CN106467894A - One plant height produces starch and the single needle algae of oils and fatss and its culture is applied - Google Patents
One plant height produces starch and the single needle algae of oils and fatss and its culture is applied Download PDFInfo
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Abstract
The invention discloses a plant height produces the single needle algae SS-06 of starch and oils and fatss, its Classification And Nomenclature is single needle algae（Monoraphidium sp.）, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, deposit number is CGMCC No. 10765.The single needle algae application potential of selection-breeding of the present invention is good, and environmental suitability is wider, under optimum conditions not only can with high yield starch but also can high Lipid-producing, can meet downstream production different demands, widen its range of application.
Description
Technical field
The invention belongs to biotechnology and field of biological energy source are and in particular to a plant height produces starch and the single needle algae of oils and fatss and its culture is applied.
Background technology
Due to the exhaustion of world petroleum resource with 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 country's pay attention to day by day bioenergies and biological-based chemicals.In numerous biomass energies and biomass chemicals, microalgae relies on the advantage of its " not striving grain with people, do not strive ground with grain " to obtain increasing concern.
Algae is one of biology of most original, and distribution is very wide, and most of algae live in water, and its structure is simple, has the features such as photosynthetic efficiency is high, growth cycle is short and speed is fast.Algae includes microalgae（Unicellular eukaryote）, large-scale algae（Sargassum）And cyanobacteria（Blue-green alge）.Different algal species can be by photosynthesis by CO2It is converted into O with water2And larger molecular organicses, such as carbohydrate and oils and fatss.Microalgae can be using the nutrient substance such as N, P in waste water, thus reducing the eutrophication of water body, saving water resource and nutritive salt cost.Therefore, using non-grain and reproducible microalgae be the raw material production energy and chemicals, compared with other biomass, to a certain extent can reduces cost, simultaneously also be microalgae downstream product exploitation provide 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 of oil-producing single needle algae LB59 and application, separates from wild environment and obtains, is resistant to high concentration sodium bicarbonate it is easy to carry out open culture, rich in oils and fatss, fat content can reach the 30.2% of dry cell weight.CN103555584A discloses a kind of acquisition of oil-producing single needle algae LB50 and application, is resistant to high concentration sodium bicarbonate, fat content can reach the 30.4% of dry cell weight.CN104073437A discloses a kind of single needle algae C29, its fast growth, and lipid-producing is high, is the strain excellent producing biodiesel；This invention additionally provides a kind of open foster method of single needle algae, and the method adopts defined medium to cultivate, the more conducively accumulation of the growth of single needle algae C29 and 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 this 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 obtain rich grease-contained biomass, carbon sequestration efficiency high, cell total lipid content accounts 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 find not only can with high yield starch but also can high Lipid-producing single needle algae.
Content of the invention
Single, the narrow deficiency of range of application for existing single needle algae product, the present invention provides a kind of high yield starch and the single needle algae of oils and fatss and its culture to apply.This algae strain under optimum conditions not only can with high yield starch but also can high Lipid-producing, can meet downstream production different demands, widen its range of application.
The single needle algae SS-06 of high yield starch of the present invention and oils and fatss, its Classification And Nomenclature is single needle algae（Monoraphidium sp.）, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, deposit number is CGMCC
No. 10765.
Frustule is in spindle to the single needle algae SS-06 that the present invention provides under the microscope, green, short axle 2-4 micron, major axis 55-65 micron.
The 18S rDNA gene sequencing analysis result of the single needle algae SS-06 that the present invention provides is shown in sequence table.According to sequence alignment, the single needle algae SS-06 of the present invention is had differences with the 18S rDNA data of the single needle algae of announcement.
The cultural method of single needle algae SS-06 of the present invention, the culture medium of employing can be the freshwater microalgae culture medium such as BG11, SE, TAP or D1, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, and intensity of illumination is 1000-10000lux, and Light To Dark Ratio is 10:14-14:10, temperature is 15-35 DEG C, pH 6-11.After culture terminates, harvest frustule, measuring its protein content is 20%-30%, and lipid content is 20%-30%, and content of starch is 10%-30%.
Application in producing starch for the single needle algae SS-06 that the present invention provides.When micro-algae culture medium rich in N element is cultivated, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10, cultivate to logarithmic (log) phase, the low N micro-algae culture medium of replacing, and add the 3-phoshoglyceric acid of 0-50mmol/L in the medium, continuous illumination is cultivated to stable phase.By above-mentioned culture, help speed up microalgae to the adaptability of environment and fast breeding, also contribute to improve the content of starch in frustule simultaneously.
Application in Lipid-producing for the single needle algae SS-06 that the present invention provides.When micro-algae culture medium rich in N element is cultivated, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10, cultivate to logarithmic (log) phase, the low N micro-algae culture medium of replacing, and add the sodium pyrophosphate of 0-10mmol/L in the medium, continuous illumination is cultivated to stable phase.By above-mentioned culture, help speed up microalgae to the adaptability of environment and fast breeding, also contribute to improve the fat content in frustule simultaneously.
Micro-algae culture medium rich in N element of the present invention refers to add excessive inorganic nitrogen in conventional micro-algae culture medium, and addition is 2.0-5.0
g/L.Described low N micro-algae culture medium refers to add small-scale inorganic nitrogen in conventional micro-algae culture medium, and addition is 0-0.5g/L.Described inorganic nitrogen is NaNO3、NH4NO3、NH4One or more of Cl etc., preferably NaNO3.
Compared with existing single needle algae, the invention has the advantages that：
1st, the single needle algae SS-06 that the present invention provides under optimum conditions not only can with high yield starch again can high Lipid-producing, the different demands that downstream produces can be met, widen its range of application.
2nd, the single needle algae SS-06 application potential of selection-breeding of the present invention is good, and environmental suitability is wider, and suitable cultivation temperature and pH scope are wider, are 15-35 DEG C respectively（Common microalgae cultivation temperature is 20-30 DEG C）It is 6-11 with pH（Common microalgae pH is 7-9）.
3rd, the single needle algae SS-06 product economy that the present invention provides is worth height, and under normal culture conditions, protein content is 20-30%, and fat content is 20-30%, and content of starch is 10-30%.Condition of culture can be changed according to different demands, thus obtaining oils and fatss or the starch of high yield.
Biomaterial preservation explanation
The single needle algae that the present invention provides（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.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but is not so limited the present invention.In the present invention, v% is volume fraction.
Embodiment 1 separation screening obtains single needle algae SS-06
（1）Enrichment culture：In October, 2011 gathers water sample from Heilungkiang Du Boerte Mongols autonomous county Shou Shan Relaxed holiday village dragon and tiger lake, after filtered through gauze, 100mL water sample is inoculated in the BG11 culture medium of 200mL and carries out enrichment culture, intensity of illumination is 7000lux, temperature is 25 DEG C, light dark period is 24h, and the brightness time, ratio was for 14:10, after culture about two weeks, culture medium assumes obvious green, and microscope observes that a large amount of fronds exist.The formula of described BG11 culture medium is shown in Table 1, table 2.
Table 1 BG11 culture medium
* in table 2 table 1 A5+Co solution composition
（2）The acquisition of pure algae strain：The water sample of enrichment culture is diluted to 10 -5 Times, aseptically it is applied to culture on BG11 solid plate, the intensity of illumination of culture is 7000 lux, and temperature is 25 DEG C, and culture green for about 10 days single algae on rear plate falls, picking list algae falls and cultivates in shaking flask, after culture 10 days, micro- sem observation determines whether for pure strain, if not purebred, repeat the above steps, till being defined as pure culture algae strain.Through repeatedly cultivating, obtain single algae and fall.
（3）The acquisition of purpose algae strain：Picking that the single algae obtaining is fallen carries out continuing culture in 50mL shaking flask, and culture intensity of illumination is 7000lux, and temperature is 25 DEG C, and light dark period is 24 hours, and the brightness time, ratio was for 14:10, it is shaken every day at least 3 times, to exponential phase, is inoculated in tubular type illumination reaction device with the inoculum concentration of 5v%, culture intensity of illumination is 7000lux, is passed through CO2
3v%（v/v）, continuous illumination.Algae strain is entered with trip temperature and pH domestication, first starts to tame from 15 DEG C, with 5 DEG C for a thermograde, improve the pH in culture medium, from pH simultaneously
6.0 beginnings, with pH 1 for a gradient, acclimation period is 3-5d；Improve temperature and pH after microalgae is stablized again simultaneously, so repeatedly, when microalgae reduces, reduce temperature and pH, extend acclimation period, until microalgae significantly reduces, domestication stops, and takes partially liq to be rule on BG11 flat board, thus obtaining target algae strain SS-06.
The identification of embodiment 2 algae strain
The extraction of the DNA of the SS-06 frustule obtaining is adopted CTAB method, and adopts 18S rDNA gene cloning, obtain 3 positive colonies are delivered to the sequencing of Shanghai Sheng Gong company.18S rDNA gene sequencing analysis result is shown in sequence table.18S rDNA sequence is signed in Genbank data base and carries out Blast comparison, result show withMonoraphidium sp.Have maximum similarity, its BLASTn value for 2591, Max index value for 99% it may be determined that SS-06 be single needle algae（Monoraphidium sp.）.
The culture application of embodiment 3 single needle algae SS-06
Single needle algae SS-06 is cultivated in tubular type illumination reaction device, 15 DEG C of cultivation temperature, pH 6.0, and intensity of illumination is 5000lux, ventilation 0.25vvm, CO2Content is 3v%, after continuous illumination is cultivated 7 days, algae solution is collected by centrifugation, and recording micro algae biomass after lyophilization is 2.0g/L, and protein content is 20%, fat content 22%, content of starch 11%.
Wherein protein detection method measures for Bradford method.Detection methods of lipid is to weigh dry algae powder, and weight is calculated as m, puts in porcelain mortar, adds appropriate amount of quartz sand and liquid nitrogen, after liquid nitrogen volatilization, grinds 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 to centrifuge tube, repeats to extract twice, united extraction liquid；Add isopyknic distilled water in extracting solution, concussion is centrifuged 4 minutes after mixing, collect upper organic phase 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, 70-80 DEG C of oven for drying, to constant weight, is calculated as W2.Calculate the total lipid content C of frustule（%）：C=100×(W2-W1)/m.Method for detecting starch takes algae powder（W）Through 600 W ultrasonications, ultrasonic time 1s, intermittent time 2s, the ultrasonic algae solution 60 times, being crushed completely；Adjust pH to 6.5, be separately added into 0.20μ l Liquozyme Supra85-95 DEG C of insulation liquefaction 1 h；Adjust pH to 4.5, be separately added into 0.4μ l Dextrozyme60 DEG C of insulation liquefaction 10h of DX, hydrolysis is centrifuged afterwards abandons precipitation completely, supernatant constant volume 100mL, glucose content C in detection hydrolyzed solution；Comparison is hydrolyzed to deionized water plus equivalent amounts of enzyme；Content of starch (g/g)=C (g/L) × V (L) × 0.9/W (g).
Application in producing starch for the embodiment 4 single needle algae SS-06
Picking flat board list algae falls in 50mL shaking flask illumination cultivation to logarithmic (log) phase, is inoculated into 5v% and cultivates rich in the micro-algae culture medium of N element, ventilation 0.25vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 14:10, cultivate to logarithmic (log) phase, replacing nitrogen-free micro-algae culture medium, and add the 3-phoshoglyceric acid of 20mmol/L in the medium, continuous illumination is cultivated to stable phase.The described micro-algae culture medium rich in N element refers to add 3.0g/L NaNO in BG11 culture medium3；No N micro-algae culture medium refers to using conventional BG11 culture medium.
Content of starch under the different condition of culture of table 3
| Sequence number | CO2Content | Intensity of illumination/lux | Light To Dark Ratio | Temperature/DEG C | pH | Starch yield/% accounts for dry cell weight |
| 1 | 1v% | 5000 | 14:10 | 15 | 6 | 15.3 |
| 2 | 2v% | 8500 | 14:10 | 25 | 8 | 29.5 |
| 3 | 3v% | 10000 | 14:10 | 35 | 11 | 20.1 |
Application in producing oils and fatss for the embodiment 5 single needle algae SS-06
Picking flat board list algae falls in 50mL shaking flask illumination cultivation to logarithmic (log) phase, is inoculated into 5v% and cultivates rich in the micro-algae culture medium of N element, ventilation 0.25vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 14:10, cultivate to logarithmic (log) phase, change low nitrogen micro-algae culture medium；And add the sodium pyrophosphate of 3mmol/L in the medium, continuous illumination cultivates to stable phase.The described micro-algae culture medium rich in N element refers to add 3g/L NaNO in BG11 culture medium3；Low N micro-algae culture medium refers to add 0.2g/L NaNO in BG11 culture medium3.
Fat content table under the different condition of culture of table 4
| Sequence number | CO2Content | Intensity of illumination/lux | Light To Dark Ratio | Temperature/DEG C | pH | Grease yield/% accounts for dry cell weight |
| 1 | 1v% | 5000 | 14:10 | 15 | 6 | 28.7 |
| 2 | 2v% | 7000 | 14:10 | 25 | 8 | 32.5 |
| 3 | 3v% | 9000 | 14:10 | 35 | 11 | 27.4 |
In summary, the single needle algae SS-06 of present invention screening can change condition of culture according to different demands, thus obtaining oils and fatss or the starch of high yield.
SEQUENCE
LISTING
<110>
Sinopec Group
China Petroleum and Chemical Corporation Fushun Petrochemical Research Institute
<120>
One plant height produces starch and the single needle algae of oils and fatss and its culture is applied
<130>
New patent application
<170> PatentIn version 3.5
<211> 1418
<212> DNA
<213> Monoraphidium sp.
<400> 1
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aagattaagc catgcatgtc taagtataaa ctgcttatac
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attaaatcag ttatagttta tttgatggta cctctacacg
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gataaccgta gtaattctag
agctaatacg tgcgtaaatc ccgacttctg gaagggacgt
180
atttattaga taaaaggccg
accgagcttt gctcgacccg cggtgaatca tgataacttc
240
acgaatcgca tagccttgtg
ctggcgatgt ttcattcaaa tttctgccct atcaactttc
300
gatggtagga tagaggccta
ccatggtggt aacgggtgac ggaggattag ggttcgattc
360
cggagaggga gcctgagaaa
cggctaccac atccaaggaa ggcagcaggc gcgcaaatta 420
cccaatcctg atacggggag
gtagtgacaa taaataacaa taccgggcat ttaatgtctg
480
gtaattggaa tgagtacaat
ctaaatccct taacgaggat caattggagg gcaagtctgg
540
tgccagcagc cgcggtaatt
ccagctccaa tagcgtatat ctaagttgtt gcagttaaaa
600
agctcgtagt tggatttcgg
gtgggttcca gcggtccgcc tatggtgagt actgctgtgg
660
ccctcctttt tgtcggggac
gggctcctgg gcttcattgt ccgggactcg gagtcgacga
720
tgatactttg agtaaattag
agtgttcaaa gcaagcctac gctctgaata ctttagcatg
780
gaatatcgcg ataggactct
ggcctatctc gttggtctgt aggaccggag taatgattaa
840
gagggacagt cgggggcatt
cgtatttcat tgtcagaggt gaaattcttg gatttatgaa
900
agacgaacta ctgcgaaagc
atttgccaag gatgttttca ttaatcaaga acgaaagttg
960
ggggctcgaa gacgattaga
taccgtcgaa gtctcaacca taaacgatgc cgactaggga
1020
ttggaggatg ttcttttgat
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1080
tccgggggga gtatggtcgc
aaggctgaaa cttaaaggaa ttgacggaag ggcaccacca
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ggcgtggagc ctgcggctta
atttgactca acacgggaaa acttaccagg tccagacata
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gtgaggattg acagattgag
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1260
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gtcaggttga ttccggtaac gaacgagacc tcagcctgct
1320
aaatatgtca cattcgcttt
ttgcggatgg ccgacttctt agagggacta ttggcgttta
1380
gtcaatggaa gtatgaggca
ataacaggtc tgtgatgc
1418
Claims (10)
1. a plant height produces the single needle algae SS-06 of starch and oils and fatss, and its Classification And Nomenclature is single needle algae（Monoraphidium sp.）, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, deposit number is CGMCC No. 10765.
2. single needle algae according to claim 1 it is characterised in that：Frustule is in spindle under the microscope for this algae strain, green, short axle 2-4 micron, major axis 55-65 micron.
3. single needle algae according to claim 1 cultural method it is characterised in that：Using BG11, SE, TAP or D1 freshwater microalgae culture medium, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, and intensity of illumination is 1000-10000lux, and Light To Dark Ratio is 10:14-14:10, temperature is 15-35 DEG C, and pH is 6-11；After culture terminates, harvest frustule, measuring its protein content is 20%-30%, in 20%-30%, content of starch is 10%-30% to lipid content.
4. application in producing starch for the single needle algae SS-06 according to claim 1.
5. according to claim 4 application it is characterised in that：When micro-algae culture medium rich in N element is cultivated, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10, cultivate to logarithmic (log) phase, the low N micro-algae culture medium of replacing, and add the 3-phoshoglyceric acid of 0-50mmol/L in the medium, continuous illumination is cultivated to stable phase.
6. application in Lipid-producing for the single needle algae SS-06 according to claim 1.
7. according to claim 6 application it is characterised in that：When micro-algae culture medium rich in N element is cultivated, ventilation 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10, cultivate to logarithmic (log) phase, the low N micro-algae culture medium of replacing, and add the sodium pyrophosphate of 0-10mmol/L in the medium, continuous illumination is cultivated to stable phase.
8. the application according to claim 5 or 7 it is characterised in that：The described micro-algae culture medium rich in N element refers to add 2.0-5.0g/L inorganic nitrogen in conventional micro-algae culture medium；Described low N micro-algae culture medium refers to add 0-0.5g/L inorganic nitrogen in conventional micro-algae culture medium.
9. according to claim 8 application it is characterised in that：Described inorganic nitrogen is NaNO3、NH4NO3、NH4One or more of Cl.
10. according to claim 9 application it is characterised in that：Described inorganic nitrogen is NaNO3.
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Cited By (1)
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| CN106867953A (en) * | 2017-03-15 | 2017-06-20 | 哈尔滨工业大学 | A kind of method that microalgae processes molasses containing waste water synchronization production capacity under cryogenic |
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