CN106635807B - One plant of oil-producing single needle algae and its culture application - Google Patents
One plant of oil-producing single needle algae and its culture application Download PDFInfo
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- 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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Abstract
The present invention relates to one plant of single needle algae SHJ-02, 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.10763.Algae strain can obtain rich grease-contained biomass in autotrophic condition with efficient growth；And strong environmental adaptability, it is still able to maintain faster growth at low temperature, fat content is high, and cell total lipid content can account for 40% of dry cell weight or more, can be used for the production of biodiesel.
Description
Technical field
The invention belongs to biotechnologys and field of biological energy source, and in particular to one plant of oil-producing single needle algae and its culture application.
Background technique
With the rapid development of society, fossil energy is increasingly in short supply, and environmental pollution is on the rise, and energy problem becomes system
The key of about entire human social development finds a kind of cleaning, economic, efficient, reproducible new energy becomes current urgent need
It solves the problems, such as, the world just gradually moves towards " rare-view set-up ".
Biomass is new energy that is a kind of sustainable, renewable, inexpensive, and can replacing fossil fuel.Microalgae, as one
Kind of aquatic biomass resource, has that photosynthetic efficiency is high, growth rate is fast, yield of biomass is high, strong environmental adaptability etc.
Advantage has comprehensive utilization value, can be used for producing biomass energy, including biodiesel, bio-fuel ethyl alcohol etc., and not with grain
Ground is striven, does not strive grain with people, can be used as biofuel feedstock, and there is preferable development prospect.Microalgae bioenergy is developed, is had
Multiple effect can both reduce the use of fossil fuel, reduce CO2Discharge can purify exhaust gas and sewage again.
Microalgae can accumulate the ingredients such as grease, starch, glycogen, certain algae during photosynthesis autotrophy into the cell
Middle starch and glycogen content account for the 30% of dry cell weight, and there are also algae under Incubation Condition, and fat content may be up to
80%.How effectively to screen acquisition have high grease or high-content of starch, strong environmental adaptability, can pilot scale culture and at
This lower new algae, has become the emphasis of various countries worker research.
(the oil-rich microalgaes such as Yang XunMonoraphidium sp.Separation and its grease Study on extraction, Agriculture of Anhui section
Learn, the 32nd phase in 2011) primary dcreening operation is carried out to the microalgae for being isolated from Hainan poisons in freshwater using Nile red fluorescent dye, obtain grease
Content is higherMonoraphidium sp., the grease of the microalgae is extracted using Bligh-Dyer ultrasonic method, and pass through orthogonal examination
Test optimization microalgae grease extract process conditions are as follows: solvent chloroform, the ratio of first alcohol and water be 10:10:9, ultrasonic power 600W,
50 DEG C of Extracting temperature, 30 min of extraction time.Oil yield is 22.95% under this condition.
CN103540533A discloses the acquisition and application of a kind of oil-producing single needle algae LB59, separates and obtains from field environment,
It is resistant to high concentration sodium bicarbonate and using it as carbon source, 30-35 DEG C of cultivation temperature, fat content can reach dry cell weight
30.2%.CN103555584A discloses the acquisition and application of a kind of oil-producing single needle algae LB50, is resistant to high concentration sodium bicarbonate
And using it as carbon source, 25-30 DEG C of cultivation temperature, fat content can reach the 30.4% of dry cell weight.CN104073437A is disclosed
A kind of single needle algae C29, the temperature of open culture is 16-40 DEG C, preferably, the temperature of open culture is 28 DEG C.
CN104611228A disclose a kind of grease-contained single needle algae of richness (Monoraphidium sp.) SS-B1 and its culture application, should
Algae strain can use containing CO2And SO2Exhaust gas or flue gas carry out illumination autophyting growth and obtain rich grease-contained biomass, culture temperature
Degree is 15-40 DEG C, and preferably 25-28 DEG C, cell total lipid content accounts for 40% of dry cell weight or more.
But the microalgae that above-mentioned separation screening comes out, what is had cannot utilize CO2Carry out autophyting growth, some lower temperature resistances
Can be poor, (it is lower than 15 DEG C) under cryogenic, the speed of growth is slow, and some is even dead.Under winter arctic weather, need
Heating culture is carried out, energy consumption is higher.
Summary of the invention
For the deficiency of existing oil-producing microalgae, the present invention provides one plant of oil-producing single needle algae and its culture applications.Algae strain
In autotrophic condition rich grease-contained biomass can be obtained with efficient growth；And strong environmental adaptability, it remains to protect at low temperature
Faster growth is held, fat content is high, can be used for the production of biodiesel.
Oil-producing single needle algae SHJ-02 provided by the invention, 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. 10763。
Single needle algae SHJ-02 provided by the invention under the microscope single algae cell be in shuttle shape, 28-32 μm of long axis, short axle 4-
6μm。
The 18S rDNA gene sequencing analysis result of single needle algae SHJ-02 provided by the invention is shown in sequence table.
The cultural method of single needle algae SHJ-02 of the present invention, includes the following steps:
(1) BG11 freshwater microalgae culture medium is prepared, is dispensed, sterilizing；
(2) it is inoculated into above-mentioned culture medium from selecting single algae on solid plate and fall, culture to exponential phase of growth；
(3) algae solution that step (2) are cultivated is forwarded in the tubular type Photoreactor equipped with BG11 culture medium, ventilation culture,
Ventilatory capacity is 0.5-3vvm(minute ventilation volume/nutrient solution volume), CO2Content is 0.5%-3%(v/v), temperature is 5-33 DEG C.
In step (1) of the present invention, prepared BG11 culture medium is divided in the conical flask of 50-250mL, per bottled liquid
Amount is 20-100mL, is sealed with ventilated membrane, wraps brown paper and sterilize, sterilize 20- under 121 DEG C of temperature, pressure 0.1MPa
30min。
In step (2) of the present invention, inoculation operation is carried out at sterile, is shaken up culture solution after inoculation, is wrapped with ventilated membrane,
It is carried out in 5-33 DEG C of constant temperature illumination reaction shaking table, intensity of illumination 1000-10000Lux, brightness reaction time is light for 24 hours
Dark time ratio is 14:10, shaking speed 80-160rpm, culture to exponential phase of growth.
In step (3) of the present invention, in tubular type Photoreactor, preferable temperature is 10-25 DEG C, ventilatory capacity 0.75-
1.5vvm, CO2Content is 1.5%-2.5%.In intensity of illumination 1000-10000Lux, brightness reaction time is for 24 hours that Light To Dark Ratio is
10:14-14:10, pH value 6-9, culture terminate culture to stationary phase is entered, harvest frustule.
The application of single needle algae SHJ-02 of the invention in production microalgae grease.The single needle algae algae strain low temperature tolerance ability is strong,
Faster growth is still able to maintain at 5-15 DEG C of low temperature, fat content is high, and shared dry cell weight is able to carry out up to 40% or more
The production of biodiesel.
Compared with existing single needle algae, the invention has the following advantages:
1, the obtained single needle algae of present invention screening, autotrophic condition can efficient growth, low temperature tolerance ability is strong, even if in 5-15
At DEG C, it is still able to maintain faster growth rate, fat content is high in cell space.
2, the economy of algae strain products obtained therefrom is high, under normal culture conditions, at 10-25 DEG C of temperature, by 14-18 days
After culture, algae dry weight ratio (dry weight of algae in every liter of culture solution) is up to 6.3g/L；The intracorporal fat content of algae is also relatively high, institute
It accounts for dry cell weight and reaches as high as 55.6%, the part material of biodiesel can be made.
Biomaterial preservation explanation
Single needle algae provided by the invention (Monoraphidium sp.) SHJ-02, it is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism is ground
Study carefully institute；Deposit number: CGMCC No. 10763；Preservation date: on April 24th, 2015.
Specific embodiment
Below with reference to embodiment, invention is further described in detail, but does not therefore cause limitation of the present invention.
1 separation screening of embodiment obtains single needle algae SHJ-02
(1) enrichment culture: in October, 2011 acquires water sample from Heilungkiang Song Hua River, and filtered through gauze removes big solid content；
The filtered water sample of 80mL being inoculated into the BG11 culture medium of 200mL, enrichment culture, the intensity of illumination of culture is 8000Lux,
Temperature is 25 DEG C, and light dark period is that for 24 hours, brightness time ratio is 14:10, and after cultivating two weeks, obvious green is presented in culture medium, is shown
Micro mirror 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, aseptically, it is solid to be applied to BG11
It is cultivated on body plate, the intensity of illumination of culture is 8000Lux, and temperature is 25 DEG C, occurs green after culture about 13 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) it the acquisition of purpose algae strain: single algae of acquisition is fallen to be inoculated into carries out continuing to cultivate in 50mL shaking flask, cultivate illumination
Intensity is 8000Lux, 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 5% inoculum concentration, culture intensity of illumination is 8000Lux, is passed through CO2
3%(v/v), continuous illumination controls cultivation temperature from 25 DEG C and is reduced to 5 DEG C step by step, every time 2 DEG C of cooling, repeatedly domestication culture 3 times.
After having tamed, partially liq is taken to cross on BG11 plate, to obtain target algae strain SHJ-02.
The identification of embodiment 2 algae strain
The extraction that the DNA of SHJ-02 frustule will be obtained uses CTAB method, and uses 18S rDNA gene cloning, will obtain
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 similar
Property, it is 99% that BLASTn value, which is 2619, Max index value, can determine SHJ-02 be single needle algae (Monoraphidium sp.).
The culture application of 3 single needle algae SHJ-02 of embodiment
(1) BG11 freshwater microalgae culture medium is prepared according to table 1, table 2, be divided in the conical flask of 250mL, per bottled liquid measure
It for 50mL, is sealed with ventilated membrane, wraps brown paper and sterilize, sterilize 20-30min under 121 DEG C of temperature, pressure 0.1MPa.
(2) it is inoculated into above-mentioned culture medium from selecting single algae on solid plate and fall SHJ-02, in 5-33 DEG C of constant temperature illumination
It is carried out in reaction shaking table, intensity of illumination 8000Lux, brightness reaction time is that for 24 hours, brightness time ratio is 14:10, shaking speed
For 100rpm, culture to exponential phase of growth.
(3) algae solution that step (2) are cultivated is forwarded in the tubular type Photoreactor equipped with BG11 culture medium, ventilation culture,
Ventilatory capacity is 1.0vvm, CO2Content is 2%(v/v), temperature is 5-30 DEG C, intensity of illumination 8000Lux, and brightness is reaction time
For 24 hours, Light To Dark Ratio 10:14-14:10, pH value 7, culture terminate culture to stationary phase is entered, harvest frustule.
Micro algae biomass and fat content are detected after the frustule of harvest is freeze-dried.Vacuum is cold under the conditions of -60 DEG C
It is lyophilized dry to measurement algae dried bean noodles weight after constant weight, calculating yield of biomass.Using n-hexane: ethyl acetate method measures total lipid content,
Specifically: dry algae powder is weighed, weight is calculated as m, is put into porcelain mortar, and appropriate amount of quartz sand and liquid nitrogen is added, and volatilizees to liquid nitrogen
Afterwards, broken wall is ground；10mL ethyl acetate: n-hexane=1:1 mixed organic solvents is added, ultrasonic cleaning instrument handles 20min, 50-
45min is extracted in 60 DEG C of waters bath with thermostatic control；It is centrifuged 15min, collects supernatant into centrifuge tube, repeats to extract twice, merges and extract
Liquid；It is added isometric distilled water into extracting solution, concussion be centrifuged 4 minutes after mixing, and collection upper organic phase is to having dried simultaneously
In the test tube of precise net weight (being calculated as W1)；Solvent 1.5h(in 70-80 DEG C of water-bath evaporation test tube is liquid molten to can't see
Agent), drying to constant weight for 70-80 DEG C of baking oven, is calculated as W2.Calculate the total lipid content C(% of frustule): C=100 × (W2-W1)/m.No
With under cultivation temperature, the cultivation results of micro algae biomass and fat content are as shown in table 3.
Micro algae biomass and fat content under 3 different temperatures of table
Seen from table 3, the single needle algae SHJ-02 that the present invention obtains also has good activity under cryogenic, and grease contains
Amount is up to 55.6%.
The application of 4 single needle algae SHJ-02 of embodiment
The algae solution of the single needle algae SHJ-02 of logarithmic growth phase is seeded in BG11 culture medium and is cultivated, BG11 culture medium
Formula see Tables 1 and 2, cultivated in tubular type bioreactor, temperature be 10 DEG C, ventilatory capacity 0.75-1.5vvm, CO2
Content is 1.5%-2.5%, and intensity of illumination 5000-8000Lux, brightness reaction time is Light To Dark Ratio 10:14-14:10, pH for 24 hours
Value is 6-9, and culture terminates culture to stationary phase is entered, harvests frustule.Microalgae is detected after the frustule of harvest is freeze-dried
Biomass and fat content.Under different condition of culture, the cultivation results of micro algae biomass and fat content are as shown in table 4.
Micro algae biomass and fat content under the different condition of culture of table 4
By table 4 as it can be seen that the single needle algae SHJ-02 that obtains of the present invention under cryogenic, is still able to maintain good growth lives
Property, and algae fat content intracellular is higher, fat content is up to 40% or more.
Comparative example 1
Using single needle algae SS-B1 described in CN104611228A as algae, using condition of culture same as Example 4,
In, selection ventilatory capacity is 1.0vvm, CO2Content is 2.0%, intensity of illumination 8000lux, maintains culture solution pH=7.Experimental result hair
The biomass of existing single needle algae SS-B1 is 1.3g/L, fat content 27.1%.It, should compared with the SHJ-02 under same culture conditions
Algae strain grows undesirable under cryogenic, and grease yield is low.
SEQUENCE LISTING
<110>Sinopec Group
Dalian Petroleum Chemical Engineering Institute, Sinopec Group
<120>one plants of oil-producing single needle algaes and its culture application
<130>new patent application
<170> PatentIn version 3.5
<211> 1418
<212> DNA
<213> Monoraphidium sp.
gtagtcatat gcttgtctca aagattaagc catgcatgtc taagtataaa ctgcttatac 60
tgtgaaactg cgaatggctc attaaatcag ttatagttta tttgatggta cctctacacg 120
gataaccgta gtaattctag agctaatacg tgcgtaaatc ccgacttctg gaagggacgt 180
atttattaga taaaaggccg accggggctt gccccgaccc gcggtgaatc atgataactt 240
cacgaatcgc atggccttgt gccggcgatg tttcattcaa atttctgccc tatcaacttt 300
cgatggtagg atagaggcct accatggtgg taacgggtga cggaggatta gggttcgatt 360
ccggagaggg agcctgagaa acggctacca catccaagga aggcagcagg cgcgcaaatt 420
acccaatcct gatacgggga ggtagtgaca ataaataaca ataccgggca tttaatgtct 480
ggtaattgga atgagtacaa tctaaatccc ttaacgagga tccattggag ggcaagtctg 540
gtgccagcag ccgcggtaat tccagctcca atagcgtata tttaagttgt tgcagttaaa 600
aagctcgtag ttggatttcg ggtgggttcc agcggtccgc ctatggtgag tactgctgtg 660
gccctccttt ttgccgggga cggtctcctg ggcttcactg tccgggaatc ggagtcggcg 720
atgatacttt gagtaaatta gagtgttcaa agcaagccta cgctctgaat actttagcat 780
ggaatatcgc gataggactc tggcctatct cgttggtctg taggaccgga gtaatgatta 840
agagggacag tcgggggcat tcgtatttca ttgtcagagg tgaaattctt ggatttatga 900
aagacgaact actgcgaaag catttgccaa ggatgttttc attaatcaag aacgaaagtt 960
gggggctcga agacgattag ataccgtcgt agtctcaacc ataaacgatg ccgactaggg
1020
attggaggat gttcttttga tgacttctcc agcaccttat gagaaatcaa agtttttggg
1080
ttccgggggg agtatggtcg caaggctgaa acttaaagga attgacggaa gggcaccacc
1140
aggcgtggag cctgcggctt aatttgactc aacacgggaa aacttaccag gtccagacat
1200
agtgaggatt gacagattga gagctctttc ttgattctat gggtggtggt gcatggccgt
1260
tcttagttgg tgggttgcct tgtcaggttg attccggtaa cgaacgagac ctcagcctgc
1320
taaatagtca cgttcgcttt ttgcggatgg ccgacttctt agagggacta ttggcgttta
1380
gtcaatggaa gtatgaggca ataacaggtc tgtgatgc 1418
Claims (8)
1. one plant of oil-producing single needle algae SHJ-02, classification naming be single needle algae (Monoraphidium sp.), in 2015 4
It is preserved within 24th China Committee for Culture Collection of Microorganisms's common micro-organisms center the moon, deposit number is CGMCC No.
10763。
2. the cultural method of single needle algae SHJ-02 according to claim 1, it is characterised in that include the following steps:
(1) BG11 freshwater microalgae culture medium is prepared, is dispensed, sterilizing；
(2) it is inoculated into above-mentioned culture medium from selecting single algae on solid plate and fall, culture to exponential phase of growth；
(3) algae solution that step (2) are cultivated is forwarded in the tubular type Photoreactor equipped with BG11 culture medium, ventilation culture, ventilation
Amount is 0.5-3vvm, CO2Content is 0.5%-3%, and temperature is 5-33 DEG C.
3. according to the method described in claim 2, it is characterized by: prepared BG11 culture medium is divided in 50- by step (1)
In the conical flask of 250mL, it is 20-100mL per bottled liquid measure, is sealed with ventilated membrane, wrap brown paper and sterilize, in temperature
121 DEG C, sterilize under pressure 0.1MPa 20-30min.
4. according to the method described in claim 2, it is characterized by: culture solution is shaken up after step (2) inoculation, with ventilated membrane packet
It is good, it is carried out in 5-33 DEG C of constant temperature illumination reaction shaking table, intensity of illumination 1000-10000Lux, brightness is reaction time
For 24 hours, brightness time ratio is 14:10, shaking speed 80-160rpm, culture to exponential phase of growth.
5. according to the method described in claim 2, it is characterized by: step (3) in tubular type Photoreactor, temperature 10-25
℃。
6. according to the method described in claim 2, ventilatory capacity is it is characterized by: step (3) is in tubular type Photoreactor
0.75-1.5vvm, CO2Content is 1.5%-2.5%.
7. according to the method described in claim 2, it is characterized by: step (3) is in intensity of illumination 1000-10000Lux, brightness
Reaction time is Light To Dark Ratio 10:14-14:10, pH value 6-9 for 24 hours, and culture terminates culture to stationary phase is entered, harvests algae
Cell.
8. application of the single needle algae SHJ-02 described in claim 1 in production microalgae grease.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109880856B (en) * | 2017-12-06 | 2022-09-09 | 中国石油化工股份有限公司 | Open type microalgae grease production method |
| CN109939540B (en) * | 2017-12-21 | 2021-08-06 | 中国石油化工股份有限公司 | Flue gas treatment method and treatment device |
| CN108611379B (en) * | 2018-05-13 | 2021-05-04 | 杭州富阳优信科技有限公司 | Method for improving yield of grease of monocladium cells |
| CN108753621A (en) * | 2018-05-30 | 2018-11-06 | 昆明理工大学 | A method of based on molasses give up mash improve single needle algae oil and fat accumulation |
| CN111100885B (en) * | 2018-10-26 | 2022-07-12 | 中国石油化工股份有限公司 | Method for improving oil production of microalgae |
| CN111100883B (en) * | 2018-10-26 | 2022-08-12 | 中国石油化工股份有限公司 | Method for producing microalgae grease by using flue gas |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540533B (en) * | 2013-06-26 | 2015-01-07 | 中国科学院水生生物研究所 | Obtaining and application of oil-producing monoraphidium LB59 |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540533B (en) * | 2013-06-26 | 2015-01-07 | 中国科学院水生生物研究所 | Obtaining and application of oil-producing monoraphidium LB59 |
| 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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Effective date of registration: 20230828 Address after: 100728 No. 22 North Main Street, Chaoyang District, Beijing, Chaoyangmen Patentee after: CHINA PETROLEUM & CHEMICAL Corp. Patentee after: Sinopec (Dalian) Petrochemical Research Institute Co.,Ltd. Address before: 100728 No. 22 North Main Street, Chaoyang District, Beijing, Chaoyangmen Patentee before: CHINA PETROLEUM & CHEMICAL Corp. Patentee before: DALIAN RESEARCH INSTITUTE OF PETROLEUM AND PETROCHEMICALS, SINOPEC Corp. |