CN110343616B

CN110343616B - Haematococcus pluvialis JNU35 with high astaxanthin yield as well as culture method and application thereof

Info

Publication number: CN110343616B
Application number: CN201910073504.9A
Authority: CN
Inventors: 张成武; 黄罗冬; 高保燕; 吴曼曼
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2022-04-26
Anticipated expiration: 2039-01-25
Also published as: CN110343616A

Abstract

The invention provides haematococcus pluvialis JNU35 with high astaxanthin yield and a culture method and application thereof. The strain is named as Haematococcus pluvialis JNU35, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, Beijing, in 2018, 9 months and 7 days, and has the preservation number of CGMCC No. 16438. Meanwhile, the culture method of the haematococcus pluvialis JNU35 is provided, and biomass can be rapidly accumulated. The invention also provides an astaxanthin induction method of the haematococcus pluvialis JNU35, which effectively balances the synchronous and rapid accumulation of biomass and astaxanthin content through the innovative optimization of a two-step strategy, greatly improves the haematococcus pluvialis biomass and astaxanthin content, can obviously reduce the cost for generating astaxanthin, has important application value in actual production, and has good industrial production prospect.

Description

Haematococcus pluvialis JNU35 with high astaxanthin yield as well as culture method and application thereof

Technical Field

The invention belongs to the technical field of microalgae biology, and particularly relates to haematococcus pluvialis JNU35 with high astaxanthin yield as well as a culture method and application thereof.

Background

Astaxanthin (astaxanthin) is a natural carotenoid of red color, and has a strong ability to scavenge oxygen radicals. There are three major recognized sources of natural astaxanthin, namely extraction from crustaceans, yeast production and microalgae production, and Haematococcus pluvialis (Haematococcus pluvialis) is the highest known organism with natural astaxanthin content, usually 1.5-3.6%, up to 5% of the dry weight, and is considered as a concentrate of natural astaxanthin. Therefore, natural astaxanthin derived from algae has a wide market prospect, and the efficient production of astaxanthin by haematococcus pluvialis has been a hot spot of international interest in the research field of algae science and production enterprises.

The commonly used culture methods for Haematococcus pluvialis are mainly one-step and two-step methods. However, the two-step method more adequately induced astaxanthin accumulation in the culture mode. Therefore, the main method for producing natural astaxanthin by haematococcus pluvialis at home and abroad is two-step culture, namely, the culture condition is optimized at the first stage to obtain higher cell density of the alga, and then induced stress (such as high light, high salt, nutrient salt deficiency and the like) is carried out at the second stage so that a large amount of astaxanthin is accumulated. Although astaxanthin synthesis is not dependent on light, strong light stimulation is an important means for inducing astaxanthin accumulation, and how to improve the induction degree of an astaxanthin accumulation stage in the two-step production process under the existing conditions is the key point for improving the astaxanthin yield of haematococcus pluvialis.

At present, the biggest problem of industrially utilizing haematococcus pluvialis to produce astaxanthin is mainly the algae seed characteristics, and most research results show that the haematococcus pluvialis is difficult to achieve higher biomass during photoautotrophic culture due to the influence of phenotype and genetic difference of different haematococcus pluvialis strain systems, and the problems of low astaxanthin yield, easy pollution, slow growth, complex induction conditions and the like exist. In the culture process, excellent algae strains need to be screened and the culture method needs to be improved. Therefore, the key to realize commercial production is to screen and obtain haematococcus pluvialis strains with excellent properties; meanwhile, the astaxanthin accumulation capacity of haematococcus pluvialis also determines the feasibility of commercial production.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a haematococcus pluvialis JNU35 with high astaxanthin yield, wherein the biomass of the haematococcus pluvialis JNU35 can reach 13.89g/L, and the highest astaxanthin content can reach 4.35%.

Another objective of the present invention is to provide a culture method of Haematococcus pluvialis JNU35, by which the Haematococcus pluvialis JNU35 grows faster and biomass accumulation is considerable.

The invention also aims to provide the astaxanthin induction method of haematococcus pluvialis JNU35, which is characterized in that an induction culture method is innovated according to the characteristics of an algal strain JUN35, the reproductive growth and astaxanthin accumulation of the algal strain are effectively balanced, the biomass and the astaxanthin content of the haematococcus pluvialis are greatly improved, and the method has high operability and practical application value.

The invention also aims to provide application of the haematococcus pluvialis.

The purpose of the invention is realized by the following technical scheme:

a Haematococcus pluvialis strain with high astaxanthin yield is named as Haematococcus pluvialis JNU35, is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, located in Beijing, China in 2018, 9 and 7 days, and has the preservation number of CGMCC No. 16438.

The invention separates and screens a haematococcus pluvialis from small sand pond beside the riverside of the riverboat bealock with elevation of 3948 m, and the haematococcus pluvialis is cultured in BG-11 culture medium or BBM culture medium (nitrogen source is 18mm/L NaNO)₃) When the zooblast algae is cultured on the medium, the zooblast algae is spherical or pear-shaped, the diameter of the zooblast algae is 10-15 mu m, the zooblast algae has two apical flagella with equal length, and the outside of a cytoplasmic membrane is coated with thicker colloid and connected with colloid. When the nitrogen nutrition is sufficient, the cells are green, and no or a small amount of astaxanthin is accumulated in the cells along with the prolonging of the culture time; the flagella of the motile cells are shed after the culture time is further prolonged, the cells are green and immobile or have a small amount of astaxanthin accumulated, when the immobile vegetative cells are transferred into a fresh culture medium, the cells can generate zoospores and show different division states, including 2, 4, 8, 16 and 32 divisions or even 64 divisions or more; the green immobile vegetative cells are transferred into a nitrogen-free medium and are subjected to high light, astaxanthin is continuously accumulated in the cells, and the whole cells are red along with the accumulation of a large amount of astaxanthin.

According to morphological identification, 18S rDNA multiple sequence alignment and phylogenetic analysis, the strain is determined to be Haematococcus pluvialis (Haematococcus pluvialis) of Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlamydomonadales (Chlamydomonadales), Haematococcuaceae (Haematococcus), Haematococcus (Haematococcus).

The culture method of haematococcus pluvialis JNU35 comprises the following steps: inoculating the haematococcus pluvialis JNU35 into BBM culture medium or BG-11 culture medium, and irradiating under the condition of containing nitrogen source at the illumination intensity of 100-200 mu mol.m^-2·s^-1Introducing 1-2% CO at 20-30 deg.C₂The air of (2) was cultured with stirring.

The culture temperature is preferably 25-30 ℃.

The nitrogen source is preferably NaNO₃、NH₄HCO₃And CO (NH)₂)₂At least one of; further preferably NH₄HCO₃And CO (NH)₂)₂At least one of (a); the initial nitrogen concentration of the nitrogen source in the culture medium is 3-18 mmol/L; further preferably 9 to 18 mmol/L; more preferably 18 mmol/L.

The illumination intensity is preferably unilateral illumination intensity.

The initial concentration of the haematococcus pluvialis JNU35 inoculation is preferably 0.50-0.90 of OD 750; more preferably, OD750 is 0.5 ± 0.01.

The haematococcus pluvialis JNU35 is preferably inoculated in a BBM culture medium.

The BG-11 culture medium is preferably a modified BG-11 culture medium, and the formula (without a nitrogen source) of the BG-11 culture medium is as follows: MgSO (MgSO)₄·7H₂O 75mg、CaCl₂·2H₂O 36mg、Na₂CO₃ 20mg、K₂HPO₄ 40mg、FeCl₃·2H₂O 3.15mg、Na₂EDTA·2H₂O4.36 mg and Citric acid 6.0mg and A5mix 1mL, add water to 1L; wherein the formula of A5mix is H₃BO₃2.86g、MnCl₂·4H₂O 1.81g、ZnSO₄·7H₂O 222mg、CuSO₄·5H₂O 79mg、NaMoO₄·2H₂O 0.39g、Co(NO₃)₂·6H₂O49.4 mg and 98% concentrated H₂SO₄1mL, add water to 1L.

The BBM culture medium comprises the following components (without nitrogen source): MgSO (MgSO)₄·7H₂O 75mg、NaCl 25mg、K₂HPO₄175mg、KH₂PO₄ 25mg、CaCl₂·2H₂O 25mg、H₃BO₃ 1.43mg、ZnSO₄.7H₂O 8.82mg、MnCl₂.4H₂O 1.44mg、MoO₃ 0.71mg、CuSO₄.5H₂O 1.57mg、Co(NO₃)₂ 0.49mg、EDTANa₂ 50mg、KOH 31mg、FeSO₄.7H₂O4.98 mg and 98% concentrated H₂SO₄mu.L, add water to 1L.

The astaxanthin induction method of haematococcus pluvialis JNU35 comprises the following steps:

(1) placing the haematococcus pluvialis JNU35 in a nitrogen-containing culture medium in a first glass columnar photobioreactor, wherein the illumination intensity is 100-200 mu mol.m^-2·s^-1Introducing air at the culture temperature of 20-30 ℃ for stirring culture to obtain vigorously growing cells;

(2) transferring the haematococcus pluvialis JNU35 cultured in the step (1) into a nitrogen-free culture medium in a second glass columnar photobioreactor in a range of 300-400 mu mol · m^-2·s^-1Introducing air at the culture temperature of 20-30 ℃ for stirring culture;

wherein the pipe diameter of the second glass column photobioreactor is smaller than that of the first glass column photobioreactor.

The pipe diameter of the second glass column photobioreactor is preferably 2/5-3/5 of that of the first glass column photobioreactor; further preferably 1/2 the diameter of the first glass cylindrical photobioreactor tube.

The pipe diameter of the first glass column-shaped photobioreactor in the step (1) is 6 cm; further preferably, the specification is

The nitrogen-containing medium in the step (1) is preferably NH-containing medium₄HCO₃BBM medium of (1), CO (NH)₂)₂BBM medium of (1), NaNO-containing medium₃BBM medium of (1), CO (NH)₂)₂BG-11 medium of (1), NaNO-containing medium₃BG-11 medium of (9) or containing NH₄HCO₃At least one of BG-11 medium of (1); more preferably containing NH₄HCO₃The BBM medium of (1).

The illumination intensity in the step (1) is preferably a unilateral illumination intensity.

The culturing time in the step (1) is preferably 15-20 days; more preferably 15 to 18 days.

The pipe diameter of the second glass column-shaped photobioreactor in the step (2) is preferably 3 cm; further preferably, the specification is

The nitrogen-free culture medium in the step (2) is preferably at least one of BBM culture medium or BG-11 culture medium without any nitrogen source; more preferably BG-11 medium.

The air in the step (1) and the step (2) preferably contains 1-2% of CO₂Of the air of (2).

The culture temperature in the step (1) and the step (2) is preferably 25-30 ℃; further preferably (25. + -. 1 ℃ C.).

The haematococcus pluvialis with high astaxanthin yield is applied to the preparation of astaxanthin.

Compared with the prior art, the invention has the following advantages and effects:

1. the Haematococcus pluvialis JNU35 with excellent properties and high astaxanthin yield is obtained by screening and separating a special high-altitude high-ultraviolet radiation habitat, the biomass of the Haematococcus pluvialis JNU35 can reach 13.89g/L, the highest astaxanthin content can reach 4.35%, and the Haematococcus pluvialis JNU has remarkable advantages compared with the existing astaxanthin-producing algal strain.

2. The invention provides a culture method suitable for the growth of a new separated algae strain JUN35 according to the characteristics of the new separated algae strain, and the culture method can quickly accumulate the haematococcus pluvialis biomass.

3. The invention also provides an astaxanthin induction method of the newly isolated alga strain JUN35, starting from two aspects of culture medium type and nitrogen source variety, optimizes the traditional Haematococcus pluvialis two-step method strategy, innovatively considers nutrition stress and light stress, designs the first step of the two-step method to culture in a reactor with larger pipe diameter, then improves the induction degree of the second step, transfers the alga cells to a reactor without nitrogen medium and with smaller pipe diameter to induce astaxanthin accumulation, and has higher operability; the induction method greatly improves the biomass of haematococcus pluvialis and the content of astaxanthin, effectively balances the synchronous and rapid accumulation of the biomass and the content of astaxanthin, finally realizes that the biomass can reach 13.89g/L and the content of astaxanthin can reach 4.35 percent at most, can obviously reduce the cost for generating astaxanthin, has important application value in actual production and has good industrial production prospect.

Drawings

FIG. 1 is a photograph of Haematococcus pluvialis JNU35 taken with an optical microscope.

FIG. 2 is a tree diagram of the 18S rDNA molecular phylogeny of Haematococcus pluvialis JNU 35; wherein ≧ represents Haematococcus pluvialis JNU35 of the present invention.

FIG. 3 is a graph showing the analysis of the effect of different initial nitrogen concentrations on JNU35 biomass accumulation in example 2.

FIG. 4 is a graph showing the analysis of the effect of different initial nitrogen concentrations on the accumulation of JNU35 astaxanthin in example 2.

FIG. 5 is a graph showing the analysis of the effect of biomass accumulation of Haematococcus pluvialis JNU35 in example 3 under different light intensities.

FIG. 6 is a graph showing the analysis of the phase change in biomass accumulation of Haematococcus pluvialis JNU35 under different culture conditions in example 4.

FIG. 7 is a graph showing the analysis of the astaxanthin content of Haematococcus pluvialis JNU35 under different culture conditions in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The media referred to in the following examples:

1. nitrogen-free modified BG-11 medium

Sequentially adding MgSO₄·7H₂O 75mg、CaCl₂·2H₂O 36mg、Na₂CO₃ 20mg、K₂HPO₄ 40mg、FeCl₃·2H₂O 3.15mg、Na₂EDTA·2H₂O4.36 mg and Citric acid 6.0mg and A5mix 1mL were added to 1L with water and sterilized.Wherein A5mix is formulated by addition of H₃BO₃ 2.86g、MnCl₂·4H₂O 1.81g、ZnSO₄·7H₂O 222mg、CuSO₄·5H₂O 79mg、NaMoO₄·2H₂O 0.39g、Co(NO₃)₂·6H₂O49.4 mg and 98% concentrated H₂SO₄1mL, followed by addition of water to 1L and sterilization.

2. Nitrogen-free BBM culture medium

Sequentially adding MgSO₄·7H₂O 75mg、NaCl 25mg、K₂HPO₄ 175mg、KH₂PO₄ 25mg、CaCl₂·2H₂O 25mg、H₃BO₃ 1.43mg、ZnSO₄.7H₂O 8.82mg、MnCl₂.4H₂O 1.44mg、MoO₃ 0.71mg、CuSO₄.5H₂O 1.57mg、Co(NO₃)₂ 0.49mg、EDTANa₂ 50mg、KOH 31mg、FeSO₄.7H₂O4.98 mg and 98% concentrated H₂SO₄3 μ L, adding water to 1L, and sterilizing.

EXAMPLE 1 isolation and characterization of 1JNU35

Firstly, collection, separation and purification of algae samples

The modified BG-11 medium in this example was prepared by adding NaNO with a nitrogen concentration of 18mmol/L to the nitrogen-free modified BG-11 medium described above₃And (4) preparing.

Taking a water sample of a small sand pond beside a riverside bealock at 3948 m Sichuan tribute Gashan bealock at the elevation into a laboratory, taking 1mL of the water sample, adding the water sample into a 100mL triangular flask containing 50mL of improved BG-11 culture medium, arranging 5 parallel samples, continuously illuminating and culturing for 5 days at 25 ℃, then taking 1mL of the culture sample under the aseptic operation every day, transferring the culture sample into a 1.5mL sterile EP tube, observing whether algae cells grow, taking 5 mu L of algae liquid containing target algae after the target algae is found, coating the algae liquid on a solid plate (20 g/L of agar is added into the solid culture medium) of the improved BG-11 culture medium, and continuously illuminating and culturing at 25 ℃. Continuously streaking a single algal colony on an improved BG-11 culture medium solid plate after the growth of the algal cells, and repeating for several times to obtain a single sterile algal colony; and (2) selecting a single algae colony, transferring the single algae colony into a small-volume improved BG-11 culture medium, after the algae grows to a certain concentration, sucking 1mL of algae liquid, transferring the algae liquid into 50mL of improved BG-11 culture medium containing antibiotics (ampicillin and chloramphenicol, the concentration is 20-100 mug/L), sucking 5 μ L of the algae liquid, coating the algae liquid on an improved BG-11 solid plate containing the antibiotics, after the algae colony grows out, selecting the single algae colony, and streaking the single algae colony on another solid plate to obtain a sterile algae colony, thus obtaining the purified sterile algae strain. The selected strain JNU35 was streaked and stored on an agar slant of modified BG-11 medium for storage.

Second, identification of algal cells

1. Microscopic feature observation

The strain JNU35 preserved on the agar slant of the modified BG-11 medium is inoculated into a 100mL triangular flask containing 50mL of the modified BG-11 medium, the culture is carried out for 3 days at 25 ℃ by continuous illumination, samples are taken at regular time every day for flaking, and the morphological characteristics, the cell division process and the process of the cell morphology changing with time are observed by using an optical microscope.

Algal strain JNU35 grew well in modified BG-11 and BBM media. The morphological characteristics of the algae are shown in figure 1, the swimming algae cells are spherical or pear-shaped, the cell diameter is 10-15 mu m, the cells have two apical flagella with equal length, and thicker colloid coating and colloid connecting filaments are arranged outside the cytoplasmic membrane. When the nitrogen nutrition is sufficient, the cells are green, the nitrogen nutrition is continuously consumed along with the prolonging of the culture time, and no or little astaxanthin is accumulated in the cells (figure 1-A, B); further extension of the culture time resulted in the shedding of the motile cell flagella, green, immobile cells or a small accumulation of astaxanthin (FIGS. 1-C, D), and when the immobile vegetative cells were transferred to fresh medium, the algal cells produced zoospores as shown in FIG. 1E; the cells generally exhibit different division states, including 2, 4, 8, 16, 32 divisions and even 64 or more divisions, as shown in FIGS. 1F-1L; FIG. 1M shows the continued release of the mother cell wall from mature cells after division; the green immobile vegetative cells are transferred into a nitrogen-free culture medium and are given high light, astaxanthin is continuously accumulated in the cells, the middle of the cells is red at first and continuously diffuses to the edges of the cells, and due to the large accumulation of the astaxanthin, the whole cells are dark green, brown, reddish brown and red after the final culture for about 10-20 days (figure 1N-1Q). From the above morphological characteristics, JNU35 was identified as haematococcus pluvialis (h.pluvialis).

2. Molecular identification of algal species

(1) DNA extraction

About 200mL of the algal solution obtained in the flask after aseptic culture for 7 days was centrifuged at 3000rpm to collect algal paste, JNU35 whole genome DNA was extracted using Takara Plant DNA Extraction Kit (MiniBEST Plant Genomic DNA Extraction Kit), and the DNA concentration and purity were measured using a Qubit Fluorometer.

(2)18S rDNA amplification, sequencing and sequence analysis

Using JNU35 algae cell whole genome DNA extracted in step (1) as template, using 18S rDNA primer (S1F: 5'-AACCTGGTTGATCCTGCCAGT-3'; S1R: 5'-TGATCCTTCTGCAGGTTCACCTAC-3') to amplify its fragment, PCR reaction system (50 uL) 10 × buffer 5 uL, 2.5 uM dNTP 4 u L, MgCl ₂3 μ L, S1F 2 μ L (10 μ M), S1R 2 μ L (10 μ M), DMSO 2.5 μ L, Taq enzyme 0.5 μ L, template DNA (concentration 100-200 ng/. mu.L) 2 μ L and ddH₂O29. mu.L. The PCR reaction program is pre-denaturation at 95 ℃ for 3 min; circulating for 35 times at 95 deg.C for 30s, 52 deg.C for 45s, and 72 deg.C for 2 min; 10min at 72 ℃; and finally 4 ℃. Detecting the amplified product by using 1% agarose gel electrophoresis, and selecting a product with a single clear band and sending the product to Shanghai Biometrics company Limited for sequencing.

Removing inaccurate head and tail sequencing parts of a sequence obtained by sequencing, performing Blast alignment on the sequenced sequence in NCBI, performing multi-sequence alignment by using a muscle program in MEGA7.0 software, constructing a Maximum Likelihood method (Maximum Likeliod) system tree by using a Kimura 2-parameter and a Gamma distribution with investment sites (G + I) substitution model, and checking the accuracy and the support rate of the tree by using a self-expanding value (Bootstrap), wherein the frequency is set to 1000 times.

The nucleotide sequence of the 18S rDNA obtained by amplification sequencing JNU35 is shown as follows (SEQ ID NO.3), the sequence is subjected to Blast alignment in NCBI, 36 homologous species 18S rRNA sequences are downloaded, and a total of 37 sequences are subjected to multi-sequence alignment and phylogenetic analysis.

GGAAATAAGCATGCATGTCTAAGTATAAACTGCTTATACGGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATGGTACTTTACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCGTATATCCCGACTTCTGGAAGGGACGTATTTATTAGATAAAAGGCCAGCCGGGCTTGCCCGACCTATGGCGAATCATGATAACTTCACGAATCGCACGGCCTTGCGCCGGCGATGTTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGCATCAATGTCTGGTAATTGGAATGAGAACAATTTAAATCCCTTAACGAGTATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGATTTCGGGTGGGTTCCAGCGGTCTGCCTCTGGTATGTACTGCTGTGGCCTACCTTTCTGCCGGGGACGTGTTCCTGGGCTTCATTGTCCGGGACTCGAATTCGGCGAGGATACTTTGAGTAAAACAGCGTGTTCAAAGCAAGCCTACGCTCTGAATGCATTAGCATGGAATATCACGATAGGACTCTGGCCTATCTTGTTGGTCTGTAGGACCGGAGTAATGATTAAGAGGGACAGTCGGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGAACTTCTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGAAGACGATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGATTGGCAGGTGTTTTATTGATGACCCTGCCAGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACACGGGAAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCAAGCGTACCTTGGTACGCGCCTGACTTCTTAGAGGGACTATTGACGTTTAGTCAGTGGAAGTGTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGATGCATTCAGCGAGCCTATCCTTGGCCGAGAGGTCCGGGTAATCTTTGAAACTGCATCGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACGAGGAATGCCTAGTAAGCGCGATCATCGCTTGCA

By constructing an ML phylogenetic tree (see FIG. 2), it was confirmed that algal strain JNU35 was Haematococcus pluvialis (H.pluvialis) of the phylum Chlorophyta (Chlorophyta), class Chlorophyceae (Chlorophyceae), order Chlamydomonadales (Chlamydomonadales), family Rhodococcaceae (Haematococcaceae) genus Haematococcus (Haematococcus).

The strain has been deposited in China general microbiological culture Collection center (CGMCC, address: No.3 of Siro 1 of Beijing, Kyoho, China academy of sciences, postal code 100101) of China Committee for culture Collection of microorganisms located in Beijing in 2018, 9.7.4.4.4.4.4.4.4.78, and is classified and named as Haematococcus pluvialis JNUL 35 with the collection number of CGMCC No. 16438.

Under the influence of genetic differences of algae strains, the problems of easy pollution, slow growth, complex induction conditions and the like exist in the culture process of haematococcus pluvialis, so that the screening of haematococcus pluvialis strains with excellent properties is always a hot spot of international research and attention.

Generally, the content of grease and secondary metabolites of algae cells is increased under the condition of nitrogen deficiency, but the biomass yield of the algae cells is obviously inhibited, and surprisingly, the inventor finds that the H.pluvialis JNU35 is easy to culture and not easy to pollute, and has stronger biomass and astaxanthin accumulation capacity; in particular, the algal strain can maintain a relatively rapid biomass accumulation while accumulating a large amount of astaxanthin after being changed to a nitrogen-free medium (under nutrient starvation conditions). The process is possibly a characteristic of long-term adaptive evolution of the algae in a high-altitude and high-radiation environment, and is worthy of deep development and utilization research.

EXAMPLE 2 Effect of different initial Nitrogen concentrations on JNU35

1. Preparation of algal species

Firstly, the sterile JNU35 algae preserved in the triangular conical flask are expanded step by step to be expanded into a glass cylindrical photobioreactor with the optical path of 10 cm. The algae cultured to logarithmic phase are settled and washed by sterile water, and then the algae cells are collected as second-level algae, and then the subsequent culture is carried out.

2. Variation of biomass at different initial nitrogen concentrations

At an initial seeding concentration of 0.50-0.90 OD750

The glass column photobioreactor adopts a two-step culture strategy, and the first stage adopts an improved BBM culture medium and CO (NH)₂)₂Setting different initial nitrogen concentrations (3mmol/L, 9mmol/L and 18mmol/L) for a nitrogen source, and setting the single-side light intensity for 24 hours to be 100-200 mu mol.m^-2·s^-1Continuously irradiating with light at the culture temperature of 25-30 ℃, and introducing 1% CO₂The culture was carried out with stirring by using compressed air. Second stage (cultivation to 1 st)5 days later), transferring the algae cells into a nitrogen-free BBM culture medium, and performing 24-hour unilateral light intensity of 300-400 mu mol.m^-2·s^-1The illumination was continued, and other conditions were kept consistent with the first phase.

During the cultivation of algae cells, the change of the biomass of algae cells was measured every day, 10mL of algae solution was taken every day, filtered with a mixed fiber filter (pore size 0.45 μm) which was previously dried and cooled to a constant weight (m1), and then dried in an oven at 105 ℃ to a constant weight (m 2). The biomass is expressed in terms of Dry Weight (DW) per unit volume of algal solution, in terms of (g/L), and is calculated as follows: DW (g/L) ═ m2-m1 × 100.

The effect of different initial nitrogen concentrations on JNU35 growth is shown in fig. 3. From day 6 onwards, algal cells grew relatively slowly at 3mmol/L nitrogen concentration, with no significant difference in growth rates at 9mmol/L and 18mmol/L nitrogen concentrations. The biomass was 4.66g/L, 5.71g/L and 5.74g/L at 3 nitrogen concentrations, respectively, after 15 days of culture. Then, the algae cells are transferred into a nitrogen-free BBM culture medium and cultured until the 25 th day, the biomass reaches the highest value, the biomass gradually levels, and the biomass respectively reaches 6.00g/L, 8.20g/L and 9.90g/L until the 30 th day.

3. Time-phase variation of astaxanthin content at different initial nitrogen concentrations

Collecting 100mL of algae solution every 3 days, centrifuging at 3000rpm for 5min, collecting algae mud, freeze drying, and storing at 4 deg.C. Adding 10mg of lyophilized algae powder into a 10mL screw-mouth glass centrifuge tube with a magnetic rotor in advance, adding 5mL dimethyl sulfoxide, heating in a constant-temperature magnetic stirring water bath at 70 deg.C for 15min, and centrifuging at 3000r/min for 5 min. The supernatant was collected and placed in a 25mL brown volumetric flask. Repeating the steps for 3-5 times until the algae residue becomes white, and uniformly avoiding light in the extraction process. All the supernatants were combined in a volumetric flask and were made to volume with dimethyl sulfoxide, shaken well and stored in the dark. Detecting and calculating the content of astaxanthin in the sample by using an ultraviolet spectrophotometer at the wavelength of 530 nm. Calculating the formula: astaxanthin content (% DW) ═ C × 0.25) × 100%/M (where C denotes the concentration (mg/L) of astaxanthin in the sample detected by the ultraviolet spectrophotometer, C ═ a530-0.0107)/0.1556, and M denotes the mass of algal powder).

The effect of different initial nitrogen concentrations on JNU35 astaxanthin accumulation is shown in FIG. 4, where astaxanthin rapidly accumulates at 3mmol/L and 9mmol/L nitrogen concentrations starting at day 6 and the astaxanthin content was 1.41% and 1.13% respectively by cultivation to day 15, in contrast to biomass accumulation. The astaxanthin content was only 0.36% by 15 days of cultivation at a high nitrogen concentration of 18mmol/L, and the astaxanthin accumulation started rapidly with the second stage shifted to nitrogen-free cultivation, and the accumulation rate was higher than the accumulation rates of astaxanthin at the nitrogen concentrations of 3mmol/L and 9mmol/L in the second stage, and finally the astaxanthin contents were 2.35%, 2.00% and 1.35% by 30 days of cultivation, respectively. The results indicate that nitrogen limitation and nitrogen-free stress significantly promote astaxanthin accumulation, but also affect algal cell growth. Therefore, it is necessary to optimize the two-step process conditions to achieve a balance between high biomass and high astaxanthin accumulation in Haematococcus pluvialis.

EXAMPLE 3 Effect of different light intensities on JNU35

In that

The glass column photobioreactor is cultured, the initial concentration of OD750 is 0.50-0.90, the first stage culture medium is an improved BBM culture medium, and the nitrogen source is CO (NH)₂)₂(the nitrogen concentration is 18mmol/L), the light intensity of 24h single side is 100-200 mu mol.m^-2·s^-1Continuously irradiating with light at the culture temperature of 25-30 ℃, and introducing 1% CO₂The culture was carried out with stirring by using compressed air. Culturing for 15 days, transferring to the second stage, transferring the algae cells to nitrogen-free BBM medium, and setting single-side light intensity of 300 μmol/m for 24 hr^-2·s^-1And a bilateral light intensity of 300. mu. mol. m^-2·s^-1The illumination was continued, and other conditions were kept consistent with the first phase. The effect of light intensity on JNU35 growth and astaxanthin accumulation was compared.

As shown in FIG. 5, JNU35 biomass reached 5.1g/L after 15 days of culture, and then transferred to nitrogen-free culture, and each was given a unilateral light intensity of 300. mu. mol. m^-2·s^-1And a bilateral light intensity of 300. mu. mol. m^-2·s^-1The biomass was 9.8g/L and 12.2g/L at two light intensities when the culture was carried out for 30 days.

The astaxanthin content was also determined from algal cells cultured on the last day by centrifugation and freeze-drying, and the astaxanthin content was determined in the same manner as in example 2. Light intensity of 300 mu mol.m on one side^-2·s^-1And a bilateral light intensity of 300. mu. mol. m^-2·s^-1The lower astaxanthin contents were 1.45% and 2.1%, respectively. The results show that the double increase of light intensity can obviously promote the accumulation of biomass and astaxanthin, and compared with the single-side high light intensity, the double-side high light intensity can increase the biomass by 2.4g/L and the astaxanthin content by 0.65 percent, but the result is still not ideal in terms of energy consumption.

Culture condition optimization of example 4JNU35

1. Selection of culture Medium and Nitrogen Source species

The cultured algal species were prepared as in example 2.

Respectively arranging modified BG-11 medium and modified BBM medium, respectively, and respectively adding NaNO in the first stage₃、NH₄HCO₃And CO (NH)₂)₂Culturing JNU35 with nitrogen source (initial nitrogen concentration is 18mmol/L), inoculating with initial concentration of OD750 ═ 0.50 + -0.01

The glass column-shaped photobioreactor is filled with 1 percent of CO₂The culture is carried out by stirring with compressed air, and the light intensity of one side is 100-200 mu mol.m for 24 hours^-2·s^-1Continuously illuminating at the culture temperature of 25 +/-1 ℃ for 15-18 days (the time for shifting to the second stage is stably determined according to the growth condition and the growth rate of the algae cells); in the second stage, the algae cells are transferred into nitrogen-free modified BG-11 medium and nitrogen-free BBM medium respectively

The single-side light intensity of the glass column-shaped photobioreactor is 300-400 mu mol.m^-2·s^-1The illumination was continued, and other conditions were kept consistent with the first phase. Measuring the change of biomass of algae cells every day during the culture of algae cells, the biomass measuring method is the same as that of example 2, the culture is stopped until the biomass is not increased after the culture reaches 40 th day, and the haematococcus pluvialis JNU3 is researched5 growth and astaxanthin accumulation in two media and three nitrogen sources.

As shown in FIG. 6, JNU35 grew well in both of the three nitrogen sources and the two media, and the 6 treatment conditions were not greatly different up to 4.6g/L after 18 days of culture, after which the medium was switched to nitrogen-free medium

The biomass of algae cells in the glass columnar photobiology can still keep rapid accumulation, wherein the biomass is higher than that of a BG-11 culture medium under the culture of the BBM culture medium, which indicates that the BBM culture medium is more suitable for the culture of haematococcus pluvialis than that of the BG-11 culture medium. The nitrogen source of the highest biomass concentration in BBM culture medium is NH₄HCO₃Obtained at 13.89 g/L; secondly, BBM culture medium nitrogen source is CO (NH)₂)₂The biomass is 13.64g/L at most; thirdly, BG-11 culture medium nitrogen source is CO (NH)₂)₂The biomass is 12.76g/L at the most. Description of NH₄HCO₃And CO (NH)₂)₂Is a stable nitrogen source suitable for the culture of haematococcus pluvialis.

2. Astaxanthin content analysis

Astaxanthin was measured in the same manner as in example 2.

Culturing under 6 culture conditions for 40 days, the astaxanthin content is shown in FIG. 7, and in contrast to biomass accumulation, the astaxanthin content in BG-11 medium is higher than that in BBM medium, and the nitrogen source is CO (NH) in BG-11 medium to the maximum₂)₂Obtained when the yield is 4.35 percent; secondly, the nitrogen source of the BG-11 culture medium is NH₄HCO₃The astaxanthin content was 4.06%.

Therefore, the invention innovatively optimizes a two-step strategy and effectively balances the synchronous and rapid accumulation of biomass and astaxanthin contents. JNU35 the best culture conditions are the first stage, with modified BG-11 medium and CO (NH) as nitrogen source₂)₂Culturing for 18 days to obtain cells with vigorous growth; in the second stage, the improved BG-11 culture medium without nitrogen source is replaced, and the tube diameter of the culture reactor is changed

Under the same light intensity, the relative unit cell light intensity is improved, and finally the biomass is 12.76g/L and the astaxanthin content is up to 4.35 percent under the condition.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> haematococcus pluvialis JNU35 with high astaxanthin yield, and culture method and application thereof

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<213> Haematococcus pluvialis JNU35 (Haematococcus pluvialis JNU35)

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ggaaataagc atgcatgtct aagtataaac tgcttatacg gtgaaactgc gaatggctca 60

ttaaatcagt tatagtttat ttgatggtac tttactcgga taaccgtagt aattctagag 120

ctaatacgtg cgtatatccc gacttctgga agggacgtat ttattagata aaaggccagc 180

cgggcttgcc cgacctatgg cgaatcatga taacttcacg aatcgcacgg ccttgcgccg 240

gcgatgtttc attcaaattt ctgccctatc aactttcgat ggtaggatag aggcctacca 300

tggtggtaac gggtgacgga ggattagggt tcgattccgg agagggagcc tgagaaacgg 360

ctaccacatc caaggaaggc agcaggcgcg caaattaccc aatcccgaca cggggaggta 420

gtgacaataa ataacaatac cgggcatcaa tgtctggtaa ttggaatgag aacaatttaa 480

atcccttaac gagtatccat tggagggcaa gtctggtgcc agcagccgcg gtaattccag 540

ctccaatagc gtatatttaa gttgttgcag ttaaaaagct cgtagttgga tttcgggtgg 600

gttccagcgg tctgcctctg gtatgtactg ctgtggccta cctttctgcc ggggacgtgt 660

tcctgggctt cattgtccgg gactcgaatt cggcgaggat actttgagta aaacagcgtg 720

ttcaaagcaa gcctacgctc tgaatgcatt agcatggaat atcacgatag gactctggcc 780

tatcttgttg gtctgtagga ccggagtaat gattaagagg gacagtcggg ggcattcgta 840

tttcattgtc agaggtgaaa ttcttggatt tatgaaagac gaacttctgc gaaagcattt 900

gccaaggatg ttttcattaa tcaagaacga aagttggggg ctcgaagacg attagatacc 960

gtcgtagtct caaccataaa cgatgccgac tagggattgg caggtgtttt attgatgacc 1020

ctgccagcac cttatgagaa atcaaagttt ttgggttccg gggggagtat ggtcgcaagg 1080

ctgaaactta aaggaattga cggaagggca ccaccaggcg tggagcctgc ggcttaattt 1140

gactcaacac gggaaaactt accaggtcca gacacgggaa ggattgacag attgagagct 1200

ctttcttgat tctgtgggtg gtggtgcatg gccgttctta gttggtgggt tgccttgtca 1260

ggttgattcc ggtaacgaac gagacctcag cctgctaaat agtcaagcgt accttggtac 1320

gcgcctgact tcttagaggg actattgacg tttagtcagt ggaagtgtga ggcaataaca 1380

ggtctgtgat gcccttagat gttctgggcc gcacgcgcgc tacactgatg cattcagcga 1440

gcctatcctt ggccgagagg tccgggtaat ctttgaaact gcatcgtgat ggggatagat 1500

tattgcaatt attagtcttc aacgaggaat gcctagtaag cgcgatcatc gcttgca 1557

Claims

1. Haematococcus pluvialis (A. pluvialis)Haematococcus pluvialis) JNU35, the method for inducing astaxanthin, comprising the steps of:

(1) placing the haematococcus pluvialis JNU35 in a nitrogen-containing culture medium in a first glass columnar photobioreactor in the presence of 100-200 [ mu ] mol/m of illumination intensity^-2·s^-1Introducing air at the culture temperature of 24-26 ℃ for stirring culture to obtain cells with vigorous growth;

(2) transferring the haematococcus pluvialis JNU35 cultured in the step (1) into a nitrogen-free culture medium in a second glass columnar photobioreactor in a range of 300-400 mu mol · m^-2·s^-1Introducing air at the culture temperature of 24-26 ℃ for stirring culture;

wherein, the haematococcus pluvialis JNU35 is preserved in 2018, 9 and 7 days in China, China general microbiological culture Collection center of China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16438;

the pipe diameter of the first glass column-shaped photobioreactor in the step (1) is 6 cm;

the pipe diameter of the second glass columnar photobioreactor in the step (2) is 3 cm;

the culture time in the step (1) is 18 days;

the nitrogen-containing culture medium in the step (1) contains CO (NH)₂)₂BG-11 medium; wherein, CO (NH)₂)₂The initial nitrogen concentration in BG-11 medium was 18 mmol/L;

the nitrogen-free culture medium in the step (2) is BG-11 culture medium without any nitrogen source;

the illumination intensity in the step (1) is the illumination intensity of a single side;

the air in the step (1) and the step (2) contains 1 percent of CO₂Of the air of (2).

2. The method for inducing astaxanthin in Haematococcus pluvialis JNU35 as claimed in claim 1, wherein:

the specification of the first glass cylindrical photobioreactor in the step (1) is that the length of the first glass cylindrical photobioreactor is 6cm multiplied by 60 cm;

the specification of the second glass cylindrical photobioreactor in the step (2) is that the length of the second glass cylindrical photobioreactor is 3cm multiplied by 60 cm.

3. The method for inducing astaxanthin in Haematococcus pluvialis JNU35 as claimed in claim 1, wherein:

in addition to the nitrogen source, each liter of BG-11 medium contains: MgSO (MgSO)₄·7H₂O 75 mg、CaCl₂·2H₂O 36 mg、Na₂CO₃20 mg、K₂HPO₄ 40 mg、FeCl₃·2H₂O 3.15 mg、Na₂EDTA·2H₂O4.36 mg and Citric acid 6.0mg and A5mix 1 mL; wherein the formula of A5mix is H₃BO₃ 2.86g、MnCl₂·4H₂O 1.81g、ZnSO₄·7H₂O 222 mg、CuSO₄·5H₂O 79 mg、NaMoO₄·2H₂O 0.39g、Co(NO₃)₂·6H₂O49.4 mg and 98% concentrated H₂SO₄1mL, add water to 1L.