CN102505026A - Method for extracting microalgae oil membrane substrate in situ - Google Patents
Method for extracting microalgae oil membrane substrate in situ Download PDFInfo
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- CN102505026A CN102505026A CN2011103504285A CN201110350428A CN102505026A CN 102505026 A CN102505026 A CN 102505026A CN 2011103504285 A CN2011103504285 A CN 2011103504285A CN 201110350428 A CN201110350428 A CN 201110350428A CN 102505026 A CN102505026 A CN 102505026A
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Abstract
The invention discloses a method for extracting a microalgae oil membrane substrate in situ. The method comprises the following steps of: culturing an oil-producing algae species in an appropriate culture medium until the algae species grows to a stable stage, adding algae liquid in which oil is accumulated into an extraction layer, establishing a two-phase system in which an extraction solvent and the algae liquid are blended by an in-situ extraction method, continuously extracting the oil from an algae body, adding into a solvent phase, gradually demixing and clarifying the extraction solvent and the algae liquid, recycling an organic solvent on the upper layer, circulating the algae liquid in the extraction layer, and continuously culturing. An integrated method for extracting the microalgae oil in situ is provided by combining a continuous microalgae culture technology, a membrane technology and a microalgae oil extraction technology, and processes of collecting, drying and crushing microalgae cells are avoided. Compared with the conventional microalgae oil extraction technology, the process has the characteristics of high oil extraction rate, low cost, environment friendliness and the like, and is simple.
Description
Technical field
The present invention relates to biotechnology and bioenergy field, relate in particular to a kind of microalgae grease process for extracting that forms that little algae cultivation, membrane technique and microalgae grease abstraction technique are integrated.
Background technology
Biofuel is the staple product of bioenergy, but receives serious restriction because of insufficient raw material makes industry development.In the raw material of preparation biofuel (the little algae of oil crops, forest and produce oil), micro algae growth is fast, and fat content is high, is a kind of biofuel bulk raw material that has prospect.The bottleneck problem of present little algae production biofuel is that cost is too high, causes its economy to can not get ensureing, particularly microalgae grease extracts and accounts for 50% of biofuel cost.Conventional microalgae grease extractive technique comprises organic solvent extractionprocess, supercritical fluid extraction, thermo-cracking etc.; It is dried powder that these methods require frond; And solids content is usually less than 1% in the algae liquid; The pre-treatment of concentrate drying not only prolongs the production of biodiesel cycle, also influences the grease extraction efficiency.
The original position extraction process is meant the two-phase system of setting up biocompatibility organic solvent and the blend of algae liquid, makes the frond grease constantly be extracted to solvent phase, evades frond and dehydrates treatment step, realizes that grease is at line drawing and the double goal that improves output.
At present the process for extracting of microalgae grease is mainly contained methods such as organic solvent extraction, overcritical and subcritical fluid extraction.For example: solvent pairs system extraction (Mixing co-solvent extraction) is meant that forming single_phase system by a kind of polar solvent and a kind of non-polar solvent extracts the frond grease.Bligh and Dyer are in nineteen fifty-nine (Bligh EG; Dyer WJ.A rapid method of total lipid extractionand purification " a kind of TL rapid extraction and purification process " .Canadian Journal ofBiochemistry and Physiology " Canadian biological chemistry and physiology journal; 1959,37 (8): the methyl alcohol/chloroform system that 911-917) proposes is still the most frequently used microalgae grease process for extracting.Based on " similar mixing " principle; Frond fully contacts with methyl alcohol/chloroform mixed solvent, and polar solvent methyl alcohol combines with the polarity fat of cytolemma, thereby destroys hydrogen bond and electrostatic interaction between lipid and protein molecule; Make non-polar solvent chloroform entering cell and dissolve neutral fat composition hydrophobic in the born of the same parents; Fully the extraction back adds entry in system, methyl alcohol promptly be dissolved in water and with the layering mutually of grease-contained chloroform, obtain thick fat extract after the chloroform volatilization.Yet because of chloroform has neural intoxicating effect, some low toxicity solvent pairs systems are also attempted being used for grease and are extracted, normal hexane/Virahol for example, DMSO/ sherwood oil, normal hexane/ethanol etc.
1996; (Richter BE such as Richter; Jones BA, Ezzell JL, Porter NL.Accelerated solvent extraction:a technique for sample preparation " accelerated solvent extraction: a kind of sample preparation technology " .Analytical Chemistry " analytical chemistry "; 1996; 68 (6): 1033-1039) propose quick solvent-extraction process (Accelerated solvent extraction, ASE), this be a kind of at comparatively high temps (50～200 ℃) and big pressure (under 10.3～20.6MPa) conditions with the treatment process of SX solid or semi-solid sample.The principle that this method is used to extract microalgae grease is; HTHP helps to increase rate of mass transfer makes solvent infiltrate frustule fast, and reduce the solvent specific inductivity simultaneously and make its polarity near grease, thus the extraction efficiency of raising solvent; The common solvent system has methyl alcohol/chloroform, Virahol/normal hexane etc.Compare with double-solvent extraction method, the ASE method has action time, and short (5～10min), solvent-oil ratio is few, the advantage that the grease extraction yield is high.For example; Traditional Folch method (chloroform/methanol; 2: 1v/v)) the grease extraction yield to green alga Rhizoclonium hieroglyphicum is 44～55%, and with the equivalent solvent at pressure 10.3MPa, only extract 5min in the time of 120 ℃ and can reach 85～95% grease extraction yield.
Supercritical fluid extraction (Supercritical fluid extraction) is a kind of emerging extractive technique; It is meant when exceeding critical temperature and pressure; Fluid is between gaseous state and liquid state; Have the diffusion mass transfer performance of gas and the solubility property of liquid simultaneously, to the greasy extraction efficiency of frond far above common organic solvent.CO
2Because advantages such as critical condition gentle (pressure 7.4MPa, 31.1 ℃ of temperature), nontoxic, unreactivenesses, use the most extensively, methyl alcohol, ethanol, water, nitrogen peroxide, sulfur hexafluoride etc. use also and report to some extent in addition.Supercritical CO
2Extraction (Supercritical carbon dioxideextraction, SCCO
2) action condition of microalgae grease is 40～50 ℃, 24.1～37.9MPa extracts the back grease and is dissolved in overcritical liquid CO
2In, only need controlled temperature and pressure to make CO during recovery
2Recover gaseous state and can separate grease.Supercritical extraction is eco-friendly green extractive technique, but because of relating to temperature and pressure control, makes the treatment process energy consumption high, and economy is relatively poor, is inappropriate for the extraction of bulk chemical.
(Subcritical water extraction SWE) is meant that water its polarity a little less than critical temperature the time reduces, and therefore has the character of similar organic solvent to Subcritical Water Extraction; Greasy solvability is also improved greatly, utilize high pressure to make water maintain liquid state simultaneously, high temperature impels water to get into cell fast; Make born of the same parents' inner lipid be extracted to water; When system was cooled to room temperature, the polarity of water raise, and the grease and the rapid layering of water that are dissolved in water are convenient to collect.In addition, subcritical ethanol also can be used in the extraction pigment, like the Serlabo among Haematococcus pluvialis and the Dunaliella salina.The advantage of this method is and can directly handles algae liquid, in system, need not to add organic solvent, and still because of the existence of high energy consumption steps such as temperature, pressure, this method industrial application is restriction to some extent also.
Above-mentioned microalgae grease Different Extraction Method comprises high energy consumption treatment steps such as frond drying, HTHP control.The dry main method of frond has daylight airing method, spraying drying and lyophilize etc. at present; When handling a large amount of frond; With the tool economy of airing method; But to tedding area and time certain requirement is arranged, therefore in drying, the technological improvement of links such as temperature, pressure control can effectively reduce grease and extract energy consumption.
The research of membrane technique is since carrying out the beginning of the eighties, and oneself is through becoming the important component part of new separation technology research field.Have the advantage of membrane technique and extraction process concurrently based on the dispersion extraction process of microporous membrane; Film medium itself does not have centrifugation to pending mixture; Mainly utilize the porousness of film, wetting ability or hydrophobicity are two to transmit mutually bigger and stable phase-contact surface is provided; Can overcome influences such as liquid flooding in conventional separation the, back-mixing, paid close attention to deeply over past ten years.Utilizing hollow-fibre membrane to promote the dispersion effect of organic solvent in algae liquid, little algae is cultivated and the coupling of grease leaching process, is to realize that microalgae grease synthesizes continuously and the novel method of on-line extraction.
Summary of the invention
The present invention combines microporous membrane technology, makes biocompatibility extraction solvent homodisperse to algae liquid, promotes the extraction efficiency of solvent to microalgae grease, has proposed a kind of brand-new little algae cultured continuously and the integrated technology of grease original position extraction.
A kind of microalgae grease film base original position method of extraction may further comprise the steps:
(1) little algae cultured continuously: produce oil algae kind is cultured to the stationary phase of growing in appropriate media, accumulates grease gradually in the cell of produce oil algae kind;
(2) original position oil and grease extracting: will accumulate the algae liquid input extraction system behind the grease, and import extraction solvent simultaneously, and utilize the original position extraction process to set up the two-phase system of extraction solvent and the blend of algae liquid, the frond grease constantly is extracted to solvent phase; Extraction solvent and the layering gradually of algae liquid, the upper strata extraction solvent recycles, and lower floor's algae liquid is circulated to the cultured continuously step; Go into to pump the flow velocity of extraction module through regulating extraction solvent and algae liquid pump, keep the volume ratio of extraction solvent in the extraction system to be no more than 10% of TV.
(3) detect fat content in the extraction solvent in real time, if grease concentration does not have remarkable increase, then oil extraction is tending towards saturated, removes the saturated extraction solvent of grease, adds new extraction solvent.
As a kind of preferred version; One hollow fiber film assembly is set in described extraction system; Algae liquid flows along the shell of hollow fiber film assembly, and extraction solvent gets in the terminal hollow fiber film tube of shutting, and the micropore through the hollow-fibre membrane tube wall is dispersed into the algae liquid that fine droplet gets into shell side again; And with algae liquid thorough mixing, make the frond grease constantly be extracted to solvent phase.
Described extraction solvent is the biocompatibility organic solvent, is generally the hydrophobic organic solvent of log P＞5.5, wherein is C especially with the carbon chain lengths
12～C
16The alkane solvents biocompatibility good.
Film disperses the method for original position extraction microalgae grease to have the advantage of membrane technique and extraction process concurrently among the present invention, is new efficient mass transfer process, has following principal feature and advantage:
(1) extraction solvent disperses to get in the algae liquid with atomic little droplet morphology through membrane micropore, and extraction solvent and algae liquid thorough mixing significantly improve extraction efficiency.
(2) this method need not broken microalgae cell, and little algae is cultivated and can carry out continuously.When produce oil algae kind was selected the algae kind of organic solvent-resistant for use, because microalgae cell and organic solvent have affinity preferably, then organic solvent was disperseing to carry out in the process of grease original position extraction with film, and activity of microalgae is influenced hardly.
(3) be in stationary phase when controlling little algae cultivation, this moment, microalgae grease was synthetic vigorous, can realize carrying out when micro algae growth and grease extract, and grease carries out online in-situ extraction.This method can break through that little algae is gathered, dry, the broken and tediously long technology extracted, thereby significantly reduces production costs.
Description of drawings
Fig. 1 is used to realize that film that the embodiment 1 of the inventive method uses disperses the synoptic diagram of organic solvent extraction plant, illustrates: 1. 2. 3. 4. 5. 6. 7. air compressor pump of peristaltic pump of airlift photobioreactor of algae liquid storage bottle of dead end hollow fiber film assembly of extract layer of extraction solvent container for storing liquid.
Fig. 2 is a fat content result of variations in time in algae liquid and the extraction agent in the embodiment of the invention 1.
Fig. 3 is that film disperses the greasy cytoactive variation tendency of organic solvent extraction Botryococcus braunii in the embodiment of the invention 1.
Embodiment
Embodiment 1:
(1) be that the airlift photobioreactor of 7L is cultivated produce oil algae Botryococcus braunii in 5. at volume, BG11 substratum (algae collective media) at 25 ± 1 ℃, was cultivated for 3 thoughtful stationary phases under continuous illumination (the light intensity 8k lux) condition.
(2) the 5. interior algae liquid of airlift photobioreactor feeds dead end PES hollow fiber film assembly shell 3. through peristaltic pump, the long-pending 400ml that is controlled to be of algae liquid.The Botryococcus braunii biocompatible solvent tetradecane is with 10ml min
-1Flow feed dead end (assembly that membrane fiber one end is shut) PES hollow fiber film assembly pipe layer 3.; The tetradecane is dispersed into fine droplet through the PES fenestra and gets into algae liquid; Intensive solvent droplets fully contacts and extracts the frond grease because of the less come-up gradually of density with frustule in uphill process.
Tetradecane solvent in the extract layer and the layering of algae liquid, the solvent phase on upper strata are passed through peristaltic pump with 10ml min
-1Flow draw back into the organic solvent container for storing liquid and 1. recycle, be the cytotoxicity that reduces solvent, keep solvent volume in the extract layer than being no more than 10% of TV.
(3) fat content of detection solvent phase in the extraction process, if grease concentration does not have remarkable increase, then oil extraction is tending towards saturated, removes the saturated extraction solvent of grease, adds new extraction solvent and keeps certain grease extraction yield.
Comparative Examples:
The extraction agent tetradecane in step (2) be directly pump in the middle of the algae liquid, through the membrane module, all the other are identical with embodiment 1.
The concrete operating method that fat content is measured part is:
The mensuration of fat content in the born of the same parents:
Algae liquid dilute in proportion and with ultraviolet spectrophotometer (GS-54, Shanghai rib light) measure its at the 680nm place absorbancy, OD680 remains on about 0.5, gets Nile red dye liquor (the concentration 1mg ml that dilution algae liquid 3ml adds 3 μ l
-1, solvent acetone), lucifuge dyeing 10min in 37 ℃ of water-baths, spectrophotofluorometer (F96-PRO, Shanghai rib light) is measured its fluorescence intensity, and excitation wavelength 480nm disperses wavelength 580nm, gains 10 grades.Fluorescence intensity/OD680 substitution computes frond fat content.
Y=0.16X+12.49 (X: fluorescence intensity, Y: fat content %)
Fat content is measured in the organic solvent (tetradecane):
(Aladdin USA) is solvent, triolein triolein (Aladdin with the tetradecane; USA) for standard substance preparation different concns solution, respectively get the Nile red dye liquor (1mgml-1 is dissolved in acetone) that 3ml adds 5 μ l respectively; Lucifuge dyeing 10min in 37 ℃ of water-baths is with its fluorescence intensity of fluorescent spectrophotometer assay, excitation wavelength 480nm; Disperse wavelength 570nm, gain 1 grade, it is following to obtain the typical curve corresponding equation:
Y=0.06X+30.07 (Y fluorescence intensity, X grease concentration mg L
-1)
When measuring the fat content in the tetradecane of extraction back, get Nile red dye liquor (the 1mg ml that the 3ml tetradecane adds 5 μ l
-1, be dissolved in acetone), lucifuge dyeing 10min in 37 ℃ of water-baths, with its fluorescence intensity of fluorescent spectrophotometer assay, excitation wavelength 480nm disperses wavelength 570nm, gains 1 grade.With the data substitution typical curve equation that records, can try to achieve its grease concentration.
The cells were tested by flow cytometry cell survival rate
Two fluorescein diacetates (FDA) are used for characterizing the cell survival rate of oil extraction process as the viable cell optical dye.When active cells had the intact cell film, the resorcinolphthalein of FDA hydrolysis can constantly accumulate in cell, made cell under blue excitation light, send green fluorescence.Work as damaged membrane, when cell lost activity, the resorcinolphthalein of FDA hydrolysis can't be at cell inner accumulation, and makes cell can't send fluorescence.Active high cell sends the intensive green fluorescence, active weak luminescence of cell a little less than.When fluorescent substance is full of whole cell, can make cell send bright green.
Botryococcus braunii algae liquid is removed impurity and cell mass through 300 order cells sieve; Get the centrifugal supernatant of abandoning of 1ml algae liquid; PBS damping fluid (phosphate buffered saline) cleans frond three times, gets an amount of PBS damping fluid suspension frond and makes algae cell density be not less than 10
6Individual ml
-1, (Sigma, USA) final concentration is 100 μ g ml to add FDA
-1Lucifuge dyeing 7min under the room temperature, and flow cytometry analysis (Beckman FC 500MCL, USA), flow velocity 100-400 cell sec
-1, total cell count is greater than 1000 during data gathering, and perhaps the time after test is accomplished, is preserved analytical results and mapping greater than 2min.
(algae liquid and organic solvent speed of circulation are 10ml min for algae liquid 400ml, tetradecane 40ml to be illustrated in figure 2 as in the extraction process in the organic solvent and Botryococcus braunii born of the same parents the variation of fat content
-1), experimental result shows, embodiment 1 extraction carries out after 2 days that grease concentration has reached peak 520mg L in the extraction agent tetradecane
-1, to 50.15%, and the control group extraction yield is 38.05% to greasy extraction rate reached.After 2 days in the tetradecane fat content change not quite, and fat content recovers gradually in the algae liquid.Can draw from interpretation, the fenestra dispersion energy effectively promotes mixing of solvent and algae liquid, has increased the contact area of frond and solvent, has improved the grease extraction yield.
Be illustrated in figure 3 as the active (100ugml of detection of film bubbling organic solvent extraction grape algae oil cells
-1The FDA 10min that dyes, the C2 quadrant is the painted viable cell of FDA district.Flow cytometer detects in the 96h original position extraction process among the embodiment 1, and cell survival rate remains on more than 90%, and presentation of results is introduced the active nothing influence of PES organic membrane pair cell in system, be applicable to greasy continuous extraction process.
Claims (6)
1. microalgae grease film base original position method of extraction may further comprise the steps:
(1) little algae cultured continuously: produce oil algae kind is cultured to the stationary phase of growing in substratum, accumulates grease gradually in the cell of produce oil algae kind;
(2) original position oil and grease extracting: the algae liquid after cultured continuously accumulates grease is imported extraction system, import extraction solvent and the blend of algae liquid simultaneously and form two-phase system, the frond grease constantly is extracted to solvent phase; Extraction solvent and the layering gradually of algae liquid, the upper strata extraction solvent recycles, and lower floor's algae liquid is circulated to the cultured continuously step;
(3) detect fat content in the extraction solvent in real time, if grease concentration does not have remarkable increase, then oil extraction is tending towards saturated, removes the saturated extraction solvent of grease, adds new extraction solvent.
2. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction is characterized in that: described produce oil algae kind is the algae kind of organic solvent-resistant.
3. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction, it is characterized in that: described solvent is the biocompatibility organic solvent.
4. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 3 position method of extraction, it is characterized in that: described biocompatibility organic solvent is the alkanes of hydrophobic value logP＞5.5.
5. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 4 position method of extraction is characterized in that: its described biocompatibility organic solvent is C
12～C
16Alkane solvents.
6. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction, it is characterized in that: the solvent volume that described extraction system contains is no more than 10% of TV.
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Cited By (4)
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| CN102839127A (en) * | 2012-08-28 | 2012-12-26 | 浙江工业大学 | Method for microalgae coupling of culturing and recovery to quickly accumulate algal oil |
| CN104046559A (en) * | 2014-06-03 | 2014-09-17 | 新奥科技发展有限公司 | Microalgae culturing apparatus and culturing method thereof |
| CN104862070A (en) * | 2015-04-24 | 2015-08-26 | 亿利资源集团有限公司 | Method for ultrasonically assisted in situ extraction of oil from microalgae |
| CN105861371A (en) * | 2016-04-26 | 2016-08-17 | 南昌大学 | Spirulina culture method based on in-situ continuous collection |
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| CN102174409A (en) * | 2011-02-23 | 2011-09-07 | 天津科技大学 | Method for quickly accumulating grease through mixotrophic sterile culture of microalgae |
| CN102191126A (en) * | 2011-03-22 | 2011-09-21 | 深圳大学 | Preparation method of biodiesel from microalgal oil by enzyme method |
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| CN102174409A (en) * | 2011-02-23 | 2011-09-07 | 天津科技大学 | Method for quickly accumulating grease through mixotrophic sterile culture of microalgae |
| CN102191126A (en) * | 2011-03-22 | 2011-09-21 | 深圳大学 | Preparation method of biodiesel from microalgal oil by enzyme method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839127A (en) * | 2012-08-28 | 2012-12-26 | 浙江工业大学 | Method for microalgae coupling of culturing and recovery to quickly accumulate algal oil |
| CN104046559A (en) * | 2014-06-03 | 2014-09-17 | 新奥科技发展有限公司 | Microalgae culturing apparatus and culturing method thereof |
| CN104862070A (en) * | 2015-04-24 | 2015-08-26 | 亿利资源集团有限公司 | Method for ultrasonically assisted in situ extraction of oil from microalgae |
| CN105861371A (en) * | 2016-04-26 | 2016-08-17 | 南昌大学 | Spirulina culture method based on in-situ continuous collection |
| CN105861371B (en) * | 2016-04-26 | 2019-07-26 | 南昌大学 | A kind of spirulina breeding method continuously harvested based on original position |
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