Polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 and its application

Abstract

The present invention provides a polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ‑S‑72 and its application, which belongs to the field of bioremediation technology for petroleum and petroleum product contaminated environments. The strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on October 31, 2024, with a deposit number of CGMCC NO: 32458. The strain is derived from the soil of the Fildes Peninsula in Antarctica, and its fermentation liquid can be directly applied to the ecological restoration of oil spills in polar or low-temperature environments. Compared with chemical surfactants, it can reduce secondary damage to the environment, and the biosurfactant produced by the strain can accelerate the emulsification of oil and accelerate the biodegradation of oil pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioremediation of environments polluted by petroleum and petroleum products, and in particular relates to a surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 originating from the Antarctic low-temperature environment and applications thereof. Background Art

[0002] Antarctica is often considered one of the most pristine environments on Earth, with perennial ice and snow and low temperatures. The Antarctic environment is also susceptible to global and local human activities. Most of the Antarctic continent is covered by glaciers (>99.5%), subject to a variety of changing environmental stresses, including temperature, humidity, light / radiation climate, and low nutrient levels. The Antarctic ecosystem is relatively fragile, experiencing relatively little direct human impact, but is susceptible to indirect human activities, such as the introduction of organic pollutants such as petroleum and inorganic pollutants such as heavy metals. These pollutants can be degraded or transformed by microorganisms, mitigating their impact on the Antarctic ecosystem. Low temperatures, as the most significant environmental factor in Antarctica, are the primary environmental factor to which indigenous microorganisms must adapt, and the production of biosurfactants is one of the strategies employed by cold-adapted Antarctic strains. Biosurfactants can enhance the bioavailability of hydrocarbon substrates and facilitate their uptake by cells. The ability to produce biosurfactants appears to be a common trait of cold-adapted strains.

[0003] Biosurfactants are a class of naturally occurring amphiphilic surface-active substances metabolized by a variety of microorganisms. They reduce surface and interfacial tension, resulting in excellent emulsification, dispersion, and foaming properties. Biosurfactants also possess advantages such as low critical micelle concentration (CMC), high biodegradability, strong temperature resistance, low toxicity, and a wide pH range. Similar to chemical surfactants, biosurfactants are composed of hydrophilic and hydrophobic groups. The hydrophilic groups are diverse, with amino acids, monosaccharides, and polysaccharides being common, while the hydrophobic groups are mostly non-polar groups composed of hydrocarbon chains. Based on the type of hydrophilic group, biosurfactants can be classified into lipopeptides, glycolipids, phospholipids, neutral lipids, and polymeric biosurfactants. Bacteria are the primary producers of surfactants, with strains primarily producing Pseudomonas, Bacillus, and Candida species.

[0004] Jiao Yabin et al. (Isolation and characterization of a high-surfactant-producing Antarctic soil bacterium [J]. Bulletin of Microbiology, 2023, 50(8): 3285-3299) isolated a new strain of Pedobacter sp. GW9-17 from the Fildes Peninsula in Antarctica. The surface tension of its culture supernatant was reduced to 42.5 mN / m, and the product was identified as flavolipids. Lamilla et al. (Streptomyces luridus So3.2 from Antarctic soil as a novel producer of compounds with bioemulsification potential [J / OL]. PLoS ONE, 2018, 13(4) [2024-08-29]) isolated a surfactant-producing strain, Streptomyces luridus So3.2, from the South Shetland Islands in Antarctica. By optimizing the culture medium and culture conditions, its emulsification rate exceeded 60%. These studies screened some Antarctic biosurfactant-producing strains from parts of Antarctica and also partially explored the composition and properties of the biosurfactants they produced. However, for Antarctica, which has unique biological resources, whether there are other biosurfactant-producing bacteria, whether their activity is higher than the reported strains, and what the composition of the biosurfactants is, still needs further development. Summary of the Invention

[0005] The present invention provides a polar low-temperature surfactant-producing bacterium, Pedobacter cryoconitis NJ-S-72, and its application. The strain is derived from the soil of the Fildes Peninsula in Antarctica. The fermentation broth can be directly applied to the ecological restoration of oil spills in polar or low-temperature environments. Compared with chemical surfactants, it can reduce secondary damage to the environment. The biosurfactant produced by the strain can accelerate the emulsification of oil and the biodegradation of oil pollution.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a polar low-temperature surfactant-producing bacterium Pedobactercryoconitis NJ-S-72, which was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit number CGMCC NO: 32458.

[0007] Preferably, the nucleotide sequence thereof is shown in SEQ ID NO: 1.

[0008] The present invention also provides a biosurfactant, which is prepared by using the polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 described in the above technical solution.

[0009] As preferably, it is prepared by the following steps:

[0010] The polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 was cultured at 15°C and 150 rpm, the culture broth was centrifuged, acidified to adjust the pH to 2.0-3.0, and incubated at 4°C overnight, followed by centrifugation to obtain a crude biosurfactant extract;

[0011] The crude extract was dispersed in aqueous solution, extracted with an equal amount of ethyl acetate, allowed to stand for stratification and the organic phase was collected. The above operation was repeated 2-3 times and the organic phases were combined and lyophilized by rotary evaporation to obtain a biosurfactant.

[0012] The present invention also provides a use of the polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to the above technical solution in the preparation of a biosurfactant.

[0013] Preferably, the biosurfactant is a biosurfactant used for ecological restoration of oil spills in low-temperature ecological environments.

[0014] As a preference, the average temperature of the low-temperature ecological environment is 10-15°C

[0015] The present invention also provides a use of the polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 according to the above technical solution in promoting petroleum emulsification and accelerating the biodegradation of petroleum pollution.

[0016] As a preference, the oil drain ring of NJ-S-72 reaches 3.1CM, and the surface tension is reduced to 33.1mN / m.

[0017] Preferably, the emulsification effect on petroleum is 25%.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are:

[0019] The present invention provides a surfactant-producing bacterium, Pedobactercryoconitis NJ-S-72, screened from the Fildes Peninsula in Antarctica. The surfactant-producing bacterium has superior activity to previously reported strains, and its fermentation broth can be directly applied to the ecological restoration of oil spills in polar or low-temperature environments. Compared with chemical surfactants, it can reduce secondary damage to the environment. In addition, the biosurfactant produced by this strain can accelerate the emulsification of oil and the biodegradation of oil pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Schematic diagram of the surface tension and oil-discharge ring of the strain at 15°C provided in an embodiment of the present invention;

[0021] Figure 2 The NJ-S-72 phylogenetic tree provided in the embodiment of the present invention;

[0022] Figure 3 The emulsification capacity of the NJ-S-72 supernatant provided in the embodiment of the present invention;

[0023] Figure 4 Fourier transform infrared spectrum of the surfactant extracted from strain NJ-S-72 provided in the embodiment of the present invention;

[0024] Figure 5 A chromatogram of a surfactant extracted from strain NJ-S-72 provided in an embodiment of the present invention;

[0025] Figure 6 The colony status of the strains Pedobacter GW9-17 and NJ-S-72 provided in the embodiments of the present invention, wherein A: GW9-17 colony, B: NJ-S-72 colony. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0027] Example 1 Experimental materials

[0028] Sample source

[0029] The polar low-temperature surfactant-producing bacteria involved were screened from soil samples collected on the Fildes Peninsula in Antarctica during the 36th Antarctic scientific expedition in 2020.

[0030] culture medium

[0031] Inorganic salt culture medium (1 L): ammonium nitrate 2.0 g, dipotassium hydrogen phosphate 1.5 g, potassium dihydrogen phosphate 3.0 g, magnesium sulfate heptahydrate 0.1 g, anhydrous calcium chloride 0.01 g, disodium ethylenediaminetetraacetic acid dihydrate 0.01 g;

[0032] M8 medium (1L): Add the following nutrients to the inorganic salt medium:

[0033] Sodium acetate 2g, peptone 0.5g, yeast extract 0.5g, potato extract powder 0.5g, glucose 0.2g, sucrose 0.2g, sodium malate 0.05g, sodium citrate 0.05g, sodium tartrate 0.05g, adjust the pH to 7.2-7.4, then sterilize at 121℃ for 15min.

[0034] Example 2 Isolation and screening of surfactant-producing bacteria

[0035] Isolation of surfactant-producing bacteria

[0036] Take 5g of soil sample collected from Antarctica and add it to a conical flask containing 100mL of petroleum culture medium. The blank control is the petroleum culture medium without sample (1% of inorganic salt culture medium is added).

[0037] (m / v) of petroleum), with 3 parallels set up in each group. 15℃, the rotation speed is 150r / min. After 14 days of enrichment culture, 1mL of enrichment culture is taken to fresh 100mL petroleum culture medium, and enrichment culture is continued for 14 days under the same conditions. After enrichment 3 times, gradient dilution is performed using the dilution plate method, and the strain is spread on M8 nutrient salt solid for strain isolation. Culture at a constant temperature of 15℃ for 7-14 days. After the colonies grow, single colonies of different morphologies are picked and streaked on fresh plates for purification, and cultured at 15℃ for 7-14 days. 32 pure cultured bacteria were obtained from them, and based on this, surfactant-producing bacteria were screened.

[0038] Screening of surfactant-producing bacteria

[0039] A bacterial culture grown on M8 medium at 15°C and 150 rpm for 5 days was centrifuged at 4°C and 9000 rpm for 10 minutes to obtain a cell-free supernatant. The supernatant was then tested using a drain ring and surface tension methods to screen for surfactant-producing bacteria.

[0040] a) Oil drain ring

[0041] The oil ring method is an indirect method for measuring surface activity. It is generally believed that the diameter of the oil ring is proportional to the surface activity of the test solution. Add 20 mL of distilled water to a glass culture dish. Then, add 500 μL of liquid paraffin containing Sudan III dye to the center of the liquid. Then, slowly add 50 μL of fermentation supernatant to the center of the paraffin. Observe and measure the size of the oil ring. Uninoculated M8 liquid culture medium serves as a negative control.

[0042] b) Surface tension

[0043] Surface tension was measured using a fully automatic surface tension meter (JYW-200B, China). 30 mL of fermentation supernatant was taken for measurement. Target strains were selected, those with large oil-discharging rings and low surface tension.

[0044] The results are as follows Figure 1 As shown, 22 of the 32 isolated strains produced an oil ring, preliminarily indicating that these 22 strains have the ability to produce surfactants. Combined with surface tension measurements, it was found that the surface tension of the majority (68%) of the strains ranged from 60mN / m to 75mN / m, which was not much different from the surface tension of water (72mN / m). However, one strain had a significantly lower surface tension, with an oil ring reaching 3.1cm and a significantly lower surface tension of 33.1mN / m. This confirmed the identification of a high-yield surfactant-producing strain, designated strain NJ-S-72.

[0045] Example 3 Identification of surfactant-producing bacteria

[0046] Molecular identification

[0047] The surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 was inoculated on a solid plate supplemented with M8 nutrients by the streak method and cultured at 15°C for two days.

[0048] The strain plates were sent to Qingke Biotech (Qingdao) for sequencing. The sequencing results were quality-checked and spliced ​​before being submitted to EZbiocloud (www.ezbiocloud.net) for 16S rRNA gene sequence alignment. 15 standard strain 16S rRNA gene sequences with high similarity to the bacteria were selected, downloaded, and imported into MEGA X for ClustalW multiple sequence alignment. A phylogenetic tree was constructed using the p-distance nucleic acid model. Figure 2 As shown, the 16S rRNA gene sequence of strain NJ-S-72 is the highest with that of Pedobacter cryoconitis DSM 14825 (Type) (Accession No.: MW928785), reaching 99.30%. The strain was deposited in the General Microbiology Center of China National Committee for Microbiological Culture Collection on October 31, 2024, with the deposit number CGMCC NO: 32458. Its nucleotide sequence is shown in SEQ ID NO: 1.

[0049] Example 4 Emulsifying ability of surfactant-producing bacteria

[0050] The emulsification index of the centrifuged supernatant of the screened strain NJ-S-72 was measured with several finished oils (diesel, motor oil, paraffin oil, petroleum, etc.) to evaluate its emulsification performance. The specific method is as follows:

[0051] Pipette 2 mL of the supernatant from the centrifugation and an equal volume of various hydrocarbons into a graduated tube, vortex for 2 minutes, and let it stand for 24 hours to calculate the 24-hour emulsification index (E24). The emulsification index formula is as follows:

[0052]

[0053] The results are as follows Figure 3 As shown, NJ-S-72 exhibited the best emulsification effect on diesel, reaching an emulsification index of 34% after 24 hours of stabilization. The surfactant produced by this strain also had a good emulsification effect on diesel, followed by petroleum oil, at 25%. However, it had poor emulsification effects on motor oil and paraffin oil, with emulsification indices of 4% and 5%, respectively, after 24 hours of stabilization.

[0054] Example 5 Extraction of biosurfactant (acid precipitation-organic solvent extraction method)

[0055] The selected strain NJ-S-72 was cultured at 15°C and 150 rpm for 5 days. The culture was centrifuged at 4°C and 9,000 rpm for 10 minutes. A 2 mol / L HCl solution was added to the supernatant, the pH adjusted to 2.0, and the supernatant was incubated at 4°C overnight. The extract was then centrifuged at 9,000 rpm for 10 minutes to obtain a crude biosurfactant extract. The crude extract was dispersed in a pH-2.0 aqueous solution, extracted with an equal amount of ethyl acetate, allowed to stand for stratification, and the organic phase collected. This operation was repeated three times, and the organic phases were combined, rotary evaporated at 45°C, and freeze-dried to obtain a biosurfactant, which was named Biosurfactant A.

[0056] Example 6 Chemical Composition Analysis of Biosurfactants

[0057] Fourier transform infrared spectroscopy (FT-IR)

[0058] The freeze-dried biosurfactant A was scanned with an attenuated total reflectance (ATR) Fourier transform infrared spectrometer (μ-FT-IR, PerkinElmer Spectrum Spotlight 400, Perkin Elmer Inc., USA) at a frequency of 2 cm and a wavelength range of 4000 cm . -1 -400cm -1 , detecting characteristic functional groups in biosurfactants.

[0059] The results are as follows Figure 4 As shown, the biosurfactant has a peak at 1725 cm -1 There is a broad absorption peak around 3270cm, which is related to the stretching vibration of the carboxyl group. -1 The strong absorption band at 1069 cm -1The absorption peaks at 2922cm are the stretching vibrations of -OH and COC, which indicates the presence of cyclic sugar structures in the molecule. -1 The absorption peaks around 1640cm are the stretching vibrations of the CH group. -1 The peaks around 1558cm indicate the presence of ester compounds. -1 、1388cm -1 and 1636cm -1 The absorption peak indicates the possible existence of amino groups. This shows that the extracted biosurfactant is a mixture of multiple substances.

[0060] HPLC-MS

[0061] Biosurfactant A was dissolved in chromatographic-grade methanol and ultrapure water (1:1) to a final concentration of 1 mg / mL. Impurities were filtered using a 0.22 μm polytetrafluoroethylene (PTFE) syringe filter, and 1 mL of the filtrate was added to a screw-capped sample vial. The sample vial was placed in an LC-MS liquid chromatograph (1100LC-MS Agilent Technologies Co., Ltd.) and detected by chromatography-mass spectrometry according to the method of Yin et al. (Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater [J]. Process Biochemistry, 2009, 44(3): 302-308).

[0062] The results are as follows Figure 5 As shown in the chromatogram, the substances shown are mainly lipids. 34 H 60 O9N4 may be a substance called flavolipid-9U, 9U, which was first discovered by Bodour et al. (Structure and Characterization of Flavolipids, a Novel Class of Biosurfactants Produced by Flavobacterium sp. Strain MTN11 [J]. Applied and Environmental Microbiology, 2004, 70 (1): 114-120) in Flavobacterium sp. strain MTN11 (Accession No. AY162137). Jiao Yabin et al. also discovered this substance in Pedobacter strains.

[0063] Table 1 Main components of surfactants extracted from strain NJ-S-72

[0064]

[0065] Performance comparison of Example 7

[0066] As mentioned above, Jiao Yabin et al. (Isolation and characteristics of a high-producing surfactant-producing Antarctic land bacterium [J]. Bulletin of Microbiology, 2023, 50(8): 3285-3299) isolated a new bacterium Pedobacter sp.GW9-17 in the Fildes Peninsula, Antarctica. In order to compare its effect with strain NJ-S-72, the two were compared in terms of colony morphology, species, and surface tension.

[0067] Colony morphology

[0068] The colony morphology of strain Pedobacter sp.GW9-17 and strain NJ-S-72 is as follows Figure 6 As shown in the figure, there is a clear difference in the colony colors of the two strains. The colony color of GW9-17 is orange, while the colony color of NJ-S-72 is light yellow.

[0069] Species

[0070] The 16S rRNA gene sequence of strain GW9-17 showed the highest similarity with Pedobacter alluvionis DSM 19624T, reaching 98.7%. The 16S rRNA gene sequence of strain NJ-S-72 showed the highest similarity with Pedobacter cryoconitis DSM 14825 (Type), reaching 99.30%. A comparison of the 116S rRNA sequences of NJ-S-72 and GW9-17 revealed a similarity of 94.29%, indicating that the two strains are not the same.

[0071] surface tension

[0072] The culture supernatant, after centrifugation at 9000 rpm for 15 minutes, was measured using an automatic surface tension meter (JYW-200B, China). The surface tension of strain NJ-S-72 was 33.1 mN / m, indicating significantly higher activity than the 42.5 mN / m obtained from the culture supernatant of Pedobacter sp. GW9-17.

[0073] Emulsifying properties

[0074] This patent application focuses on the biodegradation of petroleum pollution, so the emulsification index for petroleum is the key index. Combined with Example 4, the emulsification index of NJ-S-72 supernatant for diesel is 34%, and the emulsification index for petroleum is 25%, both of which are relatively ideal and can effectively solve the ecological restoration problem of oil leaks in environments such as polar and low-temperature environments. However, this was not recorded in the above-mentioned research by Jiao Yabin et al.

Claims

1. Application of polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 in the preparation of biosurfactants, characterized in that: The deposit number of the Pedobacter cryoconitis NJ-S-72 is CGMCCNO:32458.

2. A biosurfactant, characterized in that Prepared by using the polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 according to claim 1; The biosurfactant is prepared by the following steps: The polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 was cultured at 15°C and 150 rpm, the culture broth was centrifuged, acidified to adjust the pH to 2.0-3.0, and incubated at 4°C overnight, followed by centrifugation to obtain a crude biosurfactant extract; The crude extract was dispersed in an aqueous solution, extracted with an equal amount of ethyl acetate, allowed to stand for stratification, and the organic phase was collected. The above operation was repeated 2-3 times, and the organic phases were combined, rotary evaporated, and freeze-dried to obtain a biosurfactant. In terms of mass percentage, the biosurfactant includes C 16 H 30 O7 71%, C 34 H 60 O9N4 11%, C 18 H 34 O77%, C 20 H 36 O 11 5%, C 22 H 40 O 11 5% and C 30 H 54 O9 1%.

3. Use of the polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to claim 1 or the biosurfactant according to claim 2 in ecological restoration of oil spills in low-temperature ecological environments.

4. The use according to claim 3, characterized in that The temperature of the low-temperature ecological environment is 10-20℃.

5. Use of the fermentation broth of the polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to claim 1 in promoting the emulsification of diesel or petroleum.

6. The use according to claim 5, characterized in that The oil drainage circle of the fermentation liquid of Pedobacter cryoconitis NJ-S-72 reached 3.1CM, and the surface tension was reduced to 33.1mN / m.

7. The use according to claim 5, characterized in that The emulsification index of the fermentation liquid of Pedobacter cryoconitis NJ-S-72 to diesel is 34%, and the emulsification index to petroleum is 25%.

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