Pseudomonas CH-1 and its application and application method

Abstract

The present invention relates to a Pseudomonas CH-1 and its application and application method. The Pseudomonas CH-1 of the present invention is deposited in the China Center for Type Culture Collection, with a deposit date of May 30, 2024, and a deposit number of CCTCC M 20241107. The bacterial body of Pseudomonas CH-1 is rod-shaped, without spores, without flagella, and easy to provoke. The bacterial lawn of Pseudomonas CH-1 grows along the streak, is an aerobic bacterium, and is Gram-negative. The Pseudomonas CH-1 strain of the present invention has a wide growth temperature and pH range, can effectively degrade harmful organic substances such as octenes, n-hexane, o-xylene, nonane, ethyl acetate or o-dichlorobenzene, and is particularly excellent in degradation efficiency for high concentrations of octenes as a carbon source, providing strong support for biological purification projects for treating octenes-containing pollutants.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Pseudomonas CH-1 and an application and an application method thereof. Background Art

[0002] Octene is a colorless liquid with a petroleum-like odor. While insoluble in water, it is soluble in most organic solvents, including ethanol, ether, acetone, and petroleum ether. This characteristic makes it useful as a solvent and in the preparation of plasticizers and surfactants. Accidental inhalation or oral ingestion of octene can cause certain harm to the human body, including mild irritation to the respiratory mucosa and conjunctiva. Symptoms of poisoning include burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea, and vomiting. Furthermore, octene poses significant environmental risks, such as water pollution.

[0003] Therefore, research on the efficient degradation of octenes in the environment is essential for human health. However, there are no reports or research on the efficient degradation of octenes by Pseudomonas sp. using octenes as the sole carbon source. There is an urgent need to address the degradation of organic pollutants in biopurification projects, especially octenes. Summary of the Invention

[0004] To address the above-mentioned problems in the prior art, the research and development team has developed a Pseudomonas sp. CH-1, its applications, and application methods. The Pseudomonas sp. CH-1 of the present invention can effectively degrade harmful organic compounds such as octene, n-hexane, o-xylene, nonane, ethyl acetate, or o-dichlorobenzene. It exhibits exceptional degradation efficiency against high concentrations of octene as a carbon source, and even higher efficiency against high concentrations of octene as the sole carbon source. This provides strong support for bio-purification projects that treat such pollutants.

[0005] On the one hand, the present invention provides a Pseudomonas CH-1, wherein the Pseudomonas CH-1 is Pseudomonas sp. CH-1, which is deposited in the China Center for Type Culture Collection, with a preservation date of May 30, 2024, a preservation number of CCTCC M 20241107, and an address of Wuhan University, Wuhan, China, with a postal code of 430072.

[0006] Furthermore, the Pseudomonas CH-1 bacteria of the present invention are rod-shaped, spore-free, flagella-free, and easy to lift. The bacterial lawn of the Pseudomonas CH-1 grows along the streak. The Pseudomonas CH-1 bacteria are aerobic and Gram-negative.

[0007] Furthermore, the colonies of the Pseudomonas CH-1 described in the present invention are dot-shaped, white, opaque, plump, smooth and moist.

[0008] On the other hand, the present invention provides a use of Pseudomonas CH-1 described in any one of the above items, wherein the Pseudomonas CH-1 is used to degrade organic matter, and the organic matter includes: octene, n-hexane, o-xylene, nonane, ethyl acetate or o-dichlorobenzene.

[0009] Furthermore, in the application of Pseudomonas CH-1 described in the present invention, the organic matter is octene.

[0010] In another aspect, the present invention provides a method for using the Pseudomonas CH-1 described in any one of the above items, the method comprising:

[0011] The Pseudomonas CH-1 is expanded and cultured to obtain a bacterial liquid as an enzyme source; then, an organic matter containing octenes is used as a substrate, an inorganic salt culture medium is used as a medium, and the enzyme source is added to perform a degradation reaction on the substrate under the conditions of pH = 4-9, a rotation speed of 100-300 r / min, and a temperature of 20°C to 40°C.

[0012] Furthermore, in the method of the present invention, the initial concentration of octene in the inorganic salt culture medium is ≤200 mg / L.

[0013] Furthermore, in the application method of the present invention, when the initial bacterial concentration is OD 600 =0.02, pH=5-8, temperature of 25°C-35°C, and degradation time of 24h-48h, the degradation rate of octene with an initial concentration of 72-200 mg / L is ≥85%.

[0014] Furthermore, in the application method of the present invention, the specific preparation method of the enzyme source includes:

[0015] Step 1) Slant culture: inoculate the Pseudomonas CH-1 on a slant LB solid culture medium and culture at 30° C. for 24 to 36 hours to obtain slant cells;

[0016] The composition of the slant LB solid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, 18-20 g / L agar, and the solvent is deionized water;

[0017] Step 2) Expanding the culture: Use an inoculation loop to pick bacteria from the slant and inoculate them into LB liquid culture medium. Cultivate at 30°C for 24-36 hours to obtain the OD 600 = 0.1~0.2 bacterial solution;

[0018] The LB liquid culture medium is composed of: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, and the solvent is deionized water.

[0019] Furthermore, in the application method of the present invention, the composition of the inorganic salt culture medium is: 4.5g / L Na2HPO4·12H2O, 1.0g / L KH2PO4, 2.5g / L (NH4)2SO4, 0.2g / L MgSO4·7H2O, 0.023g / L anhydrous CaCl2, 1mL / L trace element mother solution, and the solvent is deionized water;

[0020] The trace element mother solution is composed of: 1.0 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, 0.014 g / L H3BO3, 0.10 g / L MnSO4·4H2O, 0.10 g / L ZnSO4·7H2O, 0.02 g / L Na2MoO4·2H2O, and 0.02 g / L CoCl2·6H2O, and the solvent is deionized water.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] The Pseudomonas CH-1 of the present invention can effectively degrade organic substances such as octene, n-hexane, o-xylene, nonane, ethyl acetate or o-dichlorobenzene, especially substrates with octene as carbon source and energy source have a high degradation rate. For example: at the initial bacterial cell concentration (measured in OD 600 OD 600 = 0.01, with an initial octene concentration of 72–200 mg / L, a pH of 7, and a temperature of 30°C, the degradation rate reached over 91% after 44 hours of degradation. After 48 hours, the degradation rate reached over 96%. This is of great significance for the engineering application of biological octene purification. Furthermore, Pseudomonas CH-1 strain grows over a wide range of temperatures and pH values. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a transmission electron micrograph of Pseudomonas CH-1 of the present invention;

[0024] Figure 2 is a phylogenetic tree of Pseudomonas CH-1 of the present invention;

[0025] Figure 3 is a graph showing the growth and octene degradation rate of the Pseudomonas CH-1 bacteria of the present invention;

[0026] Figure 4 The degradation rate, OD, and octene degradation rate of Pseudomonas CH-1 in inorganic salt medium at pH values ​​of 4, 5, 6, 7, 8, and 9 in Examples 8 to 13 are shown. 600 Bar chart of CO2 values;

[0027] Figure 5 The degradation rate, OD, and octene degradation rate of Pseudomonas CH-1 at 25°C, 30°C, 35°C, and 40°C in Examples 14 to 17 are shown in Table 1. 600 Bar chart of CO2 values;

[0028] Figure 6 The degradation rate curves and OD values ​​of octene by Pseudomonas CH-1 when the initial octene concentrations in Examples 18 to 22 were 14.4 mg / L, 28.8 mg / L, 43.2 mg / L, 57.6 mg / L, and 72 mg / L, respectively. 600 Column chart of values. DETAILED DESCRIPTION

[0029] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described in detail with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the specific implementation method, they shall be carried out according to conventional conditions or conditions provided by the manufacturer.

[0030] When the following description refers to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. The terms used in this disclosure are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure. Specific implementation method one:

[0032] The present invention discloses a Pseudomonas CH-1, wherein the Pseudomonas CH-1 is Pseudomonas sp. CH-1, which is deposited in the China Center for Type Culture Collection on May 30, 2024, with a deposit number of CCTCC M20241107 and an address of Wuhan University, Wuhan, China, with a postal code of 430072.

[0033] In some embodiments, the Pseudomonas CH-1 bacteria are rod-shaped, without spores, without flagella, and are easily provoked. The bacterial lawn of the Pseudomonas CH-1 grows along the streak. The Pseudomonas CH-1 bacteria are aerobic and Gram-negative.

[0034] In some embodiments, the colonies of the Pseudomonas CH-1 are dot-shaped, white, opaque, plump, smooth and moist. Specific implementation method 2:

[0036] The use of Pseudomonas CH-1 provided in any one of the first specific embodiments of the present invention is used to degrade organic matter, wherein the organic matter includes: octene, n-hexane, o-xylene, nonane, ethyl acetate or o-dichlorobenzene.

[0037] In some embodiments, the organic compound is octene. Specific implementation method three:

[0039] A method for using Pseudomonas CH-1 provided in any one of the above items of the present invention comprises:

[0040] The Pseudomonas CH-1 is expanded and cultured to obtain a bacterial solution as an enzyme source; then, an organic matter containing octenes is used as a substrate, an inorganic salt culture medium is used as a medium, and the enzyme source is added to the substrate for degradation reaction under the conditions of pH = 4-9, a rotation speed of 100-300 r / min, and a temperature of 20°C to 40°C.

[0041] In some embodiments, the initial concentration of the octene in the inorganic salt culture medium is 14-200 mg / L.

[0042] In some embodiments, when the initial bacterial cell concentration is OD 600 =0.02, pH=5-8, temperature 25℃-35℃, and degradation time ≥24h, the degradation rate of octene with an initial concentration of 72-200 mg / L is ≥85%.

[0043] In some embodiments, the conditions for the above-mentioned degradation reaction are preferably: pH = 7-8, temperature 20°C to 35°C.

[0044] In some embodiments, in the above degradation reaction, the initial concentration of octene added is preferably 110-200 mg / L.

[0045] In some embodiments, the specific preparation method of the above enzyme source includes:

[0046] Step 1) Slant culture: The Pseudomonas CH-1 was inoculated onto a slant LB solid culture medium and cultured at 30°C for 24-36 hours to obtain slant cells;

[0047] The composition of the slant LB solid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, 18-20 g / L agar, and the solvent is deionized water;

[0048] Step 2) Expanding the culture: Use an inoculation loop to pick bacteria from the slant and inoculate them into LB liquid culture medium, culture at 30°C for 24-36 hours, and obtain the OD 600 = 0.1~0.2 bacterial solution;

[0049] The composition of the LB liquid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, and the solvent is deionized water.

[0050] In some embodiments, the pH value of the above steps 1) slant culture and 2) expansion culture is 5-9.

[0051] In some embodiments, the composition of the inorganic salt culture medium is: 4.5 g / L Na2HPO4·12H2O, 1.0 g / L KH2PO4, 2.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.023 g / L anhydrous CaCl2, 1 mL / L trace element mother solution, and the solvent is deionized water;

[0052] The composition of the above-mentioned trace element mother solution is: 1.0 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, 0.014 g / L H3BO3, 0.10 g / L MnSO4·4H2O, 0.10 g / L ZnSO4·7H2O, 0.02 g / L Na2MoO4·2H2O, 0.02 g / L CoCl2·6H2O, and the solvent is deionized water.

[0053] The present invention is further described in detail below with reference to specific embodiments.

[0054] Example 1:

[0055] like Figure 1 and Figure 2 As shown, the present invention is a Pseudomonas CH-1, Pseudomonas CH-1 is Pseudomonas sp. CH-1, which is deposited in the China Center for Type Culture Collection, with a deposit date of May 30, 2024, a deposit number of CCTCC M 20241107, and an address of Wuhan University, Wuhan, China, with a postal code of 430072.

[0056] Pseudomonas CH-1 bacteria are rod-shaped, without spores or flagella, and are easy to pick up. The bacterial lawn of Pseudomonas CH-1 grows along the streak. Pseudomonas CH-1 is an aerobic bacterium and is Gram-negative.

[0057] The colonies of Pseudomonas CH-1 are dot-shaped, white, opaque, plump, smooth and moist.

[0058] Example 2:

[0059] The isolation, purification and identification methods of Pseudomonas CH-1 of the present invention are as follows:

[0060] (1) Isolation and purification of Pseudomonas CH-1

[0061] Activated sludge was collected from a sewage treatment pool at a factory in Zhejiang Province. The lower layer of sludge, after settling, was mixed with an inorganic salt culture medium at a ratio of 1:2 (v / v). 3 L of the mixture was inoculated into a 5 L sludge acclimation tank (see Jin Xiaojun. Isolation and identification of a new dioxane-degrading strain, degradation characteristics, and preparation of a bacterial agent [D]. Zhejiang University of Technology, 2012). Octene was used as a substrate, serving as a carbon source and energy source, and the strain was acclimated and cultured at room temperature. After nearly 20 days, 5 mL of sludge was taken from the acclimation tank and added to a shake flask containing 50 mL of inorganic salt culture medium. It was found that the acclimated sludge could stably degrade 110 mg / L of octenene per day in the shake flask, with a degradation rate of 80-90% (shake flask experimental conditions: 30°C, 160 r / min), thus obtaining an acclimated sample.

[0062] 1mL and 2mL of sludge samples were taken from the shake flask and transferred to other shake flasks for further performance testing. It was found that the transferred sludge could stably degrade octene at a concentration of 110mg / L per day in the shake flask. The sludge sample was further transferred for enrichment for 6 generations (1mL each time), and then the sludge after 6 generations of enrichment was transferred for 10 generations. -1 ~10 -6 The plates were spread at multiple times, and single colonies were picked. The degradation activity was determined using octene as a substrate, and the strain CH-1 with octene degradation activity was obtained after separation and purification.

[0063] (2) Identification of Pseudomonas CH-1

[0064] The morphological characteristics of the Pseudomonas CH-1 strain are as follows: the bacteria are rod-shaped, without spores and flagella; the colonies are dot-shaped, white, opaque, plump, smooth and moist, easy to pick up, and the fungus grows along the lines.

[0065] The physiological and biochemical characteristics of the strain of Pseudomonas CH-1 are: aerobic and Gram-negative.

[0066] The strain of Pseudomonas sp. CH-1 was sequenced with 16S rDNA and fully automated biolog identification was performed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA sequence of the strain (SEQ ID NO. 1) is as follows:

[0067] (Genebank accession number is PQ466000):

[0068]

[0069] The 16S rDNA sequence of Pseudomonas CH-1 strain was uploaded to the Ezbiocloud.net website and compared with the standard strains on the website. The bacterial phylogenetic tree was constructed using the Neighbor-Joining method using MEGA5.05 software and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree is shown in Figure 2. Figure 2 , thus determining that the strain CH-1 was (Pseudomonas sp.), named Pseudomonas sp. CH-1, and deposited in the China Center for Type Culture Collection on May 30, 2024, with the deposit number CCTCC No: M 20241107, address: Wuhan University, Wuhan, China, Postal Code 430072.

[0070] like Figure 1 This is a transmission electron microscope photo of Pseudomonas CH-1. Figure 2 This is the phylogenetic tree of Pseudomonas sp. CH-1.

[0071] Example 3:

[0072] The invention discloses an application of Pseudomonas CH-1, wherein the Pseudomonas CH-1 is used to degrade organic matter, and the organic matter includes octene, n-hexane, o-xylene, nonane, ethyl acetate or o-dichlorobenzene.

[0073] In this Example 3, the Pseudomonas CH-1 strain of Example 1 was used to perform degradation experiments on octene, n-hexane, o-xylene, nonane, ethyl acetate and o-dichlorobenzene to analyze the broad spectrum of substrate degradation capabilities of Pseudomonas CH-1.

[0074] The three wastes generated by actual industrial processes often contain multiple pollutants. Biodegradation is highly substrate-specific, so examining the degradation capacity of Pseudomonas CH-1 against some common pollutants is particularly important. The degradation performance of Pseudomonas CH-1 against other hydrocarbons is shown in Table 1.

[0075] Table 1 Substrate spectrum of strain CH-1

[0076]

[0077] Table 1 Description: "++" indicates that Pseudomonas CH-1 grows well and the degradation rate of the substrate reaches more than 80%; "+" indicates that Pseudomonas CH-1 can grow; "-" indicates that the substrate cannot be degraded.

[0078] As shown in Table 1, Pseudomonas CH-1 not only degrades alkanes such as nonane, n-octane, methylcyclohexane, and n-hexane, but also degrades pollutants such as octene, ethyl acetate, o-xylene, and o-dichlorobenzene to varying degrees, demonstrating its broad substrate spectrum. The ability of microorganisms to degrade organic pollutants generally depends on the molecular structure of the pollutant and its ability to lose or gain electrons. Pseudomonas CH-1 was unable to directly utilize tetrahydrofuran, likely due to steric hindrance, which makes these substances difficult to biodegrade.

[0079] Example 4:

[0080] Preparation of enzyme source for octene degradation by Pseudomonas CH-1:

[0081] Step 1) Slant culture: Pseudomonas CH-1 was inoculated on a slant LB solid medium and cultured at 30°C for 24-36 hours to obtain slant cells;

[0082] The composition of the slant LB solid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, 18-20 g / L agar, and the solvent is deionized water; the pH value of the slant LB solid culture medium is 5-9.

[0083] Step 2) Expanding the culture: Use an inoculation loop to pick bacteria from the slant and inoculate them into LB liquid culture medium, culture at 30°C for 24-36 hours, and obtain the OD 600 = 0.1~0.2 bacterial solution; the pH value of LB liquid culture medium is 5~9.

[0084] The composition of LB liquid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, and the solvent is deionized water.

[0085] Example 5:

[0086] The Pseudomonas CH-1 of the present invention is applied to the method for degrading octene, and the degradation method comprises:

[0087] The bacterial liquid obtained by expanding the culture of Pseudomonas CH-1 in Example 4 was used as an enzyme source; then, an organic matter containing octene was used as a substrate, an inorganic salt culture medium was used as a medium, and the enzyme source was added to the substrate for degradation reaction under the conditions of pH = 4-9, rotation speed 100-300 r / min, and temperature 20°C-40°C.

[0088] The initial concentration of octene in the inorganic salt culture medium is 14~200 mg / L.

[0089] The composition of the inorganic salt culture medium is: 4.5 g / L Na2HPO4·12H2O, 1.0 g / L KH2PO4, 2.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.023 g / L anhydrous CaCl2, 1 mL / L trace element stock solution, and the solvent is deionized water;

[0090] The composition of the trace element mother solution is: 1.0 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, 0.014 g / L H3BO3, 0.10 g / L MnSO4·4H2O, 0.10 g / L ZnSO4·7H2O, 0.02 g / L Na2MoO4·2H2O, 0.02 g / L CoCl2·6H2O, and the solvent is deionized water.

[0091] Example 6:

[0092] The only difference between this embodiment 6 and embodiment 5 is that:

[0093] The initial bacterial concentration in the inorganic salt medium is OD 600 =0.02, pH=7~8, temperature of 25℃~35℃, and degradation time of 24h~48h, the degradation rate of octene with an initial concentration of 110~200 mg / L is ≥85%.

[0094] Example 7:

[0095] The only difference between this embodiment 7 and embodiment 5 is that:

[0096] Octene was used as the sole carbon source, and the enzyme source bacterial solution prepared in Example 4 was taken to have an OD of 600 = 0.1, the enzyme source bacterial solution OD 600 =0.1 was inoculated into 50 mL of fresh inorganic salt medium containing 14 mg / L octenes, so that the initial concentration of Pseudomonas CH-1 cells was expressed as OD 600 =0.02, the pH value of the inorganic salt medium is 7. The cells were cultured in a shaker at 30°C and 160 r / min. The degradation rate of octene was measured at 15h, 21h, 24h, 37h, and 40h. A portion of the bacterial solution was drawn out with a 5mL syringe to measure the bacterial OD value. The results are shown in the figure. Figure 3 .

[0097] from Figure 3 It can be seen that during the experiment, as time went on, the bacterial concentration gradually increased. With octene as the substrate, the bacterial concentration reached the maximum after 40 h of cultivation. 600The concentration of octene was about 0.036, and the octene degradation rate reached over 92%. This Example 7 shows that Pseudomonas CH-1 can use octene as the sole carbon source and energy source for growth and reproduction, and its octene degradation ability is stable and efficient.

[0098] Example 8:

[0099] The only difference between this embodiment 8 and embodiment 5 is that:

[0100] The pH value of the inorganic salt medium was adjusted to 4 with 1 mol / L NaOH aqueous solution or 1 mol / L H2SO4 aqueous solution. The bacterial solution prepared by the method of Example 4 was inoculated under the condition that the initial concentration of octene in the inorganic salt medium was 110 mg / L. The initial bacterial cell concentration OD 600 = 0.01. The sample was shaken in a constant temperature shaker at 30℃ and 160r / min. After 28h of incubation, samples were taken to measure the octene degradation rate and OD 600 Values ​​and CO2 values.

[0101] Example 9:

[0102] The only difference between Example 9 and Example 8 is that the pH value of the inorganic salt culture medium is 5.

[0103] Example 10:

[0104] The only difference between Example 10 and Example 8 is that the pH value of the inorganic salt culture medium is 6.

[0105] Example 11:

[0106] The only difference between Example 11 and Example 8 is that the pH value of the inorganic salt culture medium is 7.

[0107] Example 12:

[0108] The only difference between Example 12 and Example 8 is that the pH value of the inorganic salt culture medium is 8.

[0109] Example 13:

[0110] The only difference between Example 13 and Example 8 is that the pH value of the inorganic salt culture medium is 9.

[0111] In the above examples 8 to 13, the effect of pH value on the degradation of octene by Pseudomonas CH-1 was studied, and the octene degradation rate and OD in the reaction solution were finally measured. 600 Value and CO2 value, the result is as follows Figure 4 As shown. Figure 4It can be seen that at pH values ​​of 7 and 8, Pseudomonas CH-1 has a high degradation rate of octene; especially at pH 7, the degradation rate of octene by Pseudomonas CH-1 reaches the best, about 100%; at the same time, the growth amount and CO2 value of Pseudomonas CH-1 also have a consistent trend with octene or octene degradation rate.

[0112] Example 14:

[0113] The only difference between this embodiment 14 and embodiment 5 is that:

[0114] The initial concentration of octene in the inorganic salt medium was 110 mg / L. The bacterial solution prepared by the method of Example 4 was inoculated into the inorganic salt medium. The initial bacterial concentration OD 600 = 0.01. The mixture was cultured in a shaker at a constant temperature of 160 r / min at pH = 7 and a temperature of 25°C. After 24 hours of culture, samples were taken to measure the degradation rate of octene and OD 600 Values ​​and CO2 values.

[0115] Example 15:

[0116] The only difference between Example 15 and Example 14 is that the temperature is 30°C.

[0117] Example 16:

[0118] The only difference between Example 16 and Example 14 is that the temperature is 35°C.

[0119] Example 17:

[0120] The only difference between Example 17 and Example 14 is that the temperature is 40°C.

[0121] In the above examples 14 to 17, the effect of temperature on the degradation of octene by Pseudomonas CH-1 was studied, and the octene degradation rate and OD in the reaction solution were finally measured. 600 Value and CO2 value, the result is as follows Figure 5 As shown. Figure 5 It can be seen that within the temperature range of 25-35℃, Pseudomonas CH-1 has a high degradation rate for both octenyl and octenyl; especially at 30℃, the degradation rate of octenyl by Pseudomonas CH-1 reaches the best, reaching more than 86%. At the same time, the growth amount and CO2 value produced by Pseudomonas CH-1 also have a trend consistent with the octenyl degradation rate.

[0122] Example 18:

[0123] The only difference between this embodiment 18 and embodiment 5 is that:

[0124] Under suitable culture conditions (pH=7, t=30℃), the degradation of octenes at different concentrations by Pseudomonas sp. CH-1 was studied.

[0125] The initial concentration of octene in the inorganic salt medium was 14.4 mg / L, and the bacterial solution prepared in Example 4 was inoculated, and the initial bacterial concentration OD 600 = 0.01, cultured in a shaker at 160 r / min at 30°C and pH 7, and sampled after 22.5 h of degradation to measure the octene degradation rate and OD 600 value.

[0126] Example 19:

[0127] The only difference between Example 19 and Example 18 is that the initial concentration of octene in the inorganic salt culture medium is 28.8 mg / L, and sampling is performed after 27.5 hours of degradation.

[0128] Example 20:

[0129] The only difference between Example 20 and Example 18 is that the initial concentration of octene in the inorganic salt culture medium is 43.2 mg / L, and sampling is performed after 27.5 hours of degradation.

[0130] Example 21:

[0131] The only difference between Example 21 and Example 18 is that the initial concentration of octene in the inorganic salt culture medium is 57.6 mg / L, and sampling is performed after 30 hours of degradation.

[0132] Example 22:

[0133] The only difference between Example 22 and Example 18 is that the initial concentration of octene in the inorganic salt culture medium is 72 mg / L, and sampling is performed after 35 hours of degradation.

[0134] In the above Examples 18 to 22, the effect of temperature on the degradation of octene by Pseudomonas CH-1 was studied, and the octene degradation rate and OD in the reaction solution were finally measured. 600 Value and CO2 value, the result is as follows Figure 6 As shown. Figure 6 As can be seen from the results, Pseudomonas CH-1 achieved a 100% degradation rate for octene at concentrations below 72 mg / L, converting it almost completely to CO2 and H2O. Furthermore, the bacterial population stabilized at the highest octene degradation rate at different octene concentrations.

[0135] The present invention is described by the above-mentioned specific embodiments. It should be understood by those skilled in the art that various changes and equivalent substitutions may be made to the present invention without departing from the scope of the present invention. Parts not described in detail in the present specification are well-known technologies to those skilled in the art. In addition, various modifications may be made to the present invention for specific situations or specific circumstances without departing from the scope of this new use. Therefore, the present invention is not limited to the specific embodiments disclosed, but should include all embodiments falling within the scope of the claims of the present invention.

Claims

1. A Pseudomonas sp. CH-1, characterized in that The Pseudomonas CH-1 is a Pseudomonas Pseudomonas sp.) CH-1, deposited in China Center for Type Culture Collection, the deposit date is May 30, 2024, the deposit number is: CCTCC M20241107, address: Wuhan University, Wuhan, China, Postal Code 430072.

2. A use of Pseudomonas CH-1 according to claim 1, characterized in that: The Pseudomonas CH-1 is used to degrade organic matter, and the organic matter includes: octene, nonane, n-octane, n-hexane, ethyl acetate, dichloromethane, dichloroethylene, methylcyclohexane, isopropyl alcohol, o-xylene, or o-dichlorobenzene.

3. The use of Pseudomonas CH-1 according to claim 2, characterized in that The organic matter is octene.

4. A method for using Pseudomonas CH-1 according to claim 1, characterized in that: The method comprises: The Pseudomonas CH-1 is expanded and cultured to obtain a bacterial liquid as an enzyme source; then, an organic matter containing octenes is used as a substrate, an inorganic salt culture medium is used as a medium, and the enzyme source is added to perform a degradation reaction on the substrate under the conditions of pH = 4-9, a rotation speed of 100-300 r / min, and a temperature of 20°C to 40°C.

5. The application method according to claim 4, characterized in that: The initial concentration of octene in the inorganic salt culture medium is ≤200 mg / L.

6. The application method according to claim 5, characterized in that At the initial bacterial concentration OD 600 = 0.02, pH = 5-8, temperature 25°C-35°C, and degradation time 24h-48h, the degradation rate of octene with an initial concentration of 72-200 mg / L is ≥85%.

7. The application method according to claim 6, characterized in that: The specific preparation method of the enzyme source includes: Step 1) Slant culture: inoculate the Pseudomonas CH-1 on a slant LB solid culture medium and culture at 30° C. for 24 to 36 hours to obtain slant bacteria; The composition of the slant LB solid culture medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, 18-20 g / L agar, and the solvent is deionized water; Step 2) Expanding the culture: Use an inoculation loop to pick bacteria from the slant and inoculate them into LB liquid culture medium. Cultivate at 30°C for 24-36 hours to obtain the OD 600 = 0.1~0.2 bacterial solution; The LB liquid culture medium is composed of: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder, and the solvent is deionized water.

8. The application method according to claim 7, characterized in that: The composition of the inorganic salt culture medium is: 4.5 g / L Na2HPO4·12H2O, 1.0 g / L KH2PO4, 2.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.023 g / L anhydrous CaCl2, 1 mL / L trace element mother solution, and the solvent is deionized water; The trace element mother solution is composed of: 1.0 g / L FeSO4·7H2O, 0.02 g / L CuSO4·5H2O, 0.014 g / L H3BO3, 0.10 g / L MnSO4·4H2O, 0.10 g / L ZnSO4·7H2O, 0.02 g / L Na2MoO4·2H2O, and 0.02 g / L CoCl2·6H2O, and the solvent is deionized water.

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