WO2017156701A1

WO2017156701A1 - Method for screening and culturing high-yield and low temperature-tolerant arthrospira and arthrospira

Info

Publication number: WO2017156701A1
Application number: PCT/CN2016/076352
Authority: WO
Inventors: 沈颂东; 关剑; 吴昊; 段大亮
Original assignee: 太仓薇藻生物技术有限公司
Priority date: 2016-03-15
Filing date: 2016-03-15
Publication date: 2017-09-21

Abstract

Provided is a method for screening and culturing a high-yield and low temperature-tolerant Arthrospira and the low temperature-tolerant Arthrospira obtained by this method, wherein the screening culture method comprises chemical mutagenesis and low temperature screening.

Description

Screening and cultivating method for high-yield and low-temperature resistant Arthrospira

Technical field

The invention belongs to the technical field of research and development application of Arthrospira, and in particular relates to a screening and culture method for high-yield and low-temperature-resistant Arthrospira and the low-temperature-resistant algae obtained by the method.

Background technique

Arthrospira, Cyanophyta, Cyanophyceae, Oscillatorisles, Oscillatoriaceae, Arthrospira, a photosynthetic autotrophic prokaryotic microalgal organism . Because Arthrospira and Spirulina are difficult to distinguish under microscopic light microscopy, some scholars believe that Arthrospira belongs to the genus Spirulina, and even the commercial use of Arthrospira is called Spirulina or Spirulina. Arthrospira is rich in nutrients, protein content can reach 55%-70%, its protein contains 16 kinds of amino acids, including 8 kinds of essential amino acids; and Arthrospira also contains carotene and phycocyanin which have great health care effects. Protein and γ-linolenic acid and other substances; in addition, the cell wall of Arthrospira contains no cellulose, and the human body is easily digested and absorbed. Therefore, Arthrospira is hailed as “the best ideal food and health products in the 21st century and promoted by the Food and Agriculture Organization of the United Nations (FAO) and UNESCO). As an economic microalgae, Arthrospira has entered the stage of large-scale breeding and development and utilization. Its breeding bases are mainly distributed in China, the United States, Japan, Mexico, Israel, Thailand, Vietnam and other countries and regions. As of 2012, there are more than 60 domestic algae breeding bases in China. From Hainan to Heilongjiang, there are algae-breeding algae breeding bases from Shandong to Inner Mongolia. The breeding area is about 7.5 million m ² and the annual output of dry powder is about 9600 tons. It has exceeded 4 billion yuan. In recent years, with the improvement of living standards, people pay more attention to health care. As an important economic microalgae (chlorella, green algae, arthrospira), Arthrospira is more and more popular among people, which promotes The cyanobacteria industry continues to flourish.

As a prokaryotic algae, Arthrospira is widely distributed in tropical, subtropical and warm temperate oceans, hot springs, and lakes, especially in saline-alkali lakes. More than 38 species of Arthrospira are currently known, initially from Chad Lake in Africa and Texcoco Lake in Mexico. Nowadays, the world has been widely researched and promoted in the world. It is the genus Algae, Algae and Algae. It is now promoted in the commercial production in China. The genus Algae is the most widely used.

Although the arthropods of the prior art grow fast and have strong environmental adaptability, they are still affected by factors such as light, temperature, nutrition, pH and ventilation during large-scale farming, and the biggest influencing factors are temperature and illumination. Through the statistical summary of the growth temperature of different species of Arthrospira, the results showed that the normal growth temperature of Arthrospira was 22-42 °C, the optimum breeding temperature was 28-35 °C, 16 °C is the lower limit of growth of Arthrospira, and 42 °C is the spinning cycle. The upper limit of algae growth, when the temperature is lower than 20 °C, the growth of Arthrospira is slow, but the growth is accelerated with the increase of temperature. At present, the Arthrospira used in commercial aquaculture in China is introduced from abroad, and the optimal growth temperature is 35-37 °C, and the unique low temperature resistance of Ordos sinensis in China. The optimum growth temperature is 24-25 °C, which can not meet the annual production needs of the cyanobacteria aquaculture enterprises. Therefore, the annual production cycle of Arthrospira (including shed protection) varies in different regions of China. Except for a few areas such as Hainan, Guangdong and Guangxi, which can be produced all year round, other southern regions can only produce from April to November. In the northern region, production can only be carried out from May to October. In addition, in the large-scale breeding, Arthrospira can overcome the adverse effects caused by insufficient light by adding daylighting lamps, and the temperature becomes the decisive factor affecting the production and culture of Arthrospira.

Summary of the invention

OBJECT OF THE INVENTION In order to overcome the above deficiencies, the present invention provides a screening and cultivating method for high-yield and low-temperature-resistant Arthrospira, which is reasonable and easy to apply, and is screened by chemically induced low temperature screening to screen low temperature and high quality Arthrospira mutants. It can make the Arthrospira grow normally below 20 °C, and can harvest a large amount in a short time, prolong the annual breeding cycle of Arthrospira, and improve the economic benefits of Arthrobacter.

Technical Solution: In order to overcome the deficiencies in the prior art, the present invention provides a screening and cultivating method for high-yield and low-temperature resistant Arthrospira, comprising the following steps:

<1> chemical inducer soaked in Arthrospira 20-40min;

<2> The algal strain of step <1> is repeatedly screened and cultured at a low temperature of 3000-4000 lux at 20 ° C, and a 20 ° C low temperature resistant algae strain is obtained by screening;

<3> Isolation of 20 °C low temperature resistant algal strains, repeated screening and liquid culture at 18 °C 3000-4000 lux, and screening to obtain 18 °C low temperature resistant algae strain;

<4> The 18 °C low temperature tolerance algae strain in step <3> is separated and purified in a multi-stage of 3000-4000 lux solid culture at 18 ° C, and single algae is picked and liquid cultured;

<5> Separating a liquid to culture a single algal bloom, and separating and obtaining a single algae strain;

<6> A single strain of algae was cultured in a 3000-4000 lux light liquid at 18 ° C, and a pure strain of 18 ° C low temperature tolerance was obtained.

<7> The 18 ° C low temperature tolerance pure algae strain in step <6> was cultured in a 3000-4000 lux liquid at 16 ° C, and a 16 ° C low temperature resistant algae strain was obtained by screening;

<8>16°C low temperature resistant algae strain at 3000°C lux liquid pure light at 16°C A pure strain of 16 ° C low temperature tolerance pure algae was obtained by culturing.

The invention treats Arthrospira by chemical mutagen mutagenesis, and after the mutagenesis treatment, the temperature of the Arthrospira is gradually lowered and subjected to repeated low temperature screening at 20 ° C and 18 ° C, and the normal growth algae strain at 18 ° C is screened, and then different separation methods are used. The pure strains of the algae were isolated, and the low-temperature resistant single-algae strains were isolated. Then, the low-temperature screening of the single-algae strains was carried out to screen the high-temperature-resistant algae-resistant algae at 16 °C. The method is reasonable, the application is convenient, and the low-temperature and high-quality Rhizopus sp. mutant strain can be screened, and the Arthrospira can still grow normally under the low temperature condition below 20 °C, and can be harvested in a short time, and the section is extended. The annual breeding cycle of the spirulina company will improve the economic benefits of the Cyclosporin, and the market has broad application prospects.

Further, in the screening and culture method of the above-mentioned high-yield and low-temperature-resistant Arthrospira, the repeated screening culture in the step <2> or <3> includes visually detecting the color of the culture liquid and removing the abnormal growth of Arthrospira under the microscope. The low temperature resistant algae strain can be quickly obtained by visually detecting the color of the culture medium and removing the abnormal growth of Arthrospira under the microscope.

Further, in the above screening and culture method for high-yield and low-temperature-resistant Arthrospira, the multi-stage separation and purification in the step <4> includes solid plate scribing and solid plate coating. The desired pure algae strain can be obtained quickly and accurately by multi-stage separation and purification by solid plate scribing and solid plate coating.

Further, in the above-mentioned screening and culture method for high-yield and low-temperature-resistant Arthrospira, in the step <5>, a single tube is used to separate a liquid to culture a single algal. The capillary tube is heated by an alcohol lamp, and after being melted, it is quickly pulled into a very fine tube by a forceps, and the end of the micro tube is slightly heated and blunt to prevent the terminal end sharply damage the algal body cells, which is low in cost and convenient in application.

Further, the above-mentioned screening and culture method for high-yield and low-temperature resistant Arthrospira, the liquid The culture medium was a Zarrouk medium.

Further, the method for screening and cultivating the high-yield and low-temperature-resistant Arthrospira is characterized in that the solid medium in the step <4> is a Zarrouk culture solution of 1.5% agar powder.

Zarrouk broth is rich in a variety of algae-grown nutrients and can be used for algae cultivation.

Further, in the screening and culture method of the above-mentioned high-yield and low-temperature-resistant Arthrospira, the chemical inducer in the step <1> is 0.05-0.2 mol/L ethyl methanesulfonate. Ethyl methanesulfonate (EMS) can be used to rapidly obtain low temperature tolerance mutant strains with high screening efficiency.

Further, in the screening and culture method of the above-mentioned high-yield and low-temperature-resistant Arthrospira, the single algae strain in the step <6> is cultured and screened in a 96-well plate. The 96-well plate is used for large-scale screening, with large flux and high efficiency.

As a preferred mode of the present invention, the above-described screening and culture method for high-yield and low-temperature-resistant Algae, the light intensity is 3,500 lux. As a preferred light intensity, the low temperature tolerance of Arthrospira is the highest in 3500 lux.

The invention also provides a low-temperature-resistant algae, which is obtained by the above-mentioned screening and culture method for high-yield and low-temperature-resistant algae. The ultra-low temperature-resistant Arthrospira strains screened by chemically induced low temperature can not only grow normally at 16 ° C, but also produce extremely high biomass in a short time. The ultra-low temperature resistant mutant strain induced by the culture of Caenorhabditis can be used to achieve normal production and breeding in early spring and late autumn, and the breeding cycle is extended by 2-4 months; The annual production of aquaculture in the southern festival of algae has greatly improved the economic benefits of the spine algae enterprises.

Advantageous Effects: Compared with the prior art, the present invention has the following advantages: the present invention The screening and culture method of high-yield and low-temperature-resistant Arthrospira is reasonable and easy to apply, and the obtained Arthrospira can not only grow normally at 16 °C, but also can produce extremely high biomass in a short time, and has a broad application prospect.

DRAWINGS

1 is a flow chart of a technical scheme for screening and cultivating a high-yield and low-temperature-resistant Arthrospira of the present invention;

2 is an optical microscopic morphology of Arthrospira sp. CBN2 according to the present invention;

3 is an electrophoresis pattern of a PCR amplification product of Arthrospira according to the present invention;

Figure 4 is a PCR amplification product of the genus Aspergillus liquid of the present invention

Figure 5 is an Arthrospira sp. CBN2 16S rRNA Neighbor-Joining phylogenetic tree of the present invention;

Figure 6 is an Arthrospira sp. CBN2 16S rRNA-23S rNA ITS Neighbor-Joining phylogenetic tree of the present invention;

Figure 7 is a dry biomass measurement of a 3,500 lux static light culture at 18 ° C according to the present invention;

Figure 8 is a dry biomass measurement of a pure seed culture of a 3500 lux 120 rpm/min shaker at 18 ° C according to the present invention;

Figure 9 is a dry biomass measurement of 3500 lux standing light culture at 16 ° C according to the present invention.

detailed description

The invention will be further clarified by the following specific examples, which are merely illustrative and not limiting.

Example 1

As shown in Fig. 1, a screening and culture method for high-yield and low-temperature-resistant Arthrospira includes the following steps:

<1>0.05mol/L chemical inducer ethyl sulfonate soaked in Ordos cerium 20min;

<2> The induced algae liquid of the step <1> was filtered, and the algae body was re-introduced in a liquid medium for two days. According to the amount of 5%, the flask was transferred to a flask. The algae strain was visually detected at a low temperature of 3000 lux at 20 °C, and the color of the culture solution was visually detected and the abnormal growth of Arthrospira was removed under the microscope, thereby performing repeated screening, liquid culture, and screening. 20 ° C low temperature tolerance algae strain;

<3> Isolation of 20 °C low temperature resistant algae strain, transferred to a flask at 5%, and the color of the culture medium was visually detected under the condition of 3000 lux at 18 ° C, and the abnormal growth of Arthrospira was removed under the microscope. , thereby performing repeated screening, liquid culture, screening to obtain 18 ° C low temperature tolerance algae strain;

<4> The 18°C low-temperature-tolerant algal strain in step <3> was subjected to solid-state multi-stage separation and purification by solid plate scribe and solid plate coating at a temperature of 18°C and 3000-lux, and single algae was picked. Liquid culture

<5> The capillary is heated with an alcohol lamp, and after it is dissolved, it is quickly pulled into a very fine tube by a forceps, and the end of the microtube is slightly heated and blunt (to prevent the end from sharply damaging the algal cells). Pipette a drop of algae droplets that have been diluted 10,000 times more than 10 ml in a single-algae liquid culture on a glass slide, and drop a sterilized culture solution on the other side of the algae solution for use. Gently separating the desired single algae algae body under an inverted microscope, in order to prevent the mixed bacteria from moving the separated single algae body to the side of the culture solution, and then moving from the culture solution to the 96-well plate;

<6> The Arthrospira in the 96-well plate was labeled, and the single algae strain was cultured in a 96-well plate at 18 °C 3000 lux light liquid to obtain a 18 ° C low temperature tolerance pure algae strain;

<7> The 18 ° C low temperature tolerance pure algae strain in step <6> was transferred to a flask at 1%, and cultured in a 3000 lux liquid at 16 ° C to obtain a 16 ° C low temperature resistant algae strain;

<8> 16 ° C low temperature tolerance algae strain was cultured at 16 ° C 3000 lux liquid pure light to obtain a 16 ° C low temperature tolerance pure algae strain.

The liquid culture solution described above is a Zarrouk culture solution, and the solid medium is a Zarrouk culture solution of 1.5% agar powder. Zarrouk medium is prepared as shown below

添加顺序Add order	药品名Drug name	用量/LDosage / L
11	NaHCO₃ NaHCO ₃	16.8g16.8g
22	NaNO₃ NaNO ₃	2.5g2.5g
33	NaCl NaCl		1g1g
44	KH₂PO₄ KH ₂ PO ₄	0.42g0.42g
55	K₂SO₄ K ₂ SO ₄	1g 1g
66	MgSO₄.7H₂OMgSO ₄ .7H ₂ O	0.20g0.20g
77	CaCl₂.2H₂OCaCl ₂ .2H ₂ O	0.04g0.04g
88	FeSO₄.7H₂OFeSO ₄ .7H ₂ O	0.01g0.01g

99	EDTAEDTA	0.08g0.08g
1010	A₅ A ₅	1ml1ml
1111	B₆ B ₆	10ul10ul
蒸馏水Distilled water	定容至Tolerance to	1L1L

FeSO ₄ .7H ₂ O must be proportionally integrated with EDTA before adding the solution

Note: Ti(SO ₄ ) ₂ drugs may not be added

NaHCO ₃ dosage modulation 15.0g / L

Example 2

<1>0.2mol/L chemical inducer ethyl sulfonate soaked in Ordos cerevisiae for 40min;

<2> The induced algae liquid of the step <1> was filtered, and the algae body was re-introduced in a liquid medium for two days. According to the amount of 5%, the flask was transferred to a flask. The algae strain was visually detected at a low temperature of 4000 lux, and the color of the culture solution was visually detected and the abnormal growth of Arthrospira was removed under the microscope, thereby performing repeated screening, liquid culture, and screening. 20 ° C low temperature Tolerant algal strain;

<3> Isolation of 20 °C low temperature resistant algae strain, transferred to a flask at 5%, and the color of the culture medium was visually detected under the condition of 4000 lux at 18 ° C, and the abnormal growth of Arthrospira was removed under the microscope. , thereby performing repeated screening, liquid culture, screening to obtain 18 ° C low temperature tolerance algae strain;

<4> Since the algae species are derived from the production of algae strains, the 18 °C low temperature tolerance algae strain in step <3> is subjected to solid-state multi-stage separation and purification by solid plate scribe and solid plate coating at 18 °C 4000 lux. , picking a single algae, liquid culture;

<6> The Arthrospira in the 96-well plate was labeled, and the single algae strain was cultured in a 96-well plate at 18 °C 4000 lux light liquid to obtain a 18 ° C low temperature tolerance pure algae strain;

<7> The 18 ° C low temperature tolerance pure algae strain in step <6> was transferred to a flask in an amount of 1%, and cultured in a 4000 lux liquid at 16 ° C to obtain a 16 ° C low temperature resistant algae strain;

<8> 16 ° C low temperature tolerance algae strain was cultured at 16 ° C 4000 lux liquid pure light to obtain a 16 ° C low temperature tolerance pure algae strain.

Example 3

<1>0.2mol/L chemical inducer ethyl methanesulfonate soaked in Ordos cerevisiae for 20min;

<2> The induced algae liquid of the step <1> was filtered, and the algae body was re-introduced in a liquid medium for two days. According to the amount of 5%, the flask was transferred to a flask. The algae strain was visually detected at a low temperature of 3800 lux at 20 ° C. The color of the culture solution was visually detected and the abnormal growth of Arthrospira was removed under the microscope, thereby performing repeated screening, liquid culture, and screening. 20 ° C low temperature tolerance algae strain;

<3> Isolation of 20 °C low temperature resistant algae strain, transferred to a flask at 5%, and the color of the culture medium was visually detected under the condition of 3800 lux at 18 ° C, and the abnormal growth of Arthrospira was removed under the microscope. , thereby performing repeated screening, liquid culture, screening to obtain 18 ° C low temperature tolerance algae strain;

<4> The 18°C low-temperature-tolerant algal strain in step <3> was subjected to solid-state multi-stage separation and purification by solid flat-line scribing and solid plate coating at 18°C under 3800 lux, and single algae was picked and liquid culture was carried out. ;

<5> The capillary is heated with an alcohol lamp, and after it is dissolved, it is quickly pulled into a very fine tube by a forceps, and the end of the microtube is slightly heated and blunt (to prevent the end from sharply damaging the algal cells). Pipette a drop of algae droplets that have been diluted 10,000 times more than 10 ml in a single-algae liquid culture on a glass slide, and drop a sterilized culture solution on the other side of the algae solution for use. Inverted display Gently separate the desired single algae algae body under the micro-mirror, in order to prevent the mixed bacteria, the separated single algae body is moved to the side of the culture liquid, and then moved from the culture liquid to the 96-well plate;

<6> The Arthrospira in the 96-well plate was labeled, and the single algae strain was cultured in a 96-well plate at 18 ° C in 3800 lux light, and the pure algae strain with low temperature tolerance of 18 ° C was obtained.

<7> The 18 °C low temperature tolerance pure algae strain in step <6> was transferred to a flask at 1%, and cultured at 3800 lux in 16 ° C to obtain a 16 ° C low temperature resistant algae strain;

<8> 16 ° C low temperature tolerance algae strain was cultured in 3800 lux liquid pure culture at 16 ° C to obtain a 16 ° C low temperature tolerance pure algae strain.

Example 4

<1>0.1mol/L chemical inducer ethyl methanesulfonate soaked in Ordos cerevisiae for 37min;

<2> The induced algae liquid of the step <1> was filtered, and the algae body was re-introduced in a liquid medium for two days. According to the amount of 5%, the flask was transferred to a flask. The algae strain was visually detected at a low temperature of 3500 lux at 20 ° C, and the color of the culture solution was visually detected and the abnormal growth of Arthrospira was removed under the microscope, thereby performing repeated screening, liquid culture, and screening. 20 ° C low temperature tolerance algae strain;

<3> Separate the 20°C low temperature resistant algae strain and transfer it to the flask in 5%. Under the condition of low temperature of 3500 lux at 18 ° C, the color of the culture solution was visually detected and the abnormal growth of Arthrospira was removed under the microscope, thereby performing repeated screening and liquid culture, and screening to obtain a low temperature tolerance algae strain at 18 ° C;

<4> The 18 °C low temperature resistant algae strain in step <3> was subjected to solid-state multi-stage separation and purification by solid plate scribe and solid plate coating at 18 ° C under 3500 lux, picking single algae, liquid culture. ;

<6> The Arthrospira in the 96-well plate was labeled, and the single algae strain was cultured in a 96-well plate at 18 ° C, 3500 lux light liquid, and the pure algae strain with low temperature tolerance of 18 ° C was obtained.

<7> The 18 ° C low temperature tolerance pure algae strain in step <6> was transferred to a flask at 1%, and cultured at 3 ° C 3 lux liquid at 16 ° C to obtain a 16 ° C low temperature resistant algae strain;

<8> 16 ° C low temperature tolerance algae strain was cultured at 16 ° C 3500 lux liquid pure light to obtain a 16 ° C low temperature tolerance pure algae strain.

Since Embodiment 4 is a preferred embodiment of the present invention, we pass the method of Embodiment 4. After chemical induction and repeated low temperature screening, a strain of low temperature resistant Arthrospira was isolated and screened and named as Arthrospira sp. CBN2.

1 The morphology of the algae

(1) Spirulina 20 ° C low temperature culture screening results

After induction of the algae, the culture was cultured at 3500 lux for 20 min. The algae liquid was blue-green from the color of the algae liquid. Microscopic examination revealed that the algae grew well, and it was concluded that Arthrospira grew well at a low temperature of 20 °C.

(2) Screening of algae species at 18 °C

1) 1 ml of algae solution was taken from the mixed algae liquid screened at a low temperature of 18 ° C to dilute 100 times of the plate to coat the solid culture. After the solid medium was cultured at 3,500 lux in 18 ° C for a period of time, sporadic colonies grew. From the appearance and microscopic observation, it was found that the colony of Arthrospira was dark blue-green and slightly irregular in shape; optical microscopic observation showed that the morphology and aggregation growth of different colonies were different.

2) Arthrospira was cultured in a 96-well plate at a temperature of 18 °C and 3500 lux in a 96-well plate. Some of them grew rapidly, some did not grow, and the fast-growing individual Arthrospira was screened.

3) After repeated microtubule separation and 96-well plate culture, the Arthrospira, which can grow rapidly at low temperature, was screened, and the selected Arthrospira was cultured in 3500 lux liquid light culture at 18 °C.

The growth condition of 3500 lux part of pure algae liquid light culture at 18 °C. According to the same inoculum size, the same culture condition and the same cultivation time, the growth of different algae strains was slightly different. Some algae liquids were dark blue-green, some dark green, and some dark green gray.

(3) Screening results of 16 °C algae species

1) According to the amount of 1%, transfer from 3500 lux pure culture algae solution at 18 °C to 16 °C, 3500 lux light liquid culture, and repeatedly filter the 16 °C normal growth algae strain.

The different strains of the selected strains had different growth conditions under the same growth conditions at 16 °C. Some algae liquids are blue, some are dark green, some are dark blue.

2) Observing the low temperature mutant of Arthrospira sp. CBN2 cultured in pure strain of 3500 lux at 16 °C by light microscopy, and collecting the plant length, coil circle, pitch, snail width, cell width, microscopic morphology and living habits of 30 strains. As shown in Table 1.

Table 1: Micromorphology and living habits of Arthrospira sp. CBN2

In addition, the optical microscopic morphology of the 3500 lux pure cultured Arthrospira sp. CBN2 strain was as shown in Fig. 2.

2 Molecular biological identification of Arthrospira

16S rDNA gene amplification and sequence analysis

The algae strain Arthrospira sp. CBN2 was centrifuged in a logarithmic growth phase of 60 ml of 3500 lux pure culture at 16 ° C, centrifuged at 8000 rpm for 20 minutes, centrifuged twice, and the algae was washed twice with ultrapure water. The surface of the algae mud is floated with sterilized absorbent paper, and placed in a sterile mortar to grind the liquid nitrogen to a fine powder. The Arthrospira sp.CBN2 DNA was extracted by CTAB method, and the extracted DNA product was used as a template, and the specific primers Primer1 and Primer18 were passed. Molecular biological identification was performed by cloning of Arthrospira 16S rRNA and 16S rRNA-23S rRNA internal transcribed spacer (ITS). The upstream primer Primer15'-AGAGTTTGATCCTGGCTCAG-3' downstream primer Primer185'-TTTGCGGCCGCTCTGTGTGCCTAGGTATCC-3' PCR product was sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing, and the sequencing results are shown below.

(1) Total sample sequence:

(2) 16S rRNA sequence:

(3) ITS sequence:

(4) 23S rRNA:

Result analysis:

(1) PCR amplification and positive clone detection using Arthrospira genomic DNA as template

Primrose1 and Primer18 specific primers were used for PCR amplification using Archrospira sp. CBN2 genomic DNA as a template. The PCR amplification product 1% agarose gel electrophoresis pattern is shown in 3.

From the electropherogram, the PCR product was approximately 2 kbp, which was in line with the expected results. The size of the Arthrospira sp. CBN2 PCR sequence was found to be 2014 bp by Sanger sequencing, wherein M: TakaRa DL 5000 DNA Mark; 1-2: Arthrospira sp. CBN2 PCR product.

Blue-white spot screening was carried out by picking a single colony at 37 ° C, 220 rpm LB liquid medium overnight, using M13F and M13R universal primers for bacterial PCR positive clone screening. Due to the presence of the plasmid, the PCR amplification product should be 200 bp larger than the target fragment. Three single colony liquid culture solutions were selected for each sample to carry out bacterial liquid PCR. The PCR amplification product of the bacterial liquid showed the expected result as shown in Fig. 4, and the strip size was about 2200 bp. M: TakaRa DL 5000 DNA Mark; 1-3: Arthrospira sp. CBN: 2 bacterial solution PCR product.

(2) Bioinformatics analysis of PCR amplification sequences

The sequence length of the Arthrospira sp. CBN2 was found to be 2014 bp. The amplified sequence was analyzed by NCBI website BLAST software. The results showed that the sequence includes 16S rRNA and 16S rRNA-23S rRNA internal transcribed spacer (ITS) full sequence, 23S rRNA partial sequence. Several 16S rRNA and ITS sequences were selected from the GenBank library, and the Neighbor-Joining phylogenetic tree was constructed by DNAMAN software multiple sequence alignment and using MEGA5.0 software to construct Arthrospira sp.CBN2. Sequence analysis. Bootstrap 1000 bootstrap analysis was performed when constructing the Neighbor-Joining phylogenetic tree.

The molecular results indicated that the 16S rRNA Neighbor-Joining phylogenetic tree was constructed and the algal strain Arthrospira sp. CBN2 was identified as shown in Fig. 5 as Arthrospira. The 16S rRNA-23S rRNA internal transcribed spacer (ITS) Neighbor-Joining phylogenetic tree was constructed to identify the algae Arthrospira sp. CBN2 as shown in Fig. 6.

3 Determination of the dry weight of Arthrospira thermophila under different culture conditions

60 ml of algae solution was centrifuged at 8000 rpm for 20 minutes, and the algae was washed twice with ultrapure water, then placed in a sterile fume hood overnight to dry, and then oven-dried at 78 ° C for 10 h to constant weight.

It is easy to know from Table 2 that the absolute absolute dry weight of Arthrospira sp. CBN2 is obviously increasing. The daily growth rate of algal plants rises obviously after a certain period of time and then slowly decreases and finally reaches zero. Under different culture conditions, the absolute dry biomass growth rate and daily dry biomass growth rate of Arthrospira sp. CBN2 were different in the same culture time. The overall analysis of 18 °C, 3500lux shaker light culture was better than 18 °C, 3500 lux static Light culture, 18 ° C, 3500 lux static light culture better than 16 ° C, 3500 lux static light culture. Screening of the algal strain Arthrospira sp. CBN2 at 18 ° C, 3500 lux shaker light culture preferentially reached the maximum daily growth rate, 16 ° C, 3500 lux static light culture compared to other culture conditions, the adaptation period of the algae strain is longer, but the algae strain only crossed During the adaptation period, the growth of algae plants was significantly accelerated, and the biological yield was extremely high.

It can be seen from the results of the dry biomass measurement of the 3500 lux static light culture at 18 ° C. The mixed culture was carried out for 27 days to reach the plateau stage, and its biological dry weight was 0.063 g/60 ml, that is, the biological dry weight was 1.050 g/L. When cultured alone for 27 days, the growth could continue to be continued until the plateau was reached. At 27 days, the biological dry weight of Arthrospira sp. CBN2 was 0.0808 g/60 ml, that is, the biological dry weight was 1.347 g/L. It can be seen that the pure pure culture has higher yield than the mixed culture (in the figure: CBN2 18 ° C 3500 lux pure culture, C various algae strain 18 ° C 3500 lux mixed culture).

From Fig. 8 analysis of dry biomass measurement of pure seed culture at 120 rpm/3500 lux shaker at 18 °C, shaker cultured for 13 days to reach the plateau stage. The platform dry weight of Arthrospira sp. CBN2 was 0.0723 g/60 ml, ie the biological dry weight was 1.205 g/ L. It is thus easy to know that an appropriate increase in the supply of O ₂ and CO ₂ during microalgae cultivation can significantly increase the yield and shorten the harvesting cycle.

Comparing Fig. 9 and Fig. 7, it can be found that the biomass of Alternaria algae in 3500 lux static light culture at 16 °C and 3500 lux static illumination culture at 18 °C is very different, and the selected low temperature algae sp. high. At 24d, the dry biomass yield of Arthrospira sp. CBN2 was 0.0859 g/60 ml, ie the biodry weight was 1.432 g/L.

In summary, Arthrospira sp.CBN2 belongs to the genus Algae. The algae is linear and has a slightly rounded end. The length of the plant is 800±285μm, 15±5 helix, the pitch is 58±7μm, and the helix is 7.5±2.5. Μm, cell width 7.2±2.5μm, blue-green floating growth. And Arthrospira sp. CBN2 is well tolerated at low temperature, and can still grow rapidly at a low temperature of 16 ° C and produce extremely high biomass (bio dry weight 1.432 g / L). At 16 ° C, 3500 lux static light culture, the maximum daily gain of Arthrospira sp. CBN2 was 90.83 mg / L (DW), and the biomass was less than 1.432 g / L (DW). Arthrospira sp.CBN2 can meet the annual aquaculture production of Arthrospira in China and some countries with high latitudes or low temperatures. Aquaculture production of Arthrospira. In industrial production, Arthrospira sp. CBN2 has a short culture period and long silk length, which is very suitable for aquaculture production.

Claims

A screening and culture method for high-yield and low-temperature resistant Arthrospira, characterized in that the method comprises the following steps:

<1> chemical inducer soaked in Arthrospira 20-40min;

<2> The algal strain of step <1> is repeatedly screened and cultured at a low temperature of 3000-4000 lux at 20 ° C, and a 20 ° C low temperature resistant algae strain is obtained by screening;

<3> Isolation of 20 °C low temperature resistant algal strains, repeated screening and liquid culture at 18 °C 3000-4000 lux, and screening to obtain 18 °C low temperature resistant algae strain;

<4> The 18 °C low temperature tolerance algae strain in step <3> is separated and purified in a multi-stage of 3000-4000 lux solid culture at 18 ° C, and single algae is picked and liquid cultured;

<5> Separating a liquid to culture a single algal bloom, and separating and obtaining a single algae strain;

<6> A single strain of algae was cultured in a 3000-4000 lux light liquid at 18 ° C, and a pure strain of 18 ° C low temperature tolerance was obtained.

<7> The 18 ° C low temperature tolerance pure algae strain in step <6> was cultured in a 3000-4000 lux liquid at 16 ° C, and a 16 ° C low temperature resistant algae strain was obtained by screening;

<8> 16 ° C low temperature tolerance algae strain was cultured at 16 ° C 3000-4000 lux liquid pure light to obtain 16 ° C low temperature tolerance pure algae strain.
The method for screening and cultivating high-yield and low-temperature-resistant Arthrospira according to claim 1, characterized in that the repeated screening culture in the step <2> or <3> comprises visually detecting the color of the culture liquid and removing the abnormal growth under the microscope. Arthrospira.
The method for screening and cultivating high-yield and low-temperature-resistant Arthrospira according to claim 1, characterized in that the multi-stage separation and purification in the step <4> comprises solid plate scribing and solid plate coating.
The screening and culture method for high-yield and low-temperature-resistant Arthrospira according to claim 1, characterized in that in the step <5>, a single tube is used to separate a liquid to culture a single algal.
The screening and culture method for high-yield and low-temperature-resistant Arthrospira according to claim 1, wherein the liquid culture medium is a Zarrouk culture solution.
The method for screening and cultivating high-yield and low-temperature-resistant Arthrospira according to claim 1 or 3, wherein the solid medium in the step <4> is a Zarrouk culture solution of 1.5% agar powder.
The method for screening and cultivating the high-yield and low-temperature-resistant Arthrospira according to claim 1, wherein the chemical inducing agent in the step <1> is 0.05-0.2 mol/L ethyl methanesulfonate.
The method for screening and cultivating high-yield and low-temperature-resistant Arthrospira according to claim 1, wherein the single algae strain in the step <6> is cultured and screened in a 96-well plate.
The method for screening and cultivating high-yield and low-temperature-resistant Arthrospira according to claim 1, wherein the light intensity is 3,500 lux.
A low temperature resistant Arthrospira, characterized in that the low temperature resistant Arthrospira is the low temperature resistant Arthrospira obtained by the screening culture method of the high-yield and low temperature resistant Arthrospira according to any one of claims 1-9.