CN104498361B - Amphikdrikos minutum as well as application and culture method thereof - Google Patents

Amphikdrikos minutum as well as application and culture method thereof Download PDF

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Publication number
CN104498361B
CN104498361B CN201410758297.8A CN201410758297A CN104498361B CN 104498361 B CN104498361 B CN 104498361B CN 201410758297 A CN201410758297 A CN 201410758297A CN 104498361 B CN104498361 B CN 104498361B
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minutum
amphikdrikos
algae
biodiesel
double spore
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CN104498361A (en
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韩吉昌
张琳
王松
赵璐
潘克厚
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Ocean University of China
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Ocean University of China
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract

The invention discloses amphikdrikos minutum as well as application and a culture method thereof. The amphikdrikos minutum is separated from seawater of the Yellow Sea, is named as amphikdrikos minutum in a classification mode and is preserved in Common Microbe Center of China Committee for Culture Collection of Microorganisms on October 22, 2014 with a strain preservation registry number of CGMCC No. 9815. A collection method of the pseudo-double spore algae adopts phytoplankton net detachment; a separation method of the pseudo-double spore algae adopts micropipette aspiration, stepwise dilution and separation and a spread-plate method. The amphikdrikos minutum has the beneficial effects that compared with other algae species, serving as raw materials of biodiesel, the amphikdrikos minutum is high in oil content and growth rate and is capable of effectively lowering the production cost of the biodiesel.

Description

One plant of small plan pair spore algae and its application and cultural method
Technical field
Invention belongs to field of biological energy source, is related to a kind of double spore algaes of small plan and its application, more particularly it relates to One plant of new small plan pair spore algae algae strain, its fat content are high, can be used to prepare biodiesel.
Background technology
As tellurian fossil energy reserves are just increasingly reduced, petroleum resources are likely to just rapid at short notice It is exhausted.The reserves of fossil energy are not inexhaustible, it is easy to the fossil energy reserves exploited and utilize remaining nothing Several, remaining reserves continue the value exploited so as to lose because development difficulty is increasing.In the world, energy-consuming with oil is In the case of leading, if energy consumption structure does not still change, then the energy crisis of a new generation will occur.Though coal resources It is bigger than petroleum resources storage capacity but nor inexhaustible.At present in addition to coal, can be sharp on a large scale The energy with exploitation is still little.Although the resource of solar energy be it is inexhaustible endlessly, because its exploitation is consumed It is with high costs, so can not possibly still develop rapidly being at short notice used widely.
Therefore the mankind must find a kind of new forms of energy that can replace non-renewable fossil energy in time, so as to attention is turned Move on in new energy resource structure, exploration as early as possible, research and development utilize new forms of energy resource, could so allow living in for the mankind stable Sustainable development in the case of harmony is gone down, and biodiesel just can be by this Resolving probiems.Picture biodiesel is this renewable The neutral carbon energy, be conducive to environment and economy sustainable development.It is to replace completely that current research also show microalgae For unique biodiesel of fossil diesel fuel.Biodiesel and Fossil fuel inherently derive from photosynthate.Green is planted Thing carries out photosynthesis, water, carbon dioxide and other inorganic matters is synthesized various Organic substances by luminous energy and oxygen is discharged, is moved Thing and microorganism could growth and breedings.Various biological oils and fatss are converted from the Organic substance of phytosynthesis.Any form The fatty acid from animal, plant and microorganism can be used for the raw material of biodiesel.Therefore, the high algae kind ability of fat content It is the most important raw material for preparing biodiesel.
The key for preparing biodiesel is processed using microalgae, one is the fat content for improving original algae kind, and two is by excellent Change the fat content that culture improves microalgae cell as far as possible.In the incubation of unicellular microalgae, medium component, temperature, Intensity of illumination, pH, training method, ventilation, salinity etc. can affect the content and composition of the growth of microalgae and its fatty acid. Temperature, light intensity can affect the photosynthesis and Repiration of microalgae, so as to grow on which and in vivo biochemical component produces impact.It is micro- Algae has the features such as growth rate is fast, water content in harvest is short, photosynthetic utilization rate is high, has very by the use of microalgae as biodiesel raw material High application prospect.High fat content, the algae strain of Seedling height speed are the committed steps of the link.
The content of the invention
The technical problem to be solved is：First is to provide a kind of double spore algae novel species of small plan；Second is to provide It is small to intend double applications of the spore algae in production of biodiesel；3rd be that intron in the novel species is present kind has/disappearance Multiformity, the model organism that can be evolved as research intron；4th is the cultural method for providing the double spore algaes of above-mentioned plan.
To solve above-mentioned technical problem, the technical solution used in the present invention is：
A kind of small plan pair spore algaes, isolated from Huanghai Seawater, Classification And Nomenclature intends double spore algaes for small Amphikrikos minutum, are deposited in China Committee for Culture Collection of Microorganisms on October 22nd, 2014 common Microorganism center, culture presevation registration number are CGMCC No. 9815.
The acquisition method for intending double spore algaes is phytoplankton off-grid；Separation method is drawn using micro pipette, stepwise dilution Separate and spread plate.
Cultural method, its concrete operation step are as follows：The enrichment culture microalgae in sea water F/2 culture medium, initial inoculation are close Spend for 1 × 105 Individual mL-1, it is 14h: 10h to be placed in Light To Dark Ratio, and intensity of illumination is 1100lux, and temperature is 25 DEG C, phase It is all to 1-3 is cultivated in growth cabinet of the humidity for 70% ~ 80%, the double spore algae algae solutions of small plan are centrifuged.Harvest and intend double spore algaes.It is excellent Choosing, cultivate 2 weeks in growth cabinet.
Contain following material in every 1L sea water in above-mentioned F/2 culture medium：NaNO3：75 milligrams；NaH2PO4·H2O：5 millis Gram；Na2SiO3·9H2O：20 milligrams；Na2EDTA：4.36 milligram；FeCl3·6H2O：3.16 milligram；CuSO4·5H2O：0.01 milli Gram；ZnSO4·7H2O：0.023 milligram；CoCL2·6H2O：0.012 milligram；MnCL2·4H2O：0.18 milligram；Na2MOO4· 2H2O：0.07 milligram；Vitamin B1：0.1 microgram；Vitamin B12：0.5 microgram；Biotin：0.5 microgram.
Application of the double spore algaes of described small plan on biodiesel is prepared.
Application of the double spore algaes of described small plan on the model organism evolved as research intron.
Beneficial effects of the present invention：
Compared with other algae kinds as biodiesel raw material, there are the double spore algaes of small plan of the present invention very high oils and fatss to contain Amount and growth rate, can effectively reduce the production cost of biodiesel.
Description of the drawings
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in further detail.
Fig. 1 is the double spore algae electromicroscopic photograph of small plan and Bodipy stained photographs of the present invention.
Fig. 2 is the cladogram of the double spore algaes of small plan of the present invention.
Specific embodiment
Embodiment 1
A kind of small plan pair spore algaes, isolated from Huanghai Seawater, Classification And Nomenclature intends double spore algaes for small Amphikrikos marinus, are deposited in China Committee for Culture Collection of Microorganisms on October 22nd, 2014 common Microorganism center, culture presevation registration number are CGMCC No. 9815.The acquisition method for intending double spore algaes is de- for phytoplankton Net；Separation method is drawn using micro pipette, stepwise dilution is separated and spread plate.
Cultural method, its concrete operation step are as follows：The enrichment culture microalgae in sea water F/2 culture medium, initial inoculation are close Spend for 1 × 105 Individual mL-1, it is 14h: 10h to be placed in Light To Dark Ratio, and intensity of illumination is 1100lux, and temperature is 25 DEG C, phase To cultivating 2 weeks in growth cabinet of the humidity for 70% ~ 80%, the double spore algae algae solutions of small plan are centrifuged.Harvest and intend double spore algaes.
Contain following material in every 1L sea water in above-mentioned F/2 culture medium：NaNO3：75 milligrams；NaH2PO4·H2O：5 millis Gram；Na2SiO3·9H2O：20 milligrams；Na2EDTA：4.36 milligram；FeCl3·6H2O：3.16 milligram；CuSO4·5H2O：0.01 milli Gram；ZnSO4·7H2O：0.023 milligram；CoCL2·6H2O：0.012 milligram；MnCL2·4H2O：0.18 milligram；Na2MOO4· 2H2O：0.07 milligram；Vitamin B1：0.1 microgram；Vitamin B12：0.5 microgram；Biotin：0.5 microgram.
Total fat assay method：The algae mud being collected by centrifugation is placed in vacuum freeze drier carries out lyophilization, until thoroughly It is dried algae powder.About 40 milligrams of dried algae powder are weighed with electronic balance, the sealing of clean thorough drying is carefully added into In the good screw socket teat glass (10mL) of property, and record quality m of algae powder1.2mL chloroforms, 1mL are carefully added into in screw socket pipe Methanol.10min is shaken acutely, it is to avoid liquid is spilt from screw socket pipe.Centrifugation 4000g, 10min.Supernatant is drawn to new clean In glass tubing.Repeat 2., 3..And supernatant is merged in same glass tubing.1/5 body is added in the glass tubing equipped with supernatant Long-pending NaCl solution (0.9%), acutely shakes 10min, is stored at room temperature 30min to promote layering.Thoroughly inhaled with 1mL syringes Take nethermost chloroform layer in new empty glass tubing (to weigh in advance, be designated as m2).Glass tubing is placed in 50 DEG C of water-baths and is dried It is dry, it is dried during electric heating constant-temperature blowing drying box is placed into after treating chloroform volatilization.The weight of glass tubing is finally weighed, m is designated as3。 Calculate total fat weight.Total lipid content (%)=(m3-m2)/m1。
After quiescent culture is carried out in constant temperature lighting box（Carbon dioxide is not led to）, the Biomass of the algae can reach 0.7g/L.It is logical Cross gravimetric method to be measured the microalgae grease content into plateau according to the method described above, as a result show the fat content of the algae Account for more than the 50% of dry weight.Under conditions of being not added with any rush oil synthesis material and not carrying out Stress treatment, Biomass, It is abnormal rare that fat content reaches such level.
The double spore algaes of small plan in the present embodiment carry out Electronic Speculum and Bodipy dyeing, as a result see Fig. 1.Fig. 1 can see Go out, contain 1-2 drip of grease more intracellular, and volume is extremely huge, or even more than 80% that whole cell can be accounted for；Absolutely prove The intracellular fat content of algae kind enriches.
Embodiment 2
Egg capsule algae section species sequence is chosen, using Mega4.0 softwares, cladogram is built using adjacent method.Concrete outcome is shown in Fig. 2.
DH43 is that the algae is numbered, from cladogram it can be seen that the algae withamphikrikosSp. gather for one, show The Trentepohlia is in the double spore Trentepohlias of plan.

Claims (2)

1. one plant it is small intend double spore algaes, Classification And Nomenclature intended double spore algae Amphikrikos minutum for small, in 2014 10 The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 22nd, and culture presevation registration number is CGMCC No. 9815。
2. application of the double spore algaes of small plan described in claim 1 on biodiesel is prepared.
CN201410758297.8A 2014-12-12 2014-12-12 Amphikdrikos minutum as well as application and culture method thereof Expired - Fee Related CN104498361B (en)

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CN106399168B (en) * 2016-09-12 2019-05-17 厦门大学 The method that algae microorganism forms chemotactic ring on the semisolid plate of the algae containing host is split in induction
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