CN113583901B - Perchloric acid Lei Bashi strain and application thereof in chloramphenicol and cadmium ion removal - Google Patents
Perchloric acid Lei Bashi strain and application thereof in chloramphenicol and cadmium ion removal Download PDFInfo
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Abstract
The invention discloses a strain of perchloric acid Lei Bashi bacteria and application thereof in chloramphenicol and cadmium ion removal. The strain is named as perch Lei Bashi strain (Klebsiella variicola) YB1, and the preservation number is GDMCCNO:61696 the strain was deposited at 5.27 of 2021 at Guangdong province microorganism strain collection of No. 5 building of No. 59 of No. 5 of 100 university of Mitsui, guangzhou City. The metamorphic perch Lei Bashi strain YB1 can remove chloramphenicol in a co-metabolism mode, and can also raise the pH value in a system to precipitate cadmium ions, so that the metamorphic perch Lei Bashi strain YB1 can be used for removing or reducing chloramphenicol and cadmium ions residues in the environment.
Description
Technical Field
The invention belongs to the technical field of organic pollutant microorganism restoration, and particularly relates to a strain of perchloric acid Lei Bashi and application thereof in chloramphenicol and cadmium ion removal.
Background
Chloramphenicol (CAP) is a broad-spectrum antibiotic, an anti-infective drug. The composition has effect in inhibiting bacterial growth by inhibiting synthesis of bacterial proteins, and can be used for treating infection caused by typhoid bacillus, dysentery bacillus, escherichia coli, influenza bacillus, brucella, pneumococcus, etc. Can be widely used for clinical treatment and livestock and aquaculture, and can also be used as an animal growth promoter. It is usually white or colorless needle-like or tablet-like crystals, is easily dissolved in methanol, ethanol, propanol, chloramphenicol is extremely stable, and its aqueous solution does not fail after boiling for 5 hours.
With the widespread use of chloramphenicol in people's daily production and life, some chloramphenicol hazards are increasingly prominent. The literature reports that the release of antibiotics into the environment can enhance the antibiotic resistance of pathogenic bacteria, chloramphenicol has a strong destructive effect on the blood system of human body, can induce various blood cell quantity reduction, and can also induce aplastic anemia by inhibiting protein synthesis in mitochondria in bone marrow hematopoietic stem cells.
In the traditional wastewater treatment, the main removal technology of CAP comprises chemical precipitation, activated sludge degradation, physical and chemical methods such as adsorption method, extraction method, electrochemical method, oxidation method, reduction method and the like and biological methods, the concentration of CAP after treatment is reduced, but trace level of CAP can also cause threat to an ecological system, and the methods for treating chloramphenicol wastewater at present have the defects of higher degradation efficiency, high energy consumption, secondary pollution and the like. There is a need for an economical and efficient method for treating chloramphenicol, and the research on chloramphenicol treatment by microorganisms has very broad prospects, and the microbiological method becomes a hotspot for research on chloramphenicol degradation wastewater.
Cadmium is a toxic heavy metal, has the characteristics of long decomposition period, strong mobility and difficult degradation, and can enter a human body through crops enrichment and further through a food chain. Cadmium is not an essential element of the human body, and cadmium in the human body is mainly taken in through food intake and air intake of the gas containing cadmium by respiration. Long-term life and work in cadmium-containing environments can cause chronic cadmium poisoning, which is a great hazard to kidneys and bones. Japanese reports "bone pain" events caused by consumption of cadmium contaminated water sources. The phenomenon of cadmium rice is reported in China, and the cadmium content in the rice exceeds the standard due to irrigation by water polluted by mine wastewater and wastewater of a smelting plant.
According to the national ecological environment quality profile issued by the China ecological environment department in 2020, the soil cadmium pollution in China is serious. The detailed investigation result of the soil pollution condition of the agricultural land shows that: the soil environment conditions of the agricultural land are stable but pollution is serious. The pollution of agricultural land is mainly heavy metal, wherein cadmium is the main pollutant. The safe utilization rate of polluted farmland reaches about 90%, and the safe utilization rate of polluted land reaches more than 90%.
At present, the repair treatment technology of cadmium pollution is still a research hot spot and a difficult point in the environmental field, and the repair method can be divided into physical, chemical and biological repair technologies, and the toxic effect of cadmium is reduced by reducing the content, concentration or solidification of the cadmium. Physical methods include earth-bound methods, thermal desorption repair, vitrification repair, and electrokinetic repair. Chemical methods include rinse repair and cure repair. Bioremediation can be divided into phytoremediation, animal remediation and microbial remediation. The microbial remediation technology is that cadmium is absorbed, precipitated and accumulated through the extracellular adsorption effect, extracellular precipitation effect and intracellular accumulation effect of microorganisms, so that the form of cadmium in soil is effectively changed, and the mobility and toxicity of cadmium ions are further reduced.
Disclosure of Invention
The invention aims at overcoming the defects and shortcomings of the prior art and providing a strain of perch Lei Bashi.
It is another object of the present invention to provide a culture of the gram Lei Bashi bacteria.
It is a further object of the present invention to provide the use of said perchloric Lei Bashi bacteria for the removal or reduction of Chloramphenicol (CAP) in the environment.
It is a further object of the present invention to provide the use of the g Lei Bashi strain and its cultures for the removal or reduction of cadmium ions in the environment.
The aim of the invention is achieved by the following technical scheme:
a strain of perch Lei Bashi bacteria, named perch Lei Bashi bacteria (Klebsiella variicola) YB1, deposited with the accession number GDMCC NO:61696 the strain was deposited at 5.27 of 2021 at Guangdong province microorganism strain collection of No. 5 building of No. 59 of No. 5 of 100 university of Mitsui, guangzhou City.
The 16S rDNA sequence of the metamorphic perch Lei Bashi bacteria consists of 1426 bases (bp), and the nucleotide sequence is shown as SEQ ID NO. 1.
A method for culturing the metamorphic g Lei Bashi bacteria comprises the following specific steps: inoculating the percha strain Lei Bashi into a culture medium, and culturing at 28-37 ℃.
The culture medium is LB culture medium, MSM culture medium containing Chloramphenicol (CAP), MSM culture medium containing chloramphenicol and peptone, cd 2+ And peptone in MSM medium containing Cd 2+ MSM medium of chloramphenicol and peptone; LB medium is preferred.
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is 10mg.L -1 The following are set forth; preferably 2.5 to 10 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the More preferably 10 mg.L -1 。
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is 10mg.L -1 The following (preferably 2.5 to 10 mg.L) -1 ) Concentration of peptone was 10 g.L -1 。
The Cd-containing catalyst contains Cd 2+ And Cd in peptone MSM medium 2+ The concentration is 50 mg.L -1 The following (preferably 20 mg.L) -1 The following are set forth; more preferably 2 to 10 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the More preferably 2 mg.L -1 ) Concentration of peptone was 10 g.L -1 。
The Cd-containing catalyst contains Cd 2+ Cd in MSM medium of chloramphenicol and peptone 2+ The concentration is 50 mg.L -1 The following (preferably 20 mg.L) -1 The following are set forth; more preferably 2 to 10 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the More preferably 2 mg.L -1 ) The concentration of chloramphenicol is 10mg.L -1 The following (preferably 2.5 to 10 mg.L) -1 ) Concentration of peptone was 10 g.L -1 。
The temperature of the culture is preferably 28-30 ℃; more preferably 30 ℃.
The culture time is 18-48 h; preferably 18 to 24 hours.
The culture is carried out in a shaking tableCulturing at a rotation speed of 125-150 r.min -1 。
A culture of the strain of percha Lei Bashi is prepared through inoculating the strain of percha Lei Bashi to culture medium, and culturing at 28-37 deg.C.
The inoculation amount of the metamorphic perch Lei Bashi bacteria is 1-5% by volume; preferably 2% by volume.
The culture medium is preferably one of LB culture medium, MSM culture medium and MSM culture medium containing peptone; more preferably LB medium.
The concentration of peptone in the MSM culture medium containing peptone is preferably 10 g.L -1 。
The temperature of the culture is preferably 28-30 ℃; more preferably 30 ℃.
The culture time is 18-48 h; preferably 18 to 24 hours.
The culture is carried out in a shaking table, and the rotating speed is 125-150 r.min -1 。
The application of the metamorphic perchlora Lei Bashi bacteria in removing or reducing Chloramphenicol (CAP) in the environment.
The application of the metamorphic perch Lei Bashi bacteria in removing or reducing Chloramphenicol (CAP) in the environment is that the metamorphic perch Lei Bashi bacteria are added into soil and/or water environment containing chloramphenicol, the strain can resist chloramphenicol growth, and chloramphenicol in soil or water can be eliminated.
The environment comprises soil environment and water environment.
The concentration of chloramphenicol in the water environment is 10mg.L -1 The following is given.
The time for removing is more than 0.25 days; preferably for 6 days or more.
The application of the metamorphic perch Lei Bashi bacteria and/or the metamorphic perch Lei Bashi bacteria culture in removing or reducing the cadmium ion content in the environment (reducing the cadmium content in the environment).
The application of the metamorphic perch Lei Bashi bacteria and/or the metamorphic perch Lei Bashi bacteria culture in removing or reducing the content of cadmium ions in the environment is that the metamorphic perch Lei Bashi bacteria are added into the environment of soil and/or water containing the cadmium ions, the strain can resist the growth of heavy metal cadmium, and the cadmium ions in the soil or water can be removed under the condition of cadmium ion pollution so as to remove or reduce the effective concentration of the cadmium ions in the environment.
The environment comprises soil environment and water environment.
The cadmium ion is bivalent cadmium ion (Cd) 2+ )。
The concentration of cadmium ions in the water body environment is 50mg.L -1 The following are set forth; more preferably 2 to 50 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the Even more preferably 2 to 10 mg.L -1 More preferably 10 mg.L -1 。
The time for the removal is more than 2d (days).
The metamorphic perchlora Lei Bashi is applied to the aspect of simultaneously removing or reducing cadmium ions in the environment (reducing the cadmium content in the environment) and chloramphenicol.
The application of the metamorphic gram Lei Bashi bacteria in the aspect of simultaneously removing or reducing cadmium ions and chloramphenicol in the environment is that the metamorphic gram Lei Bashi bacteria are added into the soil and/or water environment containing cadmium ions and chloramphenicol, the strain can resist heavy metal cadmium growth, raise the pH of a system, and can remove the cadmium ions and chloramphenicol in the soil or water under the condition of simultaneously polluting the chloramphenicol and the cadmium ions so as to remove or reduce the effective concentration of the cadmium ions and the chloramphenicol in the environment.
The environment comprises soil environment and water environment.
The cadmium ion is bivalent cadmium ion (Cd) 2+ )。
The concentration of chloramphenicol in the water environment is 10mg.L -1 The following is given.
The concentration of cadmium ions in the water body environment is 50mg.L -1 The following (more preferably 2 to 50 mg.L) -1 The method comprises the steps of carrying out a first treatment on the surface of the Even more preferably 2 to 10 mg.L -1 More preferably 10 mg.L -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Chloramphenicol concentration is 10mg.L -1 。
The time for the removal is more than 2d (days).
The metamorphic perch Lei Bashi strain is applied to the preparation of a cadmium ion passivating agent.
The cadmium ion is bivalent cadmium ion (Cd) 2+ )。
A biological agent for removing chloramphenicol and/or cadmium ions in environment comprises the strain of Probiotics Lei Bashi.
The cadmium ion is bivalent cadmium ion (Cd) 2+ )。
After the metamorphic gram Lei Bashi strain YB1 grows on an LB plate for 18-20 hours, bacterial plaques are round, wet and glossy, the middle bulge is beige, and after the strain is cultured for 18 hours at 30 ℃, the diameter of a bacterial colony is about 1-2 mm. Can be used for preparing chloramphenicol with concentration of 10mg.L -1 Contains 10 g.L -1 The growth was good in the MSM medium of peptone.
Compared with the prior art, the invention has the following advantages and effects:
(1) The metamorphic perch Lei Bashi strain YB1 of the invention can remove chloramphenicol in a co-metabolism mode, and the strain YB1 is inoculated to chloramphenicol with the concentration of 10 mg.L -1 Contains 10 g.L -1 In MSM culture medium of peptone at 30 deg.C and 150 r.min -1 Shake culturing for 0, 0.25, 1, 2, 3, 4, 5 and 6 days (d), the chloramphenicol removal efficiency is 0, 8.86, 8.91, 18.11, 22.99, 23.79, 25.58, 28.55, 38.09%, respectively, which shows that the strain YB1 has the performance of resisting chloramphenicol growth and removing chloramphenicol, and can be applied to the removal of low-concentration chloramphenicol in soil and water so as to remove or reduce chloramphenicol residues in the environment.
(2) The pH value of a system can be raised (amine substances are secreted) by the metamorphic perch Lei Bashi strain YB1, the strain YB1 is inoculated in an MSM culture medium containing chloramphenicol and added with peptone for shake culture for 6d, and chloramphenicol in the solution is measured, so that the following can be seen: chloramphenicol concentration in the solution of the added YB1 is 6.19 mg.L -1 While chloramphenicol in the blank sterile solution is 10mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The chloramphenicol concentration is obviously lower than that of the control non-added bacteria, which indicates that the strain YB1 can degrade chloramphenicol and has the function of reducing loopsPotential for chloramphenicol content in the environment.
(3) The metamorphic gram Lei Bashi strain YB1 can raise the pH value in the system and cause cadmium ions to precipitate. The strain YB1 was inoculated into a peptone-added MSM medium containing cadmium ions (2-50 mg/L) and chloramphenicol (10 gm/L) for shake cultivation for 2 days, and the concentration of chloramphenicol and cadmium ions in the solution was measured, so that it was found that: the concentration of chloramphenicol and cadmium ions is reduced, and Cd 2+ The YB1 growth is obviously inhibited after the concentration is more than 20 mg/L. Cadmium ion removal efficiencies of 0, 2, 5, 10, 20 and 50mg/L are respectively as follows: 0,41.05, 62.0, 69.84, 11.38, 12.4%, chloramphenicol removal efficiencies were respectively: 22.41, 10.06, 13.01, 15.1, 7.47, 3.12%. Indicating that YB1 has the simultaneous tolerance to Cd 2+ And chloramphenicol growth and simultaneous removal of Cd 2+ And chloramphenicol, can be applied to low-concentration chloramphenicol and Cd in water and soil 2+ To remove or reduce chloramphenicol and Cd 2+ Residue in the environment.
Drawings
FIG. 1 is a colony morphology of strain YB1 (dish diameter 9 cm).
FIG. 2 is a phylogenetic tree of strain YB1.
FIG. 3 shows strain YB1 in the presence of 10 g.L -1 Graph of degradation kinetics of chloramphenicol in the MSM medium of peptone.
FIG. 4 shows strain YB1 in the presence of 10 g.L -1 Graphs of cell concentration and pH change during chloramphenicol degradation in peptone MSM medium.
FIG. 5 shows strain YB1 containing 2-50mg/L Cd 2+ And 10 g.L -1 Chloramphenicol and Cd in MSM medium of peptone 2+ Degradation rate of (c), strain growth and pH profile.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Example 1 screening, isolation and purification and identification of Strain YB1
1. Chloramphenicol degradation strain YB1 screening method
1. Material preparation
Strain screening sample sources: digging a earthworm from a dry ditch beside a pig farm in Qingyuan city, tarmac, guangdong province, sealing the earthworm and surrounding soil samples by using a sampling bag, and immediately planing after the earthworm is brought back to a laboratory at 4 ℃ to obtain intestinal contents.
LB medium: 10.0g of peptone, 5.0g,NaCl 10.0g,pH 7.0-7.2 g of yeast extract powder, distilled water to 1L, and 18.0g of agar powder. Sterilizing at 121deg.C for 15min.
Inorganic salt medium (MSM): 5mL of phosphate buffer solution (KH) 2 PO 4 8.5g·L -1 、K 2 HPO 4 ·H 2 O 21.75g·L -1 、Na 2 HPO 4 ·12H 2 O 33.4g·L -1 、NH 4 Cl 5.0g·L -1 )，3.0mL 22.5g·L -1 Is MgSO of (2) 4 Solution (MgSO) 4 ·7H 2 O 46.125g·L -1 )，1.0mL 0.25g·L -1 FeCl of (C) 3 Solution (FeCl) 3 ·6H 2 O 0.42g·L -1 )，1.0mL 36.4g·L -1 CaCl of (2) 2 Solution (CaCl) 2 ·2H 2 O 48.22g·L -1 ) 1.0mL of a trace element solution (containing 39.9mg.L) -1 MnSO 4 ·H 2 O，42.8mg·L -1 ZnSO 4 ·H 2 O，34.7mg·L -1 (NH 4 ) 6 Mo 7 O 24 ·4H 2 O) mixing evenly, regulating the pH to 7.0-7.2, fixing the volume to 1L by pure water, and sterilizing for 15min at 121 ℃.
Contains 10 g.L -1 MSM medium of peptone: 10.0g of tryptone is added into the MSM culture medium solution, the pH is adjusted to 7.0-7.2, the volume is fixed to 1L by pure water, and the culture medium solution is sterilized for 15min at 121 ℃.
2. Experimental apparatus and device
Vertical pressure steam sterilization pot (B-50A, shanghai static Co., td.), portable pH meter (PHB-4, shanghai precision science Co., td.), centrifuge (Centrifuge 5810R), electrothermal oven (DGG-9070A, shanghai forest letter laboratory apparatus Co., td.), digital display thermostatic water bath (HH series, meter manufacturing Co., td.), refrigerator (RCD-205 AG7, sese:Sup>A letter electric appliance), biochemical incubator (PYX-208S-A, keli instrument), ultrse:Sup>A clean bench (SW-CJ-1F, sujing Antai air technologies Co., td.), vortex mixer (XW-80A, shanghai precision Co., td.), mycycler PCR (BIO-RAD Co., USA), electrophoresis apparatus (DYY-6C, beijing six Germany), nano Drop nucleic acid protein quantitative detector (Thermo), gel imaging system (BIO-RAD Co., USA), floor temperature oscillator (ZQ-211C).
3. Enrichment screening, separation and purification of chloramphenicol degradation strain
(1) Isolation and purification of strains
Digging a earthworm from a dry ditch beside a pig farm in Qingyuan city, tarmac, guangdong province, taking the earthworm back to a laboratory, and immediately planing the earthworm to obtain intestinal contents. 0.2g of the content is weighed and added to 10mL of the mixture containing 0.5 mg.L -1 Chloramphenicol (CAP) containing 10 g.L -1 Domestication culture in MSM culture medium of peptone, transferring to culture medium containing 1 mg.L with 2% (v/v) inoculum size after 2d of domestication -1 CAP content of 10 g.L -1 MSM medium acclimatization of peptone followed by 2% (v/v) transfer to CAP concentrations of 2.5, 5 and 10 mg.L -1 CAP content of 10 g.L -1 Respectively domesticating 3d in MSM culture medium of peptone. After domestication and screening, the bacterial liquid is diluted to 10 by a culture medium 7 Doubling, namely 0.2mL of diluent is taken and coated on LB solid medium, and the culture is carried out at 30 ℃ until colony formation; and then the single colonies are selected, separated and purified.
(2) Strain screening
And (3) inoculating the strain obtained by separation and purification in the step (1) into an LB liquid medium for expansion culture for 20-24 h. After activation, the mixture was added to a solution containing 10 mg.L in a proportion of 2% (v/v) -1 CAP content of 10 g.L -1 150 r.min in MSM culture medium of peptone -1 Culturing at 30deg.C in dark place, 6After the day, bacterial liquid was collected and CAP concentration was measured. The measurement method comprises the following steps: bacterial liquid 8000 r.min -1 Centrifuging for 1min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring filtrate by High Performance Liquid Chromatography (HPLC), and screening to obtain strain with CAP degrading capability. The HPLC conditions were: the liquid chromatographic column is CNW C18-WP (4.6X105 mm,5 μm), the A phase is water, the B phase is methanol, V (A): V (B) =40:60, the flow rate is 0.8 mL-min -1 Column temperature is 30 ℃; the sample injection amount is 20 mu L; the ultraviolet detector detects a wavelength of 278nm. Screening to obtain a strain capable of degrading CAP (after measuring the CAP concentration by HPLC, the CAP concentration is obviously reduced, and the concentration of a blank group (CK) without bacteria is basically unchanged, so as to judge whether the strain has degradation capability) and the strain is named as strain YB1.
2. Colony morphological feature observation
The strain YB1 grows faster on LB culture medium and can grow at 30-37 ℃. The plaque is circular, moist and shiny, and the central bulge is beige (fig. 1). After 20h of incubation at 30℃the colony diameter was about 2.5-3 mm. Can reach CAP concentration of 10mg.L -1 Contains 10 g.L -1 The growth was good in the MSM medium of peptone.
2.16S rDNA amplification
The total DNA of the extracted strain YB1 is used as a template, and the bacterial 16S rDNA universal primer is adopted for amplification, wherein the forward primer is 27f:5'-AGAGTTTGATCCTGGCTCAG-3' reverse primer is 1492r:5'-GGTTACCTTGTTACGACTT-3' (Stackebrandt et al, 1991) amplified the 16S rDNA gene sequence.
The total PCR reaction system was 25. Mu.L: the primers were 2. Mu.L each for the upstream and downstream primers, 0.5. Mu.L for the template DNA, 2X TaqPCR Master Mix, 12.5. Mu.L, and sterilized with ultrapure water to a total volume of 25. Mu.L.
The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 50s; annealing at 56 ℃ for 50s; extending at 72 ℃ for 50s,35 cycles; and finally, the mixture is supplemented and extended for 5min at 72 ℃. The PCR products were then detected by 1.0% agarose gel electrophoresis (DL 2000 Marker was selected). The gel was observed under a gel imaging system in which distinct bands appeared in the middle of the Marker bands of 1000bp and 2000 bp.
3.16 determination of the sequence of S rDNA
The PCR amplified product was sequenced by the Biotechnology Co., ltd. Of the Beijing Rui family (Guangzhou division) and the 16S rDNA gene sequence of the obtained strain was as follows (SEQ ID NO. 1):
TACAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAATCCATCTCTGCTAAGTTCTGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGACAAGGTTATTAACCTCACCGCCTTCCTCCCCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACCAGCTAATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGAGAGCAAGCTCTCTGTGCTACCGCTCGACTGCAGGTAGTCATCCGG。
the above sequence consists of 1426 bases (bp).
The obtained 16S rDNA gene sequence is submitted to a National Center for Biological Information (NCBI) webpage for BLAST comparison, homology comparison analysis is carried out on the obtained 16S rDNA gene sequence and the 16S rDNA gene of related mode strains in an LPSN database (http:// www.bacterio.net/index. Html), the downloaded mode strain sequence with higher homology is subjected to BLAST comparison and homology analysis on the amplified product sequence on the National Center for Biological Information (NCBI) website, and a system development tree is constructed by adopting Mega 6.0 software through a neighbor-Joining method. As a result of comparison with the 16S rDNA sequence, the strain YB1 was found to have 99.09% homology with the strain Thermus-strain Lei Bashi (Klebsiella variicola) (FIG. 2).
4. The strain YB1 is identified as a new functional strain
According to colony morphological characteristics and molecular biological identification results of the strain YB1, the strain YB1 is named as perch Lei Bashi strain (Klebsiella variicola) YB1. The strain has been stored in the Guangdong province microorganism strain collection (GDMCC) under the accession number GDMCC NO:61696, the preservation date is 2021, 5 and 27 days, and the preservation unit address is Guangzhou, first, china, no. 100, no. 59 building 5. The perchloric acid-producing strain Lei Bashi (Klebsiella variicola) has the function of removing chloramphenicol, so that perchloric acid-producing strain Lei Bashi YB1 is a new strain with the function of reducing chloramphenicol in solution.
EXAMPLE 2 Chloramphenicol removal test by Strain YB1
Culturing strain YB1 on LB plate at 30deg.C for 20 hr, inoculating single colony into LB liquid medium, culturing at 30deg.C in shaking table at 150rpm for 20 hr, and inoculating chloramphenicol at concentration of 10mg.L at ratio of 2% (v/v) -1 Contains 10 g.L -1 In MSM culture medium of peptone at 30deg.C and 150 r.min -1 Shaking culture was performed for 0, 0.25, 0.5, 1, 2, 3, 4, 5 and 6 days (d), and 3 replicates were performed using no added strain YB1 as a Control (CK). Determining the removal amount and removal efficiency of the strain YB1 on chloramphenicol; wherein the CAP content was determined by HPLC (the method is the same as in example 1).
Strain YB1 was isolated from strain YB1 at 10 g.L -1 The kinetics of chloramphenicol degradation in peptone MSM medium is shown in FIG. 3, and the rate of chloramphenicol degradation and strain growth and pH change are shown in FIG. 4. Chloramphenicol removal efficiencies were 0, 8.86, 8.91, 18.11, 22.99, 23.79, 25.5, 28.55, 38.09%, respectively. YB1 growth showed an "S-shaped" curve, and at 0.25 day (d) the cell concentration increased to OD 600 =0.81, then fluctuates between 0.74 and 0.94; and the pH of the solution was gradually raised to 8.32 and maintained at pH 8.24 to 8.77, the increase in pH being due to YB1 utilizing peptone (containing a large amount of amino groupsRadicals) are nutrients, a large amount of amine compounds are generated, resulting in an increase in the pH of the solution. The result shows that the strain YB1 has the performances of resisting the growth of chloramphenicol and removing chloramphenicol, and can be applied to the removal of chloramphenicol in soil and water.
Example 3 test of cadmium ion and Chloramphenicol Complex contamination by Strain YB1
Inoculating strain YB1 into LB liquid medium, at 125-150 r.min -1 Shaking culture at 28-30 deg.c for 18-24 hr, inoculating Cd in 2% (v/v) amount 2+ The concentration of (cadmium chloride is added) is 0, 2, 5, 10, 20, 50mg/L and the concentration of chloramphenicol is 10 mg.L -1 Contains 10 g.L -1 In the MSM culture medium of peptone, 3 repeated tests are set, and placed at 30 deg.C and 150 r.min -1 Culturing in a shaker for 2d. Measuring the removal amount and the removal efficiency of the strain YB1 on cadmium ions and chloramphenicol; wherein the CAP content was determined by HPLC (the method is the same as in example 1) and the cadmium ion content was determined by AAS.
The concentration of the strain YB1 in cadmium ions is 0, 2, 5, 10, 20 and 50mg/L and the concentration of chloramphenicol is 10 mg.L respectively -1 10 g.L of (C) -1 The graph of the removal efficiency of cadmium ions in peptone MSM medium, the degradation rate of chloramphenicol, and the strain growth and pH change are shown in FIG. 5 (experiments were performed with no strain YB1 added as a blank control, due to the control group Cd) 2+ And CAP, and therefore are not labeled in the figures): cadmium ion (Cd) 2+ ) Cadmium ion removal efficiencies of 0, 2, 5, 10, 20 and 50mg/L are respectively as follows: 0. 41.05, 62.0, 69.84, 11.38, 12.4%; the chloramphenicol removal efficiency was respectively: 22.41, 10.06, 13.01, 15.1, 7.47, 3.12%. Growth of YB1 is inhibited with increasing concentration of cadmium ions, when Cd 2+ YB1 can not grow substantially at 20mg/L or more. Cd (cadmium sulfide) 2+ At 2mg/L, the cell concentration reached the maximum OD 600 The pH of the bacterial liquid is maintained at substantially 9.1-9.2 at 0.689. The pH rise is due to the fact that YB1 grows using peptone (containing a large amount of amino groups) as a nutrient, which generates a large amount of amine compounds, resulting in an increase in the pH of the solution. In the presence of cadmium ions, the removal rate of YB1 to chloramphenicol is 3.12-15.1The degradation rate of the YB1 to the chloramphenicol is 22.41% under the condition of not adding cadmium ions, and the result shows that the strain YB1 has the performance of simultaneously tolerating the chloramphenicol and the cadmium ions and simultaneously removing the chloramphenicol and the cadmium ions, and can be applied to the treatment and the restoration of the soil and the water body polluted by the cadmium ions and the chloramphenicol.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> a strain of Probiotics Lei Bashi and its use in removal of chloramphenicol and cadmium ions
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1426
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 16S rDNA Gene sequence
<400> 1
tacagtggta gcgccctccc gaaggttaag ctacctactt cttttgcacc cactcccatg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgtagcatt ctgatctacg 120
attactagcg attccgactt catggagtcg agttgcagac tccaatccgg actacgacat 180
actttatgag gtccgcttgc tctcgcgagg tcgcttctct ttgtatatgc cattgtagca 240
cgtgtgtagc cctggtcgta agggccatga tgacttgacg tcatccccac cttcctccag 300
tttatcactg gcagtctcct ttgagttccc ggcctaaccg ctggcaacaa aggataaggg 360
ttgcgctcgt tgcgggactt aacccaacat ttcacaacac gagctgacga cagccatgca 420
gcacctgtct cacagttccc gaaggcacca atccatctct gctaagttct gtggatgtca 480
agaccaggta aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg 540
ggcccccgtc aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgattt 600
aacgcgttag ctccggaagc cacgcctcaa gggcacaacc tccaaatcga catcgtttac 660
agcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgca cctgagcgtc 720
agtctttgtc cagggggccg ccttcgccac cggtattcct ccagatctct acgcatttca 780
ccgctacacc tggaattcta cccccctcta caagactcta gcctgccagt ttcgaatgca 840
gttcccaggt tgagcccggg gatttcacat ccgacttgac agaccgcctg cgtgcgcttt 900
acgcccagta attccgatta acgcttgcac cctccgtatt accgcggctg ctggcacgga 960
gttagccggt gcttcttctg cgggtaacgt caatcgacaa ggttattaac ctcaccgcct 1020
tcctccccgc tgaaagtgct ttacaacccg aaggccttct tcacacacgc ggcatggctg 1080
catcaggctt gcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctggac 1140
cgtgtctcag ttccagtgtg gctggtcatc ctctcagacc agctagggat cgtcgcctag 1200
gtgagccgtt accccaccta ccagctaatc ccatctgggc acatctgatg gcatgaggcc 1260
cgaaggtccc ccactttggt cttgcgacgt tatgcggtat tagctaccgt ttccagtagt 1320
tatccccctc catcaggcag tttcccagac attactcacc cgtccgccgc tcgtcacccg 1380
agagcaagct ctctgtgcta ccgctcgact gcaggtagtc atccgg 1426
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Forward primer 27f
<400> 2
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> reverse primer is 1492r
<400> 3
ggttaccttg ttacgactt 19
Claims (10)
1. A strain of perch Lei Bashi, characterized in that: the name of the strain is Probiotics Lei BashiKlebsiella variicola) YB1, accession No. GDMCC NO:61696 the strain was deposited at 5.27 of 2021 at Guangdong province microorganism strain collection of No. 5 building of No. 59 of No. 5 of 100 university of Mitsui, guangzhou City.
2. A method for culturing the metamorphic g Lei Bashi bacterium according to claim 1, which comprises the following steps: and inoculating the percha Lei Bashi strain into a culture medium, and culturing at 28-37 ℃.
3. The method according to claim 2, characterized in that:
the culture medium is LB culture medium, MSM culture medium containing chloramphenicol and peptone, cd 2+ And peptone in MSM medium containing Cd 2+ MSM medium of chloramphenicol and peptone;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is mg.L -1 The following are set forth;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is mg.L -1 The concentration of peptone was 10 g.L -1 ；
The Cd-containing catalyst contains Cd 2+ And Cd in peptone MSM medium 2+ The concentration is 50 mg.L -1 The concentration of peptone was 10 g.L -1 ；
The Cd-containing catalyst contains Cd 2+ Cd in MSM medium of chloramphenicol and peptone 2+ The concentration is 50 mg.L -1 Hereinafter, the chloramphenicol concentration was 10 mg.L -1 The concentration of peptone was 10 g.L -1 ；
The culture time is 18-48 h.
4. A culture of percha Lei Bashi bacteria, characterized by: inoculating the metamorphic perch Lei Bashi bacterium of claim 1 into a culture medium, and culturing at 28-37 ℃;
the culture medium is one of LB culture medium, MSM culture medium and MSM culture medium containing peptone;
the culture time is 18-48 h.
5. Use of the perchloram Lei Bashi bacterium of claim 1 for the removal or reduction of chloramphenicol in an environment.
6. Use of a strain of percha Lei Bashi according to claim 1 and/or a strain of percha Lei Bashi according to claim 4 for removing or reducing the cadmium ion content of an environment.
7. The use according to claim 6, characterized in that:
the environment is a soil environment or a water body environment;
the cadmium ions are bivalent cadmium ions;
the concentration of cadmium ions in the water body environment is mg.L -1 The following are set forth;
the time for removing is more than 2 days.
8. Use of the metamorphic g Lei Bashi bacterium of claim 1 for simultaneously removing or reducing cadmium ions and chloramphenicol in an environment, wherein:
the environment is a soil environment or a water body environment;
the cadmium ions are bivalent cadmium ions;
the concentration of cadmium ions in the water body environment is mg.L -1 The following are set forth;
the concentration of chloramphenicol in the water environment is mg.L -1 The following is given.
9. The use of the metamorphic g Lei Bashi bacterium of claim 1 in the preparation of a cadmium ion passivating agent.
10. A biological agent for removing chloramphenicol and/or cadmium ions from an environment, characterized in that: a bacterium comprising the percha cell according to claim 1.
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