CN103540533A - 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

Acquisition and the application of produce oil single needle algae LB59

Technical field

The present invention relates to screening and the culture technique of oil-producing microalgae, relate in particular 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, by separation and purification, obtains a kind of produce oil algae strain that can be used as the fat raw material that produces oil.

Background technology

On the earth, certified algae approximately surpasses 40000 kinds, and some of them kind is rich in grease.The grease extracting from oily algae can, through reacting and be converted into biofuel with methyl alcohol, be considered to the most possible diesel oil that substitutes day by day exhausted oil source.16 carbon that it contains and the lipid acid of 18 carbon are the most favourable for the production of biofuel.The grease of algae also contains unsaturated fatty acids, can add in animal-feed and promote to grow.The algae kind that screening obtains easily and cultivates, growth is fast, produce oil is high is to 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 American National renewable energy source laboratory, just start to utilize algae to extract the R&D work of grease, preparation biofuel, from ocean and separated more than 3000 kind of algae lake, therefrom filter out kind more than 300 growth fast, containing the algae kinds such as diatom, green alga and blue-green algae of innage, but unresolved Cost Problems at that time.The investigator of other various countries also utilizes various means to screen energetically searching to adapt to the good algae kind of commercially producing in the world.

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

The present invention is exactly the good produce oil algae strain obtaining from Inner Mongol field water collection, separation and purifying based on above background.

Summary of the invention

Object of the present invention is just to overcome in prior art the problem for the production of the good algae strain deficiency of microalgae grease, acquisition and the application thereof of a kind of produce oil single needle algae LB59 are provided, in waters, field, Inner Mongol, gather water sample, separation and purification obtain a kind ofly there is resistance, the easy oil-producing microalgae of open culture, 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 ^o54 ' 4.85 ^", east longitude 116 ^o19 ' 32.71 ^".Sampling time is on July 29th, 2011, and the water temperature on the same day is 28.1 ℃, and the specific conductivity of water sample is 0.392 ms/cm, and pH is 7.45.

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

Water sample is coated with flat band method by dilution and at the BG11 that adds 8.4g/L sodium bicarbonate, cultivates, isolates single algae and fall, each algae fall a small amount of cultivate extract total fat in thin layer chromatography board relatively.Select the strain that oleaginousness is higher, universal primer amplification 18S rDNA send order-checking after clone, and its sequence is SEQ NO.1 (seeing sequence table); By phylogenetic relationship, set to build and determine that this algae is single needle Trentepohlia (Monoraphidium) member (Fig. 1); Examine under a microscope its form and meet single needle Trentepohlia feature, have sheet chloroplast(id), called after single needle algae LB59 (Monoraphidium sp.LB59) (Fig. 2); Compare with the LB50 algae strain (another patent application) belonging to together, cell is less, and 18S rDNA has 8 base differences (seeing the base in square frame in sequence table); On July 17th, 2013 is preserved in Chinese Typical Representative culture collection center (Wuhan, China Wuhan University) by this algae strain, and deposit number is CCTCC NO:M2013349.

Two, the cultivation collection method of single needle algae LB59

1. cultivate: adopt the BG11 that reduces SODIUMNITRATE content, add high density carbonic acid hydrogen sodium and cultivate, training

Support the whole compositions of base in Table 1;

By test, compare, set up 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 ℃, substratum is the BG11 with 0.75 g/L SODIUMNITRATE, adds 12.6 g/L sodium bicarbonates (the whole compositions of substratum are in Table 1).With the open photoreactor of circle, cultivate (see figure 3), pass into carbonic acid gas every day 1 hour, within every 5 days, collect 1/2 culturing cell, and supplement isopyknic fresh culture, its growth velocity can reach 23.6 g/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 activity is at 2000 μ E/m ²s reaches capacity, and can also further improve intensity of illumination 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 a large amount of cultivation, with dilute hydrochloric acid, regulate nutrient solution pH, add flocculate with chitosan sedimentation cell.

Single needle algae LB59 is transferred in collection container after a large amount of cultivation, with dilute hydrochloric acid, pH is adjusted to 6, add chitosan to 5.0 mg/L; Remove upper strata nutrient solution, collect the frustule of sedimentation.

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

1. its a large amount of oil-containing frustules that obtain of cultivating extract for grease;

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

3. a large amount of cultivations of its frustule can absorb the carbonic acid gas of industrial discharge.

The frustule of collecting is dried with freeze drier; Dry algae is extracted to 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%.The grease extracting from this algae, be rich in unsaturated fatty acids, for example, 18 carbon 3 olefin(e) acids and 18 carbon 4 olefin(e) acid summations account for the more than 20% of lipid acid.

Its purposes is mainly:

Its a large amount of oil-containing frustules that obtain of cultivating extract for grease, also can directly add in animal-feed and increase unsaturated fatty acids nutrition, in a large amount of culturing process, also can absorb the carbonic acid gas of industrial discharge.

The present invention has following advantages and positively effect:

1. the growth of single needle algae LB59, without the need for the nutrition of machine matter, can obtain continuously grease-contained biomass by open cultivation in cheap inorganic nutrient solution; In its substratum, contain higher concentration sodium bicarbonate, can prevent the infringement of protozoon and pathogenic bacterium and the pollution of other assorted algaes, in commercially producing process, 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, is maintaining 23.6 g/m ²in the growth of d and the situation of collection rate, fat content can reach 30.2% of dry cell weight, contains more than 20% 18 carbon 3 olefin(e) acids 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 is used circular light bioreactor culture single needle algae LB59;

Fig. 4 is that cultivate in duty runway pond.

On July 17th, 2013 is preserved in Chinese Typical Representative culture collection center by the algae strain of this single needle algae LB59, and deposit number is CCTCC NO:M 2013349.

Embodiment

Below in conjunction with accompanying drawing and example in detail:

Example one, semicontinuous cultivation in circular light reactor

The culture medium prescription preparation nutrient solution of as described above, at 0.1 m ²inoculation culture in circular light reactor, nutrient solution volume is 20 L.Initial OD ₆₈₀value is 0.05, and temperature is controlled as 30-35 ℃, and light intensity is 200 μ E/m ²s continuous light, lead to the CO of 1 hour every day in nutrient solution ₂, and every day, OD was surveyed in sampling ₆₈₀value, OD ₆₈₀value reaches at 6.0 o'clock, collects 1/2 algae liquid, and supplementary 1/2 fresh culture carries out semicontinuous cultivation, the later substratum of collecting a same volume of algae liquid feed supplement every 5 days.Single needle algae LB59 is adjusted to 6 with dilute hydrochloric acid by pH in collection container, adds chitosan to 5.0 mg/L, and within 30 minutes, flocculating settling efficiency reaches more than 90%; The algae liquid of flocculation is removed to supernatant liquor, obtain concentrated frustule, will after frustule lyophilize, weigh, calculate growth velocity, its growth velocity of result can reach 23.6 g/m ²d.In every 100g dry algae powder, add the ratio of 4 L chloroform/methanol (1:1) to extract total fat, then add H in the ratio of 1L ₂o, after vibration mixes, the centrifugal 5-10min of 3000 rpm makes the chloroform phase transition of solution layering ，Jiang lower floor to logical nitrogen volatilization chloroform in 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 in table 2 single needle algae LB59 forms (molar percentage)

Lipid acid kind	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 outdoor 5 m of Ordos, inner Mongolia ²raceway pond carries out open cultivation (see figure 4), and nutrient solution volume is 750 L.It is 0.04 m/s that raceway pond flow velocity take that colyliform agitator controls; Water temperature daytime is 30 ℃ of left and right, and be 24 ℃ of left and right evening; Intensity of illumination is with weather rain or shine and different, 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 to collect the frustule liquid of 1/5 volume, then adds 1/5 fresh medium to carry out semicontinuous cultivation, collects a feed supplement once every 3 days later.The algae liquid of collecting, through micro-Microscopic observation, keeps more than 99% purebred state, and seldom miscellaneous bacteria and other algae kinds are polluted.As example one, collected algae is adjusted to 6 with dilute hydrochloric acid by pH in container, add chitosan to 5.0 mg/L, flocculating settling 30 minutes, removes upper strata nutrient solution and obtains concentrated frustule for extracting grease, and total fat accounts for 24.0% of dry cell weight.

sequence table

(bases different from produce oil single needle algae LB50 have added square frame)

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

Acquisition and the application thereof of <120> produce oil single needle algae LB59

<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:

With a kind of green alga of cultivating in the BG11 that contains sodium bicarbonate, separation obtains, examine under the microscope its shape

State meets single needle Trentepohlia monoraphidiumfeature; Its 18S rDNA sequence SEQ NO.1, belongs to a kind of single needle algae through phylogenetic relationship analytical proof, called after single needle algae LB59, monoraphidiumsp. LB59; This algae kind can enduring high-concentration sodium bicarbonate, is easy to carry out open cultivation, is maintaining 23.6 g/m ²in the growth of d and the situation of collection rate, fat content can reach 30.2% of dry cell weight, has more than 20% 18 carbon 3 olefin(e) acids and 18 carbon 4 olefin(e) acids in lipid acid; On July 17th, 2013 is preserved in Chinese Typical Representative culture collection center by this algae strain, and deposit number is CCTCC NO:M 2013349.

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

1. the cultivation of single needle algae LB59: adopt the BG11 that reduces SODIUMNITRATE content, add high density carbonic acid hydrogen

Sodium is cultivated, and the whole compositions of substratum are in Table 1;

2. the flocculation of single needle algae LB59 is gathered: after a large amount of cultivation, with dilute hydrochloric acid, regulate nutrient solution pH, add shell

Glycan flocculation sediment cell.

3. by the application of cultivation collection method claimed in claim 2, it is characterized in that:

1. its a large amount of oil-containing frustules that obtain of cultivating extract for grease;

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

3. a large amount of cultivations of its frustule can absorb the carbonic acid gas of industrial discharge.

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