CN105713935A - Method for producing lipid through mixed culture of microalgae - Google Patents
Method for producing lipid through mixed culture of microalgae Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a method for producing lipid through mixed culture of microalgae. The method comprises the following steps: (1) adding a microalgae culture medium and scenedesmus obliqnus FSH-Y2 seed solution into a photo-biological reactor, and culturing for 2-5 days, wherein the pH value of a culture system is regulated to be 10-12, and the content of CO2 in the introduced gas is controlled to be 5v% or below; (2) adjusting the pH of the culture system to be 8-10, and inoculating with monoraphidium sp SS-B1 seed solution for mixed culture, wherein the content of CO2 in the introduced gas is 5v%-45v%; and (3) culturing till the stationary growth stage is finished, and harvesting microalgae cells, wherein scenedesmus obliqnus FSH-Y2 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 11, 2012, and has the preservation number CGMCC No.6551, and monoraphidium sp SS-B1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 15, 2013, and has the preservation number CGMCC No.7479. With the adoption of the method, the tolerance and solubility of the microalgae culture system to high-concentration CO2 are improved, the carbon sequestration efficiency is improved, the harvest yield of the microalgae lipid is obviously improved, and the production of biodiesel is realized.
Description
Technical field
The invention belongs to biotechnology and field of biological energy source, be specifically related to one and be capable of withstanding by high concentration CO2And the method that microalgae Mixed culture produces oils and fats under alkaline environment.
Background technology
Increasingly reducing and using fossil energy to cause the increase of greenhouse effect due to fossil energy, increasing researcher focuses on sight in the development and utilization of regenerative resource.Biomass energy is as regenerative resource most important on the earth, and it includes forestry biomass, crops, water plant, agricultural wastes etc..In many biomass energies, microalgae is important Renewable resource.They have widely distributed, Biomass is big, photosynthetic efficiency is high, adaptive capacity to environment is strong, growth cycle is short, biomass yield high.Containing unique primary or secondary metabolite, complex chemical composition in its cell.The solar conversion efficiency of microalgae can reach 3.5%, is the potential resource producing medicine, fine chemical product and New-type fuel, and the fatty acid obtained from microalgae can change into Fatty acid methyl ester, i.e. biodiesel.
Along with the development of World Economics, using and consuming of substantial amounts of fossil energy, cause the shortage of the energy and going from bad to worse of environment, particularly CO2To sharply increase the greenhouse effect caused increasingly severe, the growth cycle of microalgae is short, photosynthetic efficiency is high, CO2Fixed efficiency is high, up to more than 10 times of terrestrial plant under certain condition, is possible not only to reduce CO2Discharge, also reduces toxigenic capacity simultaneously；Except CO2Outward, the compositions such as some SOx, NOx in waste gas are cleaned process also with the metabolism of microalgae, effectively reduce noxious gas emission, be the regenerative resource most possibly meeting the required fuel of world's transport at present hence with microalgae grease as the biodiesel of raw material production.
At present more for the research of the oil-producing microalgae such as chlorella, scenedesmus.CN20110144545.6 discloses a strain Scenedesmus algal strain, and the available synthetic medium of growth of this algae strain or appropriately processed waste water grow, and is characterized in that oils and fats productivity is higher than great majority point algae strain at present, and this algae strain application includes CO2Fixing, useless water purification, oils and fats, protein, pigment, starch, polysaccharide, nucleic acid production.CN20120154470.4 disclose a micro-plan ball algae of strain rich oil marine microalgae (Nannochloropsisgaditana) algae strain and application thereof, this algae strain can under the environment of pH=4.5 normal growth, its fat content is up to 35%.CN20111019480.X disclose a strain microalgae algae strain (Mychonasessp.) and for producing the application of biodiesel, utilize this algae strain can produce the polyunsaturated fatty acid of high added value, including linolenic acid C18:3 and nervonic acid C24:1, it is while obtaining biodiesel, it is thus achieved that the side-product of high added value.These patents are all not directed to the algae kind toleration to carbon dioxide.CN102703326A discloses a kind of high CO2The microalgae of toleration and fixed rate and selection thereof, but the algae strain that provides of this patent is not directed to the fat content of this algae strain.Above-mentioned patent or can not efficiently utilize CO2Produce oils and fats, or in the biomass obtained, fat content is not high enough.Particularly in actual applications, as CO in environment2When volume fraction is more than 5v%, the growth of major part microalgae is subjected to suppress, and carbon sequestration efficiency is low；General microalgae suitable growth in neutral conditions, is unfavorable for the growth of microalgae, and microalgae utilizes CO when slant acidity or meta-alkalescence simultaneously2It is usually the HCO being dissolved in water3 -Ionic species exists, and carbon dioxide dissolubility under neutral environment is low, is unfavorable for that algae absorbs.
(organic carbon source is on the growth of single needle frustule, oil and fat accumulation and photosynthetic impact for Liu Pinghuai etc., biological engineering, 2012,33 (18): 224-246) a kind of mode of production utilizing organic carbon source to cultivate single needle algae is described, although cultivating end Biomass to have exceeded 10g/L, but this kind of mode is single needle algae Heterotrophic culture mode, utilizes the organic carbon sources such as glucose to realize Growth of Cells in incubation, and this training method does not utilize CO2Etc. inorganic carbon source economy, and the addition of organic carbon source, it is easily generated microbiological contamination problem in incubation, affects the growth of frustule.
Summary of the invention
Tolerance can not be met for existing algae kind and absorb high concentration CO2, fixing CO2Inefficient problem, the invention provides one and is capable of withstanding by high concentration CO2And the method that microalgae Mixed culture produces oils and fats under alkaline environment.The inventive method improves microdisk electrode system to high concentration CO2Toleration and dissolubility, improve carbon sequestration efficiency, the harvest yield of microalgae grease significantly improves, it is possible to carry out the production of biodiesel.
Microalgae Mixed culture of the present invention produces the method for oils and fats, including following content: micro-algae culture medium and scenedesmus obliquus FSH-Y2 seed liquor are joined in bioreactor by (1), and the pH value regulating cultivating system is 10～12, passes into CO in gas2Content control below 5%, cultivate 2～5 days；(2) pH value regulating cultivating system is 8～10, accesses single needle algae SS-B1 seed liquor and carries out Mixed culture, passes into CO in gas2Content is 5v%～45v%；(3) it is cultured to growth stable phase to terminate, harvesting microalgae cell；Described scenedesmus obliquus (Scenedesmusobliqnus) FSH-Y2 and single needle algae (Monoraphidiumsp) SS-B1, respectively at JIUYUE in 2012 11 days be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number is CGMCCNo.6551 and CGMCCNo.7479 respectively on April 15th, 2013.
FSH-Y2 algae strain frustule under the microscope of the present invention is spindle, grows thickly, has cell envelope to wrap up, and color is bottle green；Single algae cell diameter is about 6～10 μm.This algae strain can better absorb carbon dioxide at a high ph, and fast-growth is bred.
Single needle algae SS-B1 algae strain of the present invention is a kind of green algate of fresh water, and frustule be the shape that comes into leaves, green, and algae kind length is 10～20 μm, wide 2～4 μm, includes pigment, and the flat board algae form that falls is S shape, bottle green.This algae strain can the CO of enduring high-concentration2And SO2, it is possible to use containing CO2And SO2Waste gas or flue gas carry out illumination autophyting growth and obtain rich grease-contained biomass, carbon sequestration efficiency is high.
In the present invention, micro-algae culture medium adopts BG11, SE, BBM well known in the art etc. to cultivate the fluid medium of microalgae.Preparing of scenedesmus obliquus FSH-Y2 seed liquor is as follows: be 10～12 by the pH regulator of culture medium, is 20～30 DEG C in temperature, and periodicity of illumination is 24h, and the light dark time, intensity of illumination was 2000～10000Lux than for 14:10, and shaken cultivation is to exponential phase.The scenedesmus obliquus FSH-Y2 seed liquor added in bioreactor and the volume ratio 1:20～1:5 of culture medium, passing into gas is carbon dioxide and nitrogen, wherein CO2Content control at 3v%～5v%.
In the present invention, preparing of single needle algae SS-B1 seed liquor is as follows: be 7～9 by the pH regulator of culture medium, is 20～30 DEG C in temperature, and periodicity of illumination is 24h, and the light dark time, intensity of illumination was 2000～10000Lux than for 14:10, and shaken cultivation is to exponential phase；The volume ratio of single needle algae SS-B1 seed liquor and culture medium is 1:10～1:4.The cultivation temperature of Mixed culture of the present invention is 20～30 DEG C, and periodicity of illumination is 24h, and the light dark time, intensity of illumination was 2000～10000Lux than for 14:10.Pass into gas and waste gas or flue gas are preferably used, wherein CO2Content is 10v%～45v%, SO2Content is 200 × 10-6～600 × 10-6(v/v).Mixed culture terminates to growth stable phase, by mode harvesting microalgae cells such as centrifugal, sedimentations, measures dry cell weight and fat content, and dry cell weight can reach 6g/L, and fat content has exceeded the 45% of dry cell weight.
Compared with prior art, the present invention can bring following beneficial effect:
1, the present invention adopts the different microalgae seed liquor of two-step method access to carry out Mixed culture, initial reaction stage accesses the scenedesmus obliquus FSH-Y2 algae strain tolerating high pH and grows in the basic conditions, the growth of microdisk electrode initial stage miscellaneous bacteria and pest and disease damage can be suppressed, make microalgae be in growth vigor；High pH cultivates and is conducive to passing into the dissolving of high concentration carbon dioxide simultaneously, makes carbon dioxide be easier to be absorbed by microalgae, is favorably improved the fixed efficiency of carbon dioxide；
2, carbon dioxide and illumination is being utilized to obtain in the incubation of biomass, microalgae Mixed culture is not the simple combination of microalgae single culture, both algae kinds are worked in coordination, and have higher carbon sequestration efficiency than single algae kind and obtain more biomass and fat content；
3, the co-culture system of the present invention can the CO of enduring high-concentration2And SO2, it is possible to use the CO in waste gas2Carry out autophyting growth, fixing CO2, alleviate greenhouse effect and exhaust pollution problems that current industrial society brings.
Detailed description of the invention
By the examples below the present invention is described in further detail.In the present invention, wt% is mass fraction, and v% is volume fraction.
The preparation of embodiment 1 microalgae seed liquor
Microdisk electrode adopts BG11 culture medium, and culture medium prescription is as shown in Table 1 and Table 2.
Table 1BG11 culture medium
* the composition of A5+Cosolution in table 2 table 1
It is first according to table 1 and BG11 fluid medium prepared by table 2, it is 10 by the pH regulator of the culture medium of cultivation scenedesmus obliquus FSH-Y2, it is 7.5 by the pH regulator of the culture medium of cultivation single needle algae SS-B1, then scenedesmus obliquus FSH-Y2 and single needle algae SS-B1 is inoculated in above-mentioned culture medium respectively.Constant temperature illumination shaking table is cultivated, cultivation temperature is 25 DEG C, periodicity of illumination is 24h, the light dark time is than for 14:10, intensity of illumination is 5000Lux, 120rpm shaken cultivation is to exponential phase, it is thus achieved that scenedesmus obliquus FSH-Y2 seed liquor and single needle algae SS-B1 seed liquor, above-mentioned seed liquor is saved backup under 15 DEG C of low light levels.
The preparation of embodiment 2 microalgae grease
(1) in bioreactor, add FSH-Y2 seed liquor and the micro-algae culture medium of embodiment 1 preparation, the volume ratio of FSH-Y2 seed liquor and culture medium is 1:10, culture medium adopts BG11 culture medium, and it is 10 that the pH value of culture medium controls, and the intensity of illumination of cultivation is 5000Lux, periodicity of illumination is 24h, the light dark time, wherein carbon dioxide content was 5v% than for 14:10, passing into the mixing gas of nitrogen and carbon dioxide.
(2), after cultivating 2 days, the volume ratio accessing the single needle algae SS-B1 seed liquor of embodiment 1 preparation, seed liquor and culture medium is 1:10, and the pH of culture medium controls 8, passes into the mixing gas of nitrogen and carbon dioxide, wherein CO2Content is 30v%.
(3) enter stable phase after cultivating 7 days, terminate to cultivate, harvested by centrifugation microalgae cell, measure dry cell weight and fat content, under-60 DEG C of conditions, algae dried bean noodles weight is measured in vacuum lyophilization to constant weight, calculates yield of biomass, and adopts normal hexane: ethyl acetate method records total lipid content.Dry cell weight can reach 7.4g/L after testing, and fat content is the 46.7% of dry cell weight.
The preparation of embodiment 3 microalgae grease
(1) in bioreactor, add FSH-Y2 seed liquor and the micro-algae culture medium of embodiment 1 preparation, the volume ratio of FSH-Y2 seed liquor and culture medium is 1:5, culture medium adopts BG11 culture medium, and it is 12 that the pH value of culture medium controls, and the intensity of illumination of cultivation is 5000Lux, periodicity of illumination is 24h, the light dark time, wherein carbon dioxide content was 5v% than for 14:10, passing into the mixing gas of nitrogen and carbon dioxide.
(2), after cultivating 3 days, the volume ratio accessing the single needle algae SS-B1 seed liquor of embodiment 1 preparation, seed liquor and culture medium is 1:10, and the pH of culture medium controls 10, passes into the mixing gas of nitrogen and carbon dioxide, wherein CO2Content is 25v%.
(3) enter stable phase after cultivating 7 days, terminate to cultivate, harvested by centrifugation microalgae cell, measure dry cell weight and fat content, under-60 DEG C of conditions, algae dried bean noodles weight is measured in vacuum lyophilization to constant weight, calculates yield of biomass, and adopts normal hexane: ethyl acetate method records total lipid content.Dry cell weight can reach 7.1g/L after testing, and fat content is the 46.1% of dry cell weight.
The preparation of embodiment 4 microalgae grease
(1) in bioreactor, add FSH-Y2 seed liquor and the micro-algae culture medium of embodiment 1 preparation, the volume ratio of FSH-Y2 seed liquor and culture medium is 1:20, culture medium adopts BG11 culture medium, and it is 11 that the pH value of culture medium controls, and the intensity of illumination of cultivation is 5000Lux, periodicity of illumination is 24h, the light dark time, wherein carbon dioxide content was 4v% than for 14:10, passing into the mixing gas of nitrogen and carbon dioxide.
(2), after cultivating 4 days, the volume ratio accessing the single needle algae SS-B1 seed liquor of embodiment 1 preparation, seed liquor and culture medium is 1:5, and the pH of culture medium controls 9, passes into the mixing gas of nitrogen and carbon dioxide, wherein CO2Content is 40v%.
(3) enter stable phase after cultivating 8 days, terminate to cultivate, harvested by centrifugation microalgae cell, measure dry cell weight and fat content, under-60 DEG C of conditions, algae dried bean noodles weight is measured in vacuum lyophilization to constant weight, calculates yield of biomass, and adopts normal hexane: ethyl acetate method records total lipid content.Dry cell weight can reach 7.2g/L after testing, and fat content is the 46.5% of dry cell weight.
Embodiment 5 utilizes flue gas to prepare microalgae grease
Preparation condition is identical with embodiment 2, is different in that step (2) passes into gas and is replaced by containing SO2And CO2Flue gas, CO in flue gas2Content be 10v%～45v%, SO2Content is 200 × 10-6～600 × 10-6(v/v).
Inoculum concentration when scenedesmus obliquus FSH-Y2 seed liquor, single needle algae SS-B1 seed liquor single culture is 20%；During Mixed culture, the inoculum concentration of scenedesmus obliquus FSH-Y2 seed liquor and single needle algae SS-B1 seed liquor respectively 10%.Single culture condition is identical with the condition of Mixed culture, and starting to maintain pH is 10, after cultivating 3 days, passes into containing SO2And CO2Flue gas, CO in flue gas2Content be 10v%～45v%, SO2Content is 200 × 10-6～600 × 10-6(v/v) pH, maintaining reaction system is 9.
Entering stable phase after cultivating 8 days, terminate to cultivate, centrifugal collection frustule, under-60 DEG C of conditions, algae dried bean noodles weight is measured in vacuum lyophilization to constant weight, calculates yield of biomass, and adopts normal hexane: ethyl acetate method records total lipid content, and result is as shown in table 3.
The culture effect of table 3 Mixed culture and single algae
From table 3, relative to single algae, two-step method Mixed culture is possible not only to the CO of enduring high-concentration2, and certain density SO can be tolerated2, it is thus achieved that higher Biomass and fat content.As can be seen here, it is possible to use two-step method Mixed culture and flue gas prepare microalgae grease, namely achieve the production of oils and fats, waste gas can be purified simultaneously.
The preparation of comparative example 1 microalgae grease
Preparation condition is identical with embodiment 2, is different in that FSH-Y2 seed liquor and single needle algae SS-B1 seed liquor are cultivating initial addition in reactor.Entering stable phase after cultivating 7 days, terminate to cultivate, harvested by centrifugation microalgae cell, measure dry cell weight and fat content, dry cell weight can reach 6.1g/L, and fat content is the 45.6% of dry cell weight.
The preparation of comparative example 2 microalgae grease
Preparation condition is identical with embodiment 3, is different in that FSH-Y2 seed liquor and single needle algae SS-B1 seed liquor are cultivating initial addition in reactor.Entering stable phase after cultivating 7 days, terminate to cultivate, harvested by centrifugation microalgae cell, measure dry cell weight and fat content, dry cell weight can reach 5.9g/L, and fat content is the 45.1% of dry cell weight.
Claims (10)
1. the method that a microalgae Mixed culture produces oils and fats, it is characterised in that include following content: micro-algae culture medium and scenedesmus obliquus FSH-Y2 seed liquor are joined in bioreactor by (1), the pH value regulating cultivating system is 10～12, passes into CO in gas2Content control at below 5v%, cultivate 2～5 days；(2) pH value regulating cultivating system is 8～10, accesses single needle algae SS-B1 seed liquor and carries out Mixed culture, passes into CO in gas2Content is 5v%～45v%；(3) it is cultured to growth stable phase to terminate, harvesting microalgae cell；Described scenedesmus obliquus (Scenedesmusobliqnus) FSH-Y2 and single needle algae (Monoraphidiumsp) SS-B1, respectively at JIUYUE in 2012 11 days be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number is CGMCCNo.6551 and CGMCCNo.7479 respectively on April 15th, 2013.
2. in accordance with the method for claim 1, it is characterised in that: step (1) FSH-Y2 algae strain frustule under the microscope is spindle, grows thickly, has cell envelope to wrap up, and color is bottle green；Single algae cell diameter is about 6～10 μm.
3. in accordance with the method for claim 1, it is characterized in that: the preparation method of step (1) scenedesmus obliquus FSH-Y2 seed liquor is as follows: be 10～12 by the pH regulator of culture medium, it it is 20～30 DEG C in temperature, periodicity of illumination is 24h, the light dark time is than for 14:10, intensity of illumination is 2000～10000Lux, and shaken cultivation is to exponential phase.
4. the method described in claim 1 or 3, it is characterised in that: the scenedesmus obliquus FSH-Y2 seed liquor added in step (1) bioreactor and the volume ratio of culture medium are 1:20～1:5.
5. in accordance with the method for claim 1, it is characterised in that: micro-algae culture medium adopts BG11, SE or BBM fluid medium.
6. in accordance with the method for claim 1, it is characterised in that: it is carbon dioxide and nitrogen, wherein CO that step (1) passes into gas2Content control at 3v%～5v%.
7. in accordance with the method for claim 1, it is characterised in that: the algae SS-B1 algae strain of step (2) single needle is a kind of green algate of fresh water, and frustule be the shape that comes into leaves, green, and algae kind length is 10～20 μm, wide 2～4 μm, includes pigment, and the flat board algae form that falls is S shape, bottle green.
8. the method described in claim 1 or 7, it is characterized in that: the preparation method of step (2) single needle algae SS-B1 seed liquor is as follows: be 7～9 by the pH regulator of culture medium, it it is 20～30 DEG C in temperature, periodicity of illumination is 24h, the light dark time is than for 14:10, intensity of illumination is 2000～10000Lux, and shaken cultivation is to exponential phase；The volume ratio of single needle algae SS-B1 seed liquor and culture medium is 1:10～1:4.
9. in accordance with the method for claim 1, it is characterised in that: the temperature of step (2) Mixed culture is 20～30 DEG C, and periodicity of illumination is 24h, and the light dark time, intensity of illumination was 2000～10000Lux than for 14:10.
10. in accordance with the method for claim 1, it is characterised in that: the gas that step (2) passes into uses waste gas or flue gas, wherein CO2Content is 10v%～45v%, SO2Content is 200 × 10-6～600 × 10-6(v/v).
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| CN117625397A (en) * | 2023-11-30 | 2024-03-01 | 山东省农业科学院 | Vermicelli wastewater treatment process based on microalgae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109880856A (en) * | 2017-12-06 | 2019-06-14 | 中国石油化工股份有限公司 | A kind of method of open production microalgae grease |
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| CN111349680A (en) * | 2018-05-25 | 2020-06-30 | 宋庆恒 | Process for preparing biodiesel by using single needle algae cells |
| CN111349680B (en) * | 2018-05-25 | 2022-12-27 | 深圳博成知识产权有限公司 | Process for preparing biodiesel by using single needle algae cells |
| CN117625397A (en) * | 2023-11-30 | 2024-03-01 | 山东省农业科学院 | Vermicelli wastewater treatment process based on microalgae |
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