Pseudomonas and its applications

Abstract

The present application relates to the field of microorganism, disclose a kind of pseudomonas sp. And its application, the preservation number of the pseudomonas sp. CGMCC NO.31317, can survive in a wide range of alkane concentration, and with alkane as carbon source and energy source to grow and reproduce, while fully decomposing alkane.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to Pseudomonas sp. and its applications. Background Technology

[0002] As a crucial pillar of my country's economy, the petrochemical industry inevitably generates various types of wastewater during its processing and production. With increasing national emphasis on environmental protection, the Beijing-Tianjin-Hebei region has implemented stricter requirements for suspended solids, COD, and ammonia nitrogen in its wastewater discharge. Wastewater undergoing advanced treatment must meet the Class IV surface water standard, making existing wastewater treatment processes in refining and chemical enterprises insufficient to meet these new environmental demands. To satisfy the needs of water resource protection, reclaimed water utilization, and continuous pollutant reduction, the advanced removal of organic pollutants has become a key focus in the water treatment field.

[0003] Wastewater treatment in my country's refining and chemical enterprises is mainly achieved through source reduction, process control, and end-of-pipe treatment. Early wastewater treatment primarily involved a "physicochemical pretreatment + biological organic matter removal" process before discharge. With increasingly stringent wastewater discharge standards, it is necessary to add technologies such as biological total nitrogen removal and advanced oxidation for organic matter removal to the existing processes to achieve deep removal of organic matter and total nitrogen. Deep organic matter removal often employs advanced oxidation processes, first improving the biodegradability of wastewater through oxidation, followed by further biological treatment. Currently, the most researched advanced oxidation technologies include ozone catalytic oxidation, Fenton oxidation, and electrochemical oxidation.

[0004] While ozone oxidation has a strong ability to decolorize and remove organic pollutants, it suffers from high ozone consumption, low utilization rate, long hydraulic retention time, high operating costs, and the risk of ozone emission polluting the atmosphere. Fenton oxidation technology has low investment and relatively low direct operating costs, but the generated sludge is hazardous waste and produced in large quantities, with disposal costs exceeding 20 yuan / ton of water. Electrochemical oxidation is highly effective at removing organic matter from wastewater, but its disadvantages include very high investment costs, high energy consumption, rapid electrode wear, and high operating costs. Although the aforementioned advanced oxidation methods can effectively remove organic matter from wastewater, their generally high treatment costs and the risk of secondary pollution limit their widespread application.

[0005] Biochemical methods for removing organic matter are low-cost and stable, making them the primary technology used in industrial wastewater treatment. However, wastewater typically exhibits low residual pollutant concentrations after two stages of biochemical treatment, resulting in an imbalanced nutrient ratio and persistent, difficult-to-degrade organic matter, thus increasing the difficulty of advanced wastewater treatment. Therefore, conventional microbial treatment methods are insufficient to meet the requirements for advanced removal of recalcitrant organic pollutants from wastewater. It is necessary to develop efficient technologies for removing recalcitrant organic pollutants, and to achieve advanced removal of these pollutants from wastewater by screening for microorganisms with specific degradation effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and to provide a strain of Pseudomonas and its application, which can effectively degrade alkanes and remove COD from wastewater.

[0007] To achieve the above objectives, the first aspect of the present invention provides a strain of Pseudomonas, the preservation number of which is CGMCC NO.31317.

[0008] A second aspect of the present invention provides a microbial agent containing Pseudomonas as described above; preferably, the microbial agent is a liquid microbial agent.

[0009] A third aspect of the present invention provides the use of the aforementioned Pseudomonas bacteria or the aforementioned bacterial agent in the degradation of alkane.

[0010] The fourth aspect of the present invention provides the application of the aforementioned Pseudomonas or the aforementioned bacterial agent in wastewater treatment.

[0011] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with the aforementioned Pseudomonas bacteria or the aforementioned bacterial agent.

[0012] The Pseudomonas bacteria described in this invention can effectively utilize alkanes as a carbon source, thereby achieving effective removal of COD from petrochemical wastewater.

[0013] Biological Preservation

[0014] The *Pseudomonas* sp. of this invention, with accession number BHYAG-1, was deposited on July 16, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 31317. Attached Figure Description

[0015] Figure 1 The colony morphology of Pseudomonas BHYAG-1 of this invention is shown.

[0016] Figure 2 This is a scanning electron microscope image of the Pseudomonas BHYAG-1 strain of the present invention. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The first aspect of this invention provides a strain of Pseudomonas, the preservation number of which is CGMCCNO.31317. In this invention, it is designated as BHYAG-1.

[0019] According to the present invention, the 16S rDNA sequence of the Pseudomonas is shown in SEQ ID NO: 1.

[0020] SEQ ID NO: 1:

[0021]

[0022] The optimal growth pH for *Pseudomonas* described in this invention is 6-8, and the optimal growth temperature is 28-35℃. On LB agar plates, it exhibits white, irregularly raised, dry colonies. Figure 1 As shown.

[0023] The morphological characteristics of the *Pseudomonas* strain described in this invention were observed using a scanning electron microscope. Its morphology is as follows: Figure 2 As shown, the bacterial cells are rod-shaped.

[0024] A second aspect of the present invention provides a microbial agent containing Pseudomonas as described above.

[0025] Preferably, the bacterial agent is a liquid bacterial agent.

[0026] In this invention, the preparation method of the bacterial agent may include: culturing the Pseudomonas bacteria as described above in a liquid culture medium and resuspending them in a buffer solution.

[0027] Preferably, the viable count of Pseudomonas in the bacterial agent is not less than 10. 9 CFU / mL, preferably 10 9 -10 12 CFU / mL.

[0028] A third aspect of the present invention provides the use of the aforementioned Pseudomonas bacteria or the aforementioned bacterial agent in the degradation of alkane.

[0029] Preferably, the alkane can be C9-C. 34 At least one of the alkanes.

[0030] According to the present invention, when using the Pseudomonas BHYAG-1 provided by the present invention to degrade alkanes, the amount of Pseudomonas BHYAG-1-containing bacterial agent is such that the viable count in the system containing alkane is 10. 9 -10 10 CFU / mL.

[0031] According to a preferred embodiment of the present invention, the Pseudomonas BHYAG-1 provided by the present invention can stably survive in an environment with an alkane concentration of 1-1000 mg / L and grow and reproduce using alkane as a carbon source, and its ability to degrade alkane reaches more than 100 mg / (L·d).

[0032] Unless otherwise stated, the degradation rate of alkanes refers to the degradation of C9-C64 hydrocarbons. 34 The percentage of alkanes that have degraded into C8 and lower alkanes.

[0033] According to a preferred embodiment of the invention, the alkane may be derived from paraffin.

[0034] The fourth aspect of the present invention provides the application of the aforementioned Pseudomonas or the aforementioned bacterial agent in wastewater treatment.

[0035] In this invention, the wastewater can be petrochemical wastewater.

[0036] Furthermore, the pollutants in the wastewater may include at least one of the alkanes.

[0037] Preferably, the wastewater may contain C9-C 34 At least one of the alkanes.

[0038] Preferably, the alkane may be provided as diesel oil and / or paraffin.

[0039] Preferably, the alkane content in the wastewater can be 50-1000 μg / L, more preferably 100-400 μg / L.

[0040] Preferably, the COD of the wastewater can be 30-500 mg / L, and more preferably 50-200 mg / L.

[0041] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with the aforementioned Pseudomonas bacteria or the aforementioned bacterial agent.

[0042] Preferably, the contact method may be to contact the liquid bacterial agent with the wastewater to be treated.

[0043] Preferably, the contact conditions may include a temperature of 25-35°C and a time of 12-48 hours.

[0044] According to the present invention, the dosage of the *Pseudomonas* can be determined based on the degree of pollution in the wastewater. In some specific embodiments of the present invention, the dosage of the *Pseudomonas*, based on the viable count, is not less than 10 per liter of wastewater. 11 CFU, preferably 10 12 -10 13 CFU. The wastewater can be as described above.

[0045] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0046] Unless otherwise specified, all reagents and materials used in the following examples were purchased from reputable chemical reagent suppliers and were of analytical purity.

[0047] The culture medium used in this invention is as follows:

[0048] Inorganic salt liquid culture medium: ammonium chloride 0.67g, sodium nitrate 0.06g, magnesium sulfate heptahydrate 0.1g, calcium chloride 0.1g, dipotassium hydrogen phosphate trihydrate 1.5g, potassium dihydrogen phosphate 0.5g, ferric chloride hexahydrate 0.1g, deionized water 1L, pH 7-7.2.

[0049] Inorganic salt solid culture medium: Add 20 g / L of purified agar to inorganic salt liquid culture medium.

[0050] Paraffin screening solid culture medium: 1 g / L of paraffin was added to an inorganic salt solid culture medium.

[0051] LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1L deionized water, pH 7.2.

[0052] LB plate medium composition: 20 g / L of purified agar is added to LB liquid medium.

[0053] Alkane removal rate = (Alkane content before degradation / Alkane content after degradation) / Alkane content before degradation × 100%;

[0054] The COD test method refers to HJ / T 399-2007 Determination of Chemical Oxygen Demand in Water - Rapid Digestion Spectrophotometric Method.

[0055] Example 1

[0056] This embodiment is used to illustrate the ability of the Pseudomonas BHYAG-1 strain described in this invention to degrade alkanes.

[0057] The glycerol-preserved Pseudomonas BHYAG-1 bacterial suspension was inoculated onto paraffin solid selection medium using a disposable inoculation loop and cultured at 30°C for 7 days in a biochemical incubator. It was able to grow on paraffin solid selection medium with paraffin as the sole carbon source, so it can be considered that this strain has the ability to degrade alkanes.

[0058] Example 2

[0059] This embodiment is used to illustrate the bacterial agent and its preparation method described in this invention.

[0060] After activating and culturing *Pseudomonas* BHYAG-1 on a slant for 24 hours, one loopful was added to LB liquid medium. 100 mL of LB liquid medium was placed in a 250 mL Erlenmeyer flask, and the flask was incubated at 30°C and 180 rpm for 20 hours to obtain the bacterial suspension. The suspension was centrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the bacterial cells were retained. The bacterial cells were resuspended in sterile 0.01 mol / L PBS buffer to obtain the bacterial agent described in this invention. The OD600 of the bacterial agent was 2.4, and the viable count was 2.8 × 10⁻⁶. 10 CFU / ml.

[0061] Example 3

[0062] This embodiment illustrates the degradation effect of the Pseudomonas BHYAG-1 strain of the present invention on alkanes.

[0063] (1) Take 100mL of inorganic salt liquid culture medium and place it in a 250mL Erlenmeyer flask. Seal the flask with a breathable sealing film and sterilize it in an autoclave at 121℃ for 20min. After cooling, add 1g / L of paraffin to the Erlenmeyer flask.

[0064] (2) Add the above-mentioned microbial agent to make the viable bacteria count in the system 2.7 × 10⁻⁶. 9 CFU / mL, sealed with breathable sealing film, and treated on a shaker at 30℃ and 180rpm for 3d, 5d, and 7d to obtain experimental group samples. In step (1), paraffin was added to the inorganic salt culture medium, but no bacterial agent was added. The blank control group was obtained by treating it in the same way for 3d, 5d, and 7d.

[0065] (3) Add hexane at a volume ratio of 1:10 to the experimental group and blank control group samples obtained in the above steps, extract for 20 min, let stand until complete separation, take the upper organic phase, and measure the C9-C of organic compounds in the blank control group and experimental group by gas chromatography-mass spectrometry. 34 The total content of alkanes and the alkane removal rate at different reaction times are shown in Table 1.

[0066] Table 1

[0067] Reaction time (d) Blank control group (mg / L) Experimental group (mg / L) Alkane removal rate (%) 3 985 896.4 9 5 985 847.1 14 7 985 216.7 78

[0068] It can be seen that the *Pseudomonas* BHYAG-1 provided by this invention can stably survive in an environment with an alkane concentration of 985 mg / L and grow and reproduce using alkane as the sole carbon source. In contrast, existing *Pseudomonas* strains can hardly decompose alkane at an alkane concentration of 800 mg / L, meaning that the strains can hardly grow and reproduce at this concentration. These results demonstrate that the *Pseudomonas* BHYAG-1 provided by this invention can stably grow and reproduce within a wide range of alkane concentrations.

[0069] Secondly, the *Pseudomonas* BHYAG-1 provided by this invention, when decomposing alkanes at a concentration of 985 mg / L, achieved an alkane degradation rate of 109.76 mg / (L·d) after 7 days. In contrast, existing *Pseudomonas* strains, at alkane concentrations where the strain can survive (<800 mg / L), degrade alkanes at approximately 80% / d. Even if they could survive and decompose alkanes at a concentration of 985 mg / L, their degradation rate would not exceed 110 mg / L·d. -1 .

[0070] In summary, the strain provided by this invention can stably survive, grow and reproduce under high concentrations of alkanes (>900 mg / L), and fully decompose alkanes. Its ability to decompose alkanes is significantly higher than that of the Pseudomonas species mentioned in existing published texts.

[0071] Example 4

[0072] This embodiment illustrates the treatment effect of the Pseudomonas BHYAG-1 strain of the present invention on petrochemical wastewater.

[0073] The biochemical effluent containing saline wastewater from a petrochemical enterprise was treated using Pseudomonas BHYAG-1. The wastewater had a conductivity of 2800 μS / cm and a COD of 69 mg / L.

[0074] 100 mL of the above-mentioned saline wastewater from a petrochemical enterprise was placed in a 250 mL Erlenmeyer flask, and the above-mentioned bacterial agent was added to make the viable bacteria count in the reaction system 1 × 10⁻⁶. 9 CFU / mL, sealed with a breathable sealing film, and treated on a shaker at 30℃ and 180rpm for 1 day to obtain the experimental group sample. 110mL of biochemical effluent containing saline from a petrochemical plant was treated without adding any bacterial agent and treated in the same way for 1 day to obtain the blank control group.

[0075] ① Equal amounts of control and experimental group samples were taken, and organic pollutants in the control and experimental group samples were extracted with the organic solvent dichloromethane. After drying with anhydrous sodium sulfate, the samples were purged with nitrogen and analyzed by GC-MS to determine the C content in the control and experimental group samples. 12 -C 34 The amount of mixed alkanes.

[0076] ② Take equal amounts of samples from the control group and the experimental group, centrifuge at 10000 r / min for 3 min, and then filter the supernatant through a 0.22 micrometer membrane to obtain the COD result.

[0077] The mixed alkane content and COD of the control group sample were 120.73 μg / L and 69 mg / L, respectively. After degradation by the strain provided by this invention, the mixed alkane content and COD of the sample decreased to 59.16 μg / L and 35 mg / L, respectively. It can be seen that Pseudomonas BHYAG-1 has the ability to degrade pollutants in petrochemical wastewater, and the removal rates of mixed alkane and COD reached 51% and 49.3% after 24 hours, respectively.

[0078] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas sp., characterized in that, The preservation number of the Pseudomonas is CGMCC NO.31317.

2. The *Pseudomonas* strain according to claim 1, wherein, The 16S rDNA sequence of the Pseudomonas is shown in SEQ ID NO:

1.

3. A microbial agent, wherein, The bacterial agent contains the Pseudomonas aeruginosa as described in claim 1 or 2; Preferably, the bacterial agent is a liquid bacterial agent.

4. The microbial agent according to claim 3, wherein, The viable count of Pseudomonas aeruginosa in the bacterial agent is not less than 10. 9 CFU / mL, preferably 10 9 -10 12 CFU / mL.

5. The use of the Pseudomonas bacillus of claim 1 or 2 or the bacterial agent of claim 3 or 4 in the degradation of alkanes.

6. The application according to claim 5, characterized in that, The alkane is C9-C. 34 At least one of the alkanes.

7. The application of the Pseudomonas aeruginosa as described in claim 1 or 2, or the bacterial agent as described in claim 3 or 4, in wastewater treatment.

8. The application according to claim 7, wherein, The wastewater is petrochemical wastewater.

9. The application according to claim 7, wherein, The pollutants in the wastewater include at least one alkane, preferably C9-C6. 34 At least one of the alkanes; Preferably, the alkane content in the wastewater is 50-1000 μg / L, more preferably 100-400 μg / L; 1240665 I97250BHY Preferably, the COD of the wastewater is 30-500 mg / L, and more preferably 50-200 mg / L.

10. A method for treating wastewater, characterized in that, The treatment method includes contacting the Pseudomonas aeruginosa as described in claim 1 or 2, or the bacterial agent as described in claim 3 or 4, with the wastewater.

11. The processing method according to claim 10, wherein, The amount of *Pseudomonas* used, based on viable count, is not less than 10 per liter of wastewater. 11 CFU, preferably 10 12 -10 13 CFU.

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