阅读说明：本技术 制备生物表面活性剂的方法 (Process for preparing biosurfactants ) 是由 孙璟 吴隽松 于 2019-11-06 设计创作，主要内容包括：本发明属于环保技术领域,涉及一种制备生物表面活性剂的方法。该制备生物表面活性剂的方法包括以下步骤：(1)对铜绿假单胞菌(P.aeruginosa)CGMCC No1.10452进行活化培养,得到种子液；(2)将所述种子液接种于发酵培养基,进行发酵培养,获得发酵液,从所述发酵液中提取所述生物表面活性剂；其中所述发酵培养基包括以下浓度的组分：20～50g/L的甘油；3～10g/L的酵母膏；5～10g/L的磷酸一氢盐；1～5g/L的磷酸二氢盐；0.04～0.1g/L的CaCl<Sub>2</Sub>；0.02～0.1g/L的FeSO<Sub>4</Sub>；以及0.10～0.5g/L的MgSO<Sub>4</Sub>·7H<Sub>2</Sub>O。本发明提供的制备生物表面活性剂的方法,采用膜分离技术分离产物中的生物表面活性剂,相对于酸沉淀和有机溶剂萃取,不会造成环境污染问题,适合工业化推广应用。(The invention belongs to the technical field of environmental protection, and relates to a method for preparing a biosurfactant. The method for preparing the biosurfactant comprises the following steps: (1) activating and culturing pseudomonas aeruginosa (P.aeruginosa) CGMCC No1.10452 to obtain a seed solution; (2) inoculating the seed liquid to a fermentation culture medium, carrying out fermentation culture to obtain a fermentation liquid, and extracting the biosurfactant from the fermentation liquid; wherein the fermentation medium comprises the following components in concentrations: 20-50 g/L of glycerol; 3-10 g/L yeast extract; 5-10 g/L of monohydrogen phosphate; 1-5 g/L of dihydric phosphate; 0.04-0.1 g/L of CaCl 2 (ii) a 0.02-0.1 g/L of FeSO 4 (ii) a And 0.10-0.5 g/L MgSO 4 ·7H 2 And O. According to the method for preparing the biosurfactant, the biosurfactant in the product is separated by adopting a membrane separation technology, and compared with acid precipitation and organic solvent extraction, the method does not cause the problem of environmental pollution, and is suitable for industrial popularization and application.)

1. A method of preparing a biosurfactant comprising the steps of:

(1) activating and culturing pseudomonas aeruginosa (P.aeruginosa) CGMCC No1.10452 to obtain a seed solution;

(2) inoculating the seed liquid to a fermentation culture medium, carrying out fermentation culture to obtain a fermentation liquid, and extracting the biosurfactant from the fermentation liquid;

wherein the fermentation medium comprises the following components in concentrations:

20-50 g/L of glycerol;

3-10 g/L yeast extract;

5-10 g/L of monohydrogen phosphate;

1-5 g/L of dihydric phosphate;

0.04-0.1 g/L of CaCl₂；

0.02-0.1 g/L of FeSO₄(ii) a And

0.10-0.5 g/L MgSO₄·7H₂O。

2. The method according to claim 1, wherein the fermentation culture is carried out in a fermenter having an aeration rate of 1.5 to 2.5L/min and a fermentation temperature of 30 to 37 ℃.

3. The method according to claim 2, characterized in that the liquid content of the fermenter is 40-80% of the volume of the fermenter.

4. The method of claim 1, wherein the step of extracting the biosurfactant from the fermentation broth comprises the steps of:

carrying out suction filtration on the fermentation liquor by using a first membrane separation component (M1) to remove the pseudomonas aeruginosa (P. aeruginosa) in the fermentation liquor to obtain sterile fermentation liquor;

carrying out suction filtration on the sterile fermentation liquor by using a second membrane separation component (M2), and separating a water phase and an oil phase in the sterile fermentation liquor to obtain an oil phase containing the biosurfactant;

preferably, the oil phase containing the biosurfactant is dried to obtain the biosurfactant.

5. The method of claim 4, wherein step (2) is performed in a fermentation system comprising: a fermentor (C1), a first receiving tank (C2), a second receiving tank (C3), the first membrane separation module (M1), the second membrane separation module (M2), a first vacuum suction pump (P1), a second vacuum suction pump (P2), a sampling line (X1), and a separation line (X2);

the fermenter (C1) being in communication with the first receiving tank (C2) through the sampling line (X1);

the first vacuum filtration pump (P1) is disposed on the sampling line (X1);

the first receiving tank (C2) is communicated with the second receiving tank (C3) through the separation line (X2);

the second vacuum-suction pump (P2) is arranged on the separation line (X2);

the first membrane separation module (M1) is located inside the fermentor (C1) and is in communication with the feed end of the sampling line (X1);

the second membrane separation module (M2) is located inside the first receiving tank (C2) and is in communication with the feed end of the separation line (X2);

the step (2) comprises the following steps:

inoculating the seed solution into a fermentation culture medium in the fermentation tank (C1), and performing fermentation culture to obtain a fermentation liquid;

turning on the first vacuum filtration pump (P1), filtering the fermentation broth through the first membrane separation module (M1), wherein the pseudomonas aeruginosa (P. aeruginosa) in the fermentation broth is retained in the fermentation tank (C1), and the rest is introduced into the first receiving tank (C2) through the second membrane separation module (M2) and the sampling line (X1), thereby obtaining the sterile fermentation broth;

and (3) starting the second vacuum filtration pump (P2), filtering the sterile fermentation liquor by the second membrane separation assembly (M2), retaining the water phase in the sterile fermentation liquor in the first receiving tank (C2), and introducing the oil phase into the second receiving tank (C3) through the second membrane separation assembly (M2) and the separation line (X2), so as to obtain the oil phase containing the biosurfactant.

6. The method according to claim 4 or 5, wherein the filtration membrane in the first membrane separation module (M1) is a hydrophilic membrane;

the filtration membrane in the second membrane separation module (M2) is a hydrophobic membrane.

7. The method of claim 6, wherein the hydrophilic membrane is selected from at least one of a cellulose membrane, a polyethersulfone membrane, and a nylon membrane;

the hydrophobic membrane is selected from at least one of a ceramic ultrafiltration membrane, a polypropylene membrane and a polytetrafluoroethylene membrane.

8. The method according to claim 4 or 5, wherein the pore size of the filtration membrane in the first membrane separation module (M1) is 0.1-0.5 μ M;

the molecular weight cut-off of the filter membrane in the second membrane separation component (M2) is 300-1000 Da.

9. The process according to claim 4 or 5, wherein the first membrane separation module (M1) and the second membrane separation module (M2) are both of a mesh and filter membrane top-bottom stack.

10. The method according to claim 5, characterized in that the pressure of the first vacuum filtration pump (P1) is 0.1-1.0 MPa; the pressure of the second vacuum filtration pump (P2) is 0.1-1.0 MPa.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing a biosurfactant.

Background

Biosurfactants (biosurfactants) are amphoteric compounds secreted by microorganisms in the metabolic process, have surface activity and simultaneously contain hydrophilic groups and hydrophobic groups. Biosurfactants include glycolipids, lipopeptides, lipoproteins, phospholipids, and neutral lipid derivatives, among others. Biosurfactants have a number of distinct advantages over chemically synthesized surfactants: (1) low critical micelle concentration and high surface activity; (2) stability to ionic strength; (3) high biodegradability and low toxicity; (4) gradual adsorption and sustained activity; (5) excellent demulsifying performance.

At present, petroleum pollution is a prominent environmental problem faced by countries in the world, and the countries are researching how to apply bioremediation technology to repair petroleum-polluted soil, and the application of biosurfactant is a key factor of bioremediation. In addition, the biosurfactant, such as rhamnolipid, has better application prospect in the fields of heavy metal pollution treatment, biological pesticides and biological fertilizers, skin scar treatment, food preservatives, energy application and the like.

For example, patent publication No. CN102373258A discloses a method for industrially producing a lipopeptide biosurfactant. The preparation method comprises the steps of inoculating a slant solid bacillus subtilis ACCC01430 strain into a shake flask for culture, wherein the temperature is 35 +/-2 ℃, and the shake culture is carried out at 120rpm for 12-16 hours to obtain a shake flask strain; transferring the prepared shake flask strain into a primary fermentation tank filled with a culture medium to prepare a primary fermentation strain; sequentially transferring the mixture into a third-stage fermentation tank for culturing for 24-26 hours to obtain the lipopeptide biosurfactant. The lipopeptide content of the fermentation broth reaches 7-10 g/L, the success of industrial production experiments is achieved, the fermentation period is shortened to 48 hours, and the production cost is greatly reduced. And the oil is applied to the site for the first time and injected into an underground oil layer, so that water in a block which is difficult to inject water is smoothly injected, and the crude oil recovery rate is improved by 5-12%.

Also, for example, patent document No. CN104830708A discloses a crude oil degrading strain and its application. The crude oil degrading strain CGMCC 10287 is directly screened from crude oil, has the capacities of secreting rhamnolipid and degrading crude oil, and can greatly promote the degradation of the crude oil by adding a small amount of glycerol. 11.5g/L and 18.4g/L rhamnolipid can be respectively obtained by using 30g/L of glycerol or rapeseed oil, the strain is cultured in a culture medium of 1g/L of crude oil for 10 days, the degradation rate of the crude oil is 35.6%, 0.5g/L of glycerol is added in the culture medium of the crude oil, the degradation rate of the crude oil is improved to 72.5%, and the defects of the incomplete property of the existing physical method for treating the petroleum and the pollution property of the chemical method for treating the petroleum are overcome.

Further, an industrial fermentation production process for improving the yield of the biosurfactant is disclosed in the invention patent with the publication number of CN 108823250A. The process comprises the following steps: overflow fermentation and pH stage regulation are combined in the culture process of biosurfactant producing bacteria. Compared with the prior art, the biosurfactant is separated by overflow based on the physiological growth characteristics of production strains, so that the inhibition of the product on metabolism is eliminated; by controlling the pH stage by stage, the bacterial growth and metabolic environmental conditions are optimized, thereby improving the production efficiency and the yield of the biosurfactant.

The cost of the existing synthetic biosurfactant is still higher than that of the chemical surfactant, and the biological hazard degree of the pseudomonas aeruginosa is higher, which is also a great problem restricting the production of the pseudomonas aeruginosa. Therefore, the production cost is effectively reduced, and the development of a proper fermentation method by adopting low-cost raw materials is still an urgent problem to be solved in the industry.

Disclosure of Invention

The invention aims to provide a method for preparing a biosurfactant, so as to improve the yield of the biosurfactant.

In order to achieve the above object, the present invention provides a method for preparing a biosurfactant, which comprises the steps of:

(1) activating and culturing pseudomonas aeruginosa (P.aeruginosa) CGMCC No1.10452 to obtain a seed solution;

(2) inoculating the seed liquid to a fermentation culture medium, carrying out fermentation culture to obtain a fermentation liquid, and extracting the biosurfactant from the fermentation liquid;

wherein the fermentation medium comprises the following components in concentrations:

20-50 g/L of glycerol;

3-10 g/L yeast extract;

5-10 g/L of monohydrogen phosphate;

1-5 g/L of dihydric phosphate;

0.04-0.1 g/L of CaCl₂；

0.02-0.1 g/L of FeSO₄(ii) a And

0.10-0.5 g/L MgSO₄·7H₂O。

The strain used by the method for preparing the biosurfactant is pseudomonas aeruginosa (P.aeruginosa), and the preservation number is CGMCC No. 1.10452. The monohydrogen phosphate used in the fermentation medium of the present invention may be sodium monohydrogen phosphate and/or potassium monohydrogen phosphate, and the dihydrogen phosphate may be sodium dihydrogen phosphate and/or potassium dihydrogen phosphate.

In the present invention, the medium used for the activated culture of pseudomonas aeruginosa (p.aeruginosa) may be LB solid medium and LB liquid medium commonly used in the art. The LB solid culture medium has the formula as follows: 5-10 g/L tryptone, 1-5 g/L yeast extract, 5-10 g/L NaCl, 15-20 g/L agar, pH 7.0-7.2, and using deionized water for constant volume. The LB liquid medium is distinguished from the LB solid medium by the absence of agar. The specific operation of the activation culture can be that an LB solid culture medium is prepared into a slant culture medium, pseudomonas aeruginosa (P.aeruginosa) is inoculated on the slant culture medium, and the slant culture medium is cultured in an incubator at the temperature of 35-37 ℃ for 18-24 hours; and then inoculating strains cultured by the slant culture medium into an LB liquid culture medium, wherein the LB liquid culture medium is a seed culture medium, inoculating pseudomonas aeruginosa (P.aeruginosa) to about every 100mL of the seed culture medium, and culturing for 12-16 h under the conditions that the temperature is 35-37 ℃ and the rotating speed is 120-150 rpm.

In a preferred embodiment of the present invention, the fermentation culture is performed in a fermenter, wherein the aeration rate of the fermenter is 1.5 to 2.5L/min, and the fermentation temperature is 30 to 37 ℃. The liquid loading of the fermenter is 40% to 80%, for example 40%, 60%, 70% and 80% of the volume of the fermenter.

In step (2) of the present invention, the step of extracting the biosurfactant from the fermentation broth comprises the steps of:

carrying out suction filtration on the fermentation liquor by using a first membrane separation assembly, and removing the pseudomonas aeruginosa (P.aeruginosa) in the fermentation liquor to obtain sterile fermentation liquor;

and carrying out suction filtration on the sterile fermentation liquor by using a second membrane separation assembly, and separating a water phase and an oil phase in the sterile fermentation liquor to obtain the oil phase containing the biosurfactant.

In a more preferred embodiment of the present invention, the step (2) is performed in a fermentation system comprising: the device comprises a fermentation tank, a first receiving tank, a second receiving tank, a first membrane separation assembly, a second membrane separation assembly, a first vacuum pumping and filtering pump, a second vacuum pumping and filtering pump, a sampling pipeline and a separation pipeline;

the fermentation tank is communicated with the first receiving pool through the sampling pipeline;

the first vacuum filtration pump is arranged on the sampling pipeline;

the first receiving tank is communicated with the second receiving tank through the separation pipeline;

the second vacuum pumping and filtering pump is arranged on the separation pipeline;

the first membrane separation assembly is positioned in the fermentation tank and is communicated with the liquid inlet end of the sampling pipeline;

the second membrane separation assembly is positioned in the first receiving tank and communicated with the liquid inlet end of the separation pipeline.

The fermentation system also comprises a back flushing system, a flushing pipeline, and a first membrane separation component and a second membrane separation component which are repeatedly utilized, so that the cost for producing the biosurfactant is reduced.

When the method for preparing biosurfactant provided by the present invention is performed in the fermentation system as described above, the step (2) comprises the steps of:

inoculating the seed liquid into a fermentation medium in the fermentation tank, and performing fermentation culture to obtain a fermentation liquid;

starting the first vacuum filtration pump, filtering the fermentation liquor through the first membrane separation assembly, retaining the pseudomonas aeruginosa in the fermentation liquor in the fermentation tank, and leading the rest part of the pseudomonas aeruginosa into the first receiving tank through the second membrane separation assembly and the sampling pipeline to obtain the sterile fermentation liquor;

and starting the second vacuum filtration pump, filtering the sterile fermentation liquor through the second membrane separation assembly, retaining a water phase in the sterile fermentation liquor in the first receiving tank, and introducing an oil phase into the second receiving tank through the second membrane separation assembly and the separation pipeline so as to obtain the oil phase containing the biosurfactant.

In one embodiment of the present invention, the first membrane separation module is connected with the sampling pipeline as a whole; the second membrane separation assembly is connected with the separation pipeline into a whole.

In one embodiment of the present invention, the filtration membrane in the first membrane separation module is a hydrophilic membrane;

the filter membrane in the second membrane separation assembly is a hydrophobic membrane.

Specifically, the hydrophilic membrane is selected from at least one of a cellulose membrane, a polyethersulfone membrane, and a nylon membrane.

Specifically, the hydrophobic membrane is selected from at least one of a ceramic ultrafiltration membrane, a polypropylene membrane, and a Polytetrafluoroethylene (PTFE) membrane.

The first membrane separation component is used for separating strains, so the aperture of a filter membrane in the first membrane separation component is 0.1-0.5 mu m.

The second membrane separation component is used for separating an oil phase and a water phase and separating the surfactant from the sterile fermentation liquor, so that the molecular weight cutoff of a filter membrane in the second membrane separation component is 300-1000 Da.

The present invention does not limit the structural form of the first membrane separation module and the second membrane separation module as long as the above-described separation purpose is achieved. In order to improve the separation efficiency and enhance the separation effect, the first membrane separation assembly and the second membrane separation assembly are both of a structure in which a screen and a filter membrane are vertically laminated, 3-8 layers of screens and filter membranes can be vertically laminated, and the screen is arranged at the lowest part to prevent the filter membrane at the lowest part from being broken.

In the method for preparing the biosurfactant, the pressure of the first vacuum suction pump is 0.1-1.0 MPa; the pressure of the second vacuum suction pump is 0.1-1.0 MPa.

The method for preparing the biosurfactant is characterized in that the formula of pseudomonas aeruginosa (P.aeruginosa) CGMCC No. 1.10452 and a fermentation culture medium is screened, and the pseudomonas aeruginosa (P.aeruginosa) is 20-50 g/L of glycerol, 3-10 g/L of yeast extract, 5-10 g/L of monohydrogen phosphate, 1-5 g/L of dihydrogen phosphate and 0.04-0.1 g/L of CaCl₂0.02-0.1 g/L of FeSO₄And 0.10 to 0.5g/L of MgSO₄·7H₂When the biological surfactant is cultured in the fermentation medium of O, the yield of the biological surfactant is obviously increased.

According to the method for preparing the biosurfactant, the biosurfactant in the product is separated by adopting a membrane separation technology, and compared with acid precipitation and organic solvent extraction, the method does not cause the problem of environmental pollution, and is suitable for industrial popularization and application.

The method for preparing the biosurfactant optimizes the fermentation condition, and the biosurfactant is produced and separated by adopting the fermentation system in an integrated manner, so that the production and treatment cost of the biosurfactant is reduced, and the extraction rate of the biosurfactant is improved by about 20 percent compared with that of the conventional method.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings, in which like reference numerals generally represent like parts throughout.

FIG. 1 shows a schematic diagram of a fermentation system used in the method for preparing biosurfactant provided by the invention.

Figure 2 shows a schematic of a standard curve for rhamnose.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.