CN111154685B - Klebsiella variicola for degrading tetracycline and application thereof - Google Patents
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- C12R2001/22—Klebsiella
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- 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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention discloses Klebsiella variicola for degrading tetracycline and application thereof. The Klebsiella variicola strain is named Klebsiella variicola TC3 with the collection number GDMCC NO:60788, the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 building of Tokyo No. 100 college of Centraalbo Tokyo, Middleya, Guangzhou, 9.25.2019. The Klebsiella variicola TC3 can degrade tetracycline in a co-metabolism mode, so that the Klebsiella variicola TC3 can be applied to degradation of low-concentration tetracycline in soil and water to remove or reduce residue of tetracycline in the environment.
Description
Technical Field
The invention belongs to the technical field of organic pollutant microbial remediation, and particularly relates to Klebsiella variicola for degrading tetracycline and application thereof.
Background
Tetracycline (TC) is a basic broad-spectrum antibiotic which takes polycyclic tetracene carboxyl amide as a mother nucleus, influences the growth of bacteria by inhibiting the growth of protein peptide chains, and has antibacterial and bactericidal effects on certain rickettsiae, filterable viruses, protozoa and the like. Because of its advantages of good bacteriostasis and sterilization action, low price, wide antimicrobial spectrum, direct use as feed additive, etc., it has been widely used in animal husbandry and aquaculture industry. After the tetracycline antibiotics are used in animals, 35-90% of tetracycline is not absorbed by the antibiotics, but is discharged out of the body along with excrement and urine in the form of an initial parent or a metabolite and enters the environment. Besides, the nonstandard and abuse of tetracycline causes that the tetracycline enters the environment in various ways (such as municipal sewage, direct drainage of medical sewage and the like); and numerous studies have shown that tetracycline residues are also detected in various media, such as animal-derived foods (beef, pork, honey, egg, etc.), tap water and crops (wheat, barley, tobacco, etc.), etc. After entering the environment, tetracycline can cause direct threat and profound influence on soil fertility, plant biomass, soil microbial diversity and ecological safety. Therefore, it is important to reduce or eliminate the residual amount of tetracycline in the environment.
The existing microbiological method is an optimal method for degrading tetracycline in the environment, especially for reducing the residual amount of tetracycline in soil, and has wide application prospect due to the characteristics of long acting, low cost, low toxicity of degradation products and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a Klebsiella variicola strain for degrading tetracycline.
Another object of the present invention is to provide the use of said Klebsiella mutans for the degradation of tetracycline.
The purpose of the invention is realized by the following technical scheme: a Klebsiella variicola strain for degrading tetracycline, which is named Klebsiella variicola TC3 and has a collection number of GDMCC NO:60788, the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 building of Tokyo No. 100 college of Centraalbo Tokyo, Middleya, Guangzhou, 9.25.2019.
The 16S rDNA sequence of the Klebsiella variicola for degrading tetracycline consists of 1415 basic groups (bp), and the nucleotide sequence is shown as SEQ ID NO. 1.
A method for culturing the said Klebsiella variicola for degrading tetracycline comprises the following steps: inoculating Klebsiella variicola for degrading tetracycline into a culture medium, and culturing at 30-37 ℃.
The culture medium is LB culture medium.
The temperature of the culture is preferably 30 ℃.
The culture time is 18-24 h; preferably 20 h.
The cultivation is carried out in a shaking table, and the rotating speed of the shaking table is 125-150 rpm (preferably 150 rpm).
The Klebsiella variicola for degrading tetracycline is applied to degrading tetracycline.
The Klebsiella variicola for degrading tetracycline is added into water or soil containing tetracycline to degrade tetracycline in the water or soil, and the Klebsiella variicola with tetracycline degradation performance can be used for degrading tetracycline with low-concentration tetracycline in the water, so that the residue of tetracycline in the environment is removed or reduced, and the effect of treating environmental pollution is achieved.
The concentration of the tetracycline in the water containing the tetracycline is 2.5-10 mg.L-1(ii) a Preferably 10 mg.L-1。
The degradation time is preferably 3-9 days.
A tetracycline biodegradation microbial inoculum contains the Klebsiella variicola for degrading tetracycline.
After the variable-inhabitation Klebsiella TC3 grows on an LB flat plate for 24 hours, the bacterial plaque is round, the edge of the bacterial plaque is semitransparent, and the middle of the bacterial plaque is raised to be beige. Can grow well in an inorganic salt culture medium containing 10g/L peptone with a tetracycline concentration of 10 mg/L.
Compared with the prior art, the invention has the following advantages and effects:
the strain TC3 can degrade tetracycline through a co-metabolism mode, the strain TC3 is inoculated in an inorganic salt liquid culture medium containing 10g/L of peptone and having the tetracycline concentration of 10mg/L, shaking culture is carried out for 0, 3, 5 and 9 days at 30 ℃ and 150r/min, the natural hydrolysis rate after the tetracycline is 3d is 32.42%, and the degradation rate after the TC3 is added is 47.52%, wherein the microbial degradation rate is about 15%, which indicates that the strain TC3 has the tetracycline degradation performance, the strain TC3 can be applied to degradation of low-concentration tetracycline in soil and water, and the degradation effect of the strain TC3 on the low-concentration tetracycline can remove or reduce the residue of the tetracycline in the environment.
Drawings
FIG. 1 is a colony morphology of strain TC 3.
FIG. 2 is a phylogenetic tree of strain TC 3.
FIG. 3 is a graph of tetracycline degradation by strain TC3 in MSM medium containing 10g/L peptone (CK represents no added control).
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents used in the present invention are commercially available.
Example 1 screening, isolation and purification and identification of Strain TC3
Screening method of tetracycline degradation strain TC3
1. Material preparation
Screening sludge sources by degrading bacteria: the method comprises the steps of taking farmyard manure application vegetable fields of left ridge village in tea village county of Tankou city of shores in Hunan province, sealing soil samples by using sampling bags, and carrying the soil samples back to a laboratory at 4 ℃ for storage and later use.
LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 7.0-7.2 of pHs, and fixing the volume to 1L of distilled water; the solid medium was supplemented with 18.0g of agar powder. Sterilizing at 121 deg.C for 15 min.
Inorganic salt medium (MSM medium): 5mL of phosphoric acid buffer solution (KH)2PO4 8.5g·L-1、K2HPO4·H2O 21.75g·L-1、Na2HPO4·12H2O 33.4g·L-1、NH4 Cl 5.0g·L-1)，3.0mL 22.5g·L-1MgSO (2) of4Solution (MgSO)4·7H2O 46.125g·L-1)，1.0mL 0.25g·L-1FeCl of3Solution (FeCl)3·6H2O 0.42g·L-1)，1.0mL 36.4g·L-1In (C) is2Solution (CaCl)2·2H2O 48.22g·L-1) 1.0mL of a solution of trace elements (MnSO)4·H2O 39.9mg·L-1，ZnSO4·H2O 42.8mg·L-1，(NH4)6Mo7O24·4H2O 34.7mg·L-1) Mixing, adjusting pH to 7.0-7.2, adding pure water to constant volume of 1L, and sterilizing at 121 deg.C for 15 min.
Containing 10 g.L-1MSM medium for peptone: adding 10.0g of tryptone into 1L of MSM medium, and sterilizing at 121 ℃ for 15min at the pH of 7.0-7.2.
2. Laboratory apparatus and device
Vertical pressure steam sterilization pan (BL-50A, Shanghai Silique industries, Ltd.), portable pH meter (PHB-4, Shanghai precision science, Ltd.), Centrifuge (Centrifuge 5810R), electric heating oven (DGG-9070A, Shanghai Senxin experiment apparatus, Ltd.), digital display constant temperature water bath pan (HH series, Changzhou national apparatus manufacturing Co., Ltd.), refrigerator (RCD-205AG7, Hai Xin electric appliance), biochemical incubator (PYX-208S-A, Keli apparatus), clean bench (SW-CJ-1F, Sujing Antai air technology, Ltd.), voro mutex mixer (XW-80A, Shanghai Jing industries, Ltd.), MyCycler PCR (BIO-RAD, USA), electrophoresis apparatus (DYY-6C, Bei six instrument works), NaDrno nucleic acid protein quantitative detector (German rmmo), Germany, Gel imaging system (BIO-RAD, USA), floor type constant temperature oscillator (HZQ-211C).
3. Enrichment screening, separation and purification of tetracycline degradation strain
(1) Isolation and purification of the strains
Weighing 1g of farmyard soil for fertilizing vegetable in left ridge village in tea village county, shoal, Hunan province, adding into 50mL of farmyard soil containing 0.5 mg.L-1TC (Tetracycline) and 10 g.L-1Acclimatization culture of peptone in MSM medium (i.e., adding TC to a medium containing 10 g. L)-1In MSM medium containing peptone, TC was adjusted to a final concentration of 0.5 mg. multidot.L-1) Domestication for two daysThen transferred to a strain containing 1 mg.L in an amount of 2% (v/v)-1TC and 10 g.L-1Acclimatization of peptone in MSM medium, and then transfer to TC concentrations of 2.5, 5 and 10 mg.L at 2% (v/v) inoculum concentration-1TC content of 10 g.L-1Acclimatization in MSM medium of peptone. After acclimatization and screening, diluting the bacterial liquid to 10 degrees with the culture medium7Taking 0.2mL of diluent, coating the diluent on an LB solid culture medium, and culturing at 30 ℃ until bacterial colonies are formed; then selecting the grown single colony, separating and purifying.
(2) Strain screening
Inoculating the purified strain separated and purified in the step (1) into an LB liquid culture medium for amplification culture. After activation, the mixture is added to a mixture containing 10 mg.L according to the proportion of 2% (v/v)-1TC and 10 g.L-1150 r.min in MSM culture medium of peptone-1Culturing at 30 deg.C in dark, and collecting bacterial liquid after 0, 3, 5 and 9 days. The bacterial liquid is used at 10000 r.min-1Centrifuging for 10min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring filtrate with High Performance Liquid Chromatography (HPLC), and calculating degradation rate of strain to tetracycline, wherein all the above experimental steps are protected from light as much as possible. A strain which degrades tetracycline was obtained and named strain TC 3. Wherein the content of the first and second substances,
the HPLC conditions were as follows: the liquid chromatography column is CNW C18-WP (4.6X 250mm, 5 μm), phase A is methanol, phase B is acetonitrile, phase C is 0.01 mol. L-1Oxalic acid, V (A), V (B), V (C) 15:15:70, flow rate 1 mL/min-1The column temperature is 31 ℃; the sample volume is 20 mu L; the ultraviolet detector detects the wavelength of 355 nm.
Secondly, observing the morphological characteristics of the bacterial colony
The strain TC3 grows faster on an LB culture medium and can grow at the temperature of 30-37 ℃. The bacterial plaque is round, the edge is semitransparent, and the middle is raised to be beige. Can grow well in MSM medium containing 10g/L peptone with 10mg/L tetracycline concentration (FIG. 1).
2.16S rDNA amplification
And (3) amplifying by using the extracted total DNA of the bacteria as a template and adopting a bacterial 16S rDNA universal primer, wherein the forward primer is 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492 r: 5'-GGTTACCTTGTTACGACTT-3' the 16S rDNA gene sequence was amplified.
The total PCR reaction was 25 μ L: 2 mul of each of the upstream and downstream primers, 0.5 mul of template DNA, 12.5 mul of 2 XTaq PCR Master Mix, and sterilized ultrapure water to a total volume of 25 mul.
The PCR reaction program is: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 50s, annealing at 56 ℃ for 50s, extension at 72 ℃ for 50s, 35 cycles. And finally, supplementary extension is carried out for 5min at 72 ℃. After the PCR is finished, detecting a PCR product by 1.0% agarose gel electrophoresis, and selecting DL2000 Marker. And the gel imaging system is used for observing, wherein a remarkable band appears in the middle range of the Marker1000bp and the 2000bp band.
3.16 determination of the S rDNA sequence
The PCR amplified product was sequenced by Biotechnology of Boxing Ke, Beijing Rui (Guangzhou division). The 16S rDNA gene sequence of the obtained strain is as follows:
ATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGNTNAGGTTAATAACCTTGNCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTT。
the above sequence consists of 1415 bases (bp).
The obtained 16S rDNA gene sequence is submitted to a National Center for Biological Information (NCBI) webpage for BLAST comparison, and is subjected to homology comparison analysis with the 16S rDNA gene of a relevant model strain in an LPSN database (http:// www.bacterio.net/index. html), a model strain sequence with higher homology is downloaded, and an amplification product sequence is subjected to BLAST comparison and homology analysis on a National Center for Biological Information (NCBI) website, and a system development tree is constructed by a Neightbour-Joining method by adopting Mega 7.0 software. Comparison of the 16S rDNA sequence revealed that strain TC3 has 99.93% homology with Klebsiella variicola (Klebsiella variicola) (FIG. 2).
Fourthly, identifying the strain TC3 as a new functional strain
Based on the colony morphology and molecular biological identification of strain TC3, strain TC3 was identified as Klebsiella variicola (Klebsiella variicola) and was designated Klebsiella variicola TC3(Klebsiella variicola TC 3). The strain has been preserved in Guangdong province microorganism culture Collection (GDMCC), with the accession number of GDMCC No. 60788 and the preservation date of 2019, 9 months and 25 days. The address of the preservation unit is No. 59 building No. 5 building of No. 100 college of Jifura Zhonglu, Guangzhou city.
EXAMPLE 2 degradation of Tetracycline by Strain TC3 in mineral salts Medium containing Tetracycline and peptone
Streaking the strain TC3, culturing on LB plate at 30 deg.C for 20 hr, selecting single colony, inoculating in LB liquid culture medium at 30 deg.C and 150r min-1After culturing for 20h in a shaking table, inoculating the strain according to the proportion of 2% (v/v) until the tetracycline content is 10 mg.L-1Containing 10 g.L-1Culturing tryptone in MSM culture medium, and sampling after 0, 3, 5 and 9 days; control was made without addition of strain TC 3. The solution was filtered and then assayed for residual tetracycline by HPLC (tetracycline assay as in example 1).
The results show that the tetracycline without added bacteria is hydrolyzed, and the degradation of the tetracycline is obviously accelerated and enhanced after the addition of the bacteria TC 3. When the initial concentration of TC was 10ppm, the natural hydrolysis rates of TC at 3d, 5 d, and 9d were 32.42%, 39.48%, and 42.34%, respectively, and it was found that the natural hydrolysis rate increased with time. TC degradation can be promoted after the addition of TC3, and the TC degradation efficiency (hydrolysis + biodegradation) after 3, 5 and 9 days is 47.52%, 51.86% and 53.22%. The residual concentration of tetracycline after natural hydrolysis or biodegradation of TC for 3, 5, and 9 days is shown in FIG. 3.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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atgcaagtcg agcggtagca cagagagctt gctctcgggt gacgagcggc ggacgggtga 60
gtaatgtctg ggaaactgcc tgatggaggg ggataactac tggaaacggt agctaatacc 120
gcataacgtc gcaagaccaa agtgggggac cttcgggcct catgccatca gatgtgccca 180
gatgggatta gctagtaggt ggggtaacgg ctcacctagg cgacgatccc tagctggtct 240
gagaggatga ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca 300
gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc atgccgcgtg tgtgaagaag 360
gccttcgggt tgtaaagcac tttcagcggg gaggaaggcg ntnaggttaa taaccttgnc 420
gattgacgtt acccgcagaa gaagcaccgg ctaactccgt gccagcagcc gcggtaatac 480
ggagggtgca agcgttaatc ggaattactg ggcgtaaagc gcacgcaggc ggtctgtcaa 540
gtcggatgtg aaatccccgg gctcaacctg ggaactgcat tcgaaactgg caggctagag 600
tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa 660
taccggtggc gaaggcggcc ccctggacaa agactgacgc tcaggtgcga aagcgtgggg 720
agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgtcgat ttggaggttg 780
tgcccttgag gcgtggcttc cggagctaac gcgttaaatc gaccgcctgg ggagtacggc 840
cgcaaggtta aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900
taattcgatg caacgcgaag aaccttacct ggtcttgaca tccacagaac tttccagaga 960
tggattggtg ccttcgggaa ctgtgagaca ggtgctgcat ggctgtcgtc agctcgtgtt 1020
gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt atcctttgtt gccagcggtt 1080
aggccgggaa ctcaaaggag actgccagtg ataaactgga ggaaggtggg gatgacgtca 1140
agtcatcatg gcccttacga ccagggctac acacgtgcta caatggcata tacaaagaga 1200
agcgacctcg cgagagcaag cggacctcat aaagtatgtc gtagtccgga ttggagtctg 1260
caactcgact ccatgaagtc ggaatcgcta gtaatcgtag atcagaatgc tacggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttg caaaagaagt 1380
aggtagctta accttcggga gggcgcttac cactt 1415
Claims (10)
1. A Klebsiella variicola strain for degrading tetracycline is characterized in that: named Klebsiella variicola (Klebsiella variicola) TC3, deposited under the accession number GDMCC NO:60788, the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 building of Tokyo No. 100 college of Centraalbo Tokyo, Middleya, Guangzhou, 9.25.2019.
2. A method of culturing the tetracycline-degrading klebsiella alterans of claim 1, comprising: inoculating Klebsiella variicola for degrading tetracycline into a culture medium, and culturing at 30-37 ℃.
3. The method of claim 2, wherein: the culture medium is LB culture medium.
4. The method of claim 2, wherein: the culture time is 18-24 h.
5. Use of the tetracycline-degrading klebsiella alterans of claim 1 in degrading tetracycline.
6. Use according to claim 5, characterized in that: the method for degrading tetracycline of claim 1, comprising adding said Klebsiella variicola of claim 1 to a tetracycline-containing body of water or soil to degrade tetracycline in the body of water or soil.
7. Use according to claim 6, characterized in that: the concentration of the tetracycline in the water containing the tetracycline is 2.5-10 mg.L-1。
8. Use according to claim 7, characterized in that: the concentration of the tetracycline in the water body containing the tetracycline is 10 mg.L-1。
9. Use according to claim 6, characterized in that: the degradation time is 3-9 days.
10. A tetracycline biodegradation microbial inoculum is characterized in that: klebsiella variicola containing the tetracycline degradation of claim 1.
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