CN113025519B - Aeromonas intermedia and application thereof in removing chloramphenicol and dissolving phosphorus and potassium - Google Patents
Aeromonas intermedia and application thereof in removing chloramphenicol and dissolving phosphorus and potassium Download PDFInfo
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- 229960005091 chloramphenicol Drugs 0.000 title claims abstract description 73
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- 241000607534 Aeromonas Species 0.000 title claims abstract description 59
- 239000011574 phosphorus Substances 0.000 title claims abstract description 48
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 48
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 42
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 title claims abstract description 37
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
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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
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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Abstract
The invention discloses an aeromonas intermedia and application thereof in removing chloramphenicol and dissolving phosphorus and potassium. The name of the Aeromonas intermedia is Aeromonas intermedia (Aeromonas media) SZW3, the preservation number is GDMCC NO: 61503, the strain was deposited in the Guangdong provincial culture collection center of 5 th building of 59 th building of Michelia media 100, Mr. Guangzhou, 2.5.2021. The aeromonas intermedia SZW3 can remove chloramphenicol in a co-metabolism mode, and can secrete organic acid substances to reduce the pH value in a system so as to dissolve insoluble phosphorus and potassium substances. Therefore, the strain and the culture thereof can be applied to soil and water to remove chloramphenicol, and can be used for improving the content of available phosphorus and potassium in soil to promote plant growth.
Description
Technical Field
The invention belongs to the technical field of organic pollutant microbial remediation, and particularly relates to an aeromonas intermedia and application thereof in removal of chloramphenicol and dissolution of phosphorus and potassium.
Background
Chloramphenicol (CAP) was first discovered to be an antibiotic produced by Streptomyces venezuelae of the genus Actinomyces and is now produced by chemical synthesis. CAP is used as a broad-spectrum antibiotic, has an inhibition effect on both gram-negative bacteria and gram-positive bacteria, but has a stronger inhibition effect on the gram-negative bacteria than the gram-positive bacteria; the inhibition mechanism is mainly to specifically prevent the binding of mRNA to ribosomes, thereby preventing the synthesis of bacterial proteins. CAP is mainly used for treating typhoid fever, paratyphoid fever, typhus fever and the like, as well as pertussis, trachoma, bacillary dysentery, urinary tract infection and the like in clinic.
Because of low production cost and high-efficiency bacteriostasis of CAP, CAP is widely applied to agricultural cultivation and aquaculture to improve the production yield. However, only a small part of CAP is absorbed by the body after being taken by the animal, and the absorbed part is not completely metabolized and stored in the tissues and organs of the body; while most CAP is excreted by means of feces and urine and eventually into the environment. CAP is therefore detected in many environmental media and foods, such as pork, honey, shrimp, eggs, milk and fish; underwater sediments (Guiyang river), river water, livestock and poultry manure and the like. The detection limit of the pollution is also between ng/L and ug/L. CAP is stable and heat-resistant, so that the CAP is not easy to hydrolyze and photolyze in the environment; and long-term and multiple applications of CAP can impair hematopoietic function of bone marrow, causing aplastic anemia. It is therefore important to eliminate or reduce the residue of CAP in the environment.
Currently, the degradation of antibiotics in the environment is mainly focused on physical and chemical methods (such as radiation, photocatalysis, Fenton oxidation, electrochemical redox degradation and the like) and microbial degradation; the physical and chemical method needs additional reactants and relatively extreme conditions, so that the energy consumption is large and the cost is high; the microbial degradation method has the characteristics of safety, effectiveness, low cost, capability of in-situ treatment and the like, is developed in a proper environment-friendly way, and has wide application prospect.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a strain of aeromonas intermedia.
The invention also aims to provide the application of the aeromonas intermedia in removing Chloramphenicol (CAP) in soil and/or water.
It is a further object of the present invention to provide the use of said Aeromonas intermedia for solubilizing insoluble phosphorus and/or potassium compounds.
Still another object of the present invention is to provide the use of said aeromonas intermedia for increasing available phosphorus and/or available potassium in soil.
The purpose of the invention is realized by the following technical scheme:
the Aeromonas intermedia is named Aeromonas intermedia (Aeromonas media) SZW3, and the preservation number is GDMCC NO: 61503, the strain was deposited in the Guangdong province culture collection center of microbial strains of building 5 of No. 59 of Tokyo No. 100 college of Cedrury, Middy, Guangzhou, 2.5.2021.
The 16S rDNA sequence of the Aeromonas intermedia consists of 1433 basic groups (bp), and the nucleotide sequence is shown as SEQ ID NO. 1.
A method for culturing the aeromonas intermedia comprises the following specific steps: inoculating the aeromonas intermedia in a culture medium, and culturing at the temperature of 28-37 ℃.
The culture medium is one of LB culture medium, MSM culture medium containing Chloramphenicol (CAP), MSM culture medium containing chloramphenicol and peptone, NBRIP culture medium and potassium bacteria culture medium; preferably LB medium.
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is 10 mg.L-1 or less; preferably 2.5 to 10 mg.L-1(ii) a More preferably 10 mg.L-1。
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is 10 mg.L-1 or less (preferably 2.5 to 10 mg. L)-1(ii) a More preferably 10 mg.L-1) The concentration of peptone is 10 g.L-1。
The formula of the NBRIP culture medium is as follows: glucose 10 g.L-1，Ca3(PO4)2 5.0g·L-1，MgCl25g·L-1，MgSO4·7H2O 0.25g·L-1，KCl 0.2g·L-1，(NH4)2SO4 0.1g·L-1The pH was adjusted to 7.2.
The potassium bacteria culture medium comprises the following formula: potassium feldspar (K)2O·Al2O3·6SiO2)2.5g·L-1，Na2HPO40.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-1And adjusting the pH value to 6.8-7.0.
The preferable temperature of the culture is 28-30 ℃; more preferably 30 deg.c.
The culture time is 18-48 h; preferably 18-24 h.
The cultivation is carried out in a shaking table at a rotating speed of 125-150 r.min-1。
An aeromonas intermedia culture is obtained by inoculating the aeromonas intermedia in a culture medium and culturing at the temperature of 28-37 ℃.
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 medium containing peptone is preferably 10 g.L-1。
The aeromonas intermedia is used for removing or reducing Chloramphenicol (CAP) in the environment.
The environment comprises a soil environment and a water body environment.
In order to add the aeromonas intermedia into the soil and/or water environment containing chloramphenicol, the strain can tolerate the growth of the chloramphenicol and can eliminate the chloramphenicol in the soil or water.
The concentration of the chloramphenicol in the water body containing the chloramphenicol is 10 mg.L-1The following.
The removing time is more than 0.25 days; preferably for 6 days or more.
Use of said Aeromonas intermedia and/or Aeromonas intermedia culture for solubilizing insoluble phosphorus and/or potassium compounds.
The application of the aeromonas intermedia and/or the culture of the aeromonas intermedia in dissolving insoluble phosphorus and/or potassium compounds is to inoculate the aeromonas intermedia into a culture medium containing the insoluble phosphorus and/or potassium compounds and culture the aeromonas intermedia at the temperature of 28-37 ℃, wherein the aeromonas intermedia can reduce the pH value of a system to dissolve the insoluble phosphorus and/or potassium compounds; and/or adding said culture of Aeromonas intermedia to a medium containing an insoluble phosphorus and/or potassium compound to solubilize the insoluble phosphorus and/or potassium compound.
The insoluble phosphorus compound is preferably tricalcium phosphate (Ca)3(PO4)2)。
The culture medium containing the insoluble phosphorus compound is preferably an NBRIP culture medium, and the formula is as follows:
The insoluble potassium compound comprises a potassium-containing mineral; preferably potassium feldspar (K)2O·Al2O3·6SiO2)。
The culture medium containing the insoluble potassium compound is preferably a potassium bacteria culture medium, and the formula is as follows: potassium feldspar 2.5 g.L-1，Na2HPO4 0.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-1And adjusting the pH value to 6.8-7.0.
The inoculation amount of the intermediate aeromonas is 1-5% by volume percentage; preferably 2% by volume.
The temperature of the culture is preferably 28-30 ℃.
The culture time is more than 18 h; preferably 6d or more.
The use of said Aeromonas intermedia and/or Aeromonas intermedia culture for increasing available phosphorus and/or available potassium in soil.
The application of the aeromonas intermedia and/or the aeromonas intermedia culture in increasing the available phosphorus and/or the available potassium in the soil is that in order to add the aeromonas intermedia into the soil, the aeromonas intermedia can reduce the pH value of a system, so that insoluble phosphorus and/or potassium compounds in the soil are dissolved, and the content of the available phosphorus and/or the available potassium in the soil is increased (further the plant growth is promoted); and/or adding the culture of the aeromonas intermedia to a culture medium containing insoluble phosphorus and/or potassium compounds to dissolve the insoluble phosphorus and/or potassium compounds so as to improve the content of available phosphorus and/or available potassium in the soil.
The insoluble phosphorus compound is preferably tricalcium phosphate (Ca)3(PO4)2)。
The insoluble potassium compound comprises a potassium-containing mineral; preferably potassium feldspar (K)2O·Al2O3·6SiO2)。
A biological agent for removing or reducing chloramphenicol from the environment comprises the aeromonas intermedia.
The environment comprises a soil environment and a water body environment.
A biological agent for solubilizing insoluble phosphorus and/or potassium compounds, which comprises the above Aeromonas intermedia.
After the Aeromonas intermedia SZW3 grows on an LB plate for 18-20 hours, the bacterial plaque is round, moist and glossy, the middle bulge is beige, and the diameter of the bacterial colony is about 1-2 mm after the bacterial colony is cultured at 37 ℃ for 18 hours. Can be used at the concentration of 10 mg.L of chloramphenicol-1Contains 10 g.L-1Peptone grew well in an inorganic salt liquid medium.
Compared with the prior art, the invention has the following advantages and effects:
(1) the aeromonas intermedia SZW3 can remove chloramphenicol by means of co-metabolism, and the strain SZW3 is inoculated to chloramphenicol with the concentration of 10 mg.L-1Contains 10 g.L-1In inorganic salt liquid culture medium of peptone, at 30 ℃ and 150 r.min-1Shake culturing for 0, 0.25, 0.5, 1, 2, 3, 4 and 6 days (d), the removal efficiency of chloramphenicol is 0, 7.88, 20.76, 38.06, 55.86, 59.56, 64.01 and 64.28% respectively, which shows that the strain SZW3 has the performance of resisting growth of chloramphenicol and removing chloramphenicol, and can be applied to the removal of low-concentration chloramphenicol in soil and water body to remove or reduce the residue of chloramphenicol in the environment.
(2) The aeromonas intermedia SZW3 can reduce the pH value (organic acid substances are secreted) in the system, so as to dissolve insoluble phosphorus and potassium substances, the bacterial strain SZW3 is inoculated in NBRIP or potassium-solubilizing bacterial culture medium for shake culture for 6d, and the effective phosphorus and potassium contents in the solution are measured, thus the following contents can be seen: the effective phosphorus and potassium content of the solution of the added strain SZW3 is 238.62 and 7 mg.L-1While the phosphorus and potassium contents in the blank non-bacteria-added solution are 4.6 and 4.7 mg.L-1(ii) a The phosphorus-dissolving amount is obviously higher than that of a control without adding bacteria, and the potassium-dissolving content is lower, so that the strain SZW3 can dissolve insoluble phosphorus and potassium compounds, has the potential of improving the effective phosphorus and potassium content of soil, and can be used for promoting plant growth.
Drawings
FIG. 1 is a colony morphology of strain SZW3 (dish diameter 9 cm).
FIG. 2 is a phylogenetic tree diagram of strain SZW 3.
FIG. 3 shows strain SZW3 containing 10 g.L-1Kinetics of degradation of chloramphenicol in peptone MSM medium.
FIG. 4 shows strain SZW3 containing 10 g.L-1Degradation rate of chloramphenicol in peptone MSM medium and strain growth and pH change profile.
FIG. 5 is a graph showing the results of quantitative determination of potassium phosphate solubilizing of the strain SZW 3; wherein A is a quantitative determination result of available phosphorus (phosphorus); and B is the quantitative determination result of soluble potassium (potassium).
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. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. Test methods without specifying specific experimental conditions in the following examples are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Example 1 screening, isolation and purification and identification of Strain SZW3
Screening method of chloramphenicol degrading strain SZW3
1. Material preparation
Strain screening sample sources: digging a dry ditch beside a pig farm in Longping town of Yongzhou city, Qing and Yuan province to obtain a earthworm, sealing the earthworm and surrounding soil samples by using a sampling bag, and immediately removing and digging after the earthworm is brought back to a laboratory at 4 ℃ to obtain intestinal contents.
LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 7.0-7.2 of pH, constant volume of distilled water to 1L, and 18.0g of agar powder added into a solid culture medium. Sterilizing at 121 deg.C for 15 min.
Inorganic salt medium (MSM): 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-1CaCl of2Solution (CaCl)2·2H2O 48.22g·L-1) 1.0mL of a solution of trace elements (39.9 mg. L)-1MnSO4·H2O，42.8mg·L-1ZnSO4·H2O，34.7mg·L-1(NH4)6Mo7O24·4H2O), mixing, adjusting the pH value to 7.0-7.2, fixing the volume to 1L by using pure water, and sterilizing for 15min at 121 ℃.
Containing 10 g.L-1MSM medium for peptone: adding 10.0g of tryptone into the MSM culture medium solution, adjusting the pH value to 7.0-7.2, fixing the volume to 1L by using pure water, and sterilizing for 15min at 121 ℃.
2. Laboratory apparatus and device
Vertical pressure steam sterilizer (BL-50A, Shanghai Silicaceae, Inc.), portable pH meter (PHB-4, Shanghai precision science, Inc.), Centrifuge (Centrifuge 5810R), electric heat oven (DGG-9070A, Shanghai Senxin laboratories, Inc.), digital display constant temperature water bath (HH series, Changzhou national instruments manufacturing, Inc.), refrigerator (RCD-205AG7, Haixin electric appliance), biochemical incubator (PYX-208S-A, Keli apparatus), clean bench (SW-CJ-1F, Sujing Antai air technology, Inc.), voro mutex mixer (XW-80A, Shanghai Jingzike, Inc.), MyCycler PCR (BIO-Beijing, USA), electrophoresis apparatus (RAD DYY-6C, Hexagon, etc.), NaDro nucleic acid protein quantitative detector (German, Germany research, Japan, and the present, Gel imaging system (BIO-RAD, USA), floor type constant temperature oscillator (HZQ-211C).
3. Enrichment screening, separation and purification of chloramphenicol degrading strain
(1) Isolation and purification of the strains
Digging a dry water channel beside a pig farm in Longping town of Yongzhou city, Qingyuan province to obtain an earthworm, and then carrying the earthworm back to a laboratory to be immediately disassembled to obtain intestinal contents. 0.2g of the content was weighed out and added to 10mL of a solution containing 0.5 mg. L-1CAP (Chloramphenicol) content of 10 g.L-1Acclimatizing and culturing peptone in MSM culture medium, inoculating 2% (v/v) to the culture medium containing 1 mg.L- 1CAP content of 10 g.L-1MSM medium acclimatization of peptone, followed by sequential 2% (v/v) transfer to CAP concentrations of 2.5, 5 and 10 mg. L-1CAP content of 10 g.L-1And (3) respectively acclimating peptone in MSM culture medium for 3 d. After acclimatization and screening, diluting the bacterial liquid to 10 degrees with a culture medium7Taking 0.2mL of diluent, coating the diluent on an LB solid culture medium, and culturing at 30 ℃ until bacterial colonies are formed; and then selecting the grown single colony, separating and purifying.
(2) Strain screening
And (2) inoculating the strain separated and purified in the step (1) into an LB liquid culture medium for amplification culture for 20-24 h. After activation, the mixture is added to a mixture containing 10 mg.L according to the proportion of 2% (v/v)-1CAP content of 10 g.L-1150 r.min in MSM culture medium of peptone-1Culturing at 30 deg.C in dark, collecting bacterial liquid after 6 days, and measuring CAP concentration. The determination method comprises the following steps: bacterial liquid 8000r min -1Centrifuging for 1min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring filtrate with High Performance Liquid Chromatography (HPLC), and screening to obtain strain with CAP degradability. The HPLC conditions were as follows: the liquid chromatography column is CNW C18-WP (4.6 × 250mm, 5 μm), phase A is water, phase B is methanol, V (A): V (B): 40:60, and the flow rate is 0.8 mL/min-1The column temperature is 30 ℃; the sample volume is 20 mu L; the ultraviolet detector detects a wavelength of 278 nm. Screening to obtain a strain capable of degrading CAP (after the CAP concentration is measured by HPLC, the CAP concentration is obviously reduced, and the concentration of a blank control group (CK) is basically unchanged, so as to judge whether the strain has the degrading capability), and naming the strain as SZW 3.
Secondly, observing the morphological characteristics of the bacterial colony
The strain SZW3 grows faster on an LB culture medium and can grow at the temperature of 30-37 ℃. The plaque is round, moist and lustrous, and the middle of the plaque is raised to be beige. Can be used at CAP concentration of 10 mg.L-1Contains 10 g.L-1Growth was good in peptone (FIG. 1).
2.16S rDNA amplification
And (3) amplifying by using the extracted total DNA of the strain SZW3 as a template and a bacterial 16S rDNA universal primer, wherein the forward primer is 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492 r: 5'-GGTTACCTTGTTACGACTT-3' (Stackelbrandt et al, 1991) amplified the 16S rDNA gene sequence.
The total PCR reaction was 25 μ L: mu.L of each of the upstream and downstream primers, 0.5. mu.L of template DNA, 12.5. mu.L of 2 XTaq PCR Master Mix, and sterilized ultrapure water to a total volume of 25. mu.L.
The PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 50 s; annealing at 56 ℃ for 50 s; extension at 72 ℃ for 50s, 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃. The PCR product was then detected by electrophoresis on a 1.0% agarose gel (DL 2000 Marker was selected). And observing under a gel imaging system, wherein a remarkable band appears in the middle range of the Marker 1000bp and 2000bp bands.
3.16 determination of the S rDNA sequence
The PCR amplified product is sent to Beijing Rui Boxing scientific Biotechnology Limited company (Guangzhou division) for sequencing, and the 16S rDNA gene sequence of the obtained strain is as follows (SEQ ID NO. 1):
GGGACGACTACCTGCAGTCGAGCGGCAGCGGGAAAGTAGCTTGCTACTTTTGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCAGTCGAGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCGATTGGATATGCCCAGGTGGGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGATGCCTAATACGTATCAGCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAAAACTGTCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGCTGTGTCCTTGAGACGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCTGGAATCCTGCAGAGATGCGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGCTGCAAGCTAGCGATAGTGAGCGAATCCCAAAAAGCGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAAATCAGAATGTTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGGTAT。
the above sequence consists of 1433 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 6.0 software. Comparison of the 16S rDNA sequence revealed that the strain SZW3 has 99.09% homology with Aeromonas intermedia (Aeromonas media) (FIG. 2).
Fourth, the strain SZW3 is identified as a new functional strain
According to the colony morphological characteristics and molecular biological identification result of the strain SZW3, the strain SZW3 is named as Aeromonas intermedia (Aeromonas media) SZW 3. The strain is preserved in Guangdong province microbial culture collection center (GDMCC), and the preservation number is GDMCC NO: 61503, the strain is deposited at No. 59 building 5 of the Ji Dazhou No. 100 Jiedu, Guangzhou city, 2.5 days (2021). The Aeromonas intermedia (Aeromonas media) has the function of removing chloramphenicol, so that the Aeromonas intermedia SZW3 is a new strain capable of reducing the chloramphenicol in a solution.
EXAMPLE 2 Strain SZW3 Chloramphenicol removal assay
Streaking strain SZW3, culturing at 30 deg.C for 20 hr on LB plate, selecting single colony, inoculating into LB liquid culture medium, culturing at 30 deg.C and 150rpm in shaker for 20 hr, inoculating into chloramphenicol with concentration of 10 mg.L at 2% (v/v)-1Contains 10 g.L-1In inorganic salt liquid culture medium of peptone, at 30 deg.C and 150r min-1Shake cultures for 0, 0.25, 0.5, 1, 2, 3, 4 and 6 days (d) with 3 replicates with no addition of strain SZW3 as a blank (CK). The chloramphenicol removal efficiencies were 0, 7.88, 20.76, 38.06, 55.86, 59.56, 64.01 and 64.28%, respectively.
The strain SZW3 contains 10 g.L-1The degradation kinetics of chloramphenicol in peptone MSM medium are shown in FIG. 3, and the degradation rate of chloramphenicol as well as strain growth and pH change are shown in FIG. 4: the growth of SZW3 shows an S-shaped curve, and the maximum concentration of the thallus is 1.18 at OD600 at 0.5 days (d); and the pH of the solution is gradually increased to 8.7 and maintained at 8.5-8.7, wherein the pH is increased because when SZW3 uses peptone (containing a large amount of amino groups) as a nutrient substance for growth, a large amount of amine compounds are generated, so that the pH of the solution is increased. The results show that the strain SZW3 has the performance of resisting growth of chloramphenicol and removing chloramphenicol, and can be applied to the removal of chloramphenicol in soil and water.
EXAMPLE 3 qualitative test of phosphorus-dissolving and potassium-dissolving of the Strain SZW3
Bacterial strains which dissolve phosphorus and potassium (phosphorus/potassium solubilizing bacteria, meaning the conversion of insoluble phosphorus and potassium elements to soluble phosphorus and potassium) (specific references: Rawat, P., Das, S., Shankhdhar, D., et al, phosphorus-dissolving microorganisms: Mechanism and the role in phosphorus dissolution and uptake [ J ]. Journal of Soil Science and Plant Nutrition.2020, https:// doi.org/10.1007/s 42729-020-:
Inoculating the strain SZW3 into an LB liquid culture medium, and culturing at 125-150 r.min-1Shaking table culture at 28-30 deg.C for 18-24 hr, inoculating 2% (v/v) in NBRIP or potassium-decomposing bacteria liquid culture medium,setting 3 repeated tests, and placing at 30 ℃ for 150 r.min-1Culturing in shaking table for 6d, and measuring the content of available phosphorus and potassium in the solution. Wherein,
NBRIP medium formula: glucose 10 g.L-1；Ca3(PO4)2 5.0g·L-1；MgCl2 5g·L-1；MgSO4·7H2O 0.25g·L-1；KCl 0.2g·L-1；(NH4)2SO4 0.1g·L-1；pH 7.2；
Potassium bacteria culture medium: potassium feldspar (K)2O·Al2O3·6SiO2)2.5g·L-1，Na2HPO4 0.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-1，pH6.8～7.0。
The results are shown in FIG. 5: it can be seen that the strain SZW3 can reduce the pH value in the system, so as to dissolve insoluble phosphorus and potassium substances, and the effective phosphorus and potassium contents in the added strain SZW3 solution are 238.62 and 7 mg.L-1While the phosphorus and potassium contents in the blank non-bacteria-adding solution are 4.6 and 4.7 mg.L-1Wherein the phosphorus dissolving amount is obviously higher than that of a control without adding bacteria, and the content of potassium is lower. The strain SZW3 can dissolve insoluble phosphorus and potassium compounds, has the potential of improving the effective phosphorus and potassium content of soil, and can be used for promoting the growth of plants.
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 modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> one strain of aeromonas intermedia and application thereof in removal of chloramphenicol and phosphorus-dissolving potassium
<160> 3
<170> SIPOSequenceListing 1.0
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<223> 16S rDNA gene sequence
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gggacgacta cctgcagtcg agcggcagcg ggaaagtagc ttgctacttt tgccggcgag 60
cggcggacgg gtgagtaatg cctgggaaat tgcccagtcg agggggataa cagttggaaa 120
cgactgctaa taccgcatac gccctacggg ggaaagcagg ggaccttcgg gccttgcgcg 180
attggatatg cccaggtggg attagcttgt tggtgaggta atggctcacc aaggcgacga 240
tccctagctg gtctgagagg atgatcagcc acactggaac tgagacacgg tccagactcc 300
tacgggaggc agcagtgggg aatattgcac aatgggggaa accctgatgc agccatgccg 360
cgtgtgtgaa gaaggccttc gggttgtaaa gcactttcag cgaggaggaa aggttgatgc 420
ctaatacgta tcagctgtga cgttactcgc agaagaagca ccggctaact ccgtgccagc 480
agccgcggta atacggaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc 540
aggcggttgg ataagttaga tgtgaaagcc ccgggctcaa cctgggaatt gcatttaaaa 600
ctgtccagct agagtcttgt agaggggggt agaattccag gtgtagcggt gaaatgcgta 660
gagatctgga ggaataccgg tggcgaaggc ggccccctgg acaaagactg acgctcaggt 720
gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatgt 780
cgatttggag gctgtgtcct tgagacgtgg cttccggagc taacgcgtta aatcgaccgc 840
ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctggcctt gacatgtctg 960
gaatcctgca gagatgcggg agtgccttcg ggaatcagaa cacaggtgct gcatggctgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccctgtcctt 1080
tgttgccagc acgtaatggt gggaactcaa gggagactgc cggtgataaa ccggaggaag 1140
gtggggatga cgtcaagtca tcatggccct tacggccagg gctacacacg tgctacaatg 1200
gcgcgtacag agggctgcaa gctagcgata gtgagcgaat cccaaaaagc gcgtcgtagt 1260
ccggatcgga gtctgcaact cgactccgtg aagtcggaat cgctagtaat cgcaaatcag 1320
aatgttgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg 1380
ggttgcacca gaagtagata gcttaacctt cgggagggcg ttaccacggg tat 1433
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<213> Artificial Sequence (Artificial Sequence)
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<223> the forward primer was 27f
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<223> reverse primer is 1492r
<400> 3
ggttaccttg ttacgactt 19
Claims (10)
1. An aeromonas intermedia strain, which is characterized in that: named Aeromonas intermedia (Aeromonas media) SZW3, with the deposit number GDMCC NO: 61503, the strain was deposited at 2 months and 5 days 2021Guangdong province microorganism strain preservation center of No. 59 building 5 of Jie No. 100 college of Jieli Zhonglu, Guangzhou city.
2. A method for culturing Aeromonas intermedia of claim 1, comprising the steps of: inoculating the intermediate aeromonas in a culture medium, and culturing at the temperature of 28-37 ℃.
3. The method of claim 2, wherein:
the culture medium is one of LB culture medium, MSM culture medium containing chloramphenicol and peptone, NBRIP culture medium and potassium bacteria culture medium;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is 10 mg.L -1The following;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is 10 mg.L-1Hereinafter, the concentration of peptone is 10 g.L-1；
The formula of the NBRIP culture medium is as follows: glucose 10 g.L-1，Ca3(PO4)2 5.0 g·L-1，MgCl2 5 g·L-1，MgSO4·7H2O 0.25 g·L-1，KCl 0.2 g·L-1， (NH4)2SO4 0.1 g·L-1Adjusting the pH value to 7.2;
the potassium bacteria culture medium comprises the following formula: potassium feldspar 2.5 g.L-1，Na2HPO4 0.2 g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2 g·L-1，CaCO3 5.0 g·L-1，CaSO4·2H2O 0.1 g·L-1Glucose 10 g.L-1Adjusting the pH value to 6.8-7.0;
the culture time is 18-48 h.
4. A culture of Aeromonas intermedia, which is obtained by inoculating the Aeromonas intermedia of claim 1 in a medium and culturing at 28-37 ℃;
the culture medium is one of LB culture medium, MSM culture medium and MSM culture medium containing peptone.
5. Use of aeromonas intermedia in eliminating or reducing chloramphenicol in an environment according to claim 1, characterized in that: the environment comprises a soil environment and a water body environment.
6. Use of the Aeromonas intermedia of claim 1 and/or the culture of Aeromonas intermedia of claim 4 for solubilizing an insoluble phosphorus compound and/or an insoluble potassium compound.
7. Use according to claim 6, characterized in that:
the insoluble phosphorus compound is tricalcium phosphate;
The insoluble potassium compound is potassium feldspar.
8. Use of aeromonas intermedia according to claim 1 and/or an aeromonas intermedia culture according to claim 4 for increasing available phosphorus and/or available potassium in soil.
9. A biological agent for removing or reducing chloramphenicol in an environment, characterized in that: comprising Aeromonas intermedia of claim 1;
the environment comprises a soil environment and a water body environment.
10. A biological agent for dissolving an insoluble phosphorus compound and/or an insoluble potassium compound, characterized in that: comprising the Aeromonas intermedia of claim 1.
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