CN1005005B

CN1005005B - Preparation method of biosurfactant

Info

Publication number: CN1005005B
Application number: CN85104026.8A
Authority: CN
Inventors: 李祖义
Original assignee: Shanghai Institute of Organic Chemistry of CAS
Current assignee: Shanghai Institute of Organic Chemistry of CAS
Priority date: 1985-05-21
Filing date: 1985-05-21
Publication date: 1989-08-16
Also published as: CN85104026A

Abstract

A method for preparing biosurfactant comprises culturing Arthrobacter under an optimal culture condition, regulating metabolism, and controlling carbon-nitrogen ratio to obtain high-yield algal glycolipid; in addition, the resting cells of Arthrobacter can be used to produce trehalose glycolipid with low cost and convenience, and the main component of the trehalose glycolipid is trehalose tetraester. The product of the present invention may be used in tertiary petroleum exploitation, eliminating petroleum pollution, daily chemical industry, textile industry, food industry, etc.

Description

Process for preparing biosurfactant

The invention relates to a bioengineering.

The use of microorganisms for the production of surfactants is a new subject developed in the field of international bioengineering in the late seventies. The microorganism often secretes some biochemical products in the metabolic process, the molecular structure of the microorganism has two components of hydrophilicity and hydrophobicity, such as glycolipids, phospholipids, lipoproteins and the like, and the biosurfactant has the functions of emulsification, demulsification, wetting, solubilization, foaming, defoaming, static resistance, corrosion resistance and the like, so the application range is very wide, and the biological surfactant can be applied to tertiary petroleum exploitation, and can eliminate petroleum pollution, daily chemicals, food, textile industry and the like.

According to the present study, glycolipids are the most dominant type of biosurfactants, japanese flower Wang Danjian Co., ltd. Torulopsis SF (Japanese patent publication: sho 57-92785,92786), pseudomonas sp.S.ltoh laboratory (Pseudomonas sup.) to produce rhamnolipid (J.Anti biotics23,855,1971) and Rhodococcus sp.weskii (Rhodococcus erythropolis) to produce trehalose monolipid and trehalose diglyceride (Ger Pat DE-2646505, DE-2646506, DE-2646507), in which trehalose glycolipids have good surface activity and are useful in petroleum exploitation and the like, but the method for producing trehalose lipid compounds using arthrobacter growth cells has been reported in DE-3248167, but the method is complicated in culture, cumbersome in method, and has a long period, low yield and high cost. However, the glycolipid yields in the above patents are only 5-10 g/l.

In order to achieve the industrial production level of glycolipids, the culture method must be improved, the yield is increased, and the cost is reduced.

The invention relates to a method for producing trehalose lipid surfactant by microorganism, in particular to a method for producing trehalose monolipid and trehalose tetralipid by using Arthrobacter simplex (Arthrobacter simplex), which comprises the steps of preparing trehalose lipid compound by using Arthrobacter under the optimal culture condition for metabolic regulation or preparing the trehalose lipid compound by using Arthrobacter resting cells.

The method for preparing trehalose lipid compounds by using the Arthrobacter of the invention under the culture condition comprises the steps of adding acid or alkali into a culture in the presence of Arthrobacter, adjusting pH to 4-8, and introducing air or oxygen-enriched air into the culture at a volume ratio of 0.5-1.5 per minute, wherein the culture temperature is 20-40 ℃, and optimally 25-30 ℃. Ammonium salt or nitrate is contained in the culture solution as nitrogen source, and other inorganic salts and growth factors, n-alkane, distillate oil, crude oil, vegetable oil and saccharide which are necessary for propagating cells can be used as carbon source, and after fermentation, the fermentation solution is extracted by using 1-3.5 times of nitrogen imitation or dichloromethane to methanol solvent with the ratio of 1.5-3:1 (V/V) or ethyl acetate.

In the above method, the best culture condition of the invention for producing glycolipid by using arthrobacter is that corn steep liquor, denier peptone, beef extract and yeast extract can be used as organic nutrient sources, but the best use of yeast extract is that the dosage of the yeast extract is controlled to be 0.1-0.5%. In addition, the strain has special requirements on trace elements, namely Zn ⁺⁺、Mn⁺⁺、Ca⁺⁺ elements are controlled to be a certain amount, the culture solution contains 0.001-0.005 g/L ZnSO ₄·7H₂ O, and 0.01-0.05 g/L MnSO ₄·2H₂ O. CaCl ₂·2H₂ O is 0.01-0.05 g/L. The protein synthesis in the thalli is controlled to be in a certain quantity, fat metabolism is mainly carried out in vivo, carbon and nitrogen sources with a certain proportion are controlled in the culture solution, the nitrogen sources cannot be too high, the proportion of the carbon sources to the nitrogen sources is controlled to be 30-40:1, the pH value is maintained to be 4-8 in the fermentation process, ammonia water is not used, and KOH or NaOH is used for regulating.

The invention relates to a method for producing surfactant, which comprises culturing the resting cells of Arthrobacter, then producing trehalose lipid compound directly by using the resting cells, wherein the method comprises the steps of finding out which cheap carbon source can be used to produce thalli, the Arthrobacter can be longer than glucose, sucrose, mannose, fructose, waste molasses, glycerol, acetate and other carbon sources, wherein the waste molasses and glucose are more suitable carbon sources, the yield of thalli can reach 10 g/L, suspending the resting cells in carbonic acid buffer solution, physiological saline, distilled water or tap water to produce glycolipid, and using phosphoric acid buffer solution as culture solution to produce glycolipid at most, the concentration of phosphoric acid buffer solution can be 0.1-0.5M, the culture temperature of pH is 20-40 ℃, the optimal temperature is 25-30 ℃, and the glycolipid produced by the resting cells of Arthrobacter can use various saccharides, normal paraffins, distillate oil or vegetable oil as carbon sources, wherein the normal paraffin conversion rate can reach 0.4-0.6.

The method of the present invention is used to produce glycolipid compound, which is trehalose tetralipid and trehalose monolipid, with the ratio between two glycolipids being changed along with metabolic regulation, and the product is mainly trehalose monolipid when the nitrogen source is not limited, and is mainly trehalose glycolipid when the nitrogen source is limited, and the product is mainly trehalose tetralipid when the resting cell is cultured. The trehalose lipid produced by the method can be used in petroleum tertiary recovery and industries such as daily chemicals, textiles, food and the like.

Trehalose tetralipid:

Trehalose monolipid:

The metabolism regulating method of the present invention can make the yield of trehalose lipid surfactant reach 25 g/L, and the conversion rate of carbon source matrix can be doubled, and the method of producing surfactant by using resting cells is a cheap, simple and convenient method for producing surfactant.

The process according to the invention can be illustrated by the following examples:

Example 1

A5L self-controlled bioreactor is filled with 2.0L of nutrient salt solution, the ingredients are Na ₂HPO₄·2H₂ O1 g, mgSO ₄·7H₂ O0.5 g, feSO ₄·7H₂ O0.1 g, znSO ₄·7H₂ O0.005 g, KH ₂PO₄ g, (NH ₄）₂SO₄ 2.5.5 g, mnSO ₄·H₂ O0.01 g, caCl ₂·2H₂ O0.05 g, yeast extract 1g, dissolved in 1L water, added with 200ml of N-alkane, at 30 ℃, and Arthrobacter simplex (Arthrobacter simplex) (the seed culture process is to be carried out by inoculating a platinum ear to a triangular flask containing 250 ml of the culture solution from an inclined plane, the thallus concentration reaches 1-5 g dry weight/L), the rotating speed is 500 r/min, 2.5L/min is ventilated, 1N NaOH is automatically added in the culture process to maintain the pH value at 6.50, the usual fermentation time is 30-96 hours, the fermentation is carried out for 72 hours, the surfactant-containing culture solution is 2L, and the fat is extracted by using chloroform to obtain fat and fat column chromatography by using a chloroform solution (V: 2: 96 g).

Production of glycolipids by resting cells

1. Resting cell culture the arthrobacter inoculation culture method can employ the seed culture process as described in example one. Culturing Arthrobacter with 1 liter culture medium (5 liter triangular flask) of Na ₂HPO₄·2H₂ O1 g/liter, KH ₂PO₄ g/liter, feSO ₄·7H₂ O0.25 g/liter, znSO ₄·7H₂ O0.01 g/liter, KH ₂PO₄ 1 g/liter, mnSO ₄·H₂ O0.01 g/liter, caCl ₂·2H₂ O0.05 g/liter, (NH ₄）SO₄ 2.5.5 g/liter, corn steep liquor 0.5 g, glucose 30 g/liter, pH6.8,30 ℃ and shaking culture for 4 days, centrifuging the cells, washing twice with 0.9% NaCl, shaking in 0.1M phosphate buffer for 4 hours, centrifuging to obtain resting cells.

2. The resting cells are used for producing glycolipid in a fermentation tank, namely a 5 liter self-control fermentation tank is filled with 2.5 liters of 0.1M phosphate buffer solution (pH 6.5), 25 g of resting cells are inoculated, 200ml of normal alkane is added, the rotating speed is 500 rpm, the ventilation is 2.5 liters/minute, the culture is carried out for 94 hours, and finally, the extraction is carried out by using the volume ratio of the diazomethane to the methanol (2:1) which is twice, and 122 g of crude lipid is obtained after the evaporation.

3. Or placing the resting cells described in this example 1 in an underground oil layer at 15-40deg.C, introducing air, and controlling pH at 4-9. Under these conditions, a large amount of surfactant trehalose glycolipid is produced in the subsurface reservoir.

Claims

1. A method for preparing a surfactant trehalose lipid compound by metabolic regulation using arthrobacter (Arthrobacter simplex), characterized in that the method comprises the steps of:

A. Culturing Arthrobacter in a culture medium containing 0.1-0.5% corn steep liquor, peptone, beef extract or yeast extract, 5-15% carbon source of normal alkane, distillate oil, crude oil, vegetable oil or saccharide, ammonium salt or nitrate as nitrogen source, 0.001-0.005 g/L ZnSO ₄·7H₂ O, 0.01-0.05 g/L MnSO ₄·2H₂ O, 0.01-0.05 g/L CaCl ₂·2H₂ O, and Mg ⁺⁺,Fe⁺⁺,Na⁺,K⁺ ion, wherein the balance is water, controlling the carbon nitrogen ratio of the culture medium to 30-40:1, adjusting pH to 4-8 with acid or alkali, culturing at 20-40 ℃, introducing 0.5-1.5 (volume ratio) of air or enriched air, adjusting pH to 4-8 with KOH or NaOH during fermentation, fermenting for 30-96 hours,

B. extracting the culture solution with 1-3.5 times of methanol/chloroform or dichloromethane (volume ratio) of 1.5-3:1 solvent or ethyl acetate, evaporating the crude extract or subjecting to silica gel column chromatography to obtain desired sugar ester.

2. A method for preparing a surfactant trehalose lipid compound by a resting cell method using Arthrobacter (Arthrobacter simplex), characterized in that the method comprises the following steps:

A. Culturing Arthrobacter in a culture medium containing 0.1-0.5% corn steep liquor, peptone, beef extract or yeast extract, 5-15% carbon source of normal alkane, distillate oil, crude oil, vegetable oil or saccharide, ammonium salt or nitrate as nitrogen source, 0.001-0.005 g/L ZnSO ₄·7H₂ O, 0.01-0.05 g/L MnSO ₄·2H₂ O, 0.01-0.05 g/L CaCl ₂·2H₂ O, and Mg ⁺⁺,Fe⁺⁺,Na⁺,K⁺ ion, wherein the balance is water, the carbon nitrogen ratio of the culture medium is controlled to 30-40:1, pH is adjusted to 4-8 with acid or alkali, culturing is performed at 20-40 ℃, and 0.5-1.5 (volume ratio) of air or enriched air is introduced, culturing the Arthrobacter under culture conditions,

B. Arthrobacter as resting cells is suspended in phosphate buffer solution, normal saline, distilled water or tap water, saccharides, normal alkane, distillate oil, vegetable oil or acetate are used as carbon source, and culturing is carried out at 20-40 ℃, wherein the concentration of the phosphate solution is 0.1-0.5M, and the pH is 4-7.

C. Extracting with 1-3.5 times of methanol, chloroform or dichloromethane at 1.5-3:1 or ethyl acetate, evaporating the crude extract or subjecting to silica gel column chromatography to obtain desired glycolipid.

3. The method for preparing a surfactant trehalose lipid compound by using Arthrobacter as claimed in claim 2, wherein the method comprises placing Arthrobacter taken out under the culture conditions described in A as resting cells in an underground oil layer, introducing air at 15-40deg.C, and controlling pH at 4-8.

4. A process for the preparation of surfactant trehalose lipidic compounds using arthrobacter as claimed in claim 1,2 or 3 wherein said cultivation temperature is preferably 25-30 ℃.

5. A method for preparing a surfactant trehalose lipid compound as claimed in claim 1,2 or 3 wherein said organic nutrient source is yeast extract.

6. A method for preparing a surfactant trehalose lipid compound by resting cell method as claimed in claim 2 wherein said carbon source is molasses or glucose.