JP2923756B2 - Method for producing rhamnolipid using ethanol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing rhamnolipid by culturing rhamnolipid-producing bacteria in a medium containing ethanol as a carbon source.

[0002]

2. Description of the Related Art Recently, there are concerns about the effects of petroleum-based substances on ecology and depletion of petroleum energy sources. Therefore, in the field of surfactants, glycolipids, which are biodegradable and renewable surfactants, are attracting attention. Glycolipids are higher in CMC (Critical mice) than chemically synthesized surfactants.
lle concentration), low toxicity, temperature, p
It has excellent properties such as high stability against H and salt concentration.

[0003] Rhamnolipid is a glycolipid composed of rhamnose and β-hydroxydecanoic acid, and it is known that there are four types depending on the combination of sugar and fatty acid. Rhamnolipid produced by fermentation by Pseudomonas aeruginosa has a main component of 3- [3 '-(α
-L-rhamnopyranopyranosyloxy) decanoyloxy] decanoicac
id (R1) and 3- [3 '-(2 "-O-α-L-rhamnopyranosyl- α-L-rh
amnopyranosyloxy) decanolyloxy] decanoic acid (R2)
It is. Rhamnolipid has excellent surface-active ability, and is used as a growth promoter when growing microorganisms based on fats and oils, a viscosity reducing agent for heavy oil, and for cleaning containers contaminated with fats and oils. It is considered for use.

[0004] Rhamnolipid can be produced using a carbon source such as glycerin, glucose and ethanol as a fermentation raw material. However, in order to obtain high productivity, water-insoluble substances such as n-alkanes and fats are essential [Jou
rnal of Scientific & Industrial Research, 53, Augu
tst, p. 619-629 (1984)]. In addition, although inorganic salts such as sodium nitrate are effective as a nitrogen source, a low concentration is required, and the amount of phosphorus, K ⁺ , Ca ⁺⁺ , Mg ⁺⁺ , Fe ⁺⁺⁺ is restricted. It is said that a medium composition is required.

Robert et al. Examined various oils and fats and found that olive oil was the best, and 7.5 g in 60 hours by batch fermentation in a medium containing 2% olive oil as a carbon source.
/ l rhamnolipids [Biotechnology Let
ters 11, 12, p. 871-874 (1989)]. In addition, since rhamnolipid production starts during the arrest period when bacterial growth has ended, Syldatk and colleagues have reported that Pseudodom sp.
onas sp.) and collected with 10% n-tetradecane.
By reacting as a quiescent cell in a medium containing only 0.5% NaCl, a production amount of 15 g / l was obtained in 10 days [Z.
raforsch. 40C, pp. 61-67 (1986)]. It is thought that the production of rhamnolipids contributes to the emulsification and metabolism of water-insoluble substances as a cause of the suitability of water-insoluble substances as a carbon source [Microbiological Reviews, 60, 1, p.151
166 (1996)].

[0006]

The amount of rhamnolipid produced (accumulated concentration in the medium) by these methods is determined by Rhodococcus erythropolis (Rhodococcus erythropolis).
Succinoyl trehalose lipid production (10%
40 g / l from n-alkane culture medium), Candida sp.
dida sp.) to produce mannosylerythritol lipid (35 g / l from soybean oil), tolulopsis bombicola (T
orulopsis bomvicola) to produce glycolipids (glucose 1
0%, sunflower oil 9.5% to 70g / l).

[0007] According to a feasibility study of fermentation production of glycolipids, it is said that fixed costs are high and that plant costs are also high (medical publishing, bioconversion
p.146-149), it has been pointed out that industrialization of glycolipid fermentation production using water-insoluble materials as raw materials is difficult. Disadvantages of using water-insoluble materials as raw materials are: 1) Slow growth and production rate 2) The use of organic solvents that are harmful to disaster prevention and health to extract products must be used 3 ) A process may be required for the purification and separation of the fats and oils present after fermentation and the glycolipid product.

[0008] Instead of fermentative glycolipid production using a water-insoluble substance as a raw material, fermentative glycolipid production using a water-soluble carbon source is also being studied. For example, Lee et al. Have studied the optimal fermentation conditions using a water-soluble carbon source when glucose is used as a carbon source. However, only a low production amount of about 1 to 2 g / l is obtained, and it is not an efficient fermentation system [Lee et al.
of Korean Ind. & Eng. Chemistry, 4, 1, p.41-45
(1993)].

Accordingly, an object of the present invention is to provide a water-soluble carbon source capable of easily separating and purifying products, to provide high production efficiency,
It is an object of the present invention to provide a method capable of producing rhamnolipid by fermentation production at a particularly high accumulated concentration.

[0010]

The present invention provides a method for producing rhamnolipid, comprising culturing rhamnolipid-producing bacteria belonging to the genus Pseudomonas in a medium containing ethanol and collecting rhamnolipid from the culture. The present invention relates to the method, wherein ethanol is added to the medium so that the ethanol concentration in the medium is maintained at 3% or less. Yet another embodiment of the present invention is the method as described above, wherein the medium contains a yeast extract as a nitrogen source, or the medium contains a yeast extract and soybean flour as a nitrogen source.

The present inventors have reconsidered a fermentation system using ethanol, which has not been regarded as a good raw material in the past. More specifically, the present inventors have studied the medium composition such as a method of feeding ethanol and a nitrogen source, and as a result, have found that the above problems can be solved. That is, according to the present invention, 1) a total of 8 vol% (63 g / l) of ethanol produces 25 g / l of rhamnolipid and a conversion of about 40% is obtained; 2) a fermentation process that complicates the purification process Prevents the incorporation of oils and fats such as residues of raw materials. 3) In oil and fat medium, most glycolipids are produced by adhering to bacterial cells, but in ethanol medium, more than 80% of them are in the aqueous layer. It is advantageous in that it can be purified by treating the aqueous layer with a resin, avoiding dangerous organic solvent extraction.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below. In the method of the present invention, a rhamnolipid-producing bacterium belonging to the genus Pseudomonas is used. Pseudomonas (P
Rumnolipid-producing bacteria belonging to the genus seudomonas include, for example, Pseudomonas aeruginosa (Pseudomonas).
as aeruginosa) and Pseudomonas
Elginosa (Pseudomonas aeruginosa) is available as IFO 3924 strain.

The rhamnolipid-producing bacteria are cultured in a medium containing ethanol. At this time, the concentration of ethanol in the medium is maintained at 3% or less. When the ethanol concentration in the medium exceeds 3%, the fermentation ability of the rhamnolipid-producing bacterium decreases, and the concentration of rhamnolipid accumulated in the medium does not increase. In addition, since ethanol serves as a carbon source, it is consumed by the producing bacteria as the culture proceeds. Therefore, during the culture, the ethanol concentration is maintained at 3% or less by adding ethanol intermittently or continuously to the medium. Even if the ethanol concentration is too low, the production rate of rhamnolipid decreases, and therefore, for example, 1% or more is appropriate.

A nutrient source other than the above-mentioned ethanol can be appropriately added to the medium. For example, glucose, glycerin, rapeseed oil and the like can be added as a carbon source. Also,
As a nitrogen source, yeast extract, soybean flour, polypeptone, corn steep liquor (CSL) and the like can be contained. Addition of yeast extract as a nitrogen source to the medium is preferable from the viewpoint of increasing rhamnolipid production ability,
For example, 0.5-1.0% yeast extract can be used.

Further, the addition of soybean powder (Essan Meat, manufactured by Ajinomoto Co., Inc.) in addition to the yeast extract is more preferable in terms of rhamnolipid producing ability, and it is preferable to use 0.5 to 1.0% soybean powder. When two or more nitrogen sources are used, it is appropriate that the total is in the range of 0.4 to 1.2%. If the amount of the nitrogen source is too large, the productivity of rhamnolipid decreases, which is not preferable.

[0016] Culture conditions such as culture temperature and time can be appropriately determined according to the type of rhamnolipid-producing bacteria used. For example, Pseudomonas aeruginosa IFO as the aforementioned rhamnolipid producing bacterium
When using strain 3924, at room temperature (for example, 20-30 ° C),
For example, by culturing for 100 to 250 hours, rhamnolipid is accumulated in the medium.

From the culture in which rhamnolipid has accumulated, rhamnolipid can be collected by a conventional method. For harvesting and purifying rhamnolipids from cultures, for example,
Adsorption chromatography is effective. Further, since rhamnolipid has a carboxyl group, it can be collected and purified using an anion exchange resin.

The rhamnolipid obtained by the method of the present invention varies depending on the type of rhamnolipid-producing bacterium to be used. For example, Pseudodom
onasaeruginosa), 3- [3'- (α-L-rhamnopy
ranopyranosyloxy) decanoyloxy] decanoic acid (R1) and 3- [3 '-(2 "-O-α-L-rhamnopyranosyl-α-L-rhamnopyr
A rhamnolipid containing anosyloxy) decanolyloxy] decanoic acid (R2) as a main component is obtained. These rhamnolipids have excellent surface-active ability, and are used to promote growth of microorganisms and the like based on oils and fats, as well as to reduce the viscosity of heavy oil, and to wash containers contaminated with oils and fats. There are uses such as use.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. Example 1 Flask culture A 250 ml Erlenmeyer flask containing 30 ml of a seed culture medium [glucose 1%, yeast extract (Difco) 0.3%, malt extract (Difco) 0.3%, polypeptone 0.5%, pH 7.0] was prepared. , Pseudomonas aerug
inosa) Inoculated with freeze-dried powder of IFO 3924 strain at 28 ° C, 23
The cells were cultured with shaking at 0 rpm. 30 ml of production medium [ethanol 3v
ol%, yeast extract (Difco) 0.5%, soybean flour (Essan Meat, Ajinomoto Co.) 0.5%, KH ₂ PO ₄ 0.05%, MgS
_{O 4 · 7H 2 O 0.05%} , prepared 250ml Erlenmeyer flask containing pH 7.0], was added seed culture broth 2%, 28 ℃, and cultured with shaking at 230 rpm. After 24, 48, 72 and 168 hours ethanol
1 vol% was added.

Sampling was carried out over time, and the amount of rhamnolipid produced was quantified by the following TLC method. 200 μl culture solution
Was vigorously stirred with 1 ml of a 1: 1 solution of chloroform: methanol. The mixture was separated into two layers by centrifugation, and the organic solvent layer was spotted on silica gel TLC (Merck 5715, manufactured by Merck) at 5 μl. 1, 3, and 5 μg of glucose were used as quantitative standard substances.
Chloroform: methanol: water = 65: 25: 4
Orcinol sulfuric acid solution was sprayed and dried at 110 ° C. for 10 minutes. The amount of reflected light was measured at 550 nm using a TLC scanner (CS-9000, manufactured by Shimadzu Corporation), and the amount of rhamnolipid was measured using glucose as a standard. In another test, the ratio of the amount of glucose and the actual amount of rhamnolipid was determined to be 4.65, and the coefficient was multiplied to obtain the amount of rhamnolipid. As a result, production of rhamnolipids began in earnest around 50 hours, and as shown in Fig. 1, R1
(3- [3 '-(α-L-rhamnopyranopyranosyloxy) decanoylox
y] decanoic acid] and R2 [3- [3 '-(2 "-O-α-L-rhamnopyr
anosyl- α-L-rhamnopyranosyloxy) decanolyloxy] decan
oic acid], showed a production amount of 25 g / l in 7 days.

Example 2 Mini-jar culture 250 ml Erlenmeyer flask containing 30 ml of seed culture medium [glucose 1%, yeast extract (Difco) 0.3%, malt extract (Difco) 0.3%, polypeptone 0.5%, pH 7.0] And prepare Pseudomonas aerug
inosa) Inoculated with freeze-dried powder of IFO 3924 strain at 28 ° C, 23
The cells were cultured with shaking at 0 rpm. 1.5 l production medium [ethanol 3v
ol%, yeast extract (Difco) 0.5%, soybean flour (Essan Meat, Ajinomoto Co.) 0.5%, KH ₂ PO ₄ 0.05%,
31 mini jars containing 0.05% of MgSO ₄ .7H ₂ O, pH 7.0] were prepared, 2% of a seed culture was added, and the mixture was stirred and cultured at 28 ° C. at 500 rpm with aeration of 1 vvm. After 24, 48, 72, and 168 hours, ethanol was added by 1 vol%. Sampling was performed over time, and the amount of rhamnolipid produced was quantified by the TLC method. As a result, the production of rhamnolipids began in earnest from 50 hours, and as shown in Fig. 2, combined R1 and R2, 25g / g in 7 days
l Production volume is shown.

Example 3 Determination of Rhamnolipid in Culture Supernatant The culture obtained in Example 2 was centrifuged, and the amount of rhamnolipid contained in the supernatant was measured. The amount obtained by extracting chloroform and methanol from the culture was obtained. More than 80% were included. As a control, when rhamnolipid production was performed using rapeseed oil as a raw material, the results were all 10% or less.

Example 4 Purification of rhamnolipid by adsorption chromatography The culture solution of Example 2 was centrifuged to prepare 1 liter of a supernatant containing 20 g / l of rhamnolipid, and purification of rhamnolipid by adsorption chromatography was examined. The column was adsorbed on a 100 ml column of octyl sepharose or ODS resin, and eluted with 60% methanol for the former and 80% methanol for the latter. Analysis of the glycolipid fraction by liquid chromatography revealed that all samples had a relative purity of 95%.
8.8 g were obtained. Analytical conditions are columns; Shodex GS-310 7E 7.
6mm x 250mm, mobile phase; 30% methanol, detection: 203nm
.

Example 5 Purification of Rhamnolipid by Anion Exchange Chromatography One liter of the same supernatant containing 20 g / l of rhamnolipid was prepared, and purification of rhamnolipid by anion exchange chromatography was examined. The column was adsorbed on a column of 100 ml of DEAE Sepharose CL 6B resin and eluted with 100 mM NaCl, 10% ethanol. When the glycolipid fraction was analyzed by liquid chromatography, 15.3 g of a sample having a relative purity of 93% was obtained.
I got

Example 6 Production of Rhamnolipid by Alcohol Feed 30 ml of a seed culture medium [glucose 1%, yeast extract (Difco) 0.3%, malt extract (Difco) 0.3%, polypeptone 0.5%, pH 7.0] Prepare a 250 ml Erlenmeyer flask containing Pseudomonas aeruginosa
inosa) Inoculated with freeze-dried powder of IFO 3924 strain at 28 ° C, 23
The cells were cultured with shaking at 0 rpm. 30 ml of production medium [ethanol 3v
ol%, yeast extract (Difco) 0.5%, soybean flour (Essan Meat, Ajinomoto Co.) 0.5%, KH ₂ PO ₄ 0.05%, MgS
_{O 4 · 7H 2 O 0.05%} , prepared 250ml Erlenmeyer flask containing pH 7.0], was added seed culture broth 1%, 28 ℃, and cultured with shaking at 230 rpm.

According to the schedule shown in Table 1 below, ethanol having the concentration shown in Table 2 was added, and samples were taken over time, and the quantitative determination of rhamnolipid was carried out by TLC.
Ethanol was quantified by HPLC. The results (without addition of calcium carbonate and addition of calcium carbonate) are shown in Table 3.

[0027]

[Table 1] :: addition of alcohol is indicated. For 1 / 2vol, add half of each concentration.

[0028]

[Table 2] Flask No. 1 to 10: Calcium carbonate not added Flask No. 11 to 20: Calcium carbonate added

[0029]

[Table 3]

Example 7 Examination of Nitrogen Source 250 ml containing 30 ml of seed culture medium [glucose 1%, yeast extract (Difco) 0.3%, malt extract (Difco) 0.3%, polypeptone 0.5%, pH 7.0] Prepare an Erlenmeyer flask and place Pseudomonas aerug
inosa) Inoculated with freeze-dried powder of IFO 3924 strain at 28 ° C, 23
The cells were cultured with shaking at 0 rpm. 0.2% KH ₂ PO ₄ , 0.05% MgSO ₄・ 7H
Soybean flour (Essan Meat, Ajinomoto Co., Inc.) as a nitrogen source in 30 ml of production medium containing ₂ O, 0.2% CaCO ₃ , 3% alcohol
Corn steep liquor (CSL) and polypeptone at various concentrations shown in Table 4. Seed culture was added at 1%, and cultured with shaking at 230 rpm at 28 ° C. Sampling was performed over time, and the amount of rhamnolipid was determined by the TLC method.
Table 5 shows the results.

[0031]

[Table 4]

[0032]

[Table 5]

[Brief description of the drawings]

FIG. 1 shows the time course of the production of rhamnolipids R1, R2, R1 + R2 in Example 1 (flask culture).

FIG. 2 shows the time course of the production of rhamnolipids R1, R2, R1 + R2 in Example 2 (mini-jar culture).

Claims (2)

(57) [Claims] 1. Pseudomonas
s) A method for producing rhamnolipid by culturing rhamnolipid-producing bacteria belonging to the genus in a medium containing ethanol and collecting rhamnolipid from the culture, wherein the ethanol concentration in the medium is maintained at 3% or less. Ethanol is added to the medium, and the medium is used as a nitrogen source.
The above method, comprising a yeast extract and soy flour . 2. The method according to claim 1, wherein the ethanol concentration is maintained at 1 to 3% by intermittently or continuously adding ethanol to the medium.