CN108034679B - Method for extracting biosurfactant from microalgae - Google Patents
Method for extracting biosurfactant from microalgae Download PDFInfo
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- CN108034679B CN108034679B CN201711182101.5A CN201711182101A CN108034679B CN 108034679 B CN108034679 B CN 108034679B CN 201711182101 A CN201711182101 A CN 201711182101A CN 108034679 B CN108034679 B CN 108034679B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
Abstract
The invention discloses a method for extracting biosurfactant from microalgae, which comprises the following steps: preparing concentrated solution, fermenting, preparing enzyme solution, preparing, selecting screened chlorella collected on an oil spill site, culturing, concentrating, fermenting the chlorella subjected to enlarged culture, breaking the wall by using an ultrasonic crusher, extracting the enzyme solution, mixing the chlorella concentrated solution and the chlorella enzyme solution, and adding a stabilizer to obtain the biosurfactant. The beneficial effects are that: the preparation method is simple and easy to implement, low in cost, safe and environment-friendly, and does not add new pollutants; the biosurfactant extracted from the microalgae has stable emulsification indexes such as surface tension, emulsification activity, viscosity and the like, has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation and the like, has strong petroleum degradation capability, has a degradation index of 73.68-91.56 percent, and is a safe and efficient biosurfactant.
Description
Technical Field
The invention relates to the field of environmental engineering, in particular to a method for extracting a biosurfactant from microalgae.
Technical Field
At present, products which are mostly applied to oil spilling on the market comprise an oil spilling dispersing agent, an oil dissolving agent, an oil dispersing agent and the like, and the main components of the oil spilling dispersing agent are as follows: the anionic surfactant is mainly ether type, nonionic ester type, and concentrated type with low toxicity. Compared with the current chemical surfactant synthetic products, the biosurfactant has the advantages of being more beneficial to research and development. Biosurfactants are one of the natural surfactants whose molecular structure has strictly hydrophilic and hydrophobic groups. The oil-stain emulsion has the functions of emulsifying and dispersing oil stains and reducing surface tension, and has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation, simple production process and the like.
At present, the main biosurfactants are glycolipids, and specifically comprise: rhamnolipid, sophorolipid, lipopeptide, lipoprotein, lipopeptide, gramicidin, polymyxin, fatty acid, neutral fat and phospholipid, poly-biosurfactant, polysaccharide emulsifier, biological dispersant, chitin and polyester peptide chain, wherein the source of the polypeptide chain is mainly bacteria.
Disclosure of Invention
The invention aims to provide a method for extracting a biosurfactant from microalgae, which has the advantages of simple process, safety, environmental protection, low toxicity, environmental protection, mild action condition and thorough pollutant degradation.
Aiming at the problems mentioned in the background technology, the invention adopts the technical scheme that: a method of extracting biosurfactants from microalgae comprising: the preparation method comprises the following steps of preparation of concentrated solution, fermentation, preparation of enzyme solution and preparation:
preparing a concentrated solution: taking 1L of cultured chlorella liquid, centrifuging at 0-4 deg.C and 3500-; resuspending the precipitate with buffer solution, centrifuging under the previous centrifugation condition, and repeating for 3-4 times; combining all the algae solutions obtained by centrifuging the algae solutions, diluting the combined algae solutions by using a buffer solution, wherein the final volume is 200 plus 220mL, and storing the combined algae solutions at the temperature of 0-4 ℃ for later use; selecting the chlorella collected and screened on the oil spilling field for culturing, and greatly improving the activity, biological viscosity reduction, petroleum hydrocarbon degradation and other performances after concentration;
fermentation: the fermentation medium comprises the following components in percentage by weight: 38-40g/L glucose, 6-8g/L corn starch, 4-5g/L, NaCl 12.5.5-15.0 g/L peptone and (NH)4)2SO4 1.65-1.70g/L、MgSO4·7H2O 2.2-2.5g/L、KCl 0.5-0.6g/L、KH2PO4 0.5-0.6g/L、CaCl20.1-0.12g/L, sterilizing by high pressure steam at the temperature of 121-122 ℃ for 15-30 minutes, inoculating cultured chlorella according to 3.5-5.0 percent, fermenting and culturing for 72-96 hours under the conditions of the temperature of 30-33 ℃, the pH value of 6.9-7.2, the stirring speed of 320-340r/min and the ventilation volume of 0.65-0.85vvm to obtain fermentation liquor; under the condition of sufficient nitrogen source, carbon source, trace elements and other essential substances, the chlorella can be rapidly recovered to the peak activity stage, so that the dehydrogenase, oxidase, peroxidase, petroleum degrading enzyme and petrochemical enzyme of the chlorella can be rapidly recoveredAccelerating secretion of relevant organic degradation enzymes, and preparing for extracting enzyme liquid and degrading petroleum hydrocarbon;
preparing an enzyme solution: centrifuging the fermentation liquid at 0-4 deg.C and 3500-; dissolving the precipitate in buffer, and crushing the cells with an ultrasonic crusher with power of 1.0-1.3kW, probe amplitude of 38-45%, temperature of 2-4 deg.C, and time of 30-48 min; centrifuging at 0-4 deg.C and 12000r/min for 15-20 min, collecting supernatant, resuspending the precipitate with buffer solution, repeating centrifuging for 3-5 times, mixing supernatants, concentrating at low temperature to 220mL, and storing at 0-4 deg.C; the chlorella is crushed by an ultrasonic crusher, so that the wall breaking effect is good, active substances cannot be influenced, new pollutants cannot be added, and further centrifugal extraction is facilitated;
preparation: mixing the chlorella concentrated solution and chlorella enzyme solution at the temperature of 3-4 ℃ according to the volume of 1:0.5-3.0, sealing the mixed solution, placing the mixture in a triangular flask filled with ice water, mixing the mixture for 10-13 minutes under the conditions of 20-22 ℃ and 200-; the biosurfactant extracted from the microalgae has stable emulsification indexes such as surface tension, emulsification activity, viscosity and the like, has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation and the like, has strong petroleum degradation capability, has a degradation index of 73.68-91.56 percent, is simple and easy to prepare, has low cost, and can be applied to environmental engineering in a large scale.
Preferably, the chlorella is obtained by collecting and screening from an oil spill site, and has remarkable effects on biological viscosity reduction of oil, crude oil recovery improvement, biological remediation of heavy oil polluted soil and the like through domestication and passage.
Preferably, the buffer solution is Tris-HCl buffer solution with the pH value of 7.8-7.9; the Tris-HCl buffer solution does not denature the enzyme, and simultaneously can purify the enzyme and increase the enzyme content in the enzyme solution.
Preferably, the stabilizers are present in amounts of, respectively, 18-23% glycerol, 1.0-1.5mM EDTA and 0.1-0.13mM DTT, and have a pH of 7.5-7.7; the stabilizer has emulsification effect, and can enhance the stability and uniformity of biosurfactant.
Compared with the prior art, the invention has the advantages that: 1) the preparation method is simple and easy to implement, low in cost, safe and environment-friendly, the chlorella is crushed by using an ultrasonic crusher, the wall breaking effect is good, no influence is caused on active substances, no new pollutants are added, and the extraction efficiency is improved; 2) the biosurfactant extracted from the microalgae has stable emulsification indexes such as surface tension, emulsification activity, viscosity and the like, has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation and the like, has strong petroleum degradation capability, has a degradation index of 73.68-91.56 percent, and is a safe and efficient biosurfactant.
Drawings
FIG. 1 is a process flow diagram of a method of extracting biosurfactants from microalgae according to the invention.
Detailed Description
The invention is further illustrated by the following figures and examples:
example 1:
a method of extracting biosurfactants from microalgae comprising: the preparation method comprises the following steps of preparation of concentrated solution, fermentation, preparation of enzyme solution and preparation:
preparing a concentrated solution: centrifuging 1L of cultured chlorella solution at 4 deg.C and 5000r/min for 15 min, removing the supernatant, and collecting precipitate; resuspending the pellet with buffer solution, centrifuging under the previous centrifugation condition, repeating for 4 times; combining all the algae solutions obtained by centrifuging the algae solutions, diluting the combined algae solutions with a buffer solution to a final volume of 220mL, and storing the combined algae solutions at the temperature of 4 ℃ for later use; selecting the chlorella collected and screened on the oil spilling field for culturing, and greatly improving the activity, biological viscosity reduction, petroleum hydrocarbon degradation and other performances after concentration;
fermentation: the fermentation medium comprises the following components in percentage by weight: 40g/L glucose, 8g/L corn starch and 5g/L peptone、NaCl 15.0g/L、(NH4)2SO4 1.70g/L、MgSO4·7H2O 2.5g/L、KCl 0.6g/L、KH2PO4 0.6g/L、CaCl2Sterilizing with high pressure steam at 122 deg.C for 30 min at 0.12g/L, inoculating cultured Chlorella at 5.0%, fermenting at 33 deg.C, pH 7.2, stirring speed 340r/min, and ventilation amount 0.85vvm for 96 hr to obtain fermentation broth; under the condition of necessary substances such as sufficient nitrogen source, carbon source, trace elements and the like, the chlorella can be quickly recovered to the stage with peak activity, so that organic degradation related enzymes such as dehydrogenase, oxidase, peroxidase, petroleum degrading enzyme, petrochemical enzyme and the like are accelerated to secrete, and preparation is made for extracting enzyme liquid and degrading petroleum hydrocarbon;
preparing an enzyme solution: centrifuging the fermentation broth at 4 deg.C for 15 min at 5000r/min, removing the supernatant, collecting precipitate, resuspending the precipitate with buffer solution, centrifuging for 3 times, and mixing the precipitates; dissolving the precipitate in buffer, and crushing the cells with an ultrasonic crusher with power of 1.3kW, probe amplitude of 45%, temperature of 4 deg.C and time of 48 min; centrifuging at 4 deg.C and 12000r/min for 20 min, collecting supernatant, resuspending the precipitate with buffer solution, repeating centrifuging for 5 times, mixing supernatants, concentrating at low temperature to 220mL, and storing at 4 deg.C; the chlorella is crushed by an ultrasonic crusher, so that the wall breaking effect is good, active substances cannot be influenced, new pollutants cannot be added, and further centrifugal extraction is facilitated;
preparation: mixing the chlorella concentrated solution and chlorella enzyme solution at 4 deg.C according to a volume ratio of 1:3.0, sealing the mixed solution, placing in a triangular flask filled with ice water, mixing under shaking at 22 deg.C and 220r/min for 13 min, and adding stabilizer at a volume ratio of 6:1 to obtain biosurfactant; the biosurfactant extracted from the microalgae has stable emulsification indexes such as surface tension, emulsification activity, viscosity and the like, has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation and the like, has strong petroleum degradation capability, has a degradation index of 73.68-91.56 percent, is simple and easy to prepare, has low cost, and can be applied to environmental engineering in a large scale.
The buffer was Tris-HCl buffer at pH 7.8.
The stabilizer contents were 20% glycerol, 1.0mM EDTA and 0.1mM DTT, respectively, at pH 7.7.
Example 2:
a method for extracting biosurfactant from microalgae comprises the following steps:
1) preparing a concentrated solution: centrifuging 1L of cultured chlorella solution at 0 deg.C and 3500r/min for 10 min, removing the supernatant, and collecting precipitate; resuspending the precipitate with buffer solution (Tris-HCl buffer solution with pH 7.8), centrifuging under the previous centrifugation condition, and repeating for 3 times; combining all the algae solutions obtained by centrifuging the algae solutions, diluting the combined algae solutions with a buffer solution to a final volume of 200mL, and storing the combined algae solutions at the temperature of 0 ℃ for later use; selecting the chlorella collected and screened on the oil spilling field for culturing, and greatly improving the activity, biological viscosity reduction, petroleum hydrocarbon degradation and other performances after concentration;
2) fermentation: inoculating cultured Chlorella according to 3.5%, fermenting and culturing at 30 deg.C, pH 6.9, stirring speed 320r/min and ventilation amount 0.65vvm for 72 hr to obtain fermentation liquid; under the condition of necessary substances such as sufficient nitrogen source, carbon source, trace elements and the like, the chlorella can be quickly recovered to the stage with peak activity, so that organic degradation related enzymes such as dehydrogenase, oxidase, peroxidase, petroleum degrading enzyme, petrochemical enzyme and the like are accelerated to secrete, and preparation is made for extracting enzyme liquid and degrading petroleum hydrocarbon;
3) preparing an enzyme solution: centrifuging the fermentation broth at 0 deg.C and 3500r/min for 10 min, removing the supernatant, collecting precipitate, resuspending the precipitate with buffer solution, centrifuging for 2 times, and mixing the precipitates; dissolving the precipitate in buffer, and crushing the cells with an ultrasonic crusher with power of 1.0kW, amplitude of a probe of 38%, temperature of 2 ℃ and time of 30 minutes; centrifuging at 0 deg.C and 11500r/min for 15 min, collecting supernatant, resuspending the precipitate with buffer solution, repeating centrifuging for 3 times, mixing supernatants, concentrating at low temperature to 200mL, and storing at 0 deg.C; the chlorella is crushed by an ultrasonic crusher, so that the wall breaking effect is good, active substances cannot be influenced, new pollutants cannot be added, and further centrifugal extraction is facilitated;
4) preparation: mixing the chlorella concentrated solution and chlorella enzyme solution at 3 deg.C according to volume ratio of 1:0.5, sealing the mixed solution, placing in a triangular flask filled with ice water, shaking and mixing at 20 deg.C and 200r/min for 10 min, adding stabilizer at volume ratio of 5:1 to obtain biosurfactant, wherein the stabilizer content is respectively 18% glycerol, 1.0mM EDTA, 0.1mM DTT, 0.5 μ M (1S,2S,5S) -2-hydroxy pinane-3-ketone, and pH is 7.5; the (1S,2S,5S) -2-hydroxy pinane-3-ketone can be in hydrogen bond combination with Dithiothreitol (DTT), so that the strong reducibility of the DTT can be reduced under the condition of not influencing the emulsification and stabilization effects of the DTT, the stability of a biosurfactant mixture is maintained, the degradation aging is prolonged, the degradation efficiency is improved, and the cost is reduced; the biosurfactant extracted from the microalgae has stable emulsification indexes such as surface tension, emulsification activity, viscosity and the like, has the advantages of low toxicity, environmental protection, mild action conditions, thorough pollutant degradation and the like, has strong petroleum degradation capability, has a degradation index of 73.68-91.56 percent, is simple and easy to prepare, has low cost, and can be applied to environmental engineering in a large scale.
The surface tension, emulsification activity index, viscosity and oil removal test results of biosurfactants in example 1 and example 2 are shown in table 1.
TABLE 1 physicochemical indices of biosurfactants in examples 1 and 2
As can be seen from table 1, the biosurfactant in example 2 has lower surface tension and viscosity, higher emulsification activity index and oil removal rate, and higher oil removal rate of 91.56%, and has higher petroleum degradation characteristic.
Example 3:
example 3 differs from example 1 only in that the chlorella concentrate of example 3 has a Tris-HCl buffer pH of 7.0.
Example 4:
example 4 differs from example 1 only in that the time for disrupting the cells using the cell disruptor in example 4 was 15 minutes.
Example 5:
example 5 differs from example 1 only in that the time for disrupting the cells using the cell disruptor in example 5 was 1 h.
Example 6:
example 6 differs from example 1 only in that the volume ratio of the chlorella concentrate to the chlorella enzyme solution in example 6 is 0.5: 1.
Example 7:
example 7 differs from example 1 only in that the volume ratio of the chlorella concentrate to the chlorella enzyme solution in example 7 is 2: 1.
Example 8:
example 8 differs from example 1 only in that the surfactant to stabilizer volume ratio in example 8 is 5: 2.
Example 9:
example 9 differs from example 1 only in that in example 9 the surfactant to stabilizer volume ratio is 10: 1.
The results of the surface tension, emulsification activity index, viscosity and oil removal testing of the biosurfactants of examples 3-9 are shown in table 2.
The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for extracting biosurfactant from microalgae comprises the following steps:
preparing a concentrated solution: centrifuging 1L of cultured chlorella solution for 10-15 min, removing the supernatant, and collecting precipitate; resuspending the pellet with buffer solution, and centrifuging repeatedly for 3-4 times; combining all the centrifuged algae solutions, diluting the combined algae solutions with a buffer solution, wherein the final volume is 200-220mL, and storing the combined algae solutions at the temperature of 0-4 ℃ for later use; the buffer solution is Tris-HCl buffer solution with the pH value of 7.8-7.9;
fermentation: inoculating cultured chlorella according to 3.5-5.0%, and fermenting for 72-96 hr to obtain fermentation liquid; the fermentation temperature is 30-33 ℃, the pH value is 6.9-7.2, the stirring speed is 320-340r/min, and the ventilation volume is 0.65-0.85vvm in the fermentation process;
preparing an enzyme solution: centrifuging the fermentation liquid at 0-4 deg.C and 3500-; dissolving the precipitate in buffer solution, crushing cells with an ultrasonic crusher, centrifuging, reserving supernatant, resuspending the precipitate with the buffer solution, repeatedly centrifuging for 3-5 times, combining the supernatants, concentrating at low temperature to 220mL, and storing at 0-4 ℃ for later use;
preparation: mixing the chlorella concentrated solution and chlorella enzyme solution at the volume ratio of 1:0.5-3.0 at the temperature of 3-4 ℃, sealing the mixed solution, placing the mixture in a triangular flask filled with ice water, mixing the mixture in a shaking way for 10-13 minutes at the temperature of 20-22 ℃ and at the speed of 200-;
the power of an ultrasonic crusher in the enzyme liquid preparation step is 1.0-1.3kW, the amplitude of a probe is 38-45%, the temperature is 2-4 ℃, and the time is 30-48 minutes;
the stabilizers in the preparation step are 18-23% glycerol, 1.0-1.5mM EDTA, 0.1-0.13mM DTT and 0.5. mu.M (1S,2S,5S) -2-hydroxyppinan-3-one, the pH of which is 7.5-7.7.
2. The method of claim 1, wherein the biosurfactant is extracted from microalgae by the following steps: the centrifugal temperature in the preparation step of the concentrated solution is 0-4 ℃, and the rotating speed is 3500-.
3. The method of claim 1, wherein the biosurfactant is extracted from microalgae by the following steps: the fermentation medium in the fermentation step comprises the following components in percentage by weight: 38-40g/L glucose, 6-8g/L corn starch, 4-5g/L, NaCl 12.5.5-15.0 g/L peptone and (NH)4)2SO4 1.65-1.70g/L、MgSO4·7H2O 2.2-2.5g/L、KCl 0.5-0.6g/L、KH2PO4 0.5-0.6g/L、CaCl20.1-0.12g/L, and autoclaving at 121-122 ℃ for 15-30 minutes.
4. The method of claim 1, wherein the biosurfactant is extracted from microalgae by the following steps: the centrifugation temperature in the enzyme liquid preparation step is 0-4 ℃, the rotation speed is 11500-12000r/min, and the centrifugation time is 15-20 minutes.
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