CN105543099B - One plant of oily ball algae Graesiella sp.WBG-1 and separating screening method and application - Google Patents
One plant of oily ball algae Graesiella sp.WBG-1 and separating screening method and application Download PDFInfo
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Abstract
The invention discloses one plant of oily ball algaesGraesiellaSp.WBG-1 and separating screening method and application.Algae strain growth is fast, it is adaptable to change to pH, also with cell it is big, easily settled, resist protozoan and eat etc. and be appropriate for the characteristic of large-scale culture.The oily ball algaeGraesiellaSp.WBG-1 accumulates a large amount of greases in cell under low nitrogen condition of culture, it can be used as microalgae biodiesel raw material, a large amount of protein are accumulated in cell under high nitrogen condition of culture, it can be used for the production of microalgae protein, it is cultivated under higher pH condition, external source carbon dioxide can be expeditiously fixed, can be applied to the biological fixation of flue air carbon dioxide.
Description
Technical field
The present invention relates to microalgae resource, the energy and environment protection fields, relate more specifically to one plant of oily ball algae Graesiella
Sp.WBG-1 also relates to the separating screening method of one plant of oily ball algae Graesiella sp.WBG-1, further relates to one plant of oily ball
Algae Graesiella sp.WBG-1 is in microalgae biodiesel raw material, microalgae protein production, the fixed external source carbon dioxide side of microalgae
The purposes in face.
Background technique
Microalgae is extensive in distributed in nature, many kinds of, can produce diversified metabolite.Separate, screen it is excellent
Good algae researches and develops the new application of microalgae and the new process of production, is the main task of Microalgae biotechnology research.60 years 20th century
Generation, Japan realize chlorella (Chlorella) large-scale culture production (Tsukada and Kawahara, 1977,
Biological Solar Energy Conversion 363–365).To currently, spirulina Spirulina
(Shimamatsu, 2003, Hydrobiologia512:39-44), Dunaliella salina Dunaliella (Oren, 2005, Aquatic
Biosystems 1 (1): 2-15), haematococcus Haematococcus (Boussiba and Vonshak, 1991, Plant&
Cell Physiology 32(7):1077-1082；Gómez,Inostroza et al.,2013,Aob Plants 5(1):
026-038.) etc. microalgaes, which are realized, commercially produces, and Microalgae biotechnology has begun to take shape, and shows huge application potential
(Richmond,2004,Handbook of microalgal culture).In field of food, microalgae can provide excellent for the mankind
The nutriments such as matter algae source protein, unsaturated fatty acid, carotenoid (Boussiba and Vonshak, 1991, Plant&
Cell Physiology 32(7):1077-1082；Belay,Ota et al.,1993,Applied Phycology 5(2):
235-241)；In pharmaceutical sanitary field, scientist just finds the drugs such as new antibiotic, anticancer from microalgae resource abundant
(Li Jian, Zhang Xuecheng etc., 2012, Science Bulletin 1 (1): 23-31；Kim,Li et al.,2013,Bmb Reports 47(8):
433-438；Talero,Garcia-Maurino et al.,2015,Marine drugs 13(10):6152-6209).Microalgae
It is a kind of photoautotroph, the CO that microalgae is fixed every year2Account about 40% (Brown and of global net photosynthesis yield
Zeiler,1993,Energy Conversion and Management 34(9-11):1005-1013；Jansson and
Northen, 2010, Current Opinion in Biotechnology 21 (3): 365-371), in energy conversion and carbon member
Very important effect is played in element circulation, it is wide that this has microalgae in carbon dioxide discharge-reduction and industrial wastewater preprocessing technical field
General application prospect (Huntley and Redalje, 2007, Mitigation and adaptation strategies
for global change 12(4):573-608).In recent years, due to largely using non-renewable fossil in global range
Fuel declines fossil fuel storage capacity sharply, and energy crisis is approached step by step；Greenhouse gases CO simultaneously2Excess emissions
The Global Climate Changes of initiation are got worse.In this background, the research of biomass renewable energy has obtained the world
The attention of various countries.In this field, microalgae is expected to become new one after cereal crops bio-ethanol, cellulose bio-ethanol
For biomass energy raw material (Chisti, 2008, Trends in Biotechnology 26 (3): 126-131；Waltz,
2009,Nature Biotechnology 27(1):15-18；Wijffels and Barbosa,2010,Science 329
(5993):796-799.)。
Microalgae starts from the research project such as U.S. in the 1960s, representative as the research of biodiesel raw material
Aquatile kind plan (ASP), the research update method plan of the Japanese earth etc. (Hu, Sommerfeld et al., 2008,
Plant Journal54(4):621-639；Gouveia and Oliveira,2009,Journal of Industrial
Microbiology and Biotechnology 36(2):269-274).The development of the studies above project is microalgae biological energy source
Solid foundation has been established in source technology exploitation.Before and after 2005, started again in worldwide by prosperities such as the U.S., Japan
The microalgae bio-fuel research upsurge that country leads, in terms of global numerous scientific research institutions take to Microalgae biotechnology one after another
Research (Amigun, Sigamoney et al., 2008, Renewable and Sustainable Energy Reviews
12(3):690-711；Hammond,Kallu et al.,2008,Applied Energy 85(6):506-515；Schenk,
Thomas-Hall et al.,2008,Bioenergy Research 1(1):20-43；Steenberghen and López,
2008,Journal of Cleaner Production 16(5):577-590；Um and Kim,2009,Journal of
Industrial and Engineering Chemistry 15 (1): 1-7), China also successively start " 863 ", " 973 " and
The development such as " 12th Five-Year Plan " key special subjects correlative study (Li Tao, Li Aifen etc., 2011, Chinese biological engineering magazine 31 (004): 98-
105；Li Jian, Zhang Xuecheng etc., 2012, Science Bulletin 1 (1): 23-31；Mei Hong etc., 2008, plant science journal 6 (6): 650-
660)。
Nevertheless, up to the present there are no the successful cases commercially produced for microalgae biomass fuel
(Steenberghen and López,2008,Journal of Cleaner Production 16(5):577-590；
Stephens,Ross et al.,2010,Trends in Plant Science；Wijffels and Barbosa,2010,
Science 329 (5993): 796-799), leading to the bottleneck problem of this status, there are three aspects: 1, microalgae biodiesel at
This is high, seriously hinder the commercially producing of microalgae bio-fuel (Torzillo, Pushparaj et al., 2003,
Biotechnology and Bioprocess Engineering 8 (6): 338-348), 2, to microalgae grease dynamic accumulation
Genetic regulation, physiological regulating control basis understanding is not deep, engineering technology weak foundation of micro-algae large-scale culture etc. (Wijffels
and Barbosa,2010,Science 329(5993):796-799；Li Jian, Zhang Xuecheng etc., 2012, Science Bulletin 1 (1):
23-31)；3, high-quality algae (Beal, the Smith for lacking easy culture, growing fast high, the suitable large-scale culture of environmental resistance degree
et al.,2011,Bioenergy research 4(1):36-60；Liu,Chen et al.,2011,Progress in
Natural Science:Materials International 21(4):269-276；Li Tao, Li Aifen etc., 2011, China
Bioengineering magazine 31 (004): 98-105；Li,Pribyl et al.,2013,Biotechnology and
Bioengineering 110(1):97-107；Guccione,Biondi et al.,2014,Biotechnology for
Biofuels 7(1):84).In technical field of microalga biology, high-quality algae is the important foundation of all subsequent R&D works.Entirely
Ball microalgae species there are about hundreds of thousands kind is even more, but wherein for the mankind finds and record only have 3.5 ten thousand kinds (Metting,
1996,Journal of Industrial Microbiology 17(5-6):477-489；Leliaert,Smith et
Al., it 2012, Critical Reviews in Plant Sciences 31 (1): 1-46), is realized in global range at present
The microalgae commercially produced is no more than 10 kinds.High-quality algae is found from natural microalgae resource abundant, is expected to solve micro- at present
The low yield that is encountered in the production of algae large-scale culture, it is easy to pollute, harvesting energy consumption is high, environmental suitability is poor the problems such as, be reduce it is micro-
Effective way (Stephens, Ross the et al., 2010, Trends in Plant Science of algal biomass production cost；
Wijffels and Barbosa,2010,Science 329(5993):796-799)。
Utilize the CO in industrial waste gas2For carbon source culture oil-producing microalgae, can reduce oil-producing microalgae carbon source cost and
CO is realized while producing microalgae biodiesel2Biological fixation, be that the important guiding of current microalgae biodiesel research and development is thought
One of think.Oil-producing microalgae absorbs, utilizes CO2The first step, be CO2It is reacted with water, generates bicarbonate ion (HCO3 -) and hydrogen
Ion (H+), HCO3 -Ion is further dissociated into carbanion (CO3 2-) and hydrogen ion (H+), three kinds of carbon source forms in algae solution
CO2、HCO3 -And CO3 2-Relative populations relationship determine that (Lee's noctilucence etc., 1996, bioengineering journal .12 (increases by culture solution pH
Periodical): 242-248).The pH value of algae solution influences external source CO in terms of two2Utilization efficiency: (1) situation identical in other conditions
Under, pH is lower, CO in algae solution2Shared ratio is higher, and algae solution is to external source CO2Absorptivity it is lower；PH is higher, CO in algae solution2Institute
The ratio accounted for is lower, and algae solution is to external source CO2Absorptivity it is higher (Lee's noctilucence etc., 1996, Wuhan botany research .14 (3):
253-260)；(2) pH of algae solution determines CO in algae solution2The height of shared ratio (partial pressure), as CO in algae solution2Partial pressure is equal to sky
CO in gas2Partial pressure when, algae solution absorbs CO from air2Rate be equal to air discharge CO2Rate, algae solution pH at this time
Referred to as balance pH；When algae solution pH is higher than balance pH, CO in algae solution2Partial pressure is lower than CO in air2Partial pressure, algae solution are inhaled from air
Receive CO2；When algae solution pH is lower than balance pH, CO in algae solution2Partial pressure is higher than CO in air2Partial pressure, algae solution discharges into air
CO2, cause CO2Waste (Kern 1960, Journal of chemical education (1): 14；Pellet etc., 2014, water
Raw biology journal .2014 (3): 401-406).Obviously, the algae that screening can adapt to higher pH can efficiently utilize external source
CO2, in fixed flue gas CO2The potentiality of aspect are larger.
The pollution of biological pollution in microalgae mass incubation, especially protozoan (wheel animalcule, infusorian etc.), Ke Yi
Make in short time algae cell density be greatly lowered (Wang et al.2013, Bioresource Technology 128:
745-750), serious influence is caused on the growth of algae.The control technology of biological pollution during oil-producing microalgae scale evaluation is
In the world generally acknowledged one restriction microalgae biodiesel industrialization process key technology difficulty (McBride et al.2014,
Industrial Biotechnology 10(3):221-227；Rego et al.2015,Bioelectrochemistry
103:60-64).Therefore, meeting growth fastly, under the premise of target product (such as grease) content is high, screening has antibiont
The algae of pollution capacity (such as individual is larger, has chemical defence ability, tolerance extreme environment), is conducive to carry out outdoor a large amount of
Culture production.
Summary of the invention:
The purpose of the present invention is to provide a kind of oily ball algae Graesiella sp.WBG-1, algae strain is grown fastly, to pH etc.
Environmental condition has very strong adaptability；Algae strain can accumulate different target products, target under different condition of culture
Products collection efficiency is high；Meanwhile algae strain also have cell big (8~12 μm of diameter), it is easily settled, resist protozoan and eat etc. and be suitble to
Carry out the characteristic of large-scale culture.
The present invention is separated using micro pipette partition method (Andersen, 2005, Algal culturing techniques)
Oily ball algae Graesiella sp.WBG-1, this method are operated for visible microalgae cell in field of microscope, are had
With strong points, the advantages that separative efficiency is high.
It is another object of the present invention to provide a kind of oily ball algae Graesiella sp.WBG-1 in microalgae biodiesel
Application in terms of raw material production, the fixed external source carbon dioxide of microalgae protein production, microalgae.The microalgae is trained under the conditions of high nitrogen
It supports, protein content can be used for the production of microalgae protein up to 50% or more of dry weight in cell, further as protein sources
Production for food or aquatic feeds；The microalgae is cultivated under the conditions of low nitrogen, and fat content is up to dry weight in cell
50% or more, it can be used as the raw material of microalgae biodiesel；The microalgae adapts to broad culture solution pH range, in high pH item
External source CO can be efficiently utilized under part2, can be applied to CO in flue gas2Biological fixation.
In order to achieve the above purpose, the present invention uses following technical measures:
A kind of separating screening method of oily ball algae Graesiella sp.WBG-1, the steps include:
1, water sampling: sample is acquired from natural water using plankton nets, for separating microalgae cell；
2, it separates and purifies: separating microalgae cell using micro pipette partition method, and be coated with or cross by agar plate
Method purifies isolated algae；
3, algae is identified: by observing microscopic morphology (cellular morphology, size, pigment volume morphing, arrangement mode etc.), in conjunction with
The method that 18S rDNA and ITS sequence compare (NCBI Blast), carries out the taxonomic identification of algae；
4, oily ball algae Graesiella sp.WBG-1 algae culture preparation: using artificial lighting in conical flask into
Prepared by the algae culture of row oily ball algae Graesiella sp.WBG-1, algae condition of culture are as follows: 30~50 μm of ol of intensity of illumination
photon m-2s-1, 20~25 DEG C of temperature, shaking speed 100rpm, cultivation cycle 3-5 days, used medium is standard BG-11.
5, the screening comparation and assessment of oily ball algae Graesiella sp.WBG-1: column airlift reactor is utilized, in same item
(culture bore 3cm, height 35cm, cultivate 300 μm of ol m of pipe surface illumination to part-2s-1, 30 DEG C of temperature, continuous ventilation (contains 1%
CO2Air) blow rate 250mL min-1) under cultivate oily ball algae Graesiella sp.WBG-1 and other microalgaes, according to life
Substance dry weight and fat content evaluate algae；Use improvement BG-11 culture medium prescription (every liter of culture medium): sodium nitrate
(NaNO3) 0.1g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, magnesium sulfate (MgSO4·7H2O) 0.075g, calcium chloride
(CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g, disodium ethylene diamine tetraacetate (EDTANa2)
0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride (MnCl2·4H2O) 1.8mg, zinc sulfate
(ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate ((NH4)6Mo7O24·4H2O)
0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg。
A kind of oily ball algae Graesiella sp.WBG-1 obtained was preserved in Chinese Typical Representative on August 14th, 2015
Culture collection (abbreviation CCTCC, Luojiashan, Wuchang, Wuhan City, Hubei Province), deposit number are CCTCC NO:M2015486.
Contain nucleotide sequence in the oily ball algae Graesiella sp.WBG-1: sequence is shown in SEQ ID NO:1
Nucleotide sequence (18S-1722), sequence be SEQ ID NO:2 shown in nucleotide sequence (ITS1-349), sequence SEQ
Nucleotide sequence shown in ID NO:3 (ITS2-303).
The oily ball algae Graesiella sp.WBG-1 has the feature that cell to be spherical to elliposoidal, diameter 8
~12 μm, a visible pyrenoids in cell, chromatoplast cup-shaped, Zhousheng, the structures such as cell surface atrichia, protrusion are specifically shown in attached
Fig. 1.Since algae strain cell is larger, when standing, is easy to be deposited to bottom from culture solution.In addition, the oily ball algae
Graesiella sp.WBG-1 also has very strong pH adaptability, can normally give birth in extensive pH environment (pH7~10.5)
It grows and accumulates specific product.
A kind of oily ball algae Graesiella sp.WBG-1 answering in microalgae biodiesel raw material, microalgae protein production
With the steps include:
1, reactor prepares: cleaning reactor and attached device (snorkel, sand core gas distributor) first and goes out
Bacterium, culture medium pass through boiling sterilization and cooling with water, then inject 40L sterile water in incubator, are put into the dispersion of sand core gas
Device starts air-blowing.
2, culture medium is prepared: be added media components one by one according to the BG11 culture medium of improvement, it is every add a kind of component after
It is sufficiently stirred, then adds down a kind of component, prevent localized ion concentration excessively high and precipitate.Improve the chemical group of BG-11 culture medium
Divide (every liter of culture medium): sodium nitrate (NaNO3) 0.1~1.5g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, magnesium sulfate
(MgSO4·7H2O) 0.075g, calcium chloride (CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g, second two
Amine tetraacethyl disodium (EDTANa2) 0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride
(MnCl2·4H2O) 1.8mg, zinc sulfate (ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate
((NH4)6Mo7O24·4H2O) 0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg.The improvement BG-11 culture medium
Middle sodium nitrate (NaNO3) concentration be 0.1~1.5g L-1.Preferably, the oily ball algae Graesiella sp.WBG-1 is used for
When food, feed applications (microalgae albumen), sodium nitrate (NaNO3) concentration be 1.5g L-1；By the oily ball algae Graesiella
When sp.WBG-1 is used for biodiesel raw material (microalgae grease), sodium nitrate (NaNO3) concentration be 0.1g L-1。
3, batch culture: algae culture solution is accessed in above-mentioned reactor, algae solution OD is made540Reach 0.1；In natural lighting
It is cultivated under the conditions of temperature, continuous to ventilate, flow 3L/min, the gas being passed through is air/carbon dioxide gas mixture (CO2/
Air=1/99, V/V) and pass through 0.22 μm of aperture membrane filtration degerming, it cultivates 8 days.
4, biomass harvesting and analysis: algae solution is stood, oily ball frustule sinks to bottom naturally, and supernatant is discharged, dense
Algae solution 1500rpm is centrifuged 10min and harvests cell, and vacuum freeze drying obtains algae powder.The microalgae biomass of harvesting can be used as micro-
The raw material of algae biodiesel (grease) or microalgae protein.
A kind of application of oily ball algae Graesiella sp.WBG-1 in terms of the fixed external source carbon dioxide of microalgae, step
It is:
1, it oily ball algae Graesiella sp.WBG-1 batch culture: is cultivated in annular culture pond reactor
Graesiella sp.WBG-1, culture medium are the BG-11, algae solution optical density (OD after inoculation of improvement540) 0.2, culture solution stirring
Device blade rotational speed 50rpm, liquid level 20cm, 30 DEG C of temperature, 300 μm of ol m of illumination at liquid level-2s-1；
2, the supplement and fixation of external source carbon dioxide: microalgae photosynthesis is to utilize the CO in algae solution using illumination as the energy2
For carbon source, it converts inorganic carbon to the process of organic carbon.The CO in algae solution is absorbed and utilized in microalgae2, pH is caused to increase, into algae solution
Logical CO2, culture solution absorption CO2PH declines again afterwards.Using above-mentioned principle, external source CO is automatically controlled using pH on-line controller2To oil
Culture solution pH is controlled the range in setting by being passed through and closing in ball algae Graesiella sp.WBG-1 culture solution.Work as culture
When liquid pH reaches the upper limit of control range, pH on-line controller controls external source CO2It is passed into culture solution, algae solution pH decline, when
When pH drops to the lower limit of control range, stopping is passed through external source CO2.Culture solution pH is carried out again on slow with photosynthetic
It rises, when culture solution pH reaches the upper limit of control range again, external source CO2It is passed into culture solution, so constantly recycles again,
The pH of algae solution is always held in the range of suitable microalgae photosynthesis, and microalgae is by photosynthesis continuously by CO2Turn
Become organic carbon, realizes to external source CO2Biological fixation.
In order to obtain higher CO2The pH of utilization efficiency, algae solution is controlled within the scope of 9.0-10.5.
Compared with prior art, the invention patent has the advantages that
1. having a wide range of application: microalgae algae strain provided by the invention is one kind (Graesiella sp.) of oily ball Trentepohlia, algae
Kind number is WBG-1, yet there are no report, and algae strain generates different metabolites under different condition of culture, simultaneously
With extensive environmental suitability, it is resistant to high pH environment, tool is of use in many ways.
2. target product yield is high: the oily ball algae Graesiella sp.WBG-1 in laboratory conditions, initial nitric acid
Na concn 1.5g L-1, after culture 6 days, protein content reaches as high as the 53% of dry cell weight, and yield is 253mg L-1d-1(see
Embodiment 4)；The oily ball algae Graesiella sp.WBG-1 in laboratory conditions, initial sodium nitrate concentration 0.1g L-1, training
After supporting 8 days, fat content reaches as high as the 55.55% of dry cell weight, and yield is 174mg L-1d-1(see embodiment 3)；Compared to
Other algaes, the oily ball algae Graesiella sp.WBG-1 protein yields and lipid-producing of this example report all have significantly
Advantage.
3. efficiently utilizing external source CO2: common algae grows heavily suppressed (Huntley and under high ph conditions
Redalje,2007,Mitigation and adaptation strategies for global change 12(4):
573-608), the oily ball algae Graesiella sp.WBG-1 being capable of fast-growth, oil synthesis metabolism under high pH environment
Unaffected (see embodiment 4)；Since oily ball algae Graesiella sp.WBG-1 adapts to high pH environment, Liquid Culture
System can efficiently utilize external source CO2, can be applied to flue gas CO2Biological fixation
4. facilitating harvesting: 8~12 μm of cell dia of the oily ball algae Graesiella sp.WBG-1, hence it is evident that be greater than report
With application more chlorella (3~8 μm) (Guccione, Biondi et al., 2014, Biotechnology for
Biofuels 7 (1): 84), cell sinks fast (sinking 20cm or more in 12 hours) when algae solution is stood；Utilize frustule nature
Sedimentation can conveniently and efficiently harvest cellular biomass from culture solution, it is possible to reduce harvesting energy consumption.
5. having stronger resistance to protozoan pollution: under normal circumstances, often occurring during microdisk electrode primary
The pollution of animal, endangering biggish is wheel animalcule, infusorian etc., and wheel animalcule and infusorian can quickly ingest microalgae cell, the short time
Inside make microalgae cell density sharp fall, seriously affect the growth of microalgae, protozoic pollution is to restrict microalgae mass training
Feeding technical problem (McBride et al.2014, Industrial Biotechnology 10 (3): 221-227；Rego
et al.2015,Bioelectrochemistry 103:60-64).Inventor the open pond culture chlorella in outdoor, scenedesmus,
The microalgaes such as oily ball algae (Graesiella sp.WBG-1), using micro- sem observation and carry out frustule and protozoic counting；
In outdoor 5m2In open pond incubation, often it is observed that protist contamination in chlorella, scenedesmus culture solution, to microalgae
Growth and the accumulation of grease cause to seriously affect.It is wherein observed in a chlorella culture, the 4th day wheel animalcule starts a large amount of numerous
It grows, the 5th day chlorella cells quantity reduces 40%, and cell quantity continues to be greatly reduced within the 6th day, the 7th day cell quantity
10% less than the 4th day；Cilium pest pollution has occurred in another secondary chlorella culture, infusorian starts mass propagation within the 4th day, the
5 days chlorella cells quantity falls sharply 75%, and chlorella cells continue to reduce within the 6th day, and chlorella cells quantity only has the 4th within the 7th day
It 5% or so.Different from chlorella, the culture situation of scenedesmus, oily ball algae Graesiella sp.WBG-1 is trained under normal circumstances
Protozoan is not observed in nutrient solution, even if it is observed that a small amount of protozoan, protozoan will not largely ingest in algae solution
Oily ball algae Graesiella sp.WBG-1, the accumulation of growth and grease to oily ball algae have not significant impact.It can be seen that the oily ball
Algae Graesiella sp.WBG-1 has the ability for resisting protozoal predation.Oily ball algae Graesiella sp.WBG-1 is to original
Raw animal ingestion has the reason of resistivity, in addition to cell individual larger (8~12 μm of cell dia) is taken the photograph to protozoic
Food causes outside difficulty, it is also possible to by other approach, such as allelopathy, resist protozoan (Mooij, P.R.,
G.R.Stouten,et al.2015.Current Opinion In Biotechnology 33:46-51).The oily ball algae
Graesiella sp.WBG-1 has certain resistivity to protozoal predation, this has aobvious in outdoor large-scale culture
The advantage of work.
Detailed description of the invention
Fig. 1 is a kind of oily ball algae Graesiella sp.WBG-1 cell optical photograph (A) and transmission electron microscope photo (B).
Figure A is light micrograph, and display cell is spherical to elliposoidal, it is seen that a pyrenoids, chromatoplast cup-shaped, carefully
The structures such as cellular surface atrichia, protrusion；Figure B is transmission electron microscope photo, and the chromatoplast of pyrenoids and Zhousheng is high-visible.
Fig. 2 is a kind of total rouge thin-layer chromatogram of oily ball algae Graesiella sp.WBG-1 (cultivating in 40L reactor).
Lamellae matrix is silica GF254, and applied sample amount 20-40 μ g, developing agent is n-hexane/ether/acetic acid=7/3/
0.1, finally with iodo steam displaing color.Maximum ellipse spot is neutral fats TAG in figure.
Fig. 3 is that a kind of oily ball algae Graesiella sp.WBG-1 (cultivates) fatty acid total ion current color in 40L reactor
Spectrogram (gas-chromatography separation, Mass Spectrometer Method).
Horizontal axis is retention time in figure, and the longitudinal axis is total ion current (abundance), the corresponding fatty acid of mark and its rich at wave crest
Degree.
Specific embodiment
According to the following example, the invention patent can be better understood.In embodiment, described culture medium composition,
Process conditions and its result are merely to illustrate the present invention, described in detail of the invention without that should limit in claims
Range.
Embodiment 1: the separating screening method of oily ball algae Graesiella sp.WBG-1 the steps include:
A kind of separating screening method of oily ball algae Graesiella sp.WBG-1, the steps include:
1, " ∞ " word enrichment algae water sampling: is drawn below the water surface with plankton sampler made of 400 mesh yarn tulles
The sample of cell, acquisition is placed in aseptic bottle, is added the BG-11 culture medium being concentrated on a small quantity, is then stood 3 days under dim light, and
The separation of algae is carried out afterwards.Water sampling in this example is from Yunnan Province Yongsheng County.
2, it separates and purifies:
(1) searching of target frustule with separate: finding under 10 times or 20 power microscopes needs frustule (mesh to be separated
Mark frustule), then micro pipette (0.1mm internal diameter) nozzle is lifted close to target cell and rapidly, it is quick using siphonage
Ground is by target cell inspiration micro pipette.
(2) microscopy confirms: the water sample in micro pipette being blown out on glass slide, is observed under the microscope, if containing
Target frustule then adds the dilution of about 10 μ L sterile waters into water droplet, and repeats step (1).
(3) aforesaid operations (step 1-2) repeat 7-8 times, until a mesh only can be observed in operated micro liquid
Mark frustule.
(4) it culture confirmation: will move into containing the water droplet of single target frustule equipped with the sterile BG-11 culture medium of 2-5mL
In test tube, stationary culture, 20 DEG C of temperature, 30 μm of ol m of illumination-2s-1, light dark period 12h:12h.Visible green to be visually observed
When, aseptically take 10 μ L culture drop on glass slide, microscopy is confirmed whether it is target frustule；If not containing target
Frustule, or contain target frustule and other frustules simultaneously, then it is separated again by step (1-4)；If only visual target algae
Cell then carries out next step operation.
(5) it purifies: taking 100 μ L of target frustule culture solution, dilute 100 times, then 50 μ L dilution culture solution is taken to be added dropwise in BG-
On 11 agar plates, coating or four rides, thereafter at 20 DEG C of temperature, 30 μm of ol m of illumination-2s-1, light dark period 12h:12h's
Under the conditions of cultivate.When naked eyes visible green algae falls, algae of the picking spot diameter less than 1mm is fallen, and lines BG-11 fine jade again
It is cultivated on rouge plate；It is carried out continuously 3-4 plate streaking, algae of each picking spot diameter less than 1mm drops into row scribing line purifying.
Finally the algae of purifying is fallen and is lined on BG-11 agar test tubes inclined-plane, thus the algae purified.
3, algae identify: through morphological observation (cellular morphology, size, pigment volume morphing, chromatoplast arrangement mode, whether there is or not
Flagellum, structure of cell surface etc.) and after 18S rDNA and ITS sequence compare (NCBI Blast), algae strain is accredited as
Graesiella (oily ball algae) species, and numbering is Graesiella sp.WBG-1.
4, the algae culture of oily ball algae Graesiella sp.WBG-1: pure from picking on BG-11 plate under aseptic condition
The algae of change falls in access 50mL conical flask, and the sterile BG-11 fluid nutrient medium of 20mL is added, after the sealing of vapor-permeable type sealed membrane
It sets and is cultivated on shaking table, shaking speed 80rpm, 30 μm of ol m of intensity of illumination-2s-1, light dark period 14h:10h, 25 DEG C of temperature, culture
20mL culture solution is transferred in 250mL conical flask after 1 week, the sterile BG-11 fluid nutrient medium of 100mL, vapor-permeable type sealing is added
It is cultivated on the postposition shaking table of film sealing, shaking speed 100rpm, 50 μm of ol m of intensity of illumination-2s-1, light dark period 14h:10h, temperature
120mL culture solution is transferred in 1000mL conical flask by 25 DEG C of degree, culture after 3 days, and the sterile BG-11 Liquid Culture of 500mL is added
Base is cultivated on the postposition shaking table of vapor-permeable type sealed membrane sealing, shaking speed 100rpm, 50 μm of ol m of intensity of illumination-2s-1, brightness
Period 14h:10h, 25 DEG C of temperature, culture obtained the algae culture solution (OD of oily ball algae Graesiella sp.WBG-1 after 4 days540
Reach 1.0), can be used for being inoculated in next step.
5, the screening of oily ball algae Graesiella sp.WBG-1: algae is carried out using common column airlift reactor
Culture screening.The single culture tube height of column airlift reactor used is 35cm, internal diameter 3cm.With high-pressure sterilizing pot to anti-
Device and culture medium is answered to be sterilized with water, aqua sterilisa naturally cools to room temperature (20~25 DEG C) and prepares improvement BG-11 culture medium afterwards.
The algae culture solution that step 4 obtains is inoculated into prepared improvement BG-11 culture medium, culture solution OD is made540Reach 0.1.It will
Algae solution after above-mentioned inoculation is added in cylindrical reactor, is continuously passed through air-carbon dioxide of filtration sterilization (0.22 μm of filtering)
Mixed gas (CO2/ air=1/99, V/V), throughput 250mL/min sets artificial lighting (culture 300 μm of ol m of pipe surface-2s-1) under cultivate, with water-bath control culture-liquid temp be 30 DEG C, cultivate 8 days.Each algae cultivates three pipes simultaneously.Improve BG-11
Culture medium prescription 1 forms (every liter of culture medium): sodium nitrate (NaNO3) 0.1g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, sulphur
Sour magnesium (MgSO4·7H2O) 0.075g, calcium chloride (CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g,
Disodium ethylene diamine tetraacetate (EDTANa2) 0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride
(MnCl2·4H2O) 1.8mg, zinc sulfate (ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate
((NH4)6Mo7O24·4H2O) 0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg。
1500rpm is centrifuged 10min and harvests oily ball frustule, and vacuum freeze drying obtains algae powder.0.45 μm of filter membrane filtration method
Measure three cultivated pipe oily ball algae Graesiella sp.WBG-1 biomass dry weight average out to 2.51g L-1, using n-hexane/
Ethyl acetate method (Wen little Bin etc., 2012, Chinese oil .37 (11): 80-85) extracting fat, measuring total lipid content average value is
The 55.55% of dry weight.
Using the above method, the green alga obtained to other 29 plants separation carries out culture screening.The biomass of 29 plants of microalgaes is dry
The minimum 0.37g L of galassing mean value-1, up to 1.98g L-1；The 19.31% of the minimum dry weight of total lipid content average value, highest
It is the 49.83% of dry weight.With 29 plants of chloralgal facies ratios, no matter biomass dry weight or total lipid content, Graesiella sp.WBG-1
It is highest.
Oily ball algae Graesiella sp.WBG-1 after purification was submitted Chinese Typical Representative on August 14th, 2015 by applicant
Culture collection carries out preservation, deposit number are as follows: CCTCC NO:M2015486.
Contain nucleotide sequence in the oily ball algae Graesiella sp.WBG-1: its sequence is SEQ ID NO:1 institute
The nucleotide sequence (18S-1722) shown；
Contain nucleotide sequence in the oily ball algae Graesiella sp.WBG-1: its sequence is SEQ ID NO:2 institute
The nucleotide sequence (ITS1-349) shown；
Contain nucleotide sequence in the oily ball algae Graesiella sp.WBG-1: its sequence is SEQ ID NO:3 institute
The nucleotide sequence (ITS2-303) shown.
Embodiment 2: the expansion culture of oily ball algae Graesiella sp.WBG-1 and its be used for microalgae grease (biodiesel
Raw material) production.
A kind of application of oily ball algae Graesiella sp.WBG-1 in microalgae biodiesel raw material, the steps include:
1, reactor prepares: first three times with 75% ethanol incubator inner surface, being then injected into 40 liters by boiling
The culture water for sterilizing and being cooled to room temperature will be put into incubator by the snorkel and sand core gas distributor of scalding
In, it connects gas source and starts to ventilate (throughput 3L/min), the gas being passed through is air/carbon dioxide gas mixture (CO2/ air
=1/99, V/V) and pass through 0.22 μm of aperture membrane filtration degerming.
2, culture medium is prepared: media components are added one by one according to the BG11 culture medium prescription 1 of improvement, it is every to add a kind of group
It is sufficiently stirred after part, then adds down a kind of component, prevented localized ion concentration excessively high and precipitate.Improve BG-11 culture medium prescription 1
Chemical component (every liter of culture medium): sodium nitrate (NaNO3) 0.1g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, magnesium sulfate
(MgSO4·7H2O) 0.075g, calcium chloride (CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g, second two
Amine tetraacethyl disodium (EDTANa2) 0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride
(MnCl2·4H2O) 1.8mg, zinc sulfate (ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate
((NH4)6Mo7O24·4H2O) 0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg。
3, cultivate: the algae culture solution that embodiment 1 is obtained accesses in above-mentioned incubator, makes algae solution OD540Reach 0.1, together
When be inoculated with three incubators, cultivated 8 days under the conditions of natural lighting and temperature, per day intensity of illumination 30 in incubation~
45mol m-2d-1, 21.8~30.5 DEG C of daily mean temperature.
4, biomass harvesting and total rouge extraction and determination:
Algae solution is stood, oily ball frustule sinks to bottom naturally, and supernatant is discharged, and dense algae solution 1500rpm is centrifuged 10min
Cell is harvested, vacuum freeze drying obtains algae powder.
The biomass dry weight average value that 0.45 μm of filter membrane filtration method measures three incubators is 1.09gL-1, biomass yield
Average value 136.25mg L-1d-1, using n-hexane/ethyl acetate method (Wen little Bin etc., 2012, Chinese oil .37 (11): 80-
85) extracting fat measures 35.63% that total lipid content average value is dry weight.
Quantitative analysis is carried out to the fat extracted using silicon thin-layer chromatography (conventional method of analysis), wherein neutral fats
(TAGs) ratio in total rouge reaches 86.53%, sees Fig. 2.Using methanol (contain 0.5%NaOH) to above-mentioned total rouge extract into
Row is esterified and carries out the gas chromatographic analysis (conventional method of analysis) of fatty acid composition, and wherein C16, C18 etc. are suitable for refining life
The fatty acid of object diesel oil accounts for 94.62% of total fatty acids or more, sees Fig. 3.
Embodiment 3: the expansion culture of oily ball algae Graesiella sp.WBG-1 and its production for microalgae protein.
A kind of application of oily ball algae Graesiella sp.WBG-1 in microalgae protein production, the steps include:
1, reactor prepares: first three times with 75% ethanol incubator inner surface, being then injected into 40 liters by boiling
The culture water for sterilizing and being cooled to room temperature will be put into incubator by the snorkel and sand core gas distributor of scalding
In, it connects gas source and starts to ventilate (throughput 3L/min), the gas being passed through is air/carbon dioxide mix gas (CO2/ air=
1/99, V/V) and pass through 0.22 μm of aperture membrane filtration degerming.
2, culture medium is prepared: media components are added one by one according to the BG11 culture medium prescription 2 of improvement, it is every to add a kind of group
It is sufficiently stirred after part, then adds down a kind of component, prevented localized ion concentration excessively high and precipitate.Improve BG-11 culture medium prescription 2
Chemical component (every liter of culture medium): sodium nitrate (NaNO3) 1.5g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, magnesium sulfate
(MgSO4·7H2O) 0.075g, calcium chloride (CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g, second two
Amine tetraacethyl disodium (EDTANa2) 0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride
(MnCl2·4H2O) 1.8mg, zinc sulfate (ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate
((NH4)6Mo7O24·4H2O) 0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg。
3, cultivate: the algae culture solution that embodiment 1 is obtained accesses in above-mentioned reactor, makes algae solution OD540Reach 0.1, together
When be inoculated with three incubators, cultivated 8 days under the conditions of natural lighting and temperature, per day intensity of illumination 30 in incubation~
45mol m-2d-1, 21.8~30.5 DEG C of daily mean temperature.
4, biomass harvesting and protein content determination and amino acid analysis: algae solution is stood, oily ball frustule is naturally heavy
To bottom, supernatant is discharged, dense algae solution 1500rpm centrifugation 10min harvests cell, and vacuum freeze drying obtains algae powder.0.45μ
The biomass dry weight average value that m filter membrane filtration method measures three incubators is 2.85gL-1, biomass yield average value 467mg L-1d-1, it is 53.25% that Kjeldahl's method (national standard), which measures protein content average value,.Utilize amino-acid analyzer analysis of amino acid
Composition and content (Wei Wenzhi etc., 2011, Food Science 32 (5): 254-257), the results are shown in Table 1.
1 oily ball algae Graesiella sp.WBG-1 amino acid of table composition and content
*Essential amino acid
Amino acid is the basic unit for constituting protein, and oily ball algae Graesiella sp.WBG-1 contains 18 kinds of amino acid,
Including 8 kinds of essential amino acids (being marked in table 1 with " * ") needed for human body and animal.It is required that human body and animal itself cannot synthesize
Amino acid, can only be by food intake for needed for body metabolism.The World Health Organization (WHO) and FAO (Food and Agriculture Organization of the United Nation)
(FAO) propose a reference standard, the essential amino acid in protein should reach the 40% of total amino acid content, it is necessary to amino acid with
The ratio of nonessential amino acid should be greater than 60% (Wei Wenzhi etc., 2011, Food Science 32 (5): 254-257；Dong Liming etc.,
2012, Food Science 33 (3): 232-237), 8 kinds of essential amino acids contained by oily ball algae Graesiella sp.WBG-1 account for ammonia
The 41.18% of base acid total amount, it is necessary to which amino acid content and the ratio of non-essential amino acid content reach 70%, meet world health
Organize the reference standard of (WHO) and FAO (Food and Agriculture Organization of the United Nation) (FAO).In general, essential amino acids content is higher in feed, battalion
Feeding value is bigger, and oily ball algae Graesiella sp.WBG-1 essential amino acids content reaches the 21.19% of biomass dry weight, excellent
In the main feed digested tankage (essential amino acids content 16.46%) in China, soybean cake (essential amino acids content 12.64%), peanut
Benevolence cake (essential amino acids content 9.81%), maize gluten feed (essential amino acids content 5.81%), (must close to high-quality fish meal
Need amino acid content 23.97%) and brewer's yeast (essential amino acids content 23.70%).
Embodiment 4: fixed rate of the different pH culture oily ball algae Graesiella sp.WBG-1 measurements to external source carbon dioxide
Fixed rate of the different pH culture oily ball algae Graesiella sp.WBG-1 measurements to external source carbon dioxide, step
It is:
1, reactor prepares
Using annular culture pond bioreactor culture oily ball algae Graesiella sp.WBG-1, first with 75% ethanol
Annular culture pond inner surface and mixing arm three times, are then injected into 20 liters of culture use passing through scalding and being cooled to room temperature
Water.
2, culture medium is prepared
Be added media components one by one according to the BG11 culture medium prescription 1 of improvement, it is every add a kind of component after be sufficiently stirred,
A kind of component is added down again, is prevented localized ion concentration excessively high and is precipitated.Improve (every liter of 1 chemical component of BG-11 culture medium prescription
Culture medium): sodium nitrate (NaNO3) 0.1g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 0.04g, magnesium sulfate (MgSO4·7H2O)
0.075g, calcium chloride (CaCl2·2H2O) 0.036g, ferrous sulfate (FeSO4·7H2O) 0.005g, disodium ethylene diamine tetraacetate
(EDTA·Na2) 0.01g, sodium bicarbonate (NaHCO3) 1.0g, boric acid (H3BO3) 2.86mg, manganese chloride (MnCl2·4H2O)
1.8mg, zinc sulfate (ZnSO4·7H2O) 0.22mg, copper sulphate (CuSO4·5H2O) 0.08mg, ammonium molybdate ((NH4)6Mo7O24·
4H2O) 0.1104mg, cobalt nitrate (Co (NO3)2·6H2O)0.0494mg。
3, oily ball algae Graesiella sp.WBG-1 is cultivated:
The oily ball algae Graesiella sp.WBG-1 algae culture solution that embodiment 1 is obtained accesses annular culture pond reaction
In device, make OD540Reach 0.2, the algae solution liquid surface height controlling after inoculation is 20cm, the rotation speed of annular culture pond stirring blade
Rate 50rpm, 30 DEG C of algae solution temperature, 300 μm of ol m of intensity of illumination at liquid level-2s-1, light dark period 14h:10h.
4, the supply of external source carbon dioxide and algae solution pH control:
Annular culture pond reactor installs a set of CO2Supply and pH control system, the system are controlled by pH sensor, pH
Instrument, CO2Gas distributor, CO2Pipeline, solenoid valve, flowmeter and CO2Steel cylinder composition.CO2Gas distributor is located at annular culture
Bottom of pond portion, gas distributor are connected with flowmeter and solenoid valve by pipeline, then with CO2Steel cylinder connection.By adjusting flow
Meter, can accurately control CO2Ventilation Rate.PH controller can set pH control range, when pH sensor detects algae solution
PH reach the upper limit of setting range, it is open-minded that pH controller controls solenoid valve, the CO in steel cylinder2By flowmeter, solenoid valve stream
To gas distributor, become small CO2Bubble enters in algae solution, CO2During bubble floats up in algae solution gradually by
Algae solution absorbs.Algae solution absorbs CO2Afterwards, pH is reduced, when the pH that pH sensor detects algae solution reaches the lower limit of setting range, pH control
Instrument control solenoid valve processed is closed.The pH of algae solution is increased with the photosynthetic progress of microalgae, when reaching pH setting range again
The upper limit, start next round fill CO2Process.It so constantly recycles, external source CO2It is continuously passed through algae solution, is microalgae
Photosynthesis provides carbon source, and microalgae is by CO2It is changed into organic carbon, realizes external source CO2Biological fixation.
Under above-mentioned condition of culture, the culture of oily ball algae Graesiella sp.WBG-1, the pH value range point of algae solution are carried out
It Kong Zhi not be pH7-8, pH8-9, pH9-10 and pH10-10.5, repeat culture three times under every kind of pH control condition.
5, biomass harvesting and data analysis:
After culture 8 days, the average value that 0.45 μm of filter membrane filtration method measures the biomass dry weight cultivated under the conditions of four kinds of pH exists
0.987~1.152gL-1Between (table 2), biomass dry weight mean difference is not significant (p > 0.05) under different pH condition of culture.
Algae solution is stood, oily ball frustule sinks to bottom naturally, and supernatant is discharged, and dense algae solution is by 1500rpm centrifugation 10min harvesting
Cell, vacuum freeze drying obtain algae powder.Using n-hexane/ethyl acetate method (Wen little Bin etc., 2012, Chinese oil .37
(11): 80-85 total rouge) is extracted, the cell total lipid content average value cultivated three times under condition of different pH is measured and is respectively as follows: pH7-8,
44.17%；PH8-9,44.18%；PH9-10,42.94%；PH10-10.5,42.65% (table 2).As it can be seen that high pH environment is not
The growth and oil and fat accumulation of oily ball algae Graesiella sp.WBG-1 are caused significantly to inhibit.
The biomass and total lipid content of oily ball algae Graesiella sp.WBG-1 under 2 condition of different pH of table
According to (Grobbelaar J U, the 2004.Algal nutrition- of microalgae biomass dry weight carbon elements 50%
mineral nutrition.In:Richmond A.(Eds):Handbook of microalgal culture:
Biotechnology and applied phycology, 97-115), it is cultivated under the conditions of calculating 4 kinds of pH using following formula
Oily ball algae Graesiella sp.WBG-1 is to external source CO2Fixed amount (table 3).
CO2Fixed amount=(44/12) × biomass (dry weight) × 0.5
44 be CO in formula2Molecular weight, 12 be the atomic weight of carbon, and 0.5 indicates microalgae biomass (dry weight) carbon containing member
Element 50%.
In order to accurately measure CO2Consumption, use the Small-sized C O of 5L volume2Steel cylinder.By CO2Pressure reducing valve is unloaded from steel cylinder
Get off, at this point, CO2Steel cylinder is detached from air supply system, can be to CO2Steel cylinder is precisely weighed.Terminate before culture with culture every time
Afterwards, all precise CO2The weight of steel cylinder, the difference for cultivating forward and backward weight are equal to CO2Consumption.
According to CO2Fixed amount and CO2Consumption calculates CO2Fixed rate.
CO2Fixed rate=100% × CO2Fixed amount/CO2Consumption
Oily ball algae Graesiella sp.WBG-1, biomass total amount, CO are cultivated under condition of different pH2Fixed amount, CO2Consumption
Amount and CO2Fixed rate is shown in Table 3.
Fixed rate of the oily ball algae Graesiella sp.WBG-1 to external source carbon dioxide under 3 condition of different pH of table
From table 3 it can be seen that cultivating oily ball algae Graesiella sp.WBG-1 under the conditions of 4 kinds of pH, 20 liters of algae solutions are to external source
CO2Fixed amount in the range of 34.21-42.34g, pH change to external source CO2Fixed amount influence it is less big.But pH becomes
Change very big on the influence of the fixed rate of external source carbon dioxide.PH is lower, and the fixed rate of external source carbon dioxide is also lower；Culture solution pH
In 7.5-8.0, the fixed rate of external source carbon dioxide only has 3.43% for control, shows 96% or more carbon dioxide from culture solution
It overflows and wastes；Culture solution pH is controlled at 9.0 or more, and the fixed rate of external source carbon dioxide is significantly larger than pH and is lower than 9.0
Carbon dioxide fixation rate；When pH control is within the scope of 10.0-10.5, oily ball algae Graesiella sp.WBG-1 is to external source dioxy
The fixed rate average value for changing carbon reaches 69.11%, 20 times or more of the fixed rate of external source carbon dioxide when being pH7.5-8.0.Algae
Liquid pH is to external source CO2Fixed rate influence so it is big, mainly have two aspect reasons: 1, under other conditions identical situation,
PH is lower, CO in algae solution2Shared ratio is higher, and algae solution is to external source CO2Absorptivity it is lower (Lee's noctilucence etc., 1996, Wuhan plant
Object studies .14 (3): 253-260)；2, the pH of algae solution determines CO in algae solution2Partial pressure, as CO in algae solution2Partial pressure is equal to air
Middle CO2Partial pressure when, algae solution absorbs CO from air2Rate be equal to air discharge CO2Rate, algae solution pH at this time claims
To balance pH；When algae solution pH is higher than balance pH, CO in algae solution2Partial pressure is lower than CO in air2Partial pressure, algae solution are absorbed from air
CO2；When algae solution pH is lower than balance pH, CO in algae solution2Partial pressure is higher than CO in air2Partial pressure, algae solution discharges CO into air2,
Cause CO2Waste (Kern 1960, Journal of chemical education (1): 14；Pellet etc., 2014, it is aquatic
Biological journal .2014 (3): 401-406).In the identical situation of other conditions, algae solution pH is lower, and algae solution is discharged into air
CO2It is faster, cause CO2Waste it is bigger, the CO fixed by frustule2It is fewer.
1 data of table show that high pH environment does not make the growth and oil and fat accumulation of oily ball algae Graesiella sp.WBG-1
At significant impact, i.e. oily ball algae Graesiella sp.WBG-1 can adapt to high pH condition of culture.Table 2 statistics indicate that, in height
Oily ball algae Graesiella sp.WBG-1 is cultivated under the conditions of pH, can be realized the efficient utilization to external source carbon dioxide.
SEQUENCE LISTING
<110>Wuhan Botanical Garden, Chinese Acadmey of Sciences
<110>Sinopec Group
<120>one plants of oily ball algaesGraesiellaSp. WBG-1 and separating screening method and application
<130>one plants of oily ball algaesGraesiellaSp. WBG-1 and separating screening method and application
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 1722
<212> DNA
<213>oily ball algae
<400> 1
ctggtcttgt tctcagatta cgccatgcat gtctaagtat aaactgctta tactgtgaaa 60
ctgcgaatgg ctcattaaat cagttatagt ttatttggtg gtaccttact actcggataa 120
ccgtagtaat tctagagcta atacgtgcgt aaatcccgac ttctggaagg gacgtatata 180
ttagataaaa ggccgaccgg gctttgcccg acccgcggtg aatcatgata tcttcacgaa 240
gcgcatggcc ttgtgccggc gctgttccat tcaaatttct gccctatcaa ctttcgatgg 300
taggatagag gcctaccatg gtggtaacgg gtgacggagg attagggttc gattccggag 360
agggagcctg agaaacggct accacatcca aggaaggcag caggcgcgca aattacccaa 420
tcctgatacg gggaggtagt gacaataaat aacaataccg ggcatttcat gtctggtaat 480
tggaatgagt acaatctaaa tcccttaacg aggatccatt ggagggcaag tctggtgcca 540
gcagccgcgg taattccagc tccaatagcg tatatttaag ttgttgcagt taaaaagctc 600
gtagttggat ttcgggtggg ttctagcggt ccgcctatgg tgagtactgc tatggccttc 660
ctttctgtcg gggacgggct tctgggcttc actgtccggg actcggagtc gacgtggtta 720
ctttgagtaa ttagagtgtt caaagcaggc ttacgccctg aatactttag catggaataa 780
cacgatagga ctctggccta tcttgttggt ctgtaggact ggagtaatga ttaagaggga 840
cagtcggggg cattcgtatt tcattgtcag aggtgaaatt cttggattta tgaaagacga 900
actactgcga aagcatttgc caaggatgtt ttcattaatc aagaacgaag ttgggggctc 960
gaagacgatt agataccgtc gtagtctcaa ccataaacga tgccgactag ggattggcga 1020
atgttttttt aataacttcg ccagcacctt atgagaaatc aaagtttttg ggttccgggg 1080
ggagtatggt cgcaaggctg aaacttaaag gaattgacgg aagggcacca ccaggcgtgg 1140
agcctgcggc ttaatttgac tcaacacggg aaaacttacc aggtccagac atagtgagga 1200
ttgacagatt gagagctctt tcttgattct atgggtggtg gtgcatggcc gttcttagtt 1260
ggtgggttgc cttgtcaggt tgattccggt aacgaacgag acctcagcct gctaaatagt 1320
cctagttgct ttttgcagct agctgacttc ttagagggac tattggcgtt tagtcaatgg 1380
aagtatgagg caataacagg tctgtgatgc ccttagatgt tctgggccgc acgcgcgcta 1440
cactgatgca ttcaacaagc ctatccttga ccgaaaggtc cgggtaatct ttgaaactgc 1500
atcgtgatgg ggatagatta ttgcaattat tagtcttcaa cgaggaatgc ctagtaagcg 1560
caagtcatca gcttgcgttg attacgtccc tgccctttgt acacaccgcc cgtcgctcct 1620
accgattggg tgtgctggtg aagtgttcgg attggcagct tagggtggca acacctcagg 1680
tctgccgaga agttcataaa ccctccacct agagagaagc at 1722
<210> 2
<211> 349
<212> DNA
<213>oily ball algae
<400> 2
cctttgcgac tgcggaggga cattgattat taaaccacaa tgtgaacctc aacgttccgt 60
gccctggctt gccagtgggg cggctgggta ctacccggtc gtactcacag ctgggtgggc 120
attgttgcct gctcagtggc gccttggcat gatcatacac cagtgctaac cactgataaa 180
accaaactct gaagtttgat tgctattaac tggcaatctt aaccaaagac aactctcaac 240
aacggatatc ttggctctcg caacgatgaa gaacgcagcg aaatgcgata cgtagtgtga 300
attgcagaat tccgtgaacc atcgaatctt tgaacgcata ttgcgctcg 349
<210> 3
<211> 303
<212> DNA
<213>oily ball algae
<400> 3
agccttcggg caagagcatg tctgcctcag cgtcggttta caccctcacc cctccctttc 60
ttgggtgtgt tgatctttga tcaaccattg gggtggatct ggcttcccca atctgccttg 120
tagcggattg ggttggctga agcacagagg cttaagcaag gacccgatat gggcttcaac 180
tggataggta gcaacggctt gtgccgacta cacgaagttg ttgcctgtgg actttgctag 240
aggccaagca ggaacgtgct tatgcatgcc taaacttcga cctgagctca gcaagcagcc 300
cgt 303
Claims (7)
1. a kind of oily ball algae, it is characterised in that: the oily ball algae is oily ball algae Graesiella sp.WBG-1, deposit number are as follows:
CCTCC NO:M2015486。
2. oily ball algae according to claim 1, it is characterised in that: containing shown in SEQ ID NO:1 in the oily ball algae
Nucleotide sequence.
3. oily ball algae according to claim 1, it is characterised in that: containing shown in SEQ ID NO:2 in the oily ball algae
Nucleotide sequence.
4. oily ball algae according to claim 1, it is characterised in that: containing shown in SEQ ID NO:3 in the oily ball algae
Nucleotide sequence.
5. application of the oily ball algae described in claim 1 as microalgae biodiesel raw material.
6. application of the oily ball algae described in claim 1 in microalgae protein production.
7. application of the oily ball algae described in claim 1 in the fixed external source carbon dioxide of microalgae.
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Non-Patent Citations (2)
| Title |
|---|
| 一株产油微藻的筛选及分子鉴定;江丽丽等;《水生生物学报》;20130731;第37卷(第4期);606-612 * |
| 江丽丽等.一株产油微藻的筛选及分子鉴定.《水生生物学报》.2013,第37卷(第4期),606-612. * |
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