CN103952312A - Freshwater Chlorella sorokiniana GS30 and application thereof - Google Patents
Freshwater Chlorella sorokiniana GS30 and application thereof Download PDFInfo
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- CN103952312A CN103952312A CN201310753837.9A CN201310753837A CN103952312A CN 103952312 A CN103952312 A CN 103952312A CN 201310753837 A CN201310753837 A CN 201310753837A CN 103952312 A CN103952312 A CN 103952312A
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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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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention relates to the technical field of biomass energy, and in particular to a freshwater chlorella strain (i) Chlorella sorokiniana (/i) GS03 and application thereof to industrial flue gas CO2 fixation and production of microalgae biodiesel. The chlorella strain (i) C.sorokiniana (/i) GS03 provided by the invention has a preservation number of CCTCC M2013607, and higher CO2 tolerance, higher fixation efficiency of high-concentration carbon dioxide and higher biomass yield and oil yield compared with the reported microalgae strains. Therefore, the invention can greatly reduce microalgal oil production cost, and provides a good production strain for algae for efficient fixation of CO2 in typical industrial flue gas environment and biodiesel production from microalgae oil, and shows great application prospect in the field of biomass energy.
Description
Technical field
The present invention relates to biomass energy technology field, be specifically related to a strain limnetic chlorella Chlorella sorokiniana GS03 and at industrial flue gas CO
2fix and produce the application in micro-algae biofuel.
Background technology
Because of a large amount of uses of global fossil oil, " Greenhouse effect " increase the weight of day by day, the CO that combustion of fossil fuel brings
2account for total the more than 65% of " Greenhouse effect " gas.Therefore, the industry CO such as petrochemical industry, smelting, electric power
2reduction of discharging becomes the emphasis of Sustainable development.
In the last few years, some developed countries adopted various physics, chemistry and biological method research CO
2collection, concentrated and fixing and transform.But compared with conventional physical chemistry CO
2admittedly deposit the features such as method cost is high, energy consumption is large, environment friendly is poor, biological CO
2fixation method is main on the earth and carbon mode the most admittedly, and meets the most nature carbon cycle rule, is environmentally friendly and Sustainable development method resource-conserving.
Along with socio-economic development and the sustainable growth to energy demand, the scarcity of petrochemical industry resource day by day highlights, and therefore finding a kind of lasting renewable, environmentally friendly energy form becomes the emphasis that various countries scientist pays close attention to.In numerous renewable energy source (wind energy, Geothermal energy, water energy, sun power etc.), biofuel has high similarity with it at the aspect such as energy density, incendivity and petrifaction diesel, even exceed petrifaction diesel, and there is easy degraded, not sulfur-bearing and arene compounds, the characteristic such as environmentally friendly substantially, be subject to the extensive concern of countries in the world.Some developed countries have just started commercially producing of biofuel in early 1990s, and mainly taking soybean and rapeseed oil as raw materials for production.China's Biodiesel rises more late, and at present, the raw material of China's production biofuel is mainly oil plant wood fruit and the biodiesels etc. such as the oil crops such as soybean, rape, oil palm and manioca.But, this taking traditional agriculture as basic production of biodiesel mode, not only yield poorly, cost is high, and do not meet " not with agricultural strive grain strive ground " cardinal principle, the current recovery problem of waste gas grease is also difficult to good solution simultaneously, can not meet the demand of energy market in the future, therefore can not meet the demand of Biodiesel to raw material sustainable growth.Research finds that many algae can also produce a large amount of greases by Fast Growth in vivo, are called as produce oil algae.Under certain condition, micro-algae is by CO for oil-producing microalgae
2, the carbon source such as carbohydrate, hydrocarbon polymer and common grease is converted into the greases of a large amount of storages in microalgae cell, and fat content exceedes the microalgae of biological total amount 20%.Micro-algae because oleaginousness is high, the advantage such as be easy to cultivate, yield per unit is large and be regarded as a new generation or even uniquely can realize the biodiesel raw material that substitutes petrifaction diesel completely.
Reduce discharging typical industry stack gas CO by micro-algae
2in time, produces grease and has become the focus of countries in the world research with preparation biodiesel technology.The U.S., the government in Australia, Japan, West Europe, India and South Africa or enterprise drop into huge fund and carry out the research of micro-algae biofuel.The National Alliance of famous American laboratory and scientist's composition, Live Fuels company in this alliance has announced " the miniature manhattan project " supported by National Energy Board, be micro-algae energy plan, plan to realize in 2010 the industrialization that micro-algae is prepared biofuel.Japan internationality trade and the Department of Industry had once subsidized the project of a key name for " technical plan is upgraded in earth research ".This project utilizes micro-algae to carry out biological fixation of C O
2, and put forth effort to develop Closed photobioreactor technology, absorb the CO in the flue gas of fuel-burning power plant by micro-algae
2carry out production biomass energy.2008, carbon back King Company of Britain started at present the algae bio fuel project of maximum in the world, drops into 2,600 ten thousand pounds for developing correlation technique and Infrastructure, and this project expects the year two thousand twenty and realizes commercialization.AlgaeLink company of Holland is a transnational company that has industrialization algae culture equipment and algae oil processing technology, and the said firm is to its reactor of global marketing, and correlation technique support is provided.In April, 2008, the said firm and the Royal Dutch Airlines signed the agreement of utilizing algae oil exploitation Aviation Fuel.In addition, a company of Israel externally shown in 2007 utilize marine alga absorb CO
2, converting solar energy into the technology of biomass energy, every 5 kilograms of algae can produce 1 liter of fuel.
In recent years, micro-algae biodiesel technology has also caused the attention of scientific research institution of the Chinese government and enterprise, is listed in one of main project of Department of Science and Technology's 863 Program, 973 basis planning, 12 biotech development planning.Each colleges and universities and scientific research institutions have all carried out the research of this respect, mainly concentrate on the screening of algae kind, the design of micro-algae culture bioreactors and Downstream processing technology.At present, some enterprises and research institution are also carrying out the pilot scale cultivation of micro-algae production biofuel." the CO of Xinao Technology Development Co., Ltd
2-micro-algae-biofuel " gordian technique research project is by pilot scale, and build 280hm in Dalate Banner, the Inner Mongol
2both culturing microalgae base.2009, China Petrochemical Corp. and Chinese Academy of Sciences's combined launch " micro-algae biofuel complete set technology " project, objective plan to 2015 year completes ton industrial installation.
Although micro-algae biofuel is feasible at present technically, compared with fossil diesel fuel, the production of micro-algae biofuel is faced with two "bottlenecks", and the production of micro-algae biofuel is seriously obstructed.The micro-algae production of biodiesel of first cost is high, therefore product price is also higher, also cannot adapt to the current market requirement, and still limited because of the large scale culturing level of micro-algae, and this also makes holding at high price of micro-algae biofuel; The 2nd, the low following process treating processes cost that causes of micro-algal biomass autotrophy culturing cell density is high.And by " micro-algae production of biodiesel " and " CO
2reduce discharging " height Coupling Research, not only can reduce micro-algae production of biodiesel cost, and can improve microalgae cell culture density, what is more important can realize the reduction of discharging of greenhouse gases.This new concept provides new way for the situation of alleviating current energy scarcity and Greenhouse effect, also, for the production of micro-algae biofuel provides a kind of feasibility study scheme, can create a kind of situation of doulbe-sides' victory.
Summary of the invention
The object of the invention is to overcome the defect of prior art, a strain advantage characteristic limnetic chlorella is provided, this chlorella algae strain can transform industrial flue gas CO
2for starting material prepared by biofuel, the improvement that this invention can effectively the solve current industrial source atmospheric polluting material microalgae grease that is simultaneously coupled is produced the problem that reduces micro-algae production of biodiesel high cost.
First the present invention discloses a kind of chlorella strain C.sorokiniana GS03, and its preserving number is: CCTCC M2013607.
The present invention near taking from Tianshui city power station subacidity waste water sample separation screening to a strain freshwater microalgae, called after Chlorella sorokiniana GS03, this bacterial strain on November 27th, 2013 in the center preservation of Chinese Typical Representative culture collection, preserving number is CCTCC M2013607.
The algae strain C.sorokiniana GS03 that the present invention separates is when taking BG-11 substratum as basic nutritive salt culture medium culturing, this algae strain is spherical or avette, diameter, between 2～4 μ m, has the chromatoplast of Zhousheng, cup-shaped or a sheet in each cell, have 1 nucleus; Cell walls is thinner.Under common culture condition, measuring its protein content is 50% left and right, and fat content is 22%～43%, and polysaccharide content is 25% left and right, is rich in greenery element.
Through Morphological Identification and 18S rRNA, ITS-2 and rbcL extension increasing sequence are grown tree analysis, determine that this freshwater microalgae algae strain is chlorella, (according to international naming rule: generic name+kind of name+strain name is named this algae strain, generic name, kind name, strain name are respectively Chlorella, sorokiniana and GS03), called after Chlorella sorokiniana GS03(can be abbreviated as C.sorokiniana GS03), preserving number is: CCTCC M2013607.The applicable culture temperature of this algae strain and pH scope are wider, and suitable culture temperature scope is 10～45 DEG C, and appropriate pH value scope is 4.0～11, the CO that can tolerate
2concentration range is between 0.03～45v/v%, is preferably 0.03～30v/v%.
Preferably, in chlorella C.sorokiniana GS03 of the present invention, fat content is 22～43wt%.
Preferred, in described chlorella C.sorokiniana GS03, the content > 93wt% of C16 and C18 short chain fatty acid.Visible, the lipid acid composition of grease is mainly C16, C18 short chain fatty acid, is applicable to the production of biofuel.
Second aspect present invention discloses chlorella C.sorokiniana GS03 fermentation fixation of C O
2method, step is as follows:
1) preparation of seed liquor: under aseptic condition, the mono-algae of picking chlorella C.sorokiniana GS03 is fallen in the BG-11 nutrient solution of sterilizing and is cultured to logarithmic phase, obtains micro-algae seed liquor;
2) biological fermentation: micro-algae seed liquor of cultivating through logarithmic phase is inoculated in the BG-11 nutrient solution of sterilizing and is cultivated, culture temperature is 10～45 DEG C, Medium's PH Value 4.0～11, intensity of illumination is 2500～15000lux, Light To Dark Ratio is 8～24:16～0, in culturing process, pass into sterile air, air flow is 0.2～1vvm, wherein CO
2content is 0.03～30v/v%, cultivates after 10-14 days and finishes to cultivate, results chlorella C.sorokiniana GS03 fermented liquid.
Seed liquor culture condition and fermentation culture conditions are basic identical.
Preferably, described in step 1), the culture condition of algae strain seed liquor is: culture temperature is 10～45 DEG C, Medium's PH Value 4.0～11, intensity of illumination is 2500～15000lux, and Light To Dark Ratio is 8～24:16～0, passes into sterile air in culturing process, air flow is 0.2～1vvm, wherein CO
2content is 0.03v/v%.
Preferably, step 2) inoculum size of described algae strain seed liquor is 5～15v/v%.
Light To Dark Ratio of the present invention is 8～24:16～0, refers to that every day, light application time was A hour, and be B hour interlunation, and A:B is 8～24:16～0, and A+B=24 hour.
Preferably, step 2) condition of described fermentation culture is: 28 DEG C of temperature, Medium's PH Value 8.0, intensity of illumination 7500lux, Light To Dark Ratio is 12:12,, in culturing process, passing into sterile air, air flow is 0.33vvm, CO
2content is preferably 0.03～15v/v%, cultivates secondary fermentation in 14 days and finishes, and obtains fermented liquid.
Third aspect present invention discloses the technique of chlorella C.sorokiniana GS03 fermenting and preparing biological diesel oil, and processing step is as follows:
1. adopt aforementioned chlorella C.sorokiniana GS03 fermentation fixation of C O
2method prepare chlorella C.sorokiniana GS03 fermented liquid;
2. fermented liquid carries out solid-liquid separation, collects microalgae cell and obtains algae mud;
The algae mud of previous step gained is dry 3., obtain algae powder;
4. algae powder previous step being obtained is prepared fatty acid methyl ester by boron trifluoride catalysis method.
Preferably, step 2 also comprises the CO that detects algae mud
2fixed efficiency, and the detection of algae strain biomass productive rate.The detection of micro algae biomass and micro-algae productive rate is routine analysis process, and concrete grammar can be with reference to prior art.CO
2the mensuration of fixed efficiency, can utilize the average carbon content of microalgae cell and biomass productive rate indirect calculation to learn.
Preferably, dry method is 60～90 DEG C of oven dry described in step 2, or vacuum lyophilization.
Preferably, step 3 also comprises the fat content of algae powder is detected.The measuring method of fat content is conventional method of analysis, specifically can adopt chloroform methanol method to analyze fat content.
Preferably, step 4 also comprises to be analyzed the composition of fatty acid methyl ester of preparation, for analyze kind and the relative content of fatty acid methyl ester by chromatography of gases mass-spectrometric technique.
Last aspect of the present invention discloses the application of chlorella C.sorokiniana GS03 at biomass energy source domain.
Preferably, chlorella C.sorokiniana GS03 be applied as stabilizing carbon dioxide production biofuel.
Beneficial effect: limnetic chlorella C.sorokiniana GS03 provided by the invention can tolerate 0.03%～45%(v/v) CO
2, can efficiently fix 0.03%～15%(v/v) and CO
2, CO
2fixed efficiency is 110～660mg/L/d, and biomass (dry weight) concentration is 680～4000mg/L.The limnetic chlorella C.sorokiniana GS03 that the present invention separates can also be at efficient emission-reducing high-concentration industrial stack gas CO
2basis on high yield biomass and microalgae grease.This strain limnetic chlorella can, in air lift type photosynthetic organism reactor, efficiently utilize CO
2, form biomass and accumulate microalgae grease fast, fat content can reach 22%～43%, its oil fatty acid composition is mainly C16, C18 short chain fatty acid (> 93%), its oil fatty acid composition is mainly C16, C18 short chain fatty acid, is applicable to the production of biofuel.
This limnetic chlorella has higher carbonic acid gas tolerance, higher high concentration carbon dioxide fixed efficiency and biomass productive rate and grease productive rate compared with micro-algae algae strain of reporting.Therefore can greatly reduce microalgae grease production cost, be CO in efficient fixing typical industry flue gas environment
2and On Preparation of Biodiesel From Microalgal Oil provides a strain good production algae kind.
Algae strain preservation information of the present invention is as follows:
Algae strain title: chlorella C.sorokiniana GS03;
Preserving number is: CCTCC M2013607;
Preservation date: on November 27th, 2013;
Depositary institution's title: Chinese Typical Representative culture collection center;
Depositary institution is called for short: CCTCC;
Depositary institution address: wuchang, wuhan Luo Jia Shan street Wuhan University Life Science College.
brief description of the drawings
Fig. 1: the frustule stereoscan photograph of limnetic chlorella C.sorokiniana GS03
Fig. 2: the frustule optical microscope photograph of limnetic chlorella C.sorokiniana GS03
Fig. 3: the frustule fat inside of limnetic chlorella C.sorokiniana GS03 is dripped dyeing Photomicrograph
Fig. 4: the HPLC of biofuel fatty acid methyl ester composition analyzes
Embodiment
By following specific embodiment, the present invention will be further elaborated, and following examples only, for explanation, limit the scope of the invention and be not used in.
Embodiment 1
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask that contains 30ml sterilizing BG-11 nutrient solution, leave standstill and cultivate in illumination cultivation frame, 28 DEG C of temperature, under 7500lux intensity of illumination, cultivate 12 days, grow into index latter stage with 1:10 inoculative proportion enlarged culturing.
Preparation BG-11 substratum 2.4L, accesses the cell of enlarged culturing, inoculating cell optical density(OD) OD
680=1.2.Initial pH4.0,15 DEG C of temperature, Light To Dark Ratio is 8:16, under 15000lux intensity of illumination, passes into the air (CO of 0.5vvm
2content is 0.03%(v/v)), cultivate 14 days.
Centrifugal collection frustule, vacuum freeze drying is dry, claims algae powder and calculates dry weight.Algae powder biomass concentration is 680mg/L, and biomass productive rate is 70mg/L/d, CO
2fixed efficiency is 110mg/L/d, and it is 22% that chloroform methanol method is measured fat content.The composition of biofuel fatty acid methyl ester is mainly the short chain fatty acid of C16, C18, accounts for the more than 98% of total fatty acids methyl esters composition, the stereoscan photograph of microalgae cell, optical microscope photograph, and Fig. 1-3 are shown in by fat granule dyeing photo.
Embodiment 2
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask that contains 30ml sterile medium, leave standstill and cultivate in illumination cultivation frame, 28 DEG C of temperature, under 7500lux intensity of illumination, cultivate 12 days, grow into index latter stage with 5% inoculative proportion enlarged culturing.
Preparation BG-11 substratum 3.1L, accesses the cell of enlarged culturing.Initial pH7.0, temperature 45 C, Light To Dark Ratio is 24:0, under 2500lux intensity of illumination, passes into the 30%CO of 1vvm
2(v/v), cultivate 10 days.
Centrifugal collection frustule, vacuum freeze drying is dry, claims algae dried bean noodles to weigh and calculate.Algae powder biomass concentration is 2400mg/L, and biomass productive rate is 360mg/L/d, CO
2fixed efficiency is 660mg/L/d, and it is 35% that chloroform methanol method is measured fat content, and the composition of biofuel fatty acid methyl ester is mainly the short chain fatty acid of C16, C18, accounts for the more than 93% of total fatty acids methyl esters composition.Prepare fatty acid methyl ester by classical boron trifluoride catalysis method.
Embodiment 3
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask that contains 30ml sterile medium, leave standstill and cultivate in illumination cultivation frame, 28 DEG C of temperature, under 7500lux intensity of illumination, cultivate 12 days, grow into index latter stage with 1:10 inoculative proportion enlarged culturing.
Preparation BG-11 substratum 3.1L, accesses the cell of enlarged culturing.Initial pH11.0,10 DEG C of temperature, Light To Dark Ratio is 12:12, under 9500lux intensity of illumination, passes into the 45%CO of 0.2vvm
2(v/v), cultivate 14 days.
Centrifugal collection frustule, vacuum freeze drying is dry, claims algae dried bean noodles to weigh and calculate.Algae powder biomass concentration is 2500mg/L, and biomass productive rate is 260mg/L/d, CO
2fixed efficiency is 480mg/L/d, and it is 36% that chloroform methanol method is measured fat content, and the composition of biofuel fatty acid methyl ester is mainly the short chain fatty acid of C16, C18, accounts for the more than 98% of total fatty acids methyl esters composition.
Embodiment 4
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask that contains 30ml sterile medium, leave standstill and cultivate in illumination cultivation frame, 28 DEG C of temperature, intensity of illumination 7500lux, cultivate 12 days, grow into index latter stage with 1:10 inoculative proportion enlarged culturing.
Preparation BG-11 substratum, accesses the cell of enlarged culturing by 15% inoculum size.Initial pH8.0,28 DEG C of temperature, under 7500lux intensity of illumination, pass into the 15%CO of 0.33vvm
2(v/v), cultivate 14 days.
Centrifugal collection frustule, vacuum freeze drying is dry, claims algae dried bean noodles to weigh and calculate.Algae powder biomass concentration is 2700mg/L, and biomass productive rate is 250mg/L/d, CO
2fixed efficiency is 460mg/L/d, and it is 43% that chloroform methanol method is measured fat content, and the composition of biofuel fatty acid methyl ester is mainly the short chain fatty acid of C16, C18, accounts for the more than 98% of total fatty acids methyl esters composition, and stratographic analysis figure is shown in Fig. 4.
Claims (7)
1. a chlorella strain Chlorella sorokiniana GS03, its preserving number is: CCTCC M2013607.
2. an also fixation of C O of chlorella Chlorella sorokiniana GS03 fermentation
2method, step is as follows:
1) preparation of seed liquor: under aseptic condition, described in picking claim 1, the mono-algae of chlorella Chlorella sorokiniana GS03 is fallen in the BG-11 nutrient solution of sterilizing and is cultured to logarithmic phase, obtains micro-algae seed liquor;
2) biological fermentation: micro-algae seed liquor of cultivating through logarithmic phase is inoculated in the BG-11 nutrient solution of sterilizing and is cultivated, culture temperature is 10～45 DEG C, Medium's PH Value 4.0～11, intensity of illumination is 2500～15000lux, Light To Dark Ratio is 8～24:16～0, in culturing process, pass into sterile air, air flow is 0.2～1vvm, wherein CO
2content is 0.03～30v/v%, cultivates after 10～14 days and finishes to cultivate, results chlorella Chlorella sorokiniana GS03 fermented liquid.
3. method as claimed in claim 2, it is characterized in that, described in step 1), the culture condition of algae strain seed liquor is: culture temperature is 10～45 DEG C, Medium's PH Value 4.0～11, intensity of illumination is 2500～15000lux, and Light To Dark Ratio is 8～24:16～0, passes into sterile air in culturing process, air flow is 0.2～1vvm, wherein CO
2content is 0.03v/v%.
4. method as claimed in claim 2, is characterized in that step 2) condition of described fermentation culture is: 28 DEG C of temperature, Medium's PH Value 8.0, intensity of illumination 7500lux, Light To Dark Ratio is 12:12, in culturing process, pass into sterile air, air flow is 0.33vvm, CO
2content is preferably 0.03～15v/v%, cultivates secondary fermentation in 14 days and finishes, and obtains fermented liquid.
5. a technique for chlorella strain Chlorella sorokiniana GS03 fermenting and preparing biological diesel oil, step is as follows:
(1) chlorella strain Chlorella sorokiniana GS03 fermentation fixation of C O described in the arbitrary claim of employing claim 2-4
2method prepare chlorella Chlorella sorokiniana GS03 fermented liquid;
(2) fermented liquid carries out solid-liquid separation, collects microalgae cell and obtains algae mud;
(3) the algae mud of previous step gained is dry, obtain algae powder;
(4) algae powder previous step being obtained is prepared fatty acid methyl ester by boron trifluoride catalysis method.
Described in claim 1 chlorella strain Chlorella sorokiniana GS03 in the application of biomass energy source domain.
7. application as claimed in claim 6, is characterized in that, described in be applied as stabilizing carbon dioxide production biofuel.
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| CN106916747B (en) * | 2015-12-25 | 2020-06-23 | 国投生物科技投资有限公司 | Chlorella sorokiniana strain and culture method and application thereof |
| CN113481102A (en) * | 2021-08-19 | 2021-10-08 | 山西农业大学 | Chlorella sorokiniana strain as well as culture method and application thereof |
| CN113481102B (en) * | 2021-08-19 | 2022-08-26 | 山西农业大学 | Chlorella sorokiniana strain as well as culture method and application thereof |
| CN113817611A (en) * | 2021-11-03 | 2021-12-21 | 新疆金正生物科技有限公司 | Culture medium and autotrophic culture method for desert Chlorella foenum-graecum |
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