KR100458136B1 - The marine bacterium pseudomonas aeruginosa byk-2 producing rhamnolipid and methyl rhamnolipid simultaneously - Google Patents

Abstract

The present invention provides a technique for separating marine bacteria Pseudomonas aeruginosa biwaikei-2, a bioemulsifier producing strain that can be used simultaneously for the purification of effluent oil on land and in the ocean, and can be used in various industries. In the efficient separation and purification technology of the emulsifier, the strain has a unique growth characteristic of simultaneously producing a lipid lipid bioemulsifier, Rhamnolipid and Methyllamnolipid, different from Pseudomonas aeruginosa strain.

Description

MARINE BACTERIUM PSEUDOMONAS AERUGINOSA BYK-2 PRODUCING RHAMNOLIPID AND METHYL RHAMNOLIPID SIMULTANEOUSLY} Marine bacterium Pseudomonas aeruginosa biwaii producing both ramno lipid and methyl ramno lipid

The present invention is a technology for separation of marine bacteria, growth characteristics, and separation and purification of bioemulsions to develop bioemulsifiers that have excellent oil resolution and can be used in various industries on land and at sea. Bacterial Pseudomonas aeruginosa BYK-2 (April 27, 2000, date of deposit of Biotechnology Research Institute) (Microbial Accession No .: KCTC 18012P).

Typically, studies on Pseudomonas aeruginosa strains have been reported for the first time to isolate bacteria from soil and antimicrobial activity, and since 1949 it has been confirmed to produce glycolipids by FJ Jarvis et al. Much research has been done on Pseudomonas aeroginosa, its variants, and the types of glycolipid bioemulsifiers they produce. Pseudomonas aeruginosa is known as Pseudomonas aeruginosa and is mainly used for wastewater treatment and biological purification of soil oil pollution area.

Bioemulsifiers produced by Pseudomonas aeroginosa are the major component of glycolipids by many researchers, sugar is rhamnose, and lipid is composed of beta-hydroxydecanoic acid. It has been found that some mutant species appear depending on the conditions of the environment, it is reported that the form of the bioemulsifier also changes. As shown in FIG. 35, the glycolipid bioemulsifier is rhamno lipid-1 (RL-1), rhamno lipid-2 (RL-2), rhamno lipid-3 (RL-3), and lamb Four main forms of noripid-4 (RL-4) are known.

The most common of the four major forms of rhamnolipid were RL 1 and RL 3, which were found by Hisatsuka et al. (1971) and Itoh et al. (1972), respectively, and RL 2 and RL 4 are synthesized only in resting cells. Sydatk (1985) et al. As shown in FIG. 36, methyl rhamnolipids are produced by Pseudomonas aeruginosa 158 strains of Hirayama and Kato (1982), and beta-hydride at the structural sites of RL 1 and RL 3. The methyl group is bonded to the carbon position of the oxydecanoic acid. Also, as shown in FIG. 37, Yamaguchi et al. (1976) described a modified ramnori in which alpha-decanoic acid was bound to carbon positions 2, which are the lamnose structures of RL 1 and RL 3, respectively. You are reporting a feed.

However, in the case of the conventionally reported bacteria, there is an advantage that can directly decompose the oil by spraying the bacteria in the soil during the oil spill accident, it is difficult to apply directly because it is not a basophilic bacteria in the ocean.

In addition, in the case of the prior art, Pseudomonas aeruginosa was mainly separated from the soil and studied, but there is no case in which it is separated from the ocean, and in the case of soil bacteria, growth is inhibited due to salinity, which is difficult to apply in marine oil pollution. Therefore, it has been mainly used for treating soil runoff oil and wastewater.

Therefore, the present invention efficiently separates Biwai-2 from both the oil pollution area of South Korea, which produces oil and methyl rhamno lipids, which are excellent in oil resolution and different from conventional reports. Its purpose is to develop technologies that can be separated and purified.

Another object of the present invention is to develop an efficient alternative to oil spills in soil and marine oil pollution areas and to develop environmentally friendly substitutes for chemical synthetic surfactants that are used in various industries.

Pseudomonas aeruginosa biwaikei-2 of the present invention to achieve the object as described above is put in the oil contaminated area seawater sample in strain-separated oil medium and decomposed by shaking culture for 7 days, and then to the sterilized oil medium Shaking culture for 3 days with 2% inoculation; Selecting a medium having the highest resolution and smearing it on a modified seawater medium to separate single strains, and then inoculating it on a separating oil medium for shaking culture for 7 days; It is characterized by consisting of a process of screening the medium having excellent crude oil resolution again and smearing the modified seawater medium to finally isolate a single strain.

1 is a block diagram showing a method for separating oil decomposition bacteria from the marine oil pollution zone according to the present invention.

Figure 2 is a table showing the identification results of the strain of the present invention.

Figure 3 is a graph of the present invention showing the growth characteristics of Pseudomonas aeruginosa biwaikei-2 with a change in salt concentration.

Figure 4 is a graph of the present invention showing the biodegradation of refractory arabian light crude oil using Pseudomonas aeruginosa biwaikei-2.

5 is a graph of the present invention showing the biodegradability of the hardly decomposable bunker seed oil using Pseudomonas aeruginosa biwakei-2.

Figure 6 is a block diagram showing a separation and purification method of the bioemulsifier produced by the strain according to the present invention.

Figure 7 is a graph of the present invention showing the thin layer chromatography results of the bioemulsifier produced according to the type of carbon source.

8 is a graph of the present invention showing the results of thin layer chromatography of two kinds of purified bioemulsifiers (BS-1, BS-2).

9 is a graph of the present invention showing the analysis results using a conversion infrared spectroscopy apparatus of glycolipid bioemulsifier BS-1.

10 is a graph of the present invention showing the results of analysis using a fast atom bombardment mass spectrometry of glycolipid bioemulsifier BS-1.

11 is a graph of the present invention showing the analysis results using a hydrogen nuclear magnetic resonance device of the glycolipid bioemulsifier BS-1.

12 is a graph of the present invention showing the analysis results using a carbon nuclear magnetic resonance device of the glycolipid bioemulsifier BS-1.

FIG. 13 is a graph of the present invention showing the DEP spectroscopic analysis of glycolipid bioemulsifier BS-1. FIG.

14 is a graph of the present invention showing the results of NOESY spectroscopy analysis of glycolipid bioemulsifier BS-1.

15 is a graph of the present invention showing the results of TOCSY spectroscopy analysis of glycolipid bioemulsifier BS-1.

Figure 16 is a graph of the present invention showing the relay (RELAY) spectroscopic analysis of the glycolipid bioemulsifier BS-1.

FIG. 17 is a graph of the present invention showing the results of HSQC spectroscopy analysis of glycolipid bioemulsifier BS-1. FIG.

18 is a graph of the present invention showing the results of etch MBC (HMBC) spectroscopic analysis of the glycolipid bioemulsifier BS-1.

19 is a structural formula of the present invention showing the structural analysis of the glycolipid bioemulsifier BS-1.

20 is a graph of the present invention showing the analysis results using a conversion infrared spectroscopy apparatus of glycolipid bioemulsifier BS-2.

21 is a graph of the present invention showing the analysis results using the hydrogen nuclear magnetic resonance device of the glycolipid bioemulsifier BS-2.

22 is a graph of the present invention showing the analysis results using a carbon nuclear magnetic resonance device of the glycolipid bioemulsifier BS-2.

Figure 23 is a graph of the present invention showing the DEP (DEPT) spectroscopic analysis of the glycolipid bioemulsifier BS-2.

24 is a graph of the present invention showing the relay (RELAY) spectroscopic analysis of the glycolipid bioemulsifier BS-2.

Fig. 25 is a graph of the present invention showing the results of HSQC spectroscopy analysis of glycolipid bioemulsifier BS-2.

Figure 26 is a graph of the present invention showing the results of etch MBC (HMBC) spectroscopic analysis of glycolipid bioemulsifier BS-2.

Figure 27 is a structural formula of the present invention showing the structure analysis of the glycolipid bioemulsifier BS-2.

28 is a graph of the present invention showing the critical micelle concentration results of bioemulsifiers isolated from Pseudomonas aeruginosa BYK-2 strain.

29 is a graph of the present invention showing the results of measuring the hydrophilic-lipophilic balance (HLB) of bioemulsifier isolated from Pseudomonas aeruginosa BYK-2 strain.

30 is a graph of the present invention showing the results of emulsion stability analysis in fresh water of bioemulsifier and commercial surfactant isolated from Pseudomonas aeruginosa BYK-2 strain.

Figure 31 is a graph of the present invention showing the results of emulsification stability analysis in seawater of commercial emulsifiers and commercial surfactants isolated from Pseudomonas aeruginosa BYK-2 strain.

32 is a graph of the present invention showing the results of temperature stability analysis of bioemulsifier isolated from Pseudomonas aeruginosa BYK-2 strain.

FIG. 33 is a graph of the present invention showing the results of emulsion stability analysis after heat treatment for 10 minutes at 121 ° C of bioemulsifier isolated from Pseudomonas aeruginosa BYK-2 strain.

Fig. 34 is a structural formula showing the kind of rhamno lipid reported to date;

Fig. 35 is a structural formula showing the kind of methylamnolipid reported to date.

36 is a structural formula showing the kind of modified rhamno lipid reported to date.

Hereinafter will be described in detail based on a preferred embodiment according to the present invention, but is not limited to the embodiment of the present invention. In the following description, it should be noted that only parts necessary for understanding the present invention will be described, and descriptions of other parts will be omitted so as not to obscure the subject matter of the present invention.

First, in the present invention, the marine bacterium Pseudomonas aeruginosa biwaikei-2, which is a bioemulsifier producing strain that can be used simultaneously for the purification of effluent oil of land and sea and can be used in various industries, is isolated from the sea. Soil-derived Pseudomonas aeruginosa strains have unique growth characteristics that produce different glycolipid bioemulsifiers, Rhamnolipid and Methyllamnolipide simultaneously.

The above-described strain is a basophilic strain capable of growing even at 9% salt, and after 1% of crude oil and bunker seed oil, which are hardly decomposable oils, are incubated in a modified Elbi medium containing seawater and fresh water for 7 days. In the case of crude oil, 92.1% (sea water) and 96% (fresh water) were biodegradable, and 76.0% (sea water) and 80% (fresh water), respectively. The bioemulsifier extracted by incubating each carbon source (hydrocarbon compounds, carbohydrates, and sugars) with substrates was analyzed by thin layer chromatography, and all of them were glycolipids.Together, the transfer rate (Rf) was 0.48, 0.52, 0.65, 0.70, 0.82, 0.91 was confirmed to produce a total of seven kinds of glycolipid bioemulsifier.

Among the seven kinds of glycolipid bioemulsifiers, two materials (BS-1 and BS-2) with the highest yields of 0.48 and 0.65 were separated and analyzed using various analytical equipments such as nuclear magnetic resonance (MRI). 664 Rhamnolipid (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyldecanoyl-β-hydroxydecanoic acid) and methyl ramnolipid (2-O-α-L-rhamnopyranosyl-α-L -rhamnopyranosyl-β-hydroxyldecanoyl-β-hydroxydecanoic acid methyl ester), and Pseudomonas aeruginosa, which produces both rhamnolipid and methyl rhamnolipid, has not been found.

In addition, when looking at the physical properties of the glycolipid bioemulsifier produced by the strain of the present invention, the critical micelle concentration is 10mg / L, the previously reported critical micelle concentration of the glycolipid bioemulsion derived from Pseudomonas aeruginosa (15mg / L, 50mg / The critical micelle concentration is formed in much smaller amount than L), and it is an eco-friendly material that is stable to the ecosystem because of its excellent emulsification / dispersion ability and low usage. The surface tension was 29.5 mN / m and the interface tension was 0.98 mN / m.

On the other hand, the present invention found that the emulsion stability in the physical properties of the bioemulsifier was found to be very efficient in treating freshwater or seawater effluent oil by maintaining excellent emulsion stability in both freshwater and seawater than commercially available surfactants compared to commercially available surfactants in freshwater and seawater. The temperature stability and thermal stability after autoclaving at 121 ° C. for 10 minutes are also very good, and it can be stably used in industries such as the food industry having a heat treatment process.

Figure 1 is a schematic diagram of a process for separating the strain to be used in the present invention in sea water.

When the strain is separated by the method as shown in FIG. 1, strains related to oil degradation can be easily separated, and in the case of the present inventors, it was possible to secure oil-degrading bacteria of about 80 strains by the present method. Therefore, among the strains having excellent oil resolution and bioemulsifier production ability of the BWK-2 (BYK-2) was used in the present invention and the medium and method related thereto are the same as in Example 1.

{Example 1}

Seawater collected from 30 areas of the southern coast, including the southern coastal oil contaminated area, for separation on site (non-sterile arabian light crude oil, 0.5 g; yeast extract, 5.0 mg; K ₂ HPO _4, 0.5 mg; (NH ₄ ) ₂ 50 ml of SO _4, 50 mg) was added directly to the contaminated oil in the oil-contaminated area seawater, and transferred to a laboratory, and then incubated at 25 ° C. at 180 rpm for 7 days using a shaker incubator.

After 7 days of incubation, the seawater from the area where the Arabian Light crude oil was completely decomposed was selected, and 2% of each oil digestion medium was added to the separating oil medium containing the re-sterilized seawater and incubated for 3 days. Selected medium with the highest resolution was modified (peptone, 5.0 g; yeast extract, 1.0 g; ferric citrate, 0.1 g; NH ₄ NO _3, 1.6 mg; Na ₂ HPO _4, 8.0 mg; agar, 0.13) g; sea water, 1.0 L; pH 7.0), single strains were isolated, inoculated in a separate oil medium, incubated under the same conditions for 7 days, and the medium having excellent crude oil resolution was reselected to a modified seawater medium. Finally, single strains were isolated. The strain produced by this method was named BYK-2 strain.

Identification of the BW-2 strain isolated according to the present invention, Pseudomonas having a gram negative motility by first examining the morphological observation and various physiological and biochemical characteristics through gram staining as shown in FIG. It was identified as aeruginosa and was named Pseudomonas aeruginosa biwaikei-2.

Meanwhile, FIG. 3 examined growth characteristics according to the salinity concentration of Pseudomonas aeruginosa biwaikei-2 according to the present invention. That is, in general, the Pseudomonas aeruginosa strain isolated from the soil is reported that the bacteria do not grow at all in the salinity concentration of 6.5% or more, but in the case of the strain of the present invention isolated from the sea even in the range of 0 to 9% This was confirmed to be possible. In particular, the salt concentration of the seawater was 3%, and in the case of the strain of the present invention, it was found that it was not affected by growth at all until the salt concentration of 4% was found to be available in both soil and ocean. Details related to this are the same as in Example 2.

{Example 2}

In order to investigate the effect of salinity concentration on the growth of Pseudomonas aeruginosa biwaike-2, incubated for 48 hours with the salt concentration of 0-9% in modified Elbe medium, the concentration of the culture solution was measured using a spectrophotometer. Measured at 660 nm.

4 and 5 is a 7-day fresh water and seawater is added whether the strain of the present invention has excellent oil resolution in seawater and fresh water, and how much oil resolution for the refractory oils Arabian Light crude oil and bunker seed oil As a result of culturing in modified Elbe medium, 92.1% and 96% were biodegraded in seawater and fresh water, respectively.In case of bunker seed oil, 76.2% and 80% were biodegraded, respectively. It was confirmed that the medium and method used here are the same as in Example 3.

{Example 3}

Gravimetric method (Mulkins-Phillips et al., 1974) was used to investigate the biodegradability of egg-degradable oils. Gravimetrically, in a 250 ml Erlenmeyer flask, 50 ml of sterile modified Elbi medium (tryptone, 10.0 g; yeast extract, 5.0 g; seawater or fresh water, 1 liter) added with 1% Arabian Light crude oil and bunker seed oil was added. Incubated 1% of the culture culture before time and shake culture at 180 ℃ at 25 ℃. In order to investigate the biodegradability of the oil, the Erlenmeyer flask containing 50 mL of the culture solution was recovered by incubation time (24, 48, 72 hours), 50 mL of benzene was added to the recovered Erlenmeyer flask, and the remaining oil was extracted. Moisture was removed and used for quantitative analysis. The container used for the measurement was first weighed as a scale capable of quantifying up to 0.1 mg after drying for 1 hour in a 100 ° C. dryer, and then dried and cooled in a 80 ° C. dryer for 6 hours after putting the benzene extract dehydrated in the container. The biodegradation was measured by quantification on a balance.

6 is a method of extracting, concentrating, and separating and purifying glycolipid bioemulsion according to the present invention, and when recovering by such a method, it is easy to remove hydrocarbon compounds or fatty acids remaining in the extracted bioemulsion stock solution, and high-performance liquid chromatography. It has the advantage of recovering sugar lipids having a purity of 95% or more in a short time without using the back. Specific matters related to this are the same as in Example 4.

{Example 4}

After culturing the strain using various carbon sources as a substrate, the culture solution was centrifuged at 8,000 g for 10 minutes at 4 ° C, and the culture supernatant was recovered. The recovered culture supernatant was adjusted to 2.0 with 5 normal hydrochloric acid, and then stirred and extracted with the same amount of chloroform methanol mixed solvent (CM, chloroform: methanol = 1: 1, volume / volume) for 10 minutes and concentrated using a rotary vacuum evaporator. It was. Next, the concentrated sample was placed in a separation tube (2.5 × 100 cm) filled with silica gel and chloroform, and then the remaining substrate was first removed with chloroform. The glycolipid bioemulsifier was first separated and concentrated as a chloroform methanol mixed solvent. The bioemulsifier thus obtained was checked for purity by thin layer chromatography, and then separated by using a silica gel-filled cartridge (Waters Co., USA) to confirm purity after concentration.

On the other hand, as a bioemulsifier separation method using a silica cartridge, the silica cartridge is first washed with 20 ml of chloroform, and then the sample dissolved in chloroform is put into the silica cartridge to adsorb the sample. Samples adsorbed are purified by elution of chloroform and methanol in a concentration ratio. Purity is confirmed by thin layer chromatography and high performance liquid chromatography.

FIG. 8 shows the types of sugar lipid bioemulsifier according to the type of carbon source, which is cultured by changing the carbon source used as a substrate among the components of the medium, followed by extraction and concentration with an organic solvent, and several types of sugar lipids by thin layer chromatography. It was confirmed that the bioemulsifier produced.

According to the types of glycolipids produced by each carbon source, four types of glycolipids (0.48, 0.52, 0.65, 0.82) were produced in Kuwait crude oil, bunker seed oil, hexadecane and tetradecane when hydrocarbon compounds were used as substrates. Five types (moving rate 0.48, 0.52, 0.65, 0.70, 0.82) were produced in the floating paraffin.

Three kinds (migration rate 0.48, 0.65, 0.82) were produced from fatty acid-based oleic acid, olive oil and lecithin. Four kinds of sugar-based arabinose, lactose, and fructose (moving rate 0.48, 0.65, 0.75, 0.82), and five kinds of trihalose, dextrose, maltose (moving rate 0.48, 0.52, 0.65, 0.75, 0.82), and 5 types (moving rate 0.48, 0.65, 0.70, 0.75, 0.82) were produced in galactose, and 5 types (moving rate 0.48, 0.65, 0.75, 0.82, 0.91) were produced in sucrose, respectively. Based on these results, it was confirmed that the bioemulsifier produced by the strain of the present invention produces various kinds of sugar lipids according to the type of carbon source, and also three kinds regardless of the type of substrate (migration rate 0.48, 0.65, 0.82). Glycolipids were confirmed to be commonly produced.

Therefore, in the strain of the present invention, seven types of glycolipids that can be produced according to the type of substrate (migration rate 0.48, 0.52, 0.65, 0.70, 0.75, 0.82, 0.91) among the Pseudomonas aeruginosa strains reported to date The maximum number of sugar lipids that a strain can produce is 4 (RL 1, RL 2, RL 3, and RL 4), whereas the ability of producing 7 types of sugar lipids is superior to Pseudomonas spp. It is interpreted as the Luginosa variant. Specific matters related to this are the same as in Example 5.

{Example 5}

Bioemulsifier components were analyzed by thin layer chromatography. For thin layer chromatography analysis, a silica gel 60 coated glass plate (silica gel 60 F ₂₅₄ glass plate) was used, and the developing solvent was a mixture of chloroform: methanol: water 60: 25: 4 (volume / volume). Reagents containing 20% sulfuric acid and alpha-naphtoresorcinol reagent were used as detection reagents.

8 is isolated and purified for two types of migration rate 0.48 (BS-1) and 0.65 (BS-2), the most bioemulsifier production in the culture medium, the purity was 95% and 96.1%, respectively. 500 ml and 420 ml were obtained. This sample was used to analyze the structures of glycolipid bioemulsifiers BS-1 and BS-2.

Figure 9 is the result of analysis using the absorption wavelength conversion infrared spectrometer has a ^{3450cm -1, 2920cm -1, 1750cm -1} , 1450cm -1, 1050- 1120cm -1 to determine the glycolipid emulsifier biological functionality of the BS-1 sample , Hydroxyl groups (-OH), long chain hydrocarbons (-CH _2- ), esters (-CH2-CO-OR) and methyl groups (-methyl, -CH ₃ ) for each wavelength , -Vibration (CH _2- ).

FIG. 10 is a molecular weight of 650.37 and a chemical formula of C ₃₂ H ₅₈ O ₁₃ as a result of analysis using a fast atom impact mass spectrometer of the glycolipid bioemulsifier BS-1.

11 to 17 are nuclear magnetic resonance analysis performed for the structural analysis of the glycolipid bioemulsifier BS-1 was performed simultaneously with the one-dimensional analysis and two-dimensional analysis. FIG. 11 is a hydrogen nuclear magnetic resonance analysis, FIG. 12 is a carbon nuclear magnetic resonance analysis, FIG. 13 is a DEPT spectroscopy, FIG. 14 is a NOESY spectroscopy, FIG. 15 is a TOCSY spectroscopy, FIG. 16 shows a relay spectroscopic analysis, and FIG. 17 shows an HSQC spectroscopic analysis.

18 is a result of HMBC spectroscopic analysis, when these data are combined and analyzed, the structure of FIG. 19 is completed. The structure of the glycolipid bioemulsifier BS-1 is 2-o-alpha-el- with two alpha-L-rhamnose and two beta-hydroxydecanoic acid bound together. Rhamnopyranosyl-alpha-el-lamnopyranosyl-beta-hydroxydecanoyl-betahydroxydecanoic acid (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid). This structure is derived from four types of lamnolipids (RL1, RL2, RL3, and RL4) (Gruber et al., 1993) produced by Pseudomonas aeruginosa DSM 2659, which is derived from soil. Matches.

20 is a glycolipid emulsifier BS-2 biological sample using a transform infrared spectrometer to determine the functionality of the analysis result of BS-1 to the absorption wavelength of 3450cm ^-1, 2920cm ^-1 in the same position, 1750cm ^-1, 1450cm ^-1, 1050 -1120 cm ^-1 was found. The molecular weight was 664 and the chemical formula was C ₃₃ H ₆₀ O ₁₃ as a result of analysis using the fast atomic bomb mass spectrometer of the glycolipid bioemulsifier BS-2.

21 to 27 are nuclear magnetic resonance analysis performed for the structural analysis of the glycolipid bioemulsifier BS-2 was performed simultaneously with the one-dimensional analysis and two-dimensional analysis Figure 21 is hydrogen nuclear magnetic resonance analysis, Figure 22 carbon nuclear magnetic resonance analysis 23 shows DEPT spectroscopy, 24 shows relay spectroscopy, 25 shows HSQC spectroscopy, and 26 shows HCMC spectroscopy. When the structure is analyzed as a whole, the structure of FIG. 27 is completed.

The structure of the glycolipid bioemulsifier BS-2 consists of two alpha-L-rhamnose, one beta-hydroxydecanoic acid and one beta-hydroxydecanoic acid. 2-O-alpha-L-lamnopyranosyl-alpha-L-lamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoic acid methyl ester (β-hydroxydecanoic acid methyl ester) bound to 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxy-decanoyl-β-hydroxydecanoic acid methyl ester has been identified by Hirayama et al. (1982) in Pseudomonas, Japan. Methyl rhamnolipids (GL1, GL2), first reported separately from Peudomonas aeruginosa 158, were found to be consistent with GL2 of the two structures, but reported by Gruber et al, 1993. The GL2 reported by Ramno lipid RL3 and Hirayama et al, 1982 Each of the strains produced rhamnolipid or methyl rhamnolipid , but in the case of the strain of the present invention, the production of the rhamnolipid and the methylramnolipid at the same time in one strain was considered in the study of Pudomonas aeruginosa ( Pudomonas aeruginosa) . It is considered to be a mutant species with new mycological characteristics that has not been reported to date.

FIG. 28 shows the critical micelle concentration for Pseudomonas aeruginosa biwaikei-2 bioemulsifier. In general, surfactants are involved in lowering the surface tension and the interfacial tension in the form of adsorption at low concentrations, and when the concentration increases, the adsorption of the surfactant reaches saturation to form an aggregate, which is called a critical micelle concentration (CMC). Unlike the particle colloid, the important property of the icell is very stable in water, so it plays an important role in maintaining the emulsion stability, which is the original purpose of the emulsifier.

The critical micelle concentration was measured by diluting the bioemulsifier separated from the culture supernatant by different concentration using a surface tension meter. The critical micelle concentration of the glycolipid bioemulsifier produced by the strain was 10 mg / l, and the surface tension was measured. Was 29.5 mN / m. This concentration was produced by 15 mg / L of critical micelles (Mulligan et al., 1993) and Pseudomonas aeruginosa ATCC9027 of Pseudomonas aeruginosa isolated from soil. The critical micelles were formed even at a concentration less than 50 mg / L (Zhang et al., 1992) of the critical micelle concentration of rhamno lipid, and the emulsion stability was also excellent. This is a result of the characteristics of this strain to produce the lamnolipids and the methyllamnolipids at the same time. Details thereof are the same as in Example 6.

{Example 6}

The surface tension meter was prepared by diluting and extracting the concentrated sample separated from the culture supernatant by 10 ml of each concentration (0, 1, 2, 3, 4, 5, 7.5, 10, 25, 50, 100 mg / l). Surface tension was measured by (TD-1, Lauda, German) to determine the critical micelle concentration.

The hydrophilic lipophilic balanced biosurfactant (HLB) shown in FIG. 29 is a value indicating whether the glycolipid bioemulsifier produced by the strain of the present invention has strong hydrophilicity or strong hydrophobicity, and is an index indicating an emulsification form or characteristic. . Hydrophilic lipophilic balance values of 1 to 7 are reported as water / oil (W / O) and 10-13 are reported as oil / water (Oter). , 1994). Its characteristic is the hydrophilic lipophilic balance of 1-4 is insoluble, 4-7 is inferior in stability and dispersibility, 7-9 is stable but opaque, 10-13 is slightly cloudy, and 13 or more is present as a clear solution. It is known.

Therefore, the hydrophilic lipophilic balance of the glycolipid bioemulsifier (C. BS, crudebiosurfactant) produced by the strain of the present invention and purified BS-1 bioemulsifier was measured, and commercially available surfactants (emulsan, Triton X-100, SDS, Tween 80, Tween 40, and Tween 20) were measured. The hydrophilic lipophilic balance of the glycolipid bioemulsifier is 11.1, the purified BS-1 is 10.6, the emulsan is 11.4, the Triton X-100 is 10.0, the SDS is 10.6, the Tween 80 Tween 80 was 15.0, Tween 40 was 13.4, Tween 20 was 12.1, and all the surfactants used in the experiment were identified as oil / water (O / W). Details thereof are the same as in Example 7.

{Example 7}

Determination of the hydrophilic lipophilic balance value of the glycolipid bioemulsifier (C. BS) and BS-1 and commercially available surfactants (Triton X-100, SDS, Tween 80, Tween 40, Tween 20, Emulsan) produced by the strain of the present invention It was measured by the titration method by Porter (1994). In the measurement, 0.01 g of various surfactants were dissolved in 10 ml of distilled water, and then titrated with an organic solvent mixed with 4% benzene and 96% 1,4-dioxane (1,4-dioxane). 10 ml of solution in which various surfactants were dissolved was titrated by mixing with a stirrer, and the hydrophilic lipophilic balance value was continuously obtained by dropping an organic solvent dropwise until a clear end point was obtained.

Figures 30 and 31 analyze the emulsification stability of the freshwater and seawater in the physical properties of the bioemulsifier produced by the strain of the present invention, and the commercially available surfactant as the amount of change in the slope with the change of time after emulsification by the emulsification activity measurement method Compared with. As a result, in fresh water, the bioemulsifier produced by the strain of the present invention has a decay constant (Kd) = -26. In seawater, the emulsifier and Tween 40, and the bioemulsifier produced by the strain of the present invention have a decay constant (Kd) = -36.6 , -40.3, -43.8 showed a similar stability, although somewhat higher in emulsification. These results have been found that the bioemulsifier produced by the strain of the present invention can be used for both oil treatment for seawater or fresh water. Details thereof are the same as in Example 8.

{Example 8}

The bioemulsifier produced by the strain of the present invention and various commercially available surfactants (Triton X-100, SDS, Tween 80, Tween 40, Tween 20, Emulsan) were added in 0.01% of each, and the Rosenberg et al. (Rosenberg E. et al 1979) The emulsion activity was measured by the method. The emulsification stability was measured in the same manner as the emulsification activity measurement method, and the absorbance was measured at 620 nm at intervals of 10 minutes to calculate the decay constant (Kd value) from the slope of the changed value.

Decay Constant (Kd) = (logX ₂ -logX ₁ ) / 10

(X ₁ : absorbance value after standing for 10 minutes after shaking, X ₂ : absorbance value measured every 10 minutes)

32 and 33 examined the temperature stability and thermal stability of the bioemulsifier produced by the strain of the present invention. The temperature stability was measured at 10 minutes intervals for 60 minutes in each temperature range (20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 100 ℃) from 20 ℃ to 100 ℃. The temperature stability was the most stable at 30 ℃ as the disintegration constant (Kd) was -6.07, and for each temperature (20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 100 ℃), and the disintegration constant ( The Kd) values were -6.82, -6.79, -8.73, -9.04, -9.45, and -10.7, showing even emulsification stability over the entire temperature range of 20 ° C to 100 ° C. In order to measure the thermal stability, high pressure autoclaving at 121 ° C. for 10 minutes, the emulsion stability was compared with that without sterilization. As a result, after 60 minutes of heat treatment, the disintegration constant (Kd) value was -6.13, and when heat-treated, it was confirmed that the thermal stability was very good at -6.53. Details thereof are the same as in Example 9.

{Example 9}

Temperature and thermal stability of the bioemulsifier produced by the strain of the present invention was carried out in the same manner as the emulsion stability measurement method, the emulsion stability was measured by temperature (20, 30, 40, 50, 60, 70, 80, 100 ℃) . In addition, for thermal stability experiments, the bioemulsifier was heat-treated at 121 ° C. for 10 minutes using a high pressure autoclave, and thermal stability was compared with that of the unheated sample. The experiment was used by adding 0.01% (weight / volume) of bioemulsifier to 20mM Tris-HCl buffer (pH 7.0).

As described above, the present invention is supplied to the triangular flask containing the components of the separating oil medium at the seawater sample collection site in the oil contaminated area to supply nutrition, so that the oil-degrading bacteria that occur during the transfer to the laboratory after sampling It is possible to reduce the number of oils, and to preserve many different kinds of oil-degrading bacteria, and to shake and culture them in the laboratory, it is possible to separate many kinds of oil-degrading bacteria in a short time. It can be used for both, and has developed an indigenous bacterium, which is a domestic dominant species, and treats the spilled oil.

In addition, the present invention can be efficiently used for the removal of poorly soluble and hardly decomposable hydrocarbons contained in wastewater or sewage, as well as the outflow oil treatment, by simultaneously using the land and ocean for effluent treatment.

In order to obtain a high-purity sample, the inventors of the present invention mainly used a high-speed, high-performance liquid chromatography method to separate a large amount of time and a large amount of time due to the limitation of the resolution of the sample. Using the proposed separation method, a large amount of high purity (more than 95%) of sugar lipids can be easily separated at low cost in a short time.

In addition, in the case of the strain of the present invention, Pseudomonas aeruginosa is separated from the seawater and the production of rhamnolipid and mechilamnolipid at the same time in one strain It has new fungal properties that have not been reported to date in the study. That is, the strains of the present invention, in which two types of substances are mixed, have lower critical micelle concentrations than those of Pseudomonas aeruginosa, which have been reported so far. It can be formed in a concentration to maintain the emulsion stability even in a small amount, and the amount of use can be reduced, there is a much more environmentally friendly effect.

In addition, the emulsion stability of the bioemulsifier is also stable in both fresh water and sea water compared to commercial surfactants, and because of the temperature or thermal stability, it can be used in a variety of industries in place of the chemical synthetic surfactant.

Claims (4)

Pseudomonas aeruginosa BYK-2, characterized in that it produces lamnolipid and methyl rhamnolipid simultaneously as a glycolipid bioemulsion producing strain isolated from the ocean Number KCTC 18012P). Samples of seawater samples in oil-contaminated areas are placed in oil medium for strain isolation, shaken for 7 days (25 ℃, shake culture at 180 rpm) for degradation, and then inoculated with 2% in sterilized oil medium for 3 days shaking. A first step of culturing (shaking cultivation at 25 ° C., 180 rpm); A second step of selecting the strain having the highest resolution in the first step and smearing the modified seawater medium to separate single strains, and then inoculating the same in a separation oil medium and shaking culture for 7 days at 25 ° C. at 180 rpm; Pseudomonas aeruginosa BYK-2, characterized in that the second step is characterized in that the third step of separating the strain with excellent crude oil resolution and smeared on modified seawater medium to finally isolate a single strain. -2: separation method of Accession No. KCTC 18012P) strain. The method for extracting and concentrating a glycolipid bioemulsifier of claim 1, followed by separation and purification, comprising: culturing the strain using various carbon sources as a substrate, and then centrifuging the culture solution to recover the culture supernatant; The recovered culture supernatant was adjusted to pH 2.0 with 5 normal hydrochloric acid, and then extracted with the same amount of chloroform methanol mixed solvent (CM, chloroform: methanol = 1: 1, V / V), followed by rotary vacuum evaporation. Concentration process, The concentrated sample was placed in a separation tube filled with silica gel and chloroform, and then the substrate remaining with chloroform was first removed, and then the glycolipid biomaterial was formed by first separating the glycolipid bioemulsifier as a chloroform methanol mixed solvent and then concentrating. Process for separating and purifying emulsifiers. delete