A method for improving the surface hydrophobicity of petroleum-degrading bacteria and its application

Abstract

This invention discloses a method for improving the surface hydrophobicity of petroleum-degrading bacteria, comprising the following steps: adding hydrophobic growth substrates and / or surfactants during the cultivation of petroleum-degrading bacteria, such that the mass concentration of surfactants in the culture medium is less than the critical micelle concentration of surfactants; collecting bacterial cells by centrifugation during the late logarithmic growth phase of the petroleum-degrading bacteria strain to obtain the petroleum-degrading bacteria with high surface hydrophobicity; this invention improves the surface hydrophobicity of petroleum-degrading bacteria during the large-scale cultivation process, thereby enabling the added petroleum-degrading bacteria to effectively adhere to petroleum pollutants when added to petroleum-polluted seawater environments for bioremediation, reducing microbial loss, reducing the mass transfer distance of petroleum hydrocarbons from the water body to the cell, and improving the bioremediation effect.

Description

Technical Field

[0001] This invention belongs to the field of petroleum pollution environmental remediation technology, specifically, it relates to a method for improving the surface hydrophobicity of petroleum-degrading bacteria and its application. Background Technology

[0002] Despite continuous advancements in modern industrial technology, the extraction, drilling, and transportation of oil inevitably result in large quantities of oil and related pollutants flowing into the ocean, causing marine pollution. These pollutants cause lasting damage to the ocean, poison marine life, and ultimately affect human health through the food chain. Therefore, addressing marine oil spill pollution is of paramount importance.

[0003] Microbial remediation of oil pollution is an effective method, but its effectiveness in actual oil spill remediation is not ideal. In marine environments, the poor bioavailability of petroleum hydrocarbons (PHH) is a major reason why microorganisms released into the marine environment are easily lost. The slow mass transfer process of PHH from the oil phase to the microbial cell is one of the bottlenecks affecting degradation efficiency and a significant reason for the poor bioavailability of PHH. Direct adhesion to PHH surfaces is an effective way for microorganisms to overcome this poor bioavailability. Cell-surface hydrophobicity (CSH) is one of the most important factors determining the non-specific adhesion of microorganisms to various biological and non-biological surfaces and interfaces, and is also one of the main factors affecting the absorption and degradation of hydrophobic organic matter by microorganisms. Microorganisms can utilize their surface hydrophobicity to adhere to PHH surfaces at the oil-water interface, thereby effectively reducing the mass transfer distance of PHH, improving its bioavailability, greatly reducing the loss of microorganisms due to seawater dilution, and facilitating their contact with oxygen, thus increasing the oil degradation rate.

[0004] Patent application CN103523928A discloses a microbial agent for bioremediation of oil-polluted waters and its preparation method. This invention discloses a microbial agent for petroleum bioremediation, comprising petroleum-degrading bacteria and a bioimmobilization carrier. The bacteria are immobilized in a floating, degradable biocarrier capable of slowly releasing nutrients such as nitrogen and phosphorus. The bioimmobilization carrier of this invention uses a slow-release fertilizer capable of slowly releasing nutrients such as nitrogen and phosphorus as its core, surrounded by a biocarrier immobilized with petroleum-degrading bacteria. The biocarrier has a porous, hollow structure to maintain its buoyancy; the slow-release fertilizer can continuously provide nitrogen, phosphorus, and other nutrients for the petroleum-degrading bacteria to utilize, without the need for external nutrient supplementation. The petroleum-degrading microbial agent involved in this invention can be used for the bioremediation of oil-polluted water bodies.

[0005] Patent application CN104388312A discloses a method for screening petroleum-degrading bacteria. The enrichment medium, screening medium, and acclimatization medium used in this method are media in which petroleum is the sole carbon source. Specifically, petroleum and polysorbate-80 are added to the media. The beneficial effects of this invention are: This invention uses the nonionic surfactant polysorbate-80 as a solubilizer, which improves the solubility of petroleum in water, solving the problem of difficulty in dissolving petroleum in media; this invention truly achieves the sole carbon source of petroleum, with the media using a petroleum-polysorbate-80 solubilizer as the carbon source throughout the initial screening, acclimatization, and purification processes of the petroleum-degrading bacteria; the screening of petroleum-degrading bacteria in this patent includes processes such as initial screening, acclimatization, and purification. The strains are gradually acclimatized in an acclimatization medium with a gradually increasing petroleum concentration gradient, adapting to growth and reproduction with petroleum as the sole carbon source, thus improving their tolerance to petroleum and their degradation efficiency.

[0006] Although the relationship between microbial surface hydrophobicity and adhesion has been extensively studied, research on enhancing the surface hydrophobicity of petroleum-degrading bacteria to promote their adhesion to petroleum hydrocarbons and increase biodegradation efficiency for application in the bioremediation of marine oil pollution is scarce. The regulation of the surface hydrophobicity of petroleum-degrading bacteria and its adhesion mechanism to petroleum hydrocarbons remains unclear. Many chemical substances have been reported to inhibit the adhesion of microorganisms to hydrophobic interfaces, but substances that promote the adhesion of microorganisms to the oil-water interface are rarely reported. Improving the surface hydrophobicity of petroleum-degrading bacteria during microbial culture, thereby enabling the added petroleum-degrading bacteria to effectively adhere to petroleum pollutants during bio-enhanced remediation of marine oil pollution, reducing microbial loss, and shortening the mass transfer distance of petroleum hydrocarbons from the water body to the cell, is crucial for improving bioremediation effectiveness. Summary of the Invention

[0007] The purpose of this invention is to provide a method for improving the surface hydrophobicity of petroleum-degrading bacteria. This method involves adding hydrophobic growth substrates and / or surfactants during the large-scale cultivation of petroleum-degrading bacteria with hydrophobic surfaces, and monitoring the bacterial growth cycle to obtain petroleum-degrading bacteria with high surface hydrophobicity. These bacteria are then added to the surface of petroleum-polluted seawater, which promotes the adhesion of petroleum-degrading bacteria to petroleum hydrocarbons, reduces the mass transfer distance of petroleum hydrocarbons from the water body to the cell, and improves the bioremediation effect of marine oil spill pollution.

[0008] Another object of the present invention is to provide an application of the petroleum-degrading bacteria prepared by the method in the preparation of bioremediation materials for marine oil pollution.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A first aspect of the present invention provides a method for improving the surface hydrophobicity of petroleum-degrading bacteria, comprising the following steps:

[0011] Hydrophobic growth substrates and / or surfactants are added during the cultivation of petroleum-degrading bacteria, such that the mass concentration of the surfactant in the culture medium is less than the critical micelle concentration of the surfactant. During the late logarithmic growth phase of the petroleum-degrading bacteria strain, the bacterial solution is centrifuged to collect the bacterial cells, thereby obtaining the petroleum-degrading bacteria with high surface hydrophobicity.

[0012] Preferably, the method for improving the surface hydrophobicity of petroleum-degrading bacteria includes the following steps: dissolving a surfactant in deionized water and sterilizing it by filtration through a filter membrane; adding the surfactant aqueous solution to LB medium and mixing thoroughly so that the mass concentration of the surfactant in the LB medium is less than the critical micelle concentration of the surfactant; inoculating the petroleum-degrading bacteria into the above LB medium and culturing it to obtain the petroleum-degrading bacteria with high surface hydrophobicity.

[0013] The surfactant is selected from at least one of nonionic surfactants, anionic surfactants, and amphoteric surfactants.

[0014] The amount of the hydrophobic growth substrate used is 1% to 2% (v / v, volume ratio).

[0015] The nonionic surfactant is selected from Tween20 or Tween80.

[0016] The anionic surfactant is selected from rhamnolipids.

[0017] The zwitterionic surfactant is selected from lecithin.

[0018] The critical micelle concentration of Tween20 is 60 mg / L.

[0019] The critical micelle concentration of Tween80 is 14 mg / L.

[0020] The critical micelle concentration of the rhamnolipid is 40 mg / L.

[0021] The critical micelle concentration of the lecithin is 164 mg / L.

[0022] The petroleum-degrading bacteria are selected from at least one of the following genera: Pseudomonas, Acinetobacter, Gordon's, Flavobacterium, Aeromonas, Achromobacter, Alcaligenes, Enterobacteriaceae, Corynebacterium, Arthrobacter, and Bacillus.

[0023] The hydrophobic growth substrate is selected from at least one of diesel, engine oil, crude oil, gasoline, etc.

[0024] A second aspect of the present invention provides the application of the petroleum-degrading bacteria with high surface hydrophobicity prepared by the method described above in the preparation of bioremediation materials for marine oil pollution.

[0025] The application includes the following steps:

[0026] The prepared petroleum-degrading bacteria with high surface hydrophobicity were inoculated onto the surface of petroleum hydrocarbon-contaminated seawater.

[0027] The petroleum hydrocarbons are selected from at least one of diesel, engine oil, crude oil, gasoline, etc.

[0028] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The method for improving the surface hydrophobicity of petroleum-degrading bacteria provided by this invention involves adding hydrophobic growth substrates and / or surfactants during large-scale culture, monitoring the bacterial growth cycle, and collecting the bacterial cells by centrifugation during the late logarithmic growth phase of the strain, thus obtaining petroleum-degrading bacteria with high surface hydrophobicity. When these highly hydrophobic petroleum-degrading bacteria are inoculated into petroleum-contaminated environments for bioremediation, they promote adhesion between the bacteria and petroleum hydrocarbons, thereby reducing the mass transfer distance of petroleum hydrocarbons from the oil phase to the intracellular environment, improving the bioavailability of petroleum pollutants, and simultaneously reducing the loss of petroleum-degrading bacteria, significantly improving the bioremediation effect. Attached Figure Description

[0030] Figure 1 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 over culture time.

[0031] Figure 2 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with the concentration of Tween20.

[0032] Figure 3 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with Tween80 concentration.

[0033] Figure 4 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with the concentration of rhamnolipin.

[0034] Figure 5 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with lecithin concentration.

[0035] Figure 6 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with the concentration of diesel fuel as a hydrophobic growth substrate.

[0036] Figure 7 This is a schematic diagram showing the effect of the hydrophobic growth substrate diesel and surfactant on the surface hydrophobicity of strain Y9.

[0037] Figure 8 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 over culture time.

[0038] Figure 9 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with Tween20 concentration.

[0039] Figure 10 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with Tween80 concentration.

[0040] Figure 11 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with lecithin concentration.

[0041] Figure 12 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with the concentration of diesel fuel as a hydrophobic growth substrate.

[0042] Figure 13 This is a schematic diagram showing the effect of the hydrophobic growth substrate diesel and surfactant on the surface hydrophobicity of strain X1.

[0043] Figure 14 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 over time.

[0044] Figure 15 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with the growth of diesel fuel as a hydrophobic substrate.

[0045] Figure 16 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with Tween20 concentration.

[0046] Figure 17 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with Tween80 concentration.

[0047] Figure 18 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with the concentration of rhamnolipin.

[0048] Figure 19 This is a schematic diagram showing the changes in the surface hydrophobicity and diesel degradation rate of strain Y9 after the addition of oil and surfactants.

[0049] Figure 20 This is a schematic diagram showing the changes in the surface hydrophobicity and diesel degradation rate of strain S2 after the addition of oil and surfactant. Detailed Implementation

[0050] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] A method for improving the surface hydrophobicity of the petroleum-degrading bacterium Acinetobacter sp. Y9 includes the following steps:

[0053] I. Methods

[0054] (1) Effect of growth cycle on surface hydrophobicity of strain Y9

[0055] Under aseptic conditions, single colonies were picked from a plate and placed into 3 mL of LB liquid medium. The culture was incubated overnight at 30°C and 180 rpm. The culture was then inoculated into 200 mL of LB liquid medium and incubated at 30°C and 180 rpm. Every 4 hours, 10 mL of the bacterial culture was taken to measure OD600 and the surface hydrophobicity of the strain. Growth curves and curves showing the change in surface hydrophobicity over time were plotted.

[0056] (2) Effect of surfactant on the hydrophobicity of the surface of strain Y9 in expanded culture

[0057] The effects of Tween20 and Tween80 as nonionic surfactants, rhamnolipin as anionic surfactant, and lecithin as a zwitterionic surfactant on the surface hydrophobicity of the strain were studied.

[0058] Accurately weigh the surfactant and dissolve it in deionized water. Sterilize the solution by filtering it through a filter membrane using a sterile syringe. Add the surfactant solution to 50 mL of LB medium and mix thoroughly to ensure that the surfactant concentration in the medium is equal to the critical micelle concentration of the surfactant. Set up an experimental mass concentration gradient (as shown in Table 1). Set up three parallel samples for each mass concentration, with a blank control without surfactant.

[0059] Single colony strains were inoculated onto the above-mentioned LB medium and incubated overnight at 37°C and 120 rpm for 12 h. 4 mL of the bacterial suspension was then taken and the OD value was measured. 600 After reaching 1.0, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, and the surface hydrophobicity of the strain was measured.

[0060] Table 1. Types and mass concentrations of added surfactants

[0061]

[0062] (3) Effect of diesel concentration, a hydrophobic growth substrate, on the hydrophobicity of the surface of strain Y9 in expanded culture.

[0063] Single colony strain Y9 was inoculated from the plate into LB liquid medium and incubated overnight at 37°C and 180 rpm. Diesel fuel was added to 50 ml of LB medium at concentrations of 0.25 ml, 0.5 ml, 0.75 ml, and 1 ml, respectively, to achieve oil concentrations of 0.5%, 1%, 1.5%, and 2%. 0.15 ml of Y9 bacterial suspension was added to each suspension and incubated at 37°C and 120 rpm for 4 hours. 4 mL of the bacterial suspension was then used to measure the OD value.600 After the concentration was reduced to 0.5, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0064] (4) Effects of hydrophobic growth substrates diesel oil and surfactants on the hydrophobicity of the surface of strain Y9 in expanded culture

[0065] Single colony strain Y9 was inoculated from LB agar plates into 50 ml LB liquid medium containing 0.5 ml diesel oil, 0.5 ml diesel oil and 60 mg / L Tween 20, 0.5 ml diesel oil and 7 mg / L Tween 80, and 0.5 ml diesel oil and 10 mg / L rhamnolipid. LB medium without added oil and surfactant served as a blank control. The experiment was divided into 5 groups, with 3 replicates per group. Incubation was performed overnight at 37℃ and 120 rpm for 12 h. 4 mL of the bacterial culture was collected, and the OD was measured. 600 After reaching 1.0, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, and the surface hydrophobicity of the strain was measured.

[0066] (5) Method for determining the hydrophobicity of bacterial surface

[0067] Take 50 mL of bacterial culture obtained in steps (2) and (3) respectively, centrifuge at 3500 rpm for 10 min to collect bacterial cells, wash twice with 50 mL of sterile water, resuspend the bacterial cells in phosphate buffer, and measure the absorbance of the bacterial culture at 600 nm using a UV spectrophotometer. Dilute with phosphate buffer to OD. 600 Approximately 1.0, denoted as OD 600前 Take 5 mL of the above diluted solution into a 15 mL centrifuge tube, add 0.5 mL of diesel oil, incubate at 30 °C for 10 min, then vortex thoroughly for 2 min and let stand for 10 min. Use a pipette tip to aspirate the lower bacterial layer and measure its absorbance at 600 nm, recording it as OD. 600后 Repeat the experiment three times and take the average value. Calculate the hydrophobicity of the bacterial cell surface using the following formula:

[0068] CSH% (cell surface hydrophobicity of the strain) = (OD) 600前 -OD 600后 ) / OD 600前 ×100%

[0069] II. Results

[0070] (1) Effect of growth cycle on surface hydrophobicity of strain Y9

[0071] The surface hydrophobicity of strain Y9 changes with the growth cycle as follows: Figure 1 As shown, Figure 1This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 over culture time. The diagram shows that the surface hydrophobicity of strain Y9 increases with growth time during the lag phase and early logarithmic growth phase, and then slowly decreases with increasing growth time after entering the late logarithmic and stationary phases.

[0072] (2) The nonionic surfactant Tween20 enhances the hydrophobicity of the surface of strain Y9.

[0073] The effect of adding different concentrations of Tween20 on the surface hydrophobicity of strain Y9 during cultivation is as follows: Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the change in surface hydrophobicity of strain Y9 with Tween20 concentration. The results in the figure show that when the Tween20 concentration is below the critical micelle concentration of 60 mg / L, the surface hydrophobicity of the strain is significantly increased (P<0.05), reaching a maximum of 66.40% at the critical micelle concentration.

[0074] (3) The nonionic surfactant Tween80 enhances the hydrophobicity of the surface of strain Y9.

[0075] The effect of adding different concentrations of Tween80 on the surface hydrophobicity of strain Y9 during cultivation is as follows: Figure 3 As shown, Figure 3 This is a schematic diagram illustrating the change in surface hydrophobicity of strain Y9 with Tween80 concentration. The results show that, compared to the blank control group, changes in Tween80 concentration significantly increased hydrophobicity (P<0.05). When the Tween80 concentration was below the critical micelle concentration of 14 mg / L, the surface hydrophobicity of the strain was higher than when it was above the critical micelle concentration, reaching its maximum at the critical micelle concentration of 14 mg / L.

[0076] (4) The anionic surfactant rhamnolipin enhances the hydrophobicity of strain Y9.

[0077] The effect of adding different concentrations of rhamnolipin on the surface hydrophobicity of strain Y9 during cultivation is as follows: Figure 4 As shown, Figure 4 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with rhamnolipin concentration. The results in the figure show that when the rhamnolipin concentration is below the critical micelle concentration of 40 mg / L, the surface hydrophobicity of the strain is significantly increased (P<0.05).

[0078] (5) The amphoteric surfactant lecithin enhances the hydrophobicity of the surface of strain Y9.

[0079] The effect of adding different concentrations of lecithin on the surface hydrophobicity of strain Y9 during cultivation, as follows: Figure 5 As shown, Figure 5This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with lecithin concentration. The results in the figure show that when the lecithin concentration is below the critical micelle concentration of 164 mg / L, the surface hydrophobicity of the strain is significantly increased (P<0.05).

[0080] (6) The hydrophobic growth substrate diesel fuel enhances the hydrophobicity of strain Y9.

[0081] The effect of oil concentration on the surface hydrophobicity of strain Y9 in expanded culture, such as Figure 6 As shown, Figure 6 This is a schematic diagram showing the change in surface hydrophobicity of strain Y9 with the concentration of diesel fuel as a hydrophobic growth substrate. The experimental results show that when the added diesel fuel concentration is higher than 1.0%, the surface hydrophobicity of strain X1 increases significantly (P<0.05).

[0082] (7) Hydrophobic growth substrates diesel oil and surfactants enhance the hydrophobicity of strain Y9 surface.

[0083] The effects of adding oil and surfactants on the surface hydrophobicity of strain Y9 during cultivation are as follows: Figure 7 As shown, Figure 7 This diagram illustrates the effects of hydrophobic growth substrates (diesel oil) and surfactants on the surface hydrophobicity of strain Y9. The results show that the hydrophobicity of the strain cultured with added oil (79.19%) was significantly higher than that of the control (64.20%). The hydrophobicity of strains cultured with oil and the surfactant rhamnolipin, and strains cultured with oil and Tween20, was significantly higher than that of the control (P<0.05). The strain cultured with oil and rhamnolipin exhibited the highest hydrophobicity (89.66%), an increase of 25.44% compared to the control. These results indicate that adding oil and Tween20, or adding oil and rhamnolipin, can improve the hydrophobicity of the strain.

[0084] Example 2

[0085] The surface hydrophobicity of the petroleum-degrading bacterium Gordonia sp. X1 is enhanced.

[0086] I. Methods

[0087] (1) Effect of culture time on the surface hydrophobicity of strain X1

[0088] Under aseptic conditions, single colonies were picked from a plate and transferred to 50 mL of LB liquid medium, and incubated overnight at 37°C and 180 rpm. 0.2 mL of the bacterial culture was then inoculated into 200 mL of LB liquid medium and incubated at 37°C and 180 rpm. Every 4 hours, 15 mL of the bacterial culture was collected to determine the OD (occurrence density). 600The cells were centrifuged at 3500 rpm for 15 min, the bacterial cells were collected, the surface hydrophobicity of the strain was measured, and the growth curve and the curve of surface hydrophobicity change over time were plotted.

[0089] (2) Effect of surfactant on the hydrophobicity of the surface of strain X1 in the expansion culture

[0090] The effects of Tween20 and Tween80 as nonionic surfactants and lecithin as an amphoteric surfactant on the surface hydrophobicity of the bacterial strain were studied.

[0091] Accurately weigh the surfactant and dissolve it in deionized water. Sterilize the solution by filtering it through a filter membrane using a sterile syringe. Add the surfactant solution to 50 mL of LB medium and mix thoroughly to ensure that the surfactant concentration in the medium is equal to the critical micelle concentration of the surfactant, thus setting up an experimental mass concentration gradient (as shown in Table 2). Set up three parallel samples for each mass concentration, with a blank control without surfactant.

[0092] Single colony strain X1 was inoculated from the plate into LB liquid medium and incubated overnight at 37°C and 180 rpm. 0.15 ml of the bacterial suspension was then incubated in a surfactant-containing medium at 37°C and 180 rpm for 4 hours. 4 mL of the bacterial suspension was then used to measure the OD value. 600 After reaching 1.0, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0093] Table 2. Types and mass concentrations of added surfactants

[0094]

[0095] (3) Effect of diesel concentration, a hydrophobic growth substrate, on the hydrophobicity of the surface of strain X1 in its expanded culture.

[0096] Single colony strain X1 was inoculated from the plate into LB liquid medium and incubated overnight at 37°C and 180 rpm. Diesel fuel was added to 50 ml of LB medium at concentrations of 0.25 ml, 0.5 ml, and 0.75 ml, respectively, to achieve oil concentrations of 0.5%, 1%, and 1.5%. 0.15 ml of X1 bacterial suspension was added to each suspension and incubated at 37°C and 120 rpm for 4 hours. 4 mL of the bacterial suspension was then used to measure the OD value. 600 After the concentration was reduced to 0.5, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0097] (4) Effects of hydrophobic growth substrates diesel oil and surfactants on the hydrophobicity of the surface of strain X1 in expanded culture

[0098] Single colony strain X1 was inoculated from LB agar plates into 50 ml LB liquid medium and cultured overnight. 0.15 ml of the bacterial suspension was then inoculated into 50 ml LB liquid medium in the following concentrations: blank, 0.75 ml diesel oil (this concentration showed the highest hydrophobicity of strain X1), 0.75 ml diesel oil and 60 mg / L Tween 20 (this concentration showed the highest hydrophobicity of strain X1), 0.75 ml diesel oil and 14 mg / L Tween 80 (this concentration showed the highest hydrophobicity of strain X1), and 0.75 ml diesel oil and 320 mg / L lecithin (this concentration showed the highest hydrophobicity of strain X1). The experiment consisted of 5 groups, with 3 replicates per group. The culture was incubated overnight at 37°C and 180 rpm for 4 hours. 4 mL of the bacterial suspension was then used to measure the OD (exposure potential). 600 After the concentration was reduced to 0.5, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0099] The method for determining the surface hydrophobicity of the above strains was performed according to the method in Example 1.

[0100] II. Results

[0101] (1) Effect of culture time on the surface hydrophobicity of strain X1

[0102] The effect of culture time on the surface hydrophobicity of strain X1 is as follows: Figure 8 As shown, Figure 8 This is a schematic diagram illustrating the change in surface hydrophobicity of strain X1 over culture time. The experimental results show that the surface hydrophobicity of strain X1 first decreases and then increases with increasing culture time. The highest hydrophobicity (17.15%) was observed after 4 hours of culture; the lowest hydrophobicity (1.62%) was observed after 12 hours of culture.

[0103] (2) The nonionic surfactant Tween20 enhances the surface hydrophobicity of strain X1.

[0104] The effect of nonionic surfactant Tween20 on the surface hydrophobicity of strain X1 is as follows: Figure 9 As shown, Figure 9 This diagram illustrates the change in surface hydrophobicity of strain X1 with Tween 20 concentration. The experimental results show that the addition of Tween 20 during cultivation improved the surface hydrophobicity. As the Tween 20 concentration increased, the hydrophobicity of strain X1 first increased and then decreased. When the Tween 20 concentration was the critical micelle concentration of 60 mg / L, the surface hydrophobicity of strain X1 reached its highest value of 38.63%, significantly higher than the blank control group.

[0105] (3) The nonionic surfactant Tween80 enhances the hydrophobicity of the surface of strain X1 during its expanded culture.

[0106] The effect of nonionic surfactant Tween80 on the surface hydrophobicity of strain X1 during expansion culture is as follows: Figure 10 As shown, Figure 10 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with Tween 80 concentration. The experimental results show that when the added Tween 80 concentration is higher than 7 mg / L, the hydrophobicity of strain X1 is significantly improved. When the added Tween 80 concentration is the critical micelle concentration of 14 mg / L, the surface hydrophobicity of strain X1 reaches its highest level of 35.44%, which is 22.97% higher than the hydrophobicity of the blank control group (12.47%).

[0107] (4) The amphoteric surfactant lecithin enhances the hydrophobicity of the surface of strain X1 during its expansion culture.

[0108] The effect of zwitterionic surfactant lecithin on the surface hydrophobicity of strain X1 in expanded culture, as follows: Figure 11 As shown, Figure 11 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with lecithin concentration. The experimental results show that the surface hydrophobicity of strain X1 after the addition of lecithin was significantly higher than that of the control group without lecithin (P<0.01), and increased with increasing concentration.

[0109] (5) Diesel fuel, a hydrophobic growth substrate, enhances the hydrophobicity of the surface of strain X1 during its expansion culture.

[0110] The effect of oil concentration on the surface hydrophobicity of strain X1 in expanded culture, as follows: Figure 12 As shown, Figure 12 This is a schematic diagram showing the change in surface hydrophobicity of strain X1 with the concentration of diesel fuel as a hydrophobic growth substrate. The experimental results show that when the added oil concentration is higher than 1.0%, the surface hydrophobicity of strain X1 increases significantly (P<0.01).

[0111] (6) Hydrophobic growth substrates diesel oil and surfactants enhance the hydrophobicity of strain X1 surface.

[0112] The effects of oil and surfactants on the surface hydrophobicity of strain X1 are as follows: Figure 13 As shown, Figure 13 This is a schematic diagram showing the effects of the hydrophobic growth substrate diesel oil and surfactant on the surface hydrophobicity of strain X1. The experimental results show that the addition of oil, surfactant, and oil all significantly increased the surface hydrophobicity of strain X1 (P<0.01).

[0113] Example 3

[0114] The surface hydrophobicity of the petroleum-degrading bacterium Pseudomonas sp. S2 is increased.

[0115] I. Methods

[0116] (1) Effect of culture time on the surface hydrophobicity of strains

[0117] Pick a single colony of strain S2 from a plate and add it to 3 mL of LB liquid medium. Incubate overnight at 30°C and 180 rpm. Inoculate the bacterial culture into 200 mL of LB liquid medium and incubate at 30°C and 180 rpm. Every 4 hours, take 10 mL of the bacterial culture and measure the OD. 600 The cells were centrifuged at 3500 r / m for 10 min, the bacterial cells were collected, the surface hydrophobicity of the strain was measured, and the growth curve and the surface hydrophobicity change curve over time were plotted.

[0118] (2) Effect of oil (hydrophobic growth substrate diesel) concentration on the hydrophobicity of the surface of strain S2 in expanded culture

[0119] Single colony strain S2 was inoculated from the plate into LB liquid medium and incubated overnight at 37°C and 180 rpm. Diesel fuel was added to 50 ml of LB medium at concentrations of 0.25 ml, 0.5 ml, and 0.75 ml, respectively, to achieve oil concentrations of 0.5%, 1%, and 1.5%. 0.15 ml of S2 bacterial suspension was added to each suspension and incubated at 37°C and 120 rpm for 4 hours. 4 mL of the bacterial suspension was then used to measure the OD value. 600 After the concentration was reduced to 0.5, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0120] (3) Effect of surfactant on the hydrophobicity of the surface of strain S2 in expanded culture

[0121] The effects of Tween20 and Tween80 as nonionic surfactants and rhamnolipid as anionic surfactant on the surface hydrophobicity of the strain were studied.

[0122] Accurately weigh the surfactant and dissolve it in deionized water. Sterilize the solution by filtering it through a filter membrane using a sterile syringe. Add the surfactant solution to 50 mL of LB medium and mix thoroughly to ensure that the surfactant concentration in the medium is equal to the critical micelle concentration of the surfactant, thus setting up an experimental mass concentration gradient (Table 3). Set up three parallel samples for each mass concentration, with a blank control without surfactant.

[0123] Single colony strain S2 was inoculated from the plate into LB liquid medium and incubated overnight at 37°C and 180 rpm. 0.15 ml of the bacterial suspension was then added to a medium containing surfactant and incubated overnight at 37°C and 180 rpm. 4 mL of the bacterial suspension was then used to measure the OD value. 600 After reaching 1.0, the bacterial cells were collected by centrifugation at 3500 rpm for 15 min, and the surface hydrophobicity of the strain was measured.

[0124] Table 3. Types and mass concentrations of added surfactants

[0125]

[0126] The method for determining the surface hydrophobicity of the above strains was performed according to the method in Example 1.

[0127] II. Results

[0128] (1) Effect of culture time on the surface hydrophobicity of strains

[0129] The effect of culture time on the surface hydrophobicity of strain S2 is as follows: Figure 14 As shown, Figure 14 This is a schematic diagram illustrating the change in surface hydrophobicity of strain S2 over culture time. The experimental results show that during the logarithmic growth and stationary phases, the surface hydrophobicity of strain S2 gradually increases with increasing culture time; however, during the senescence phase, the surface hydrophobicity decreases.

[0130] (2) The hydrophobic growth substrate diesel fuel enhances the hydrophobicity of the surface of strain S2.

[0131] The effect of oil concentration on the surface hydrophobicity of strain S2 in expanded culture, such as Figure 15 As shown, Figure 15 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with the hydrophobic growth substrate diesel. The experimental results show that when the added oil concentration is higher than 1.0%, the surface hydrophobicity of strain S2 increases significantly (P<0.05).

[0132] (3) The nonionic surfactant Tween20 enhances the hydrophobicity of the surface of strain S2.

[0133] The effect of nonionic surfactant Tween20 on the surface hydrophobicity of strain S2 is as follows: Figure 16 As shown, Figure 16 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with Tween 20 concentration. The experimental results show that after adding Tween 20 during cultivation, the surface hydrophobicity of the bacteria increased with increasing Tween 20 concentration when the concentration was below the critical micelle concentration of 60 mg / L. The surface hydrophobicity reached its highest value of 46.41% at the critical micelle concentration of 60 mg / L, which was significantly higher than the blank control group (P<0.05).

[0134] (4) The nonionic surfactant Tween80 enhances the hydrophobicity of strain S2.

[0135] The effect of nonionic surfactant Tween80 on the surface hydrophobicity of strain S2 during expansion culture is as follows: Figure 17 As shown, Figure 17 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with Tween80 concentration. The experimental results show that when the added Tween80 concentration is below its critical micelle concentration of 14 mg / L, the hydrophobicity of strain S2 increases. At the critical micelle concentration of 14 mg / L, the surface hydrophobicity reaches its highest level of 48.62%, which is significantly higher than the blank control group (P<0.05).

[0136] (5) The anionic surfactant rhamnolipin enhances the hydrophobicity of strain S2.

[0137] The effect of adding different concentrations of rhamnolipin on the surface hydrophobicity of strain S2 during cultivation is as follows: Figure 18 As shown, Figure 18 This is a schematic diagram showing the change in surface hydrophobicity of strain S2 with rhamnolipin concentration. The results in the figure show that the surface hydrophobicity of the strain was significantly increased at rhamnolipin concentrations of 40 mg / L and 80 mg / L (P<0.05).

[0138] Example 4

[0139] Enhancing the surface hydrophobicity of the petroleum-degrading bacterium Acinetobacter sp. Y9 has a promoting effect on the bioremediation of diesel-contaminated seawater.

[0140] I. Methods

[0141] (1) Straw culture and determination of surface hydrophobicity

[0142] Prepare 3 L of basic inorganic salt solution (NaCl 24 g / L; KCl 0.7 g / L; MgSO4·7H2O 0.7 g / L; NH4Cl 1.325 g / L; KH2PO4 2 g / L; Na2HPO4·12H2O 6.037 g; distilled water 1 L; pH 7.5); add 2% trace element solution (MgSO4·7H2O 4 g / L; CuSO4·5H2O 1 g / L; MnCl4 1.169 g / L; FeSO4·7H2O 1 g / L; CaCl2 1 g / L; distilled water 1 L), filter and sterilize. Pour 100 ml into each Erlenmeyer flask.

[0143] Strain Y9 was inoculated from the plate into 50 ml of LB liquid medium and cultured at 37°C and 180 rpm for approximately 9 hours. 0.15 ml of Y9 bacterial suspension was then cultured overnight at 37°C and 180 rpm in 50 ml of LB liquid medium in the following solutions: blank, 0.5 ml diesel oil, 60 mg / L Tween 20, 7 mg / L Tween 80, 10 mg / L rhamnolipin, 0.5 ml diesel oil and 60 mg / L Tween 20, 0.5 ml diesel oil and 7 mg / L Tween 80, and 0.5 ml diesel oil and 10 mg / L rhamnolipin. 15 ml of the bacterial suspension was used to determine the surface hydrophobicity of the strain, following the method described in Example 1.

[0144] Centrifuge 5 ml of bacterial culture at 3500 rpm for 15 min, discard the supernatant, resuspend in sterile inorganic salt culture medium, and inoculate into Erlenmeyer flasks, adding 1 ml of diesel oil to each flask. Set up 3 replicates per group, with an uninoculated seawater culture medium containing the same oil concentration (1%) as a blank control. Incubate at 37℃ and 180 rpm for 7 days.

[0145] (2) Determination of petroleum degradation rate

[0146] Pour the seawater culture medium into a 250 ml separatory funnel, add 35 ml of petroleum ether, and shake vigorously for 3 minutes. Allow the mixture to separate into layers, collect the supernatant, and return the lower water layer to the separatory funnel. Repeat the extraction three times and combine the organic phases. Transfer 1 ml of the supernatant to a 15 ml centrifuge tube, add 10 ml of petroleum ether, and measure the absorbance at 255 nm using a UV-Vis spectrophotometer, with petroleum ether as a blank control.

[0147] Accurately weigh 1g of diesel oil and dissolve it in a volumetric flask containing 100ml of petroleum ether. Shake thoroughly; the diesel oil concentration is now 10g / L. Use a pipette to transfer 5ml of this solution to a 20ml volumetric flask, and dilute to the mark with petroleum ether; the solution concentration is now 2.5mg / L. Take 0.4ml, 0.8ml, 1.6ml, 3.2ml, and 4.8ml of this solution into 10ml volumetric flasks, and dilute to the mark with petroleum ether, respectively. The solution concentrations are now 100mg / L, 200mg / L, 400mg / L, 800mg / L, and 1200mg / L. Set λ = 255nm and measure the absorbance. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis.

[0148] The concentration of residual oil was calculated using standard curve analysis. The petroleum degradation rate was calculated using the following formula:

[0149] Petroleum degradation rate = (Petroleum concentration in control group - residual concentration in inoculated group) / Petroleum concentration in control group × 100%.

[0150] II. Results

[0151] like Figure 19 As shown, Figure 19 This diagram illustrates the effects of adding oil and surfactants on the surface hydrophobicity and diesel degradation rate of strain Y9. Adding diesel, Tween20, Tween80, rhamnolipid, Tween20 + oil, Tween80 + oil, or rhamnolipid + oil during the cultivation of Y9 significantly improved the surface hydrophobicity of strain Y9.

[0152] The petroleum-degrading bacteria with high surface hydrophobicity prepared in step (1) were inoculated into artificial seawater culture medium containing diesel. After 7 days of culture, the petroleum degradation rate was significantly increased compared with the control group (P<0.05).

[0153] Example 5

[0154] The hydrophobic surface of the petroleum-degrading bacterium Pseudomonas sp. S2 promotes the bioremediation of diesel fuel contamination.

[0155] I. Methods

[0156] (1) Straw culture and determination of surface hydrophobicity

[0157] Prepare 3 L of basic inorganic salt solution (NaCl 24 g / L; KCl 0.7 g / L; MgSO4·7H2O 0.7 g / L; NH4Cl 1.325 g / L; KH2PO4 2 g / L; Na2HPO4·12H2O 6.037 g; distilled water 1 L; pH 7.5); add 2% trace element solution (MgSO4·7H2O 4 g / L; CuSO4·5H2O 1 g / L; MnCl4 1.169 g / L; FeSO4·7H2O 1 g / L; CaCl2 1 g / L; distilled water 1 L), filter and sterilize. Pour 100 ml into each Erlenmeyer flask.

[0158] Strain S2 was inoculated from the plate into 50 ml of LB liquid medium and cultured at 37°C and 180 rpm for approximately 9 hours. 150 μl of S2 bacterial suspension was then incubated overnight at 37°C and 180 rpm in 50 ml LB liquid medium containing blank, 0.5 ml diesel oil, 30 mg / L Tween 20, 10 mg / L rhamnolipid, and both 0.5 ml diesel oil and 30 mg / L Tween 20. The surface hydrophobicity of the strain was measured using 15 ml of the bacterial suspension, following the method described in Example 1.

[0159] Centrifuge 5 ml of bacterial culture at 3500 rpm for 15 min, discard the supernatant, resuspend the culture in sterile inorganic salt medium, and inoculate into Erlenmeyer flasks, adding 1 ml of diesel oil to each flask. Set up three replicates per group, with an uninoculated seawater culture medium containing the same oil concentration (1%) as a blank control. Incubate at 37℃ and 180 rpm for 7 days, and then determine the petroleum degradation rate. The method for determining the petroleum degradation rate is the same as in Case Study 4.

[0160] II. Results

[0161] like Figure 20 As shown, Figure 20 This diagram illustrates the effects of adding oil and surfactants on the surface hydrophobicity and diesel degradation rate of strain S2. Adding diesel, Tween20, Tween80, rhamnolipid, Tween20 + oil, Tween80 + oil, or rhamnolipid + oil during the S2 culture process significantly improved the surface hydrophobicity of strain S2.

[0162] The petroleum-degrading bacteria with high surface hydrophobicity prepared in step (1) were inoculated into artificial seawater culture medium containing diesel. After 7 days of culture, the petroleum degradation rate was significantly increased compared with the control group (P<0.05).

[0163] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for improving the surface hydrophobicity of petroleum-degrading bacteria, characterized in that, Includes the following steps: Hydrophobic growth substrates and / or surfactants are added during the cultivation of petroleum-degrading bacteria, such that the mass concentration of surfactants in the culture medium is less than the critical micelle concentration of surfactants. During the late logarithmic growth of the petroleum-degrading bacteria strain, the bacterial solution is centrifuged to collect the bacterial cells, thereby obtaining the petroleum-degrading bacteria with high surface hydrophobicity. The surfactant is selected from at least one of Tween20 or Tween80; The hydrophobic growth substrate is selected from diesel fuel; The petroleum-degrading bacteria were selected from Acinetobacter sp. Y9. The amount of the hydrophobic growth substrate used is 1% to 2% by volume; The critical micelle concentration of Tween20 is 60 mg / L; The critical micelle concentration of Tween80 is 14 mg / L.

2. The method for improving the surface hydrophobicity of petroleum-degrading bacteria according to claim 1, characterized in that, The surfactant is selected from lecithin; The critical micelle concentration of the lecithin is 164 mg / L.

3. The application of the petroleum-degrading bacteria with high surface hydrophobicity prepared by the method of claim 1 in the preparation of marine oil pollution bioremediation materials.

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