Patent Application
20080020947
The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
a to 5d are graphic diagrams showing the degree of biodegradation by the novel microbial strain EN3 of Example 25 as a function of time (day) at initial diesel oil concentrations of 1,000 ppm, 5,000 ppm, 10,000 ppm and 20,000 ppm, respectively;
a to 6d are graphic diagrams showing the degrees of biodegradation by the novel microbial strain EN67 of Example 26 as a function of time (day) at initial diesel oil concentrations of 1,000 ppm, 5,000 ppm, 10,000 ppm and 20,000 ppm, respectively;
a to 7d are graphic diagrams showing the degrees of biodegradation by the novel microbial strain EN96 of Example 27 as a function of time (day) at initial diesel oil concentrations of 1,000 ppm, 5,000 ppm, 10,000 ppm and 20,000 ppm, respectively;
Rhodococcus baikoneurensis EN3, Acinetobacter johnsonii EN67 and Acinetobacter haemolyticus EN96, which are novel microbial strains according to the present invention, were isolated from soils which have been contaminated with various types of oil over a long period of time in the Kyungki-do province of Korea. The novel microbial strain Rhodococcus baikoneurensis EN3 is a gram-positive coccus or bacillus, the Acinetobacter johnsonii EN67 and the Acinetobacter haemolyticus EN96 are gram-positive cocci. These microbial strains have excellent biodegradability for various oil spectra.
The base sequences of 16S rRNA genes for the identification and classification of the novel microbial strain Rhodococcus baikoneurensis EN3, Acinetobacter johnsonii EN67 and Acinetobacter haemolyticus EN96, isolated according to the present invention, are shown in the accompanying SEQ ID NOS: 1 to 3, respectively, and showed a homology of 99% to Rhodococcus baikoneurensis DSM44587T, Acinetobacter johnsonii ATCC 17909T and Acinetobacter haemolyticus ATCC17906T, respectively.
Said novel microbial strains classified according to the present invention were named “Rhodococcus baikoneurensis EN3”, “Acinetobacter johnsonii EN67” and “Acinetobacter haemolyticus EN96”, respectively, and were deposited in the Korean Collection for Type Cultures (KCTC) under accession numbers KCTC 19082, KCTC 12360 and KCTC 12361, respectively, on Dec. 8, 2004.
Thus, the novel strains according to the present invention are specified as “Rhodococcus baikoneurensis EN3 KCTC19082”, “Acinetobacter johnsonii EN67 KCTC12360”, and “Acinetobacter haemolyticus EN96 KCTC12361”, respectively.
The morphological, physiological and biochemical characteristics of the Rhodococcus baikoneurensis EN3 KCTC19082 according to the present invention are shown in Table 1 below.
Rhodococcus baikoneurensis
The morphological, physiological and biochemical characteristics of Acinetobacter johnsonii EN67 KCTC12360 and Acinetobacter haemolyticus EN96 KCTC12361 according to the present invention are shown in Table 2 below.
Acinetobacter
Acinetobacter
johnsonii
haemolyticus
A phylogenetic tree of Rhodococcus baikoneurensis EN3 KCTC19082, the novel microbial strain according to the present invention, is shown in
Microorganisms, which can be used in the inventive method for the bioremediation of oil-contaminated soils, are not limited to said novel microorganisms, and any microorganisms can be used in any particular limitation as long as they have having excellent oil biodegradability.
Such microorganisms include those having biodegradability for various oil contaminants isolated from soils, which have been contaminated with oils over a long period of time.
More specifically, Nocardia sp., Gordonia sp., Rhodococcus sp. and Acinetobactor sp. microorganisms can be used, and microorganisms belonging to these species have excellent biodegradability for various poorly-degradable substances such as diesel oil, bunker C oil, high-boiling-point aromatic compounds, chlorine compounds and the like. Also, such microorganisms are widely present in nature and degrade various contaminants as carbon sources and energy sources.
Specific examples of microbial strains, which were found to be capable of being used in the bioremediation of oil-contaminated soils by present invention, include Rhodococcus baikoneurensis EN3, Acinetobacter johnsonii EN67, and Acinetobacter haemolyticus EN96, which are the novel microbial strains, as well as Nocardia transvalensis DSM43405T (hereinafter, depository authority and accession number), Nocardia asteroides ATCC19247T, Gordonia sputi DSM43896T, Gordonia rhizosphera IFO16068T, Gordonia nitida LE31T, Gordonia hirsuta DSM44140T, Gordonia bronchialis CIP1780.88T, Gordonia amarae DSM43392T, Gordonia desulfuricans NCIMB40816T, Rhodococcus zopfii ATCC51349T, Rhodococcus wratislaviensis NCIMB13082T, Rhodococcus tukisamuensis Mb8T, Rhodococcus ruber DSM43338T, Rhodococcus rhodochrous CIP1759.88T, Rhodococcus rhodnii DSM43336T, Rhodococcus pyridinovorans KCTC0647BPT, Rhodococcus percolatus MBS1T, Rhodococcus opacus DSM43205T, Rhodococcus marinonascens DSM43752T, Rhodococcus koreensis DNP505T type2, Rhodococcus jostii IF016295T, Rhodococcus globerulus DSM43954T, Rhodococcus fascians DSM20669T, Rhodococcus erythropolis ATCC4277T, Rhodococcus erythreus DSM43066T, Rhodococcus equi DSM20307T, Rhodococcus coprophilus ATCC29080T, Rhodococcus baikonurensis GTC 1041T, Acinetobacter towneri AB1110T, Acinetobacter baylyi B2T, Acinetobacter calcoaceticus DSM30006T, Acinetobacter grimontii 17A04T, Acinetobacter lwoffii DSM2403T, Acinetobacter radioresistens ATCC17909T, Acinetobacter tandoii 4N13T, Acinetobacter towneri AB1110T, Acinetobacter baumannii ATCC19606T, Acinetobacter bouvetii 4B02T, Acinetobacter gerneri 9A01T, Acinetobacter junii ATCC17908T, Acinetobacter parvus LUH4616T, Acinetobacter schindleri NIPH1034T, Acinetobacter tjernbergiae 7N16T, and Acinetobacter ursingii NIPH137T. Although these microbial strains can also be used alone, these can preferably be used in suitable combinations with each other depending on needs (i.e., effective treatment according to the kind of contaminated oils, etc.).
The inventive method for the bioremediation of oil-contaminated soil comprises inoculating the oil-contaminated soil with at least one microbial strain selected from among the above-described microbial strains and proliferating the inoculated microbial strain so as to remove the oil from soil by biodegradation.
Said microbial strains all show a degradation rate of 100% at a diesel oil concentration of 1,000 ppm, and also show a degradation rate of more than 90% even at a diesel oil concentration of 20,000 ppm (see Table 3 below).
The inoculation level (including medium weight) of said microorganisms, which can be used in the inventive method for the bioremediation of oil-contaminated soil, is about 0.001-8% (v/v or v/w), and preferably about 0.1-3% (v/v or v/w), but is not limited thereto.
Also, in the inventive method for bioremediation of oil-contaminated soil, the biodegradation rate of oils can be significantly increased by about 20-70% by adding biosurfactant 2-alkyl-3-hydroxylic acid represented by Formula 1 below, or its derivative:
wherein R1 and R2 each independently represents a C4-C50 straight or branched-chain alkyl group including hydroxy, methoxy, keto, carbonyl, carboxy, epoxy, ester or a cyclopropane ring, and R3 represents —OR4, monoethanolamine, diethanolamine, D-glucosamine, glucamine, N-methylglucamine, glucose, ramnose, mannose, galactose, lactose, sucrose, maltose, arabinose, cellobiose, or polysaccharide including said monosaccharide or disaccharide, wherein R4 represents hydrogen, sodium, potassium, magnesium, calcium, ammonium or triethanolamine.
Hereinafter, 2-alkyl-3-hydroxylic acid and its derivative, which can be effectively used in the inventive method for the bioremediation of oil-contaminated soil, will be described in detail.
Said compound can be synthesized through a reaction pathway as described below. The synthetic reaction according to the present invention consists of a two-step process comprising a step of hydrogenating an alkyl ketene dimer in the presence of a 5% Pd/C, 10% Pd/C or 0.5% Pd/Al2O3 catalyst in a hydrogen atmosphere to form β-lactone, and a step of either subjecting β-lactone to ring-opening reaction to prepare 2-alkyl-3-hydroxylic acid or allowing the β-lactone to react with sugar and a nucleophile to form a derivative of 2-alkyl-hydroxylic acid.
The alkyl ketene dimer has been widely used as a sizing agent for selectively preventing liquid from penetrating into paper in the papermaking industry. As the alkyl ketene dimer, commercially available compounds, for example, products available from BASF AG, Nippon Oil & Fat Co., Ltd., Hercules, Inc., etc., can be used in the present invention. Alternatively, it can also be synthesized by reacting acyl chloride with triethylamine to have the desired alkyl group and alkenyl group. The synthesis of the alkyl ketene dimmer is described in detail in several papers [J. Amer. Chem. Soc., 87, 5191 (1965)/ibid., 72, 1461 (1950)/ibid., 69, 2444 (1947)].
The alkyl ketene dimmer obtained as described above reacts with a 5% Pd/C, 10% Pd/C or 0.5% Pd/Al2O3 catalyst in a hydrogen atmosphere to form β-lactone, which is then converted into 2-alkyl-3-hydroxylic acid or its derivative by ring-opening reaction or a reaction with saccharide.
Hereinafter, a process for preparing said 2-alkyl-3-hydroxy fatty acid and its derivative will be described in detail with reference to Reaction Scheme 1 below.
The first-step reaction in reaction scheme 1 above is a reaction of hydrogenating the alkyl ketene dimmer with a 5% Pd/C, 10% Pd/C or 0.5% Pd/Al2O3 catalyst in a hydrogen atmosphere to produce β-lactone, and the second-step reaction is a reaction of subjecting the β-lactone to ring-opening reaction in the presence of alkali or allowing the β-lactone to react with a nucleophile having sugar, thus making 2-alkyl-3-hydroxy fatty acid or its derivative.
For the hydrogenation in the first-step reaction according to the present invention, it is possible to use various catalysts known to be effective for hydrogenation. Examples of such catalysts may include a palladium-alumina complex (Pd/Al2O3), a ruthenium chloride (II)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl complex (RuCl(2,2′-bis(diphenylphosphino)-1,1′-binaphthyl)), Raney-Ni, a rhodium aluminum oxide complex (Rh/Al2O3), a palladium complex (Pd/BaSO4, Pd/C), etc. Among said reaction catalysts, 5% Pd/C, 10% Pd/C and 0.5% Pd/Al2O3 catalysts, which are easily separated and purified, can preferably be used. Also, the amount of catalyst used is 0.01-4 wt % based on the weight of the alkyl ketene dimer. The preferred amounts of catalyst used is 0.05-1.0 wt % for 5% Pd/C, 0.025-0.5 wt % for 10% Pd/C, and 0.5-2 wt % for 0.5% Pd/Al2O3. Although the hydrogenation in the first-step reaction can also be carried out at high pressure, it is preferably conducted at about 1-50 atm, and more preferably about 2-40 atm. The 0.5% Pd/Al2O3 catalyst has advantages in that, because it is a supported catalyst which takes the form of a pellet, not a powder, unlike general catalysts, it is easy to separate after reaction, and it enables to obtain 2-alkyl-3-hydroxy-alkanoic β-lactone with high yield, even when it is recycled more than 10 times.
However, the reaction yield can be decreased due to the formation of 2-alkyl fatty acid caused by the ring opening reaction of the alkyl ketene dimer depending on parameters, including reaction time, a solvent used and reaction temperature. This side reaction can be confirmed by NMR, and it can be actually confirmed that 2-octyl-octanoic acid, 2-pentadecyl-pentadecanoic acid, etc. are formed from a C8 alkyl ketene dimer, a C16 alkyl ketene dimer, etc. [1H-NMR CDCl3, δ] 0.87 (t, 6H), 1.25-1.64 (m), 2.23-2.38 (m, 1H)]. Thus, in order to suppress these undesirable side reactions, it is important to suitably maintain the reaction conditions. For this purpose, it is preferable that the reaction be carried out between room temperature and 50° C., and be terminated just after the alkyl ketene dimer disappears as measured by thin layer chromatography (TLC). Particularly when a suitable mixed solvent selected depending on the carbon atom number of the alkyl ketene dimer is used, the side reactions will not occur and 2-alkyl-3-hydroxy-alkanoic acid β-lactone can be obtained with high yield without a special purification process.
Examples of a solvent, which can be used in the first step of reduction, may include methanol, ethanol, ethyl acetate, n-propanol, n-butanol, isopropanol, tetrahydrofuran, dimethylformamide, dimethylsulfoxide and the like. Preferred are methanol, ethanol and a mixed solvent thereof. The most preferable solvent for the first step reaction is a mixture of ethyl acetate and ethyl alcohol (5%:95%-95%:5% v/v).
In the ring-opening reaction of the second step, the β-lactone is ring-opened by reaction with a base, thus obtaining 2-alkyl-3-hydroxylic acid salt. Also, the obtained salt can be isolated in the presence of an organic solvent, and the isolated salt can be acidified and extracted, thus obtaining a high purity of 2-alkyl-3-hydroxylic acid.
It is possible to allow the β-lactone produced in the first step to react with a nucleophile to prepare a new derivative. If 2-alkyl-3-hydroxylic acid is substituted with sugar as a hydrophilic group, it is expected to have increased hydrophilicity and various surfactant abilities. Also, it is possible to prepare new derivatives using glucose, ramnose, mannose, galactose, lactose, sucrose, maltose, arabinose, cellobiose, saccharide including such monosaccharide or disaccharide, amine-containing sugar such as D-glucosamine, glucamine or N-methylglucamine, monoethanolamine or diethanolamine.
Said 2-alkyl-3-hydroxy fatty acid or its derivative can also be used as an effluent water treatment agent due to its excellent surfactant action and an eco-friendly property of low secondary contamination. Also, it has a relatively simple chemical structure, and thus is synthesized in an easy and economical manner.
The inventive method for the bioremediation of oil-contaminated soil may comprise isolating and identifying microbial strains by: a step of collecting soil contaminated with oils; a step of isolating oil-degrading microbial strains from the collected soil; a step of separately culturing a few hundred kinds of the isolated microbial strains; a first screening step of screening microbial strains having excellent oil biodegradability from the cultured microbial strains; and a second screening step of screening microbial strains, the ability of which to degrade a high concentration of oil contaminants is increased through the use of biosurfactants such as said 2-alkyl-3-hydroxy acid and its derivative, from said microbial strains screened in the first screening step.
The biosurfactants are 2-alkyl-hydroxy fatty acid and its derivative, secreted by microorganisms. These are present in the cell wall of acetmital microorganisms and function to facilitate the microbial use of oils by lowering the interfacial tension between microorganisms and oil carbon sources, such that the microorganisms can grow using oils as carbon sources in sites having a high concentration of oils. In the inventive bioremediation method, said biosurfactants serve to increase the ability of the identified and isolated microorganisms to biodegrade oil contaminants.
The biosurfactant 2-alkyl-3-hydroxylic acid or its derivative is used in an amount of 0.0001-10 wt %, and preferably 0.001-10 wt %, based on the total weight of the microbial strain and the medium.
Hereinafter, the identification, isolation and classification of said microbial strains, preparation methods of biosurfactant 2-alkyl-3-hydroxy fatty acid and its derivative, and oil biodegradation rates, will be described with reference to examples and comparative examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
Isolation of Microorganisms having Oil Degradation Activity
Soils, which have been contaminated with various types of oil over a long period of time in the Kyungki-do province of Korea, were used as inoculation samples. Oil-contaminated soils were collected from portions of 0-2 m depth from the ground surface, and then immediately placed into bottles and freeze-stored at 4° C. Specific information on the collection of the samples is shown in Table 3 below.
To screen microorganisms having excellent oil biodegradability, the samples were spread onto plate media consisting of LB (Luria-Bertani) agar media or tryptic soy agar media and were cultured at 30° C. for 48 hours. After the culture step, the formed single colonies were observed with a microscope, and 500 cocci and 500 bacilli were selected, spread again onto the plate media and then isolated. The isolated microbial strains were inoculated into liquid media containing 1,000 ppm, 5,000 ppm, 10,000 ppm and 20,000 ppm of diesel oil and mineral components (NH4NO3, 1 g/L; MgSO4, 0.2 g/L; CaCl2, 0.02 g/L; FeCl2, 0.05 g/L; KH2PO4, 1 g/L; K2HPO4, 1 g/L; pH 7.0) in triangular flasks, and then cultured at 30° C. for 7 days.
Identification of Isolated Microorganisms
Three microbial strains showing the most excellent growth in the liquid media containing diesel oil and mineral components were selected and subjected to microbial identification. For the identification of the isolated strains, the base sequences of the 16S rRNA genes were analyzed. For this purpose, the strains were first cultured in MRS broths (Difco) for 7 days with shaking at 30° C. and 150 rpm. To collect microbial cells from the culture media, the media were centrifuged at 6,000 rpm for 20 minutes, and microbial cells at the lower layer were collected. Following this, the chromosomal DNA of the cells was extracted and purified using the DNeasy Tissue Kit (Qiagen). 16S rRNA genes were amplified by PCR using the chromosomal DNA as a template.
Then, the phylogenetic positions of the isolated EN3, EN67 and EN96 strains were analyzed. For the phylogenetic analysis, the full sequences of the amplified 16S rRNA genes of the EN3, EN67 and EN96 strains were analyzed using SeqMan software, BioEdit program and CLUSTAL X program, and then the related sequences of associated strains were searched in the GenBank database using the BLAST search program. Then, the distance matrix between the sequences was analyzed using the BioEdit program, and a phylogenetic tree for the EN-3 strain was completed using the Mega2 program according to the neighbor-joining method.
The base sequence of the 16S rRNA gene of the novel microbial strain EN3 is shown in SEQ ID NO: 1, and the base sequences of the 16S rRNA genes of the novel microbial strains EN67 and EN96 are shown in SEQ ID NOS: 2 and 3, respectively.
In addition,
After analyzing the base sequences of the amplified genes, the classification of the novel isolated strains was performed using the MEGA version 2.1 program. As a result, the isolated strains were identified as novel microorganisms. Also, the morphological, physiological and biochemical characteristics of the novel microbial strains were analyzed, and the analysis results are shown in Tables 1 and 2 above.
Also, the scanning electron microscope (SEM) photographs (X10,000) of the novel strains EN3 and EN96 cultured in TSA media to the exponential-growth phase for the morphological observation of the strains are shown in
Measurement of Oil Contaminant Degradation Activity of Isolated Microorganisms
Diesel oil used in this experiment was purchased from GS-Caltex Oil Corporation (Taejon, Korea), and composed of 42.7% alkanes, 33.4% cycloalkanes and 23.9% aromatics. Numerical values shown on a technical data sheet obtained from GS-Caltex Oil Corporation were recorded.
In the experiment, 100 ml of mineral media (NH4NO3, 1 g/L; MgSO4, 0.2 g/L; CaCl2, 0.02 g/L; FeCl2, 0.05 g/L; K2HPO4, 1 g/L; KH2PO4, 1 g/L; pH 7.0) containing no carbon and energy sources were added to 500-ml flasks, to which 100, 500, 1000, 1500 and 2000 mg of diesel oil were then added. Thereafter, each of the strains cultured in tryptic soy agar media (Difco) was inoculated into the flasks, which were then sealed with polytetrafluoroethylene. Herein, each of the microbial strains was inoculated at a concentration of 6×106 cfu/ml to a final concentration ratio of 1% (v/v) and then adjusted to pH 6.9-7.1. Thus, the diesel oil was the sole carbon/energy source, and the microorganisms would degrade the diesel oil during the growth thereof. Also, for use as a control group in this experiment, a diesel oil-containing mineral salt medium non-inoculated with the strains was cultured and measured for TPH, in order to compensate a reduction in the concentration of the diesel oil, caused by the volatilization of low-boiling-point compounds among various compounds contained in the diesel oil, with the passage of time. Also, to prevent the error of measurement, three samples were measured and the average of the measurements was recorded, and to minimize the amount of diesel oil, which would be attached to the flasks and thus not measured, the sample contained in each of the flasks was measured after extracting it with 100 ml of normal hexane. The microbial strains were cultured at 30° C. and 300 rpm for 7 days, sampled at one-day intervals and measured for diesel oil degradation rate by gas chromatography.
In the analysis of the diesel oil, gas chromatography (HP 5890 series II, Hewlett-Packard, USA) equipped with FID detector (HP 5890 series II, Hewlett-Packard, USA), and an HP-1 column (30 m×0.32 mm×1 μm, J&W Scientific, USA), were used, helium was used as a carrier gas, and an injector and a detector were set at 280° C. and 300° C. The temperature of the column was maintained at 40° C. for 2 minutes, elevated to 300° C. at a rate of 10° C./min, and then maintained at 300° C. for 15 minutes. In this experiment, 2 μl of each of the samples was injected and the amount of remaining diesel oil was measured as the total of all the peak areas of the gas chromatogram, i.e., TPH (total petroleum hydrocarbon). The extraction of the remaining oil contaminants was performed using N-hexane, and degradation rate (%) was calculated according to equation 1 below.
Degradation rate (%)=TPH area of microorganism-inoculated group/TPH area of uninoculated group×100 [Equation ]
The results of oil biodegradability tests for the microbial strains, isolated and obtained by the present inventors, are shown in Table 4 below.
Pseudomonas aeruginosa
Klebsiella pneumonia
Bacillus subtilis
Streptomyces speibonae
Bacillus pumilus
Bacillus licheniformis
Rodococcus baikoneurensis EN3
Acinetobacter johnsonii EN67
Acinetobacter haemolyticus EN96
Acinetobacter junii EN105
Gordonia nitida NP1
Gordonia amicalis NP2
Gordonia desulfuricans NP3
Gordonia SRP2 NP4
Gordonia westfalica NP6
Gordonia namibiensis NP7
As can be seen in Table 4 above, Pseudomonas aeruginosa of Comparative Example 1, Klebsiella pneumonia of Comparative Example 2, Bacillus subtilis of Comparative Example 3, Streptomyces speibonae of Comparative Example 4, Bacillus pumilus of Comparative Example 6, and Bacillus licheniforimis of Comparative Example 6, all showed an oil degradation rate of 50-60% on the average at initial diesel concentrations of 1000 ppm and 5000 ppm, and an oil degradation rate of 10-20% on the average at an initial diesel concentration of 10,000 ppm, and did not show diesel degradation at an initial diesel concentration of 20,000 ppm. Particularly, Bacillus licheniformis of Comparative Example 6 showed a degradation rate of 10% on the average at initial diesel concentrations of 1000 ppm and 5000 ppm, suggesting that the oil degradation ability thereof was very low.
Meanwhile, Rodococcus baikoneurensis EN3 of Example 2, Acinetobacter johnsonii EN67 of Example 3, Acinetobacter haemolyticus EN96 of Example 4, Acinetobacter junii EN105 of Example 5, Gordonia nitida NP1 of Example 6, Gordonia amicalis NP2 of Example 7, Gordonia desulfuricans NP3 of Example 8, Gordonia SPR2 NP4 of Example 9, Gordonia westfalica NP6 of Example 10, Gordonia namibiensis NP-7 of Example 11, and the mixed strain of Example 12, had an excellent degradability of 82-100% at initial diesel concentrations of 1,000 ppm and 5,000 ppm, but the oil degradability thereof was relatively reduced at initial diesel concentrations of 10,000 ppm and 20,000 ppm. However, the mixed cultured strain of Example 12, consisting of a mixture of Example 2, Example 6 and Example 7, showed an increase in diesel oil degradability at initial diesel concentrations of 10,000 ppm and 20,000 ppm, compared to those of the individual strains.
The results of diesel oil degradation tests for Rodococcus baikoneurensis EN3-inoculated media and uninoculated media, conducted at initial diesel concentrations of 1000 ppm, 5000 ppm, 10,000 ppm and 20,000 ppm, are shown in
In
As can be seen in
Meanwhile, the results of diesel oil degradation tests for Acinetobacter johnsonii EN67-inoculated media and uninoculated media, conducted at initial diesel concentrations 1000 ppm, 5000 ppm, 10,000 ppm and 20,000 ppm, are shown in
In
As can be seen in
Meanwhile, the results of diesel oil degradation tests for Acinetobacter haemolyticus EN96-inoculated media and uninoculated media, conducted at initial diesel concentrations of 1000 ppm, 5000 ppm, 10,000 ppm and 20,000 ppm, are shown in
In
As can be seen in
Meanwhile, the results of diesel oil degradation tests for Gordonia nitida NP1-inoculated media and uninoculated media, conducted at initial diesel concentrations of 1000 ppm, 5000 ppm, 10,000 ppm, 15,000 ppm and 20,000 ppm, are shown in
In
As can be seen in
Measurement of Biosurfactant's Ability to Increase Oil Degradation Activity of Microorganisms
In the cases of Rodococcus baikoneurensis EN3 of Example 2, Acinetobacter junii EN105 of Example 5, Gordonia nitida NP1 of Example 6, Gordonia amicalis NP2 of Example 7, Gordonia desulfuricans NP3 of Example 8, Gordonia westfalica NP6 of Example 10, and Gordonia namibiensis NP7 of Example 11, except for Acinetobacter johnsonii EN67 of Example 3, Acinetobacter haemolyticus EN96 of Example 4 and Gordonia SPR2 NP4 of Example 9, the oil biodegradability of the microorganisms was reduced at high diesel oil concentrations of more than 10,000 ppm.
For this reason, 2-hexyl-3-hydroxy-decanoic acid having 16 carbon atoms according to the present invention was added liquid media each containing 10,000 ppm or 20,000 ppm of diesel oil and mineral medium (NH4NO3, 1 g/L; MgSO4, 0.2 g/L; CaCl2, 0.02 g/L; FeCl2, 0.05 g/L; KH2PO4, 1 g/L; K2HPO4, 1 g/L; pH 7.0). Then, the media were inoculated with each of Rodococcus baikoneurensis EN3 of Example 2 and Gordonia nitida NP1 of Example 6 and cultured at 30° C. for 7 days. Then, an increase in the oil biodegradability of each of the microbial strains was measured using gas chromatography.
The critical micelle concentration (cmc) of 2-hexyl-3-hydroxy-decanoic acid was 37.1 ppm, and the concentrations of 2-hexyl-3-hydroxy-decanoic acid were 10 ppm, 50 ppm and 100 ppm, respectively. At all the acid concentrations, the microbial strain showed an oil biodegradation rate of about 70%, which was increased compared to about 30%, which was the biodegradation rate in the case in which 2-hexyl-3-hydroxy-decanoic acid was not added. Also, regardless of a change in the concentration of 2-hexyl-3-hydroxy-decanoic acid, the highest values of degradation of diesel oil were approximately the same.
The concentrations of 2-hexyl-3-hydroxy-decanoic acid were 9 ppm, 90 ppm and 900 ppm. At all the acid concentrations, the microbial strain showed a great increase in its diesel oil degradation rate, but did not show an increase in the oil degradation rate at an initial diesel oil concentration of 20,000 ppm and a concentration of 2-hexyl-3-hydroxy-decanoic acid of 9 ppm. However, at concentrations of 2-hexyl-3-hydroxy-decanoic acid of 90 ppm and 900 ppm, the highest values of degradation of diesel oil were approximately the same, regardless of changes in the concentration.
Microbial Growth Promotion of Biosurfactant 2-hexyl-3-hydroxy-decanoic Acid
These examples relate to an experiment of confirming whether the growth of microorganisms having oil biodegradability is promoted due to the addition of biosurfactant 2-hexyl-3-hydroxy-decanoic acid.
In this experiment, the mixed cultured strain of Examples 2, 6 and 7 was added to tryptic soy broth (TSB) and then cultured for 13 hours. In the culture process, the microbial strain was added into a 100-ml flask at an initial concentration of 4.9×106 cfu/ml to a work volume of 10 ml, and the inoculation level of the strain was 1% (w/v), i.e., 0.1 ml.
As shown in Table 5 below, each of the media used in this experiment contained a suitable mixture of glucose, yeast extract, malt extract, Na2HPO4, KH2PO4, K2HPO4, MgSO4, CaCl2, FeSO4, CoCl2, ZnSO4, CuSO4 and MnSO4, and the biosurfactant 2-hexyl-3-hydroxy-decanoic acid was used in Examples 16 and 17.
In Comparative Examples 7-10 and Examples 16 and 17, the microbial strain was cultured in media (10 g/L) at 30° C. for 24 hours, and then measured for colony-forming units (cfu) per ml to determine the degree of proliferation of microorganisms.
Specifically, a composition used in Comparative Examples 7-10 consisted of glucose, yeast extract, malt extract, Na2HPO4, KH2PO4, K2HPO4, MgSO4, CaCl2, FeSO4, CoCl2, ZnSO4, CuSO4 and MnSO4, and a composition used in Examples 16 and 17 consisted of glucose, yeast extract, malt extract, Na2HPO4, KH2PO4, K2HPO4, MgSO4, CaCl2, FeSO4, CoCl2, ZnSO4, CuSO4 and MnSO4, to which the biosurfactant 2-hexyl-3-hydroxy-decanoic acid was added.
Hereinafter, the present invention will be described in further detail with reference to preferred embodiments.
As can be seen Table 5 above, Comparative Examples 7-10, to which 2-hexyl-3-hydroxy-decanoic acid was not added, showed a total number of microorganisms of 1.1×108 to 2.2×109 cfu/ml. On the other hand, the total microorganism numbers in Examples 16 and 17, to which 2-hexyl-3-hydroxy-decanoic acid was added, were 1.7×1011 cfu/ml and 3.1×1011 cfu/ml. This suggests that the addition of 2-hexyl-3-hydroxy-decanoic acid leads to the promotion of growth of the microorganisms.
Examples below illustrate the synthesis of biosurfactants from an alkyl ketene dimer having 8, 12 or 16-18 carbon atoms, but the scope of the present invention is not limited thereto.
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EA:EtOH=3:1 and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 96 g (95% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EA:EtOH=3:1 and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 98 g (97% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EtOH and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 30 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (EA:Hex=1:20), and the solvent was evaporated under reduced pressure, yielding 91 g (90% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EA:EtOH=3:1 and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 96 g (95% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 900 ml of EA:EtOH=2:1 and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 96 g (95% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EtOH and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 40 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (EA:Hex=1:20), and the solvent was evaporated under reduced pressure, yielding 90 g (89% yield) of 2-hexyl-3-hydroxy-decanoic ⊖-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EA:EtOH=3:1 and stirred well. Then, 2 g (2 wt %) of 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure, yielding 96 g (95% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step b 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EA:EtOH=1:1 and stirred well. Then, 2 g (2 wt %) of onetime recycled 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure, yielding 95 g (94% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.39 mol) of a C8 alkyl ketene dimer was added to 800 ml of EtOH and stirred well. Then, 2 g (2 wt %) of twice recycled 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 50 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure, The residue was separated by column chromatography (EA:Hex=1:20), and the solvent was evaporated under reduced pressure, yielding 89 g (88% yield) of 2-hexyl-3-hydroxy-decanoic β-lactone as a light yellow liquid.
Rf (EA:Hex=1:20): 0.58
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 3.60 (m, 1H, CH2CHCO), 4.51 (m, 1H, CHCH—O).
Step b 2: 12 g (0.3 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.20 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 51 g (95% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.27 mol) of a C12 alkyl ketene dimer was added to 900 ml of EA:EtOH=2:1 and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 94 g (94% yield) of 2-decyl-3-hydroxy-tetradecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 35.97° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 3.60 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 3.2 g (0.075 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 20 g (0.05 mol) of 2-decyl-3-hydroxy-tetradecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 19.9 g (95% yield) of 2-decyl-3-hydroxy-tetradecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 2.48 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.27 mol) of a C12 alkyl ketene dimer was added to 900 ml of EA:EtOH=2:1 and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 96 g (96% yield) of 2-decyl-3-hydroxy-tetradecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 35.97° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 3.60 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 3.2 g (0.075 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 20 g (0.05 mol) of 2-decyl-3-hydroxy-tetradecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 19.9 g (95% yield) of 2-decyl-3-hydroxy-tetradecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 2.48 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.27 mol) of a C12 alkyl ketene dimer was added to 800 ml of EA:EtOH=1:1 and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 93 g (93% yield) of 2-decyl-3-hydroxy-tetradecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 35.97° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 3.60 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 3.2 g (0.075 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 20 g (0.05 mol) of 2-decyl-3-hydroxy-tetradecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 19.9 g (95% yield) of 2-decyl-3-hydroxy-tetradecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 2.48 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.27 mol) of a C12 alkyl ketene dimer was added to 900 ml of EA:EtOH=1:2 and stirred well. Then, 2 g (2 wt %) of five times recycled 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 15 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (EA:Hex=1:20), and the solvent was evaporated under reduced pressure, yielding 89 g (89% yield) of 2-decyl-3-hydroxy-tetradecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 35.97° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 3.60 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 3.2 g (0.075 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 20 g (0.05 mol) of 2-decyl-3-hydroxy-tetradecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 19.9 g (95% yield) of 2-decyl-3-hydroxy-tetradecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 2.48 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.27 mol) of a C12 alkyl ketene dimer was added to 800 ml of EtOH and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 40 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure, yielding 90 g (90% yield) of 2-decyl-3-hydroxy-tetradecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 35.97° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 3.60 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 3.2 g (0.075 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 20 g (0.05 mol) of 2-decyl-3-hydroxy-tetradecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 19.9 g (95% yield) of 2-decyl-3-hydroxy-tetradecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 38H, CH2), 2.48 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.19 mol) of a C16-18 alkyl ketene dimer was added to 800 ml of EA:EtOH=1:1 to be heated and completely melted and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 92 g (92% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 60.89° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 3.59 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 5.6 g (0.14 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.09 mol) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 49 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.19 mol) of a C16-18 alkyl ketene dimer was added to 800 ml of EtOH to be heated and completely melted and stirred well. Then, 0.1 g (0.1 wt %) of 10% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 2 atm in a H2 atmosphere for 3-4 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 94 g (94% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 60.89° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 3.59 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 5.6 g (0.14 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.09 mol) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 49 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.19 mol) of a C16-18 alkyl ketene dimer was added to 800 ml of EtOH to be heated and completely melted and stirred well. Then, 0.2 g (0.2 wt %) of 5% Pd/C was added thereto, and the mixture was sealed well, and stirred well at a pressure of 30 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through celite, and the solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 94 g (94% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 60.89° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 3.59 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 5.6 g (0.14 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.09 mol) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 49 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.19 mol) of a C16-18 alkyl ketene dimer was added to 800 ml of EtOH to be heated and completely melted and stirred well. Then, 2 g (2 wt %) of ten times recycled 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 50 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 91 g (91% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 60.89° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 3.59 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 5.6 g (0.14 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.09 mol) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 49 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
Step 1: 100 g (0.19 mol) of a C16-18 alkyl ketene dimer was added to 800 ml of EtOH to be heated and completely melted and stirred well. Then, 2 g (2 wt %) of a new 0.5% Pd/Al2O3 was added thereto, and the mixture was sealed well, and stirred well at a pressure of 50 atm in a H2 atmosphere for 4-5 hours. After completion of the reaction, the reaction product was filtered through 0.5% Pd/Al2O3, and the solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 95 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone as a white solid.
Rf (EA:Hex=1:20): 0.58 mp: 60.89° c
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 3.59 (m, 1H, CH2CHCO), 4.56 (m, 1H, CHCH—O).
Step 2: 5.6 g (0.14 mol, 1.5 eq) of NaOH was dissolved in 600 ml of EtOH:H2O=3:1, and 50 g (0.09 mol) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic β-lactone was added thereto and stirred well for 3-4 hours. Then, the stirred solution was acidified with 6N HCl. The acidic solution was extracted with methylene chloride (MC), and the MC layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. It was recrystallized from EtOH, yielding 49 g (95% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
20 g (0.078 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone and 17 g (0.94 mol, 1.2 eq) of glucose were added to 200 ml of DMF (N,N-dimethylformamide). A catalytic amount of sulfuric acid was added thereto, and the solution was heated under reflux for 10-24 hours. The reaction solution was cooled to room temperature, 100 ml of water was added thereto, and the aqueous solution was neutralized with 1N NaOH. The neutral solution was extracted with methylene chloride (MC), the MC layer was dried with Na2SO4 and filtered, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=20:1), and the solvent was evaporated under reduced pressure, yielding 21.5 g (63% yield) of 2-hexyl-3-hydroxy-decanoic acid glucose ester as a white solid.
Rf (MC:EtOH=10:1): 0.41
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.40 (m, 1H, CH2CHOCH(OH)CH(OH)), 3.48-3.52 (m, 2H, (OH)CHCH(OH)CH(OH)), 3.76 (m, 1H, CH2CH(OH)CH), 4.21 (d, 2H, OCH2CH), 4.27 (m, 1H, CH2CHOCH(OH)), 5.41 (d, 1H, OCH(OH) CH(OH)).
20 g (0.078 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone and 17 g (0.94 mol, 1.2 eq) of mannose were added to 200 ml of DMF (N,N-dimethylformamide). A catalytic amount of sulfuric acid was added thereto, and the solution was heated under reflux for 10-24 hours. The reaction solution was cooled to room temperature, 100 ml of water was added thereto, and the aqueous solution was neutralized with 1N NaOH. The neutral solution was extracted with methylene chloride (MC), the MC layer was dried with Na2SO4 and filtered, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=20:1), and the solvent was evaporated under reduced pressure, yielding 20.5 g (60% yield) of 2-hexyl-3-hydroxy-decanoic acid mannose ester as a white solid.
Rf (MC:EtOH=10:1): 0.41
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.40 (m, 1H, CH2CHOCH(OH)CH(OH)), 3.48-3.52 (m, 2H, (OH)CHCH(OH)CH(OH)), 3.76 (m, 1H, CH2CH(OH)CH), 4.21 (d, 2H, OCH2CH), 4.27 (m, 1H, CH2CHOCH(OH)), 5.41 (d, 1H, OCH(OH)CH(OH)).
20 g (0.078 mol) of 2-hexyl-3-hydroxy-decanoic β-lactone and 20.3 g (0.94 mol, 1.2 eq) of D-glucosamine hydrochloride were added to 200 ml of toluene. The solution was heated under reflux for 10-24 hours. The reaction solution was cooled to room temperature, 100 ml of water was added thereto, and the aqueous solution was neutralized with 1N NaOH. The neutral solution was extracted with methylene chloride (MC), the MC layer was dried with Na2SO4 and filtered, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (MC:EtOH=20:1), and the solvent was evaporated under reduced pressure, yielding 14.3 g (42% yield) of N-(3′-D-glucosyl)-2-hexyl-3-hydroxy-decaneamid.
Rf (MC:EtOH=10:1): 0.38
1H NMR (CDCl3): δ 0.88 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.50 (m, 1H, CHCHCO), 3.40 (m, 1H, (OH)CHCH(OH)CHO CH2), 3.43 (m, 1H, CH2CH(OH)CH), 3.66 (d, 2H, CHCHOCH2OH), 3.76 (m, 1H, CHCHOCH2), 3.86 (m, 1H, (OH)CHCH(NH)CH(OH)), 4.03 (m, 1H, (OH)CHCH(OH)CH(NH)CH), 5.95 (m, 1H, CHCH(NH)CHO(OH)).
10 g (39.7 mmol) of a C8 alkyl ketene dimer was added to a solution of 3.18 g (79.4 mmol) of NaOH in 100 ml of 90% EtOH, and the mixture was allowed to react at 30-50° C. for 3 hours. Then, 3.26 g (89.3 mmol) of NaBH4 was added thereto and the mixture was allowed to react at 30-40° C. for 10-24 hours. The reaction mixture was acidified with 1N hydrochloric acid and extracted with methylene chloride (MC). The MC layer was separated by column chromatography (MC:EtOH=45:1), and the solvent was evaporated under reduced pressure, yielding 6.8 g (63% yield) of 2-hexyl-3-hydroxy-decanoic acid as a light yellow syrup.
Rf (MC:EtOH=45:1): 0.48
1H NMR (CDCl3): δ 0.87 (t, 6H, CH3), 1.12-1.91 (br, 22H, CH2), 2.45 (m, 1H, CHCHCO), 3.76 (m, 1H, CH2CH(OH)CH).
10 g (18.8 mmol) of a C16-18 alkyl ketene dimer was added to a solution of 1.50 g (37.6 mmol) of NaOH in 50 g of 90% ethanol, and the mixture was allowed to react 30-50° C. for 3 hours. Then, 1.00 g (26.3 mmol) of NaBH4 was added thereto and the mixture was allowed to react at 30-40° C. for 10-24 hours. The reaction mixture was acidified by addition of 1N hydrochloric acid and extracted with methylene chloride (MC). The resulting white solid was recrystallized from EtOH, yielding 6.2 g (60% yield) of 2-hexadecyl/tetradecyl-3-hydroxy-eicosanoic/octadecanoic acid as a white solid.
Rf (MC:EtOH=45:1): 0.49
1H NMR (CDCl3): δ 0.86 (t, 6H, CH3), 1.12-1.91 (br, 62H, CH2), 2.46 (m, 1H, CHCHCO), 3.85 (m, 1H, CH2CH(OH)CH).
The typical results are shown in Table 6 below.
In this Example, 2-hexyl-3-hydroxy-decanoic acid was selected from among 2-alkyl-hydroxylic acids, biosurfactants which can be effectively used in the inventive bioremediation method. The selected compound was evaluated with respect to emulsification activity (E24 %) for n-tetradecane and cyclohexane, in comparison with synthetic nonionic surfactant Tween 80 and another biosurfactant rhamnolipid secreted by microorganisms.
In this experiment, 5 ml of n-tetradecane or cyclohexane was added to 5 ml of alkaline distilled water (pH 9.5) containing each of 10 ppm, 100 ppm and 1000 ppm of 2-hyxyl-3-hydroxy-decanoic acid in test tubes, and the mixture was strongly vortexed for 2 minutes, left to stand at 30° C. for 24 hours and then measured for emulsion stability. Tween 80 and rhamnolipid were also treated in the same manner as described above and were measured for emulsification activity, and the measured emulsification activities were compared with that of 2-hexyl-3-hydroxy-decanoic acid.
The emulsion stability value (E24 %) was evaluated in the same manner as the emulsification activity value and calculated by dividing the height of an emulsion layer in the test tube by the total weight of the test tube and then multiplying the divided value by 100%.
In
As shown in
As described above, the novel microorganisms according to the present invention has excellent biodegradability for a wide spectrum of oils. Also, according to the inventive method for the bioremediation of oil-contaminated soils, the contaminated soils can be purified in an effective, economical and eco-friendly manner compared to the prior bioremediation methods, through the use of at least one selected from among various microbial strains having oil degradability isolated from oil-contaminated soils, including the novel microorganisms, in combination with biosurfactant 2-alkyl-3-hydroxylic acid or its derivative, which serves to increase these microbial strains' biodegradability and can be prepared with high yield in an effective and easy manner.
Although the preferred embodiment of the present invention has been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2006-0067042 | Jul 2006 | KR | national |
