CN112608863A

CN112608863A - Pandora pandora pnomenusa S2-2 strain and application thereof

Info

Publication number: CN112608863A
Application number: CN202011521924.8A
Authority: CN
Inventors: 陈进军; 聂芳红; 陈蒙蒙; 杨俊玲; 照那木拉; 陈志宝; 林红英; 刘素青; 康丹菊; 朱棣华; 何敏
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-04-06
Anticipated expiration: 2040-12-21
Also published as: CN112608863B

Abstract

The invention discloses a Pandora Pandoraea pnomenusa S2-2 strain and application thereof, wherein the strain is deposited in Guangdong province microorganism culture collection center (GDMCC) at 11-12 th month in 2020, and the strain preservation number is GDMCC No: 61284. the research of the invention shows that the Pandoraea pnomenusa S2-2 strain has the function of efficiently degrading polychlorinated biphenyl, and the Pandoraea pnomenusa S2-2 strain has good potential and application prospect for the restoration of polychlorinated biphenyl environmental pollution as an effective and green polychlorinated biphenyl pollution degrading strain.

Description

Pandora pandora pnomenusa S2-2 strain and application thereof

Technical Field

The invention relates to the technical field of microbial degradation, and particularly relates to a Pandora Pandoraea pnomenusa S2-2 strain and application thereof.

Background

With the rapid development of industrial modernization, the natural environment is inevitably polluted by various industrial wastes, and Polychlorinated biphenyls (PCBs) are one of them. As early as 1881, German scientists Sehmid and Sehult firstly synthesized PCBs in laboratories, and in 1929 PCBs were first produced in the United states, and then the commercial and large-scale production of polychlorinated biphenyl was started in various countries, and the polychlorinated biphenyl is widely applied to industrial and agricultural production. PCBs have various excellent properties such as chemical inertness, thermal stability, low conductivity, and flame retardancy, and are highly hydrophobic and insoluble in water and various organic solvents, and are widely used in the manufacture of hydraulic oils, flame retardants, sealants, and plasticizers, and are distributed worldwide in various commercial forms. While mass production and application of PCBs are proceeding, PCBs gradually accumulate in the environment and living bodies, and thus the risk of PCBs gradually begins to emerge. In the last 30 th century, potential risks related to PCBs have been proposed, and the public safety of society is not really attracted until Japanese outbreak of rice bran oil event and American Monsanto event seriously jeopardize the public safety in 1968.

At present, the most applied methods for treating the pollution caused by degrading the polychlorinated biphenyl mainly comprise a physical repair method, a chemical repair method and a biological repair method. The physical repair technology such as adsorption, precipitation and landfill does not fundamentally eliminate PCBs, in addition, the solubility of polychlorinated biphenyl is enhanced by adding a surfactant, the polychlorinated biphenyl is further and better eluted, and chemical bonds of chemical substances are broken by high-energy electrons through the technology of micro-ion isoelectric and microwave radiation, so that the effect of instantly decomposing pollutants is achieved, but the popularization and the use of the technology are limited by expensive ion equipment. The chemical method for treating PCBs has fast reaction, and the common methods at present include an incineration method, an oxidation method, a reduction method, a photolysis catalysis method, an electrolysis method and the like, can thoroughly eliminate PCBs pollution, but have harsh reaction conditions, large investment in treatment equipment and cannot be widely applied. The method for bioremediation treatment of polychlorinated biphenyl pollution is a popular technology for environmental treatment gradually because the method is more green, environment-friendly and low in cost, plays an important role in environmental ecological function while thoroughly eliminating environmental pollution. The invention patent of China with the publication number of CN102618457B provides Rhodococcus WB-1 which has the advantages of high growth speed, simple culture method, high efficiency and stable degradation of polychlorinated biphenyl, not only can degrade polychlorinated biphenyl in a solution, but also can degrade polychlorinated biphenyl residues in soil, and the degradation efficiency is as high as 80-90%. However, there are still few engineering cases to date in which such microorganisms are used to remediate polychlorinated biphenyl contaminated sites. The fundamental reason is that the polychlorinated biphenyl degrading bacteria are not abundant enough. The types of the polychlorinated biphenyls which can be degraded by the microorganisms are limited, the degradation efficiency is divided into high and low levels, 60-90 types of the common polychlorinated biphenyls in the environment cannot be completely degraded by one or two degrading bacteria.

Therefore, polychlorinated biphenyl degrading bacteria still need to be continuously screened from the environment in the future to accumulate more excellent degrading bacteria, so that sufficient microbial resources are provided for the biological strengthening technology, and meanwhile, gene resources can be provided for the construction of efficient engineering bacteria.

Disclosure of Invention

The invention aims to overcome the problem of limited varieties of the existing polychlorinated biphenyl degrading bacteria and provides a Pandora pandora pnomenusa S2-2 strain with polychlorinated biphenyl degrading capability.

The second purpose of the invention is to provide the application of the Pandora Pandoraea pnomenusa S2-2 strain in degrading polychlorinated biphenyl or repairing polychlorinated biphenyl polluted natural environment.

A third object of the present invention is to provide a method for degrading polychlorinated biphenyl.

The fourth purpose of the invention is to provide the application of the Pandora Pandoraea pnomenusa S2-2 strain in preparing the microbial preparation for degrading polychlorinated biphenyl.

The fifth object of the present invention is to provide a microbial preparation for degrading polychlorinated biphenyl.

The sixth purpose of the invention is to provide the application of the microbial preparation in degrading polychlorinated biphenyl or repairing polychlorinated biphenyl polluted natural environment.

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

the Pandora pandora pnomenusa S2-2 strain is characterized in that the strain is deposited in Guangdong province microorganism culture collection center (GDMCC) at 11-12 th month in 2020, and the strain deposit number is GDMCC No: 61284.

specifically, the nucleotide sequence of the 16s rDNA of the strain is shown as SEQ ID No. 1.

The pandoramea pnomenusa S2-2 strain is subjected to 4-chlorobiphenyl degradation test, and the result shows that the pandoramea pnomenusa S2-2 strain has degradation rates of 97.12%, 99.54%, 99.44%, 99.66% and 99.72% for C1(5mg/L), C2(10mg/L), C3(20mg/L), C4(40mg/L) and C5(60mg/L) of 4-chlorobiphenyl with different concentrations respectively, and the degradation rate gradually increases with the increase of the concentration of the 4-chlorobiphenyl, which indicates that the pandoramea pnomenusa S2-2 strain has a good degradation effect on PCBs.

Therefore, the invention provides the application of the Pandora Pandoraea pnomenusa S2-2 strain in degrading polychlorinated biphenyl or repairing polychlorinated biphenyl polluted natural environment.

Preferably, the concentration of the polychlorinated biphenyl is 5-60 mg/L.

Most preferably, the polychlorinated biphenyl is 4-chlorobiphenyl.

The invention also provides a method for degrading polychlorinated biphenyl, which comprises the step of inoculating Pandora Pandoraea pnomenusa S2-2 strain and/or bacterial liquid thereof to polychlorinated biphenyl pollutants.

The invention also provides application of the Pandora Pandoraea pnomenusa S2-2 strain in preparation of a microbial preparation for degrading polychlorinated biphenyl.

The invention also provides a microbial preparation for degrading polychlorinated biphenyl, which comprises Pandora Pandoraea pnomenusa S2-2 strain and/or bacterial liquid thereof.

The application of the microbial preparation in degrading polychlorinated biphenyl or repairing polychlorinated biphenyl polluted natural environment is also within the protection scope of the invention.

Preferably, the bacterial liquid is a culture liquid obtained by fermenting Pandoraea pnomenusa S2-2 strain.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a Pandora Pandoraea pnomenusa S2-2 strain and application thereof, wherein the strain is deposited in Guangdong province microorganism culture collection center (GDMCC) at 11-12 th month in 2020, and the strain preservation number is GDMCC No: 61284. the research of the invention shows that the Pandoraea pnomenusa S2-2 strain has the function of efficiently degrading polychlorinated biphenyl, and the Pandoraea pnomenusa S2-2 strain has good potential and application prospect for the restoration of polychlorinated biphenyl environmental pollution as an effective and green polychlorinated biphenyl pollution degrading strain.

Drawings

FIG. 1 is a photograph showing the colony morphology of MM30 medium.

FIG. 2 is a photograph showing colony morphology of LB medium.

FIG. 3 is a gram-stained microscopic photograph of S2-2.

FIG. 4 is a scanning electron micrograph of S2-2.

FIG. 5 shows the residual amounts of S2-2 degraded PCBs at different times.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

EXAMPLE 1 screening of Strain S2-2

(1) Preparation of a selective separation culture medium:

texture 44 solution (mg/100 mL): na (Na)₂B₄O₇·10H₂O 17.7，CuSO₄·5H₂O 39.2，CaCl₂·6H₂O 20.1，ZnSO₄·7H₂O1095, EDTA 250, 100-₂SO₄To prevent the generation of precipitates.

Trace metal salt solution (mg/100 mL): EDTA 0.5, CaCO₃ 1，FeSO₄·7H₂O 0.5，MgSO₄·7H₂O 10，MnSO₄·H₂O 10，Mixture44 10mL。

Macroelement solution (g/L): (NH)₄)₂SO₄ 1，KH₂PO₄ 3，Na₂PO 6, 0.5mL of trace metal salt solution, adjusted to pH 7.1 (adjusted by dropwise addition of dilute HCl and NaOH).

MM30 liquid medium: (NH)₄)₂SO₄ g/L、1KH₂PO₄ 3g/L、Na₂PO₄6g/L, 0.5mL of trace metal salt solution and pH 7.1 (when preparing MM30 culture medium, vitamin B120.0002mg/L is added to promote the induction of microbial dechlorination.)

MM30 solid medium: adding 1.8% agar into MM30 liquid culture medium, sterilizing with high pressure steam (121 deg.C, 0.1MPa) for 20min, pouring into culture dish, cooling, solidifying, adding biphenyl or polychlorinated biphenyl solution, and blow drying.

LB culture medium: 10g/L of tryptone and 5g/L, NaCl 10g/L, pH 7.0.0 of yeast extract.

(2) Screening of strains

(2-1) adding biphenyl mother liquor into the MM30 liquid culture medium to enable the final concentration to be 200mg/L, standing to enable the organic solvent to be volatilized, adding a proper amount of fresh mangrove forest bottom mud samples, and culturing for 5-7 days in a constant-temperature shaking table at 30 ℃ and 180 rpm.

(2-2) adding 1mL of the relatively clear bacterial suspension into an MM30 culture medium with the BP concentration of 200mg/L, and carrying out passage for 5-7 times by taking biphenyl as a unique carbon source.

(2-3) taking 100 mu L of bacterial liquid, uniformly coating the bacterial liquid on an MM30 solid culture medium with BP concentration of 200mg/L after continuous gradient dilution, carrying out inverted culture in an incubator at 37 ℃ for 48-72 h, selecting bacterial colonies with different forms, streaking the bacterial colonies on an MM30 solid culture medium containing biphenyl, and continuing purification culture until a single bacterial colony is obtained.

(2-4) amplifying the obtained single strain by using specific primers (PcbA1, PcbA4 and PcbA5), wherein the sequences of the primers are shown in Table 1, the sample adding system is shown in Table 2, the PCR amplification program is shown in Table 3, the gel electrophoresis check shows that the strain with the target size fragment is transferred to MM30 liquid culture medium which takes PCB3 (4-chlorobiphenyl) as a unique carbon source for continuous generation for 5-7 times.

(2-5) streaking the dipped bacterial liquid, inoculating the streaked bacterial liquid to an MM30 solid culture medium with PCB3 as a unique carbon source, and carrying out inverted culture for 48-72 h to obtain a single bacterial colony.

TABLE 1 primer sequences

TABLE 2 sample application System

TABLE 3 PCR amplification procedure

Example 2 identification of Strain S2-2

1. Conventional biological assays

(1) Appearance characteristics of Strain S2-2

During passaging, growth was slower on MM30 medium, and S2-2 formed a macroscopic tip-sized, partially transparent colony around 72h after inoculation, as shown in FIG. 1.

On LB solid medium, S2-2 appeared to be visually translucent spherical colonies after 72-96 h, as shown in FIG. 2.

FIG. 3 is a gram-stained microscopic image (magnification 1000X) of S2-2, S2-2 is a gram-negative strain, and pink long rods, multiple arranged in a chain or Y shape, are observed.

FIG. 4 is a scanning electron microscope image of S2-2, and the strain morphology was observed at 5K times and 45K times respectively under an electron microscope, and it was found that S2-2 is a long and thin rod-shaped bacterium under an electron microscope, and is slightly bent, the strain width is about 0.3 μm, the length is between 0.5-0.9 μm, and S2-2 is a long and thin rod-shaped bacterium which is splitting under 45K times, and is slightly bent (red arrow).

(2) Biochemical characterization of Strain S2-2

The growth conditions of the strains in different carbon-containing and nitrogen-containing compound culture media, the decomposition and utilization conditions of the strains on different carbon sources and nitrogen sources and metabolites thereof are observed, and the test uses a novel biochemical identification tube (microorganism of Kjeldahl, novel biochemical identification tube of 070060) for biochemical identification.

As shown in Table 4, S2-2 can ferment glucose to produce gas, but can not utilize 4 kinds of sugar sources for metabolizing sucrose, mannose, arabinose and inositol, and in the biochemical identification of lysine decarboxylase and arginine double hydrolase, the S2-2 control tube and the test tube both turn into blue-green, which is probably caused by that certain alkaline substances are produced in the growth process of the strain.

TABLE 4

2. Molecular biological identification

(1) Sequencing of the Strain S2-2 Gene

The separated bacterial strain is cultured in an LB broth culture medium for 36-48 h, the growth is vigorous, the DNA of the degradation-simulating bacteria is extracted by a water extraction method in the experiment, and the specific extraction steps are as follows:

sucking 1.5mL of bacterial liquid into a centrifuge tube, centrifuging at 1200rpm/min for 5min, discarding the supernatant, adding 1mL of sterile water, shaking, mixing uniformly, centrifuging again, cleaning, and repeating the above operations for 2 times;

and after 2 times of cleaning, removing the supernatant, adding 100 mu L of sterile water, shaking and uniformly mixing, carrying out boiling water bath for 10min, carrying out ice bath for 5min, centrifuging again, wherein the supernatant is dissolved bacterial DNA, sending the DNA to Guangzhou Biotechnology limited company for sequencing, and carrying out homology comparison on a part of sequence obtained by determination and Genebank data.

(2) Identification results

The nucleotide sequence of 16S rDNA of S2-2 is shown in SEQ ID NO:1, the homology of S2-2 and NZ _ CP009553.3(Pandoraea pnomenusa strain DSM 16536) is as high as 99.09%, and the strain belongs to the Pandora genus, so that the degrading bacteria obtained by screening is identified as Pandora (Pandoraea pnomenusa).

The strain is preserved in Guangdong province microorganism culture collection center (GDMCC) in 11-12 months in 2020, and the strain preservation number is GDMCC No: 61284, classified and named as Pandoraea pnomenusa S2-2, deposited at Tomliki Zhonglu 100, Guangzhou, Guangdong province.

Example 3 verification of PS2-2 on PCB3 (4-chlorobiphenyl) degradation system

(1) Reagent

The inorganic salt culture medium used in the test is MM30 inorganic salt liquid culture medium and PCB3 (4-chlorobiphenyl) single standard.

(2) PCB3 (4-chlorobiphenyl) degradation verification system

Degradation validation experiments occurred in a 50mL volume black-capped wide-mouth screw-triangular flask with a reaction volume of 5 mL. The degradation test sets PCB3 as 5 concentration gradients which are respectively C1(5mg/L), C2(10mg/L), C3(20mg/L), C4(40mg/L) and C5(60mg/L), firstly, an MM30 inorganic salt liquid culture medium is added into a degradation system, then, a PCB3 mother liquor is added, after the solvent is volatilized, a prepared degradation bacterium liquid is added in an inoculation amount of 10%, shaking culture is carried out at a rotating speed of 180rpm at 30 ℃, the residual quantity of the PCB3 is detected at 0 hour, 24 hours, 48 hours, 72 hours and 96 hours after inoculation, and the degradation effect is verified and observed.

(3) Quantitative detection of PCBs

PCBs have strong hydrophobicity, are difficult to dissolve in polar solutions such as water and the like, and are easy to dissolve in organic solvents. Therefore, the extraction of PCBs in the solution of the degradation system is crucial, and in order to fully extract PCBs in the system, a demulsifier is added for treatment before the test detection:

2mL of dehydrated n-hexane was added to the degradation system, and 2g of (NH) was added₄)2SO₄Taking the extract as a demulsifier, performing vortex oscillation for 1min, centrifuging for 2min at the rotating speed of 120 Xg, and extracting polychlorinated biphenyl in a solution system as far as possible;

taking out the glass bottle, transferring the upper layer organic phase into a dry glass centrifuge tube by using a dry glass dropper, adding 1mL of normal hexane into the original glass bottle, repeating the steps of shaking and centrifuging, and transferring the upper layer liquid into the glass centrifuge tube;

adding a small amount of anhydrous sodium sulfate, drying to remove water, sucking the upper layer liquid by a dry glass dropper, transferring to a new dry glass centrifuge tube, sucking a proper amount of the upper layer liquid into a sample injection vial, storing at 4 ℃, and preparing for GC-u ECD sample loading.

The test directly compares the polychlorinated biphenyl by an external standard method to carry out quantitative detection on the polychlorinated biphenyl. The amount of the substrate substance represented by the unit chromatographic peak area is different, the concentration of the PCB3 in the degradation system liquid is calculated by comparing the peak area of the degradation system sample chromatogram with the peak area of the standard product and combining the known concentration of the standard product PCB3 according to the peak area ratio of the two peak areas during data processing, and the calculation formula is as follows:

A_PCB3: peak area for added PCB3 in the degradation system;

A_PCB3the standard is as follows: peak area for purchased PCB3 standard;

C_PCB3the standard is as follows: concentration of PCB3 standard (10 ng/. mu.L).

(4) Degradation capability of S2-2 on PCB3 (4-chlorobiphenyl)

FIG. 5 and Table 5 show the residual amounts of PCBs at different degradation periods, and it can be seen that the degradation rates of the S2-2 strain are respectively 98.23%, 98.85%, 99.65%, 99.71% and 99.75% at the concentrations of PCB3 (4-chlorobiphenyl) in five groups of C1(5mg/L), C2(10mg/L), C3(20mg/L), C4(40mg/L) and C5(60mg/L), and the residual rate of only C5 group is higher and close to 40% after 24h of the S2-2 strain, the residual quantity of the C5 group is suddenly reduced after 48 hours, the residual rates of the C3 group and the C4 group are both lower than 10 percent compared with the 4 group, the residual rates of the C5 group are both lower than 1 percent close to the C3 group and the C4 group, only a small amount of degradation exists in the C1 group within 24-48 h, the residual rate remains about 6%, the degradation residual amounts of all concentration groups are relatively close after 72h, and the C5 group reaches the lowest, the degradation rate of the final 5 concentration groups is about 1%, and the PCBs residue of the C3, the C4 and the C5 groups is less than 0.35%.

TABLE 5S 2-2 residual concentration of PCBs at different periods in the degradation System

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

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<120> Pandora Pandoraea pnomenusa S2-2 strain and application thereof

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gggcgtaaag cgtgcgcagg gcggttttgt aagacggatg tgaaatcccc gggcttaacc 600

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tagcagtgaa atgcgtagag atgtggagga ataccgatgg gaaggcagcc ccctgggcca 720

atactgacgc tcatgcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 780

acgccctaaa cgatgtcaac tagttgttgg ggattcattt ccttagtaac gtagctaacg 840

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cacgtcatac aatggtcggt acagagggct gccaaaccgc gaggtggagc taaccccaga 1260

aaaccgatcg tagtccggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320

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Claims

1. The Pandora pandora pnomenusa S2-2 strain is characterized in that the strain is deposited in Guangdong province microorganism culture collection center (GDMCC) at 11-12 th month in 2020, and the strain deposit number is GDMCC No: 61284.

2. the Pandora pandora pnomenusa S2-2 strain according to claim 1, wherein the nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID No: 1.

3. Use of the Pandora Pandoraea pnomenusa S2-2 strain according to claim 1 for degrading polychlorinated biphenyl or restoring a natural environment contaminated with polychlorinated biphenyl.

4. The use according to claim 3, wherein the concentration of the polychlorinated biphenyl is 5 to 60 mg/L.

5. Use according to claim 3, wherein the polychlorinated biphenyl is 4-chlorobiphenyl.

6. A method for degrading polychlorinated biphenyl is characterized in that Pandora Pandoraea pnomenusa S2-2 strain and/or bacterial liquid thereof is inoculated to polychlorinated biphenyl pollutants.

7. Use of the Pandora Pandoraea pnomenusa S2-2 strain according to claim 1 for the preparation of a microbial preparation for the degradation of polychlorinated biphenyls.

8. A microbial preparation for degrading polychlorinated biphenyl, which comprises Pandora Pandoraea pnomenusa S2-2 strain and/or its bacterial liquid.

9. Use of the microbial preparation according to claim 8 for degrading polychlorinated biphenyl or restoring natural environment polluted by polychlorinated biphenyl.

10. The method of claim 6 or the preparation of claim 8, wherein the bacterial liquid is a culture liquid obtained by fermenting Pandoraea pnomenusa S2-2 strain.