A strain of Pseudomonas and its application in degradation of pineneol and remediation of contaminated soil

Abstract

The present invention discloses a strain of Pseudomonas and its application in degrading pinene alcohol and remediating contaminated soil. The deposit number of the Pseudomonas sp. S4 is GDMCC No: 63948, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 31, 2023. The present invention provides a strain of Pseudomonas sp. S4 with the function of efficiently degrading pinene alcohol. The degradation rate of 350 mg / L pinene alcohol after culturing for 3 days under laboratory conditions is as high as 95%, and a composite surfactant containing monorhamnolipid and dirhamnolipid is produced, which can be used to repair soil contaminated by pinene alcohol and increase the diversity of microbial species in the soil. The combination with rhamnolipid or biochar can play a synergistic effect, and the repair effect is more significant, which provides a new technical option for the treatment of pinene alcohol pollution and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Pseudomonas and application thereof in degrading pineneol and remediating contaminated soil. Background Art

[0002] Flotation reagents are drugs used for mineral sorting. They perform flotation by changing the physical and chemical properties and hydrophobicity of different mineral surfaces. With the changing times, due to the increasing demand for mineral resources, the scale of mines and the intensity of mining are also expanding, resulting in a large number of organic flotation reagents being widely used in the mining process. There are many types of mining flotation reagents, and the commonly used ones include collectors (xanthate, black medicine, white medicine, fatty acids, mineral oils, etc.), foaming agents (pineneol, cresol oil, alcohols, etc.) and adjusters (lime, copper sulfate, sodium sulfide, sodium carbonate, sulfur dioxide). During the mining process, flotation reagents can penetrate into the ground and pollute groundwater and farmland.

[0003] Terpineol, commonly known as terpineol, is one of the most widely used foaming agents in flotation because of its simple preparation method, low cost, strong foam stability formed in mining, and obvious collection effect. Terpineol is mainly made of turpentine oil as a raw material, added with an emulsifier, and hydrated at room temperature and dehydrated. Terpineol has a ring structure and has three isomers (α-terpineol, β-terpineol and γ-terpineol). It is usually a light yellow oily transparent liquid with a pungent odor, flammable, slightly soluble in water and glycerin, easily soluble in ethanol, and oxidizable in the air.

[0004] Terpineol has medium to strong toxicity. Due to its ring structure and side chain, it can stably exist in the environment, is difficult to degrade, and a large amount of use in mining and mineral processing has caused serious soil pollution, seriously threatening ecological safety. How to economically and effectively remove these harmful substances from the environment is a difficult problem that current technicians in this field are still working on. In the current prior art, terpineol in the soil is mainly removed by methods such as physical methods (ozonolysis, ultraviolet irradiation, activated carbon adsorption), chemical methods (lime sedimentation, sodium hypochlorite oxidation) and biological methods (plant enrichment), but the risks and problems such as high processing cost, large difficulty in realizing industrialized large-scale processing, and introduction of other pollutants are common. Microbiological method has the advantages of simple operation, economical and applicable, no secondary pollution, but terpineol has a wide spectrum of antifungal and antibacterial capabilities, and screening the microorganisms that can degrade terpineol is very challenging, and there are few reports on the biodegradation pathway and mechanism of terpineol at present. Summary of the invention

[0005] In order to solve the problem in the prior art of lack of efficient microorganisms for repairing pinene alcohol-contaminated soil and related repair methods, the present invention provides a strain of Pseudomonas and its application in degrading pinene alcohol and repairing contaminated soil.

[0006] The first object of the present invention is to provide a Pseudomonas sp. S4.

[0007] The second object of the present invention is to provide the use of Pseudomonas sp. in degrading terpineol.

[0008] The third object of the present invention is to provide the use of Pseudomonas sp. in remediating soil contaminated by pinene alcohol.

[0009] The fourth object of the present invention is to provide an application of a combination of Pseudomonas sp. and a biosurfactant in remediating soil contaminated by pinene alcohol.

[0010] A fifth object of the present invention is to provide an application of Pseudomonas sp. in combination with biochar in remediating soil contaminated by pinene alcohol.

[0011] A sixth object of the present invention is to provide a preparation for degrading terpineol.

[0012] The seventh object of the present invention is to provide a method for remediating soil contaminated by terpineol.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] In this study, pinolene alcohol was used as the target pollutant, and mining soil was used as the source medium of microorganisms. A surfactant-producing Pseudomonas sp. S4 was screened and isolated through concentration gradient domestication. The degradation conditions of pinolene alcohol were optimized by single factor and response surface curve analysis. The crude surfactant extract was obtained from the fermentation broth of Pseudomonas sp. S4 using acid precipitation and extraction methods, and the types of biosurfactants were identified by Fourier transform infrared spectroscopy (FTIR) and high performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF-MS / MS). The application effect of Pseudomonas sp. S4 immobilized with rhamnolipid and biochar in the remediation of pinolene alcohol contaminated soil was explored, and high-throughput sequencing technology was used to reveal the structural changes of microbial communities in contaminated soil under remediation.

[0015] The present invention requests protection for the following contents:

[0016] A Pseudomonas sp. S4, the preservation number of the Pseudomonas sp. S4 is GDMCC No: 63948, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0017] The use of Pseudomonas sp. in degrading pinene alcohol, the use of Pseudomonas sp. in treating pinene alcohol pollution, and the use of Pseudomonas sp. in repairing soil contaminated by pinene alcohol should be within the protection scope of the present invention.

[0018] Preferably, the Pseudomonas sp. is Pseudomonas sp. S4, with a preservation number of GDMCC No: 63948, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0019] More preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: pH 5-10.

[0020] Further preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: pH 7-9.

[0021] More preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: pH 7-8.

[0022] Still more preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: pH 7.

[0023] More preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: a temperature of 20°C to 40°C.

[0024] Further preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: a temperature of 25°C to 40°C.

[0025] More preferably, the conditions for degrading terpineol by Pseudomonas sp. S4 include: a temperature of 30°C.

[0026] More preferably, the conditions for the degradation of terpineol by Pseudomonas sp. S4 include: the inoculation amount is OD 600nm =0.2~1.

[0027] Further preferably, the conditions for the degradation of terpineol by Pseudomonas sp. S4 include: the inoculation amount is OD 600nm =0.4~1.

[0028] More preferably, the conditions for the degradation of terpineol by Pseudomonas sp. S4 include: the inoculation amount is OD 600nm =0.7.

[0029] Most preferably, the conditions for degradation of terpineol by Pseudomonas sp. S4 include: pH 7, temperature 30°C, inoculation amount OD 600nm =0.7.

[0030] Preferably, the soil comprises paddy soil and / or mine soil.

[0031] The application of Pseudomonas sp. in combination with biosurfactants in the degradation of pineneol, the application of Pseudomonas sp. in combination with biosurfactants in the treatment of pineneol pollution, and the application of Pseudomonas sp. in combination with biosurfactants in the repair of pineneol-contaminated soil should be within the scope of protection of the present invention.

[0032] Preferably, the Pseudomonas sp. is Pseudomonas sp. S4, with a preservation number of GDMCC No: 63948, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0033] Preferably, the biosurfactant comprises rhamnolipid, and the rhamnolipid comprises monorhamnolipid and disrhamnolipid.

[0034] Preferably, the soil comprises paddy soil and / or mine soil.

[0035] The application of Pseudomonas sp. in combination with biochar in the restoration of soil contaminated by pinene alcohol should be within the scope of protection of the present invention.

[0036] Preferably, the Pseudomonas sp. is Pseudomonas sp. S4, with a preservation number of GDMCC No: 63948, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0037] Preferably, the biochar is loaded with the Pseudomonas sp. S4.

[0038] More preferably, the Pseudomonas sp. S4 is adsorbed on the surface of the biochar, that is, the preparation is a biochar-immobilized bacterial agent containing Pseudomonas sp. S4.

[0039] Further preferably, the method for preparing the biochar immobilized bacterial agent comprises the following steps: 600nm The bacterial suspension of Pseudomonas sp. S4 with a pH of 0.5 to 1.5 is fully reacted with biochar to obtain the product.

[0040] More preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0041] Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =1.

[0042] Further preferably, the ratio of the bacterial suspension of Pseudomonas sp. S4 to biochar is (4-6) mL: (20-30) g.

[0043] More preferably, the ratio of the bacterial suspension of Pseudomonas sp. S4 to the biochar is 5 mL:25 g.

[0044] Further preferably, the method for preparing the biochar immobilized bacterial agent further comprises: after sufficient reaction, performing solid-liquid separation on the obtained reaction product.

[0045] More preferably, the solid-liquid separation method comprises: centrifugation and / or drying.

[0046] Specifically, the preparation method of the biochar immobilized bacterial agent includes the following steps: 5 mL of a concentration of OD 600nm =1 Pseudomonas sp. S4 bacterial suspension is fully reacted with 25 g of biochar, the obtained reaction product is centrifuged at 1000 r / min for 10 min, the precipitate is collected and dried in an oven at 30°C to obtain the product.

[0047] The use of a biochar-immobilized bacterial agent containing Pseudomonas sp. in degrading pine alcohol, the use of a biochar-immobilized bacterial agent containing Pseudomonas sp. in treating pine alcohol pollution, and the use of a biochar-immobilized bacterial agent containing Pseudomonas sp. in repairing soil contaminated by pine alcohol should be within the scope of protection of the present invention.

[0048] Preferably, the Pseudomonas sp. is Pseudomonas sp. S4, with a preservation number of GDMCC No: 63948, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0049] Preferably, the method for preparing the biochar immobilized bacterial agent comprises the following steps: 600nm The bacterial suspension of Pseudomonas sp. S4 with a pH of 0.5 to 1.5 is fully reacted with biochar to obtain the product.

[0050] More preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0051] Further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =1.

[0052] More preferably, the ratio of the bacterial suspension of Pseudomonas sp. S4 to biochar is (4-6) mL: (20-30) g.

[0053] Further preferably, the usage ratio of the bacterial suspension of Pseudomonas sp. S4 to the biochar is 5 mL:25 g.

[0054] More preferably, the method for preparing the biochar-immobilized bacterial agent further comprises: after sufficient reaction, performing solid-liquid separation on the obtained reaction product.

[0055] Further preferably, the solid-liquid separation method comprises: centrifugation and / or drying.

[0056] Specifically, the preparation method of the biochar immobilized bacterial agent comprises the following steps: 5 mL of a concentration of OD 600nm=1 Pseudomonas sp. S4 bacterial suspension is fully reacted with 25 g of biochar, the obtained reaction product is centrifuged at 1000 r / min for 10 min, the precipitate is collected and dried in an oven at 30°C to obtain the product.

[0057] A preparation for degrading pineneol, the preparation comprising Pseudomonas sp. S4, with a preservation number of GDMCC No: 63948, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

[0058] Preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =0.5~1.5.

[0059] More preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0060] Further preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =1.

[0061] Preferably, the formulation further comprises a biosurfactant or biochar.

[0062] More preferably, the biosurfactant comprises rhamnolipid. The rhamnolipid includes monorhamnolipid and dirhamnolipid.

[0063] More preferably, the concentration of rhamnolipid in the preparation is 50 mg / L to 200 mg / L.

[0064] Still further preferably, the concentration of the rhamnolipid in the preparation is 100 mg / L.

[0065] More preferably, the biochar is loaded with the Pseudomonas sp. S4.

[0066] Further preferably, the Pseudomonas sp. S4 is adsorbed on the surface of the biochar, that is, the preparation is a biochar-immobilized bacterial agent containing Pseudomonas sp. S4.

[0067] More preferably, the method for preparing the biochar immobilized bacterial agent comprises the following steps: 600nm The bacterial suspension of Pseudomonas sp. S4 with a pH of 0.5 to 1.5 is fully reacted with biochar to obtain the product.

[0068] Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0069] Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =1.

[0070] Still more preferably, the ratio of the bacterial suspension of Pseudomonas sp. S4 to biochar is (4-6) mL: (20-30) g.

[0071] Still more preferably, the usage ratio of the bacterial suspension of Pseudomonas sp. S4 to the biochar is 5 mL:25 g.

[0072] Still more preferably, the method for preparing the biochar immobilized bacterial agent further comprises: after sufficient reaction, performing solid-liquid separation on the obtained reaction product.

[0073] Still further preferably, the solid-liquid separation method comprises: centrifugation and / or drying.

[0074] Specifically, the preparation method of the biochar immobilized bacterial agent comprises the following steps: 5 mL of a concentration of OD 600nm =1 Pseudomonas sp. S4 bacterial suspension is fully reacted with 25 g of biochar, the obtained reaction product is centrifuged at 1000 r / min for 10 min, the precipitate is collected and dried in an oven at 30°C to obtain the product.

[0075] A method for repairing soil contaminated by pinene alcohol, treating the soil contaminated by pinene alcohol with a preparation, wherein the preparation contains Pseudomonas sp. S4, the preservation number of which is GDMCC No: 63948, and has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 31, 2023.

[0076] Preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =0.5~1.5.

[0077] More preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0078] Further preferably, the concentration of Pseudomonas sp. S4 is OD 600nm =1.

[0079] Preferably, the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL: (20-30) g.

[0080] More preferably, the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL:25 g.

[0081] More preferably, the concentration is OD 600nm =0.5-1.5, and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL: (20-30) g.

[0082] More preferably, the concentration is OD 600nm = =0.8 to 1.2 and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL: (20 to 30) g.

[0083] Still more preferably, the concentration is OD 600nm =1 and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL: (20-30) g.

[0084] More preferably, the concentration is OD 600nm =0.5 to 1.5, and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL:25 g.

[0085] More preferably, the concentration is OD 600nm = =0.8 to 1.2 and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL: 25 g.

[0086] Still more preferably, the concentration is OD 600nm =1 and the volume mass ratio of the bacterial suspension of Pseudomonas sp. S4 to the soil contaminated with pinene alcohol is 1 mL:25 g.

[0087] Preferably, the formulation further comprises a biosurfactant or biochar.

[0088] More preferably, the biosurfactant comprises rhamnolipid. The rhamnolipid includes monorhamnolipid and dirhamnolipid.

[0089] Further preferably, the biosurfactant is rhamnolipid.

[0090] More preferably, the concentration of the rhamnolipid in the preparation is 50 mg / L to 200 mg / L.

[0091] Still further preferably, the concentration of the rhamnolipid in the preparation is 100 mg / L.

[0092] More preferably, the mass ratio of the rhamnolipid to the soil contaminated by terpineol is 1:(1×10 5 ~1.5×10 5 ).

[0093] Still more preferably, the mass ratio of the rhamnolipid to the soil contaminated by terpineol is 1:(1.25×10 5 ).

[0094] More preferably, the biochar is loaded with the Pseudomonas sp. S4.

[0095] Further preferably, the Pseudomonas sp. S4 is adsorbed on the surface of the biochar, that is, the preparation is a biochar-immobilized bacterial agent containing Pseudomonas sp. S4.

[0096] More preferably, the method for preparing the biochar immobilized bacterial agent comprises the following steps: 600nm The bacterial suspension of Pseudomonas sp. S4 with a pH of 0.5 to 1.5 is fully reacted with biochar to obtain the product.

[0097] Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =0.8~1.2.

[0098] Still further preferably, the concentration of the bacterial suspension of Pseudomonas sp. S4 is OD 600nm =1.

[0099] Still more preferably, the ratio of the bacterial suspension of Pseudomonas sp. S4 to biochar is (4-6) mL: (20-30) g.

[0100] Still more preferably, the usage ratio of the bacterial suspension of Pseudomonas sp. S4 to the biochar is 5 mL:25 g.

[0101] Still more preferably, the method for preparing the biochar immobilized bacterial agent further comprises: after sufficient reaction, performing solid-liquid separation on the obtained reaction product.

[0102] Still further preferably, the solid-liquid separation method comprises: centrifugation and / or drying.

[0103] Specifically, the preparation method of the biochar immobilized bacterial agent comprises the following steps: 5 mL of a concentration of OD 600nm =1 Pseudomonas sp. S4 bacterial suspension is fully reacted with 25 g of biochar, the obtained reaction product is centrifuged at 1000 r / min for 10 min, the precipitate is collected and dried in an oven at 30°C to obtain the product.

[0104] More preferably, the mass ratio of the biochar-immobilized bacterial agent to the pinene alcohol-contaminated soil is 1:(20-30).

[0105] Still more preferably, the mass ratio of the biochar-immobilized bacterial agent to the pinene alcohol-contaminated soil is 1:25.

[0106] Preferably, the soil contaminated by pinene alcohol includes paddy soil contaminated by pinene alcohol and / or mining soil contaminated by pinene alcohol.

[0107] Compared with the prior art, the present invention has the following beneficial effects:

[0108] The present invention provides a strain of Pseudomonas sp. S4 with the function of efficiently degrading pineneol. The degradation rate of 350 mg / L pineneol after culturing for 3 days under laboratory conditions is as high as 95%, and the strain can also produce a composite surfactant containing mono-rhamnolipid and di-rhamnolipid. Pseudomonas sp. S4 can be used to repair soil contaminated by pineneol, increase the diversity of microbial species in the soil, and can play a synergistic effect when used in combination with rhamnolipid or biochar, and the repair effect is more significant, providing a new technical option for treating pineneol pollution, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 These are the colony morphological characteristics of strain S4 in nutrient agar medium.

[0110] Figure 2 The morphological characteristics of strain S4 under scanning electron microscopy (5000 times).

[0111] Figure 3 is the 16S rDNA phylogenetic tree of strain S4.

[0112] Figure 4Figure 3 shows the effects of different factors on the degradation of pineneol by Pseudomonas sp. S4. A is the degradation rate of pineneol at different pH values, B is the degradation rate of pineneol at different temperatures, and C is the degradation rate of pineneol at different inoculation amounts.

[0113] Figure 5 are the response surface test results, A is the response analysis surface of pineol degradation rate, temperature and pH, and B is the contour line of pineol degradation rate, temperature and pH.

[0114] Figure 6 This is the degradation kinetic curve of pineneol with different initial concentrations by Pseudomonas sp. S4.

[0115] Figure 7 A is the result of the blue gel plate, and B is the emulsification effect of Pseudomonas sp. S4

[0116] Figure 8 This is the infrared absorption spectrum of Pseudomonas sp. S4 metabolites.

[0117] Fig. 9 This is the mass spectrum of the HPLC-TOF-MS / MS analysis of the surfactant of Pseudomonas sp. S4, A to C are C 14 H 26 O 7 , C 14 H 22 O 7 and C 20 H 36 O 11 .

[0118] Fig.10 The results of quantitative analysis of rhamnolipid production by Pseudomonas sp. S4 are shown in Figure 1. A is the standard curve of rhamnose content and absorbance, and B is the production of rhamnolipid by Pseudomonas sp. S4 during 24 h of culture.

[0119] Fig.11 These are scanning electron microscope images of biochar before and after bacterial fixation, A is before bacterial fixation, and B is after bacterial fixation.

[0120] Fig.12 These are the results of the pinene alcohol contaminated soil remediation experiment. A is the statistical degradation rate of pinene alcohol in the contaminated paddy soil of group CK, group A, group B, group C and group D; B is the statistical degradation rate of pinene alcohol in the contaminated paddy soil of group CK and the contaminated mining soil of group E and group F. DETAILED DESCRIPTION

[0121] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0122] The reagents used in the embodiments of the present invention include: terpineol, purchased from Shanghai Aladdin Chemical Co., Ltd., CAS No.: 8000-41-7, purity> 95%; dichloromethane purchased from Sigma-Aldrich, USA, CAS No.: 75-09-2, purity> 98%; rhamnose, purchased from Shanghai McLean Biochemical Technology Co., Ltd., CAS No.: 6155-35-7; biochar, purchased from Henan Lize Environmental Protection Technology Co., Ltd., item No.: LZ-04-05; rhamnolipid, purchased from Shanghai McLean Biochemical Technology Co., Ltd., CAS No.: 869062-42-0.

[0123] The culture medium used in the embodiment of the present invention includes: liquid basic salt (MSM) culture medium (K 2 HPO 4 , 5.8g / L; KH 2 PO 4 , 4.5 g / L; (NH 4 ) 2 SO 4 , 2.0g / L; MgCl 2 , 0.15g / L; CaCl 2 , 0.03g / L; Na 2 MoO 4 ·2H 2 O, 0.0025 g / L; FeCl 3 , 0.0016g / L; MnCl 2 ·2H 2 O, 0.0014 g / L; double distilled water, 1000 mL; pH value is 7); LB medium (peptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 5 g / L;; double distilled water, 1000 mL; pH value is 7); nutrient agar (LB solid) medium (peptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 5 g / L; agar, 20 g / L; double distilled water, 1000 mL; pH value is 7).

[0124] Example 1 Screening, Isolation and Identification of Pseudomonas

[0125] 1. Gradient domestication

[0126] Soil samples from a contaminated mining area in Nandan County, Guangxi were used as target pollutants, and the concentration gradient method was used to domesticate the strain.

[0127] First, 10 g of the mine soil sample was inoculated into 100 mL of MSM medium containing 1% (v / v) pineneol, and then cultured in a shaker at 25°C and 150 rpm for 5 days; then, the sample was inoculated into MSM medium containing 2% pineneol at a 10% (v / v) inoculation amount, and cultured under the same conditions for 5 days; the sample was inoculated into MSM medium containing 3% pineneol at a 10% (v / v) inoculation amount, and cultured under the same conditions for 5 days; the sample was inoculated into MSM medium containing 4% pineneol at a 10% (v / v) inoculation amount, and cultured under the same conditions for 5 days; the sample was inoculated into MSM medium containing 5% pineneol at a 10% (v / v) inoculation amount, and cultured under the same conditions for 5 days to obtain a culture solution. Each concentration was repeated three times.

[0128] The obtained culture solution was separated and purified by LB solid medium to obtain a single colony, the corresponding strain number was S4, the strain S4 was inoculated into a test tube slant medium and stored in a refrigerator at 4°C for use.

[0129] 2. Identification of strains

[0130] 1. Morphological identification

[0131] (1) Colony characteristics

[0132] The strain S4 was inoculated onto the LB plate and cultured in a constant temperature incubator for 24 hours. During the culture process, the shape, size, surface, transparency, edge and color of the strain S4 were observed.

[0133] like Figure 1 As shown, after strain S4 was cultured on LB solid medium for 24 hours, the colonies appeared as round, transparent protrusions with a brown-yellow color.

[0134] (2) Scanning electron microscopy observation of strain morphology

[0135] A single colony of strain S4 was picked from LB solid culture medium with an inoculating loop and added to LB liquid culture medium, and cultured in a shaker at 25°C and 160rpm for 24 hours; 1mL of bacterial solution was drawn from the conical flask and placed in a 2.5mL centrifuge tube, and centrifuged at 6000rpm for 6 minutes, the supernatant was discarded, and the bacteria were collected; 1mL of sterilized 0.9% NaCl solution was added and mixed, and the bacteria were collected by centrifugation (repeated 3 times); then 1mL of 2.5% glutaraldehyde was added, stirred evenly, and placed in a 4°C refrigerator for fixation for 4 hours; after fixation, ethanol was added for dehydration; the bacteria were collected by centrifugation, and after freeze-drying, the morphological characteristics of strain S4 were observed using a scanning electron microscope.

[0136] like Figure 2 As shown, the strain S4 was observed under a scanning electron microscope to be straight or slightly curved rod-shaped, arranged singly or in pairs, and about 1 μm in length.

[0137] 2. Identification of physiological and biochemical characteristics

[0138] The identification was carried out with reference to "Bergey's Manual of Determinative Bacteriology" (Lippincott Williams & Wilkins, 1994).

[0139] (1) Gram staining

[0140] Use an inoculation loop to take a loop of distilled water and place it on a glass slide; burn the inoculation loop with an alcohol lamp to sterilize it, pick strain S4 from the LB solid culture medium and mix it with the distilled water on the glass slide, then place the smear next to the alcohol lamp flame to fix it. Add a drop of crystal violet staining solution, wait for 1 minute, and wash with water; add Gram iodine solution and wait for 1 minute, then rinse with water; then add a large amount of 95% ethanol, decolorize for 10 seconds, and rinse with water; finally add counterstain solution, after 1 minute, wash with water; dry and examine under a microscope.

[0141] (2) Catalase test

[0142] Use an inoculation loop to pick up strain S4 from the LB solid culture medium and place it in a beaker containing 3% hydrogen peroxide to observe whether bubbles are generated. If there are no bubbles, it is negative, otherwise it is positive.

[0143] (3) Methyl red test

[0144] Strain S4 was inoculated into universal culture medium. After culturing for 3 days, 3 drops of methyl red indicator were added and the color change was observed. Bright red was positive and yellow was negative.

[0145] (4) Oxidase test

[0146] Use an inoculation loop to pick up strain S4 from the LB solid culture medium, put it into a solution containing 1% dimethyl-p-phenylenediamine hydrochloride, and observe the color change. Purple is positive.

[0147] (5) Indole experiment

[0148] Inoculate strain S4 into peptone culture medium containing tryptophan, culture for 48 hours, add indole reagent, and observe the results. If the contact point between the two liquid surfaces turns red, it is positive; if there is no color change, it is negative.

[0149] (6) Urease test

[0150] Inoculate strain S4 into a culture medium containing urea, culture for 48 hours, and observe the color change. Red is positive, and no color change is negative.

[0151] (7) Hydrogen sulfide experiment

[0152] Use an inoculation loop to pick strain S4 from LB solid culture medium and inoculate it onto triple sugar iron agar medium. After culturing for 24 hours, observe the color change of the culture medium. If black is produced, it is positive, otherwise it is negative.

[0153] (8) Citrate experiment

[0154] Use an inoculation loop to pick strain S4 from LB solid culture medium and inoculate it into citrate LB culture medium. Culture for 3 days and observe the color change of the culture medium. If blue is produced, it is positive, otherwise it is negative.

[0155] (9) Experimental results

[0156] Table 1 Physiological and biochemical characteristics of strain S4

[0157]

[0158] Note: “+” means positive, “-” means negative.

[0159] As shown in Table 1, strain S4 is a Gram-positive bacterium that can grow using citrate. The results of the strain catalase, urease, hydrogen sulfide and oxidase tests were all positive. The results of the methyl red and indole tests were all negative.

[0160] 3. 16S rDNA sequence homology analysis

[0161] The 16S rDNA fragment of strain S4 was amplified using a universal primer pair (27F and 1492R). The PCR reaction system was: TaqMixture 25 μL; primer 27F 1 μL; primer 1492R 1 μL; DNA template 1 μL; ddH 2 O 22μL; total volume 50μL. PCR reaction conditions are: 95℃ initial preheating for 5min; melting at 94℃ for 45s, annealing at 55℃ for 45s, extension at 72℃ for 1min 15s, 32 cycles, and finally keeping at 72℃ for 10min. After PCR amplification, 1.5% agarose gel electrophoresis was performed and photographed in a gel imaging system to detect the size and specificity of the amplified fragment.

[0162] The PCR product was recovered and sent to Bioengineering Company for sequencing. The 16S rDNA sequence of strain S4 was obtained as follows:

[0163]

[0164] The 16S rDNA sequence of strain S4 (SEQ ID NO.1) was compared with the 16S rDNA of similar microorganisms in the GenBank database. The results showed that strain S4 was closely related to Pseudomonas putida strain J15. Some sequences with high homology were selected from the BLAST comparison results, and multiple sequence alignment was achieved using the biological software Clustal W. The relationship was analyzed using the neighborhood-joining method, and the following was constructed: Figure 3 The phylogenetic tree shown in the figure shows that strain S4 is most closely related to Pseudomonas putida strain J15 (MH814578.1) by comparing the differences between different species.

[0165] 4. Degradation of pineneol

[0166] (1) Preparation of standard solution

[0167] Accurately measure the quantitative terpineol and add dichloromethane to mix evenly to obtain a 10 mg / L terpineol standard mother solution. Dilute the terpineol mother solution with chromatographically pure dichloromethane to obtain terpineol standard solutions with concentrations of 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L, respectively.

[0168] (2) Cultivation of strain S4 and preparation of test samples

[0169] The strain S4 was inoculated into MSM medium containing 350 mg / L pineneol, and cultured continuously for 3 days in a shaker at 30°C and 150 rpm. After the culture was completed, the culture medium was collected to obtain sample A.

[0170] The MSM culture medium containing 350 mg / L pineneol was placed in a shaker at 30° C. and 150 rpm and cultured continuously for 3 days to obtain sample B, which was used as a control culture medium.

[0171] The culture medium A and the culture medium B were respectively placed in a conical flask, 50 mL of dichloromethane was added to each, and the mixture was shaken at 150 rpm for 10 min to mix thoroughly; then ultrasonicated for 10 min, and repeated twice; allowed to stand at room temperature for 10 min, and then the organic dichloromethane phase in the lower layer was collected, 1 mL of each was taken, filtered through a 0.45 μm microporous filter membrane, and the filtrate was collected to obtain the sample A and the sample B to be tested, respectively, and then placed in 2.5 mL injection bottles respectively, and the terpineol content was determined using a gas chromatography-mass spectrometer (GC-MS).

[0172] (3) GC-MS detection

[0173] Splitless injection was used with an injection volume of 1.0 μL.

[0174] Detection conditions: chromatographic column, DB-5ms; carrier gas is helium, injection port temperature is 230°C, injection is by automatic injector; initial pressure is 55 kPa, flow rate is 1.5 mL / min.

[0175] Temperature program: initial temperature maintained at 60°C for 1 min; raised to 120°C at 10°C / min and maintained for 2 min, then raised to 200°C at 10°C / min and maintained for 1 min. Mass spectrometry qualitative and quantitative analysis: Mass spectrometry used EI ion source, temperature was 200°C, target ion of terpineol was 59m / z, reference ions were 93m / z and 121m / z, retention time was 6.393min.

[0176] The recovery rate of blank spiked samples of terpineol compounds was determined by the detection method established above, and the recovery rate was between 96.1% and 109.7%.

[0177] Determine the terpineol standard solution of different concentrations, establish a standard curve of concentration (X) and peak area (Y), and obtain a regression equation. Determine the peak area of ​​the sample to be tested, substitute it into the regression equation, calculate the terpineol content of the sample to be tested, and then calculate the degradation rate of terpineol according to formula (1):

[0178] Formula (1): Degradation rate (%) = (1-D 1 / D 0 )×100%;

[0179] Among them, D 1 is the pinoleol content of medium A after the culture of strain S4, and D 0 is the pinoleol content of the control medium (medium B).

[0180] (4) Test results

[0181] The degradation of terpineol by strain S4 was determined by GC-MS, and the results showed that strain S4 could effectively degrade terpineol at a concentration of 350 mg / L, and the degradation rate of terpineol after 3 days of cultivation was as high as 95%, indicating that strain S4 could efficiently degrade terpineol.

[0182] In summary, the strain finally screened by the present invention is a Pseudomonas (Pseudomonassp.) S4 capable of degrading pineol, and was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on October 31, 2023, with a deposit number of GDMCC No: 63948, and a storage location of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and the storage name is Pseudomonassp. S4.

[0183] Example 2 Degradation characteristics of terpineol by Pseudomonas sp. S4

[0184] 1. Experimental Methods

[0185] 1. Preparation of bacterial suspension

[0186] A small amount of Pseudomonas sp. S4 was selected from the test tube of the preserved strain, added to a triangular flask containing LB liquid medium and cultured at constant temperature for 24 hours. The bacterial precipitate was collected by centrifugation for 5 minutes. After removing the supernatant, the bacterial cells were washed three times with 0.9% sterile saline and resuspended to obtain the OD 600nm =1 bacterial suspension.

[0187] 2. Preparation of pineneol mother liquor

[0188] Accurately measure the quantitative terpineol and add dichloromethane to mix and dilute to obtain 1×10 4 The 100 mg / L pineneol mother solution was filtered with a 0.45 μm filter membrane, placed in a brown constant volume flask, and stored in a 4°C refrigerator away from light.

[0189] 3. Effects of different factors on the degradation of terpineol by Pseudomonas sp. S4

[0190] (1) Effect of pH

[0191] The pH value of 100 mL of the prepared MSM medium was adjusted to 5, 6, 7, 8, 9 and 10 respectively using a pH meter, and then sterilized by high pressure in an autoclave (121°C, 20 min); 1 mL of pine oil mother solution was added to the sterilized MSM medium, and then Pseudomonas sp. S4 was inoculated (OD 600nm =0.6); after sealing with sealing film, the cells were placed in a shaking incubator (30°C, 150 rpm) and cultured for 2 days.

[0192] After the culture was completed, the culture fluid was collected and the degradation rate of terpineol by Pseudomonas sp. S4 was detected according to the method of "GC-MS detection" in Example 1. MSM culture medium of each pH without inoculation was used as a blank control, and 3 replicates were set for each treatment.

[0193] (2) Effect of temperature

[0194] The prepared 100 mL MSM medium was sterilized in an autoclave (121°C, 20 min); then 1 mL of pine oil mother solution was added to the sterilized MSM medium in a clean bench, and Pseudomonas sp. S4 was inoculated (OD 600nm =0.6); after sealing with sealing film, the culture medium was placed in a shaker at 20°C, 25°C, 30°C, 35°C and 40°C, shaken at 150 rpm, and cultured for 2 days.

[0195] After the culture was completed, the culture fluid was collected and the degradation rate of terpineol by Pseudomonas sp. S4 was detected according to the method of "GC-MS detection" in Example 1. MSM medium without bacteria was used as a blank control, and 3 replicates were set for each treatment.

[0196] (3) Effect of inoculum dose on the degradation capacity of pine oil-degrading bacteria

[0197] The prepared 100 mL MSM medium was sterilized in an autoclave (121°C, 20 min); then, 1 mL of pine oil mother solution was added to the sterilized MSM medium in an ultra-clean workbench, and Pseudomonas sp. S4 was inoculated at OD 600nm =0.2, OD 600nm =0.4, OD 600nm =0.6, OD 600nm =0.8 and OD 600nm = 1, sealed with a sealing film, and placed in a shaking incubator (30°C, 150 rpm) for 2 days.

[0198] After the culture was completed, the culture fluid was collected and the degradation rate of terpineol by Pseudomonas sp. S4 was detected according to the method of "GC-MS detection" in Example 1. The MSM medium without bacteria was used as a blank control, and 3 replicates were set for each treatment.

[0199] (4) Response surface experiment

[0200] Box-Behnken Design was used to analyze the degradation rate of pineol based on the results of the above three single-factor experiments. 1 )、Temperature(X 2 ) and inoculum dose (X 3 ) on it, and the specific grouping is shown in Table 2. The optimal combination conditions for Pseudomonas sp. S4 to degrade pineneol were determined by response surface methodology.

[0201] Table 2 Box-Behnken Design experimental factor level table and coding

[0202]

[0203] Design-Expert 8.0 software was used to draw a response surface diagram to study the effect of single factors and the interaction of various factors on the degradation rate of pineneol. 600nm The inoculum amount was fixed at 0.8, and the effects of temperature and pH on the degradation rate of pineneol were investigated. The corresponding three-dimensional response surface and contour map were made.

[0204] 4. Kinetic study on the degradation of terpineol by Pseudomonas sp. S4

[0205] The prepared 100 mL MSM culture medium was sterilized in an autoclave (121°C, 20 min); then, different amounts of pineol mother liquor were added to the sterilized inorganic salt culture medium in an ultra-clean workbench to make the final concentrations of pineol 100 mg / L, 300 mg / L, 600 mg / L and 1000 mg / L, respectively. Then, Pseudomonas sp. S4 was cultured according to the optimal combination conditions obtained by the response surface experiment and the culture was continued for 3 days.

[0206] In order to analyze the kinetics of terpineol degradation by Pseudomonas sp. S4, the culture medium was collected at 0 h, 6 h, 12 h, 48 h and 72 h of culture, and the content of terpineol in the culture medium was detected according to the method of "GC-MS detection" in Example 1. The MSM culture medium without bacteria was used as a blank control, and 3 replicates were set for each treatment.

[0207] The first-order degradation kinetic model was used to fit the content (C) and time (t) of pineneol in the culture medium of Pseudomonas sp. S4 at different culture times, and the degradation kinetic curves of pineneol at different concentrations were drawn. The calculation method of the first-order degradation kinetic model is shown in formula (2), and the calculation method of the degradation half-life model is shown in formula (3):

[0208] Formula (2): lnC = a + kt;

[0209] Formula (3): t 1 / 2 =0.693 / k;

[0210] Where C is the content of terpineol in the culture medium at time t (h), k is the first-order rate constant, a is a constant, t is the culture time, t 1 / 2 is the biodegradation half-life of terpineol.

[0211] 2. Experimental Results

[0212] 1. Degradation rate of pineneol by Pseudomonas sp. S4 under different factors

[0213] (1) pH

[0214] like Figure 4 As shown in A in the figure, in the pH range of 5 to 10, the ability of Pseudomonas sp. S4 to degrade pinene alcohol is significantly affected by pH. When the pH is 5 to 8, the rate of degradation of pinene alcohol by Pseudomonas sp. S4 increases with the increase of pH. When the pH is 8 to 10, the degradation rate of pinene alcohol by Pseudomonas sp. S4 decreases with the increase of pH. When the pH is 8, the degradation rate of pinene alcohol by Pseudomonas sp. S4 is the highest, up to 92%. When the pH is 5 and 10, the degradation rates of pinene alcohol are as low as 61% and 73%, respectively. When the pH is 7 to 9, the degradation rates of pinene alcohol are all higher than 75%.

[0215] The above results show that the degradation efficiency of pineneol by Pseudomonas sp. S4 is inhibited in acidic and strongly alkaline environments, but it has good degradation ability for pineneol in weakly alkaline environments, and the most suitable pH range for the degradation of pineneol is 7-9.

[0216] (2) Temperature

[0217] like Figure 4 As shown in B, when the temperature is 20℃~40℃, the ability of Pseudomonas sp. S4 to degrade pinene alcohol is significantly affected by temperature. When the temperature is 20℃~30℃, the rate of degradation of pinene alcohol by Pseudomonas sp. S4 increases with the increase of temperature. When the temperature is 30℃~40℃, the degradation rate of pinene alcohol by Pseudomonas sp. S4 decreases with the increase of temperature. When the temperature is 30℃, the degradation rate of pinene alcohol by Pseudomonas sp. S4 is the highest, up to 93%. When the temperature is 20℃, the degradation rate of pinene alcohol is as low as 63%. When the temperature is 25℃~40℃, the degradation rate of pinene alcohol is higher than 75%.

[0218] The above results show that the most suitable temperature range for Pseudomonas sp. S4 to degrade terpineol is 25℃~40℃.

[0219] (3) Inoculation amount

[0220] like Figure 4 As shown in C, the ability of Pseudomonas sp. S4 to degrade terpineol was significantly affected by the inoculum amount, which was OD 600nm =0.2~0.8, the degradation rate increases with the increase of inoculum amount. When the inoculum amount is OD 600nm =0.8, Pseudomonas sp. S4 had the highest degradation rate of terpineol, up to 93%; the inoculation amount was OD 600nm =0.8~1, the degradation rate of pineneol by Pseudomonas sp. S4 was slightly reduced; when the inoculation amount was OD 600nm =0.4~1, the degradation rate of pineol is higher than 75%.

[0221] The above results show that the most suitable inoculation amount of Pseudomonas sp. S4 for degrading terpineol is OD 600nm =0.4~1.

[0222] 2. Response surface test results

[0223] Table 3 Box-Behnken test design and pineneol degradation results

[0224]

[0225]

[0226] Note: The data in the table are mean ± standard deviation (n = 3); the same letters in the same column indicate no significant difference (P>0.05).

[0227] Table 4 Box-Behnken test design and pineneol degradation results

[0228]

[0229] Note: R 2 =0.9916(corrected R 2 =0.9807), coefficient of variation (CV) =1.98%; P<0.05 indicated that the investigated factors had significant effects.

[0230] The response surface test results are shown in Table 3. The degradation rate of pineol was used as the response value to perform quadratic polynomial model regression analysis on the response surface results, and the results shown in Table 4 were obtained. The regression equation of the degradation rate of pineol was fitted as shown in formula (4):

[0231] Formula (4): Y=94.00+3.20×X 1 +0.0150×X 2 -19.58×X1 2 -7.45×X 2 2 -2.30×X 3 2 ;

[0232] Among them, Y represents the response value (degradation rate of pinene alcohol), X 1 , X 2 and X 3 They represent the three independent variables: pH, temperature and inoculum amount.

[0233] The results in Table 4 show that the coefficient R 2 =0.9916, indicating that there is a high correlation between the measured degradation rate of pineneol by Pseudomonas sp. S4 and the predicted degradation rate; the corrected determination coefficient is 0.9807, which further explains the change in the response value of Pseudomonas sp. S4; the coefficient of variation (CV) is 1.98%, indicating that the obtained regression equation can better reflect the measured degradation rate of pineneol, and the fitting effect of the model is very significant (P<0.0001). In addition, it can be seen that pH (X 1 ) and temperature (X 2 ) had a significant linear effect on the degradation rate of pineneol by Pseudomonas sp. S4 (P<0.05), and the quadratic coefficient X 11 and X 22 The results showed that the linear effect of Pseudomonas sp. S4 on the degradation rate of pineol reached a very significant level (P < 0.0001), while the linear effects of other factors on the degradation rate of Pseudomonas sp. S4 were not significant (P > 0.05).

[0234] The three-dimensional response surface and contour map are shown in Figure 5 As shown in A and B in the figure, it can be seen that as the temperature rises from 20℃ to 30℃, the degradation rate of pineneol by Pseudomonas sp. S4 increases; however, when the temperature exceeds 30℃, the degradation rate of pineneol by strain S4 begins to gradually decrease, which is consistent with the previous experimental results; when the pH is maintained between 7 and 8, the degradation efficiency of pineneol by strain S4 is the highest, which is also consistent with the previous experimental results. The optimal combination conditions for Pseudomonas sp. S4 to degrade pineneol are finally predicted to be: pH = 7, temperature 30℃, inoculation amount OD 600nm=0.7, the predicted degradation rate of pineneol is 95%. Under the best combination conditions, after 3 repeated experiments, the average degradation rate of pineneol measured is 95%, indicating that the prediction result is true and reliable.

[0235] 3. Kinetics of terpineol degradation by Pseudomonas sp. S4

[0236] like Figure 6 As shown in the figure, in general, with the increase of the initial concentration of pineneol in the MSM medium, the degradation rate of Pseudomonas sp. S4 decreased. The reason may be that high concentration of pineneol can inhibit the growth of Pseudomonas sp. S4 to a certain extent. Specifically, when the concentration of pineneol is ≤300 mg / L, Pseudomonas sp. S4 can completely degrade pineneol when it is cultured for 24 hours. When the initial concentration of pineneol increases to 1000 mg / L, the degradation rate of pineneol by Pseudomonas sp. S4 decreases to only 79% at 24 hours. However, it can still fully utilize pineneol as the only carbon source and energy source for growth, and there is no obvious delay effect in the degradation process. This shows that Pseudomonas sp. S4 has a certain tolerance to high concentrations of pineneol.

[0237] Table 5 Degradation kinetic parameters of pineneol at different initial concentrations

[0238]

[0239] Note: C is the content of terpineol in the culture medium at time t (h), t is the culture time, R 2 The coefficient of determination indicates the degree of correlation; the data in the table are the average of 3 replicates.

[0240] In order to explore the degradation kinetics of pineneol by Pseudomonas sp. S4, the first-order degradation kinetic model was used to fit the content (C) and culture time (t) of pineneol in the culture medium at different initial concentrations. The fitting results of the degradation kinetic fitting model and the half-life model are shown in Table 5. It can be seen that when Pseudomonas sp. S4 degrades pineneol, the half-life is proportional to the substrate concentration. The half-lives corresponding to the substrate concentrations of 100 mg / L, 300 mg / L, 600 mg / L and 1000 mg / L are 4.46 h, 7.18 h, 10.5 h and 12.73 h, respectively, and the degradation rate constants (k) are 0.1553 d, respectively. -1 , 0.0965d -1 , 0.066d-1 and 0.0544d -1 .

[0241] Example 3 Identification of biosurfactants produced by Pseudomonas sp. S4 metabolism

[0242] 1. Experimental Methods

[0243] 1. Blue gel plate experiment

[0244] Hexadecyltrimethylammonium bromide (CTAB) 0.5% (w / v), methylene blue 0.002% (w / v), beef extract 0.5% (w / v), peptone 1.0% (w / v), NaCl 0.5% (w / v) and agar powder 2.0% (w / v) were diluted to 1L with sterile water, the pH was adjusted to 7, and the plate was poured after sterilization at 120℃ for 25min to obtain a blue gel plate. The blue gel plate contains CTAB and methylene blue, which can react with glycolipid surfactants secreted by microorganisms.

[0245] A small amount of Pseudomonas sp. S4 was picked from the preserved strain test tube, diluted and spread on a blue gel plate, placed in a constant temperature incubator at 37°C for 2 days, and the color change of the colonies on the plate was observed.

[0246] 2. Preparation of fermentation broth

[0247] The optimal combination conditions for degradation of terpineol by Pseudomonas sp. S4 determined in Example 2 (pH = 7, temperature 30°C, inoculation amount OD 600nm =0.7) to prepare the fermentation liquid, the specific steps are as follows:

[0248] Pseudomonas sp. S4 was inoculated with OD 600nm =0.7 was inoculated into MSM medium (pH=7) containing 100 mg / L pineneol, and cultured continuously for 3 days in a shaking incubator at 30°C and 150 rpm. After the culture was completed, the culture medium was collected to obtain the fermentation broth.

[0249] 3. Emulsification performance determination

[0250] Liquid paraffin and fermentation liquid were added to a 10 mL centrifuge tube at a volume ratio of 1:1, cultured at 30°C for 24 hours, then vortexed for 1 minute, placed at room temperature for 24 hours, and the height of the emulsion layer was measured. The emulsification rate of Pseudomonas sp. S4 was calculated according to the formula: emulsification rate (%) = emulsion layer height / total liquid height × 100%. Liquid paraffin without fermentation liquid was used as a control.

[0251] 3. Extraction of surfactant

[0252] Take 100 mL of fermentation liquid, centrifuge at 6000 rpm for 5 minutes, collect the supernatant and put it into a conical flask, add hydrochloric acid to adjust the pH to 2, seal it and put it in a 4°C refrigerator overnight for precipitation. After precipitation, take the supernatant and centrifuge at 8500 rpm for 10 minutes, discard the supernatant, collect the precipitate, resuspend the precipitate with dichloromethane, and obtain the surfactant produced by Pseudomonas sp. S4 by rotary evaporation.

[0253] 4. Fourier transform infrared spectrometer (FTIR) analysis

[0254] 4000~400cm -1 A sample of 1 mg surfactant and 100 mg KBr was analyzed using FTIR within the spectral range to detect the characteristic functional groups in the surfactant.

[0255] 5. HPLC-TOF-MS / MS analysis

[0256] The extracted surfactant was identified by HPLC-TOF-MS / MS. The sample was dissolved in acetonitrile and the sample concentration was 15 mg / mL. It was filtered with a 0.22 μm microporous filter membrane and 10 μL was injected. The chromatographic column was DP-5 (0.25 μm×0.25 mm×30 m). The mobile phase A was methanol: water = 1:100, and the mobile phase B was formic acid: acetonitrile = 1:100. The flow rate was 0.5 mL / min.

[0257] 6. Quantitative analysis of rhamnolipids

[0258] The rhamnolipids in the extracted surfactants were analyzed using the anthrone-sulfuric acid colorimetric method. The basic principle is that under the action of concentrated sulfuric acid, rhamnolipids will lose water and generate furfural, and then anthrone and furfural will further react to produce blue-green furfural derivatives.

[0259] Take 0.2g of anthracene and add it to 100mL of concentrated sulfuric acid as a color developer; prepare 100ppm of rhamnose mother solution; inject 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.9mL of rhamnose mother solution into test tubes respectively, then add enough water to make the liquid volume reach 1mL, and finally add 4mL of color developer, and mix on ice; put the test tube into boiling water and boil for 8min, then mix on ice; use ultraviolet spectrophotometer to measure the OD value of each sample at a wavelength of 620nm, and make a correlation between rhamnose concentration and OD 620nm The standard curve of

[0260] Pseudomonas sp. S4 was inoculated into an MSM medium with pineneol as the sole carbon source, and cultured continuously for 24 h in a shaker at 30 ° C and 150 rpm. The culture medium was collected every 2 h, and the surfactant was extracted according to the method of this example. Then, the OD value at a wavelength of 620 nm was measured by anthrone-sulfuric acid colorimetry, and the standard curve was substituted to obtain the corresponding rhamnose concentration. Then, according to the formula: rhamnolipid content = rhamnose concentration × 3.4 (rhamnolipid to rhamnose conversion coefficient), the rhamnolipid yield produced by Pseudomonas sp. S4 at each time point was obtained.

[0261] 2. Experimental Results

[0262] 1. Blue gel plate test results

[0263] like Figure 7 As shown in A, after culturing on the blue gel plate for 2 days, blue spots appeared around the colonies, indicating that Pseudomonas sp. S4 can produce glycolipid biosurfactants.

[0264] 2. Emulsification performance test results

[0265] like Figure 7 As shown in Figure B, compared with the control group, the fermentation liquid of Pseudomonas sp. S4 has a significant emulsification effect on pineneol, and a stable emulsion layer is formed after 24 hours of cultivation. According to the measurement results, the height of the emulsion layer is 3.9 cm and the total liquid phase height is 6 cm. Based on this, the emulsification rate of Pseudomonas sp. S4 is calculated to be 65%, indicating that the emulsification effect of the surfactant secreted by it is high.

[0266] 3. FTIR analysis results

[0267] The infrared absorption spectrum of the surfactant produced by the growth of Pseudomonas sp. S4 is shown in Figure 8 As shown, 3422.51cm -1 is the stretching vibration peak of NH bond; 2929.04cm -1 For aliphatic -CH 2 -Symmetrical tensile vibration; 1638.9cm -1 It is the stretching vibration of the CO-N bond of amide, a type of lipopeptide; 1543.24cm -1 Existence-CH 3 Group; 1229cm -1 The presence of sugar ring acetal structure COC at 1066 cm -1The above results show that the surfactant produced by Pseudomonas sp. S4 during metabolism belongs to glycolipids.

[0268] 4. HPLC-TOF-MS / MS analysis results

[0269] HPLC-TOF-MS / MS was used to detect the active substances generated by Pseudomonas sp. S4 during metabolism. The negative ion mass spectrometry signal was shown in the figure below. Fig. 9 As shown in A to C in the figure, the main mass spectrum fragments are m / z 306, 302 and 452, C 14 H 26 O 7 and C 14 H 22 O 7 All belong to monorhamnolipids; C 20 H 36 O 11 It belongs to disrhamnolipid. It shows that the surfactant produced by Pseudomonas sp. S4 includes monorhamnolipid and disrhamnolipid.

[0270] 5. Quantitative analysis results of rhamnolipids

[0271] Rhamnose concentration and OD 620nm The standard curve is as follows Fig.10 As shown in A, the rhamnose concentration and OD 620nm There is a significant linear relationship, the regression equation is y=0.0072x+0.3556, and the correlation coefficient is R 2 As high as 0.9902.

[0272] At the beginning of the culture, pineneol appeared as a transparent oil distributed on the surface of the liquid, but as time went on, it was gradually decomposed and emulsified into small oil droplets; at the same time, the color of the liquid also changed. After 24 hours of shaking flask culture, the fermentation liquid of pineneol turned milky white, and no oil was seen floating on the liquid surface.

[0273] The production of rhamnolipids by Pseudomonas sp. S4 during 24 h of culture Fig.10 As shown in B, during the degradation of pineneol, rhamnolipids accumulated in large quantities during the stable period of bacterial growth, reaching 2.57 g / L at 18 h. After 18 h, even though the growth of Pseudomonas sp. S4 had entered the decay phase, it was still able to accumulate a certain amount of rhamnolipids.

[0274] In summary, Pseudomonas sp. S4 uses pineneol as a carbon source and breaks the interface between pineneol and water by secreting rhamnolipids during its growth, thereby increasing its contact with pineneol, promoting the uptake and metabolism of pineneol, and self-proliferation.

[0275] Example 4 Pseudomonas sp. S4 remediation of contaminated soil

[0276] 1. Experimental Methods

[0277] 1. Test soil

[0278] The test soils used in this example include: 1. paddy soil from Guangzhou Experimental Farm, used to study the degradation rate of Pseudomonas sp. S4 under experimental conditions; 2. mine soil from the mining area of ​​Nandan County, Guangxi, used to simulate the actual soil remediation environment. Some physical and chemical properties of the two soils are shown in Table 6.

[0279] Table 6 Physical and chemical properties of paddy soil and mine soil

[0280]

[0281] 2. Preparation of pineneol-contaminated soil

[0282] Quantitatively measure pinene alcohol and dissolve it in dichloromethane. Put 250g of paddy soil or 250g of mining soil into a 500mL conical flask and mix well. Place it in a dark place and let the dichloromethane evaporate naturally for 3 days (turn it over and over to let the solvent evaporate). The concentration of pinene alcohol is 100mg kg -1 The contaminated paddy soil or pinene alcohol concentration is 100 mg kg -1 of contaminated mine soil.

[0283] 3. Preparation of bacterial suspension of Pseudomonas sp. S4

[0284] Pseudomonas sp. S4 was picked from the LB solid plate with an inoculation loop and inoculated into the liquid LB medium, and cultured on a shaking platform for 24 h (30°C, 150 rpm); the bacterial liquid was collected and centrifuged (4°C, 3500 rpm), the precipitate was collected to obtain the bacterial cells, and the bacterial cells were resuspended in 0.9% sterile saline to make a concentration of OD 600nm =1 bacterial suspension.

[0285] 4. Preparation of biochar immobilized bacterial agent

[0286] A single colony of Pseudomonas sp. S4 was picked up and inoculated into 100 mL of LB medium for 24 h. The bacterial solution was collected and centrifuged (4 °C, 3500 rpm) to remove the supernatant. The precipitate was collected to obtain the bacterial cells, which were washed with 0.9% sterile saline, centrifuged again, and resuspended with 5 mL of saline to make a concentration of OD 600nm =1 bacterial suspension.

[0287] Weigh 25 g of biochar and add it to a sterilized tissue culture bottle, pour in 5 mL of bacterial suspension, mix well, and after the biochar fully adsorbs Pseudomonas sp. S4, centrifuge at 1000 r / min for 10 min, collect the precipitate and dry it in a 30°C oven to obtain the biochar immobilized bacterial agent for use.

[0288] The morphology of Pseudomonas sp. S4 before and after fixation on biochar was examined by scanning electron microscopy. Fig.11 As shown in A in the figure, the surface of biochar before solidification is not smooth, but has irregular wrinkles and gaps; Fig.11 As shown in B, after fixation, the surface morphology of biochar did not change significantly, while the degrading bacteria were captured in the wrinkles and gaps on the surface of biochar. Under a high-power electron microscope, the morphology of Pseudomonas sp. S4 on the surface of biochar was clearly visible and abundant, and the biochar itself did not undergo significant changes, indicating that Pseudomonas sp. S4 had been successfully fixed to the biochar.

[0289] 5. Remediation test of soil contaminated by pinene alcohol

[0290] Table 7 Grouping settings for pineneol contaminated soil remediation experiments

[0291]

[0292] Note: The concentration of Pseudomonas sp. S4 bacterial suspension is OD 600nm =1; the initial concentrations of rhamnolipid and CTAB were both 200 mg / L, and the solvents were both water.

[0293] A total of seven treatment groups were set up in this experiment, using rhamnolipid, CTAB, the contaminated soil (contaminated paddy soil or contaminated mining soil) prepared in this example, a bacterial suspension of Pseudomonas sp. S4 and a biochar immobilized bacterial agent, and each treatment was prepared according to the information shown in Table 7, and 6 replicates were set for each treatment group.

[0294] Each treatment group was incubated at 30°C, and sterile water was added to each treatment group to keep the soil moisture content at 20% (v / w), and the incubation was carried out for 11 days. During the incubation process, samples were taken once every 2 days, and the sampling amount was 1 g. The pineneol content in the contaminated soil of each treatment group was detected by referring to the method of "pineneol degradation" in Example 1, and the degradation rate of pineneol by Pseudomonas sp. S4 was counted.

[0295] 6. Microbial diversity analysis

[0296] After the cultivation, soil samples from CK, A, B, C, E and F were collected. Based on the Illumina NovaSeq sequencing platform, paired-end sequencing was used to construct a small fragment library for high-throughput sequencing of 16S rDNA of the microbial community. Reads were spliced ​​and screened, clustered or denoised, and species annotation and abundance analysis were performed. Mothur software and R language tools were used to perform alpha diversity analysis according to the sequencing volume and sequencing depth of different samples, and the Simpson's index, Shannon's index and Chao1 index were calculated to evaluate the richness of soil microbial communities and the completeness of community diversity.

[0297] 2. Experimental Results

[0298] 1. Results of the pinene alcohol contaminated soil remediation test

[0299] like Fig.12As shown in A in the figure, after 11 days of co-incubation, the pineol content in the contaminated paddy soil without any treatment (CK group) decreased from 100 mg / kg to 78 mg / kg, and the total degradation rate of pineol was 22%; in the contaminated paddy soil only with Pseudomonas sp. S4 for remediation (Group A), the pineol content was reduced from 100 mg / kg to 39 mg / kg, and the total degradation rate of pineol was 61%; in the contaminated paddy soil only with rhamnolipid for remediation (Group B), the pineol content was reduced from 100 mg / kg to 72 mg / kg, and the total degradation rate of pineol was 28%; in the contaminated paddy soil with Pseudomonas sp. S4 and rhamnolipid for co-remediation (Group C), the pineol content was reduced from 100 mg / kg to 13 mg / kg, and the total degradation rate of pineol was 87%; In the contaminated paddy soil (Group D) co-remediated by Pseudomonas sp.) S4 and cationic surfactant CTAB, the growth of Pseudomonas sp. S4 was inhibited by the film-dissolving toxicity of CTAB, and the total degradation rate of pineneol was only 33%. This shows that Pseudomonas sp. S4 can significantly reduce the content of pineneol in contaminated paddy soil, and can produce a synergistic effect with rhamnolipids, and the effect of co-remediation of contaminated paddy soil is further significantly improved, while CTAB inhibits the degradation of pineneol in contaminated paddy soil by Pseudomonas sp. S4.

[0300] like Fig.12 As shown in Figure B, after 11 days of co-incubation, the total degradation rate of pineneol in the contaminated paddy soil without any treatment (CK group) was only 17.5%; compared with the CK group, the pineneol content in the contaminated mining soil (E group) that was remediated by only adding Pseudomonas sp. S4 was significantly reduced, and the degradation rate of pineneol showed an upward trend over time. After 11 days of co-incubation, the total degradation rate of pineneol reached 48.5%; compared with the E group, the pineneol content in the contaminated mining soil (F group) that was remediated by adding biochar immobilized bacteria was further significantly reduced. Although the degradation rate was low in the first few days, with the increase of time, the physical adsorption and mass transfer properties of biochar gradually emerged, reducing the time for Pseudomonas sp. S4 to adapt to the environment, promoting its efficient degradation ability, and the degradation ability remained stable, so that the degradation rate of pineneol gradually increased and surpassed, and the total degradation rate of pineneol reached 67.8%. The results showed that Pseudomonas sp. S4 could significantly reduce the content of pineneol in contaminated mining soil, and the effect of repairing contaminated paddy soil was further significantly improved after biochar adsorption and fixation.

[0301] The above results show that Pseudomonas sp. S4 can repair soil contaminated by pineneol, and its combination with rhamnolipid or biochar can produce synergistic effects, and the repair effect is more significant.

[0302] 2. Microbial diversity analysis results

[0303] Table 8 Analysis results of Alpha diversity index of bacteria in each treatment group

[0304]

[0305] As shown in Table 8, compared with the CK group, the Shannon's index, Chao1's index, and Simpson's index of soil microorganisms in the other treatment groups increased significantly, among which the Shannon's index and Chao1's index of group E, where Pseudomonas sp. S4 was used to repair the contaminated mine soil, were the highest, and the Simpson's index of group F, where biochar-immobilized bacteria were used to repair the contaminated mine soil, was the highest. This indicates that Pseudomonas sp. S4 improves the abundance and uniformity of bacteria in the contaminated soil and increases microbial diversity while degrading pine ethanol.

[0306] 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 the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Pseudomonas species ( Pseudomonas sp.) S4, characterized in that The Pseudomonas Pseudomonas sp.) The deposit number of S4 is GDMCC No: 63948, and it was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 31, 2023.

2. Pseudomonas ( Pseudomonas sp.) in the degradation of terpineol, characterized in that: The Pseudomonas Pseudomonas sp.) is the Pseudomonas ( Pseudomonas sp.) S4.

3. Pseudomonas ( Pseudomonas sp.) in the restoration of soil contaminated by pinene alcohol, characterized in that: The Pseudomonas Pseudomonas sp.) is the Pseudomonas ( Pseudomonas sp.) S4.

4. Pseudomonas ( Pseudomonas sp.) and a biosurfactant in the restoration of soil contaminated by pinene alcohol, characterized in that: The Pseudomonas Pseudomonas sp.) is the Pseudomonas ( Pseudomonas sp.) S4.

5. Pseudomonas ( Pseudomonas sp.) and biochar in the restoration of soil contaminated by pinene alcohol, characterized in that: The Pseudomonas Pseudomonas sp.) is the Pseudomonas ( Pseudomonas sp.) S4.

6. A preparation for degrading terpineol, characterized in that: The preparation contains the Pseudomonas ( Pseudomonas sp.) S4.

7. The preparation according to claim 6, characterized in that The formulation may also include a biosurfactant or biochar.

8. The preparation according to claim 7, characterized in that The biosurfactant includes rhamnolipid.

9. A method for repairing soil contaminated by pinene alcohol, characterized in that: The soil contaminated by pinene alcohol is treated with the preparation according to any one of claims 6 to 8.

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