CN106467894B - One plant height produces single needle algae and its culture application of starch and grease - Google Patents
One plant height produces single needle algae and its culture application of starch and grease Download PDFInfo
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Abstract
The invention discloses a plant height produce starch and grease single needle algae SS-06, classification naming be single needle algae (Monoraphidium sp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on April 24th, 2015, deposit number is CGMCC No.10765.The single needle algae application potential of 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 satisfy downstream production different demands, widen its application range.
Description
Technical field
The invention belongs to biotechnologys and field of biological energy source, and in particular to a plant height produce starch and grease single needle algae and
It cultivates application.
Background technique
Since the exhausted and price of world petroleum resource is gradually high, environmental issue is increasingly serious and global climate becomes
Change and causes a series of influence, since the 1970s, many country's pay attention to day by day bioenergies and biological-based chemicals
Development.In numerous biomass energies and biomass chemicals, advantage of the microalgae by its " not striving grain with people, do not strive ground with grain "
More and more concerns are obtained.
Algae is one of biology of most original, and distribution is very wide, and in water, structure is simple, has for most of algae lives
The features such as photosynthetic efficiency is high, growth cycle is short and speed is fast.Algae includes microalgae (unicellular eukaryote), large-scale algae (seaweed)
With cyanobacteria (blue-green alge).Different algal species can be by photosynthesis by CO2O is converted into water2And larger molecular organics, such as carbon water
Compound and grease.Microalgae can use the nutriments such as N, P in waste water, to reduce the eutrophication of water body, save water money
Source and nutritive salt cost.It therefore, is that raw material produces the energy and chemicals using non-grain and reproducible microalgae, with other biomass
It compares, can reduce cost to a certain extent, while also providing a new approach for the exploitation of microalgae downstream product.
Single needle algae is one kind of green alga, is irregular spindle, and presently found single needle algae is only used for accumulation grease.
CN103540533A discloses the acquisition and application of a kind of oil-producing single needle algae LB59, separates and obtains from field environment, is resistant to
High concentration sodium bicarbonate is easy to carry out open culture, is rich in grease, and fat content can reach the 30.2% of dry cell weight.
CN103555584A discloses the acquisition and application of a kind of oil-producing single needle algae LB50, is resistant to high concentration sodium bicarbonate, grease
Content can reach the 30.4% of dry cell weight.CN104073437A discloses a kind of single needle algae C29, and the speed of growth is fast, and grease produces
Rate is high, is the strain excellent for producing biodiesel；The invention additionally provides a kind of open feeding method of single needle algae, and the method is adopted
With defined medium culture, the more conducively growth of single needle algae C29 and the accumulation of grease.CN104611228A discloses one kind and is rich in
Grease single needle algae (Monoraphidium sp) SS-B1 and its culture application, deposit number CGMCCNo.7479, the algae strain
It is resistant to the CO of high concentration2And SO2, can use containing CO2And SO2Exhaust gas or flue gas carry out illumination autophyting growth obtain it is rich
Grease-contained biomass, carbon sequestration is high-efficient, and cell total lipid content accounts for 40% of dry cell weight or more, is able to carry out biodiesel
Production.
But above-mentioned single needle algae is only used for production grease, product is single, and application range is narrow.There is presently no discoveries both may be used
With high yield starch again can high Lipid-producing single needle algae.
Summary of the invention
Single for existing single needle algae product, the narrow deficiency of application range, the present invention provides a kind of high yield starch and grease
Single needle algae and its culture application.The algae strain under optimum conditions not only can with high yield starch but also can high Lipid-producing, can satisfy
The different demands of downstream production, widen its application range.
The single needle algae SS-06 of high yield starch and grease of the present invention, classification naming be single needle algae (Monoraphidium sp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on April 24th, 2015, preservation is compiled
Number be CGMCC No. 10765.
Frustule is in spindle to single needle algae SS-06 provided by the invention under the microscope, green, and 2-4 microns of short axle, long axis
55-65 microns.
The 18S rDNA gene sequencing analysis result of single needle algae SS-06 provided by the invention is shown in sequence table.According to sequence ratio
Right, the 18S rDNA data of single needle algae SS-06 and the single needle algae announced of the invention have differences.
The cultural method of single needle algae SS-06 of the present invention, it is light that the culture medium of use can be BG11, SE, TAP or D1 etc.
Water micro-algae culture medium, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 1000-10000lux, Light To Dark Ratio
For 10:14-14:10, temperature is 15-35 DEG C, pH 6-11.After culture, frustule is harvested, measuring its protein content is
20%-30%, lipid content 20%-30%, content of starch 10%-30%.
Single needle algae SS-06 provided by the invention is producing the application in starch.It is cultivated in the micro-algae culture medium rich in N element
When, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10,
Culture replaces low N micro-algae culture medium to logarithmic phase, and adds the 3-phoshoglyceric acid of 0-50mmol/L in the medium, continuously
Illumination cultivation is to stationary phase.By above-mentioned culture, microalgae is helped speed up to the adaptability and fast breeding of environment, while also being had
Help improve the content of starch in frustule.
Application of the single needle algae SS-06 provided by the invention in Lipid-producing.It is cultivated in the micro-algae culture medium rich in N element
When, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 10:14-14:10,
Culture replaces low N micro-algae culture medium to logarithmic phase, and adds the sodium pyrophosphate of 0-10mmol/L, continuous illumination in the medium
It cultivates to stationary phase.By above-mentioned culture, microalgae is helped speed up to the adaptability and fast breeding of environment, is also contributed to simultaneously
Improve the fat content in frustule.
Micro-algae culture medium of the present invention rich in N element refers to that addition is excessive inorganic in conventional micro-algae culture medium
Nitrogen, additional amount are 2.0-5.0 g/L.The low N micro-algae culture medium refers to the addition small-scale inorganic nitrogen in conventional micro-algae culture medium,
Additional amount is 0-0.5g/L.The inorganic nitrogen is NaNO3、NH4NO3、NH4One or more of Cl etc., preferably NaNO3。
Compared with existing single needle algae, the invention has the following advantages:
1, single needle algae SS-06 provided by the invention under optimum conditions not only can with high yield starch but also can high Lipid-producing, can
To meet the different demands of downstream production, its application range is widened.
2, the single needle algae SS-06 application potential of breeding of the present invention is good, and environmental suitability is wider, is suitble to cultivation temperature and pH model
It encloses relatively wide, is 15-35 DEG C (common microalgae cultivation temperature is 20-30 DEG C) respectively and pH be 6-11(common microalgae pH is 7-9).
3, single needle algae SS-06 product economy value provided by the invention is high, and under normal culture conditions, protein content is
20-30%, fat content 20-30%, content of starch 10-30%.Condition of culture can be changed according to different demands, to obtain
The grease or starch of high yield.
Biomaterial preservation explanation
Single needle algae provided by the invention (Monoraphidium sp.) SS-06, it is preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research
Institute；Deposit number: CGMCC No. 10765；Preservation date: on April 24th, 2015.
Specific embodiment
Below with reference to embodiment, invention is further described in detail, but is not so limited the present invention.In the present invention, v%
For volume fraction.
1 separation screening of embodiment obtains single needle algae SS-06
(1) enrichment culture: in October, 2011 is from the Relaxed holiday village Heilungkiang Du Boerte Mongols autonomous county Shou Shan dragon and tiger
100mL water sample is inoculated into the BG11 culture medium of 200mL after filtered through gauze and is carried out enrichment culture by lake acquisition water sample, light
It is 7000lux according to intensity, temperature is 25 DEG C, and light dark period is that for 24 hours, brightness time ratio is 14:10, after culture about two weeks, training
It supports base and obvious green is presented, microscope observes that a large amount of fronds exist.The formula of the BG11 culture medium is shown in Table 1, table 2.
1 BG11 culture medium of table
* in 2 table 1 of table A5+Co solution composition
(2) water sample of enrichment culture the acquisition of pure algae strain: is diluted to 10-5Times, it is solid to be aseptically applied to BG11
It is cultivated on body plate, the intensity of illumination of culture is 7000 lux, and temperature is 25 DEG C, occurs green after culture about 10 days on plate
Single algae falls, and picking list algae, which falls in shaking flask, to be cultivated, and after culture 10 days, micro- sem observation determines whether for pure strain, if not pure
Kind, it repeats the above steps, until being determined as pure culture algae strain.By cultivating repeatedly, obtains single algae and fall.
(3) acquisition of purpose algae strain: picking is fallen to single algae of acquisition and carries out continuing to cultivate into 50mL shaking flask, cultivates illumination
Intensity is 7000lux, and temperature is 25 DEG C, and light dark period is 24 hours, and brightness time ratio is 14:10, is shaken every day at least 3 times,
It to logarithmic growth phase, is inoculated into tubular type illumination reaction device with the inoculum concentration of 5v%, culture intensity of illumination is 7000lux, is passed through
CO23v%(v/v), continuous illumination.Temperature and pH domestication are carried out to algae strain, first tamed since 15 DEG C, with 5 DEG C for a temperature
Gradient, while the pH in culture medium is improved, since pH 6.0, with pH 1 for a gradient, acclimation period 3-5d；Work as microalgae
Temperature and pH are improved simultaneously again after stabilization, repeatedly, when microalgae is reduced, temperature and pH is reduced, extends acclimation period, until
Microalgae significantly reduces, and domestication stops, and partially liq is taken to cross on BG11 plate, to obtain target algae strain SS-06.
The identification of embodiment 2 algae strain
The extraction of the DNA of the SS-06 frustule of acquisition is used into CTAB method, and uses 18S rDNA gene cloning, will
To 3 positive colonies send to Shanghai Sangon Biotech Company be sequenced.18S rDNA gene sequencing analysis result is shown in sequence table.By 18S
RDNA sequence log on to Genbank database carry out Blast comparison, as the result is shown withMonoraphidium sp.Have maximum
Similitude, BLASTn value are that 2591, Max index value is 99%, can determine SS-06 be single needle algae (Monoraphidium sp.).
The culture application of 3 single needle algae SS-06 of embodiment
Single needle algae SS-06 is cultivated in tubular type illumination reaction device, and 15 DEG C of cultivation temperature, pH 6.0, intensity of illumination is
5000lux, ventilatory capacity 0.25vvm, CO2Content is that after continuous illumination culture 7 days algae solution is collected by centrifugation, after freeze-drying in 3v%
Measuring micro algae biomass is 2.0g/L, protein content 20%, fat content 22%, content of starch 11%.
Wherein protein detection method is the measurement of Bradford method.Detection methods of lipid is to weigh dry algae powder, weight
It is calculated as m, is put into porcelain mortar, appropriate amount of quartz sand and liquid nitrogen is added, after liquid nitrogen volatilization, grinds broken wall；10mL acetic acid second is added
Ester: n-hexane=1:1 mixed organic solvents, ultrasonic cleaning instrument handle 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, combined extract；It is added into extracting solution isometric
Distilled water, concussion be centrifuged 4 minutes after mixing, and collection upper organic phase is to having dried and the examination of precise net weight (being calculated as W1)
Guan Zhong；Solvent 1.5h in 70-80 DEG C of water-bath evaporation test tube, drying to constant weight for 70-80 DEG C of baking oven, is calculated as W2.Calculate frustule
Total lipid content C(%): C=100 × (W2-W1)/m.Method for detecting starch takes algae powder (W) through 600 W ultrasonications, ultrasonic time
1s, intermittent time 2s, ultrasound 60 times, the algae solution being crushed completely；PH to 6.5 is adjusted, is separately added into 0.20μl Liquozyme Supra85-95 DEG C of 1 h of heat preservation liquefaction；PH to 4.5 is adjusted, is separately added into 0.4μl Dextrozyme DX
60 DEG C of heat preservations liquefaction 10h, centrifugation abandoning precipitating, supernatant constant volume 100mL detect glucose content C in hydrolyzate after hydrolyzing completely；
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).
Application of the 4 single needle algae SS-06 of embodiment in production starch
Picking plate list algae falls in illumination cultivation in 50mL shaking flask and is inoculated into the microalgae rich in N element to logarithmic phase with 5v%
It is cultivated in culture medium, ventilatory capacity 0.25vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 14:
10, nitrogen-free micro-algae culture medium is replaced in culture to logarithmic phase, and adds the 3-phoshoglyceric acid of 20mmol/L in the medium, even
Continue illumination cultivation to stationary phase.The micro-algae culture medium rich in N element, which refers to, is added 3.0g/L in BG11 culture medium
NaNO3；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
| Serial 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 of the 5 single needle algae SS-06 of embodiment in production grease
Picking plate list algae falls in illumination cultivation in 50mL shaking flask and is inoculated into the microalgae rich in N element to logarithmic phase with 5v%
It is cultivated in culture medium, ventilatory capacity 0.25vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio 14:
10, it cultivates to logarithmic phase, replaces low nitrogen micro-algae culture medium；And the sodium pyrophosphate of 3mmol/L, continuous illumination are added in the medium
It cultivates to stationary phase.The micro-algae culture medium rich in N element refers to the addition 3g/L NaNO in BG11 culture medium3；Low N
Micro-algae culture medium refers to the addition 0.2g/L NaNO in BG11 culture medium3。
Fat content table under the different condition of culture of table 4
| Serial 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 that the present invention screens can change condition of culture according to different demands, to obtain height
The grease or starch of yield.
SEQUENCE LISTING
<110>Sinopec Group
China Petroleum and Chemical Corporation Fushun Petrochemical Research Institute
<120>one plant heights produce single needle algae and its culture application of starch and grease
<130>new patent application
<170> PatentIn version 3.5
<211> 1418
<212> DNA
<213> Monoraphidium sp.
<400> 1
gtagtcatat gcttgtctca aagattaagc catgcatgtc taagtataaa ctgcttatac 60
tgtgaaactg cgaatggctc attaaatcag ttatagttta tttgatggta cctctacacg 120
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 gacttctcca gcaccttatg agaaatcaaa gtttttgggt 1080
tccgggggga gtatggtcgc aaggctgaaa cttaaaggaa ttgacggaag ggcaccacca 1140
ggcgtggagc ctgcggctta atttgactca acacgggaaa acttaccagg tccagacata 1200
gtgaggattg acagattgag agctctttct tgattctatg ggtggtggtg catggccgtt 1260
cttagttggt gggttgcctt gtcaggttga ttccggtaac gaacgagacc tcagcctgct 1320
aaatatgtca cattcgcttt ttgcggatgg ccgacttctt agagggacta ttggcgttta 1380
gtcaatggaa gtatgaggca ataacaggtc tgtgatgc 1418
Claims (8)
1. a plant height produce starch and grease single needle algae SS-06, classification naming be 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 SS-06 according to claim 1, it is characterised in that: frustule is in spindle under the microscope for algae strain
Shape, green, 2-4 microns of short axle, 55-65 microns of long axis.
3. the cultural method of single needle algae SS-06 described in claim 1, it is characterised in that: use BG11, SE, TAP or D1 fresh water
Micro-algae culture medium, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 1000-10000lux, and Light To Dark Ratio is
10:14-14:10, temperature are 15-35 DEG C, pH 6-11；After culture, frustule is harvested, measuring its protein content is 20%-
30%, lipid content is in 20%-30%, content of starch 10%-30%.
4. single needle algae SS-06 described in claim 1 is producing the application in starch, it is characterised in that: in the microalgae for being rich in N element
When being cultivated in culture medium, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio
10:14-14:10, culture to logarithmic phase, replaces low N micro-algae culture medium, and adds the 3- phosphoric acid of 0-50mmol/L in the medium
Glyceric acid, continuous illumination culture to stationary phase.
5. application of the single needle algae SS-06 described in claim 1 in Lipid-producing, it is characterised in that: in the microalgae for being rich in N element
When being cultivated in culture medium, ventilatory capacity 0.1-1.0vvm, CO2Content is 1v%-3v%, intensity of illumination 5000-10000lux, Light To Dark Ratio
10:14-14:10, culture to logarithmic phase, replaces low N micro-algae culture medium, and adds the pyrophosphoric acid of 0-10mmol/L in the medium
Sodium, continuous illumination culture to stationary phase.
6. application according to claim 4 or 5, it is characterised in that: the micro-algae culture medium rich in N element refers to
2.0-5.0g/L inorganic nitrogen is added in conventional micro-algae culture medium；The low N micro-algae culture medium refers in conventional micro-algae culture medium
0-0.5g/L inorganic nitrogen is added.
7. application according to claim 6, it is characterised in that: the inorganic nitrogen is NaNO3、NH4NO3、NH4One in Cl
Kind is several.
8. application according to claim 7, it is characterised in that: the inorganic nitrogen is NaNO3。
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| CN103540533A (en) * | 2013-06-26 | 2014-01-29 | 中国科学院水生生物研究所 | Obtaining and application of oil-producing monoraphidium LB59 |
| CN103555584A (en) * | 2013-06-26 | 2014-02-05 | 中国科学院水生生物研究所 | Acquisition and application of grease-producing monoraphidium LB50 |
| CN104073437A (en) * | 2014-05-20 | 2014-10-01 | 海南大学 | Single needle alga, as well as culture method and recovery method and application thereof |
| CN104611228A (en) * | 2013-11-05 | 2015-05-13 | 中国石油化工股份有限公司 | Highly oil-containing monoraphidium and culture and application thereof |
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| CN103540533A (en) * | 2013-06-26 | 2014-01-29 | 中国科学院水生生物研究所 | Obtaining and application of oil-producing monoraphidium LB59 |
| CN103555584A (en) * | 2013-06-26 | 2014-02-05 | 中国科学院水生生物研究所 | Acquisition and application of grease-producing monoraphidium LB50 |
| CN104611228A (en) * | 2013-11-05 | 2015-05-13 | 中国石油化工股份有限公司 | Highly oil-containing monoraphidium and culture and application thereof |
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