CN106554980A - A kind of method that utilization single needle algae produces starch - Google Patents
A kind of method that utilization single needle algae produces starch Download PDFInfo
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Abstract
The invention discloses a kind of method that utilization single needle algae produces starch, including（1）Microalgae seed liquor is prepared, described microalgae is single needle algae（Monoraphidium sp.）SS-06, deposit number are CGMCC No. 10765；（2）Single needle algae seed liquor is accessed in the micro-algae culture medium rich in N element, exponential phase is cultivated and is stopped, after standing sedimentation, discharging supernatant, obtain frustule；（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and adds ADP glucose pyrophosphatase activator in the medium, and continuous illumination culture to stable phase obtains the frustule of rich in starch.The inventive method contributes to the propagation and starch accumulation of frustule, can obtain the frustule of high-content of starch, the features such as with process is simple, low cost, high output.
Description
Technical field
The invention belongs to biotechnology and field of biological energy source, and in particular to a kind of method that utilization single needle algae produces starch.
Background technology
Due to the exhaustion of world petroleum resource and since price is gradually high, environmental issue is increasingly serious, and Global climate change causes a series of impact, 20 century 70s, the development of many national pay attention to day by day bioenergies and biological-based chemicals.In numerous biomass energies and biomass chemicals, microalgae has obtained increasing concern by the advantage of its " grain not being striven with people, ground is not striven with grain ".
Algae is one of biology of most original, and distribution is very wide, and most of algae are lived in water, its simple structure, with photosynthetic efficiency height, the short and fireballing feature of growth cycle.Algae includes microalgae（Unicellular eukaryote）, large-scale algae（Sargassum）And cyanobacteria（Blue-green alge）.Different algal species can pass through photosynthesis by CO2O is converted into water2And larger molecular organicses, such as carbohydrate and oils and fatss.The culture of microalgae does not strive water with striving with crops, it is possible to use the nutrition such as N, P in waste water, so as to reduce the eutrophication of water body, saving water resource and nutritive salt cost.Therefore, using non-grain and reproducible microalgae is the raw material production energy and chemicals, compared with other biomass, to a certain extent can reduces cost, while also the exploitation for microalgae downstream product provides a new approach.
Single needle algae is one kind of chlorella, is irregular spindle, and the single needle algae having now been found that is only used for accumulating oils and fatss.CN103540533A discloses a kind of acquisition and application of oil-producing single needle algae LB59, separates from wild environment and obtains, is resistant to high concentration sodium bicarbonate, it is easy to carry out open culture, and rich in oils and fatss, fat content can reach the 30.2% of dry cell weight.CN103555584A discloses a kind of acquisition and application of oil-producing single needle algae LB50, is resistant to high concentration sodium bicarbonate, and fat content can reach the 30.4% of dry cell weight.CN104073437A discloses a kind of single needle algae C29, and its fast growth, lipid-producing are high, is the strain excellent for producing biodiesel；The invention additionally provides a kind of open foster method of single needle algae, and the method adopts defined medium culture, the more conducively growth of single needle algae C29 and the accumulation of oils and fatss.CN104611228A discloses a kind of rich grease-contained single needle algae（Monoraphidium sp）SS-B1 and its culture application, deposit number is CGMCCNo.7479, and the algae strain is resistant to the CO of high concentration2And SO2, it is possible to use containing CO2And SO2Waste gas or flue gas carry out illumination autophyting growth and obtain rich grease-contained biomass, carbon sequestration efficiency high, cell total lipid content account for more than the 40% of dry cell weight, can carry out the production of biodiesel.
But, above-mentioned single needle algae is only used for producing oils and fatss, and product is single, and range of application is narrow.There is presently no discovery can be with the single needle algae of high yield starch and production method.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of method that utilization single needle algae produces starch.The inventive method contributes to the propagation and starch accumulation of frustule, can obtain the frustule of high-content of starch, the features such as with process is simple, low cost, high output.
The method that the present invention produces starch using single needle algae, including following content：
（1）Microalgae seed liquor is prepared, described microalgae is single needle algae（Monoraphidium sp.）SS-06, was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, and deposit number is CGMCC No. 10765；
（2）Single needle algae seed liquor is accessed in the micro-algae culture medium rich in N element, exponential phase is cultivated and is stopped, after standing sedimentation, discharging supernatant, obtain frustule；
（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and adds ADP glucose pyrophosphatase activator in the medium, and continuous illumination culture to stable phase obtains the frustule of rich in starch.
Step of the present invention（1）Using the single needle algae described in CN201510503226.8（Monoraphidium sp.）SS-06, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；Deposit number：CGMCC
No. 10765；Preservation date：On April 24th, 2015.
Step of the present invention（1）Prepare micro-algae culture medium that microalgae seed liquor adopts for cellar culture microalgae culture medium, can such as be the freshwater microalgae culture medium such as BG11, SE, TAP or D1.Microalgae seed liquor is referred to cultivates to exponential phase the algae solution for obtaining after microalgae is accessed culture medium, and concrete condition of culture is as follows：Temperature 25-30 DEG C, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, speed of agitator 50-200rpm, pH 6.0-9.0, and intensity of illumination is 1000-10000lux, Light To Dark Ratio 10:14 -14:10.
Step of the present invention（2）2%-10% of the inoculum concentration of middle microalgae seed liquor for culture volume.Condition of culture of the concrete condition of culture with microalgae seed liquor.
Step of the present invention（2）The described micro-algae culture medium rich in N element refers to and excessive inorganic nitrogen is added in conventional micro-algae culture medium, can such as be NaNO3、NH4NO3、NH4One or more in Cl etc., addition are 2.0-5.0g/L.
Step of the present invention（3）The described low micro-algae culture medium containing N element refers to the addition small-scale inorganic nitrogen in conventional micro-algae culture medium, can such as be NaNO3、NH4NO3、NH4One or more in Cl etc., addition are 0-0.5g/L.Step of the present invention（3）Described ADP glucose pyrophosphatases（AGPase）Activator preferably adopts 3-phoshoglyceric acid, and addition is 1-50mmol/L, preferably 5-30mmol/L.AGPase is added in micro-algae culture medium, the synthesis of starch is helped lend some impetus to, microalgae starch yield is improved.Acetyl-CoA carboxylase is added in the medium simultaneously more preferably（ACCase）Inhibitor, can such as be one or more in fragrant phenoxy phenoxy propionic acid, cyclohexyl diketone, phenyl pyrazoles or plant perfume extract etc., the pinke needle oil in diclofop-methyl, the methoxyphenone of cyclohexanedione apoplexy due to endogenous wind or plant perfume extract in preferably fragrant phenoxy phenoxy propionic acid, addition is 1-100 μm of ol/L, preferably 30-50 μm ol/L.Add AGPase activator and ACCase inhibitor in micro-algae culture medium simultaneously, help lend some impetus to Starch synthesis, and can effectively suppress lipogenesis, so as to improve microalgae starch yield.
Step of the present invention（3）Described microdisk electrode can adopt conventional training method, can such as adopt the condition of culture of microalgae seed liquor.It is preferred that cultivating in the following ways：Temperature 25-30 DEG C, ventilation 0.1-1.0vvm, CO2Content is 3v%-5v%, and speed of agitator 50-200rpm, pH are 7.5-8.5, and intensity of illumination is 1000-10000lux.By above-mentioned culture, adaptability and fast breeding of the microalgae to environment are helped speed up, so as to be favorably improved content of starch in frustule.
Compared with prior art, the invention has the advantages that：
1st, produce starch under given conditions using the single needle algae SS-06 of new selection-breeding, with content of starch is high, process is simple the features such as.
2nd, by the Discrete control to single needle algae SS-06 nitrogen content, carbon content, illumination and pH in incubation, contribute to the propagation and starch accumulation of single needle algae SS-06 cells, the frustule of high-content of starch can be obtained.
3rd, in the second stage addition AGPase activator of single needle algae SS-06 subsection filters, there is certain improvement to single needle algae SS-06 starch yields；Add AGPase activator and ACCase inhibitor simultaneously, the content of starch of single needle algae SS-06 can be greatly improved.
Specific embodiment
With reference to embodiment, the invention will be further described, but is not so limited the present invention.In the present invention, v% is volume fraction.
The present invention is using the single needle algae described in CN201510503226.8（Monoraphidium sp.）SS-06, was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, and deposit number is CGMCC No. 10765.The algae strain is collection water sample of in the October, 2011 from Heilungkiang Du Boerte Mongols autonomous county Shou Shan Relaxed holiday village dragon and tiger lake, and screening domestication is obtained.
The micro-algae culture medium that the embodiment of the present invention is used is BG11 culture medium, and Ju Ti Pei Fang is shown in Table 1, table 2.
1 BG11 culture medium of table
* in 2 table 1 of table A5+Co solution composition
Embodiment 1
（1）Single needle algae on picking flat board（Monoraphidium sp.）SS-06 is accessed in BG11 culture medium, in 30 DEG C of temperature, ventilation 0.25vvm, CO2Content is 2v%, and speed of agitator 100rpm, pH are 7.0, intensity of illumination 7000lux, Light To Dark Ratio 14:Cultivated under conditions of 10, cultivate to exponential phase and obtain microalgae seed liquor.
（2）Microalgae seed liquor is accessed in the BG11 culture medium rich in N element according to inoculum concentration 5v%, is referred to rich in N element and NaNO is added according to 3.0g/L in BG11 culture medium3, cultivate exponential phase and stop, after standing sedimentation, discharging supernatant, obtain frustule.The same step of condition of culture（1）.
（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and the low micro-algae culture medium containing N element is the NaNO that 0.05 g/L is added in BG11 culture medium3, and add the 3-phoshoglyceric acid of 20mmol/L in the medium, in 30 DEG C of temperature, ventilation 0.25vvm, CO2Content is 2v%, and speed of agitator 100rpm, pH are 7.0, and under the conditions of intensity of illumination is 7000lux, continuous illumination culture to stable phase harvests algae solution.
Micro algae biomass will be measured after above-mentioned algae solution lyophilization for 2.3
G/L, fat content 20.5%, content of starch 29.0%.Wherein detection methods of lipid is as follows：Dry algae powder is weighed, weight is calculated as m, is put in porcelain mortar, add appropriate amount of quartz sand and liquid nitrogen, after liquid nitrogen volatilization, grind breaking cellular wall；Add 10mL ethyl acetate:Normal hexane=1:1 mixed organic solvents, are cleaned by ultrasonic instrument and process 20min, extract 45 minutes in 50-60 DEG C of water bath with thermostatic control；Centrifugation 15 minutes, collects supernatant into centrifuge tube, repeats to extract twice, united extraction liquid；Add isopyknic distilled water in extracting solution, concussion is centrifuged 4 minutes after mixing, upper organic phase is collected to drying and precise net weight（It is calculated as W1）Test tube in；Solvent 1.5h in 70-80 DEG C of water-bath evaporation test tube（To can't see liquid solvent）, 70-80 DEG C of oven for drying be calculated as W2 to constant weight.Calculate the total lipid content C of frustule（%）：C=100×（W2-W1）/m.Method for detecting starch is enzyme hydrolysis method, takes algae powder（W）600 W ultrasonications of Jing, ultrasonic time 1s, intermittent time 2s, ultrasound 60 times, the algae solution for being crushed completely；PH is adjusted to 6.5,0.20 is separately added intoμ l Liquozyme Supra85-95 DEG C of insulation 1 h of liquefaction；PH is adjusted to 4.5,0.4 is separately added intoμ l Dextrozyme 60 DEG C of insulation liquefaction 10h of DX, centrifugation after hydrolysis completely abandon precipitation, supernatant constant volume 100mL, glucose content C in detection hydrolyzed solution；Control is hydrolyzed to deionized water plus equivalent amounts of enzyme；Content of starch (g/g)=C (g/L) × V (L) × 0.9/W (g).
Embodiment 2
（1）Single needle algae on picking flat board（Monoraphidium sp.）SS-06 is accessed in BG11 culture medium, in 25 DEG C of temperature, ventilation 0.25vvm, CO2Content is 1v%, and speed of agitator 100rpm, pH are 6.0, intensity of illumination 5000lux, Light To Dark Ratio 14:Cultivated under conditions of 10, cultivate to exponential phase and obtain microalgae seed liquor.
（2）Microalgae seed liquor is accessed in the BG11 culture medium rich in N element according to inoculum concentration 5v%, is referred to rich in N element and NH is added according to 2.0g/L in BG11 culture medium4NO3, cultivate exponential phase and stop, after standing sedimentation, discharging supernatant, obtain frustule.The same step of condition of culture（1）.
（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and the low micro-algae culture medium containing N element is referred to and add 0.1g/L to add NH in BG11 culture medium4NO3, and add the 3-phoshoglyceric acid of 5mmol/L in the medium, in 25 DEG C of temperature, ventilation 0.25vvm, CO2Content is 1v%, and speed of agitator 100rpm, pH are 6.0, and under the conditions of intensity of illumination is 5000lux, continuous illumination culture to stable phase harvests algae solution.
Micro algae biomass will be measured after above-mentioned algae solution lyophilization for 2.3
G/L, fat content 19.6%, content of starch 25.3%.
Embodiment 3
（1）Single needle algae on picking flat board（Monoraphidium sp.）SS-06 is accessed in BG11 culture medium, in 30 DEG C of temperature, ventilation 0.25vvm, CO2Content is 3v%, and speed of agitator 100rpm, pH are 8.0, intensity of illumination 8000lux, Light To Dark Ratio 14:Cultivated under conditions of 10, cultivate to exponential phase and obtain microalgae seed liquor.
（2）Microalgae seed liquor is accessed in the BG11 culture medium rich in N element according to inoculum concentration 5v%, is referred to rich in N element and NH is added according to 2.5g/L in BG11 culture medium4Cl, cultivates exponential phase and stops, supernatant is discharged after standing sedimentation, obtain frustule.The same step of condition of culture（1）.
（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and the low micro-algae culture medium containing N element is referred to and add 0.05g/L to add NH in BG11 culture medium4Cl, and add the 3-phoshoglyceric acid of 30mmol/L in the medium, in 30 DEG C of temperature, ventilation 0.25vvm, CO2Content is 3v%, and speed of agitator 100rpm, pH are 8.0, and under the conditions of intensity of illumination is 8000lux, continuous illumination culture to stable phase harvests algae solution.
Micro algae biomass will be measured after above-mentioned algae solution lyophilization for 2.5
G/L, fat content 18.5%, content of starch 33.7
%。
Embodiment 4
Incubation and operating condition are with embodiment 1.Difference is：Step（3）Add the methoxyphenone of 50 μm of ol/L in the medium simultaneously.Micro algae biomass is measured after lyophilization for 2.3g/L, fat content 14.3%, content of starch 36.7%.
Embodiment 5
Incubation and operating condition are with embodiment 2.Difference is：Step（3）Add 30 μm of ol/L diclofop-methyls in the medium simultaneously.Micro algae biomass is measured after lyophilization for 2.3g/L, 13.4 % of fat content, content of starch 32.1%.
Embodiment 6
Incubation and operating condition are with embodiment 3.Difference is：Step（3）Add the pinke needle oil of 40 μm of ol/L in the medium simultaneously.Micro algae biomass is measured after lyophilization for 2.5g/L, fat content 12.4%, content of starch 40.7%.
Embodiment 7
Incubation and operating condition are with embodiment 1.Difference is：Step（3）By CO2Content is improved to 4v%, and pH is improved to 8.5.Micro algae biomass is measured after lyophilization for 2.5 g/L, 19.4 % of fat content, content of starch 34.1%.
Comparative example 1
Incubation and operating condition are with embodiment 1.Difference is：Step（1）Using the single needle algae described in CN104611228A.Micro algae biomass is measured after lyophilization for 2.2 g/L, fat content 32.3
%, content of starch 20.1%.
Comparative example 2
Incubation and operating condition are with embodiment 1.Difference is：Step（3）3-phoshoglyceric acid is added without in culture medium.Micro algae biomass is measured after lyophilization for 2.4
G/L, 30.3 % of fat content, content of starch 17.8%.
Comparative example 3
Incubation and operating condition are with embodiment 1.Difference is：Culture medium is changed without, all the time using BG11 culture medium.Micro algae biomass is measured after lyophilization for 2.2 g/L, 20.6 % of fat content, content of starch 27.1%.
Claims (10)
1. a kind of method that utilization single needle algae produces starch, it is characterised in that including following content：
（1）Microalgae seed liquor is prepared, described microalgae is single needle algae（Monoraphidium sp.）SS-06, was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, and deposit number is CGMCC No. 10765；
（2）Single needle algae seed liquor is accessed in the micro-algae culture medium rich in N element, exponential phase is cultivated and is stopped, after standing sedimentation, discharging supernatant, obtain frustule；
（3）By step（2）The frustule for obtaining is accessed in the low micro-algae culture medium containing N element, and adds ADP glucose pyrophosphatase activator in the medium, and continuous illumination culture to stable phase obtains the frustule of rich in starch.
2. method according to claim 1, it is characterised in that：Step（1）Culture medium that microalgae seed liquor adopts is prepared for BG11, SE, TAP or D1 freshwater microalgae culture medium, microalgae seed liquor refer to will microalgae access culture medium after cultivate to exponential phase the algae solution for obtaining.
3. method according to claim 1 and 2, it is characterised in that：Step（1）Concrete condition of culture it is as follows：Temperature 25-30 DEG C, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, speed of agitator 50-200rpm, pH 6.0-9.0, and intensity of illumination is 1000-10000lux, Light To Dark Ratio 10:14 -14:10.
4. method according to claim 1, it is characterised in that：Step（2）The described micro-algae culture medium rich in N element refers to the addition inorganic nitrogen NaNO in conventional micro-algae culture medium3、NH4NO3、NH4One or more in Cl, addition are 2.0-5.0g/L.
5. method according to claim 1, it is characterised in that：Step（3）The described low micro-algae culture medium containing N element to be referred to and add inorganic nitrogen NaNO in conventional micro-algae culture medium3、NH4NO3、NH4One or more in Cl, addition are 0-0.5g/L.
6. method according to claim 1, it is characterised in that：Step（3）Described ADP glucose pyrophosphatase activator adopts 3-phoshoglyceric acid, and addition is 1-50mmol/L.
7. the method according to claim 1 or 6, it is characterised in that：Step（3）Add acetyl-CoA carboxylase inhibitor in the medium simultaneously.
8. method according to claim 7, it is characterised in that：Described acetyl-CoA carboxylase inhibitor is one or more in fragrant phenoxy phenoxy propionic acid, cyclohexyl diketone, phenyl pyrazoles or plant perfume extract, and addition is 1-100 μm of ol/L.
9. method according to claim 8, it is characterised in that：Described acetyl-CoA carboxylase inhibitor is the pinke needle oil in diclofop-methyl in fragrant phenoxy phenoxy propionic acid, the methoxyphenone of cyclohexanedione apoplexy due to endogenous wind or plant perfume extract, and addition is 30-50 μm of ol/L.
10. method according to claim 1, it is characterised in that：Step（3）Described microdisk electrode is in the following ways：Temperature 25-30 DEG C, ventilation 0.1-1.0vvm, CO2Content is 3v%-5v%, and speed of agitator 50-200rpm, pH are 7.5-8.5, and intensity of illumination is 1000-10000lux.
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Cited By (1)
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| CN114058514A (en) * | 2021-11-29 | 2022-02-18 | 华东理工大学 | Method for accumulating starch by using marine green algae, Qingdao and Pantoea galbana |
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