CN105602933A

CN105602933A - Screening and culturing method of high-yield and low-temperature-resistant arthrospira and arthrospira

Info

Publication number: CN105602933A
Application number: CN201610130604.7A
Authority: CN
Inventors: 沈颂东; 关剑; 吴昊; 段大亮
Original assignee: Taicang Weizao Biotechnology Co Ltd
Current assignee: Taicang Weizao Biotechnology Co Ltd
Priority date: 2016-03-09
Filing date: 2016-03-09
Publication date: 2016-05-25

Abstract

The invention provides a screening and culturing method of high-yield and low-temperature-resistant arthrospira, a preparation method of the screening and culturing method and arthrospira obtained through the screening and culturing method. The screening and culturing method is reasonable and feasible, low-temperature-resistant high-quality arthrospira mutant strains are screened out by chemically inducing low-temperature screening, arthrospira can normally grow at a temperature of 20 DEG C or below, a large amount of arthrospira can be harvested within a short period of time, the annual cultivation period of arthrospira enterprises is prolonged, and economic benefits of arthrospira enterprises are improved.

Description

Screening and culturing method and the arthrospira of the low temperature resistant arthrospira of a kind of high yield

Technical field

The technical field that the invention belongs to arthrospira research and development application, particularly, relates to the low temperature resistant joint of a kind of high yieldRevolve the screening and culturing method of algae and the low temperature resistant arthrospira that application the method obtains.

Background technology

Arthrospira (Arthrospira) rule belongs to Cyanophyta (Cyanophyta) Cyanophyceae (Cyanophyceae) Oscillatoriales(Oscillatorisles) Oscillariaceae (Oscillatoriaceae) Arthrospira (Arthrospira), be one can be photosynthetic fromThe micro-algae biology of foster protokaryon. Because arthrospira and spirulina are not easily distinguishable under micro-light microscopic, once there is scholar to think ArthrospiraIn Spirullina, so that business is still referred to as spirulina or spirulina (arthrospira) arthrospira custom. Arthrospira nutritional labelingAbundant, protein content can reach 55%-70%, contains 16 seed amino acids in its albumen, comprising 8 kinds of essential amino acids; AndAnd arthrospira also contains and has the materials such as carrotene, phycocyanin and the gamma-Linolenic acid of medical care effect; Joint revolves in additionNot containing cellulose of frustule wall, human body is is very easily digested and assimilated. Therefore food and agricultural organization of arthrospira united state (FAO) and section of the United NationsReligion literary composition tissue (UNESCO) is described as " 21 century best ultimate food and health products promote ". Arthrospira is as the micro-algae of economyEntered large-scale farming and exploitation stage, its cultivation base is mainly distributed in China, the U.S., Japan, Mexico, with lookThe countries and regions such as row, Thailand, Vietnam. End China's arthrospira cultivation base in 2012 and just reach more than 60, from Hainan to blackAll there is arthrospira cultivation base in Longjiang from Shandong to Inner Mongol, cultured area approximately 7,500,000 m², the about 9600t of dry powder annual production, produces per yearValue is estimated to have exceeded 4,000,000,000 yuan. Along with growth in the living standard, people focus on health care more in recent years, and arthrospira is as onePlant the important micro-algae of economy (chlorella, green alga, arthrospira), be more and more subject to people's favor, it is promoting arthrospira industryConstantly flourish.

Arthrospira, as a kind of protokaryon algae, is extensively distributed in the torrid zone, subtropical zone and ocean, warm temperate zone, hot spring, He HuPool, particularly in salt alkali lake. Originally the current known arthrospira kind more than 38 that has is all from African Chad lake and MexicanTexcoco lake. What now obtain in the world broad research and popularization is Obtusatus arthrospira, Gas Vesicle Protein From Arthrospira maxima and salt pool arthrospira,Now Obtusatus arthrospira and Gas Vesicle Protein From Arthrospira maxima for what do one's utmost to promote, the widest with Obtusatus arthrospira application in China's commodity productionGeneral.

Although the arthrospira growth and breeding of prior art is fast, environmental suitability is strong, in the time of large-scale cultivation, be still subject to illumination,The impact of the factors such as temperature, nutrition, pH and ventilation, its maximum effect factor is exactly temperature and illumination. By to different arthrospirasBreeding growth temperature statistical summary, result shows that arthrospira normal growth temperature is 22-42 DEG C, the suitableeest the bread worm is 28-35DEG C, 16 DEG C is arthrospira growth lower limit, 42 DEG C is the arthrospira growth upper limit, and arthrospira poor growth during lower than 20 DEG C, but along withTemperature raises and growth quickening. China is all Obtusatus arthrospira and the utmost points from external introduction for the arthrospira of commercial aquaculture at presentLarge arthrospira, its optimum growth temperature is 35-37 DEG C, and China oneself distinctive low temperature resistant Erdos arthrospira the most suitable growthTemperature is 24-25 DEG C, also can not meet arthrospira breeding enterprise whole year production needs. Therefore at China's different regions arthrospira(include canopy protection) year production cycle has nothing in common with each other, can whole year production cultivation except minority areas such as Hainan, Guangdong and Guangxi,Other southern areas only can produce cultivation April to November, and northern area only can be produced cultivation May to October. In addition, joint revolvesAlgae can turn off the light to overcome the harmful effect that illumination deficiency is brought by adding day in the time of large-scale cultivation, and temperature just becomes impactArthrospira is produced the decisive factor of cultivation.

Summary of the invention

Goal of the invention: in order to overcome above deficiency, the invention provides the screening and culturing of the low temperature resistant arthrospira of a kind of high yieldMethod, it is rationally easily gone, and it is convenient to apply, and screens by chemical induction low temperature, filters out low temperature resistant high-quality arthrospira mutant strain,Can make arthrospira normal growth below 20 DEG C, and can gather in a large number in the short time, extend the arthrospira enterprise culture-cycle in year,Improve the economic benefit of arthrospira enterprise.

Technical scheme: in order to overcome the deficiencies in the prior art, the invention provides the low temperature resistant joint of a kind of high yield and revolveThe screening and culturing method of algae, comprises the following steps:

<1>chemical inducer soaks arthrospira 20-40min;

<2>step<1>algae strain in the repeated screening of 20 DEG C of 3000-4000lux low temperature, Liquid Culture, screening obtain 20 DEG C lowThe strain of temperature tolerance algae;

<3>separate the strain of 20 DEG C of cold tolerance algaes, in the repeated screening of 18 DEG C of 3000-4000lux low temperature, Liquid Culture, screeningObtain the strain of 18 DEG C of cold tolerance algaes;

<4>step<3>in the strain of 18 DEG C of cold tolerance algaes in 18 DEG C of 3000-4000lux solid culture multi-stage separation purifying,The single algae of picking falls, Liquid Culture;

<5>separating liquid is cultivated single algae and is fallen, and separates and obtains single algae strain;

<6>single algae strain is in 18 DEG C of 3000-4000lux illumination Liquid Culture, and screening obtains 18 DEG C of pure algae strains of cold tolerance;

<7>step<6>in the pure algae strain of 18 DEG C of cold tolerances in 16 DEG C of 3000-4000lux Liquid Culture, screening obtain 16DEG C cold tolerance algae strain;

<8>strain of 16 DEG C of cold tolerance algaes obtains 16 DEG C of low temperature tolerances in 16 DEG C of purebred illumination cultivation of 3000-4000lux liquidThe pure algae strain of property.

The present invention, by chemical mutagen mutagenic treatment arthrospira, reduces step by step temperature to arthrospira after mutagenic treatment and carries out20 DEG C and 18 DEG C of low temperature screenings repeatedly, filter out the strain of 18 DEG C of normal growth algaes, then uses different separation methods purebred to algae strainSeparate, isolate low temperature resistant single algae strain, then the further low temperature of single algae strain is filtered out to high resistance to of 16 DEG C of biological yieldsLow temperature arthrospira. Its method is reasonable, and it is convenient to apply, and filters out low temperature resistant high-quality arthrospira mutant strain, can make arthrospira 20Under DEG C following cryogenic conditions, arthrospira still can normal growth, and can gather in a large number in the short time, extends arthrospira enterpriseCulture-cycle in industry year, improve the economic benefit of arthrospira enterprise, market application foreground is wide.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described step<2>or<3>in anti-Multiple screening and culturing comprises naked eyes detection nutrient solution color and rejects under the microscope the arthrospira of growth failure. Detect by naked eyesNutrient solution color and the arthrospira of rejecting under the microscope growth failure can obtain the strain of cold tolerance algae fast.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described step<4>in multi-stage separation pureChange and comprise solid plate line and solid plate coating. By the multi-stage separation purifying that solid plate is rule and solid plate is coated withCan quick and precisely obtain required purebred algae strain.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described step<5>the middle microcapillary that adoptsSeparating liquid is cultivated single algae and is fallen. Capillary heats with alcolhol burner, pulls into superfine microtubule rapidly, simultaneously after it dissolves with tweezersMicrotubule end slightly thermal change is blunt, in case end sharply damages frond cell, its cost is low and application is convenient.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, the nutrient solution of described Liquid Culture isZarrouk nutrient solution.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, is characterized in that: described step<4>In solid medium be the Zarrouk nutrient solution of 1.5% agar powder.

Zarrouk nutrient solution is rich in the nutriment of multiple algal grown, can be preferably for the cultivation of algae.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described step<1>in chemical inductionAgent is 0.05-0.2mol/L ethylmethane sulfonate. Ethylmethane sulfonate (EMS) can be for obtaining the sudden change of low temperature tolerance fastAlgae strain, screening effeciency is high.

Further, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described step<6>in single algae strainIn 96 orifice plates, cultivate screening. 96 orifice plates are for Large-scale Screening, and flux is large, and efficiency is high.

As a kind of optimal way of the present invention, the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield, described lightBe 3500lux according to intensity. As intensity of illumination preferably, the life of the arthrospira of cold tolerance under the intensity of illumination of 3500luxLong most effective

The present invention also provides a kind of low temperature resistant arthrospira, for the screening and culturing method of the above-mentioned low temperature resistant arthrospira of high yield obtainsLow temperature resistant arthrospira. The super low-temperature resistant arthrospira filtering out by chemical induction low temperature, they can not only be at 16 DEG C of normal growths, andAnd can produce high biomass in the short time. Arthrospira enterprise is not in the situation that increasing any production equipment, and cultivation induction is sievedThe super low-temperature resistant mutant strain of selecting can make northern arthrospira enterprise realize early spring and normal produce cultivation late fall, makes its culture-cycleExtend the 2-4 month; Can make southern arthrospira enterprise realize whole year production cultivation, greatly improve the economic benefit of arthrospira enterprise.

Beneficial effect: compared with prior art, the present invention has the following advantages: the low temperature resistant joint of high yield of the present invention revolvesRationally easy row of the screening and culturing method of algae, it is convenient to apply, and the arthrospira of acquisition can not only be at 16 DEG C of normal growths, and the short timeIn can produce high biomass, have a extensive future.

Brief description of the drawings

Fig. 1 is the technical scheme flow chart of the screening and culturing method of the low temperature resistant arthrospira of high yield of the present invention;

Fig. 2 is Arthrospirasp.CBN2 optical microscopic morphology of the present invention;

Fig. 3 is arthrospira pcr amplification product electrophoretogram of the present invention;

Fig. 4 is arthrospira bacterium liquid pcr amplification product of the present invention

Fig. 5 is Arthrospirasp.CBN216SrRNANeighbor-Joining phylogenetic tree of the present invention;

Fig. 6 is Arthrospirasp.CBN216SrRNA-23SrNAITSNeighbor-of the present inventionJoining phylogenetic tree;

Fig. 7 is that 18 DEG C of 3500lux of the present invention leave standstill illumination cultivation dry biomass mensuration;

Fig. 8 is that the purebred cultivation dry biomass of 18 DEG C of 3500lux120rpm/min shaking tables of the present invention is measured;

Fig. 9 is that 16 DEG C of 3500lux of the present invention leave standstill illumination cultivation dry biomass mensuration.

Detailed description of the invention

To, by several specific embodiments, further illustrate the present invention below, these embodiment are in order to describe the problem,It is not a kind of restriction.

Embodiment 1

As shown in Figure 1, the screening and culturing method of the low temperature resistant arthrospira of a kind of high yield, comprises the following steps:

<1>0.05mol/L chemical inducer ethylmethane sulfonate soaks Erdos arthrospira 20min;

<2>by step<1>induction after algae liquid filter, frond is reentered in fluid nutrient medium recovers two days. By 5% amountBe transferred in triangular flask, algae strain, under 20 DEG C of 3000lux cryogenic conditions, detects nutrient solution color and at microscope by naked eyesThe arthrospira of lower rejecting growth failure, thus carry out repeated screening, Liquid Culture, and screening obtains the strain of 20 DEG C of cold tolerance algaes;

<3>separate the strain of 20 DEG C of cold tolerance algaes, be transferred in triangular flask by 5% amount, in 18 DEG C of 3000lux cryogenic conditionsUnder, detect nutrient solution color and reject under the microscope the arthrospira of growth failure by naked eyes, thereby carry out repeated screening, liquidBody is cultivated, and screening obtains the strain of 18 DEG C of cold tolerance algaes;

<4>step<3>in the strain of 18 DEG C of cold tolerance algaes under 18 DEG C of 3000-lux conditions, rule by solid plate andSolid culture multi-stage separation purifying is carried out in solid plate coating, and the single algae of picking falls, Liquid Culture;

<5>capillary heats with alcolhol burner, after it dissolves, pulls into superfine microtubule rapidly with tweezers, slightly heating of microtubule end simultaneouslyRust (in case end sharply damages frond cell). Inhale one and diluted the more than ten thousand times 10ml centrifuge tube list algae Liquid Culture that fallsAlgae drop on slide, algae liquid another other to drip sterilized nutrient solution for subsequent use. Under inverted microscope, separate gentlyGo out needed single arthrospira frond, for preventing that miscellaneous bacteria from sneaking into first, the single frond separating moved in nutrient solution on one side,And then move to 96 orifice plates from nutrient solution;

<6>arthrospira in 96 orifice plates is carried out to mark, single algae strain is 18 DEG C of 3000lux illumination Liquid Culture in 96 orifice plates,Screening obtains 18 DEG C of pure algae strains of cold tolerance;

<7>step<6>in the pure algae strain of 18 DEG C of cold tolerances be transferred in triangular flask by 1% amount, in 16 DEG C of 3000lux liquidBody is cultivated, and screening obtains the strain of 16 DEG C of cold tolerance algaes;

<8>strain of 16 DEG C of cold tolerance algaes obtains 16 DEG C of pure algaes of cold tolerance in 16 DEG C of purebred illumination cultivation of 3000lux liquidStrain.

Wherein, above-mentioned liquid medium is Zha Luke (Zarrouk) nutrient solution, and solid medium is 1.5% agar powderZarrouk nutrient solution. Shown in Zarrouk nutrient solution is formulated as follows

Order of addition	Medicine name	Consumption/L
			1	NaHCO₃	16.8g
2	NaNO₃	2.5g
			3	NaCl	1g
4	KH₂PO₄	0.42g
			5	K₂SO₄	1g
6	MgSO₄.7H₂O	0.20g
			7	CaCl₂.2H₂O	0.04g
8	FeSO₄.7H₂O	0.01g
			9	EDTA	0.08g
10	A₅	1ml
			11	B₆	10ul
Distilled water	Be settled to	1L

FeSO₄.7H₂O adds solution after must integrating in proportion with EDTA again

A₅Micro solution B6 micro solution

Medicine name	Consumption g/L
		NH₄VO₃	2.29g
NiSO₄.7H₂O	4.78g
		Na₂WO₄.2H₂O	1.79g 4 -->
Ti(SO₄)₂	4.00g
		Co(NO3)₂.6H₂O	0.44g
Distilled water	1000ml

Medicine name	Consumption g/L
		H₃BO₃	2.86
MnCl₂.4H₂O	1.80
		ZnSO_4.7H₂O	0.22
MoO₃	0.01
		CuSO₄.5H₂O	0.08
Distilled water	1000ml

Note: Ti (SO₄)₂Medicine can not add

NaHCO₃Consumption modulation 15.0g/L

Embodiment 2

<1>0.2mol/L chemical inducer ethylmethane sulfonate soaks Erdos arthrospira 40min;

<2>by step<1>induction after algae liquid filter, frond is reentered in fluid nutrient medium recovers two days. By 5% amountBe transferred in triangular flask, algae strain, under 20 DEG C of 4000lux cryogenic conditions, detects nutrient solution color and at microscope by naked eyesThe arthrospira of lower rejecting growth failure, thus carry out repeated screening, Liquid Culture, and screening obtains the strain of 20 DEG C of cold tolerance algaes;

<3>separate the strain of 20 DEG C of cold tolerance algaes, be transferred in triangular flask by 5% amount, in 18 DEG C of 4000lux cryogenic conditionsUnder, detect nutrient solution color and reject under the microscope the arthrospira of growth failure by naked eyes, thereby carry out repeated screening, liquidBody is cultivated, and screening obtains the strain of 18 DEG C of cold tolerance algaes;

<4>produce algae strain, step<3 because algae kind derives from>in the strain of 18 DEG C of cold tolerance algaes in 18 DEG C of 4000lux conditionsUnder, to rule and be coated with and carry out solid culture multi-stage separation purifying with solid plate by solid plate, the single algae of picking falls, liquid trainingSupport;

<5>capillary heats with alcolhol burner, after it dissolves, pulls into superfine microtubule rapidly with tweezers, and microtubule end slightly simultaneouslyThermal change blunt (in case end sharply damages frond cell). Inhale one and diluted the more than ten thousand times 10ml centrifuge tube list algae Liquid Culture that fallsAlgae drop on slide, algae liquid another other to drip sterilized nutrient solution for subsequent use. Under inverted microscope, divide gentlyFrom going out needed single arthrospira frond, for preventing that miscellaneous bacteria from sneaking into first, the single frond separating is moved to nutrient solution on one sideIn, and then move to 96 orifice plates from nutrient solution;

<6>arthrospira in 96 orifice plates is carried out to mark, single algae strain is 18 DEG C of 4000lux illumination Liquid Culture in 96 orifice plates,Screening obtains 18 DEG C of pure algae strains of cold tolerance;

<7>step<6>in the pure algae strain of 18 DEG C of cold tolerances be transferred in triangular flask by 1% amount, in 16 DEG C of 4000lux liquidBody is cultivated, and screening obtains the strain of 16 DEG C of cold tolerance algaes;

<8>strain of 16 DEG C of cold tolerance algaes obtains 16 DEG C of pure algaes of cold tolerance in 16 DEG C of purebred illumination cultivation of 4000lux liquidStrain.

Embodiment 3

<1>0.2mol/L chemical inducer ethylmethane sulfonate soaks Erdos arthrospira 20min;

<2>by step<1>induction after algae liquid filter, frond is reentered in fluid nutrient medium recovers two days. By 5% amountBe transferred in triangular flask, algae strain, under 20 DEG C of 3800lux cryogenic conditions, detects nutrient solution color and at microscope by naked eyesThe arthrospira of lower rejecting growth failure, thus carry out repeated screening, Liquid Culture, and screening obtains the strain of 20 DEG C of cold tolerance algaes;

<3>separate the strain of 20 DEG C of cold tolerance algaes, be transferred in triangular flask by 5% amount, in 18 DEG C of 3800lux cryogenic conditionsUnder, detect nutrient solution color and reject under the microscope the arthrospira of growth failure by naked eyes, thereby carry out repeated screening, liquidBody is cultivated, and screening obtains the strain of 18 DEG C of cold tolerance algaes;

<4>step<3>in the strain of 18 DEG C of cold tolerance algaes under 18 DEG C of 3800lux conditions, rule and consolidate by solid plateSolid culture multi-stage separation purifying is carried out in the dull and stereotyped coating of body, and the single algae of picking falls, Liquid Culture;

<6>arthrospira in 96 orifice plates is carried out to mark, single algae strain is 18 DEG C of 3800lux illumination Liquid Culture in 96 orifice plates,Screening obtains 18 DEG C of pure algae strains of cold tolerance;

<7>step<6>in the pure algae strain of 18 DEG C of cold tolerances be transferred in triangular flask by 1% amount, in 16 DEG C of 3800lux liquidBody is cultivated, and screening obtains the strain of 16 DEG C of cold tolerance algaes;

<8>strain of 16 DEG C of cold tolerance algaes obtains 16 DEG C of pure algaes of cold tolerance in 16 DEG C of purebred illumination cultivation of 3800lux liquidStrain.

Embodiment 4

<1>0.1mol/L chemical inducer ethylmethane sulfonate soaks Erdos arthrospira 37min;

<2>by step<1>induction after algae liquid filter, frond is reentered in fluid nutrient medium recovers two days. By 5% amountBe transferred in triangular flask, algae strain, under 20 DEG C of 3500lux cryogenic conditions, detects nutrient solution color and at microscope by naked eyesThe arthrospira of lower rejecting growth failure, thus carry out repeated screening, Liquid Culture, and screening obtains the strain of 20 DEG C of cold tolerance algaes;

<3>separate the strain of 20 DEG C of cold tolerance algaes, be transferred in triangular flask by 5% amount, in 18 DEG C of 3500lux cryogenic conditionsUnder, detect nutrient solution color and reject under the microscope the arthrospira of growth failure by naked eyes, thereby carry out repeated screening, liquidBody is cultivated, and screening obtains the strain of 18 DEG C of cold tolerance algaes;

<4>step<3>in the strain of 18 DEG C of cold tolerance algaes under 18 DEG C of 3500lux conditions, rule and consolidate by solid plateSolid culture multi-stage separation purifying is carried out in the dull and stereotyped coating of body, and the single algae of picking falls, Liquid Culture;

<6>arthrospira in 96 orifice plates is carried out to mark, single algae strain is 18 DEG C of 3500lux illumination Liquid Culture in 96 orifice plates,Screening obtains 18 DEG C of pure algae strains of cold tolerance;

<7>step<6>in the pure algae strain of 18 DEG C of cold tolerances be transferred in triangular flask by 1% amount, in 16 DEG C of 3500lux liquidBody is cultivated, and screening obtains the strain of 16 DEG C of cold tolerance algaes;

<8>strain of 16 DEG C of cold tolerance algaes obtains 16 DEG C of pure algaes of cold tolerance in 16 DEG C of purebred illumination cultivation of 3500lux liquidStrain.

Because embodiment 4 is optimum embodiment of the present invention, we to the method by embodiment 4 through chemical induction and repeatedlyLow temperature screening, the low temperature resistant arthrospira mutant strain of a strain is isolated in final screening, temporarily called after Arthrospirasp.CBN2.

1 low temperature arthrospira mode of appearance

(1) 20 DEG C of low temperature resistant cultivation the selection result of spirulina

Arthrospira induction is cultivated a period of time at 20 DEG C of 3500lux after processing, and sees and finds algae liquid blue-green, mirror from algae liquid colorArthrospira frond well-grown is found in inspection, can conclude arthrospira well-grown under 20 DEG C of low temperature.

(2) 18 DEG C of Strain selection situations of arthrospira

1) the mixed algae liquid filtering out from 18 DEG C of low temperature, drawing 100 times of plate streaking applying solids of 1ml algae liquid dilution cultivates. GuBody culture medium, after 18 DEG C of 3500lux illumination cultivation a period of times, grows fragmentary bacterium colony. Observe discovery from outward appearance and microscopy:Arthrospira bacterium colony outward appearance dark blue green, slightly irregular cycle; Optical microscope, its form of different bacterium colonies and gathering growth patternHave nothing in common with each other.

2) arthrospira is separated in 18 DEG C of 3500lux illumination cultivation of 96 orifice plate by microcapillary, and some Fast Growths, haveDo not grow, filter out the individual plant arthrospira of Fast Growth.

3) separate and 96 orifice plates cultivation finishing screen is selected the arthrospira of Fast Growth at low temperatures through microcapillary repeatedly,The purebred arthrospira filtering out is trained to purebred cultivation in the illumination of 18 DEG C of 3500lux liquid.

18 DEG C of 3500lux part pure strain liquid illumination cultivation upgrowth situations. Different algae strains are according to same inoculum concentration, with trainingSupport condition, same to incubation time, different algae strain growing states are slightly variant, the dense dark blue green of some algae liquid, the dirty-green having, haveDark green colour cast grey.

(3) 16 DEG C of Strain selection results of arthrospira

1) from the 18 DEG C of purebred cultivation algae of 3500lux liquid, be transferred to 16 DEG C, 3500lux illumination Liquid Culture by 1% amount, repeatedly lowTemperature filters out the strain of 16 DEG C of energy normal growth algaes.

The different algae strains that filter out are each variant with its upgrowth situation of growth conditions at 16 DEG C. The sky blue that algae liquid has, hasStrong green, some azure blacks etc.

2) the Arthrospirasp.CBN2 low temperature sudden change to 16 DEG C of purebred cultivations of 3500lux by light microscopeStrain is observed, and to 30 algae strains carry out strain length, bung flange, pitch, spiral shell is wide, cell is wide, microscopic pattern is observed and life habit statistics remittanceTotal as table 1.

Table 1:Arthrospirasp.CBN2 microscopic pattern and life habit

Algae strain name strain length/μ m bung flange/pitch/μ m spiral shell is wide/and μ m cell is wide/μ m form color end form life habit
	The slightly thin circle of the linear blue-green of Arthrospira sp.CBN2 800 ± 285 15 ± 5 58 ± 7 7.5 ± 2.5 7.2 ± 2.5 waves type of swimming

In addition, the Arthrospirasp.CBN2 algae strain optical microscopic morphology of the purebred cultivation of 3500lux as shown in Figure 2.

2 low temperature arthrospira molecular biology identification

16SrDNA gene magnification and sequence analysis

Get the exponential phase algae liquid 60ml of the Arthrospirasp.CBN216 DEG C of purebred cultivation of 3500lux of algae strain,Centrifugal 20 minutes of 8000rpm, centrifugal twice, twice, ultra-pure water washing algae mud. Suck algae mud surface with sterilizing blotting paper and swim, putIn sterilizing mortar, liquid nitrogen grinding is to fine and smooth Powdered. By CTAB method, Arthrospirasp.CBN2DNA is extracted,Taking the DNA product that extracts as template, by Auele Specific Primer Primer1 and Primer18 clone arthrospira 16SrRNA and 16SRRNA-23SrRNAinternaltranscribedspacer (ITS), carries out molecular biology identification. Upstream primerPrimer15 '-AGAGTTTGATCCTGGCTCAG-3 ' downstream primer Primer185 '-TTTGCGGCCGCTCTGTGTGCCTAGGTATCC-3 ' PCR product is served Hai Shenggong bioengineering Co., Ltd and is checked order, and surveysOrder result is as follows.

(1) the total sequence of sample:

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTCTGCTTAACACATGCAAGTCGAACGGGCTCTTCGGAGCTAGTGGCGGACGGGTGAGTAACACGTGAGAATCTGGCTCCCGGTCGGGGACAACAGAGGGAAACTTCTGCTAATCCCGGATGAGCCGAAAGGTAAAAGATTTATCGCCGGGAGATGAGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGCAAGCCTGACGGAGCAAGACCGCGTGGGGGAGGAAGGCTCTTGGGTTGTAAACCCCTTTTCTCAAGGAAGAACACAATGACGGTACTTGAGGAATAAGCCTCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGAGGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGTAGGTGGCAGTTCAAGTCTGCTGTCAAAGACAGTAGCTCAACTACTGAAAGGCAGTGGAAACTGAACAGCTAGAGTACGGTAGGGGCAGAGGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGGAAGAACACCGGTGGCGAAAGCGCTCTGCTGGGCCGTAACTGACACTGAGGGACGAAAGCTAGGGGAGCGAATGGGATTAGATACCCCAGTAGTCCTAGCCGTAAACGATGGAAACTAGGTGTAGCCTGTATCGACCCGGGCTGTGCCGAAGCTAACGCGTTAAGTTTCCCGCCTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAGGGCTTGACATGTCCGGAATCTTGGTGAAAGCCGAGAGTGCCTTCGGGAGCCGGAACACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTCCTTAGTTGCCATCATTCAGTTGGGCACTTTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTACGTCCTGGGCTACACACGTACTACAATGGGGGGGACAAAGGGTAGCCAAGACGCGAGTCTGAGCCAATCCCGTAAACCTCTCCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGGAGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATCCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTTAGCCACGCCCGAAGTCGTTACTCTAACCGTTCGCGGAGGAGGATGCCGAAGGCAGGGCTGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCACCTCCTTTTTAGGGAGACCTACTTCGAGATATCGCGCCTTAACAACTATAGCCGTGTCTTGAGGTCATCCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTATTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGGATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCACGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTTTGTGATGGTGCTAGTTGGGGTGAGATGAGATGAGATGACCTCTGATAGATAATTTATCACTGTACAGCTCCTAAATCTTTAGATGTTAGTCTGAGATTGGATAGCTGGACATCTGTTCCAGTCAGAACCTTGAAAACTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTAGGTCAAGCTACAAAGGGCTAACGGTGGATACCTAGGCACACAGAGCGGCCGCAAA2014bp

(2) 16SrRNA sequence:

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTCTGCTTAACACATGCAAGTCGAACGGGCTCTTCGGAGCTAGTGGCGGACGGGTGAGTAACACGTGAGAATCTGGCTCCCGGTCGGGGACAACAGAGGGAAACTTCTGCTAATCCCGGATGAGCCGAAAGGTAAAAGATTTATCGCCGGGAGATGAGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGCAAGCCTGACGGAGCAAGACCGCGTGGGGGAGGAAGGCTCTTGGGTTGTAAACCCCTTTTCTCAAGGAAGAACACAATGACGGTACTTGAGGAATAAGCCTCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGAGGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGTAGGTGGCAGTTCAAGTCTGCTGTCAAAGACAGTAGCTCAACTACTGAAAGGCAGTGGAAACTGAACAGCTAGAGTACGGTAGGGGCAGAGGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGGAAGAACACCGGTGGCGAAAGCGCTCTGCTGGGCCGTAACTGACACTGAGGGACGAAAGCTAGGGGAGCGAATGGGATTAGATACCCCAGTAGTCCTAGCCGTAAACGATGGAAACTAGGTGTAGCCTGTATCGACCCGGGCTGTGCCGAAGCTAACGCGTTAAGTTTCCCGCCTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAGGGCTTGACATGTCCGGAATCTTGGTGAAAGCCGAGAGTGCCTTCGGGAGCCGGAACACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTCCTTAGTTGCCATCATTCAGTTGGGCACTTTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTACGTCCTGGGCTACACACGTACTACAATGGGGGGGACAAAGGGTAGCCAAGACGCGAGTCTGAGCCAATCCCGTAAACCTCTCCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGGAGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATCCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTTAGCCACGCCCGAAGTCGTTACTCTAACCGTTCGCGGAGGAGGATGCCGAAGGCAGGGCTGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCACCTCCTTT1482bp

(3) ITS sequence:

TTAGGGAGACCTACTTCGAGATATCGCGCCTTAACAACTATAGCCGTGTCTTGAGGTCATCCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTATTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGGATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCACGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTTTGTGATGGTGCTAGTTGGGGTGAGATGAGATGAGATGACCTCTGATAGATAATTTATCACTGTACAGCTCCTAAATCTTTAGATGTTAGTCTGAGATTGGATAGCTGGACATCTGTTCCAGTCAGAACCTTGAAAACTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA477bp

(4)23SrRNA:

GGTCAAGCTACAAAGGGCTAACGGTGGATACCTAGGCACACAGAGCGGCCGCAAA55bp

Interpretation of result:

(1) taking arthrospira genomic DNA as template pcr amplification and positive colony detect

Utilize Primer1 and Primer18 Auele Specific Primer PCR to expand taking Arthrospirasp.CBN2 genomic DNA as templateIncrease. Pcr amplification product 1% agarose gel electrophoresis figure is as shown in 3.

Find out the about 2kbp of PCR product from electrophoretogram, meet expected results. Learn through the order-checking of Sanger methodArthrospirasp.CBN2PCR sequence size is 2014bp, wherein M:TakaRaDL5000DNAMark; 1-2:Arthrospirasp.CBN2PCR product.

Blue hickie screening picking list bacterium colony, in 37 DEG C, 220rpmLB fluid nutrient medium incubated overnight, adopts M13F and M13RUniversal primer carries out the screening of bacterium liquid PCR positive colony. Due to the existence of plasmid, electrophoresis detection pcr amplification product should be than object sheetThe large 200bp of section. Three single bacterium colony liquid mediums of each Sample selection carry out bacterium liquid PCR, and bacterium liquid pcr amplification product accords with as Fig. 4Close expected results, stripe size is in 2200bp left and right. M:TakaRaDL5000DNAMark; 1-3:ArthrospiraSp.CBN:2 bacterium liquid PCR product.

(2) pcr amplification sequence bioinformatic analysis

Learn that through order-checking Arthrospirasp.CBN2 sequence length is 2014bp. By the comparison point of NCBI website BLAST softwareAnalyse this extension increasing sequence, result shows that this sequence comprises 16SrRNA and 16SrRNA-23SrRNAinternalTranscribedspacer (ITS) complete sequence, 23SrRNA partial sequence. From GenBank storehouse, select several algae strain 16SRRNA and ITS sequence, by DNAMAN software Multiple Sequence Alignment with utilize MEGA5.0 software building Neighbor-Joining systemSystem is grown tree Arthrospirasp.CBN2 is carried out to sequence analysis. While building Neighbor-Joining phylogenetic tree, doBootstrap1000 bootstrapping analyzed.

Molecule result shows: build 16SrRNANeighbor-Joining phylogenetic tree, qualification algae strain ArthrospiraSp.CBN2 as Fig. 5 be arthrospira. Build 16SrRNA-23SrRNAinternaltranscribedspacer (ITS)Neighbor-Joining phylogenetic tree, identify algae Arthrospirasp.CBN2 as Fig. 6 be Obtusatus arthrospira.

Under 3 different condition of culture, the biological dry weight of low temperature arthrospira is measured

Get centrifugal 20 minutes of 60ml algae liquid 8000rpm centrifugal twice, ultra-pure water washing 2 times for algae mud, after put into sterile hoodSpend the night and dry, then dry and put 10h to constant weight at 78 DEG C of baking ovens.

Easily know from table 2, the absolute biological dry weight of Arthrospirasp.CBN2 is the trend of obvious rising, algae strain generallyAfter day growth rate first obviously rises in certain hour section, slow decreasing finally goes to zero. Under different condition of culture, identical trainingIn the time of supporting, the absolute dry biomass growth rate of Arthrospirasp.CBN2 and day dry biomass growth rate are each different,18 DEG C of aggregate analyses, 3500lux shaking table illumination cultivation are better than 18 DEG C, 3500lux and leave standstill illumination cultivation, and 18 DEG C, 3500lux leave standstillIllumination cultivation is better than 16 DEG C, the standing illumination cultivation of 3500lux. Arthrospirasp.CBN218 DEG C of screening algae strain, 3500luxShaking table illumination cultivation preferentially reaches maximum day growth rate, when 16 DEG C, 3500lux leave standstill illumination cultivation than algae under other condition of cultureThe laundering period of strain is longer, as long as but algae strain tide over its algae strain day growth of laundering period and obviously accelerate, biological yield is high.

Leaving standstill illumination cultivation dry biomass measurement result from 8 DEG C of 3500lux of Figure 71 can find out, Mixed culture 27d reachesTo plateau, its biological dry weight 0.063g/60ml is biological dry weight 1.050g/L. Mixed culture 27d does not reach plateau separatelyStill can continue breeding growth, the biological dry weight of the biological dry weight 0.0808g/60ml of Arthrospirasp.CBN2 in the time of 27d1.347g/L. Independent purebred cultivation is higher (in figure: CBN218 DEG C of 3500lux is purebred than Mixed culture cultivation productive rate as can be seen hereCultivate 18 DEG C of 3500lux Mixed culture of many kinds of algae strains of C).

Analyze shaking table training from the 8 DEG C of purebred cultivation dry biomass of 3500lux shaking table 120rpm/min measurement results of Figure 81Foster 13d reaches plateau, and the biological dry weight of plateau Arthrospirasp.CBN2 is that 0.0723g/60ml is biological dry weight1.205g/L. Easily know thus the appropriate O of increasing in the time of both culturing microalgae₂And CO₂Supply can obviously improve output and shortening is gathered the cycle.

Comparison diagram 9 and Fig. 7 can find, low temperature arthrospira leaves standstill illumination cultivation and 18 DEG C at 16 DEG C of 3500luxIt is widely different that 3500lux leaves standstill its biomass of illumination cultivation, and the low temperature arthrospira filtering out on the contrary at low temperature than high temperature output moreHigh. In the time of 24d, the dry biological yield of Arthrospirasp.CBN2 is the biological dry weight 1.432g/L of 0.0859g/60ml.

To sum up, Arthrospirasp.CBN2 belongs to Obtusatus arthrospira, algae strain wave linearity, the slightly thin blunt round of end, strainLong 800 ± 285 μ m, 15 ± 5 spiral numbers, pitch long 58 ± 7 μ m, wide 7.5 ± 2.5 μ m of spiral, wide 7.2 ± 2.5 μ m of cell, indigo plantGreen floating growth. And Arthrospirasp.CBN2 cold tolerance is good, still can be under 16 DEG C of low temperature Fast Growth alsoProduce high biomass (biological dry weight 1.432g/L). 16 DEG C, the standing illumination cultivation of 3500lux, ArthrospiraThe maximum daily gain 90.83mg/L of sp.CBN2 (DW), does not reach plateau biomass and reaches 1.432g/L (DW). ArthrospiraSp.CBN2 can meet annual breeding production and some high latitude or the temperature low countries and regions pair of enterprise of China to arthrospiraThe breeding production of arthrospira. In industrial production cultivation, short, strain filament length of Arthrospirasp.CBN2 culture-cycle, very suitableIn enterprise's breeding production.

Claims

1. a screening and culturing method for the low temperature resistant arthrospira of high yield, is characterized in that: comprise the following steps:

<1>chemical inducer soaks arthrospira 20-40min;

2. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described step < 2>or<3>in repeated screening cultivate and comprise that naked eyes detect nutrient solution color and reject under the microscope the arthrospira of growth failure.

3. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described step < 4> in multi-stage separation purifying comprise that solid plate line and solid plate are coated with.

4. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described step < 5> in adopt microcapillary separating liquid to cultivate single algae to fall.

5. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described liquid trainingFoster nutrient solution is Zarrouk nutrient solution.

6. according to the screening and culturing method of the low temperature resistant arthrospira of high yield described in claim 1 or 3, it is characterized in that: described stepSuddenly the solid medium<4>is the Zarrouk nutrient solution of 1.5% agar powder.

7. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described step < 1> in chemical inducer be 0.05-0.2mol/L ethylmethane sulfonate.

8. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described step < 6> in single algae strain in 96 orifice plates, cultivate screening.

9. the screening and culturing method of the low temperature resistant arthrospira of high yield according to claim 1, is characterized in that: described illumination is strongDegree is 3500lux.

10. a low temperature resistant arthrospira, is characterized in that: described low temperature resistant arthrospira is described in claim 1-9 any oneThe low temperature resistant arthrospira that the screening and culturing method of the low temperature resistant arthrospira of high yield obtains.