Polar region low-temperature surfactant producing strain Pedobacter cryconitis NJ-S-72 and application thereof

Abstract

The invention provides a polar region low-temperature surfactant producing strain Pedobacter cryconiis NJ-S-72 and application of the polar region low-temperature surfactant producing strain Pedobacter cryconiis NJ-S-72, and belongs to the technical field of bioremediation of petroleum and petroleum product polluted environments. The strain is preserved in the China General Microbiological Culture Collection Center on October 31, 2024, and the preservation number is CGMCC NO: 32458. The bacterial strain is derived from soil in Peninsula of the Antarctic Field, fermentation liquor of the bacterial strain can be directly applied to ecological restoration of petroleum leakage in a polar region or a low-temperature environment, and compared with a chemical surfactant, secondary damage to the environment can be reduced, and a biological surfactant generated by the bacterial strain can accelerate and promote petroleum emulsification, so that the petroleum leakage rate is increased. The biodegradation of petroleum pollution is accelerated.

Description

Technical Field

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

[0002] Antarctica is often considered one of the most pristine environments on Earth, with perennial ice and low temperatures. The Antarctic environment is also susceptible to human activities both globally and locally. Most of the Antarctic continent is covered by glaciers (>99.5%) and is subject to multiple changing environmental stresses, such as temperature, humidity, light / radiation climate, and low nutrient conditions. The Antarctic ecosystem is relatively fragile and is relatively less affected by direct human impacts, 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, reducing their impact on the Antarctic ecosystem. Low temperature, as the most significant environmental factor in the Antarctic environment, is the first environmental factor that indigenous microorganisms must adapt to, and the production of biosurfactants is one of the strategies adopted by cold-adapted Antarctic strains. Biosurfactants can increase the bioavailability of hydrocarbon substrates and promote their uptake by cells. The ability to produce biosurfactants appears to be a common feature of cold-adapted strains.

[0003] Biosurfactants are a class of natural amphiphilic surface active substances that can be produced by the metabolism of a variety of microorganisms. They can reduce surface and interfacial tension and produce good emulsification, dispersion and foaming properties. In addition, biosurfactants also have advantages such as low critical micelle concentration (CMC), high biodegradability, strong temperature resistance, low toxicity and wide pH tolerance range. Similar to chemical surfactants, biosurfactants are composed of hydrophilic groups and hydrophobic groups. The hydrophilic groups are relatively diverse, and the common ones are amino acids, monosaccharides and polysaccharides, while the hydrophobic groups are mostly non-polar groups composed of hydrocarbon chains. According to the type of hydrophilic group, biosurfactants can be divided into lipopeptides, glycolipids, phospholipids, neutral lipids and polymer biosurfactants. Bacteria are the main producers of surfactants, and the strains that produce surfactants are mainly Pseudomonas, Bacillus, Candida, etc.

[0004] Jiao Yabin et al. (Isolation and characterization of an Antarctic soil bacterium with high surfactant production [J]. Bulletin of Microbiology, 2023, 50(8): 3285-3299) isolated a new bacterium 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 bacterium 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 invention provides a polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 and an application thereof. 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, the secondary damage to the environment can be reduced, and the biological surfactant 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 Microbiological Center of China Microbiological Culture Collection Administration on October 31, 2024, with a deposit number of CGMCC NO: 32458.

[0007] Preferably, the nucleotide sequence 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] Preferably, it is prepared by the following steps:

[0010] The polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 were cultured at 15°C and 150 r / min, the culture solution was centrifuged, the pH was adjusted to 2.0-3.0 by acidification and placed at 4°C overnight, and then centrifuged to obtain a crude biosurfactant extract;

[0011] The crude extract was dispersed in an aqueous solution, and an equal amount of ethyl acetate was added for extraction. The organic phase was collected after standing for stratification. The above operation was repeated 2-3 times, and the organic phases were combined, and then subjected to rotary evaporation and freeze-drying to obtain a biosurfactant.

[0012] 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 the preparation of biological surfactants.

[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 an application 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] Preferably, the oil drainage 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, which has better activity than 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, and the biological surfactant produced by the strain can accelerate the emulsification of oil and accelerate the biodegradation of oil pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A schematic diagram of the surface tension and oil drainage 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 states of 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 technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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] 2g sodium acetate, 0.5g peptone, 0.5g yeast extract, 0.5g potato extract powder, 0.2g glucose, 0.2g sucrose, 0.05g sodium malate, 0.05g sodium citrate, 0.05g sodium tartrate, adjust the pH to 7.2-7.4, and 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. For the blank control, choose petroleum culture medium without sample (add 1%

[0037] (m / v) of petroleum), with 3 parallels in each group. 15℃, the rotation speed is 150r / min. After 14 days of enrichment culture, 1mL of enrichment culture was taken into fresh 100mL of petroleum culture medium, and enrichment culture was continued for 14 days under the same conditions. After enrichment 3 times, gradient dilution was performed using the dilution plate method, and the strains were spread on M8 nutrient salt solids for strain separation. Constant temperature culture was carried out at 15℃ for 7-14 days. After the colonies grew out, single colonies of different morphologies were picked and streaked on fresh plates for purification, and cultured at 15℃ for 7-14 days. 32 pure cultured bacteria were obtained, and surfactant-producing bacteria were screened based on this.

[0038] Screening of surfactant-producing bacteria

[0039] The bacterial liquid cultured on M8 medium at 15°C and 150 r / min for 5 days was centrifuged at 4°C and 9000 r / min for 10 min to obtain the cell-free supernatant. The cell-free supernatant after centrifugation was tested using the oil ring and surface tension method to screen for surfactant-producing bacteria.

[0040] a) Oil drain ring

[0041] The oil-discharging ring method is an indirect method for measuring surface activity. It is generally believed that the diameter of the oil-discharging ring is proportional to the surface activity of the test solution. Add 20 mL of distilled water to a glass culture dish, then drop 500 uL of liquid paraffin with Sudan III dye in the center of the liquid surface, slowly drop 50 uL of fermentation supernatant in the center of the paraffin, and observe and measure the size of the oil-discharging ring. Use the uninoculated M8 liquid culture medium as a negative control.

[0042] b) Surface tension

[0043] The surface tension was measured by a fully automatic surface tension meter (JYW-200B, China). 30 mL of fermentation supernatant was taken for measurement. The strains with large oil-discharging ring and small surface tension were selected as the target strains.

[0044] The results are as follows Figure 1 As shown, 22 of the 32 isolated strains produced oil rings, which preliminarily indicated that these 22 strains had the ability to produce surfactants. Combined with surface tension measurements, it was found that the surface tension of most strains (68%) was between 60mN / m and 75mN / m, which was not much different from the surface tension of water (72mN / m), but the surface tension of one strain was significantly reduced, with an oil ring of 3.1CM and a significantly reduced surface tension of 33.1mN / m. It was thus determined that a high-yield surfactant-producing strain was screened and named 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 streaking 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, and the p-distance nucleic acid model was used to construct a phylogenetic tree. Figure 2 As shown, the 16SrRNA gene sequence similarity between strain NJ-S-72 and Pedobactercryoconitis DSM 14825 (Type) (Accession No.: MW928785) is the highest, reaching 99.30%. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on October 31, 2024, with the deposit number CGMCC NO: 32458, and its nucleotide sequence table 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, engine oil, paraffin oil, petroleum, etc.) to evaluate its emulsification performance. The specific method is as follows:

[0051] Pipette 2 mL of the supernatant and equal volumes of various hydrocarbons into a graduated tube, vortex at high speed for 2 minutes, and let 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 has the best emulsification effect on diesel, and the emulsification index reaches 34% after standing for 24 hours. The surfactant produced by this strain has a good emulsification effect on diesel, followed by 25% for petroleum. The emulsification effect on motor oil and paraffin oil is poor, and the emulsification index is 4% and 5% respectively after standing for 24 hours.

[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 150r / min for 5 days, the culture solution was centrifuged at 4°C and 9000r / min for 10min, 2mol / L HCl solution was added to the centrifugal supernatant, the pH was adjusted to 2.0 and placed at 4°C overnight. Then centrifuged at 9000r / min for 10min to obtain a crude biosurfactant extract. The crude extract was dispersed in an aqueous solution at pH = 2.0, and then an equal amount of ethyl acetate was added for extraction, the organic phase was allowed to stand and the stratification was collected. The above operation was repeated 3 times and the organic phases were combined, and the biosurfactant was obtained by rotary evaporation at 45°C and freeze-drying, and it 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 at a frequency of 2 cm with an attenuated total reflectance (ATR) Fourier transform infrared spectrometer (μ-FT-IR, PerkinElmer Spectrum Spotlight 400, Perkin Elmer Inc., USA) over 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 on the left and right are the stretching vibrations of the CH group. -1 The peaks on the left and right 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 out using a 0.22 μm polytetrafluoroethylene (PTFE) syringe filter, and 1 mL of the filtrate was added to a screw-capped sample bottle. The sample bottle was placed in an LC-MS liquid chromatograph (1100LC-MS Agilent Technologies, Inc.) and chromatographic mass spectrometry was performed according to the method of Yin et al. (Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6isolated 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 found this substance in Pedobacter strains.

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

[0064]

[0065] Example 7 Performance Comparison

[0066] As mentioned above, Jiao Yabin et al. (Isolation and Characterization of a High-Producing Surfactant Pedobacter sp. from Antarctica [J]. Bulletin of Microbiology, 2023, 50(8): 3285-3299) isolated a new bacterium Pedobacter sp.GW9-17 from the Fildes Peninsula in Antarctica. In order to compare its effect with that of 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, there is a clear difference in the colony colors of the two. The colony color of GW9-17 is orange-yellow, while the colony color of NJ-S-72 is light yellow.

[0069] Species

[0070] The 16S rRNA gene sequence of strain GW9-17 and Pedobacter alluvionis DSM 19624T has the highest similarity, reaching 98.7%; the 16S rRNA gene sequence of strain NJ-S-72 and Pedobacter cryoconitis DSM 14825 (Type) has the highest similarity, reaching 99.30%. Comparison of 116S rRNA of NJ-S-72 and GW9-17 showed that the similarity between the two was 94.29%, and it was inferred that the two were not the same strain.

[0071] Surface tension

[0072] The supernatant of the culture solution after centrifugation at 9000r for 15min was measured with an automatic surface tension meter (JYW-200B, China). 30mL of the fermentation supernatant was taken for measurement, and the surface tension of strain NJ-S-72 was 33.1mN / m, which was much higher than the 42.5mN / m of the culture supernatant of Pedobacter sp.GW9-17.

[0073] Emulsification performance

[0074] This patent application focuses on the biodegradation of petroleum pollution, so the emulsification index of 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. Polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 31, 2024, with the deposit number CGMCC NO: 32458.

2. The surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to claim 1, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

1.

3. A biosurfactant, characterized in that The method is prepared by using the polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 described in claim 1.

4. The biosurfactant according to claim 3, characterized in that Prepared by the following steps: The polar low-temperature surfactant-producing bacteria Pedobacter cryoconitis NJ-S-72 were cultured at 15°C and 150 r / min, the culture solution was centrifuged, the pH was adjusted to 2.0-3.0 by acidification and placed at 4°C overnight, and then centrifuged to obtain a crude biosurfactant extract; The crude extract was dispersed in an aqueous solution, and an equal amount of ethyl acetate was added for extraction. The organic phase was collected after standing for stratification. The above operation was repeated 2-3 times, and the organic phases were combined, and then subjected to rotary evaporation and freeze-drying to obtain a biosurfactant.

5. Use of the polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to claim 1 or 2 in the preparation of biosurfactants.

6. The use according to claim 5, characterized in that: The biosurfactant is a biosurfactant used for ecological restoration of oil spills in low-temperature ecological environments.

7. The use according to claim 6, characterized in that: The temperature of the low temperature ecological environment is 10-20℃.

8. Use of the fermentation liquid of the polar low-temperature surfactant-producing bacterium Pedobacter cryoconitis NJ-S-72 according to claim 1 or 2 in promoting petroleum emulsification and accelerating the biodegradation of petroleum pollution.

9. The use according to claim 8, characterized in that: The oil drain ring of NJ-S-72 reaches 3.1CM, and the surface tension is reduced to 33.1mN / m.

10. The use according to claim 8, characterized in that: The emulsification effect on petroleum is 25%.

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