaction monitoring (MRM) is quantitative, and HPLC/MS/MS is determined, with reference to text
Offer：Ma Lili, Guo Changsheng, Hu Wei, Sha Jian, Zhu Xingwang, Ruan Yuefei, Wang Yuqiu, SPE-high performance liquid chromatography-series connection matter
Spectrometry determines fluoquinolone, tetracycline and sulfa antibiotics, analytical chemistry, 2010,38 (1), 21-26 in soil simultaneously.It is logical
The degradation rate of terramycin in different time soil can be obtained by crossing residual quantity, as a result referring to table 3, wherein degradation rate=(control soil
Oxytetracycline residues in oxytetracycline residues-processing soil in earth)/compare oxytetracycline residues × 100% in soil.
This degradation bacterial agent can effectively remove residual of the terramycin in agricultural sludge soil, and removal effect is preferable.Soil is mould
When plain concentration is 5mg/kg and 10mg/kg, bacterial strain JZB42C005 is respectively 55.6% He to its degradation rate after handling 5 days
48.8%.
Degradation rates of the bacterial strain JZB42C005 of table 3 to various concentrations terramycin in agricultural sludge soil
SEQUENCE LISTING
<110>Beijing City Agriculture and Forestry Institute
<120>Tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application
<130> 160671
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1447
<212> DNA
<213> Acinetobacter calcoaceticus
<400> 1
agccctgggc caacgtggtt accgccctct ttgcagttag gctagctact tctggtgcaa 60
caaactccca tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca 120
ttctgatccg cgattactag cgattccgac ttcatggagt cgagttgcag actccaatcc 180
ggactacgat cggctttttg agattagcat cctatcgcta ggtagcaacc ctttgtaccg 240
accattgtag cacgtgtgta gccctggccg taagggccat gatgacttga cgtcgtcccc 300
gccttcctcc agtttgtcac tggcagtatc cttaaagttc ccgacattac tcgctggcaa 360
ataaggaaaa gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420
cgacagccat gcagcacctg tatgtaagtt cccgaaggca ccaatccatc tctggaaagt 480
tcttactatg tcaaggccag gtaaggttct tcgcgttgca tcgaattaaa ccacatgctc 540
caccgcttgt gcgggccccc gtcaattcat ttgagtttta gtcttgcgac cgtactcccc 600
aggcggtcta cttatcgcgt tagctgcgcc actaaagcct caaaggcccc aacggctagt 660
agacatcgtt tacggcatgg actaccaggg tatctaatcc tgtttgctcc ccatgctttc 720
gcacctcagc gtcagtgtta ggccagatgg ctgccttcgc catcggtatt cctccagatc 780
tctacgcatt tcaccgctac acctggaatt ctaccatcct ctcccacact ctagctaacc 840
agtatcgaat gcaattccca agttaagctc ggggatttca catttgactt aattagccgc 900
ctacgcgcgc tttacgccca gtaaatccga ttaacgcttg caccctctgt attaccgcgg 960
ctgctggcac agagttagcc ggtgcttatt ctgcgagtaa cgtccactat ctctaggtat 1020
tatctaaagt agcctcctcc tcgcttaaag tgctttacaa ccataaggcc ttcttcacac 1080
acgcggcatg gctggatcag gcttgcgccc attgtccaat attccccact gctgcctccc 1140
gtaggagtct gggccgtgtc tcagtcccag tgtggcggat catcctctca gacccgctac 1200
agatcgtcgc cttggtaggc ctttacccca ccaactagct aatccgactt aggctcatct 1260
attagcgcaa ggtccgaaga tcccctgctt tctcccgtag gacgtatgcg gtattagcat 1320
tcctttcgaa atgttgtccc ccactaatag gcagattcct aagcattact cacccgtccg 1380
ccgctaggtg atagtgcaag caccatcacc ccgctcgact tgcatgtgta aagccgccgc 1440
cccgcga 1447
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Universal primer 27F
<400> 2
gagagtttga tcctggctca g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Universal primer 1492R
<400> 3
acggatacct tgttacgact t 21
Claims (10)
1. a kind of tetracycline antibiotics degradation bacteria strains, the bacterial strain is acinetobacter calcoaceticus (Acinetobacter
Calcoaceticus) JZB42C005, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
Tibetan number is CGMCC No.12812.
2. a kind of microbial inoculum containing bacterial strain described in claim 1.
3. microbial inoculum according to claim 2, it is characterised in that acinetobacter calcoaceticus in the microbial inoculum
(Acinetobacte calcoaceticus) JZB42C005 concentration is 0.5-5 × 107CFU/mL。
4. the preparation method of the microbial inoculum described in Claims 2 or 3, it is characterised in that by acinetobacter calcoaceticus after purification
JZB42C005 is inoculated in common liq culture medium or LB fluid nutrient mediums, is centrifuged after culture and is collected sediment, Ran Houxi
The sediment is washed, is finally diluted using diluent and obtains the microbial inoculum.
5. preparation method according to claim 4, it is characterised in that the culture is specially：In 25-35 DEG C, 180r/
24-48h is cultivated under the conditions of min.
6. preparation method according to claim 4, it is characterised in that the centrifugal condition is：4-5 DEG C of temperature, rotating speed
10000-12000rpm, centrifugation time is 5-12min.
7. preparation method according to claim 4, it is characterised in that the diluent is 0.1mol/L Na2HPO4-
KH2PO4Buffer solution.
8. application of the bacterial strain in terms of tetracycline antibiotics of degrading described in claim 1.
9. application of the microbial inoculum in terms of tetracycline antibiotics of degrading described in claim 2.
10. application according to claim 8 or claim 9, it is characterised in that the tetracycline antibiotics are terramycin.
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| CN107557315A (en) * | 2017-09-12 | 2018-01-09 | 中国农业科学院农业资源与农业区划研究所 | One plant of tylosin degradation bacteria and its application |
| CN110591936A (en) * | 2019-07-02 | 2019-12-20 | 湖南农业大学 | High-efficiency terramycin degradation strain and application thereof |
| CN110904002A (en) * | 2019-11-25 | 2020-03-24 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN111172144A (en) * | 2019-12-27 | 2020-05-19 | 华南农业大学 | Microbial agent for efficiently degrading tetracycline antibiotics and application of microbial agent in soil pollution remediation |
| CN114433621A (en) * | 2022-01-27 | 2022-05-06 | 北京市农林科学院 | Method for reducing abundance of antibiotic resistance genes in soil by using edible fungi |
| CN115232758A (en) * | 2021-04-25 | 2022-10-25 | 江苏省农业科学院 | Acinetobacter and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557315A (en) * | 2017-09-12 | 2018-01-09 | 中国农业科学院农业资源与农业区划研究所 | One plant of tylosin degradation bacteria and its application |
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| CN110904002A (en) * | 2019-11-25 | 2020-03-24 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN110904002B (en) * | 2019-11-25 | 2022-04-29 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN111172144A (en) * | 2019-12-27 | 2020-05-19 | 华南农业大学 | Microbial agent for efficiently degrading tetracycline antibiotics and application of microbial agent in soil pollution remediation |
| CN115232758A (en) * | 2021-04-25 | 2022-10-25 | 江苏省农业科学院 | Acinetobacter and application thereof |
| CN114433621A (en) * | 2022-01-27 | 2022-05-06 | 北京市农林科学院 | Method for reducing abundance of antibiotic resistance genes in soil by using edible fungi |
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