CN102373258B - Industrialized preparation method of lipopeptide biosurfactant - Google Patents
Industrialized preparation method of lipopeptide biosurfactant Download PDFInfo
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Abstract
The invention provides an industrialized preparation method of a lipopeptide biosurfactant and relates to a production method of a biosurfactant which is prepared from bacillus subtilis ACCC01430 through fermentation. The method comprises the following steps: slope solid bacillus subtilis strains are joined into a shaking bottle for cultivation at the temperature of 35+/-2 DEG C, and are cultured by shaking at 120 revolutions per minute for 12-16 hours so as to obtain shaking bottle strains; the prepared shaking bottle strains are joined into a primary fermentation tank provided with a culture medium to obtain a primary fermentation strain; the primary fermentation strain is joined into a secondary fermentation tank provided with a culture medium to prepare a secondary fermentation strain; the secondary fermentation strain is joined into a third-level fermentation tank provided with a culture medium to be fermented for 24-26 hours, and then the lipopeptide biosurfactant is obtained. In the industrialized preparation method of the lipopeptide biosurfactant disclosed by the invention, the content of lipopeptid achieves 7-10%g/L, an industrialized production experiment is successful, the fermentation cycle is shortened to 48 hours, and the production cost is greatly reduced. According to the invention, industrially produced lipopenic fermentation liquor is applied on site firstly, and is injected into an underground oil layer, so water is smoothly injected into the original blocks in which water is hard to be injected, the recovery ratio of crude oil is increased by 5-12%, and the efficiency is high.
Description
Technical field
The present invention relates to a kind of production method of bio-surfactant, especially relate to a kind of industrialized process for preparing of lipopeptide biosurfactant.
Background technology
Lipopeptid has another name called lipopeptide, belongs to and contains the amino acid lipoid.Lipopeptide Biosurfactants is generally the metabolite of gram-positive genus bacillus.The lipopeptid molecule is comprised of fat hydrocarbon chain two portions of hydrophilic peptide bond and oleophylic, due to its special chemical constitution and amphiphilic molecules structure, Lipopeptide Biosurfactants has important application prospect in fields such as medicine, food, makeup and microbe oil productions.Lipopeptid class tensio-active agent is generally produced by gram-positive genus bacillus metabolism, the bacterial strain that can produce lipopeptid class tensio-active agent has a lot, as subtilis (Bacillussu btilis), Bacillus licheniformis (Bacilluslic heniformis) etc.Except bacterium can produce lipopeptid class tensio-active agent, yeast, fungi also can produce.
Lipopeptide Biosurfactants can be divided into ring-type lipopeptid and the large class of linear lipopeptid two by its textural classification.
The ring-type lipopeptid.The ring-type lipopeptid refers in molecule the class lipopeptid with ring texture, and the amino in the carboxyl of the C-terminal amino acid of peptide chain and peptide or lipid acid or hydroxyl are connected and form ring texture.According to the difference of the composition that forms ring, the ring-type lipopeptid can be divided into again to the ring-type lipopeptid of ring-type lipopeptid, lipid acid T-Ring of lipid acid Cheng Huan and the lipid acid ring-type lipopeptid from ring.Contain the amino acid lipoid and be and take the bio-surfactant of low-shrinkage amino acid as hydrophilic radical.Typical product has lipopeptid, lipoprotein, lipoamino acid.This is the good tensio-active agent of a class surface property, and its emulsifying property is good, and soil removability is strong, also fine with the consistency of other various tensio-active agents, has simultaneously good anti-microbial property.
Many biomolecules have hydrophilic and lipophilic group, and they are closely similar with the tensio-active agent of chemosynthesis on performance.Usually those are had amphipathicly, show the natural surface active agent that high surface produces by microorganism, animals and plants or plant very much and be called bio-surfactant (Biosurfactant is called for short BS).Wherein the bio-surfactant activity by microorganisms is higher, has well hydrophilic, oleophylic performance and interface priority allocation ability, comparatively is suitable for large-scale industrialization production.But existing tensio-active agent, owing to yielding poorly, defects such as the cycle is long, coefficient is low, complex process, can scale operation also few.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, a kind of industrialized process for preparing of lipopeptide biosurfactant is provided, this production technique has that output is high, efficiency is high, coefficient is high, fermentation period is short, technique is simple, the product comprehensive cost is reduced, be applicable to suitability for industrialized production fully, popularization and the large-scale application of lipopeptide biosurfactant played an important role.
The industrialized process for preparing of a kind of lipopeptide biosurfactant of the present invention, be to be made through fermentation culture by subtilis ACCC01430, and its concrete steps are as follows:
A, by inclined-plane solid subtilis (ACCC01430) bacterial classification access shake-flask culture, every 500ml substratum access 1 ring bacterial classification in shaking flask, temperature is that 35 ± 2 ℃, 120 rev/mins concussions were cultivated 12～16 hours, obtains the shaking flask bacterial classification;
B, one grade fermemtation are cultivated: the shaking flask strain transfer that the A step is made enters to be equipped with in the one grade fermemtation tank of substratum, the volume ratio of shaking flask bacterial classification and substratum is 1:19～20, in temperature, be that 35 ± 2 ℃, pH6～7.5, ventilating ratio 1:0.3～0.5 and stirring velocity are that the condition bottom fermentation of 100～150 rev/mins was cultivated 10～14 hours, obtain the one grade fermemtation bacterial classification;
C, second order fermentation are cultivated: the one grade fermemtation strain transfer that the B step is made enters to be equipped with in the second order fermentation tank of substratum, the volume ratio of one grade fermemtation bacterial classification and substratum is 1:10, in temperature, be that 35 ± 2 ℃, pH 6～7.5, ventilating ratio 1:0.3～0.5 and stirring velocity are that the condition bottom fermentation of 100～110 rev/mins was cultivated 10～14 hours, obtain the second order fermentation bacterial classification;
D, fermentation culture: the second order fermentation strain transfer that the C step is made enters to be equipped with in the three grade fermemtation tank of substratum, the volume ratio of second order fermentation bacterial classification and substratum is 1:10, in temperature, be that 35 ± 2 ℃, pH 6～7.5, ventilating ratio 1:0.3～0.5, stirring velocity are 80～100 rev/mins, at thalline, be in the decline phase and carry out feed supplement, fermentation culture 24～26 hours, obtain lipopeptide biosurfactant.
As a further improvement on the present invention, shaking flask and fermentor cultivation based formulas at different levels are: Rice pollard oil 2～5%, urea 0.2～1.5%, molasses 10～15%, KCl 0.05～1.0%, KH
2PO
40.05～1.2%, K
2HPO
40.05～1.5%, yeast extract paste 0.005～0.1%, composite trace element 0.005～0.01%, surplus is water, and pH is 6.0-7.5;
Described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms.
As a further improvement on the present invention, described feed supplement is Rice pollard oil, and each feed supplement amount is added by 3%～5% of the overall accumulated amount of fermented liquid.
As a further improvement on the present invention, the coefficient of fermentor tanks at different levels is 60～80%.
A kind of industrialized process for preparing of lipopeptide biosurfactant, whole manufacture process are the processes that enlarges step by step the fermentation pure culture.The lipopeptide biosurfactant fermented liquid obtained, after sterilization and centrifugation, becomes the product of different grades according to the purifying that needs of various uses, is widely used in the industries such as tertiary oil recovery, daily use chemicals, agricultural chemicals, food, soil remediation.
The industrialized process for preparing of a kind of lipopeptide biosurfactant of the present invention, have the characteristics such as output is high, the cycle is short, coefficient is high, technique is simple, realized the suitability for industrialized production of lipopeptide biosurfactant.
The industrialized process for preparing of a kind of lipopeptide biosurfactant of the present invention, its lipopeptid content reaches 7～10%g/L, and has obtained the success of suitability for industrialized production experiment, and fermentation period shortens to 48 hours, greatly reduces production cost; The present invention by the lipopeptid fermented liquid site of deployment of suitability for industrialized production, is injected into oilbearing stratum first, and the block water that makes originally to be difficult to water filling injects smoothly, and makes oil recovery factor improve 5～12%; Efficiency is high.
The accompanying drawing explanation
Fig. 1 is surface tension;
The interfacial tension value of the biological lipopeptid tensio-active agent of Fig. 2 when different concns.
Embodiment
The invention will be further described below in conjunction with embodiment:
The title of the bacterium that the present invention adopts and bacterial classification source are as follows:
Subtilis (ACCC01430), Chinese agriculture microbial strains preservation center.
The standard of the decline phase of thalline in the embodiment of the present invention 1～4, be shown in that Higher Education Publishing House's in May, 2002 second edition " microbiology study course " Zhou Deqing writes.In the following embodiments, after entering three grade fermemtation, per hour detect once.
Shaking flask and fermentor cultivation based formulas are: Rice pollard oil 2%, urea 1%, molasses 11%, KCl 0.1%, KH2PO4 0.05%, K2HPO4 0.3%, yeast extract paste 0.005%, composite trace element 0.008%, all the other are water, pH is 7.0, and described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms.
Shaking flask and fermentor cultivation:
Inclined-plane subtilis (ACCC01430) is linked in 7 3L shaking flasks, pack into the substratum of 1L of shaking flask, each shaking flask access 2 ring bacterial classification, temperature is that 35 ± 2 ℃, 120 rpm are cultivated 14 h, blood counting chamber detects the bacterium number and reaches more than 200,000,000, is and obtains the shaking flask bacterial classification, transfer into the one grade fermemtation tank of 200 liters, coefficient is 70% again, and inoculum size 5%, temperature are that 35 ± 2 ℃, ventilating ratio are that 1:0.5, pH are 7.0, mixing speed is that 110 rpm cultivate 12 h, obtain the one grade fermemtation bacterial classification, transfer again into 2 tons of second order fermentation tanks, coefficient is 70%, inoculum size 10%, temperature is 35 ± 2 ℃, ventilating ratio is 1:0.5, pH is 7.0, mixing speed is that 100 rpm cultivate 12 h, obtain the second order fermentation bacterial classification, transfer again into 20 tons of three grade fermemtation tanks, charge amount is 14 tons, the processing parameter of three grade fermemtation tank is: 35 ± 2 ℃ of temperature, 0.1 ton of solid NaOH of sterilizing front use is transferred to 7.0 by pH, ventilating ratio is 1:0.5, mixing speed is 80 rpm, totally add during this time 0.5 ton of Rice pollard oil, it is 7.5g/L that fermentation culture detected lipopeptid content by the anti-pushing manipulation of micelle-forming concentration after 24 hours.
Embodiment 2
Shaking flask and fermentor cultivation based formulas are: Rice pollard oil 3%, urea 1.5%, molasses 17%, KCl 1.0%, KH
2PO
40.2%, K
2HPO
41.5%, yeast extract paste 0.02%, composite trace element 0.01%, all the other are water, pH is 7.0, and described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms.
Shaking flask and fermentor cultivation:
By inclined-plane solid subtilis (ACCC01430) bacterial classification, to transfer into 4 3L shaking flasks (the 1L substratum of respectively packing into), every 500ml substratum access 1 ring bacterial classification, be 35 ± 2 ℃ in temperature, 120 rpm cultivate 13.5 h, after blood counting chamber detects the bacterium number and reaches more than 200,000,000, transfer into the one grade fermemtation tank of 200 liters, and coefficient is 67%, inoculum size 3%, temperature is 35 ± 2 ℃, ventilating ratio is 1:0.4, pH is 7.0, mixing speed is that 120 rpm cultivate 12 h, transfers into 2 tons of second order fermentation tanks, and coefficient is 67%, inoculum size 10%, temperature is 35 ± 2 ℃, ventilating ratio is 1:0.4, pH is 7.0, mixing speed is that 100 rpm cultivate 12 h, transfers into 20 tons of three grade fermemtation tanks, and charge amount is 14 tons, and the processing parameter of three grade fermemtation tank is: 35 ± 2 ℃ of temperature, 0.10 ton of NaOH of sterilizing front use is controlled at 7.0 by pH, and ventilating ratio is 1:0.4, mixing speed is 80 rpm, during accumulative total add 0.6 ton of Rice pollard oil, it is 8.8g/L that fermentation culture detected lipopeptid content by the anti-pushing manipulation of micelle-forming concentration after 25 hours.
Embodiment 3
Shaking flask and fermentor cultivation based formulas are: Rice pollard oil 5%, urea 0.2%, molasses 20%, KCl 0.05%, KH
2PO
41.2%, K
2HPO
40.05%, yeast extract paste 0.1%, composite trace element 0.005%, all the other are water, pH is 7.5, and described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms.
Shaking flask and fermentor cultivation:
By inclined-plane solid subtilis (ACCC01430) bacterial classification, transfer into 6 3L shaking flasks (the 1L substratum of packing into), 35 ± 2 ℃, 120 rpm cultivate 12h, blood counting chamber detects the bacterium number and reaches more than 200,000,000, transfer into the one grade fermemtation tank of 200 liters, coefficient is 60%, inoculum size 5%, temperature is 35 ± 2 ℃, ventilating ratio is 1:1, pH is 7.5, mixing speed is that 120rpm cultivates 12 h, transfer into 2 tons of second order fermentation tanks, coefficient is 60%, inoculum size 10%, temperature is 35 ± 2 ℃, ventilating ratio is 1:1, pH is 7.5, mixing speed is that 100rpm cultivates 12 h, transfer into 20 tons of three grade fermemtation tanks, charge amount is 16 tons, the processing parameter of three grade fermemtation tank is: 35 ± 2 ℃ of temperature, sterilizing front pH is transferred to 7.5 left and right, later stage is controlled at 7.0 with 0.12 ton of NaOH by pH, ventilating ratio is 1:1, mixing speed is 100 rpm, totally add during this time 0.7 ton of Rice pollard oil, it is 10.4g/L that fermentation culture detected lipopeptid content by the anti-pushing manipulation of micelle-forming concentration after 24 hours.
Embodiment 4
Shaking flask and fermentor cultivation based formulas are: Rice pollard oil 3%, urea 1%, molasses 20%, KCl 0.5%, KH
2PO
40.5%, K
2HPO
40.5%, yeast extract paste 0.05% and composite trace element 0.005%, all the other are water, pH is 6.5, and described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms.
Shaking flask and fermentor cultivation:
By inclined-plane solid subtilis (ACCC01430) bacterial classification, transfer into 7 3L shaking flasks (the 1L substratum of packing into), inoculum size 5%, 35 ± 2 ℃, 120 rpm cultivate 12h, transfer into the one grade fermemtation tank of 200 liters, coefficient is 65%, inoculum size 5%, 35 ± 2 ℃, ventilating ratio is 1:0.3, pH is 6.5, mixing speed is that 120 rpm cultivate 12 h, transfer into 2 tons of second order fermentation tanks, coefficient is 65%, inoculum size 10%, temperature is 35 ± 2 ℃, ventilating ratio is 1:0.3, pH is 6.5, mixing speed is that 120 rpm cultivate 12 h, transfer into 20 tons of three grade fermemtation tanks, charge amount is 15 tons, the processing parameter of three grade fermemtation tank is: 35 ± 2 ℃ of temperature, sterilizing front pH is transferred to 6 left and right, later stage is controlled at 7.0 with 0.23 ton of NaOH by pH, ventilating ratio is 1:0.3, mixing speed is 120 rpm, totally add during this time 0.6 ton of Rice pollard oil, it is 9.7g/L that fermentation culture detected lipopeptid content by the anti-pushing manipulation of micelle-forming concentration after 25 hours.
By above-mentioned several examples, can find out and adopt this production technique fermentation lipopeptid content can reach 7-10 g/L.
The lipopeptid tensio-active agent that will make through embodiment 1-4, measure lipopeptid content, fermented liquid surface tension and oil displacement efficiency, and result is as follows:
(1), the anti-pushing manipulation detection level of the micelle-forming concentration of lipopeptid tensio-active agent in fermented liquid: as shown in Figure 1;
(2), the interfacial tension of Bio-surface active detects: as shown in Figure 2.
A. laboratory apparatus: an interfacial tension survey meter, full-automatic surface tension instrument, ten thousand/electronic balance, electronic scales, beaker, suction pipe etc. are dripped in the rotation of JJ2000B type.
B. experiment material: bio-surfactant: the fermented liquid concentration of subtilis (ACCC01430) is 1～5%, water: Daqing oil field local water, crude oil: Daqing crude oil, density are 0.8806g/m
3, sodium hydroxide: the solid sheet;
C. detection method is referring to the oil and gas industry standard SY/T 5370-1999 of the People's Republic of China (PRC).
D. detected result:
Interfacial tension value when bio-surfactant concentration is 1.0% and between Daqing crude oil, be shown in Fig. 2.
Sum up: its lipopeptid content after fermentation of subtilis ACCC01430 can be reached to 5～10%g/L, 35 ℃, the lipopeptid fermented liquid of 0.5～1% concentration are when PH is 8-9, can make crude oil and local water interfacial tension lowering to 0.07～0.2mN/m, in water effective permeability 50-400md, residual oil saturation 30～50%, injection rate, be that 0.1pv lipopeptid fermented liquid concentration is under 1-5%, the condition of 50 ℃, oil recovery factor has improved the 5-10% left and right than water drive.
Claims (2)
1. the industrialized process for preparing of a lipopeptide biosurfactant, it is characterized in that: this lipopeptide biosurfactant is to be made through fermentation culture by subtilis ACCC01430, and its concrete steps are as follows:
A, by inclined-plane solid subtilis ACCC01430 bacterial classification access shake-flask culture, every 500ml substratum access 1 ring bacterial classification in shaking flask, temperature is that 35 ± 2 ℃, 120 rev/mins concussions were cultivated 12～16 hours, obtains the shaking flask bacterial classification;
B, one grade fermemtation are cultivated: the shaking flask strain transfer that the A step is made enters to be equipped with in the one grade fermemtation tank of substratum, the volume ratio of shaking flask bacterial classification and substratum is 1:19～20, in temperature, be that 35 ± 2 ℃, pH6～7.5, ventilating ratio 1:0.3～0.5 and stirring velocity are that the condition bottom fermentation of 100～150 rev/mins was cultivated 10～14 hours, obtain the one grade fermemtation bacterial classification;
C, second order fermentation are cultivated: the one grade fermemtation strain transfer that the B step is made enters to be equipped with in the second order fermentation tank of substratum, the volume ratio of one grade fermemtation bacterial classification and substratum is 1:10, in temperature, be that 35 ± 2 ℃, pH 6～7.5, ventilating ratio 1:0.3～0.5 and stirring velocity are that the condition bottom fermentation of 100～110 rev/mins was cultivated 10～14 hours, obtain the second order fermentation bacterial classification;
D, fermentation culture: the second order fermentation strain transfer that the C step is made enters to be equipped with in the three grade fermemtation tank of substratum, the volume ratio of second order fermentation bacterial classification and substratum is 1:10, in temperature, be that 35 ± 2 ℃, pH 6～7.5, ventilating ratio 1:0.3～0.5, stirring velocity are 80～100 rev/mins, at thalline, be in the decline phase and carry out feed supplement, fermentation culture 24～26 hours, obtain lipopeptide biosurfactant;
Wherein, described shaking flask and fermentor cultivation based formulas at different levels are: Rice pollard oil 2～5%, urea 0.2～1.5%, molasses 10～15%, KCl 0.05～1.0%, KH
2PO
40.05～1.2%, K
2HPO
40.05～1.5%, yeast extract paste 0.005～0.1%, composite trace element 0.005～0.01%, surplus is water, and pH is 6.0-7.5;
Described composite trace element is zinc sulfate 1.1 g/L, stannic oxide 1.3 g/L, Manganous chloride tetrahydrate 1.0 g/L, copper sulfate 1.0 g/L, cobalt oxide 1.0g/L and aqueous solvent is composite forms;
Feed supplement described in step D is Rice pollard oil, and each feed supplement amount is added by 3%～5% of the overall accumulated amount of fermented liquid.
2. the industrialized process for preparing of a kind of lipopeptide biosurfactant according to claim 1, it is characterized in that: the coefficient of fermentor tanks at different levels is 60～80%.
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