CN103540533B - Obtaining and application of oil-producing monoraphidium LB59 - Google Patents

Abstract

The invention discloses an obtaining and an application of an oil-producing monoraphidium LB59, and relates to a screening and culturing technology of an oil-producing micro-alga. Through subjecting alga seeds separated from a field environment to 18SrDNA sequence analysis and morphological identification, the alga is determined to be a member of Monoraphidium, and the sequence is SEQ ID No.1. The oil-producing monoraphidium LB59 disclosed by the invention can tolerate high-concentrate sodium bicarbonate, is suitable for open type culture, is rich in grease, the grease content can reach 30.2% of dry cell weight under the condition that a growth and collection rate of 23.6g/m<2>d is held, and the fatty acid contains more than 20% of C-18 3-olefine acid and C-18 4-olefine acid.

Description

The acquisition of produce oil single needle algae LB59 and application

Technical field

The present invention relates to screening and the culture technique of oil-producing microalgae, particularly relate to acquisition and the application of a kind of produce oil single needle algae LB59.Specifically, the present invention is from field acquisition water sample, obtains a kind of produce oil algae strain that can be used as the fat raw material that produces oil by separation and purification.

background technology

On the earth, certified algae is about more than 40000 kinds, and some of them kind is rich in grease.The grease extracted from oily algae can be converted into biofuel through reacting with methyl alcohol, is considered to the most possible diesel oil substituting day by day exhausted petroleum resources.The lipid acid of its 16 carbon contained and 18 carbon is the most favourable for the production of biofuel.The grease of algae, also containing unsaturated fatty acids, can add growth promoting effects in animal-feed to.Screening obtains and easily cultivates, grows the algae kind fast, produce oil is high is produce the key of the oily algal biomass with the market competitiveness.As far back as the seventies in last century, the water biological species plan in National Renewable Energy laboratory, just start to utilize algae to extract the R&D work of grease, preparation biofuel, more than 3000 kind of algae has been separated from ocean and lake, therefrom filter out and 300, to plant that growth is fast more, diatom containing innage, the algae kind such as green alga and blue-green algae, but unresolved Cost Problems at that time.The investigator of other various countries also utilizes various means to screen the excellent algae kind found and can adapt to commercially produce energetically in the world.

Current needs following problem urgently to be resolved hurrily: the screening of micro-algae algae strain that, fat content fast to growth velocity is high and genetic improvement; The foundation of low cost, high-density culture system; Efficiently, the exploitation of low cost optical bio-reactor; The harvesting technique of high-level efficiency, low cost; The high efficient energy sources transformation technology etc. of micro-algae.As can be seen from the above, fast for growth, easily mass propgation, micro-algae that fat content is high seed selection is the current matter of utmost importance needing to solve.In recent years, domestic Duo Jia laboratory started respectively the Department of Science and Technology, Natural Science Fund In The Light entrust and large-lot producer subsidy under, carry out the work gathering and screen the strain of high produce oil algae on a large scale, and have made some progress.The micro-algae being selected from natural habitat often has certain resistance in temperature, illumination and salinity adaptation, is suitable for mass propgation in open raceway pond and produces.

The present invention is exactly from Inner Mongol field water collection, abstraction and purification and one that obtains excellent produce oil algae strain based on above background.

summary of the invention

Object of the present invention is just to overcome the problem for the production of the excellent algae strain deficiency of microalgae grease in prior art, acquisition and the application thereof of a kind of produce oil single needle algae LB59 are provided, namely water sample is gathered in waters, field, Inner Mongol, a kind of oil-producing microalgae with resistance, easily open culture that separation and purification obtains, and set up and cultivate and the method such as collection.

One, the acquisition of single needle algae LB59

1. sample collecting

Water sample picks up from the Inner Mongol cylinder Ge Le alliance Xilinhot City peach woods tower pulling field G303 national highway puddles of water, and exact position is north latitude 43 ° 54 ' 4.85 ", east longitude 116 ° 19 ' 32.71 ".Sampling time is on July 29th, 2011, and the water temperature on the same day is 28.1 DEG C, and the specific conductivity of water sample is 0.392ms/cm, pH is 7.45.

2. the separation of algae kind, 18S rDNA sequential analysis and identification of morphology

Water sample is cultivated by diluting the BG11 that painting flat band method is adding 8.4g/L sodium bicarbonate, isolate single algae falls, and each algae falls to cultivating on a small quantity the total fat of extraction and compares in thin layer chromatography board.The strain selecting oleaginousness higher, universal primer amplification 18S rDNA, after clone, send order-checking, its sequence is SEQ NO.1 (see sequence table); Determine that this algae is single needle Trentepohlia (Monoraphidium) member (Fig. 1) by phylogenetic relationship tree structure; Examine under a microscope its form and meet single needle Trentepohlia feature, there is sheet chloroplast(id), called after single needle algae LB59 (Monoraphidium sp.LB59) (Fig. 2); Compared with the LB50 algae strain (another patent application) belonged to together, cell is less, and 18S rDNA has 8 base differences (base see in square frame in sequence table); This algae strain is preserved in China typical culture collection center (Wuhan, China Wuhan University) by July 17th, 2013, and deposit number is CCTCC NO:M2013349.

Two, the cultivation collection method of single needle algae LB59

1. cultivate: adopt the BG11 reducing SODIUMNITRATE content, add high density sodium bicarbonate and cultivate, the whole composition of substratum is in table 1;

Compared by test, establish single needle algae LB59 culture condition, single needle algae LB59 culture condition is: intensity of illumination 200 μ E/m ²s, temperature condition 30-35 DEG C, substratum is the BG11 with 0.75g/L SODIUMNITRATE, adds 12.6g/L sodium bicarbonate (the whole composition of substratum is in table 1).Carry out cultivation (see figure 3) with circular open formula photoreactor, pass into carbonic acid gas every day 1 hour, within every 5 days, collect the culturing cell of 1/2, and supplement isopyknic fresh culture, its growth velocity can reach 23.6g/m ²the d(dry-matter that every day, every square meter obtained), can continue for a long time like this to cultivate and collect; Because its photosynthetic oxygen evolution is active in 2000 μ E/m ²s reaches capacity, and can also improve intensity of illumination further and promote its growth and produce oil.

Table 1 medium component

Chemical composition	Consumption (mg/L)
		K 2HPO 4·3H 2O	40
MgSO 4·7H 2O	75
		CaCl 2·2H 2O	36
C 6H 8O 7(citric acid)	6
		FeC 6H 5O 7·NH 4OH(ferric ammonium citrate)	6
EDTANa 2	1
		Na 2CO 3	20
NaNO 3	750
		NaHCO 3	12600
H 3BO 3	2.8
		MnCl 2·4H 2O	1.86
ZnSO 4·7H 2O	0.22
		Na 2MoO 4·2H 2O	0.39
CuSO 4·5H 2O	0.08
		CoCl 2·6H 2O	0.01

2. flocculation is gathered

After mass propgation, regulate nutrient solution pH with dilute hydrochloric acid, add flocculate with chitosan sedimentation cell.

Single needle algae LB59 is transferred in collection container after mass propgation, with dilute hydrochloric acid, pH is adjusted to 6, add chitosan to 5.0mg/L; Remove culture supernatants, collect the frustule of sedimentation.

Three, the application of the cultivation collection method of single needle algae LB59

1. the oil-containing frustule that its mass propgation obtains is used for grease and extracts;

2. its frustule directly adds in animal-feed increases unsaturated fatty acids nutrition;

3. the carbonic acid gas of the mass propgation Absorbable rod industrial discharge of its frustule.

The frustule freeze drier collected carries out drying; Dry algae is extracted total fat with the chloroform/methanol of 1:1, and calculating the per-cent that total fat accounts for dry cell weight is 30.2%.From the grease that this algae is extracted, be rich in unsaturated fatty acids, such as 18 carbon 3 olefin(e) acids and 18 carbon 4 olefin(e) acid summations account for more than 20% of lipid acid.

Its purposes is mainly:

The oil-containing frustule that its mass propgation obtains is used for grease and extracts, and also directly can add in animal-feed and increase unsaturated fatty acids nutrition, go back the carbonic acid gas of Absorbable rod industrial discharge in mass propgation process.

The present invention has following advantages and positively effect:

1. the growth of single needle algae LB59 is without the need for the nutrition of machine matter, can obtain grease-contained biomass continuously in the inorganic nutrient solution of cheapness by open cultivation; Containing higher concentration sodium bicarbonate in its substratum, the pollution of the infringement of protozoon and pathogenic bacterium and other assorted algaes can be prevented, commercially producing in process and can reduce costs.

2. this algae strain is collected easily, and adding flocculation agent can rapid precipitation.

3. grease is rich in this algae strain, at maintenance 23.6g/m ²when the growth of d and collection rate, fat content can reach 30.2% of dry cell weight, containing 18 carbon 3 olefin(e) acids of more than 20% and 18 carbon 4 olefin(e) acids in lipid acid.

accompanying drawing explanation

Fig. 1 is the position of single needle algae LB59 in phylogenetic relationship tree;

Fig. 2 is the Photomicrograph of single needle algae LB59;

Fig. 3 uses circular light bioreactor culture single needle algae LB59;

Fig. 4 is that duty runway pond is cultivated.

The algae strain of this single needle algae LB59 is preserved in China typical culture collection center by July 25th, 2013, and deposit number is CCTCC NO:M2013349.

embodiment

Below in conjunction with accompanying drawing and example in detail:

Example one, in circular light reactor Semi-continuous cultivation

The culture medium prescription preparation nutrient solution of as described above, at 0.1m ²inoculation culture in circular light reactor, nutrient solution volume is 20L.Initial OD ₆₈₀value is 0.05, and temperature controls as 30-35 DEG C, and light intensity is 200 μ E/m ²s continuous light, leads to the CO of 1 hour every day in nutrient solution ₂, and every day, OD was surveyed in sampling ₆₈₀value, OD ₆₈₀when value reaches 6.0, collect the algae liquid of 1/2, and the fresh culture of supplementary 1/2 carries out Semi-continuous cultivation, collect the substratum of the same volume of algae liquid feed supplement later every 5 days.PH is adjusted to 6 with dilute hydrochloric acid by single needle algae LB59 in collection container, adds chitosan to 5.0mg/L, and within 30 minutes, flocculating settling efficiency reaches more than 90%; The algae liquid of flocculation is removed supernatant liquor, and obtain concentrated frustule, weigh after frustule lyophilize, calculate growth velocity, its growth velocity of result can reach 23.6g/m ²d.The ratio adding 4L chloroform/methanol (1:1) in every 100g dry algae powder extracts total fat, then adds H in the ratio of 1L ₂o, after vibration mixing, the centrifugal 5-10min of 3000rpm makes solution layering, by nitrogen volatilization chloroform logical in lower floor's chloroform phase transition to another container, obtains total grease, and weighing and calculating the per-cent that total fat accounts for dry cell weight is 30.2%.Through gas Chromatographic Determination, in the lipid acid of total fat, 16 carbonic acid and 18 carbonic acid account for 99.10%, and wherein 18 carbon 3 diluted acids and 18 carbon 4 diluted acids account for 20.25%(in table 2).

Lipid acid composition (molar percentage) in table 2 single needle algae LB59

Fatty acid species	Fatty acid ratio
		14:0	0.72±0.19
14:1	0.17±0.14
		16:0	28.42±2.23
16:1	4.27±0.52
		18:0	1.73±0.63
18:1	35.08±8.11
		18:2	9.25±1.53
γ-18:3	1.00±0.30
		α-18:3	12.90±2.59
18:4	6.35±1.79
		20:0	0.10±0.14

Example two, outdoor raceway pond are cultivated

According to this algae strain culture medium prescription, utilize the underground water preparation nutrient solution of locality, Inner Mongol, at the outdoor 5m of Ordos, inner Mongolia ²raceway pond carries out open cultivation (see figure 4), and nutrient solution volume is 750L.Raceway pond flow velocity controls as 0.04m/s with colyliform agitator; Water temperature daytime is about 30 DEG C, and be about 24 DEG C in the evening; Intensity of illumination is rain or shine different with weather, and the average intensity of illumination on daytime is 600 μ E/m ²s, night is in dark state; Every day, OD was surveyed in sampling ₆₈₀value, when being cultured to OD ₆₈₀value reaches 2.0, starts the frustule liquid collecting 1/5 volume, and the fresh medium then adding 1/5 carries out Semi-continuous cultivation, collects a feed supplement once every 3 days later.The algae liquid collected, through basis of microscopic observation, keeps the purebred state of more than 99%, and little miscellaneous bacteria and other algae kinds are polluted.As example one, pH is adjusted to 6 with dilute hydrochloric acid by collected algae in container, adds chitosan to 5.0mg/L, flocculating settling 30 minutes, the frustule that namely removal culture supernatants obtains concentrating is for extracting grease, and total fat accounts for 24.0% of dry cell weight.

sequence table

(base different from produce oil single needle algae LB50 has added square frame)

<110> Inst. of Hydrobiology, Chinese Academy of Sciences; Sinopec Group

The acquisition of <120> produce oil single needle algae LB59 and application thereof

<140>

<141>

<160> 1

<210> 1

<211> 1786

<212> DNA

<400>

TGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAACTGCTTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATGGTACTTTTACACGGATAACCGTAGTAAATCTAGAGCTAATACGTGCGTAAATCCCGACTTCTGGAAGGGACGTATTTATTAGATAAAAGGCCGACCGGGCTCTGCCCGACCCGCGGTGAATCATGATAACTTCACGAATCGCATAGCCTCGTGCTGGCGATGTTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGATACGGGGAGGTAGTGACAATAAATAACAATACCGGGCATTCAATGTCTGGTAATTGGAATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGATTTCGGGTGGGTTCCAGCGGTCCGCCTATGGTGAGTACTGCTGTGGCCCTCCTTTCTGTCGGGGACGGGCTCCTGGGCTTCACTGTCCGGGACTCGGATTCGACGATGATACTTTGAGTAAATTAGAGTGTTCAAAGCAAGCCTACGCTCTGAATACTTTAGCATGGAATATCGCGATAGGACTCTGGCCTATCTCGTTGGTCTGTAGGACCGGAGTAATGATTAAGAGGGACAGTCGGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGAAGACGATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGATTGGAGGATGTTCTTTTGATGACTTCTCCAGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGTGAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCACTGCCGCTTTTTGCGGTTGGCCGACTTCTTAGAGGGACAGATGCTACAAAAGCATCGGAAGTATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACGCGTTCAACAAGCCTATCCTTGACCGAGAGGTCTGGGTAATCTTTGAAACCGCGTCGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACGAGGAATGCCTAGTAGGCGCAAGTCATCAGCTTGCGCCGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGGGTGTGCTGGTGAAGTGTTCGGATTGGCGAAGCGGGTGGCAACACTTGCTTTTGCCGAGAAGTTCATTAAACCCTCCCACCTAGAGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCAGAAGGATCA。

Claims (3)

1. a produce oil single needle algae LB59, is characterized in that:

This algae strain is preserved in China typical culture collection center by July 17th, 2013, and deposit number is CCTCC NO:M 2013349.

2., by the cultivation collection method of a kind of produce oil single needle algae LB59 according to claim 1, it is characterized in that:

1. the cultivation of single needle algae LB59: adopt the BG11 reducing SODIUMNITRATE content, add high density sodium bicarbonate and cultivate, the whole composition of substratum and consumption as follows, consumption presses mg/L;

K ₂hPO ₄3H ₂o, 40; MgSO ₄7H ₂o, 75; CaCl ₂2H ₂o, 36; Citric acid, 6;

FeC ₆H ₅O ₇·NH ₄OH，6； EDTANa ₂，1； Na ₂CO ₃，20； NaNO ₃，750；

NaHCO ₃，12600； H ₃BO ₃，2.8； MnCl ₂·4H ₂O，1.86； ZnSO ₄·7H ₂O，0.22；

Na ₂MoO ₄·2H ₂O，0.39； CuSO ₄·5H ₂O，0.08； CoCl ₂·6H ₂O，0.01；

2. the flocculation of single needle algae LB59 is gathered: after mass propgation, regulate nutrient solution pH with dilute hydrochloric acid, add flocculate with chitosan sedimentation cell.

3., by the application of the frustule of cultivation collection method acquisition according to claim 2, it is characterized in that:

1. the oil-containing frustule that its mass propgation obtains is used for grease and extracts;

2. its frustule directly adds in animal-feed increases unsaturated fatty acids nutrition;

3. the carbonic acid gas of the mass propgation Absorbable rod industrial discharge of its frustule.