CN113025519B - Aeromonas intermedia and application thereof in removing chloramphenicol and dissolving phosphorus and potassium - Google Patents

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

A strain of Aeromonas intermedia and its application in removing chloramphenicol and dissolving phosphorus and potassium

technical field

The invention belongs to the technical field of microbial restoration of organic pollutants, and particularly relates to a strain of Aeromonas intermedia and its application in removing chloramphenicol and dissolving phosphorus and potassium.

Background technique

Chloramphenicol (CAP) was first discovered as an antibiotic produced by Actinomycete Streptomyces venezuela, and now it has been produced by chemical synthesis. As a broad-spectrum antibiotic, CAP has inhibitory effect on both Gram-negative and positive bacteria, but the inhibitory effect on Gram-negative bacteria is stronger than that of positive bacteria; its inhibitory mechanism is mainly to specifically prevent mRNA and ribosomes. bind, thereby preventing bacterial protein synthesis. CAP is mainly used for the treatment of typhoid, paratyphoid and typhus, as well as pertussis, trachoma, bacillary dysentery and urinary tract infection.

Due to the low production cost and high bacteriostatic efficiency of CAP, CAP is widely used in agriculture and aquaculture to increase production yield. However, after CAP is ingested by the body, only a small part is absorbed by the body, and the absorbed part is not completely metabolized and stored in the body tissues and organs; while most CAP is excreted through feces and urine, and finally enters the environment. Therefore, CAP has been detected in many environmental media and foods, such as pork, honey, prawns, eggs, milk and fish; underwater sediments (Guiyang River), river water and livestock and poultry manure. The detection limit of its contamination is also ng/L-ug/L. CAP is stable and heat-resistant, so it is not easily hydrolyzed and photolyzed in the environment; and long-term and repeated application of CAP can damage the hematopoietic function of the bone marrow and cause aplastic anemia. Therefore, it is crucial to eliminate or reduce CAP residues in the environment.

At present, the degradation of antibiotics in the environment mainly focuses on physicochemical methods (such as radiation, photocatalysis, Fenton oxidation, and electrochemical redox degradation, etc.) and microbial degradation; due to the need for additional reactants to be added to physicochemical methods and relatively extreme conditions, energy consumption The large cost is high; and the microbial degradation method is safe, effective, inexpensive and can be processed in situ, which is suitable for environment-friendly development and has broad application prospects.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a strain of Aeromonas intermedia.

Another object of the present invention is to provide the application of the Aeromonas intermedia in removing chloramphenicol (CAP) in soil and/or water.

Another object of the present invention is to provide the application of the Aeromonas intermedia in dissolving insoluble phosphorus and/or potassium compounds.

Another object of the present invention is to provide the application of the Aeromonas intermedia in increasing available phosphorus and/or available potassium in soil.

The object of the present invention is achieved through the following technical solutions:

A strain of Aeromonas intermedia, the name is Aeromonas media SZW3, the preservation number is GDMCC NO: 61503, the strain was preserved on February 5, 2021 at No. 59, Yard, No. 100, Middle Xianlie Road, Guangzhou The Guangdong Provincial Microbial Culture Collection Center on the 5th floor of the building.

The 16S rDNA sequence of Aeromonas intermedius consists of 1433 bases (bp), and its nucleotide sequence is shown in SEQ ID NO.1.

A method for culturing the Aeromonas intermedia, the specific steps are: inoculating the Aeromonas intermedia in a culture medium and culturing at 28°C to 37°C.

Described substratum is LB substratum, MSM substratum, the MSM substratum containing chloramphenicol (CAP), the MSM substratum containing chloramphenicol and peptone, a kind of in NBRIP substratum and potassium bacterial substratum; LB medium is preferred.

The concentration of chloramphenicol in the MSM medium containing chloramphenicol is 10 mg·L -1 or less ^; preferably 2.5-10 mg·L ^-1 ; more preferably 10 mg·L ^-1 .

The concentration of chloramphenicol in the MSM medium containing chloramphenicol and peptone is 10 mg·L -1 or less ⁽ preferably 2.5-10 mg·L ^-1 ; more preferably 10 mg·L ^-1 ), and the concentration of peptone It is 10g·L ^-1 .

The formula of the NBRIP medium is as follows: glucose 10g·L ^-1 , Ca ₃ (PO ₄ ) ₂ 5.0g·L ^-1 , MgCl ₂ 5g·L ^{-1 ,} MgSO ₄ ·7H ₂ O 0.25g·L -1 ¹ , KCl 0.2g·L ^-1 , (NH ₄ ) ₂ SO ₄ 0.1g·L ^-1 , adjust pH to 7.2.

The formula of the potassium bacterial culture medium is as follows: potassium feldspar (K ₂ O·Al ₂ O ₃ ·6SiO ₂ ) 2.5g·L ^-1 , Na ₂ HPO ₄ 0.2g·L ^-1 , MgSO ₄ ·7H ₂ O 0.02g·L ^-1 , NaCl 0.2g·L ^-1 , CaCO ₃ 5.0g·L ^-1 , CaSO ₄ ·2H ₂ O 0.1g·L ^-1 , glucose 10g·L ^-1 , pH adjusted to 6.8～ 7.0.

The temperature of the culture is preferably 28-30°C; more preferably 30°C.

The culturing time is 18-48h; preferably 18-24h.

The culturing is carried out in a shaker, and the rotation speed is 125-150 r·min ^-1 .

An Aeromonas intermedia culture is obtained by inoculating the above Aeromonas intermedia in a culture medium and culturing at 28°C to 37°C.

The medium is preferably LB medium, one of MSM medium and peptone-containing MSM medium; more preferably LB medium.

The concentration of peptone in the MSM medium containing peptone is preferably 10 g·L ^-1 .

The application of Aeromonas intermedia in removing or reducing chloramphenicol (CAP) in the environment.

The environment includes soil environment and water body environment.

The application of the Aeromonas intermedia in removing or reducing chloramphenicol (CAP) in the environment is to add the Aeromonas intermedia to the soil and/or water environment containing chloramphenicol. The strain can tolerate chloramphenicol growth and can eliminate chloramphenicol in soil or water.

The concentration of chloramphenicol in the water body containing chloramphenicol is 10 mg·L ^-1 or less.

The removal time is more than 0.25 days; preferably more than 6 days.

Use of the Aeromonas intermedius and/or Aeromonas intermedius cultures in dissolving insoluble phosphorus and/or potassium compounds.

The application of the Aeromonas intermedia and/or the Aeromonas intermedius culture in dissolving insoluble phosphorus and/or potassium compounds is to inoculate the Aeromonas intermedia to contain insoluble phosphorus and/or potassium compounds. Or the medium of potassium compound, cultivate under the condition of 28 ℃～37 ℃, this Aeromonas intermedia can reduce the pH of the system to dissolve insoluble phosphorus and/or potassium compound; and/or the described Aeromonas intermedia The bacteria culture is added to a medium containing insoluble phosphorus and/or potassium compounds to dissolve the insoluble phosphorus and/or potassium compounds.

The insoluble phosphorus compound is preferably tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ).

Described medium containing insoluble phosphorus compound is preferably NBRIP medium, and its formula is as follows:

Glucose 10g·L ^-1 , Ca ₃ (PO ₄ ) ₂ 5.0g·L ^-1 , MgCl ₂ 5g·L ^-1 , MgSO ₄ ·7H ₂ O 0.25g·L ^-1 , KCl 0.2g·L ^-1 , (NH ₄ ) ₂ SO ₄ 0.1 g·L ⁻¹ , pH 7.2.

The insoluble potassium compound includes potassium-containing minerals; preferably potassium feldspar (K ₂ O·Al ₂ O ₃ ·6SiO ₂ ).

The medium containing insoluble potassium compounds is preferably potassium bacteria medium, and its formula is as follows: potassium feldspar 2.5g·L ^-1 , Na ₂ HPO ₄ 0.2g·L ^-1 , MgSO ₄ 7H ₂ O 0.02 g·L ^-1 , NaCl 0.2g·L ^-1 , CaCO ₃ 5.0g·L ^-1 , CaSO ₄ ·2H ₂ O 0.1g·L ^-1 , glucose 10g·L ^-1 , pH adjusted to 6.8-7.0.

The inoculation amount of the Aeromonas intermedia is 1-5% by volume; preferably 2% by volume.

The temperature of the culture is preferably 28-30°C.

The culturing time is more than 18h; preferably more than 6d.

Use of the Aeromonas intermedius and/or Aeromonas intermedius cultures in 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 potassium in the soil, in order to add the Aeromonas intermedia into the soil, the Aeromonas intermedia bacteria capable of lowering the pH of the system, thereby dissolving insoluble phosphorus and/or potassium compounds in the soil to increase the available phosphorus and/or potassium content in the soil (thereby promoting plant growth); and/or the Aeromonas intermedia culture Adding to a medium containing insoluble phosphorus and/or potassium compounds dissolves the insoluble phosphorus and/or potassium compounds to increase the available phosphorus and/or potassium content in the soil.

The insoluble phosphorus compound is preferably tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ).

The insoluble potassium compound includes potassium-containing minerals; preferably potassium feldspar (K ₂ O·Al ₂ O ₃ ·6SiO ₂ ).

A biological bacterial agent for removing or reducing chloramphenicol in the environment contains the above Aeromonas intermedia.

The environment includes soil environment and water body environment.

A biological bacterial agent for dissolving insoluble phosphorus and/or potassium compounds, comprising the above Aeromonas intermedia.

Aeromonas intermedius SZW3 in the present invention grows on LB plate for 18-20 hours, and the plaque is round, moist and shiny, and the middle bulge is beige. It can grow well in the inorganic salt liquid medium containing 10g·L ^-1 peptone with a concentration of 10mg·L ^-1 chloramphenicol.

Compared with the prior art, the present invention has the following advantages and effects:

(1) Aeromonas intermedius SZW3 in the present invention can remove chloramphenicol through co-metabolism, and the strain SZW3 is inoculated into an inorganic chloramphenicol containing 10 g·L ^-1 peptone with a concentration of 10 mg·L ^-1 of chloramphenicol. In saline liquid medium, 30℃, 150r·min ^-1 shaking culture for 0, 0.25, 0.5, 1, 2, 3, 4 and 6 days (d), the removal efficiency of chloramphenicol was 0, 7.88, 20.76, 38.06, respectively , 55.86, 59.56, 64.01 and 64.28%, indicating that the strain SZW3 has the ability to tolerate chloramphenicol growth and remove chloramphenicol, and can be applied to the removal of low concentrations of chloramphenicol in soil and water to remove or reduce chloramphenicol residues in the environment.

(2) Aeromonas intermedia SZW3 in the present invention can reduce the pH (secreting organic acids) in the system, thereby dissolving insoluble phosphorus and potassium substances, and inoculating the strain SZW3 in NBRIP or potassium-solubilizing bacteria medium and shaking After culturing for 6 days, the content of available phosphorus and potassium in the solution was measured. It can be seen that the content of available phosphorus and potassium in the SZW3 solution with bacteria was 238.62 and 7 mg·L ^-1 , while the contents of phosphorus and potassium in the blank solution without bacteria were 4.6 and 4.7. mg·L ^-1 ; the amount of dissolved phosphorus was significantly higher than that of the control without the addition of bacteria, while the content of dissolved potassium was lower, indicating that the strain SZW3 could dissolve insoluble phosphorus and potassium compounds, and had the potential to increase the content of available phosphorus and potassium in the soil. Can be used to promote plant growth.

Description of drawings

Figure 1 is a diagram of the colony morphology of strain SZW3 (Petri dish diameter 9 cm).

Figure 2 is a phylogenetic tree diagram of strain SZW3.

Figure 3 is a graph showing the degradation kinetics of chloramphenicol by strain SZW3 in MSM medium containing 10 g·L ^-1 peptone.

Figure 4 is a graph showing the degradation rate of chloramphenicol, strain growth and pH changes of strain SZW3 in MSM medium containing 10 g·L ^-1 peptone.

Fig. 5 is a graph showing the quantitative determination result of soluble potassium dissolved in the strain SZW3; wherein, A is the quantitative determination result of available phosphorus (phosphorus); B is the quantitative determination result of soluble potassium (potassium).

Detailed ways

The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

Example 1 Screening, isolation, purification and identification of strain SZW3

1. Screening method of chloramphenicol degrading strain SZW3

1. Material preparation

Strain screening sample source: An earthworm was excavated from a dry canal next to a pig farm in Longping Town, Lianzhou City, Qingyuan City, Guangdong Province. The earthworm and surrounding soil samples were sealed in a sampling bag, and brought back to the laboratory at 4°C. content.

LB medium: 10.0 g of peptone, 5.0 g of yeast extract powder, 10.0 g of NaCl, pH 7.0-7.2, dilute to 1 L with distilled water, and add 18.0 g of agar powder to the solid medium. Sterilize at 121°C for 15min.

Inorganic salt medium (MSM): 5 mL 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 in MgSO ₄ solution (MgSO ₄ ·7H ₂ O 46.125g·L ^-1 ) ^, 1.0mL 0.25g·L -1 ¹ of FeCl ₃ solution (FeCl ₃ ·6H ₂ O 0.42g·L ^-1 ), 1.0mL of 36.4g·L ^-1 of CaCl ₂ solution (CaCl ₂ ·2H ₂ O 48.22g·L ^-1 ), 1.0mL trace Elemental solution (containing 39.9 mg·L ^-1 MnSO ₄ ·H ₂ O, 42.8 mg·L ^-1 ZnSO ₄ ·H ₂ O, 34.7 mg·L ^-1 (NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O) Mix well, adjust pH to 7.0-7.2, dilute to 1L with pure water, and sterilize at 121°C for 15min.

MSM medium containing 10 g·L ^-1 peptone: Add 10.0 g of tryptone to the above MSM medium solution, adjust the pH to 7.0-7.2, make up to 1 L with pure water, and sterilize at 121 °C for 15 min.

2. Experimental instruments and equipment

Vertical pressure steam sterilizer (BL-50A, Shanghai Jingke Industrial Co., Ltd.), portable pH meter (PHB-4, Shanghai Precision Science Co., Ltd.), centrifuge (Centrifuge 5810R), electric oven (DGG-9070A, Shanghai Senxin Experimental Instrument Co., Ltd.), digital constant temperature water bath (HH series, Changzhou Guoyu Instrument Manufacturing Co., Ltd.), refrigerator (RCD-205AG7, Hisense Electric), biochemical incubator (PYX-208S-A, Keli Instrument) , ultra-clean workbench (SW-CJ-1F, Sujing Antai Air Technology Co., Ltd.), vortex mixer (XW-80A, Shanghai Jingke Industrial Co., Ltd.), MyCycler PCR (BIO-RAD, USA), electrophoresis instrument (DYY-6C, Beijing Liuyi Instrument Factory), Nano Drop nucleic acid and protein quantitative detector (Thermo, Germany), gel imaging system (BIO-RAD, USA), floor-standing constant temperature oscillator (HZQ-211C).

3. Enrichment screening and isolation and purification of chloramphenicol-degrading strains

(1) Isolation and purification of strains

An earthworm was excavated from a dry canal next to a pig farm in Longping Town, Lianzhou City, Qingyuan City, Guangdong Province. Weigh 0.2 g of the content and add it to 10 mL of MSM medium containing 0.5 mg·L ^-1 CAP (chloramphenicol) and 10 g·L ^-1 peptone for acclimatization and culture. The inoculum amount was transferred to MSM medium containing 1 mg·L ^-1 CAP ^for acclimatization, and then 2% (v/v) was transferred to CAP concentrations of 2.5, 5 and 10 mg·L ^-1 in turn ^{. 1} CAP was acclimated in MSM medium containing 10 g·L ^-1 peptone for 3 days. After domestication and screening, the bacterial liquid was diluted to 10 ⁷ times with medium, 0.2 mL of the diluted solution was spread on LB solid medium, and cultured at 30°C until colonies formed; then single colonies that grew out were picked out, separated and purified.

(2) strain screening

The strains separated and purified in step (1) are inoculated into LB liquid medium for expanded culture for 20-24 hours. After activation, it was added to MSM medium containing 10 mg·L ^-1 CAP and 10 g·L ^-1 peptone at a ratio of 2% (v/v), cultured at 30 °C for 150 r·min ^-1 in the dark, and the bacteria were collected after 6 days. solution to measure the CAP concentration. The determination method is as follows: the bacterial solution is centrifuged at 8000 r·min ^-1 for 1 min, the supernatant is collected and passed through a 0.22 μm organic filter membrane, the filtrate is determined by high performance liquid chromatography (HPLC), and the strains with CAP degrading ability are obtained by screening. The HPLC conditions are: the liquid chromatography column is CNW C18-WP (4.6×250mm, 5μm), the A phase is water, the B phase is methanol, V(A):V(B)=40:60, and the flow rate is 0.8mL· min ^-1 , the column temperature is 30℃; the injection volume is 20μL; the detection wavelength of the UV detector is 278nm. A strain capable of degrading CAP was obtained by screening (after the CAP concentration was determined by HPLC, the CAP concentration decreased significantly, and the concentration of the blank control group (CK) was basically unchanged, so as to determine whether the strain had the ability to degrade), and named the strain SZW3.

2. Observation of the morphological characteristics of the colony

Strain SZW3 grows faster on LB medium and can grow at 30～37℃. The plaque is round, moist and shiny, with a beige-yellow bulge in the middle. It grows well in peptone containing 10 g·L ^-1 at a CAP concentration of 10 mg·L ^-1 (Figure 1).

2.16S rDNA amplification

Using the total DNA of the extracted strain SZW3 as a template, the bacterial 16S rDNA universal primer was used for amplification, the forward primer was 27f: 5'-AGAGTTTGATCCTGGCTCAG-3', and the reverse primer was 1492r: 5'-GGTTACCTTGTTACGACTT-3' (Stackebrandt et al., 1991) amplified the 16S rDNA gene sequence.

The total PCR reaction system was 25 μL: 2 μL of upstream and downstream primers, 0.5 μL of template DNA, 12.5 μL of 2×Taq PCR MasterMix, and sterilized ultrapure water to a total volume of 25 μL.

The PCR reaction program was as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 50 s; annealing at 56 °C for 50 s; extension at 72 °C for 50 s, 35 cycles; and final extension at 72 °C for 5 min. The PCR products were detected by 1.0% agarose gel electrophoresis (DL2000Marker was selected). Observed under the gel imaging system, a clear band appeared in the middle range of Marker 1000bp and 2000bp bands.

3. Determination of 16S rDNA sequence

The product after PCR amplification was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. (Guangzhou Branch) for sequencing, and the 16S rDNA gene sequence of the obtained strain was as follows (SEQ ID NO.1):

GGGACGACTACCTGCAGTCGAGCGGCAGCGGGAAAGTAGCTTGCTACTTTTGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCAGTCGAGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCGATTGGATATGCCCAGGTGGGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGATGCCTAATACGTATCAGCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAAAACTGTCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGCTGTGTCCTTGAGACGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCTGGAATCCTGCAGAGATGCGGGAGTGCCTTCGGGAATCAGAA CACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGCTGCAAGCTAGCGATAGTGAGCGAATCCCAAAAAGCGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAAATCAGAATGTTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGGTAT。

The above sequence consists of 1433 bases (bp).

The obtained 16S rDNA gene sequence was submitted to the National Center for Biological Information (NCBI) website for BLAST comparison, and was compared with the 16S rDNA gene of the relevant type strain in the LPSN database (http://www.bacterio.net/index.html). Source alignment analysis, download the sequence of the type strain with higher homology, and perform BLAST alignment and homology analysis on the amplified product sequence on the website of the National Center for Bioinformatics (NCBI), using Mega 6.0 software, Neighbour- Joining method to construct phylogenetic tree. By comparing the 16S rDNA sequences, it was found that the strain SZW3 had 99.09% homology with Aeromonas media (Fig. 2).

4. Strain SZW3 was identified as a new functional strain

According to the morphological characteristics of the strain SZW3 colony and the identification results of molecular biology, the strain SZW3 was named as Aeromonas media SZW3. The strain has been preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), the preservation number is GDMCC NO: 61503, the strain was deposited on February 5, 2021, and the address of the preservation unit is Building 59, No. 100, Xianlie Middle Road, Guangzhou City. building. The Aeromonas media has the function of removing chloramphenicol. Therefore, Aeromonas media SZW3 is a new strain with reduced mycin in solution.

Example 2 Bacterial strain SZW3 removes chloramphenicol test

After streaking the strain SZW3 and culturing it on LB plate at 30°C for 20h, pick a single colony and inoculate it in LB liquid medium, cultivate it in a shaker at 30°C and 150rpm for 20h, and then inoculate it at a ratio of 2% (v/v). Chloramphenicol concentration was 10mg·L ^-1 in inorganic salt liquid medium containing 10g·L ^-1 peptone, 30℃, 150r·min ^-1 shaking culture 0, 0.25, 0.5, 1, 2, 3, 4 and On day 6 (d), with no addition of strain SZW3 as blank control (CK), three replicate experiments were set. The removal efficiencies of chloramphenicol were 0, 7.88, 20.76, 38.06, 55.86, 59.56, 64.01 and 64.28%, respectively.

The degradation kinetics of chloramphenicol by strain SZW3 in MSM medium containing 10 g·L ^-1 peptone is shown in Figure 3, and the degradation rate of chloramphenicol as well as strain growth and pH changes are shown in Figure 4: Growth of SZW3 Showing an "S-shaped" curve, the maximum bacterial concentration was OD600=1.18 at 0.5 days (d); and the pH of the solution gradually increased to 8.7, and was maintained at pH 8.5-8.7. The increase in pH is because SZW3 utilizes peptone ( When it grows as nutrients, it will produce a large amount of amine compounds, which will increase the pH of the solution. The results indicated that the strain SZW3 had the ability to tolerate chloramphenicol growth and remove chloramphenicol, and could be applied to the removal of chloramphenicol from soil and water.

Qualitative test of dissolving phosphorus and potassium of embodiment 3 strain SZW3

Strain phosphate/potassium solubilizing bacteria (phosphate/potassium solubilizing bacteria, meaning converting insoluble phosphorus and potassium into soluble phosphorus and potassium) (specific references: Rawat, P., Das, S., Shankhdhar, D., et al., Phosphate-solubilizing microorganisms: Mechanism and their role in phosphate solubilization and uptake[J]. Journal of Soil Science and Plant Nutrition. 2020, https://doi.org/10.1007/s42729- 020-00342-7.) Functional test:

Strain SZW3 was inoculated into LB liquid medium, cultured at 125-150r·min ^-1 and shaken at 28-30°C for 18-24h, and then 2% (v/v) was added to NBRIP or potassium-solubilizing bacterial liquid medium , set up 3 repeated experiments, placed in 30 ℃, 150r·min ^-1 shaker for 6 days, and measured the content of available phosphorus and potassium in the solution. in,

NBRIP medium formula: glucose 10g·L ^-1 ; Ca ₃ (PO ₄ ) ₂ 5.0g·L ^-1 ; MgCl ₂ 5g·L ^-1 ; MgSO ₄ ·7H ₂ O 0.25g·L ^-1 ; KCl 0.2g · L ⁻¹ ; (NH ₄ ) ₂ SO ₄ 0.1 g·L ⁻¹ ; pH 7.2;

Potassium bacteria medium: potassium feldspar (K ₂ O·Al ₂ O ₃ ·6SiO ₂ ) 2.5g·L ^-1 , Na ₂ HPO ₄ 0.2g·L ^{-1 ,} MgSO ₄ ·7H ₂ O 0.02g·L -1 ¹ , NaCl 0.2g·L ^-1 , CaCO ₃ 5.0g·L ^-1 , CaSO ₄ ·2H ₂ O 0.1g·L ^-1 , glucose 10g·L ^-1 , pH 6.8～7.0.

The results are shown in Figure 5: it can be seen that the strain ^SZW3 can reduce the pH in the system, thereby dissolving insoluble phosphorus and potassium substances. The contents of phosphorus and potassium in the bacteria-added solution were 4.6 and 4.7 mg·L ^-1 , and the dissolved phosphorus content was significantly higher than that of the control without bacteria, while the potassium-solubilized content was lower. It shows that the strain SZW3 can dissolve insoluble phosphorus and potassium compounds, and has the potential to increase soil available phosphorus and potassium content, and can be used in promoting plant growth.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> A strain of Aeromonas intermedius and its application in removing chloramphenicol and dissolving phosphorus and potassium

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1433

<212> DNA

<213> Aeromonas media

<220>

<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 acggggggccc 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

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer is 27f

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse primer is 1492r

<400> 3

ggttaccttg ttacgactt 19

Claims (10)

1. a strain of Aeromonas intermedius, characterized in that: the name is Aeromonas intermedia ( Aeromonas media ) SZW3, the preservation number is GDMCC NO: 61503, and the bacterial strain was preserved in Xianlie Middle Road, Guangzhou City on February 5, 2021 Guangdong Provincial Microbial Culture Collection Center on the 5th floor of Building 59, No. 100 Compound. 2 . A method for culturing Aeromonas intermedia according to claim 1 , wherein the specific steps are: inoculating Aeromonas intermedia in a culture medium, and culturing at 28° C. to 37° C. 3 . 3. method according to claim 2, is characterized in that: Described substratum is a kind of in LB substratum, MSM substratum, MSM substratum containing chloramphenicol, MSM substratum containing chloramphenicol and peptone, NBRIP substratum and potassium bacterial substratum; The concentration of chloramphenicol in the MSM medium containing chloramphenicol is below 10 mg·L ^-1 ; The concentration of chloramphenicol in the MSM medium containing chloramphenicol and peptone is below 10 mg·L ^-1 , and the concentration of peptone is 10 g·L ^-1 ; The formula of the NBRIP medium is as follows: glucose 10 g·L ^-1 , Ca ₃ (PO ₄ ) ₂ 5.0 g·L ^-1 , MgCl ₂ 5 g·L ^-1 , MgSO ₄ 7H ₂ O 0.25 g· L ^-1 , KCl 0.2 g·L ^-1 , (NH ₄ ) ₂ SO ₄ 0.1 g·L ^-1 , adjust pH to 7.2; The formula of the potassium bacterial culture medium is as follows: potassium feldspar 2.5 g·L ^-1 , Na ₂ HPO ₄ 0.2 g·L ^-1 , MgSO ₄ ·7H ₂ O 0.02 g·L ^-1 , NaCl 0.2 g·L -1 . ^-1 , CaCO ₃ 5.0 g·L ^-1 , CaSO ₄ ·2H ₂ O 0.1 g·L ^-1 , glucose 10 g·L ^-1 , pH adjusted to 6.8～7.0; The culturing time is 18-48 h. 4. A culture of Aeromonas intermedia, characterized in that, the Aeromonas intermedia according to claim 1 is inoculated into a culture medium, and obtained by culturing at 28°C to 37°C; The medium is one of LB medium, MSM medium and peptone-containing MSM medium. 5. The application of Aeromonas intermedia according to claim 1 in removing or reducing chloramphenicol in the environment, wherein the environment includes soil environment and water body environment. 6. Use of the Aeromonas intermedius of claim 1 and/or the Aeromonas intermedius culture of claim 4 for dissolving insoluble phosphorus compounds and/or insoluble potassium compounds. 7. application according to claim 6, is characterized in that: Described insoluble phosphorus compound is tricalcium phosphate; The insoluble potassium compound is potassium feldspar. 8. Use of the Aeromonas intermedius of claim 1 and/or the Aeromonas intermedius culture of claim 4 for increasing available phosphorus and/or available potassium in soil. 9. A biological bacterial agent for removing or reducing chloramphenicol in the environment, characterized in that: containing Aeromonas intermedia according to claim 1; The environment includes soil environment and water body environment. 10. A biological inoculant for dissolving insoluble phosphorus compounds and/or insoluble potassium compounds, comprising the Aeromonas intermedia according to claim 1.

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