Pseudomonas putida L4 and its application

Abstract

The present invention provides a Pseudomonas putida L4 and an application thereof, belonging to the field of microbial technology, and capable of achieving degradation of polycyclic aromatic hydrocarbon derivatives, especially 1-hydroxypyrene. The Pseudomonas putida L4 was deposited in the China Center for Type Culture Collection on July 8, 2024, with a deposit number of CCTCC NO: M20241492, and the 16S rDNA gene sequence of Pseudomonas putida L4 is shown in SEQ ID NO: 1.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pseudomonas putida L4 and applications thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are composed of two or more benzene rings fused together. They can be transmitted over long distances and are persistent organic pollutants that are carcinogenic, teratogenic, and mutagenic.

[0003] Oxygenated PAHs (OPAHs) are common PAHs derivatives in the environment. They can be directly emitted through fossil fuel combustion along with PAHs. They can also be generated by photolysis of PAHs or by reactions with atmospheric oxidants (HO·, O3, NO3 − OPAHs are secondary products produced by chemical reactions with NO2. Compared with PAHs, OPAHs have lower vapor pressure and higher molecular weight, so they are more easily adsorbed by particulate matter.

[0004] The degradation process of PAHs and OPAHs in the natural environment mainly involves hydrolysis, photodegradation and microbial degradation, but in the natural environment, hydrolysis and photodegradation are weak and the degradation rate is slow. At present, scholars have screened a large number of PAHs efficient degradation strains from environmental media, but there is no relevant literature reporting OPAHs degradation strains. Summary of the invention

[0005] The present invention provides a Pseudomonas putida ( Pseudomonas putida ) L4 and its application can achieve the degradation of OPAHs.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Pseudomonas putida ( Pseudomonas putida ) L4, Pseudomonas putida was deposited in the China Center for Type Culture Collection (CCTCC) on July 8, 2024, with the deposit number CCTCC NO: M20241492, and the 16S rDNA gene sequence of Pseudomonas putida L4 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides an application of Pseudomonas putida L4, based on the above-mentioned Pseudomonas putida L4, which is applied to degrading polycyclic aromatic hydrocarbon derivatives.

[0009] In a third aspect, the present invention provides a bacterial suspension for degrading polycyclic aromatic hydrocarbon derivatives, wherein the bacterial suspension is prepared by the following steps:

[0010] Step 1, activating Pseudomonas putida L4 overnight and shaking culturing to the logarithmic phase;

[0011] Step 2: Collect Pseudomonas putida L4 cells and adjust the OD of the cells 600 The value reached 1.0 to obtain a bacterial suspension.

[0012] In an implementation of the second aspect or the third aspect, the polycyclic aromatic hydrocarbon derivative includes 1-hydroxypyrene.

[0013] The Pseudomonas putida provided by the present invention ( Pseudomonas putida ) L4, which can use 1-OH PYR as the sole carbon source and energy source for growth and reproduction. Under pure culture conditions, the bacteria can degrade 2 mg / L 1-OHPYR in inorganic salt culture medium by 45% in 7 days. The present invention has great application prospects in the biological treatment of environmental pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic diagram of the growth morphology of Pseudomonas putida L4 provided in the examples of the present application;

[0015] Figure 2 is a transmission electron micrograph of Pseudomonas putida L4 provided in the examples of the present application;

[0016] Figure 3 is a schematic diagram of the phylogenetic tree of 16S rDNA of Pseudomonas putida L4 provided in the examples of the present application;

[0017] Figure 4 Schematic diagram of the degradation ability of Pseudomonas putida L4 provided in the examples of the present application. DETAILED DESCRIPTION

[0018] The terms "first" and "second" etc. in the description and claims of the present invention are used to distinguish different objects rather than to describe a specific order of the objects.

[0019] The “and / or” in the embodiments of the present application indicates the relationship between objects. For example, A and / or B may indicate the following three situations: A exists alone, B exists alone, and A and B exist at the same time.

[0020] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0021] Example 1: In order to solve the problem that there is no OPAHs-degrading strain reported in the relevant literature in the background technology, this example provides a Pseudomonas putida ( Pseudomonas putida )L4, which can achieve the degradation of OPAHs.

[0022] Pseudomonas putida Pseudomonas putida L4 strain, classified and named as Pseudomonas putida L4 Pseudomonas putida L4, the deposit number is CCTCC NO: M20241492, the deposit date is July 8, 2024, the depositor is China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0023] The 16S rDNA gene sequence of Pseudomonas putida L4 is shown in SEQ ID NO:1.

[0024] The above-mentioned Pseudomonas putida ( Pseudomonas putida ) The isolation and identification process of L4 is as follows.

[0025] 1. Separation

[0026] Weigh about 5 g of test soil and add it to a 250 mL conical flask, add 100 mL of ultrapure water, place it in a shaker at 30 °C and 150 rpm in the dark for 8 h, then take it out and let it stand for about two hours. The supernatant is the enriched initial indigenous microorganisms.

[0027] Take 5 mL of supernatant and transfer it to 95 mL of inorganic salt medium containing polycyclic aromatic hydrocarbon derivatives (containing 0.2 mg / L 1-OH PYR, hereinafter referred to as SPAH). After shaking culture at 30 °C and 150 rpm for 5 days, take 5 mL of inoculum each time for continuous enrichment culture, transfer 5 times, and increase the SPAH content in the inorganic salt medium to 2 mg / L accordingly (i.e., containing 1-OHPYR, the SPAH content in the inorganic salt medium after the first transfer is 0.2 mg / L; the SPAH content in the inorganic salt medium after the second transfer is 0.5 mg / L; the SPAH content in the inorganic salt medium after the third transfer is 1 mg / L; the SPAH content in the inorganic salt medium after the fourth transfer is 1.5 mg / L; the SPAH content in the inorganic salt medium after the fifth transfer is 2 mg / L). Then dilute the culture medium that has been acclimated 5 times by 10 4 ~10 5 Spread on MSM solid medium containing 1 mg / L SPAH contaminant, and invert and culture at 30℃ for 1-3 days. After a single colony grows on the MSM solid medium, pick a single colony and purify it by streaking multiple times to isolate a bacterial strain, numbered L4. Then inoculate the strain on LB solid plate and invert and culture at 30℃ for 5 days to observe its colony morphology.

[0028] Among them, the formulas of the above-mentioned inorganic salt culture medium, MSM solid culture medium and LB solid plate (also called LB solid culture medium) are as follows.

[0029] Mineral salt medium (MSM): (NH4)2SO4: 1.5 g / L; KH2PO4: 0.5 g / L; K2HPO4·3H2O: 1.91 g / L; NaCl: 0.5 g / L; MgSO4·7H2O: 0.2 g / L; adjust the final pH of the inorganic salt culture medium to 7.0; add 1.5% (W / V) agar powder to the solid culture medium.

[0030] LB liquid medium: Yeast extract: 5.0 g; Tryptone: 10.0 g; Sodium chloride (NaCl): 10.0 g; add ultrapure water to 1 L, adjust pH = 7.0, and sterilize at 121°C for 20 minutes; LB solid medium is based on LB liquid medium and added with 1.5% (W / V) agar powder.

[0031] MSM solid culture medium: (NH4)2SO4: 1.5 g / L; KH2PO4: 0.5 g / L; K2HPO4·3H2O: 1.91 g / L; NaCl: 0.5 g / L; MgSO4·7H2O: 0.2 g / L; agar powder 15 g / L.

[0032] The above strain L4 was streaked and cultured on LB solid medium for 5 days. The colonies were white, round, mucous and opaque, with irregular edges, raised surfaces, and moist and smooth. Figure 1 shown.

[0033] 2. Identification

[0034] (1) Transmission electron microscopy observation and identification

[0035] Inoculate the purified strain L4 into LB liquid medium for overnight activation. Take 1 mL of bacterial solution and centrifuge at 8000 rpm for 3-5 min, remove the supernatant, and add MSM bacteria. Repeat the above operation three times, add 1 mL of 2.5% (v / v) glutaraldehyde to the harvested bacterial pellet, mix thoroughly, and let stand at 4°C overnight.

[0036] Pour away the fixative solution, rinse the sample three times with 0.1 M, pH 7.0 phosphate buffer, 15 min each time; fix the sample with 1% osmium acid solution for 1-2 h; carefully remove the osmium acid waste solution, rinse the sample three times with 0.1 M, pH 7.0 phosphate buffer, 15 min each time; dehydrate the sample with gradient concentrations (including 30%, 50%, 70%, 80%, 90% and 95%) of ethanol solution, treat each concentration for 15 min, then treat with 100% ethanol for 20 min; finally, transition to pure acetone for 20 min.

[0037] Treat the sample with a mixture of embedding agent and acetone (V / V=1 / 1) for 1 h; treat the sample with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 h; treat the sample with pure embedding agent overnight; embed the infiltrated sample and heat it at 70°C overnight to obtain the embedded sample.

[0038] Optionally, the embedding agent is a spurr low-viscosity embedding agent. The embedding agent may also be other embedding agents that can achieve the above functions, which is not further limited in the embodiments of the present application.

[0039] The sample was sliced ​​in an ultra-thin slicer to obtain 70-90 nm slices. The slices were stained with a 1% lead citrate solution and a 50% ethanol saturated solution of uranyl acetate for 5-10 min each. After drying, they could be observed in a transmission electron microscope. The morphological characteristics of the slices under transmission electron microscope were elliptical, such as Figure 2 shown.

[0040] (2) 16S rDNA molecular identification of strains

[0041] The total bacterial DNA was extracted and the bacterial genome was amplified by PCR using the universal primers for bacterial 16S rDNA. After the PCR product was sequenced (sequencing was completed by Nanjing Qingke Biological), the sequencing results were compared with the 16S rDNA sequences reported in GenBank for homology, and related bacterial species were selected for phylogenetic tree analysis. The results are as follows: Figure 3 As shown, the 16S rDNA gene sequence of the strain L4 isolated and purified by the present invention is similar to that of Pseudomonas putida Pseudomonas putida (GenBank accession number CP101910.1) has the highest homology (homology rate reaches 99%).

[0042] In the above PCR amplification, the PCR program is: pre-denaturation: temperature 98°C time 2 minutes; cycle phase: (temperature 98°C time 10 seconds, temperature 57°C time 10 seconds, temperature 72°C time 45 seconds) cycle number 35; extension phase: temperature 72°C time 5 minutes; storage phase: temperature 4°C.

[0043] The PCR system includes: total system: 20ul; primer 27F (10P) 2ul, primer 1492R (10P) 2ul, DNA template 1ul, ddH2O 15ul.

[0044] The strain obtained by screening in this example was identified as Pseudomonas putida ( Pseudomonas putida ), named Pseudomonas putida L4.

[0045] The 16S rDNA gene sequence of the Pseudomonas putida L4 (i.e. the above sequencing result) is as follows:

[0046] SEQ ID NO: 1:

[0047]

[0048] Example 2: This example provides a Pseudomonas putida ( Pseudomonas putida ) L4 application, based on the Pseudomonas putida provided in Example 1 ( Pseudomonas putida )L4, and applied it to the degradation of polycyclic aromatic hydrocarbons derivatives.

[0049] For the Pseudomonas putida provided in Example 1 ( Pseudomonas putida ) The degradation characteristics of L4 for polycyclic aromatic hydrocarbon derivatives are studied as follows.

[0050] The strain Pseudomonas putida After L4 was inoculated in an inorganic salt medium containing 2 mg / L 1-hydroxypyrene (hereinafter referred to as 1-OH PYR) and cultured for 7 days, it degraded 45% of 1-OH PYR.

[0051] The above-mentioned inorganic salt culture medium uses 1-OH PYR as the sole carbon source, and its formula is: (NH4)2SO4: 1.5 g / L; KH2PO4: 0.5 g / L; K2HPO4·3H2O: 1.91 g / L; NaCl: 0.5 g / L; MgSO4·7H2O: 0.2 g / L; 1-OHPYR: 2 mg / L; the final pH of the inorganic salt culture medium is adjusted to 7.0.

[0052] In one implementation, the above use is for Pseudomonas putida ( Pseudomonas putida ) L4 is used to prepare preparations for degrading polycyclic aromatic hydrocarbon derivatives.

[0053] Specifically, the above preparation is a bacterial suspension.

[0054] The bacterial suspension is prepared by the following steps:

[0055] Step 1, activating Pseudomonas putida L4 overnight and shaking culturing to the logarithmic phase;

[0056] Specifically, Pseudomonas putida L4 strain was inoculated into 100 ml LB medium, cultured at 30°C and 150 rpm for 24 h, and then centrifuged at 8000 rcf for 5 min to obtain a primary suspension;

[0057] It should be noted that the above temperature, rotation speed, incubation time, centrifugal speed and centrifugal time can be adjusted within a reasonable range according to actual conditions, and this embodiment does not limit the values ​​of the above temperature, rotation speed, incubation time, centrifugal speed and centrifugal time;

[0058] Step 2: Collect Pseudomonas putida L4 cells and adjust the OD of the cells 600 The value reaches 1.0, and a bacterial suspension is obtained;

[0059] Specifically, the cells of Pseudomonas putida L4 were obtained from the primary suspension, and the OD of the cells was calculated after washing the cells twice with MSM. 600 The value was adjusted to 1.0 to obtain a bacterial suspension, and the bacterial suspension was temporarily stored at 4 °C for later use;

[0060] It should be noted that the number of times the bacteria are washed and the OD of the bacteria 600 The value and the temporary storage standby temperature of the bacterial suspension can be adjusted within a reasonable range according to the actual situation. This embodiment does not adjust the number of times the bacterial cells are washed, the OD of the bacterial cells, etc. 600 The value and the temporary storage temperature of the bacterial suspension are limited.

[0061] In order to verify the degradation performance of the bacterial suspension obtained in the above steps on PAH derivatives, the strain Pseudomonas putida L4 was inoculated into 19 mL MSM culture medium containing 2 mg / L 1-hydroxypyrene (hereinafter referred to as 1-OH PYR). 1 mL of the above bacterial suspension was inoculated, and the culture medium without L4 bacterial suspension was used as a blank control, and its pH was adjusted to 7. Three replicates were set for each of the above two groups of samples. The culture was cultured at 30°C and 150 rpm constant temperature shaker for 7 days, and samples were taken at regular intervals every day. 10 mL of dichloromethane was added to the taken conical flask, and ultrasonic vibration was performed for 5 min; after the ultrasonication, the lower layer of liquid was collected with a rotary evaporator, and the above operation was repeated three times. The vacuum rotary evaporation was carried out at a constant temperature of 40°C until it was nearly dry, and the volume was adjusted to 2 mL with acetonitrile. After passing through a 0.22 μm pore size organic phase filter, the sample to be tested containing 1-OH PYR was obtained, and its sample was detected and analyzed by HPLI / UV-FLD (high performance liquid chromatography / ultraviolet-fluorescence detector).

[0062] The chromatographic conditions of HPLC / UV-FLD analysis were as follows: LC-20AT high performance liquid chromatograph (equipped with SPD-2A ultraviolet detector) was used for HPLC / UV-FLD analysis. The detection time was 50 min, and the injection volume of the injection system was 20 μL. The separation system used acetonitrile-water as the mobile phase, the initial flow rate was 0.6 mL / min, and 1-OH PYR was separated by gradient elution. The chromatographic column was a Φ4.6×250 mm Inertsil ODS-P liquid chromatography column, and the column temperature was 40 ℃. The detection system used an ultraviolet detector and turned on the dual wavelength detection mode, which were 220 nm and 254 nm respectively.

[0063] After HPLC / UV-FLD analysis, Pseudomonas putida The degradation effect of L4 on 1-OH PYR is as follows Figure 4 As shown. Figure 4It can be seen that on the first day, L4 only removed 11.5% of 1-OH PYR; on the second to fourth days, the effect of L4 in removing 1-OH PYR remained stable; as the culture time increased, its degradation effect increased significantly. On the seventh day, L4 could remove 46.5% of 1-OH PYR. It can be seen that the bacteria has a significant degradation effect on 1-OH PYR under 7-day shaking culture. Under pure culture conditions, the bacteria can degrade 2 mg / L 1-OH PYR in the inorganic salt culture medium by 45% in 7 days, indicating that Pseudomonas putida The L4 strain has a significant ability to degrade 1-OH PYR, which also shows that the above bacterial suspension is a Pseudomonas putida L4 preparation is able to achieve the degradation of 1-OH PYR.

[0064] It should be noted that in the above process of verifying the degradation ability of the bacterial suspension,

[0065] Based on the above, due to the above-mentioned Pseudomonas putida ( Pseudomonas putida ) The use of L4 in the degradation of polycyclic aromatic hydrocarbon derivatives is still under exploration, therefore, this embodiment does not further limit the above use.

[0066] Example 3: This example provides a bacterial suspension for degrading polycyclic aromatic hydrocarbon derivatives, based on the Pseudomonas putida ( Pseudomonas putida ) L4. The preparation process of the bacterial suspension is similar to that of the bacterial suspension in Example 2.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A strain of Pseudomonas putida ( Pseudomonas putida ) L4, characterized in that, The Pseudomonas putida L4 was deposited in the China Center for Type Culture Collection on July 8, 2024, with a deposit number of CCTCC NO: M20241492. The 16S rDNA gene sequence of the Pseudomonas putida L4 is shown in SEQ ID NO:

1.

2. The use of Pseudomonas putida L4 according to claim 1, characterized in that: The Pseudomonas putida L4 is applied to degrade 1-hydroxypyrene.

3. A bacterial suspension for degrading polycyclic aromatic hydrocarbon derivatives, characterized in that: The bacterial suspension contains the Pseudomonas putida L4 described in claim 1.

4. The bacterial suspension for degrading polycyclic aromatic hydrocarbon derivatives according to claim 3, characterized in that: The bacterial suspension is prepared by the following steps: Step 1, activating Pseudomonas putida L4 overnight and shaking culturing to the logarithmic phase; Step 2: Collect the cells of Pseudomonas putida L4 and adjust the OD of the cells. 600 The value reached 1.0 to obtain a bacterial suspension.

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