CN109355237B

CN109355237B - Burkholderia HQL1813 and separation and screening method and application thereof

Info

Publication number: CN109355237B
Application number: CN201811504043.8A
Authority: CN
Inventors: 喻婵; 李俊红; 刘厚权
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2021-01-15
Anticipated expiration: 2038-12-10
Also published as: CN109355237A

Abstract

The invention relates to Burkholderia HQL1813 and a separation and screening method and application thereof, belonging to the technical field of microorganisms. The Burkholderia HQL1813 disclosed by the invention is obtained by separating and screening soil of a Hainan cherry tomato planting base, is Burkholderia HQL1813, and has a preservation number as follows: CCTCC NO: m2018708, and the 16S rRNA sequence of the Burkholderia HQL1813 is shown as SEQ ID No. 1. The Burkholderia (Burkholderia) HQL1813 provided by the invention can effectively degrade methyl phosphonic acid and convert the methyl phosphonic acid into orthophosphate, so that the Burkholderia (Burkholderia) HQL can be used for degrading organophosphorus polluted waste and researching the phosphorus circulation in the biological environment.

Description

Burkholderia HQL1813 and separation and screening method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to Burkholderia, in particular to Burkholderia HQL1813, a separation and screening method and application thereof.

Background

With the development of modern science and technology, the usage amount of organic phosphorus is increased, and the conditions of water eutrophication and organic phosphorus pesticide residue in soil are increasingly serious. Especially the residue of organophosphorus pesticide in soil, which can be poisoned by ingestion, inhalation or skin absorption. Mistakenly eating food polluted by the organophosphorus pesticide (including fruits, vegetables, dairy foods, poisoned livestock and poultry aquatic products and the like); misuse of a toy or pesticide container contaminated with a pesticide; inappropriately using organophosphorus pesticide to kill mosquito, fly, louse, bedbug, cockroach, skin disease and expelling parasite; and children can be poisoned by playing near the fields sprayed with organophosphorus pesticides.

In water environment, the geochemical process of converting and releasing organic phosphorus to inorganic phosphorus is mainly influenced by two aspects of photocatalytic degradation and microbial action. Under the action of microorganisms, organic phosphorus is decomposed to form inorganic phosphate and other products, and cell substances or mineral substances are synthesized at the same time, so that a phosphorus circulation process in the water environment is formed. Part of organic phosphorus in the water body can be decomposed to generate inorganic phosphate and other products under the action of microbial intracellular/exoenzyme; the part of inorganic phosphorus and inorganic phosphorus from other sources in the water body can be directly utilized by microorganisms under the conditions of diffusion and membrane protein transportation, participate in microbial metabolism in the microorganisms, synthesize phosphorus-containing biomass or polymeric phosphorus, and are released into the water body along with the life activities of the microorganisms to participate in a new cycle.

Therefore, the applicant finds that microorganisms are important participants in the research on the cyclic migration of organic phosphorus in water, and some strains can effectively degrade the organic phosphorus into inorganic phosphorus so as to meet the needs of the strains. By researching the metabolism of the organic phosphorus, the Burkholderia HQL1813 is separated from the Hainan cherry tomato planting soil, has the capacity of degrading the organic phosphorus into inorganic phosphorus, can effectively decompose the organic phosphorus in the environment into the inorganic phosphorus and other organic matters, is released into the environment, and participates in the circulation of phosphorus in the biological environment.

Disclosure of Invention

The first purpose of the invention is to provide a Burkholderia HQL1813, the second purpose of the invention is to provide the separation and screening method of the Burkholderia HQL1813, and the third purpose of the invention is to provide the application of the Burkholderia HQL1813, which can be used for degrading organic phosphorus.

In order to achieve the first object of the present invention, the present invention adopts the following technical solutions: burkholderia HQL1813, which is classified and named: burkholderia (Burkholderia) HQL1813, deposited in the chinese collection of type cultures (CCTCC), address: the preservation number of the Wuhan university in Wuhan, China is as follows: CCTCC NO: m2018708, date of deposit: 24 days 10 and 2018; the 16S rRNA sequence of the Burkholderia HQL1813 is shown as SEQ ID No. 1.

The Burkholderia HQL1813 is obtained by separating and screening soil in a Hainan cherry tomato planting base.

The Burkholderia (Burkholderia) HQL1813 disclosed by the invention also has the following properties:

1. microbiological characteristics

The Burkholderia HQL1813 is characterized in that a single bacterial colony is light yellow after streak culture on an inorganic salt solid plate, the bacterial colony is circular, and the morphology of the bacterial colony is shown in figure 1; in addition, the bacteria liquid is diluted and then subjected to gram staining experiment to find that the bacteria are stained to be purple red, and the bacteria are gram-negative bacteria and are rod-shaped, as shown in figure 2.

In order to achieve another object of the present invention, the present invention provides the method for separating and screening the burkholderia HQL1813, comprising the steps of:

collecting soil of a Hainan cherry tomato culture base, adding sterile water, uniformly dispersing by shaking in a shaking table at 28 ℃, standing for layering, taking supernatant, and continuously shaking in the shaking table at 28 ℃ for 3-5 days; then inoculating the culture medium into a new sterilization enrichment culture medium for culturing for 2-4 d; diluting the enriched culture solution with sterile water, coating the diluted culture solution on the surface of an inorganic salt solid plate containing methylphosphoric acid, inoculating the diluted culture solution on the surface of a new inorganic salt solid plate containing methylphosphoric acid after bacterial colonies grow out, and carrying out streak separation until single bacterial colonies are obtained; and finally, inoculating the single grown colony to an inorganic salt liquid culture medium containing methyl phosphate, carrying out shake culture on a shaker at 28 ℃, and screening a strain with better growth according to the growth condition of the colony to obtain the Burkholderia HQL 1813.

Further, the concentration of the methyl phosphate in the inorganic salt solid plate or the inorganic salt liquid culture medium in the above technical scheme is preferably 5 mmol/L.

In order to achieve the third object of the invention, the invention also provides the application of the Burkholderia HQL 1813.

The invention relates to application of Burkholderia HQL1813 in degradation of organic phosphorus pollutants.

Further, in the above technical solution, the organophosphorus contaminant is preferably methyl phosphonic acid.

An organophosphorus degrading agent, wherein the degrading agent comprises the Burkholderia HQL 1813.

An organophosphorus degrading pesticide, which comprises the Burkholderia HQL 1813.

A flame retardant comprising the burkholderia HQL1813 of the present invention described above.

The Burkholderia HQL1813 disclosed by the invention is applied to migration and transformation of phosphorus in a research environment and a biogeochemical environment.

The Burkholderia HQL1813 disclosed by the invention is applied to research on inorganic pyrophosphatase (PPase).

Compared with the prior art, the Burkholderia HQL1813, the separation and screening method and the application thereof have the following beneficial effects:

(1) the Burkholderia HQL1813 is gram-negative bacteria, has the capability of efficiently degrading organophosphorus, can be used for degrading organophosphorus pesticide residues in soil or water, reduces the toxicity and ecological hazards of organophosphorus in the soil or water, finally realizes effective treatment of organophosphorus pollution and can also prevent water eutrophication.

(2) The methyl phosphonic acid is considered to be a possible source for generating methane in the ocean, and the invention can effectively degrade the methyl phosphonic acid in the ocean, convert the methyl phosphonic acid into orthophosphate and reduce the harm of methane to fishes and water bodies.

(3) The Burkholderia HQL1813 can be used for researching migration and conversion of phosphorus in the environment and the biogeochemical environment, is a strain which is environment-friendly and has good application prospect, and has important significance for the development of a technology for degrading organic phosphorus pollution by microorganisms.

(4) Since the Burkholderia HQL1813 of the invention contains inorganic pyrophosphatase, the Burkholderia of the invention can also be used for researching the effect of the expression of the inorganic pyrophosphatase (PPase) on the degradation of methyl phosphonic acid by strains.

Drawings

FIG. 1 is a morphological diagram of a single colony of Burkholderia HQL1813 of example 1 of the present invention after streaking culture on an inorganic salt solid plate;

FIG. 2 is a graph showing the results of gram staining test of Burkholderia HQL1813 according to example 1 of the present invention;

FIG. 3 is a graph showing the growth curves of Burkholderia HQL1813 inoculated in a liquid medium containing 5mmol/L of methylphosphonic acid (methylphosphonic acid) as an inorganic salt in example 2 of the present invention;

FIG. 4 is a graph showing the change in inorganic phosphorus concentration measured by phosphomolybdic blue method, in which Burkholderia HQL1813 according to example 2 of the present invention is inoculated in a liquid medium containing 5mmol/L of methylphosphonic acid;

FIG. 5 is a diagram showing the results of gel electrophoresis detection of the PCR-amplified fragment in example 3 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation mode and a specific operation process are given, but the protection scope of the invention is not limited to the following embodiment.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

Example 1 isolation screening and molecular characterization of Burkholderia HQL1813

The method comprises the following steps of separating, screening and identifying Burkholderia (Burkholderia) HQL1813 from a soil sample retrieved from a Hainan cherry tomato culture base by adopting an inorganic salt solid flat plate containing 5mmol/L of methyl phosphate, and specifically comprises the following steps:

(1) soil sample collection: soil of cherry tomato medium in Hainan province.

(2) Sample enrichment: respectively placing the collected soil samples into sterile triangular flasks, adding appropriate amount of sterile water, shaking and scattering uniformly at 28 deg.C and 250rpm/min, and standing for 30 min. And adding 5mL of the supernatant after standing into a triangular flask containing 50mL of enrichment medium, shaking for 3-5 d at 28 ℃ and 250rpm/min by using a shaking table, and then inoculating 5mL of the supernatant into a new 50mL of sterile enrichment medium for culturing for 3 d.

(3) Screening and culturing organophosphorus degrading bacteria: respectively diluting the enriched culture solution to 10 with sterile water^-4、10^-5、10^-6And (3) coating the mixture on an inorganic salt solid plate containing 5mmol/L of methyl phosphate, and after the bacteria grow out at the temperature of 28 ℃, picking the bacteria on the culture medium on a new inorganic salt solid plate containing 5mM of methyl phosphate, and streaking and separating the bacteria until a single colony is obtained. The single colonies were inoculated into an inorganic salt liquid medium containing 5mmol/L of methylphosphoric acid, and shake-cultured at 28 ℃ and 250rpm/min with a shaker. And selecting strains with better growth according to the growth condition of colonies.

(4) And (3) strain identification: a single colony on a methylphosphonate plate was picked, dissolved in 10. mu.L of sterile water, and the 16S sequence was amplified using the bacterial solution as a template and a bacterial 16S universal primer. The upstream sequence: 27F (5 '-AGA GTT TGA TCM TGG CTC AG-3') as shown in SEQ ID No. 2; and the downstream sequence: 1525R (5 '-AAG GAG GTG WTC CAR CC-3'), as shown in SEQ ID No.3, a fragment of the bacterium is amplified. The PCR system is a 50 mu L system, 2 mu L of bacterial liquid, 1.5 mu L of upstream primer, 1.5 mu L of downstream primer, (the concentration of the primer is 10 mu mol/L), 5 mu L of 10 XPCR buffer, 1 mu L of dNTPs, 2 mu L of pfu enzyme, ddH₂O37. mu.L. PCR reaction conditions of 98 ℃ for 3 min; 30s at 98 ℃, 30s at 57 ℃, 80s at 72 ℃ and 35 cycles; 5min at 72 ℃. The PCR reaction product is detected by 0.7% agarose gel electrophoresis, and sent to sequencing analysis after detection. The sequencing result of the amplified fragment is shown as SEQ ID No. 1.

The strain Burkholderia (Burkholderia) HQL1813 belongs to Burkholderia by BLAST comparison, and the homology is 99%. The 16S rRNAGEnBank accession number is MK 044746.

(5) Morphological feature observation of the strain: the bacterial strain belongs to Burkholderia, and a single bacterial colony is faint yellow after streak culture on an inorganic salt solid plate, and is circular (as shown in figure 1); after being diluted, the bacteria liquid is taken and subjected to gram staining experiment to find that the bacteria are stained into purple red, which shows that the bacteria are gram-negative bacteria and are rod-shaped (as shown in figure 2).

Example 2 ex vivo culture of Burkholderia (Burkholderia) HQL 1813.

Burkholderia (Burkholderia) HQL1813 was streaked and activated on a solid plate containing 5mmol/L of an inorganic salt of methylphosphoric acid, and a single colony on the plate was inoculated into a flask containing 50mL of a liquid medium containing 5mmol/L of an inorganic salt of methylphosphoric acid. As a control, 3 groups of the parallel experiment groups, in which the inoculum size was one percent, and a group of non-inoculated blank medium were set. The culture temperature of 4 groups of culture medium was 28 deg.C, the rotation speed of shaker was 250rpm, and the culture time was 60h, to obtain fermentation broth containing Burkholderia (Burkholderia) HQL 1813. Wherein: the formulation of inorganic salt liquid medium containing 5mmol/L of methylphosphonic acid was as follows:

NaCl 0.2078g、KCl 0.273g、(NH₄)₂SO₄ 3g、MgSO₄·7H₂dissolving O0.5340 g, methylphosphonic acid 0.4801g and glucose 10g in 800ml of water, adding water to a constant volume of 1L, and sterilizing at 108 deg.C for 20min with high pressure steam. 1mL (1 vol% of the amount added) of a filter-sterilized solution of trace elements was added before use.

5mmol/L of methylphosphoric acid (methylphosphoric acid) in solid medium and 15g/L of agar in liquid medium.

The formula of the trace element solution is as follows:

FeCl₃ 0.03g、FeSO₄·7H₂O 0.03g、MnSO₄·H₂O 0.015g、ZnSO₄ 0.014g、CoCl₂0.2g of the extract was sterilized by filtration through a 0.22 μm filter membrane and stored at 4 ℃ in the dark.

From the growth graph (FIG. 3) and the concentration change graph of inorganic phosphorus in the culture medium (FIG. 4) (note: both contrast in FIG. 3 and FIG. 4 and experiment are average values of 3 parallel experimental groups), it can be seen that Burkholderia (Burkholderia) HQL1813 in the present application can grow well in the culture medium containing organic phosphorus, and has the ability to degrade organic phosphorus. The curve is measured after 1% of inoculum in a 50mL system. The strain starts logarithmic growth after 20h, and the maximum OD value reaches 2.0. After the culture is carried out for 80 hours, the concentration of inorganic phosphorus in the culture medium reaches 95 mu mmol/L, and organic phosphorus can be effectively degraded into inorganic phosphorus. Therefore, the strain can be applied to the degradation related application of organic phosphorus, such as the application in degrading organic phosphorus pollution, the application in preparing an organic phosphorus pollution degradation agent, the application in degrading organic phosphorus pesticides and flame retardants, the application in relieving water eutrophication, the application in researching the migration and transformation of phosphorus in the environment and the application in the biogeochemical environment.

Example 3 determination of inorganic pyrophosphatase (PPase) in Burkholderia (Burkholderia) HQL 1813.

Primer F (5'-ACT TTA AGA AGG AGA TAT ACC ATG ACC TAC ACT ACC AGA CA-3') of an inorganic pyrophosphatase gene reported in the literature is used, and is shown as SEQ ID No. 4; r (5'-ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TTA AAC AGA ACC GGA GAT GAA G-3') as shown in SEQ ID No. 5; the bacterial solution of the bacterium is amplified. The PCR system is a 50-mu-L system, 2 mu-L of bacterial liquid, 1.5 mu-L of upstream primer, 1.5 mu-L of downstream primer, (the concentration of the primer is 10 mu mol/L), 5 mu-L of 10 XPCR buffer, 1 mu-L of dNTPs, 2 mu-L of pfu enzyme and 37 mu-L of ddH2O 37. PCR reaction conditions of 98 ℃ for 3 min; 30s at 98 ℃, 30s at 57 ℃, 90s at 72 ℃ and 35 cycles; 5min at 72 ℃. The PCR reaction product was detected by 0.7% agarose gel electrophoresis to obtain a single bright band, and the detection result is shown in FIG. 5, which is a band of an inorganic pyrophosphatase sequence. Therefore, the Burkholderia (Burkholderia) HQL1813 in the present application contains inorganic pyrophosphatase (PPase), and the effect of the expression of inorganic pyrophosphatase (PPase) on the degradation of methylphosphonic acid by the strain can be studied by using the Burkholderia of the present invention.

Sequence listing

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gtgcatccgc tgcttaccat gcaagtcgaa cggcagcacg ggtgcttgca cctggtggcg 60

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gcaggcggtg atgtaagacc gatgtgaaat ccccgggctt aacctgggaa ctgcattggt 600

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Claims

1. A Burkholderia HQL1813, which is characterized in that: the classification is named as: burkholderia (B) (BBurkholderia) HQL1813, already deposited in the chinese culture collection center (CCTCC), address: the preservation number of the Wuhan university in Wuhan, China is as follows: CCTCC NO: m2018708, date of deposit: 24 days 10 and 2018; the 16S rRNA sequence of the Burkholderia HQL1813 is shown as SEQ ID No. 1.

2. The use of Burkholderia HQL1813 as claimed in claim 1 for the degradation of organophosphorus contaminants, wherein: the organophosphorus contaminant is methylphosphonic acid.

3. An organophosphorus degrading agent, which is characterized in that: the degrading agent comprises the Burkholderia HQL1813 of claim 1.

4. An organophosphorus degradation pesticide, which is characterized in that: the pesticide comprises the Burkholderia HQL1813 of claim 1.