CN111662839B

CN111662839B - Bacillus belgii for degrading tetracycline, magnetic immobilized microorganism composite material and application

Info

Publication number: CN111662839B
Application number: CN202010482942.3A
Authority: CN
Inventors: 徐会娟; 李永涛; 郭桂芳; 吴祥瑶; 张玉龙; 龚贝妮; 谭泽文; 刘永林
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2022-03-25
Anticipated expiration: 2040-06-01
Also published as: CN111662839A

Abstract

The invention discloses a Bacillus belgii strain for degrading tetracycline, a magnetic immobilized microorganism composite material and application. The strain is named as Bacillus velezensis N32, is preserved in Guangdong province microorganism strain preservation center of No. 59 building 5 of No. 100 college of Xieli Zhongluo, Guangzhou city in 11 and 6 days of 2019, and has the preservation number of GDMCC NO: 60887. the strain has the ability of degrading tetracycline, can be applied to the degradation of tetracycline in soil and water, thereby removing or reducing the residue of tetracycline in the environment and achieving the effect of treating environmental pollution. The magnetic immobilized microorganism composite material prepared by the strain can improve the tetracycline degradation effect of the Bacillus belgii N32, has more thorough tetracycline removal effect, is easy to recover, and is more convenient and environment-friendly in actual use.

Description

Bacillus belgii for degrading tetracycline, magnetic immobilized microorganism composite material and application

Technical Field

The invention belongs to the technical field of microorganism and organic pollution microorganism remediation and removal of environmental organic pollutants such as water, soil and the like, and particularly relates to a bacillus beiLeisi for degrading tetracycline, a magnetic immobilized microorganism composite material and application thereof.

Background

Antibiotics are indispensable medicines in agricultural production and breeding industries and are also one of the human medical medicines with the largest global use amount. However, long-term use inevitably results in drug residues, causing serious environmental pollution problems. China is the biggest world antibiotic producing country and the largest world using country, and the total usage amount of 36 antibiotics in China in 2013 is estimated to reach 92700 tons, about 53800 tons of antibiotics enter the environment, and serious environmental pollution is caused, wherein the pollution is the most serious by tetracycline antibiotics. The tetracycline antibiotics are relatively high in solubility in water, cannot be completely absorbed after entering animals, are mostly discharged out of bodies in the form of raw medicines, can exist in sewage and soil applied with organic fertilizers for a long time, and are continuously accumulated in the environment, so that serious harm is caused to the environment.

At present, the treatment methods for antibiotic pollution are roughly divided into three categories, namely physical methods, chemical methods and biological methods. The physical method utilizes the technologies of filtration, adsorption, sedimentation, aeration and the like to achieve the effect of purifying water quality, but mainly aims at the wastewater polluted by antibiotics, and has limitation on the application range; the chemical method is characterized in that strong oxidizing oxides such as ozone, hydrogen peroxide and potassium permanganate are added to oxidize and mineralize the antibiotics, so that the purpose of degrading the antibiotics is achieved, but the chemical method is difficult to practically apply due to the fact that the cost is relatively high, the treatment effect is not thorough, and the possibility of secondary degradation products to pollute the environment exists. In recent years, bioremediation techniques have been developed to hope for environmental pollution control, and biodegradation is a process of decomposing antibiotics into small molecular compounds without toxic effects by using various plants and microorganisms having a degrading effect. The method can change the structure and physicochemical properties of the antibiotic, has low cost and strong specificity, does not cause secondary pollution, and has become a current research hotspot. In the early 80 s, people utilized immobilized microorganism technology to the water treatment direction, and the technology is the hot spot of research in various countries. The technology can not only improve the concentration of microbial cells in a limited area, but also enable the microorganisms to keep higher activity, but the effectiveness of the microorganisms in the immobilization process is difficult to guarantee, and the effective recovery of the immobilized material after use is inconvenient, so that the application range is not wide. Therefore, the application of suitable methods or materials for microorganism immobilization to achieve the effective degradation of tetracycline by microorganisms and the magnetic recovery of the immobilized materials thereof in the environment is a big problem that must be overcome at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a strain of Bacillus belgii for degrading tetracycline.

Another object of the present invention is to provide the use of said Bacillus belgii for the degradation of tetracyclines.

It is still another object of the present invention to provide a magnetic immobilized microorganism composite obtained by using the above tetracycline-degrading Bacillus belgii.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a strain of Bacillus belgii for degrading tetracycline is named as Bacillus belgii (Bacillus velezensis) N32, is preserved in Guangdong province microbial strain preservation center of No. 59 building and No. 5 building of Miehuo No. 100 college of Middleyao, Guangzhou, 11 and 6 days in 2019, and has the preservation number of GDMCC NO: 60887.

the 16S rDNA gene sequence of the bacillus beleisi for degrading tetracycline consists of 1450 basic groups (bp), and the nucleotide sequence is shown as follows (also shown as SEQ ID NO. 1):

GGCTGGGGGTGCTATAATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGACAGAAC。

the bacillus belgii for degrading tetracycline has the following taxonomic characteristics: the growth morphology on LB solid medium plates was: the bacterial plaque is round, the edge is semitransparent, and the color is beige.

The Bacillus belgii for degrading tetracycline grows well in a culture medium with the tetracycline concentration of 10 mg/L.

The culture medium is preferably an inorganic salt culture medium containing 10g/L of peptone.

The inorganic salt culture medium is MSM culture medium.

The culture method of the bacillus belgii for degrading tetracycline preferably comprises the following steps: inoculating the Bacillus belgii for degrading tetracycline into a culture medium, and culturing.

The culture medium is preferably LB culture medium.

The culture temperature is preferably 30-37 ℃; more preferably 30 deg.c.

The culture time is preferably 12-24 h; more preferably 14 to 20 hours.

The Bacillus belgii for degrading tetracycline is applied to degrading tetracycline.

The application is that the Bacillus belgii for degrading tetracycline is directly placed in an environment containing tetracycline for culture; or further processing the Bacillus belgii for degrading tetracycline to obtain a product, and culturing in an environment containing tetracycline.

The environment includes a body of water and soil.

The article is preferably a magnetically immobilized microbial composite.

A magnetic immobilized microorganism composite material contains the Bacillus belgii for degrading tetracycline.

The magnetic immobilized microorganism composite material is preferably prepared by the following steps: in a sterile environment, mixing the liquid of the Bacillus belgii for degrading tetracycline and a sterile sodium alginate solution, stirring uniformly, and adding sterile nano Fe₃S₄Vortex and mix evenly to obtain solution A; then add solution A to sterile CaCl₂And in the solution, crosslinking, fixing and cleaning to obtain the magnetic immobilized microorganism composite material.

The sterile environment is preferably an ultra-clean workbench after sterilization treatment.

The concentration of the sodium alginate solution is preferably 0.08-0.12 g/mL; more preferably 0.1 g/mL.

The pH value of the sodium alginate solution is 7 +/-0.02.

The sodium alginate solution is preferably prepared by the following steps: dissolving sodium alginate in water, and adjusting pH to 7 + -0.02 to obtain sodium alginate solution.

The CaCl is₂The concentration of (b) is preferably 0.07-0.09 g/mL; more preferably 0.08 g/mL.

The CaCl is₂The pH of the solution was 7 ± 0.02.

The CaCl is₂The solution is preferably prepared by: taking CaCl₂Adding into water, dissolving in water, adjusting pH to 7 + -0.02 to obtain CaCl₂And (3) solution.

The pH is preferably adjusted by an acid; more preferably by hydrochloric acid solution; most preferably, the concentration is adjusted by a hydrochloric acid solution with 6 mol/L.

The dosage of each raw material in the magnetic immobilized microorganism composite material is preferably selected from bacterial liquid, sodium alginate solution and Fe₃S₄20 mL: 20mL of: 0.4 g.

The CaCl is₂The solution is used in excess, preferably in an amount of at least 2 times, preferably at least 5 times the volume of solution a.

The sterilization condition is preferably sterilization at 121 ℃ for 20 min.

The solution A is preferably added dropwise; more preferably, it is dropped dropwise with a pipette.

The time for crosslinking and fixing is preferably 2-5 min; more preferably for 2 min.

The washing is preferably performed with sterile water.

The number of cleaning is preferably 2-4; more preferably 3 times.

The magnetic immobilized microorganism composite material is preferably a magnetic immobilized microorganism composite material which has magnetism, a spherical shape and beige color.

The magnetic immobilized microorganism composite material is applied to degradation of tetracycline; preferably comprising the steps of: and (2) placing the magnetic immobilized microorganism composite material in an environment containing tetracycline for tetracycline removal, and recovering the magnetic immobilized microorganism composite material by magnetic force after the treatment is finished.

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

1. the invention separates to obtain Bacillus velezensis (Bacillus velezensis) N32 for degrading tetracycline for the first time, the strain can degrade the tetracycline through a co-metabolism mode, the strain is inoculated into MSM culture medium containing 10g/L peptone and the concentration of the tetracycline is 10mg/L for culture, the concentration of the tetracycline in the solution is reduced to 5.29mg/L on the third day, and the concentration of the tetracycline in the solution without the strain is 7.30 mg/L; on the sixth day, the concentration of tetracycline in the solution inoculated with the strain was 3.16mg/L, while the concentration of tetracycline in the solution not inoculated with the strain was 5.71 mg/L; the Bacillus velezensis N32 has the ability of degrading tetracycline, and can be applied to the degradation of tetracycline in soil and water, thereby removing or reducing the residue of tetracycline in the environment and achieving the effect of treating environmental pollution.

2. According to the invention, the magnetic immobilized microorganism composite material is prepared by immobilizing Bacillus velezensis (Bacillus velezensis) N32, so that the tetracycline degradation effect of the Bacillus velezensis (Bacillus velezensis) N32 can be improved, the tetracycline removal effect is more thorough, the tetracycline residue in the environment can be removed more stably and efficiently, and the effect of treating environmental pollution can be achieved; meanwhile, the preparation method of the magnetic immobilized microorganism composite material is simple, the practical application and the operation are convenient, no secondary pollution is caused, and the magnetic immobilized microorganism composite material is more environment-friendly.

Drawings

FIG. 1 is a colony morphology of strain N32.

FIG. 2 is a phylogenetic tree of 16SrDNA of strain N32.

FIG. 3 is a graph showing the results of the validation of the effect of strain N32 on tetracycline degradation.

FIG. 4 is a schematic representation of a magnetically immobilized microbial composite based on strain N32.

FIG. 5 is a graph showing the results of the validation of the effect of the magnetically immobilized microbial composite based on strain N32 on tetracycline degradation.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 screening, isolation, purification and characterization of Bacillus belgii for tetracycline degradation

Screening of Bacillus beleisi for degrading tetracycline

1. Material preparation

Strain screening soil: collected from a grassland in an inner Mongolia autonomous region, the soil sample is sealed by a valve bag and is brought back to a refrigerator in a laboratory for cold storage and standby at the temperature of 4 ℃.

LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 7.0-7.2 of pHs, and sterilizing at 121 ℃ for 20min after constant volume of distilled water is up to 1L.

LB solid medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 18.0g of agar powder, pH 7.0-7.2, constant volume of distilled water to 1L, and sterilizing at 121 ℃ for 20 min.

Inorganic salt medium (MSM): 5mL of phosphoric acid buffer solution (KH)₂PO₄ 8.5g/L、K₂HPO₄·H₂O 21.75g/L、Na₂HPO₄·12H₂O 33.4g/L、NH₄Cl 5.0g/L), 3.0mL MgSO 22.5g/L₄Solution (MgSO)₄·7H₂O46.125 g/L), 1.0mL of FeCl with a concentration of 0.25g/L₃Solution (FeCl)₃·6H₂O0.42 g/L), 1.0mL of CaCl with the concentration of 36.4g/L₂Solution (CaCl)₂·2H₂O48.22 g/L), 1.0mL of trace element solution (containing 39.9 mg/LMnSO)₄·H₂O、42.8mg/LZnSO₄·H₂O、34.7mg/L(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 20min at 121 ℃.

MSM medium containing 10g/L peptone: adding 10.0g of bacteriological peptone 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 20min at 121 ℃.

Screening a culture medium: adding 10g of peptone into the MSM culture medium, adding 18g of agar, adjusting the pH value to 7.0-7.2, and fixing the volume to 1L by using pure water.

Pressure culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 18.0g of agar powder, pH 7.0-7.2, constant volume of distilled water to 1L, sterilization at 121 ℃ for 20min, cooling to about 60 ℃, adding 25mL of tetracycline solution with the concentration of 1g/L and sterilization by filtration to ensure that the tetracycline concentration in the culture medium is 25mg/L (the step is carried out in an ultraclean workbench in a dark place).

2. Separation, purification and screening of tetracycline degradation bacteria

(1) Enrichment, separation and purification of the strain (the strain separation and purification processes are all carried out under the condition of keeping out of the sun)

Adding 10g of soil sample into 90mL of an inorganic salt culture medium with 20mg/L tetracycline concentration, and performing acclimation culture in a constant-temperature shaking table at 30 ℃ and 150rpm in the dark for 5 days to obtain an acclimation culture solution; then adding 10mL of domestication culture solution into 90mL of inorganic salt culture medium with tetracycline concentration of 40mg/L, and placing the medium in a constant-temperature shaking table with the temperature of 30 ℃ and the speed of 150rpm to be domesticated and cultured for 5 days in a dark place; by analogy, domestication culture is carried out in an inorganic salt culture medium with the tetracycline concentration of 60mg/L, 80mg/L and 100mg/L in sequence in a dark place (the conditions are the same as the domestication conditions in the inorganic salt culture medium with the tetracycline concentration of 20 and 40mg/L, and the difference is only that the tetracycline concentration is different), so as to obtain domestication liquid. After enrichment and domestication, adding 1mL of domestication solution into 9mL of sterile water for dilution, and continuously diluting until 10%^-70.1mL of the diluted solution was applied to a solid medium containing tetracycline at a concentration of 25mg/LLB, incubated at 30 ℃ until colonies grew, and morphological characteristics of the colonies were visually observed.

Respectively selecting colonies with different forms, inoculating the colonies onto an LB solid culture medium with tetracycline concentration of 25mg/L by adopting a plate-streaking method, and carrying out constant-temperature dark culture at 30 ℃ until single colonies grow out. Inoculating the single colony obtained by separation and purification into 20mL of LB culture medium to obtain an inoculation liquid; inoculating the inoculation liquid into MSM culture medium containing 10g/L peptone with tetracycline concentration of 10mg/L according to the proportion of 2% (v/v), simultaneously making blank control of non-inoculated strain, placing in constant temperature shaking table with 30 deg.C and 150rpm, and culturing in dark for 0, 2, 4, 6, 8 days, and measuring tetracycline residual concentration in the bacterial liquid. The determination steps are as follows: centrifuging the filtrate at 10000rpm for 1min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring tetracycline residue concentration with High Performance Liquid Chromatography (HPLC), screening to obtain strain with tetracycline degradation ability, and storing in-80 deg.C refrigerator for use.

(2) Strain mutagenesis screening

Activating the strains which are preliminarily screened at the early stage and have tetracycline degradation capability on an LB solid culture medium at 30 ℃ to enable the strains to grow lawn; and then adding 1mLLB solid culture medium into a 2mL corning tube, after the LB solid culture medium is solidified, punching a cylindrical small hole with the depth of about 0.3cm and the diameter of about 0.5cm at the center of the corning tube by using a puncher, then taking a lawn with the thickness of 0.3cm and the diameter of about 0.5cm from a flat plate after the bacterial strain is cultured for 24 hours by using the puncher, and placing the lawn in the small hole in the corning tube to prepare a lawn sample. Carrying out mutation experiments on the lawn through microgravity biology experiments, wherein the vibration conditions are as follows: temperature: 20 ℃; rotating speed: 9 revolutions per minute; the rotation mode is as follows: three-dimensional rotation; the length of the experiment was: for 72 hours. Cleaning the strain subjected to the microgravity biology experiment with sterile water to obtain a bacterial suspension, and diluting to 10%^-7Then 0.1mL of bacterial liquid is taken to be coated on a pressure culture medium and cultured for 20h at the constant temperature of 30 ℃, then a single colony is selected and inoculated into a 20mLLB culture medium and cultured for 18h at the temperature of 30 ℃ and 150rpm, and a culture solution is obtained. The culture broth was inoculated at an inoculum size of 2% (v/v) into MSM medium containing 10mg/L TC (tetracycline), 10g/L peptone, and cultured at 150rpm and 30 ℃ in the dark, and the bacterial solution was harvested after 3 and 6 days of culture. Centrifuging the bacterial liquid at 10000rpm for 1min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring the residual concentration of tetracycline in the filtrate by High Performance Liquid Chromatography (HPLC), and screening to obtain the strain with stable tetracycline degradation capability. All experimental steps to complete the microgravity biology experiments were performed protected from light. Finally, the strain with the best tetracycline degradation effect is obtained through screening and is named as the strain N32.

HPLC conditions: liquid chromatography column: CNW Athena C18-WP (4.6 mm. times.250 mm, 5 μm); mobile phase: phase A is methanol (purity is 99.9%), phase B is acetonitrile (purity is 99.9%), phase C is 0.01mol/L oxalic acid, V (A), V (B), V (C) 15:15:70, flow rate is 1mL/min, column temperature is 31 ℃; the sample volume is 20 mu L; the ultraviolet detector detects the wavelength of 355 nm.

Secondly, colony morphological characteristics and identification

1. And (3) observing colony morphological characteristics: the strain N32 is inoculated in a 20mLLB culture medium according to the inoculation amount of 2% (V/V), the strain grows faster in a shaker at 30 ℃ and 150rpm, in addition, the strain N32 is placed in a constant-temperature incubator at 37 ℃ for culture, the strain grows well after 20 hours of culture, and the fact that N32 can grow at 30-37 ℃ is known. By observing the turbidity of the LB medium solution and determining the OD600, it was found that N32 grew faster in LB medium. N32 in tetracycline concentration of 10mg/L containing 10g/L peptone MSM medium, at 30 degrees C, 150rpm shaking table in light-shielding culture, OD600 results show, N32 in tetracycline, peptone MSM medium growth well. The morphology of the strain N32 is shown in FIG. 1, and when the strain is cultured in LB solid medium for 24h, the bacterial plaque is round, the edge is translucent, and the bacterial plaque is beige.

2. Molecular biological identification of bacterial species

Taking the total DNA of the extracted strain N32 as a template, adopting a bacterial 16S rDNA gene universal primer, and taking a forward primer of 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' the 16S rDNA gene sequence was amplified.

The total PCR reaction was 25 μ L: mu.L of each of the upstream and downstream primers (primer concentration: 10. mu. mol/L), 1. mu.L of template DNA (template concentration: 75 ng/. mu.L), 12.5. mu.L of 2 XTaq PCR Master Mix, and 25. mu.L of sterilized ultrapure water.

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 1min for 20s, 34 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃.

The product obtained by PCR amplification is sent to Guangzhou Tianyihui gene technology limited company for sequencing, the 16S rDNA gene of the obtained strain N32 consists of 1450 basic groups (bp), and the gene sequence is shown as SEQ ID NO. 1.

The obtained 16S rDNA gene sequence was submitted to EZ BioCloud (https:// www.ezbiocloud.net) web page for comparison, and homology comparison analysis was performed with the 16S rDNA gene of the relevant model strain in LPSN database (http:// www.bacterio.net/index. html), and the model strain sequence with higher homology was downloaded, and phylogenetic tree was constructed by Neightbour-Joining method using Mega 6.0 software, and the results are shown in FIG. 2: strain N32 has 99.78% homology with Bacillus velezensis (Bacillus velezensis).

According to the colony morphological characteristics and molecular biological identification results of the strain N32, the strain N32 is identified as Bacillus subtilis (Bacillus velezensis) and named as Bacillus subtilis N32. The strain is stored in Guangdong province microbial culture collection center (GDMCC) in 2019 at 11/6, and the preservation number is GDMCC NO: 60887, the address of the preservation unit is No. 59 building No. 5 building of No. 100 Dazhong-Lianlu, Guangzhou city.

At present, no document reports that Bacillus beleisi has the function of degrading tetracycline at home and abroad. Therefore, Bacillus velezensis N32 is a new strain with tetracycline degradation function.

EXAMPLE 2 degradation of Tetracycline by Strain N32 in MSM Medium containing Tetracycline and peptone

The strain N32 is streaked on an LB plate and is cultured for 24 hours in the dark at the temperature of 30 ℃, a single colony is selected and inoculated in a 20mLLB liquid culture medium, and the single colony is cultured for 18 hours in the dark in a shaking table at the temperature of 30 ℃ and at the speed of 150 rpm. Inoculating to MSM medium containing 10mg/L tetracycline and 10g/L bacteriological peptone at a ratio of 2% (v/v), culturing in dark place, and sampling after 3 and 6 days; MSM medium without strain N32 was used as a Control (CK). The residual amount of tetracycline TC was measured by HPLC after filtration of the suspension (the tetracycline assay method was the same as in example 1).

The result shows that after 3 days, the tetracycline control without inoculated strain N32 is hydrolyzed, and after inoculated strain N32, the degradation rate of tetracycline is increased; after 6 days, the TC residual concentration in the control group was 5.71mg/L, and the TC residual concentration in the N32-inoculated experimental group was 3.16mg/L, which was lower than that in the control group (FIG. 3).

Example 3 preparation of magnetic immobilized microorganism composite material based on strain N32 and verification of tetracycline adsorption and degradation capability thereof

Preparation of magnetic immobilized microorganism composite material

(1) Mixing sodium alginate with ultrapure water, magnetically stirring until completely dissolved to obtain sodium alginate solution with concentration of 10% (g: mL). Adding CaCl₂Mixing with ultrapure water, magnetically stirring until completely dissolving to obtain CaCl₂Solution, concentration 8%.

(2) Adding the sodium alginate solution obtained in the step (1) and CaCl₂Adding hydrochloric acid (concentration of 6mol/L) into the solution, adjusting pH to 7, placing into autoclave, sterilizing at 121 deg.C for 20min, taking out, and cooling to room temperature.

(3) Streaking a strain N32 on an LB plate, culturing for 24h at 30 ℃ in a dark place, selecting a single colony, inoculating the single colony in 20mL of LB liquid culture medium, culturing for 18h at 30 ℃ in a shaking table at 150rpm in a dark place, adding 20mL of bacterial liquid and 20mL of sodium alginate solution into a sterile 100mL conical flask, and uniformly stirring to obtain a mixed solution A; then sterile nano Fe with the total mass of 1 percent of the mixed solution A is added₃S₄Vortex and mix evenly to make nano Fe₃S₄And dispersing uniformly to obtain a mixture B.

(4) Using a liquid-transferring gun in a super-clean workbench to suck the uniformly mixed solution in the step (3) and dropwise adding the uniformly mixed solution into excessive CaCl₂And (3) performing crosslinking fixation for 2min in a solution (the volume of which is 5 times that of the mixed solution B), and finally washing with sterile water for 3 times to obtain the magnetic immobilized microorganism composite material (figure 4).

Second, determination of tetracycline adsorption degradation capability of magnetic immobilized microorganism composite material

The 12 magnetic immobilized microorganism composite pellets were added to 20mL MSM medium containing 10mg/L TC and 10g/L peptone, and blank control experiment with direct inoculation of strain N32 (inoculum size was 2%, i.e., 0.4mL bacterial liquid was inoculated in 20mL MSM medium, the same as the inoculum size of the magnetic immobilized microorganism composite) and no addition of any substance was carried out simultaneously, and the mixture was placed in a shaker at 30 ℃ and 150rpm, and after 0 day and 6 days of incubation, the concentration of residual tetracycline in the solution was measured by High Performance Liquid Chromatography (HPLC) to obtain the degradation effect (the tetracycline measurement method was the same as in example 1).

The result shows that the tetracycline control of the non-inoculated strain N32 is hydrolyzed after 6 days, the tetracycline catalytic degradation rate is increased after the strain N32 is inoculated, the tetracycline catalytic degradation rate of the experimental group added with the magnetic immobilized microorganism composite material is higher, the tetracycline catalytic degradation rate is increased by 32.2% compared with the non-inoculated strain N32, the tetracycline catalytic degradation rate of the experimental group added with the magnetic immobilized microorganism composite material is increased by 19.7% compared with the experimental group inoculated with the strain N32, and the tetracycline removing effect of the magnetic immobilized microorganism composite material is more thorough. (FIG. 5)

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> tetracycline degradation bacillus belgii, magnetic immobilized microorganism composite material and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1450

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SEQ ID NO.1

<400> 1

ggctgggggt gctataatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggcttc 180

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960

ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1020

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1140

ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200

tgctacaatg gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc 1260

tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1320

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tatggagcca gccgccgaag 1440

gtgacagaac 1450

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 27F

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 1492R

<400> 3

ggttaccttg ttacgactt 19

Claims

1. The bacillus beleisi for degrading tetracycline is characterized in that: the name of the Bacillus belgii for degrading tetracycline is Bacillus belgii (B) ((B))Bacillus velezensis) N32, deposited at 6.11.2019 in the Guangdong province microbial strain collection center located in the No. 59 building 5 of the Mieli Zhou No. 100 college, Guangzhou city, with the collection number GDMCC NO: 60887.

2. use of the Bacillus belgii for degrading tetracycline of claim 1 in degrading tetracycline.

3. Use according to claim 2, characterized in that it comprises the following steps: directly placing the bacillus beilesensis for degrading tetracycline in an environment containing tetracycline for culturing; or further processing the Bacillus belgii for degrading tetracycline to obtain a product, and culturing in an environment containing tetracycline.

4. Use according to claim 3, characterized in that: the environment includes a body of water and soil.

5. Use according to claim 3, characterized in that: the product is a magnetic immobilized microorganism composite material.

6. A magnetic immobilized microorganism composite material, which is characterized in that: a Bacillus belgii bacterium containing the tetracycline degradation product of claim 1.

7. The magnetically immobilized microorganism composite according to claim 6, prepared by the steps of: mixing the bacterial solution of Bacillus belgii for degrading tetracycline and the sterile sodium alginate solution in the sterile environment, stirring, adding sterile nano Fe₃S₄Vortex and mix evenly to obtain solution A; then add solution A to sterile CaCl₂And in the solution, crosslinking, fixing and cleaning to obtain the magnetic immobilized microorganism composite material.

8. The magnetically immobilized microorganism composite of claim 7, wherein:

the concentration of the sodium alginate solution is 0.08-0.12 g/mL;

the pH value of the sodium alginate solution is =7 +/-0.02;

the CaCl is₂The concentration of (A) is 0.07-0.09 g/mL;

the CaCl is₂pH =7 ± 0.02;

the dosage of each raw material in the magnetic immobilized microorganism composite material is determined by bacterial liquid, sodium alginate solution and Fe₃S₄= 20 mL: 20mL of: 0.4g of proportioning;

the CaCl is₂The solution was in excess, which was 5 times the volume of solution a.

9. The magnetically immobilized microorganism composite of claim 7, wherein:

the solution A is added dropwise;

the time for crosslinking and fixing is 2-5 min;

the cleaning is performed by using sterile water.

10. Use of the magnetic immobilized microbial composite of any one of claims 6 to 9 for degrading tetracycline.