A strain of Pseudomonas P13 and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a strain of Pseudomonas P13 for degrading α-pinene and its application. After treating (+)-α-pinene or (-)-α-pinene with the Pseudomonas P13 of the present invention for 4 days, the degradation rates of (+)-α-pinene and (-)-α-pinene are 34.96% and 15.70% respectively, and α-pinene can be effectively degraded in a short time.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Pseudomonas P13 and application thereof. Background Art

[0002] Alpha-pinene (α-pinene) is a naturally occurring cyclic olefin compound with good chemical stability, hydrophobicity and volatility. It is currently widely used in processing and manufacturing industries. However, with the increase in industrial use, untreated α-pinene is directly discharged into the atmosphere, reacting with ozone, hydroxyl and other free radicals in the atmosphere to form photochemical smog and destroy the ozone layer. When humans are exposed to an environment containing α-pinene, it will irritate organs such as the skin, mucous membranes or eyes, posing a threat to human health. In recent years, the effective treatment of α-pinene in industrial waste has become a focus of attention for society and enterprises.

[0003] Traditional treatment methods for industrial waste including α-pinene mainly include physical absorption and chemical incineration. Although these traditional methods can effectively purify waste and reduce air pollution, they still have the problems of high economic cost and secondary pollution of the treated materials. Therefore, there is an urgent need for safer, more economical and non-secondary pollution methods to treat α-pinene in industrial waste. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Pseudomonas P13 and an application thereof. The Pseudomonas P13 provided by the present invention can effectively degrade α-pinene, and has low degradation cost and is pollution-free.

[0005] The invention provides a strain of Pseudomonas sp. P13, and the preservation number of the Pseudomonas sp. P13 is CCTCC NO: M2022814.

[0006] The invention also provides the use of the Pseudomonas P13 in degrading α-pinene.

[0007] The present invention also provides a reagent for degrading alpha-pinene, comprising the Pseudomonas P13 described in the above technical solution.

[0008] The present invention also provides a method for preparing the reagent described in the above technical solution, comprising the following steps:

[0009] Pseudomonas P13 was inoculated into LB liquid medium and cultured until the logarithmic growth phase, and the cells were collected;

[0010] The cells were diluted with solvent to an OD of 600 The value is 0.5-1.5, and the reagent is obtained.

[0011] Preferably, each 1L of the LB liquid culture medium comprises the following components: 10g of peptone, 5g of yeast extract and 10g of sodium chloride; the pH of the LB liquid culture medium is 7.2-7.4.

[0012] Preferably, the solvent comprises sterile water.

[0013] Preferably, the culture temperature is 28°C and the rotation speed is 200r / min.

[0014] The present invention also provides application of the reagent described in the above technical solution in degrading α-pinene.

[0015] The present invention provides a Pseudomonas P13 for degrading alpha-pinene, which has been biologically preserved and has a preservation number of CCTCC NO: M 2022814. The present invention screens and obtains a strain of Pseudomonas P13 from one-year-old fresh branches of Masson pine after being fed by Monochamus alternatus, and the strain can effectively degrade alpha-pinene in a short time. The results of the embodiment show that after the Pseudomonas P13 of the present invention is used to treat (+)-alpha-pinene or (-)-alpha-pinene for 4 days, the degradation rates of the strain for (+)-alpha-pinene and (-)-alpha-pinene are 34.96% and 15.70%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is the colony morphology of Pseudomonas P13;

[0018] Figure 2 is the phylogenetic tree of Pseudomonas P13;

[0019] Figure 3 This is the degradation result of α-pinene by Pseudomonas sp. P13.

[0020] Biological deposit information

[0021] Pseudomonas sp. P13 was deposited in China Center for Type Culture Collection (CCTCC) on June 7, 2022, at Room 211, China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Edit The number is CCTCC NO: M 2022814. DETAILED DESCRIPTION

[0022] The invention provides a strain of Pseudomonas P13 capable of degrading alpha-pinene. The Pseudomonas P13 has been biologically preserved, and the preservation number is CCTCC NO: M 2022814.

[0023]

[0024] The Pseudomonas P13 strain of the present invention is a rod-shaped, Gram-negative bacterium that is strictly aerobic. On the LB solid culture medium, the colonies are off-white, round, opaque, convex in the middle, with irregular edges, and the colony surface is smooth and shiny. The Pseudomonas P13 of the present invention can effectively degrade alpha-pinene in a short time. Degradation experiments show that after the Pseudomonas P13 is used to treat (+)-alpha-pinene or (-)-alpha-pinene for 4 days, the degradation rates of the strain to (+)-alpha-pinene and (-)-alpha-pinene are 34.96% and 15.70% respectively.

[0025] According to the physiological and biochemical characteristics of Pseudomonas P13 described in the present invention, the application of Pseudomonas P13 in the degradation of α-pinene also falls within the protection scope of the present invention.

[0026] The present invention also provides a reagent for degrading α-pinene, comprising the Pseudomonas P13 described in the above technical solution. The OD of the reagent of the present invention is 600 The value is preferably 0.5 to 1.5, and more preferably 1.5. The Pseudomonas sp. P13 of the present invention can effectively degrade α-pinene in a short time.

[0027] The present invention also provides a method for preparing the reagent described in the above technical solution, comprising the following steps:

[0028] Pseudomonas P13 was inoculated into LB liquid medium and cultured until the logarithmic growth phase, and the cells were collected;

[0029] The cells were diluted with solvent to an OD of 600 The value is 0.5-1.5, and the reagent is obtained.

[0030] The present invention inoculates Pseudomonas P13 into LB liquid culture medium and cultures it to the logarithmic growth phase to obtain a culture solution containing Pseudomonas P13. The culture temperature of the present invention is preferably 28°C, and the culture speed is preferably 200r / min. The present invention has no strict requirements on the culture time, and the culture of Pseudomonas P13 can be terminated when it is in the logarithmic growth phase. In the present invention, each 1L of the LB liquid culture medium preferably includes the following components: 10g of peptone, 5g of yeast extract, and 10g of sodium chloride; the pH of the LB liquid culture medium is 7.2-7.4. The present invention has no strict requirements on the source of the LB liquid culture medium, and it can be purchased conventionally or prepared by itself. The present invention uses LB liquid culture medium to culture the Pseudomonas P13 to the logarithmic growth phase to obtain vigorous bacteria.

[0031] After obtaining the culture solution containing Pseudomonas P13, the present invention preferably centrifuges the culture solution containing Pseudomonas P13 to collect the bacterial cells, i.e., the precipitate obtained by centrifugation. The speed of the centrifugation in the present invention is preferably 10000 rpm, and the centrifugation time is preferably 5 min. The present invention has no strict requirements on the temperature of the centrifugation, and room temperature centrifugation is sufficient.

[0032] After obtaining the bacterial cells, the present invention preferably uses sterile water to centrifuge and wash the bacterial cells 2 to 3 times, and uses a solvent to dilute the washed bacterial cells OD 600 The value is 0.5-1.5, and the reagent is obtained. The solvent of the present invention is preferably sterile water.

[0033] In the embodiment of the present invention, after treating 150 mg (+)-α-pinene or (-)-α-pinene with 100 μL of the reagent for 4 days, the degradation rates of (+)-α-pinene and (-)-α-pinene were 34.96% and 15.70%, respectively. The reagent of the present invention can effectively degrade α-pinene in a short time.

[0034] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] The culture medium and reagents used in the embodiments of the present invention are as follows:

[0036] Bushnell-Haas broth medium: 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 1 g / L ammonium nitrate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L ferric chloride, 0.02 g / L calcium chloride dihydrate and the balance water, pH 7.2-7.4, high temperature moist heat sterilization at 121°C for 20 min.

[0037] LB liquid culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L and the balance water, pH 7.2-7.4, high temperature moist heat sterilization at 121℃ for 20min.

[0038] LB solid culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 18g / L and the balance water, pH 7.2-7.4, high temperature moist heat sterilization at 121℃ for 20min.

[0039] Example 1

[0040] 1. Isolation of strains

[0041] 1.1 Sample processing

[0042] Select one-year-old fresh branches of Masson pine after being fed by Monochamus alternatus, use sterile dissection tools to extract 1-2 mm of bark around the feeding wound, and then transfer the bark sample into a 5 mL sterile centrifuge tube containing 3 mL of phosphate buffer (10 mM PBS). After the sample in the centrifuge tube is treated with ultrasound (50 / 60 Hz, 117 V, 1.0 A) for 60 seconds, a sterile electric grinding pestle is immersed in the liquid surface of the tube and ground for 30 seconds to separate the bacterial cells from the bark tissue. Finally, centrifuge at a low speed of 1000 rpm for 2 minutes, collect the supernatant in the tube, and then centrifuge the supernatant at 6000 rpm for 10 minutes to retain the precipitate. Resuspend the precipitate with 0.5 mL of sterile PBS as the bacterial suspension to be screened.

[0043] 1.2 Preliminary screening of strains that degrade α-pinene

[0044] (1) Add 50 μL of the bacterial suspension to be screened in step 1.1 to a 5 mL sterile centrifuge tube containing 3 mL of Bushnell-Haas broth medium, and add 30 μL of (+)-α-pinene or (-)-α-pinene as the sole carbon source. After mixing, place in a shaker at 28°C and culture at 180 r / min for 4 days to obtain the primary screening culture medium;

[0045] (2) Dilute the primary screening culture medium in a gradient manner, specifically 10 3 times dilution, 10 4 times dilution, 10 5 times dilution and 10 6 The strains were diluted 100 μL each time, and 100 μL of the dilutions were evenly spread on LB solid medium plates, and incubated in an incubator at 28°C for 48 h. Single colonies were selected as α-pinene-degrading strains.

[0046] In this experiment, one-year-old fresh branches of Masson pine that had been fed by Monochamus alternatus were processed and then diluted and spread on LB solid culture medium. After preliminary screening, an α-pinene-degrading strain was obtained and marked as P13.

[0047] 1.3 Strain purification and preservation

[0048] Add the α-pinene-degrading strain (P13) selected in step 1.2 to the LB liquid culture medium, place it in a 28°C shaker, and culture it at 200r / min for 24h. Use a sterile inoculation loop to dip the culture solution of the α-pinene-degrading strain, and streak it on the LB solid culture medium plate for 2 to 3 times until a purified single colony can be picked out. Select a single colony and add it back to the LB liquid culture medium for 24h. Pipette the bacterial solution and mix it with 30% sterile glycerol in a 1:1 ratio, and store it in a -80°C refrigerator for later use.

[0049] 2. Morphological identification

[0050] The single colonies of P13 picked out in step 1.2 were activated in LB liquid medium, and then streaked on LB solid medium plates for purification. The morphology of the purified P13 single colonies was observed. The morphological observation results of P13 are as follows: Figure 1 As shown. Figure 1 It can be seen that the P13 colony is off-white, round, opaque, convex in the middle, with irregular edges, and a smooth and shiny surface. The morphological characteristics are consistent with those of Pseudomonas sp. bacteria.

[0051] 3.16S rDNA Identification

[0052] The genomic DNA of α-pinene-degrading bacteria P13 was extracted and used as a template for PCR amplification using universal primers of 16S rDNA, wherein the primers were 27F (SEQ ID NO. 2: 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (SEQ ID NO. 3: 5′-GGTTACCTTGTTACGACTT-3′).

[0053] The PCR reaction system (25 μL) was: 1 μL DNA template, 12.5 μL 2× Taq PCR Master Mix, 0.5 μL upstream and downstream primers, and 10.5 μL ddH2O. The PCR amplification program was: 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 5 min, and the PCR instrument used was Bio-Rad T100.

[0054] The PCR product was sent to Shanghai Jie Li Biotechnology Co., Ltd. for purification and sequencing, and the 16S rDNA gene sequence of the P13 strain was obtained as shown in SEQ ID NO.1. The obtained sequence was submitted to NCBI, compared with the sequences in the GenBank database, and the homologous sequences were downloaded for the construction of the phylogenetic tree. All sequences were aligned using Clustal W software, and the strains were constructed using MEGA 7.0 software and the neighbor-joining method. The constructed phylogenetic tree is shown in Figure 2 .

[0055] The morphological identification results of α-pinene-degrading bacteria P13 are as follows: Figure 1 Combined with the results of morphological identification, sequence alignment and phylogenetic analysis showed that the morphological characteristics of α-pinene-degrading bacteria P13 were consistent with those of Pseudomonas sp. bacteria and were highly homologous to Pseudomonas sp. bacteria. Finally, the α-pinene-degrading bacteria P13 was named Pseudomonas sp. P13.

[0056] Example 2

[0057] Application effect of Pseudomonas sp. P13 in degradation of α-pinene:

[0058] (1) Sample preparation: The preserved Pseudomonas P13 was inoculated into LB liquid medium, cultured in a shaking incubator at 28°C and 200 rpm until the logarithmic growth phase, centrifuged at 10,000 rpm for 5 min at room temperature, discarded the medium and retained the bacteria, and washed with sterile water for 2 to 3 times by centrifugation. The bacterial solution was adjusted to OD 0. 600 The value was adjusted to 1.5 to obtain Pseudomonas P13 bacterial solution.

[0059] 150 mg of (+)-α-pinene or (-)-α-pinene and 100 μL of Pseudomonas P13 bacterial liquid were added to 15 mL of Bushnell-Haas broth medium as experimental group 1; the Bushnell-Haas broth medium without Pseudomonas P13 bacterial liquid but only 150 mg of (+)-α-pinene or (-)-α-pinene was added as blank control 1; all treatments were repeated 7 times, and cultured at 28°C and 180 r / min shaking for 4 days. After the culture, 3 mL of chromatographic grade n-hexane was added to each tube for extraction, and the tube was sealed and shaken for 3 hours, and placed upright at -20°C overnight. After thawing at room temperature, 1 mL of n-hexane extract was transferred to a 2 mL injection bottle, and 2 μL of isobutylbenzene was added to each injection bottle as an internal standard before testing. Quantitative analysis was performed by gas chromatography, and the samples were stored at -20°C before analysis.

[0060] (2) Gas chromatography analysis method: The chromatographic column is an HP-5 quartz capillary column (30m×0.32mm×0.25μm); the detector is a hydrogen flame ionization detector (FID); the carrier gas is high-purity nitrogen; the injection port temperature is 250℃, without splitting; the detector temperature is 280℃; the column flow rate is 1mL / min; the injection volume is 1μL. The heating program is: 40℃ for 3min, 20℃ / min to 120℃, 1min, 20℃ / min to 180℃, 2min.

[0061] (3) Calculation of degradation rate: First, the relative correction factor f is calculated based on the internal standard (isobutylbenzene) and the standard (chromatographic grade (+)-α-pinene or (-)-α-pinene):

[0062] f=(A i / m i ) / (A s / m s )

[0063] Among them A i and A s are the peak areas of internal standard and standard, m iand m s are the amounts of internal standard and standard added, respectively.

[0064] Then, the content m of the component to be measured is calculated based on the gas chromatographic peak response value of the component to be measured containing the internal standard. t :

[0065] m t =f×(A t / A i / m i )

[0066] Among them A t and A i are the peak areas of the component to be tested and the internal standard, m i is the amount of internal standard added.

[0067] Finally, the degradation rates of (+)-α-pinene and (-)-α-pinene by the strains after different treatment samples were cultured for different time were calculated using the content of (+)-α-pinene or (-)-α-pinene in each degradation sample and the content of (+)-α-pinene or (-)-α-pinene in the control sample. Figure 3 As shown. Figure 3 It can be seen that after Pseudomonas P13 treated (+)-α-pinene and (-)-α-pinene for 4 days, the degradation rates of (+)-α-pinene and (-)-α-pinene by the strain were 34.96% and 15.70%, respectively, indicating that Pseudomonas P13 of the present invention can effectively degrade α-pinene in a short time.

[0068] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas sp. P13, characterized in that: The deposit number of the Pseudomonas sp. P13 is CCTCC NO: M 2022814; The Pseudomonas P13 is screened and obtained from one-year-old fresh branches of Masson pine after being fed by Monochamus alternatus.

2. Use of the Pseudomonas sp. P13 according to claim 1 in the degradation of α-pinene; The α-pinene includes (+)-α-pinene and (-)-α-pinene.

3. A reagent for degrading α-pinene, characterized in that The Pseudomonas sp. P13 according to claim 1; The α-pinene includes (+)-α-pinene and (-)-α-pinene.

4. The method for preparing the reagent according to claim 3, characterized in that: The steps include: Pseudomonas P13 was inoculated into LB liquid medium, cultured to the logarithmic growth phase, and the cells were collected; The cells were diluted with solvent to an OD 600 The value is 0.5-1.5, and the reagent is obtained.

5. The preparation method according to claim 4, characterized in that: Each 1L of the LB liquid culture medium includes the following components: 10g of peptone, 5g of yeast extract and 10g of sodium chloride; the pH of the LB liquid culture medium is 7.2-7.

4.

6. The preparation method according to claim 4, characterized in that: The solvent includes sterile water.

7. The preparation method according to claim 4, characterized in that: The culture temperature is 28° C. and the rotation speed is 200 r / min.

8. Use of the reagent according to claim 3 or the reagent obtained by the preparation method according to any one of claims 4 to 7 in the degradation of α-pinene; The α-pinene includes (+)-α-pinene and (-)-α-pinene.

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