KR101673058B1 - Culture method for increasing the biosurfactant production and growth of oil-degradable strain Bacillus pumilus IJ-1 - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a novel strain, Bacillus pumilus IJ-1, which has oil-degrading ability, and more particularly to a method for culturing Bacillus faecillus IJ-1 which produces a biosurfactant with oil resolution. Since the Bacillus pumilus IJ-1 strain of the present invention has excellent oil-resolving ability such as crude oil, gasoline, kerosene and light oil and further has ability to produce a biological surfactant, the growth of B. pumilus IJ-1 strain and the biological surfactant The present invention, which clarifies the optimum cultivation conditions capable of producing, is very useful for the environmental industry because it can be used for prevention of diffusion of oil pollution and efficient decomposition of oil at the time of marine oil accidents or soil oil contamination.

Description

[0001] The present invention relates to a culture method for increasing the productivity of biosurfactant production and growth of an oil-degradable strain Bacillus pumilus IJ-1,

The present invention relates to a novel strain, Bacillus fumillus IJ-1, which has oil-degrading ability, and more particularly to a culture method for increasing the productivity of a novel strain Bacillus fumillus IJ-1 having oil resolution and an activity of producing a biosurfactant .

Worldwide, oil transportation is largely dependent on ocean transportation, and marine pollution is increasing due to oil spills and various waste oil emissions caused by oil tanker accidents, etc., causing serious damage to marine ecosystems. In recent years, huge oil pollution accidents, ranging from tens of thousands to hundreds of thousands of tons of marine accidents, have occurred due to the large size, automation and speeding of oil transportation vessels. In 1989, Exxon Valdez Pollution accident, 1991 Gulf War accident, The amount of oil spills out into the oceans around the world, such as the outbreak of the Braer in Britain, is enormous. In Korea, marine oil pollution accidents occur frequently, causing serious damage to the destruction of natural ecosystem due to marine pollution and aquaculture in coastal waters. Many efforts have been made by many researchers to solve the oil pollution problem as well as to increase the occurrence of marine oil pollution.

In particular, a technique for decomposing and removing various hydrocarbon source substrates for petroleum products such as hydrocarbons such as n-, iso-, and cyclo-alkanes, which are marine oil pollutants, and crude oil, diesel oil and kerosene, have. In general, to remove oil pollutants, an oil fence may be installed, an oil solidifying agent may be used, or a boom type absorbent may be connected to a limited extent. Leaked oil is either mechanically recovered using oil skimmer (trawl skimmer) or recovered by directly oiling the effluent using an adsorbent. After that, the remaining thin oil film is sprayed with an oil treatment agent to disperse the offshore outflow oil into fine particles to remove the contact area so as to effectively utilize the oil droplet in the natural environment.

However, these methods should be used based on the guidelines for the use of oil treatment agents, and the method of incinerating the oil without recovering oil is not considered to be a good method due to the influence of air pollution or incineration residues.

Therefore, there is a need to develop new technologies for early detection of oil pollution, prevention of spread of contamination, and effective control in marine oil accidents. Especially, there is an increasing interest in biological treatment methods by this method.

This is an urgent need to find a useful microorganism having excellent oil resolution in nature because it has an advantage of being environmentally friendly when using a biological treatment method, for example, a useful microorganism existing in nature, rather than a physicochemical method.

The microorganisms producing the biosurfactant with the oil-splitting ability are Pseudomonas , Achromobacter , Flavobacterium , Nocardia , Aeromonas , Arthrobacter , Vibrio , Bacillus , Micrococcus , Corollospora , Dendryphiella , Mycobacterium , Torulopsis , Acinetobacter genus Are reported.

On the other hand, a biological surfactant lipopeptide sequence is peptide-lipid, which is arthrofactin, B. licheniformis to the Arthrobacter sp. Production Production Serratia There are viscosins produced by serrawettin and Pseudomonas fluorescens produced by marcescens .

The biosurfactants produced in Bacillus are gramicidin S and tyrocidine, which are simple peptides, surfactants in which β-hydroxy fatty acid is ester-bonded to seven amino acid cyclic peptides, seven α-amino acids and one β lt; RTI ID = 0.0 > iturin < / RTI >

Since such a biosurfactant is a substance produced by an organism unlike a conventional chemical synthetic surfactant, it has excellent biodegradability and low toxicity, and has a variety of chemical structures and specificities for each structure. Therefore, detergent, environment, food and beverage, Related products, medicines, cosmetics industry, paint industry, and the like.

Among the biosurfactants, interest in biosurfactants due to microorganisms is gradually increasing because microorganisms are easier to handle than other animal and plant cells, and various kinds of biosurfactants can be obtained depending on microorganism species. This is because the rate is much faster than other organisms and a large amount of biosurfactants can be produced using relatively simple devices in a short period of time. In addition, it is relatively simple to cultivate, it can be easily produced using simple raw materials such as glucose, hydrocarbon, and food-grade oil, and it can be advantageously produced by applying genetic engineering or biochemical techniques.

The interest in bio-surfactants is basically due to environmental concerns, and it is intended to replace the existing chemical synthetic surfactants. However, due to the relatively high cost and the difficulties to deal with organisms, Which is not produced in large quantities. Therefore, in order to solve this problem, it is necessary to secure a highly efficient strain, to develop an effective production method and separation method, and to develop a high-performance biosurfactant that will benefit the biologically- It is true.

Korean Patent No. 10-0641834 Korean Patent No. 10-52063

Hur SH, Yang JS, and Hong JH (2002) Production of biosurfactant using Bacillus spp. JKorean Soc Food Sci Nutr 31, 389-93

Disclosure of the Invention The object of the present invention is to provide a culture method for culturing a strain of B. pumilus IJ-1 in order to improve the production yield of a biological surfactant capable of removing marine crude oil contamination will be.

(A) seeding B. pumilus strain IJ-1 in an LB liquid medium; seed culture; And (B) Bacillus Pew milreoseu (B. pumilus) IJ-1, comprising the step of adding the culture was inoculated to be the species of 0.1 ~ 5.0% of the culture of B. pumilus IJ-1 strain in the medium A- (v / v) The present invention provides a method for culturing an IJ-1 strain for producing a biosurfactant from a strain.

In one embodiment of the present invention, the step (A) is performed at a temperature of 33 to 37 ° C at 150 to 250 rpm for 14 to 18 hours using a liquid medium having a pH of 6.5 to 7.5, and the step (B) To 37 < 0 > C at 150-250 rpm for 72-120 hours.

In one embodiment of the present invention, in step (B), the carbon source of the carboxymethyl cellulose (CMC), dextrose, fructose, galactose But are not limited to, galactose, glucose, glycerol, lactose, maltose, mannitol, mannose, raffinose, sucrose, but are not limited to, sucrose, trehalose, xylose, nonane, decane, tetradecane, hexadecane, crude oil, benzene, , Toluene, and paraffin. In addition, the present invention is not limited thereto.

In one embodiment of the present invention, in step (B), the A-medium further contains glycerol as a carbon source, and the glycerol may be contained in an amount of 0.1 to 1.5% (v / v) have.

In one embodiment of the present invention, the step (B) may further include an organic nitrogen source in the A-medium.

In one embodiment of the present invention, the organic nitrogen source is selected from the group consisting of beef extract, casein, malt extract, peptone, skim milk, soytone, Tryptone, yeast extract, and the like.

In one embodiment of the present invention, in step (B), KNO ₃ may be further added to the A-medium as an inorganic nitrogen source.

In one embodiment of the present invention, the organic nitrogen source is tryptone, and the tryptone may be contained in an amount of 0.3 to 1.5% (w / v) of the total culture composition.

In one embodiment of the present invention, in step (B), the A-medium may further contain 0 to 2% (w / v) NaCl.

In one embodiment of the present invention, the pH of the A-medium may be 5 to 10 in the step (B).

In one embodiment of the present invention, the culturing temperature in step (B) may be 15 to 40 ° C.

Since the microorganism B. pumilus IJ-1 of the present invention is excellent in oil resolution, such as crude oil, gasoline, kerosene and light oil, and has further ability to produce a biosurfactant, the growth of B. pumilus strain IJ-1 and the biological surfactant Can be used for prevention of the spread of oil pollution and efficient decomposition of oil during marine oil accidents or pollution of oil in the soil, and thus it is very useful in environmental industry.

Figure 1 is a graph showing the effect of NaCl concentration on the growth of B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
Figure 2 is a graph showing the effect of NaCl concentration on the surface tension of biosurfactants produced by B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
Figure 3 is a graph showing the effect of initial pH on the growth of B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
Figure 4 is a graph showing the effect of initial pH on the surface tension of biosurfactants produced by B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
Figure 5 is a graph showing the effect of incubation temperature on the growth of B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
6 is a graph showing the influence of the culture temperature on the surface tension of the biosurfactant produced by B. pumilus IJ-1 in A-medium containing 0.5% (w / v) tryptone.
FIG. 7 shows a comparison of the surface tension reduction rates of the experimental plot versus the predicted plot.
FIG. 8 is a graph showing the reaction surface state of the response variable for the independent variable according to the present invention in a three-dimensional graph and contour line analysis.

The present invention is characterized by providing a culturing method capable of increasing the production efficiency of biological surfactant of Bacillus pneumylus IJ-1 having oil resolution.

Oil spills with petroleum hydrocarbon compounds cause natural environmental pollution and harm to human health. In particular, oil spills into the oceans and the soil are polluted with ecosystem pollution, There is a problem that time and cost are consumed.

Accordingly, although many techniques for removing oil pollution have been conventionally studied, there is a problem that most of them use chemicals to cause pollution of other natural environments.

In order to develop a method for using microorganisms as a method for eliminating oil contamination through the application No. 10-2013-0087007 of the present invention, the present inventors have isolated and identified a microorganism existing in a natural environment having excellent oil resolution, Bacillus pumilus IJ-1 strain ", and the strain identified was deposited with the Korean Society for Microbiological Protection (KCCM) on Nov. 2, 2012 and received the deposit number KCCM11319P.

The present invention relates to a culture method for maximizing the growth, oil resolution and biosurfactant producing ability of the strain, and in order to understand the present invention more clearly, it is possible to refer to Application No. 10-2013-0087007 .

Step of the invention (A) Bacillus Pew milreoseu (B. pumilus) was inoculated IJ-1 strain in LB broth seed culture (seed culture); And (B) further culturing the seed cultured B. pumilus strain IJ-1 in an amount of 0.1 to 5.0% (v / v) in the A-medium for further production of the biosurfactant Thereby providing a culture method.

In this case, the step (A) may be performed at a temperature of 33 to 37 ° C at 150 to 250 rpm for 14 to 18 hours using a liquid medium having a pH of 6.5 to 7.5, and the step (B) It is preferable to culture at 250 rpm for 72 to 120 hours.

In this case, it is more preferable that the step (A) is carried out at 35 rpm for 16 hours using a liquid medium of pH 7, and the step (B) is carried out at 35 rpm for 200 rpm for 96 hours .

On the other hand, the medium A- agarose (c arboxymethyl cellulose, CMC), dextromethorphan Oz (dextrose), fructose Oz (fructose), galactose (galactose), glucose (glucose), glycerol as carboxymethyl cellulose as a carbon source (s), glycerol, lactose, maltose, mannitol, mannose, raffinose, sucrose, trehalose, xylose, It is made up of xylose, nonane, decane, tetradecane, hexadecane, crude oil, benzene, toluene and paraffin. if further comprises at least one selected from the group media supplemented with a bar, the carbon atom identified as the surface tension is reduced relative to the control group is the Bacillus Pew milreoseu (B. pumilus) IJ-1 increases the production efficiency of biological surfactants strain (See Table 1).

In this case, glycerol is preferably added as the carbon source, and the glycerol is preferably added in an amount of 0.1 to 1.5% (v / v) of the total culture composition. For the best strain growth, the glycerol concentration is preferably 1.0 % (v / v), and it is preferable to add the glycerol concentration to 0.5% (v / v) for the best biological surfactant production efficiency (see Table 2).

In addition, it is preferable to add an organic nitrogen source or an inorganic nitrogen source to the A-medium. Examples of the organic nitrogen source include beef extract, casein, malt extract, peptone, It is preferable to add at least one selected from the group consisting of skim milk, soytone, tryptone and yeast extract, and the A-medium of step (B) It is preferable to further add KNO ₃ as a nitrogen source (see Table 3).

In this case, it is more preferable to add tryptone to the organic nitrogen source. For the best strain growth, the tryptone is added so that the concentration of the tryptone is 1.5% (w / v) It is preferable to add glycerol so that the concentration of glycerol is 0.5% (w / v) (see Table 4).

Meanwhile, in order to increase the productivity of the strain Bacillus besylus IJ-1 of the present invention and the production efficiency of the biosurfactant, the strain (B) further contains NaCl at a concentration of 0 to 2% (w / v) , And it is more preferable to further include NaCl at a concentration of 1% (w / v) (see Figs. 1 and 2).

In order to increase the productivity of the Bacillus subtilis IJ-1 strain of the present invention and to increase the production efficiency of the biosurfactant, the pH of the A-medium is preferably 5 to 10 at the time of the step (B) It is more preferable that the pH is 9 (see Figs. 3 and 4).

In order to increase the productivity of the Bacillus subtilis IJ-1 strain of the present invention and to increase the production efficiency of the biosurfactant, the culture temperature in the step (B) is preferably 15 to 40 ° C. For the best growth efficiency, More preferably 35 DEG C, and more preferably 20 DEG C for the best biological surfactant production efficiency (see Figs. 5 and 6).

Furthermore, the inventors of the present invention confirmed by using a reaction surface methodology to more accurately and precisely find the optimal conditions of the culture conditions for the production of the biosurfactant of Bacillus faecillus IJ-1 having oil resolution, and found that the optimum culture conditions for the production of biosurfactants The NaCl concentration was estimated to be 0.745% (w / v), the incubation temperature was 19.29 ℃, and the tryptone concentration was estimated to be 0.787% (w / v) It is expected to be useful for prediction of biosurfactant production.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The present invention is intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these embodiments.

< Example  1>

Oil-resolving Bacillus Pumilus IJ -1 of Biological surfactant  For optimization of culture conditions for production, OFAT ( One Factor at  a Time ) Confirmation using method

<1-1> Used strain and medium

In the present invention, as a biosurfactant producing strain, Bacillus pumilus IJ-1 was used. B. pumilus IJ-1 was cultured in Luria-Bertani (LB) [1.0% (w / v) tryptone (Difco, USA), 0.5% (w / v) yeast extract (Bioshop, Canada) NaCl (Bioshop), and 1.5% agar (w / w) (Difco), pH 7.0]. The medium for the production of the biosurfactant was A-medium [0.3% (w / v) KNO ₃ , 0.22% (w / v) Na ₂ HPO ₄ , 0.014% (w / v) KH ₂ PO ₄ , 0.001% (w / v) NaCl, 0.06% (w / v) MgSO 4, 0.004% (w / v) CaCl 2, 0.002% (w / v) FeSO 4, and 0.1 mL of trace element solution (g / L) (2.32 g ZnSO ₄ H ₂ O, 1.78 g MnSO ₄ H ₂ O, 0.56 g H ₃ BO ₃ , 1 g CuSO ₄ H ₂ O, 0.39 g Na ₂ MoO ₄ H ₂ O, 0.42 g CoCl ₂ H ₂ O and 1 g EDTA) was used.

Meanwhile, the nitrogen sources used in the present invention are Difco as a beef extract, casein as a usb (USA), malt extract as a Difco, peptone as a Difco, skim milk as a Difco, soytone as a Bio Basic, tryptone as a Difco, yeast extract as a Bioshop (NH ₄ ) H ₂ PO ₄ is Junsei (Japan), NH ₄ Cl is Bio Basic, NH ₄ NO ₃ is Junsei, (NH ₄ ) O ₂ SO ₄ is Sigma, KNO ₃ is Sigma, NaNO ₂ is Junsei, NaNO ₃ Junsei and CH ₃ COONH ₄ were purchased from Yakuri.

The carbon sources used in the present invention are as follows: arabinose, carboxymethyl cellulose (CMC) Sigma, dextrose Difco, fructose Daejung (Korea), galactose Daejung, glucose Bioshop, glycerol Junsei, lactose Daejung, maltose Hexane, pentane, Alfa Aesar (USA), hexane, Alfa Aesar (USA), and mannitol is Junsei, mannose is Acros (USA), raffinose is Wako (Japan), soluble starch is Yakuri, sucrose is Daejung, trehalose is Wako, xylose is Junsei , heptane is Carlo Erba (Italy), octane is Carlo Erba, nonane is Alfa Aesar, decane is Carlo Erba, tetradecane is Alfa Aesar, hexadecane is Alfa Aesar and crude oil (Kuwait) benzene was purchased from Alfa Aesar, toluene was purchased from Carlo Erba, and paraffin was purchased from Carlo Erba.

On the other hand, the A-medium is referred to as reference Hur SH, Yang JS, and Hong JH (2002) Production of biosurfactant using Bacillus spp .. JKorean Soc Food Sci Nutr 31, 389-93. The KNO ₃ used _was Junsei (Japan), Na ₂ HPO ₄ was Junsei, KH ₂ PO ₄ was Yakuri (Japan), NaCl was Promega, MgSO ₄ was Yakuri, CaCl ₂ is Yakuri, FeSO ₄ is Sigma, ZnSO ₄ H ₂ O Lt; RTI ID = _0.0 &gt; MnSO4H2O &lt; / _RTI & Are Yakuri, H ₃ BO ₃ Is Sigma, CuSO ₄ H ₂ O , Sigma, Na ₂ MoO ₄ H ₂ O, Yakuri, CoCl ₂ H ₂ O Sigma and EDTA were purchased from Bioshop.

<1-2> Culture conditions

B. pumilus IJ-1 was inoculated into LB liquid medium (pH 7.0) for the production of biosurfactants and seed culture was carried out at 200 rpm at 35 ° C, the optimal temperature for strain growth, for 16 hours. The grown strain was inoculated 1.0% (v / v) in A-medium and cultured at 35 ° C at 200 rpm for 96 hours.

<1-3> Strain growth measurement

The growth of B. pumilus IJ-1 was measured at 600 nm using an indirect counting method using BioPhotometer 6131 spectrophotometer (Eppendorf AG, Eppendorf, Germany).

<1-4> Biological surfactant  detach

The seed culture was inoculated 1.0% (v / v) in the biosurfactant production medium and incubated at 35 ° C for 4 days with shaking. The supernatant was filtered (0.45, Minisart, Sartorius Stedium Biotech GmbH, Goettingen, Germany) as a biosurfactant after centrifugation (4 ° C, 10,000 rpm, 20 min)

<1-5> Surface tension measurement

When microorganisms produce biosurfactants, surface tension drops. Using this fact, we confirmed the surface tension activity of biosurfactants produced by microorganisms with surface tension values. The surface tension was measured repeatedly at 25 ° C three times using the Surface Tension Analyzer DST-60 (Surface Electro Optics Co., Suwon, Korea) by the Ring method and the average value thereof was calculated.

<1-6> Biological surfactant  Optimal for production Carbon source  Research

In order to investigate the effect of carbon sources on the production of biosurfactants, sugars or carbohydrates such as arabinose, carboxymethyl cellulose (CMC), dextrose, fructose, galactose, glucose , pentane (C ₅ ), hexane (C ₆ ), heptane (C ₇ ), octane (C ₈ ), nonane (C ₆ ), glycerol, lactose, maltose, mannitol, mannose, raffinose, soluble starch, sucrose, trehalose, _{C 9), decane (C 10} ), tetradecane (C 14), hexadecane (C 16), crude oil (produced in Kuwait), polyaromatic hydrocarbons (PAH) giant amphipods _{benzene (C 6 H 6),} toluene (C 7 H 8 ) and paraffin (C _n H _{2n + 2} ) were added at 1.0% (w / v or v / v) at 35 ° C and 200 rpm for 96 hours, and the growth and surface tension of the strain were measured. The growth and surface tension of the strains were measured by incubating for 96 hours at 35 ℃ and 200 rpm while changing the optimum carbon source concentration to 0.5 1.0, 1.5 and 2.0% (w / v). Then, 0.5% (w / v) or v / v of each carbon source was added to the A-medium supplemented with 0.5% (w / v) tryptone to determine the optimum carbon source for the determined optimum nitrogen source. And cultured at 200 rpm for 96 hours to measure the growth and surface tension of the strain. Since the initial surface tension value differs according to each carbon source, the difference between the initial surface tension value and the final surface tension value, that is, the surface tension reduction rate, is calculated and expressed.

As a result, as shown in Table 1, the strain growth was higher in the medium supplemented with sugars or carbohydrates than the hydrocarbons. The addition of glycerol, mannose, tetradecane and crude oil as a carbon source resulted in a high rate of surface tension reduction. Among them, glycerol was the best carbon source for the production of biosurfactants with the highest surface tension reduction rate of 43.3%. In particular, the surface tension reduction rate of tetradecane was 40.3%, which showed the highest surface tension reduction rate among hydrocarbons and the surface tension reduction rate of crude oil was 39.6%. On the other hand, surface tension reduction rate of arabinose, soluble starch, pentane, hexane and octane was lower than that of control.

On the other hand, the optimum concentration of glycerol determined as an optimal carbon source for the production of biosurfactants was investigated. As a result, as shown in Table 2, when the concentration of glycerol was 1.0% (v / v), the strain showed the best growth. When 0.5% (v / v) glycerol was added, the surface tension reduction rate was the highest at 45.2%.

<1-7> Biological surfactant  Optimum nitrogen source investigation for production

To investigate the effect of nitrogen source for biosurfactant production, various organic nitrogen sources (beef extract, casein, malt extract, peptone, skim milk, soytone, tryptone and yeast extract) and inorganic nitrogen source [(NH ₄ ) H _{2 PO 4, NH 4 Cl,} NH 4 NO 3, (NH 4) O 2 SO 4, KNO 3, NaNO 2, NaNO 3, CH 3 COONH 4] was added per each 0.5% (w / v) to 35 ℃ , And cultured at 200 rpm for 96 hours to measure the growth and surface tension of the strain. Growth and surface tension of the strains were measured by culturing at 35 ℃ and 200 rpm for 96 hours while varying the optimum concentration of nitrogen source at 0.3, 0.5 1.0 and 1.5% (w / v). To determine the optimum nitrogen source for the determined optimum carbon source, 0.5% (w / v) of each of the above nitrogen sources was added to the A-medium supplemented with 0.5% (v / v) glycerol, And the growth and surface tension of the strain were measured. Since the initial surface tension value differs according to each nitrogen source, the difference between the initial surface tension value and the final surface tension value, that is, the surface tension reduction rate, is calculated and indicated.

As a result, as shown in Table 3, OD ₆₀₀ value of peptone was the highest at 2.853, followed by skim milk (OD ₆₀₀ = 2.772) and beef extract (OD ₆₀₀ = 2.432). Inorganic nitrogen source showed relatively lower growth than organic nitrogen source. When tryptone was used as a nitrogen source, surface tension reduction rate was the highest at 52.0%, followed by yeast extract (46.1%), soytone (45.9%) and peptone (43.9%). Organic nitrogen source was found to be more suitable for the production of biosurfactants than inorganic nitrogen sources. Among them, tryptone was determined to be the optimum nitrogen source.

In addition, as shown in Table 4, strain growth and surface tension were measured by adding tryptone determined to be the optimum nitrogen source to the medium at 0.3, 0.5, 1.0 and 1.5% (w / v). The highest growth rate of tryptone was obtained at the highest concentration of tryptone, and the decrease of surface tension was highest at tryptone of 0.5% (w / v).

The optimum nitrogen source for the determined optimum carbon source 0.5% (v / v) glycerol was investigated. As a result, as shown in Table 5, strain growth was highest in tryptone. Growth in organic nitrogen source was higher than inorganic nitrogen source. On the other hand, the reduction rate of surface tension was lower than that of the control. Especially, in the media supplemented with 0.5% (w / v) tryptone, which is the optimum nitrogen source, the surface tension reduction rate was the lowest at 2.2%. Therefore, it was shown that medium supplemented with 0.5% (v / v) glycerol, which is a carbon source, with various nitrogen sources was not suitable for the production of biosurfactants.

The optimal carbon source for the determined optimal nitrogen source 0.5% (w / v) tryptone was found to be higher in crude oil (OD ₆₀₀ = 6.728) and soluble starch (OD ₆₀₀ = 5.290) as shown in Table 6 below. The surface tension reduction rate was lower than that of the control. Therefore, it was shown that medium supplemented with various carbon sources to tryptone containing 0.5% (w / v) nitrogen source was not suitable for the production of biosurfactants.

<1-8> Biological surfactant  Investigation of optimal inorganic salts for production

To investigate the effect of inorganic salts for biosurfactant production, a total of 15 kinds of inorganic salts (CaCl 2) were added to A-medium supplemented with 0.5% (w / v) tryptone, which has the greatest effect on the production of biosurfactants, _{2, CaCO 3, CoCl 2,} CuSO 4, K 2 HPO 4, KCl, KH 2 PO 4, MgSO 4, MnSO 4, NaCl, KNO 3, Na 2 HPO 4, FeSO 4, H 3 BO 3, Na 2 MoO ₄ ) was added to each 0.1% (w / v) concentration, and cultured at 35 ° C and 200 rpm for 96 hours to measure the strain growth and surface tension. Since the initial surface tension value differs according to each inorganic salt, the difference between the initial surface tension value and the final surface tension value, that is, the surface tension reduction rate, is calculated and indicated.

As a result, as you can see in Table 7, B. pumilus IJ-1 showed low growth on medium supplemented with various inorganic salts. In addition, in the medium containing various inorganic salts, the surface tension reduction rate was lower than that of the control, and the medium containing inorganic salts was found to be unsuitable for the production of biosurfactants. Therefore, B. pumilus The optimal medium composition for IJ-1 biosurfactant production was determined with A-medium containing 0.5% (w / v) tryptone.

<1-9> Investigation of the effect of NaCl concentration on biosurfactant production

In order to investigate the production of biosurfactants according to various NaCl concentrations, NaCl concentrations in the A-medium supplemented with 0.5% (w / v) tryptone were 0, 1, 2, 3, 4, 5, 6, , 9, and 10% (w / v), respectively. The seed culture was inoculated at various concentrations of NaCl at 1.0% (v / v), and the growth of the strain and the surface tension of the biosurfactant were measured at intervals of 12 hours while shaking at 35 ° C and 200 rpm.

As a result, the strain showed the best growth when cultured at a concentration of 0-2% (w / v) NaCl, and the best growth was obtained at 48 hours after culturing. The higher the NaCl concentration in the medium, the slower the growth of the strain and the lowest at 10% (w / v) NaCl (see FIG. 1). It was confirmed that the growth of the strain and the production of biosurfactant by the concentration of NaCl added to the culture medium were almost the same. Also NaCl concentration B. pumilus IJ-1 was found to affect the production of biosurfactants. The surface tension value of the culture medium containing 0-1% (w / v) NaCl was 32.3-32.7 dyne / cm at 72 hours of culture. The maximum reduction surface tension value of 33.6-34.3 dyne / cm was maintained at 2-3% (w / v) NaCl concentration and 34.9-35.5 dyne / cm at 4-5% (w / v) NaCl concentration (See Fig. 2).

<1-10> Effect of pH on Biological Surfactant Production

1.0 N HCl and 1.0 N NaOH were added to the medium supplemented with 0.5% (w / v) tryptone at pH 1.0 to pH 10, respectively, to investigate the production of biosurfactants according to various initial pHs of the medium Respectively. The seed culture was inoculated at various concentrations of 1.0% (v / v) in various pH ranges, and the growth of the strain and the surface tension of the biosurfactant were measured at intervals of 12 hours while shaking at 35 ° C and 200 rpm.

Microorganisms applied in the field are exposed to various pH environments. B. pumilus IJ-1 was grown in a medium of pH 5-10, and the highest growth was observed in a medium of pH 9. Whereas it hardly grows in the medium of initial pH 3-4 (see FIG. 3). B. pumilus IJ-1 exhibited high surface tension activity over a wide pH range from pH 5 to 10. Especially, at pH 9, the best surface tension activity was confirmed to be 30.5 dyne / cm at 72 hours of culture. On the contrary, when cultured at pH 3 and 4, the surface tension value did not decrease with the lapse of incubation time, and it was judged that the acid condition was not suitable for the production of biosurfactant (see FIG. 4).

<1-11> Investigation of temperature effects for biosurfactant production

B. pumilus IJ-1 was inoculated with 1.0% (v / v) in 0.5% (w / v) tryptone supplemented medium at 15-50 ℃ to investigate biosurfactant production according to various culture temperatures. The growth of the strain and the surface tension of the biosurfactant were measured every 12 hours while shaking at 200 rpm for 96 hours at 5 ° C intervals.

It is known that the biodegradation efficiency of microorganisms is greatly affected by the temperature change since the activity of degrading microorganisms decreases below or below the appropriate temperature. B. pumilus IJ-1 grew at a relatively wide range of temperatures (20-45 ℃), with the best growth at 35 ℃. Similar growth patterns were observed at 20, 25 and 30 ℃, and relatively low growth was observed at 40 ℃ and 45 ℃. On the other hand, it was confirmed that it hardly grows at 15 and 50 ° C (see FIG. 5). As a result of the investigation of the surface tension according to the incubation temperature, the surface tension activity was high at 20-35 ℃, where the strain growth was good. In particular, the surface tension at 20 ° C was 28.4 dyne / cm at 72 hours of culture, indicating the best surface activity. The minimum surface tension values at 25, 30, and 35 ℃ were measured as 29.4 dyne / cm (72 h), 32.1 dyne / cm (60 h) and 35.5 dyne / cm (72 h), respectively. On the other hand, temperatures of 15 and 50 ° C were found to be unsuitable for biosurfactant production (see FIG. 6).

&Lt; Example 2 >

In order to optimize culture conditions for the production of biosurfactants of Bacillus pumilus IJ-1 with oil resolution, confirmation using reaction surface methodology

The present inventors confirmed the optimization of the culture conditions for the production of the biosurfactant of Bacillus subtilis IJ-1 by using the OFAT (One Factor at a Time) method of Example 1 above.

However, this method can be used to specify one of the factor variables (carbon source, nitrogen source, inorganic salt, incubation temperature, initial pH of the medium and NaCl concentration) and constantly changing the remaining factor parameters to establish conditions according to one factor Method, the inventors of the present invention use response surface methodology (RSM), which can widely evaluate the change of reaction while minimizing the number of experiments when multiple independent variables affect the dependent variable by performing a complex action To confirm the optimal culture condition suitable for the production of biosurfactant of Bacillus subtilis IJ-1 more accurately and accurately.

<2-1> Used strain and medium

In the present invention, Bacillus pumilus IJ-1 was used as a biosurfactant producing strain. B. pumilus IJ-1 was cultured in Luria-Bertani (LB) [1.0% (w / v) tryptone (Difco, USA), 0.5% (w / v) yeast extract (Bioshop, Canada) NaCl (Bioshop), and 1.5% agar (w / w) (Difco), pH 7.0]. The medium for the production of the biosurfactant was A-medium [0.3% (w / v) KNO ₃ , 0.22% (w / v) Na ₂ HPO ₄ , 0.014% (w / v) KH ₂ PO ₄ , 0.001% (w / v) NaCl, 0.06% (w / v) MgSO 4, 0.004% (w / v) CaCl 2, 0.002% (w / v) FeSO 4, and 0.1 mL of trace element solution (g / L) (2.32 g ZnSO ₄ H ₂ O, 1.78 g MnSO ₄ H ₂ O, 0.56 g H ₃ BO ₃ , 1 g CuSO ₄ H ₂ O, 0.39 g Na ₂ MoO ₄ H ₂ O, 0.42 g CoCl ₂ H ₂ O and 1 g EDTA) was used.

Various media of tryptone and NaCl were added to the medium of the present invention for the production of biosurfactants.

<2-2> Culture conditions

B. pumilus IJ-1 was inoculated into 5 mL of A-medium (pH 9.0) for the production of biosurfactants and seeded for 6 to 14 hours at 35 rpm at 200 rpm, the optimal temperature for strain growth. The grown strains were inoculated 1.0% (v / v) in medium containing various concentrations of factor (tryptone and NaCl) and cultured at 72 rpm at various temperatures (20-30 ° C, Lt; / RTI &gt;

<2-3> Biological surfactant  detach

Biosurfactants were isolated by modifying the method of Ghojavand et al. (2008). The culture of the shake cultured strain was centrifuged at 10,000 rpm at a temperature of 4 ° C for 20 minutes to remove the cells. The pH was adjusted to 2.0 while gradually adding 6.0 M HCl to the supernatant. The mixture was allowed to stand at 4 ^° C overnight, The active agent was precipitated. The precipitate was collected by centrifugation, dissolved in an alkaline aqueous solution (0.1 N NaOH) to a final pH of 8.0, and lyophilized. The dried material was extracted three times with 10 mL of methanol and concentrated to use as a biosurfactant.

<2-4> Surface tension measurement

Surface tension is a tension that acts to reduce the surface at the free surface of the liquid when two phases of different nature are adjacent to each other. When the microorganism produces a biosurfactant, the surface tension decreases. Using this fact, surface tension activity of microbial biosurfactants was confirmed by surface tension (Lang, 2002). Surface tension was measured three times at 25 ^° C using the Surface Tension Analyzer DST-60 (Surface Electro Optics Co., Suwon, Korea) and the average value was calculated (Pagilla et al., 2002).

<2-5> 3 ³ Factor experiment planning

The present inventors embodiment NaCl and incubation temperature, tryptone, resulting in a 1 B. pumilus IJ-1 was the most influential factor in the production of biosurfactants.

In order to verify the validity of the model, the range of the variables was selected based on the results of Example 1, and the experiment was designed using the ^three- factorial experimental design method. The independent variables were NaCl concentration (0, 0.5, 1%, w / v), incubation temperature (20, 25, 30 ^o C) and tryptone concentration (0, 0.5, 1%, w / v) And the level of each factor was coded in three levels of -1, 0, and 1 (see Table 8).

To make a regression equation, each factor was coded according to the following equation.

For the i-th variable, X _i is the standardized experimental point, X _i is the actual experimental point, x _i ⁰ is the center of the experimental point, and Δ x _i is the range. X _i is the range of 1≤ X _i ≤1.

In order to prepare the culture conditions, the experiment was designed by the ^3- factorial experimental design method. The experiment was repeated three times or more with 30 different culturing conditions by combining the three factors and the surface tension reduction rate (%) according to each culturing condition The results are shown in Table 9.

Run
order Factor  One:
NaCl
(w / v) Factor  2:
Temperature
( ^o C ) Factor  3:
Tryptone
(w / v) Response Reduction ratio of 표면 tension Experimental  (%) Predicted  (%) One 0.0 30 0.0 0.00 1.2682 One 0.0 30 0.0 0.41 1.2682 One 0.0 30 0.0 3.35 1.2682 2 1.0 25 0.5 47.45 48.8031 2 1.0 25 0.5 49.83 48.8031 2 1.0 25 0.5 50.20 48.8031 3 0.5 30 0.0 2.42 1.8815 3 0.5 30 0.0 4.06 1.8815 3 0.5 30 0.0 4.58 1.8815 4 1.0 20 0.0 1.03 3.5476 4 1.0 20 0.0 1.31 3.5476 4 1.0 20 0.0 2.39 3.5476 4 1.0 20 0.0 3.75 3.5476 5 1.0 25 1.0 47.70 49.1804 5 1.0 25 1.0 48.20 49.1804 5 1.0 25 1.0 49.24 49.1804 6 0.5 30 0.5 42.11 44.6031 6 0.5 30 0.5 43.02 44.6031 6 0.5 30 0.5 43.31 44.6031 7 0.0 25 0.5 46.60 48.5098 7 0.0 25 0.5 46.70 48.5098 7 0.0 25 0.5 47.98 48.5098 8 1.0 30 1.0 40.47 40.9706 8 1.0 30 1.0 42.07 40.9706 8 1.0 30 1.0 43.58 40.9706 9 0.5 20 0.5 49.68 50.4565 9 0.5 20 0.5 52.47 50.4565 9 0.5 20 0.5 52.85 50.4565 10 0.5 20 0.0 2.16 3.0429 10 0.5 20 0.0 2.88 3.0429 10 0.5 20 0.0 3.01 3.0429 11 0.0 25 0.0 1.52 3.0298 11 0.0 25 0.0 5.25 3.0298 11 0.0 25 0.0 5.46 3.0298 12 1.0 30 0.0 1.15 0.6303 12 1.0 30 0.0 2.56 0.6303 12 1.0 30 0.0 4.07 0.6303 13 0.0 20 0.5 46.02 48.4995 13 0.0 20 0.5 50.48 48.4995 13 0.0 20 0.5 50.84 48.4995 14 0.0 30 0.5 41.59 44.4021 14 0.0 30 0.5 41.68 44.4021 14 0.0 30 0.5 44.42 44.4021 15 0.5 25 1.0 49.37 50.3785 15 0.5 25 1.0 50.80 50.3785 15 0.5 25 1.0 50.83 50.3785 16 1.0 25 0.0 2.78 4.1479 16 1.0 25 0.0 4.31 4.1479 16 1.0 25 0.0 4.69 4.1479 17 0.0 20 1.0 48.97 52.0477 17 0.0 20 1.0 51.14 52.0477 17 0.0 20 1.0 51.54 52.0477 17 0.0 20 1.0 52.65 52.0477 18 0.5 25 0.5 50.00 49.5888 18 0.5 25 0.5 50.77 49.5888 18 0.5 25 0.5 51.45 49.5888 19 1.0 20 1.0 51.92 53.2721 19 1.0 20 1.0 52.35 53.2721 19 1.0 20 1.0 52.87 53.2721 19 1.0 20 1.0 53.79 53.2721 20 0.5 25 0.5 50.20 49.5888 20 0.5 25 0.5 51.43 49.5888 20 0.5 25 0.5 51.51 49.5888 21 0.0 30 1.0 43.52 43.2582 21 0.0 30 1.0 44.98 43.2582 21 0.0 30 1.0 45.91 43.2582 22 0.0 25 1.0 50.36 49.7119 22 0.0 25 1.0 50.53 49.7119 22 0.0 25 1.0 51.03 49.7119 23 1.0 20 0.5 53.22 50.5488 23 1.0 20 0.5 53.50 50.5488 23 1.0 20 0.5 53.88 50.5488 24 0.5 25 0.0 2.00 4.5212 24 0.5 25 0.0 2.67 4.5212 24 0.5 25 0.0 4.26 4.5212 25 0.5 20 1.0 52.37 53.5922 25 0.5 20 1.0 52.42 53.5922 25 0.5 20 1.0 53.10 53.5922 26 0.5 25 0.0 2.17 4.5212 26 0.5 25 0.0 3.60 4.5212 26 0.5 25 0.0 4.18 4.5212 27 1.0 30 0.5 39.01 42.9394 27 1.0 30 0.5 40.34 42.9394 27 1.0 30 0.5 41.28 42.9394 28 0.5 30 1.0 40.06 43.0467 28 0.5 30 1.0 43.23 43.0467 28 0.5 30 1.0 45.82 43.0467 29 0.0 20 0.0 0.78 0.6735 29 0.0 20 0.0 1.11 0.6735 29 0.0 20 0.0 1.43 0.6735 30 0.5 25 1.0 49.25 50.3785 30 0.5 25 1.0 51.36 50.3785 30 0.5 25 1.0 51.40 50.3785

As a reaction item, the surface tension reduction rate (%) was set as a measurement item. Since the initial surface tension value differs according to each culturing condition, the difference between the initial surface tension value and the final surface tension value, that is, the surface tension reduction rate, was calculated. The equation for obtaining the surface tension reduction rate is as follows.

<2-6> Optimization of culture conditions using reaction surface methodology

B. pumilus IJ-1 was used to determine optimal culture conditions for biosurfactant production. The relationship between each independent variable and the experimental variables, ie, the first linear effect, the quadratic curve effect, and the interplay between the factors, was examined by the second polynomial regression equation. The surface condition was optimized by three - dimensional graph and contour line analysis.

At this time, the independent variable X _i and response variables Y _i to X _j (surface tension reduction rate,%) is showed as a secondary regression equation such as the following, β ₀ is a constant and β _i, β _ii, β _ij is the coefficient to be.

In the Y _i is a value between the k value at the predictive value of the response variable, subscript i and j are 1, β ₀ is a constant, β _i are the linear regression coefficient, β _ii are quadratic regression coefficients, β _ij are the effects of the interaction, and X _i and X _j are the standardization experimental points.

Statistical analysis system (SAS) 9.2 software (SAS Institute, Cray, NC, USA) was used for the regression analysis of model equations. The fitness of the model was verified by ANOVA test. The response surface condition of the response variable to the independent variables was analyzed by three - dimensional graph and contour analysis.

Analysis of variance (ANOVA) The effect list and statistical values of each variable are shown in Table 10. In the analysis of variance, the coefficient of determination (R ² ) is 0.9938, which is very close to 1, indicating that the reaction model is reliable. As a result of confirming the significance of the overall experimental model, the probability value (P value) And the hypothetical model response is appropriate for the data.

the first order of the response variables Y, based on _{t -statistic (X 1, X 2} , X3), wherein the second ^{_{(X 1 2, X 2 2}} , X 3 2), wherein interaction _{(X 1 X 2, X 1} X ₃ and X ₂ X ₃ ) (see Table 11), and all of the interactions (except for X ₁ X ₃ ) were significant (<0.05).

The relationship between the three factors, NaCl concentration (%, w / v), culture temperature ( ^o C), and tryptone concentration (%, w / v) The quadratic equation obtained from the reaction surface analysis is as follows.

In addition, the formula derived using the converted coded factors is as follows.

The significance of the significance (P-value) value is generally referred to as 'statistically significant' if it is less than 0.05 and 'highly significant' if it is less than 0.01 It says. The term 'note' here means that the assumed model is meaningful in interpreting the data.

The most important factors affecting the production of biosurfactants of B. pumilus IJ-1 were tryptone ( p-value <0.0001), culture temperature ( p-value <0.0001) and NaCl ( p-value = 0.0036) (See Table 12).

Experimental plot versus surface tension reduction ratio In the predicted plot, the actual values were distributed near the straight line (see FIG. 7), which means that the actual value is very close to the predicted value (R ² = 0.9938).

Therefore, it can be seen that Equation 4 is a suitable model for predicting the production efficiency of biological surfactant under the experimental conditions described above.

The reaction surface state of the response variable to the independent variable is shown by a three-dimensional graph and a contour line analysis in FIG. 2, and the concentration of NaCl in the biosurfactant production at the tryptone concentration of 0.5% (w / v) As a result of the correlation between the incubation temperature and the reaction surface and the contour plot, the surface tension reduction rate tended to increase with decreasing temperature. NaCl concentration had no significant effect on the biosurfactant production compared to the temperature. NaCl concentration and the highest temperature at low culture temperature. The optimum NaCl concentration for the production of the biosurfactant was found to be 0.73% (w / v) and the incubation temperature was 21.0 ° C (see FIG. 8A).

The correlation between the NaCl concentration and the tryptone concentration on the biosurfactant production at the incubation temperature of 20 ° C was shown by the reaction surface and the contour plot. There was no significant difference in the biosurfactant production depending on the NaCl concentration in the low tryptone concentration range In the range of high tryptone concentration, the biosurfactant production tended to increase as the NaCl concentration increased. It was found that trytone concentration had more effect on NaCl concentration. The optimum NaCl concentration for producing biosurfactants was 0.71% (w / v) and the tryptone concentration was 0.76% (w / v) (see FIG. 8B).

  The correlation between the incubation temperature and the tryptone concentration for biosurfactant production at 0.5% (w / v) NaCl concentration was shown by the reaction surface and contour plot. As the incubation temperature increased, The surfactant production was lowered, and it was found that the range of high tryptone concentration was not significantly affected by the incubation temperature (see FIG. 8C).

The optimum culture temperature for the production of biosurfactants was 20 ℃ and the tryptone concentration was 0.79% (w (w)). / v).

The biosurfactant production is not affected by NaCl concentration. The lower the incubation temperature and the higher the concentration of tryptone, the higher the biosurfactant production. appear.

Further, the comparison between the predicted values of the regression equation obtained from the reaction surface analysis and the experimental values is shown in Table 13. In the optimal culture conditions for producing the biosurfactant, the NaCl concentration was 0.745% (w / v), the incubation temperature was 19.29 ° C , And the tryptone concentration was estimated to be 0.787% (w / v). There was a slight difference between the experimental values for confirmation, but it could be used for predicting biosurfactant production.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

(A) seeding culture of B. pumilus strain IJ-1 of accession number KCCM11319P in an LB liquid medium; And
(B) To the medium containing 0.3 to 1.5% (w / v) of tryptone and 0 to 2% (w / v) of NaCl, the seed cultured B. pumilus strain IJ- To 5.0% (v / v).
A method for culturing an IJ-1 strain for producing a biosurfactant from a strain of B. pumilus IJ-1. The method according to claim 1,
In the step (A), the medium is cultured at 33 to 37 ° C at 150 to 250 rpm for 14 to 18 hours using a liquid medium having a pH of 6.5 to 7.5,
Wherein the step (B) is performed at a temperature of 33 to 37 DEG C at a temperature of 150 to 250 rpm for 72 to 120 hours. delete The method according to claim 1,
Wherein the culture medium further comprises glycerol as a carbon source in the culture medium, and the glycerol is contained in an amount of 0.1 to 1.5% (v / v) of the total culture medium. delete delete The method according to claim 1,
Wherein the culture medium further comprises KNO ₃ as an inorganic nitrogen source in the culture medium. delete delete The method according to claim 1,
Wherein the culture medium has a pH of 5 to 10 in the step (B). The method according to claim 1,
Wherein the culturing temperature in step (B) is 15 to 40 ° C.

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