CN103952312B - One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof - Google Patents

One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof Download PDF

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CN103952312B
CN103952312B CN201310753837.9A CN201310753837A CN103952312B CN 103952312 B CN103952312 B CN 103952312B CN 201310753837 A CN201310753837 A CN 201310753837A CN 103952312 B CN103952312 B CN 103952312B
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algae
chlorella
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microalgae
biodiesel
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CN103952312A (en
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缪晓玲
巩三强
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Shanghai Jiaotong University
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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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Abstract

The present invention relates to biomass energy technology field, specifically related to a strain limnetic chlorella <i>Chlorella? sorokiniana? </i>GS03 and at fixing industrial stack gas CO 2with the application in production microalgae biodiesel.Does is preserving number of the present invention CCTCC? the chlorella algae strain <i>C.sorokinianaLEssT.LTssT.L T/i> of M2013607? GS03, has higher carbonic acid gas tolerance, higher high concentration carbon dioxide fixed efficiency and Biomass yield and grease productive rate compared with the micro-algae algae strain reported.Therefore microalgae grease production cost can greatly be reduced, for efficiently fixing CO in typical industry flue gas environment 2and On Preparation of Biodiesel From Microalgal Oil provides the excellent production algae kind of a strain, has great application prospect in field of biological energy source.

Description

One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof
Technical field
The present invention relates to biomass energy technology field, be specifically related to a strain limnetic chlorella ChlorellasorokinianaGS03 and at industrial flue gas CO 2application in fixing and production microalgae biodiesel.
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 more than 65% of total " Greenhouse effect " gas.Therefore, the industry CO such as petrochemical industry, smelting, electric power 2reduce discharging the emphasis becoming Sustainable development.
In the last few years, some developed countries adopted various physics, chemistry and biological method to study 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 the most effectively consolidates carbon mode, and meets nature carbon cycle rule the most, is Sustainable development method that is environmentally friendly and resource-conserving.
Along with socio-economic development and the sustainable growth to energy demand, the scarcity of fossil resources day by day highlights, and therefore finds the emphasis that a kind of lasting renewable, environmentally friendly energy form becomes various countries scientist concern.In numerous renewable energy sources (wind energy, Geothermal energy, water energy, sun power etc.), biofuel has high similarity with petrifaction diesel with it in energy density, incendivity etc., even exceed petrifaction diesel, and there is easy degraded, substantially not sulfur-bearing and arene compounds, the characteristic such as environmentally friendly, be subject to the extensive concern of countries in the world.Some developed countries just start commercially producing of biofuel in early 1990s, and mainly with soybean and rapeseed oil for raw materials for production.China Biodiesel rises more late, and at present, the raw material of China's production biofuel is oil plant wood fruit and the biodiesel etc. such as the oil crops such as soybean, rape, oil palm and manioca mainly.But, this production of biodiesel mode based on traditional agriculture, not only yield poorly, cost is high, and do not meet the cardinal principle of " do not strive grain with agricultural and strive ground ", the recovery problem that waste gas grease is current simultaneously is also difficult to good solution, the demand of energy market in the future can not be met, therefore can not meet the demand of Biodiesel to raw material sustainable growth.Research finds that many algae can grow fast and produce a large amount of greases in vivo, is called as produce oil algae.Namely under certain condition, micro-algae is by CO for oil-producing microalgae 2, carbohydrate, the carbon source such as hydrocarbon polymer and common grease be converted into the grease of a large amount of storage in microalgae cell, and fat content exceedes the microalgae of biological total amount 20%.Micro-algae is regarded as biodiesel raw material that is of new generation or even that uniquely can realize substituting completely petrifaction diesel because of advantages such as oleaginousness are high, be easy to cultivation, yield per unit is large.
Typical industry stack gas CO is reduced discharging by micro-algae 2while produce grease with prepare biodiesel technology become countries in the world study focus.The U.S., the government in Australia, Japan, West Europe, India and South Africa or enterprise drop into the research that huge fund carries out microalgae biodiesel.The NationalAlliance of famous American laboratory and scientist's composition, LiveFuels company in this alliance announces " the miniature manhattan project " supported by National Energy Board, i.e. micro-algae energy plan, plans the industrialization realizing micro-algae prepare biofuel in 2010.Japan internationality trade and the Department of Industry had once subsidized the project that a key name is " plan of earth research update method ".This project utilizes micro-algae to carry out biological fixation of C O 2, and put forth effort exploitation 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 algae bio fuel project maximum in the world at present, and drop into 2,600 ten thousand pounds for developing correlation technique and Infrastructure, this project expects the year two thousand twenty and realizes commercialization.AlgaeLink company of Holland is a transnational company having industrialization algae culture equipment and algae oil processing technology, and the said firm to its reactor of global marketing, and provides correlation technique support.In April, 2008 the said firm and the Royal Dutch Airlines signed and utilize algae oil to develop the agreement of Aviation Fuel.In addition, a company of Israel externally illustrated in 2007 and utilizes marine alga to absorb CO 2, convert solar energy into the technology of biomass energy, every 5 kilograms of algae can produce 1 liter of fuel.
In recent years, microalgae biodiesel technology also result in the attention of scientific research institution of the Chinese government and enterprise, is listed in one of Department of Science and Technology's 863 Program, 973 main projects that basis is planned, 12 biotech developments are planned.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, microdisk electrode bioreactor design and down stream processing technique.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 " key technology research project by pilot scale, and builds 280hm in Dalate Banner, the Inner Mongol 2both culturing microalgae base.2009, China Petrochemical Corp. and Chinese Academy of Sciences's combined launch " microalgae biodiesel complete set technology " project, objective plan completed ton industrial installation by 2015.
Although microalgae biodiesel is feasible at present technically, compared with fossil diesel fuel, the production of microalgae biodiesel is faced with two "bottlenecks", and the production of microalgae biodiesel is seriously obstructed.First microalgae biodiesel production cost is high, therefore product price is also higher, also cannot adapt to the current market requirement, and because of the large scale culturing level of micro-algae still limited, this also makes holding at high price of microalgae biodiesel; Two is that the low following process treating processes cost that causes of microalgae biomass autotrophy culturing cell density is high.And by " microalgae biodiesel production " and " CO 2reduce discharging " height Coupling Research, not only can reduce microalgae biodiesel production cost, and can improve microalgae cell culture density, what is more important can realize the reduction of discharging of greenhouse gases.This new concept is alleviate the in short supply and situation that is Greenhouse effect of current energy source to provide new way, also for the production of microalgae biodiesel 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 the defect overcoming prior art, provide a strain advantage characteristic limnetic chlorella, this chlorella algae strain can conversion industry stack gas CO 2for starting material prepared by biofuel, the improvement that this invention effectively can the solve current industrial source atmospheric polluting material microalgae grease that is simultaneously coupled is produced and is reduced the too high problem of microalgae biodiesel production cost.
First the present invention discloses a kind of chlorella strain C.sorokinianaGS03, and its preserving number is: CCTCCM2013607.
The present invention is from taking near Tianshui city power station subacidity waste water sample separation screening to a strain freshwater microalgae, called after ChlorellasorokinianaGS03, this bacterial strain on November 27th, 2013 in China typical culture collection center preservation, preserving number is CCTCCM2013607.
The algae strain C.sorokinianaGS03 that the present invention is separated is based on BG-11 substratum during nutritive salt culture medium culturing, this algae strain is spherical or avette, diameter, between 2 ~ 4 μm, has the chromatoplast of a Zhousheng, cup-shaped or sheet, has 1 nucleus in each cell; Cell walls is thinner.Under normal culture conditions, measuring its protein content is about 50%, and fat content is 22% ~ 43%, and polysaccharide content is about 25%, is rich in greenery element.
Through Morphological Identification and 18SrRNA, ITS-2 and rbcL extension increasing sequence grows tree analysis, determine that this freshwater microalgae algae strain is chlorella, (according to Intemational Nomenclature 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 ChlorellasorokinianaGS03 (can be abbreviated as C.sorokinianaGS03), preserving number is: CCTCCM2013607.This algae strain is applicable to culture temperature and pH scope is comparatively wide, and suitable culture temperature scope is 10 ~ 45 DEG C, and optimum pH 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.sorokinianaGS03 of the present invention, fat content is 22 ~ 43wt%.
Preferred, in described chlorella C.sorokinianaGS03, 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.sorokinianaGS03 and ferments and fixation of C O 2method, step is as follows:
1) preparation of seed liquor: under aseptic condition, the mono-algae of picking chlorella C.sorokinianaGS03 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 cultivates, 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, pass into sterile air in culturing process, air flow is 0.2 ~ 1vvm, wherein CO 2content is 0.03 ~ 30v/v%, cultivates after 10-14 days and terminates to cultivate, results chlorella C.sorokinianaGS03 fermented liquid.
Seed liquor culture condition and fermentation culture conditions are substantially identical.
Preferably, step 1) culture condition of described 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, and refer to that every day, light application time was A hour, interlunation is B hour, A:B is 8 ~ 24:16 ~ 0, and A+B=24 hour.
Preferably, step 2) condition of described fermentation culture is: temperature 28 DEG C, Medium's PH Value 8.0, intensity of illumination 7500lux, Light To Dark Ratio is 12:12, pass into sterile air in culturing process, air flow is 0.33vvm, CO 2content is preferably 0.03 ~ 15v/v%, cultivates secondary fermentation in 14 days and terminates, and obtains fermented liquid.
Third aspect present invention discloses the technique of chlorella C.sorokinianaGS03 fermenting and preparing biological diesel oil, and processing step is as follows:
1. adopt aforementioned chlorella C.sorokinianaGS03 to ferment and fixation of C O 2method prepare chlorella C.sorokinianaGS03 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. the algae powder that previous step obtains is prepared fatty acid methyl ester by boron trifluoride catalysis method.
Preferably, step 2 also comprises the CO detecting algae mud 2fixed efficiency, and the detection of algae strain Biomass yield.Micro algae biomass and micro-algae productive rate be detected as routine analytical procedures, concrete grammar can with reference to prior art.CO 2the mensuration of fixed efficiency, can utilize microalgae cell mean carbon content and Biomass yield indirect calculation to learn.
Preferably, method dry described in step 2 is 60 ~ 90 DEG C of oven dry, or vacuum lyophilization.
Preferably, step 3 also comprises and detecting the fat content of algae powder.The measuring method of fat content is conventional method of analysis, and chloroform methanol method specifically can be adopted to analyze fat content.
Preferably, step 4 also comprises to be analyzed the composition of the fatty acid methyl ester of preparation, for being analyzed 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.sorokinianaGS03 at biomass energy source domain.
Preferably, chlorella C.sorokinianaGS03 be applied as stabilizing carbon dioxide and production biofuel.
Beneficial effect: limnetic chlorella C.sorokinianaGS03 provided by the invention can tolerate 0.03% ~ 45% (v/v) CO 2, efficiently can fix 0.03% ~ 15% (v/v) CO 2, CO 2fixed efficiency is 110 ~ 660mg/L/d, and biomass (dry weight) concentration is 680 ~ 4000mg/L.The limnetic chlorella C.sorokinianaGS03 that the present invention is separated can also 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 photosynthesis physiological target, efficiency utilization CO 2, form biomass 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 yield and grease productive rate compared with the micro-algae algae strain reported.Therefore microalgae grease production cost can greatly be reduced, for efficiently fixing CO in typical industry flue gas environment 2and On Preparation of Biodiesel From Microalgal Oil provides the excellent production algae kind of a strain.
Algae strain preservation information of the present invention is as follows:
Algae strain title: chlorella C.sorokinianaGS03;
Preserving number is: CCTCCM2013607;
Preservation date: on November 27th, 2013;
Depositary institution's title: China typical culture collection center;
Depositary institution is called for short: CCTCC;
Depositary institution address: wuchang, wuhan Luo Jia Shan street Wuhan University Life Science College.
Accompanying drawing explanation
The frustule stereoscan photograph of Fig. 1: limnetic chlorella C.sorokinianaGS03
The frustule optical microscope photograph of Fig. 2: limnetic chlorella C.sorokinianaGS03
The frustule fat inside of Fig. 3: limnetic chlorella C.sorokinianaGS03 drips dyeing Photomicrograph
Fig. 4: the HPLC of Biodiesel Fatty acid methyl esters composition analyzes
Embodiment
By following specific embodiment, the present invention will be further elaborated, following examples only for illustration of, and be not used in and limit the scope of the invention.
Embodiment 1
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask containing 30ml sterilizing BG-11 nutrient solution, in illumination cultivation frame quiescent culture, temperature 28 DEG C, 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, temperature 15 DEG C, Light To Dark Ratio is under 8:16,15000lux intensity of illumination, passes into the air (CO of 0.5vvm 2content is 0.03% (v/v)), cultivate 14 days.
Collected by centrifugation frustule, vacuum freeze drying is dry, claims algae powder and calculates dry weight.Algae powder biomass concentration is 680mg/L, and Biomass yield is 70mg/L/d, CO 2fixed efficiency is 110mg/L/d, and it is 22% that chloroform methanol method measures fat content.The composition of Biodiesel Fatty acid methyl esters is mainly the short chain fatty acid of C16, C18, account for more than 98% of total fatty acids methyl esters composition, the stereoscan photograph of microalgae cell, optical microscope photograph, and fat granule stained photographs is shown in Fig. 1-3.
Embodiment 2
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask containing 30ml sterile medium, in illumination cultivation frame quiescent culture, temperature 28 DEG C, 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 under 24:0,2500lux intensity of illumination, passes into the 30%CO of 1vvm 2(v/v), cultivate 10 days.
Collected by centrifugation frustule, vacuum freeze drying is dry, claims algae dried bean noodles weigh and calculate.Algae powder biomass concentration is 2400mg/L, and Biomass yield is 360mg/L/d, CO 2fixed efficiency is 660mg/L/d, and it is 35% that chloroform methanol method measures fat content, and the composition of Biodiesel Fatty acid methyl esters is mainly the short chain fatty acid of C16, C18, accounts for more than 93% of total fatty acids methyl esters composition.Fatty acid methyl ester is prepared by classical boron trifluoride catalysis method.
Embodiment 3
Under aseptic condition, on solid plate, picking list algae is fallen in the 100ml triangular flask containing 30ml sterile medium, in illumination cultivation frame quiescent culture, temperature 28 DEG C, 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, temperature 10 DEG C, Light To Dark Ratio is under 12:12,9500lux intensity of illumination, passes into the 45%CO of 0.2vvm 2(v/v), cultivate 14 days.
Collected by centrifugation frustule, vacuum freeze drying is dry, claims algae dried bean noodles weigh and calculate.Algae powder biomass concentration is 2500mg/L, and Biomass yield is 260mg/L/d, CO 2fixed efficiency is 480mg/L/d, and it is 36% that chloroform methanol method measures fat content, and the composition of Biodiesel Fatty acid methyl esters is mainly the short chain fatty acid of C16, C18, accounts for 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 containing 30ml sterile medium, in illumination cultivation frame quiescent culture, temperature 28 DEG C, 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, temperature 28 DEG C, under 7500lux intensity of illumination, passes into the 15%CO of 0.33vvm 2(v/v), cultivate 14 days.
Collected by centrifugation frustule, vacuum freeze drying is dry, claims algae dried bean noodles weigh and calculate.Algae powder biomass concentration is 2700mg/L, and Biomass yield is 250mg/L/d, CO 2fixed efficiency is 460mg/L/d, and it is 43% that chloroform methanol method measures fat content, and the composition of Biodiesel Fatty acid methyl esters is mainly the short chain fatty acid of C16, C18, and account for more than 98% of total fatty acids methyl esters composition, stratographic analysis figure is shown in Fig. 4.

Claims (6)

1. a chlorella strain ChlorellasorokinianaGS03, its preserving number is: CCTCCM2013607; The temperature range that described algae strain is suitable for cultivating is 10 ~ 45 DEG C; The CO that can tolerate 2concentration range is 0.03 ~ 45%v/v.
2. a chlorella ChlorellasorokinianaGS03 ferments and fixation of C O 2method, step is as follows:
1) preparation of seed liquor: under aseptic condition, described in picking claim 1, the mono-algae of chlorella ChlorellasorokinianaGS03 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 cultivates, 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, pass into sterile air in culturing process, air flow is 0.2 ~ 1vvm, wherein CO 2content is 0.03 ~ 30%v/v, cultivates after 10 ~ 14 days and terminates to cultivate, results chlorella ChlorellasorokinianaGS03 fermented liquid.
3. method as claimed in claim 2, it is characterized in that, step 1) culture condition of described 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 0.03%v/v.
4. method as claimed in claim 2, is characterized in that, step 2) condition of described fermentation culture is: temperature 28 DEG C, Medium's PH Value 8.0, intensity of illumination 7500lux, Light To Dark Ratio is 12:12, pass into sterile air in culturing process, air flow is 0.33vvm, CO 2content is 0.03 ~ 15%v/v, cultivates secondary fermentation in 14 days and terminates, and obtains fermented liquid.
5. a technique for chlorella strain ChlorellasorokinianaGS03 fermenting and preparing biological diesel oil, step is as follows:
(1) chlorella strain ChlorellasorokinianaGS03 described in the arbitrary claim of claim 2-4 is adopted to ferment and fixation of C O 2method prepare chlorella ChlorellasorokinianaGS03 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) the algae powder that previous step obtains is prepared fatty acid methyl ester by boron trifluoride catalysis method.
6. chlorella strain ChlorellasorokinianaGS03 described in claim 1 is in the application of biomass energy source domain; Describedly be applied as stabilizing carbon dioxide and production biofuel.
CN201310753837.9A 2013-12-31 2013-12-31 One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof Expired - Fee Related CN103952312B (en)

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