RU2710131C1 - Bracteacoccus aggregatus microalgae strain - producer of a mixture of natural bioantioxidant of astaxanthin and provitamin a - Google Patents

Abstract

FIELD: photobiotechnology.SUBSTANCE: invention relates to photobiotechnology. Disclosed is a strain of microalgae Bracteacoccus aggregatus (syn. Cohaerens), which is a producer of a mixture of natural bioantioxidant astaxanthin and provitamin A (β-carotene). Strain is deposited in Russian Collection of Microalgae at establishment of "Timiryazev Institute of Plant Physiology of Russian Academy of Sciences (IPPAS)" with assigned identifier IPPAS C-2045.EFFECT: strain is able to simultaneously accumulate high levels of astaxanthin and β-carotene.1 cl, 4 dwg

Description

Application area

The invention relates to photobiotechnology and is a new strain of microalgae Bracteacoccus aggregatus (syn. Cohaerens), intended for the simultaneous production of natural bioantioxidants astaxanthin and β-carotene (provitamin A). This invention can be used to obtain raw materials for the manufacture of cosmetics, dietary supplements, functional nutrition, feed and food additives, as well as in biotechnological wastewater treatment with the associated production of valuable carotenoids (astaxanthin and β-carotene).

State of the art

The genus Bracteacoccus Tereg 1923 is known to be typical of the Bracteacoccaceae family located in the system at the base of the phylogenetic tree of the Scenedesmales order. A remarkable feature of the order is the presence in all, without exception, 9 families of species that have a specific, genetically determined trait - the ability to secondary carotenogenesis. As a rule, these are the inhabitants of brackish and temporary freshwater reservoirs, as well as soil and aerophytic algae [Chubchikova I.N. et al. Chlorococcal microalgae as a potential source of natural ketocarotinoids. Ecology of the sea. 2009, Vol. 77, p. 77-83; Abe K., Hattori N., Hirano M. Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. multistriata. Food Chem. 2007, t. 100, No. 2, p. 656-661; Fujii K., Imazato E., Nakashima H. et al. Isolation of non fastidious microalgae with astaxanthin-accumulation property and its potential for application to aquaculture. Aquaculture. 2006, v. 261, p. 285-293]. Green microalgae Bracteacoccus minor and Bracteacoccus giganteus are known in which the average daily yield of total carotenoids from a liter of the original culture can be 2.3 mg⋅ ^-1 ⋅ day ^-1 and the relative content of all forms of astaxanthin in them reaches 50% of the total [Chubchikova I.N. . et al. Screening of unicellular green algae as potential sources of natural ketocarotenoids. Features of growth and secondary carotenogenesis in representatives of the genus Bracteacoccus (Chlorophyceae). Morsk. eco-friendly. journal 2011, t. 10, No. 1, p. 91-97].

It is known that the green microalga Coelastrella rubescens (formerly Scotiellopsis rubescens Vinatzer) [Kaufnerova V., Elias M. The demise of the genus Scotiellopsis Vinatzer (Chlorophyta). Nova Hedwigia. 2013, 97, pp. 415-428] belongs to the group of extremobiont unicellular eukaryotes, capable of under adverse environmental conditions from the vegetative state to the dormant stage and to accumulate in the resulting aplanospores a commercially significant amount (1-2% dry weight) of specific protective ketocarotenoids - products of multistage enzymatic oxidation of β- carotene in astaxanthin [Lemoine, Y., Schoefs, B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth. Res. 2010, 106, pp. 155-177; Chubchikova I.N. Effect of medium composition on the content of secondary carotenoids in the microalga Scotiellopsis rubescens (Chlorophyceae). Morsk. eco-friendly. journal 2012, t. 11, No. 4, p. 95-101; Minyuk G.S. et al. Effect of pH and CO ₂ on the growth and metabolism of the microalga Coelastrella (Scotiellopsis) rubescens. Plant physiology. 2016, t. 63, p. 601-610].

Astaxanthin is a red ketocarotinoid, the most effective natural antioxidant that has a beneficial effect on the human and animal body. Astaxanthin is widely in demand as a component of cosmetic formulas, drugs, food and feed additives [Guerin, M. et al. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 2003, 21, pp. 210-216 .; Han, D. et al. Biology and commercial aspects of Haematococcus pluvialis. In Handbook of Microalgal Culture: Applied Phycology and Biotechnology. 2 ed .; Richmond, A .; Hu, Q., Eds. Blackwell: 2013; pp. 388-405]. The only source of astaxanthin in animals is food [Lorenz, RT, Cysewski, GR Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol. 2000, 18, 160-167]. Currently, a significant proportion of astaxanthin is synthesized chemically, but synthetic pigment, unlike natural, is a racemate with a high content of stereoisomers that do not have biological activity. Strains of green microalgae Haematococcus pluvialis Flotow, capable of accumulating astaxanthin up to 3-6% dry weight [Lemoine, Y., Schoefs, B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress, are mainly used for the industrial production of natural astaxanthin. Photosynth. Res. 2010, 106, pp. 155-177] due to the ability to form astaxanthin-rich motionless coccoid cells resistant to adverse environmental conditions [Boussiba, S. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol. Plant. 2000, 108, pp. 111-117; Solovchenko, A. Physiology and adaptive significance of secondary carotenogenesis in green microalgae. Russ. J. Plant Physiol. 2013, 60, pp. 1-13]. The invention is known [RU 2573944 C1, 01/27/2016]: Haematococcus pluvialis BM1 microalgae strain suitable for producing astaxanthin and characterized by the ability to grow on media containing up to 27% sodium chloride, high astaxanthin accumulation ability - up to 3-6% dry biomass and having high productivity (up to 300 mg of dry biomass per day with 1 liter of culture). A known method of cultivating Haematococcus pluvialis for the industrial production of astaxanthin-enriched biomass [US Pat. No. 6,022,701 C16P] (cultivation under illumination in the range of 30-140 μE PAR / (m ² ⋅ s) and a temperature of 15-28 ° C using tap water as medium and bubbling the culture with a gas-air mixture with 1.5% CO ₂ ). Also, a method is known for the efficient production of carotenoids, including astaxanthin, by obtaining mutant strains of Haematococcus pluvialis [EP 1995325 A1, 11/26/2008] SSAP 34/8 and SAG 34-1b with increased ability to accumulate carotenoids.

However, the known microalgae strains Haematococcus pluvialis are characterized by a low growth rate, a narrow range of physicochemical parameters of cultivation and a high risk of contamination, requiring the use of expensive closed cultivation systems that excessively increase the price of natural astaxanthin.

Also known is the yeast strain Phaffia rhodozyma [RU 2385925 C1, 04/10/2010] VKPM Y-2982, capable of producing astaxanthin (7.0-8.0 mg / g dry biomass under optimal (60 lux) illumination). The proportion of astaxanthin in the total pool of carotenoids can be up to 70%. A strain of unicellular green alga Chlorella zofingiensis ATCC 30412 [US 7063957 B2, 06/20/2006], which is capable of heterotrophic growth in the dark and after 2 weeks of growth, is capable of accumulating up to 10 mg / l astaxanthin, is described.

β-carotene is an orange pigment, an unsaturated hydrocarbon from the group of carotenoids. It is found in all green plants and has long been used as a dye in the food industry and animal feed [L.J. Borowitzka, M.A. Borowitzka, T.R. Moulton. The mass culture of Dunaliella salina for fine chemicals: From laboratory to pilot plant. Hydrobiologia. 1984, 1, pp. 115-121]. In addition, β-carotene is used as a component of dietary supplements and vitamin complexes. The total world market for β-carotene is estimated at approximately 270-300 million US dollars, of which 20-30% is the share of natural β-carotene [Borowitzka M.A. High-value products from microalgae-their development and commercialization. Journal of Applied Phycology. 2013, V. 25, Issue 3, pp. 743-756].

Currently, microalgae are known as promising producers of natural β-carotene, especially Dunaliella salina (up to 10% β-carotene by dry cell weight) [Oren, A. Adaptation to life at high salt concentration in Archaea, Bacteria, and Eukarya (Gunde- Cimerman, N., Oren A., and Plemenitas, A., eds), Springer. N.Y., 2005, pp. 493-502]. A known strain of algae Dunaliella salina Teod. CALU - 834, isolated from the coastal zone of Sasyk Estuary [SU 1324627 A1], which is used in the microbiological industry as a producer of protein and β-carotene, as feed, vitamin and biostimulating additives in animal diets, as well as for waste disposal. Also known is a strain of unicellular algae Dunaliella salina, a producer of bioantioxidants [RU 2497945 C2, 06/20/2012] and a method for culturing Dunaliella salina [RU 2541446 C1, 02/10/2015]. An invention is known that encompasses several new halotolerant hyperaccumulative carotenogenic strains of the microalgae Dunaliella salina DF15, DF17 and DF40 [WO 2018141978, 08/09/2018] with a commercially significant potential for β-carotene accumulation. In recent years, the Blakeslea trispora mushroom has become an important alternative source of natural β-carotene competing with the algal product [EP 1367131 A1, 03/03/2003].

The main disadvantages of the above examples are the lack of ability to simultaneously accumulate commercially significant amounts of astaxanthin and β-carotene.

накопливающая смесь вторичных кетокаротиноидов с доминированием диацильных эфиров астаксантина (37-42% суммы) [Г.С. Минюк и др. Особенности вторичного каротиногенеза у Bracteacoccus minor (Chlorophyta) в условиях двухстадийной культуры. Algologia. 2015, 25(1): 21-34].Known green soil microalga Bracteacoccus minor (Chodat)

an accumulating mixture of secondary ketocarotenoids with dominance of diacyl astaxanthin esters (37-42% of the total) [G.S. Minyuk et al. Peculiarities of secondary carotenogenesis in Bracteacoccus minor (Chlorophyta) in a two-stage culture. Algologia. 2015, 25 (1): 21-34].

et al. The role of light and nitrogen in growth and carotenoid accumulation in Scenedesmus sp. Algal Research. 2016, 16, pp. 69-75]. Сумма каротиноидов на ранней стационарной фазе роста культуры Scenedesmus sp. CCALA 1074 составляла 2,3% от сухого веса, при этом доля астаксантина составляла 9%, а β-каротина 5,4% от суммы каротиноидов. Scenedesmus sp. CCALA 1074 успешно выращивают в открытом тонкослойном фотобиореакторе объемом 150 л., при этом, он демонстрирует превосходный рост, высокую продуктивность каротиноидов и термотолерантность, что делает этот штамм перспективным кандидатом для биотехнологического производства богатой каротиноидами биомассы.Recently described strain of Scenedesmus sp. CCALA 1074 which was isolated from the plankton of the River Sil (Switzerland, 47 ° 10'47.982''N, 8 ° 41'33.504''E) [Pavel

et al. The role of light and nitrogen in growth and carotenoid accumulation in Scenedesmus sp. Algal Research. 2016, 16, pp. 69-75]. The amount of carotenoids in the early stationary phase of growth of the culture of Scenedesmus sp. CCALA 1074 was 2.3% of dry weight, with astaxanthin accounting for 9% and β-carotene for 5.4% of the total carotenoid. Scenedesmus sp. CCALA 1074 is successfully grown in an open thin-layer photobioreactor with a volume of 150 liters, while it shows excellent growth, high carotenoid productivity and thermal tolerance, which makes this strain a promising candidate for biotechnological production of carotenoid-rich biomass.

Known microalgae strain Coelastrella striolata var. multistriata, which was isolated from a rock surface in Shizuoka, Japan [Katsuya Abe et al. Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. multistriata. Food Chemistry. 2007, 100, pp. 656-661]. The authors state the ability of this microalgae to synthesize a significant amount of a mixture of carotenoids, such as canthaxanthin, astaxanthin and β-carotene. Reddish-orange microalgae cells can accumulate up to 56.0 mg of secondary carotenoids per 1 g of biomass. The canthaxanthin, astaxanthin, and β-carotene carotenoids in the cells were 47.5, 1.5, and 7.0 mg / g dry weight, respectively.

The closest analogue (prototype) is the microalgae Coelastrella rubescens and the method of their cultivation for the production of carotenoids and lipids [RU 2661086 C1, 07/11/2018]. To implement the proposed method for producing biomass Coelastrella rubescens using a strain of Coelastrella (Scotiellopsis) rubescens IPPAS H-350 (Vinatzer / Innsbruck V195 = CCALA475), which is grown by the method of two-stage storage culture. Obtained at the end of the “green” stage, C. rubescens biomass, consisting of metabolically active dividing cells, the authors propose to use (partially or in whole) as an independent raw material for obtaining a mixture of xanthophylls (lutein, zeaxanthin, neoxanthin and β-carotene) and irreplaceable FSW18: 2ω and C18: 3ω for food purposes, and also (partially or in whole) as an inoculum for carrying out the II (“red”) cultivation stage in order to obtain astaxanthin group ketocarotinoids and technical lipids. The content of total carotenoids in the biomass at the end of the "green" stage of growth of the culture collected on the 11th day reached 2.1 ± 0.17% CB with a productivity of 2.2 ± 0.08 mg⋅ ^{-1 -1} day ^-1 . The composition of carotenoids was dominated by lutein / zeaxanthin (51.9% of the total), neoxanthin (18.1%), α- and β-carotenes (10.6%). Astaxanthin and its biosynthesis intermediates were absent in the biomass. Provided that the “green” biomass is used to carry out the “red” stage, the yield of total carotenoids from a liter of the initial culture with an initial density of 1.1 × 10 ⁶ cells⋅ ^-1 (or 0.17 g / l ST) for 19 days is 52 , 3 mg⋅l ^-1 , average productivity - 2.75 mg⋅l ^-1 ⋅ days ^-1 , the content of total carotenoids in the biomass is 0.9-1.0% SV, the proportion of astaxanthin was 26%. The disadvantages of the prototype include the low content of β-carotene in the biomass at the “red” stage of cultivation and the insufficiently high proportion of astaxanthin, as well as very modest indicators for the content of total carotenoids.

The technical problem to which the claimed invention is directed is to obtain a microalgae strain with a high level of simultaneous accumulation of astaxanthin and β-carotene (not less than 55 wt.% Astaxanthin and 35 wt.% Β-carotene) while maintaining high crop productivity (not less than 3 , 6 mg⋅l ^-1 ⋅ days ^-1 in the sum of carotenoids), the ability to bioabsorb phosphates from the medium (not less than 2.3 mg⋅l ^-1 ⋅ days ^-1 ).

Disclosure of invention

The technical problem was solved by the microalgae strain Bracteacoccus aggregatus (syn. Cohaerens) BM 5/15 IPPAS C-2045 isolated from a reservoir in the north of Russia, the Republic of Karelia, the village of Louhi, Protok between the White Sea and Lower Ershov Lake, 66 ° 32'07 '' N, 33 ° 03'59''E deposited in the Russian Collection of Microalgae at the establishment of the Institute of Plant Physiology K.A. Timiryazev Russian Academy of Sciences (IPPAS) with assigned IPPAS identifier S-2045.

For the first time, a strain of microalgae was obtained, capable of a high level of simultaneous accumulation of a mixture of astaxanthin and β-carotene, providing high productivity during cultivation, and having the ability to bioabsorb phosphates from the medium.

The essence of the invention lies in the fact that to achieve the goal, they use the green microalga Bracteacoccus aggregatus (syn. Cohaerens) strain IPPAS C-2045, isolated and identified by the authors of the application from a reservoir in the north of Russia, the Republic of Karelia, the village of Loukhi, The channel between the White Sea and the Lower Ershov Lake (66 ° 32'07``N, 33 ° 03'59''E). As a result, the obtained strain is characterized by a high ability to simultaneously accumulate astaxanthin and β-carotene (55-60 wt.% And 35-40 wt.%, Respectively, of the sum of carotenoids at the "red" stage of cultivation), while the amount of carotenoids is estimated at 2, 4-2.6% of dry weight, the culture has high productivity (100-200 mg of dry biomass and 3.6-3.8 mg of carotenoids per day with 1 liter of culture), the ability to bioabsorb phosphates from the medium (2.3-2 , 6 mg⋅l ^-1 day ^-1 ).

To achieve the technical result of the invention, the culture of the alga Bracteacoccus aggregatus (syn. Cohaerens) strain IPPAS C-2045 is grown by a two-stage method aimed at achieving a commercially significant yield of target products (a mixture of astaxanthin and β-carotene).

As a result, 100-200 mg⋅ ^-1 biomass per day (by dry weight) and 3.6-3.8 mg carotenoids per day with 1 liter of culture are obtained; the biomass contains 2.4-2.6% carotenoids, in which the proportion of astaxanthin 55-60 mass. % and β-carotene 35-40 wt. % The proposed strain of microalgae culture is characterized by the ability to bioabsorb phosphates from the medium (2.3-2.6 mg ⋅ ^-1 ⋅ day ^-1 ).

Brief Description of the Drawings

The invention is illustrated by the following drawings.

In FIG. Figure 1 shows the microalgae cells of Bracteacoccus aggregatus (syn. Cohaerens) strain IPPAS C-2045 under conditions of vegetative cultivation and accumulation of carotenoids - bright field microscopy.

In FIG. 2 The accumulation of dry mass of Bracteacoccus aggregatus (syn. Cohaerens) IPPAS C-2045 upon cultivation on BG-11 medium under vegetative growth conditions shows the average values and standard deviations (2 biological replicates).

In FIG. Figure 3 shows the carotenoid content in the cells of the Bracteacoccus aggregatus culture (syn. Cohaerens) strain IPPAS C-2045.

In FIG. Figure 4 shows the phylogenetic tree of the strain Bracteacoccus aggregatus (syn.cohaerens) IPPAS C-2045 based on the sequence of the internal transcribed spacer 2 (ITS2) of the nuclear ribosomal gene cluster, obtained using the maximum likelihood algorithm. The accuracy of various elements of the topology, determined using the bootstrap test, is indicated near them (in percent). Scale - the number of nucleotide substitutions per number of positions in multiple alignment.

The implementation of the invention

The strain Bracteacoccus aggregatus (syn.cohaerens) IPPAS C-2045 was isolated from a reservoir in the north of Russia, the Republic of Karelia, the village of Louhi, the Protok between the White Sea and Lower Yershov Lake (66 ° 32'07``N, 33 ° 03'59 ' 'E). Selected as a result of screening for the biosynthesis of carotenoids (a mixture of astaxanthin and β-carotene).

The isolation method is from an accumulative culture obtained by inoculating BG-11 medium with selected cells, followed by sieving on solid BG-11 medium.

Morphological signs.

Growth pattern on storage medium: homogeneous, aggregation is possible in the later stages. Cytological description:

cell shape, size: strain B. aggregatus (syn. cohaerens) IPPAS C-2045 is represented by small coccoid cells (up to 9 μm in diameter) or elliptic (Fig. 1). When growing on a BG-11 medium, multicellular clusters are often formed. With a decrease in the growth rate or transfer to adverse conditions, the cells significantly increase in size (up to 20 μm) and acquire an orange color (Fig. 1). The pyrenoid is absent.

a) chromatophore (shape, color): one large parietal chromatophore, with an increase in size, smaller green chloroplasts appear.

et al., 2013]).b) during cultivation, flagellum stages were not detected (however, according to the literature, in rare cases zoospores with two flagella are found [

et al., 2013]).

c) morphological features under optimal growth conditions: the cells are small, have a green color, coccoid (Fig. 1).

g) with prolonged storage, the cells increase in size, acquire an orange color (Fig. 1).

e) morphological features under conditions ensuring the maximum yield of the product: cells acquire an orange color. There is a reduction in the photosynthetic apparatus, a decrease in the volume occupied by plastids, numerous plastoglobules appear in the stroma of the plastids.

Physiological properties of the strain.

Optimum cultivation conditions:

For cultivation using a liquid nutrient medium BG-11 [Stanier, R. et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Microbiology and Molecular Biology Reviews. 1971, 35 (2): 171-205], prepared from stock solutions; All components can be autoclaved.

Environment BG-11 of the following composition:

K ₂ HPO ₄ - 0.04 g / l, NaNO ₃ - 0.3 g / l, MgSO ₄ ⋅ 7H ₂ O - 0.075 g / l,

CaCl ₂ ⋅ 2H ₂ 0 - 0.036 g / l, citric acid - 0.006 g / l,

FeSO ₄ ⋅7H ₂ 0 - 0.006 g / l, Na ₂ CO ₃ - 0.2 g / l, EDTA - 0.001 g / l,

FeSO ₄ ⋅7H ₂ 0 solution (7.45 g / l) + EDTA (5.57 g / l) - 1 ml / l,

trace element solution (H ₃ BO ₃ - 2.86 g / l, MnCl ₂ ⋅ 4Н ₂ 0 - 1.86 g / l, ZnSO ₄ ⋅ 7H ₂ 0 - 0.22 g / l, CuSO ₄ ⋅ 5H ₂ 0 - 0.08 g / l, Na ₂ MoO ₄ ⋅ 7H ₂ 0 - 0.39 g / l, Co (NO ₃ ) ₂ ⋅ 6H ₂ 0 - 0.05 g / l) - 1 ml / l,

pH at the beginning of cultivation - 7.4-7.7

pH at the end of cultivation 6.9-7.2

air sparging rate 0.8-1.2 l / min

temperature 23-25 ° С

round-the-clock lighting, illumination of 60-80 mol quanta / m ² / s

type of lamps: LED, 4700 K

To induce carotenogenesis and transition to the “red” stage of cultivation, cells at the exponential growth phase are washed twice with nitrogen-free medium BG-11 ₀ [Rippka, R. et al. Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Journal of General Microbiology. 1979, 111, 1-61), are resuspended in 300-400 ml of this medium and incubated under illumination of 460-480 μmol of photons PHAR m ^-2 s ^-1 for 7-10 days.

After that, the biomass is separated from the medium by centrifugation with an acceleration of at least 3000 g for 4-5 minutes.

Productivity under optimal cultivation conditions:

the accumulation of biomass (dry weight, mg / l per day): 100-200;

yield of a useful product: the amount of carotenoids is estimated at 2.4-2.6% dry weight, carotenoid productivity 3.6-3.8 mg per day with 1 liter of culture, the simultaneous accumulation of astaxanthin and β-carotene (55-60 wt. % and 35-40 wt.%, respectively, of the sum of carotenoids).

Biotechnological characteristics of the strain.

The microalgae strain Bracteacoccus aggregatus (syn. Cohaerens) IPPAS C-2045, deposited in the Russian Collection of Microalgae at the Institute of Plant Physiology K.A. Timiryazev Russian Academy of Sciences (IPPAS) with the assigned identifier IPPAS C-2045, producer of a mixture of natural bioantioxidant astaxanthin and β-carotene (provitamin A) in a ratio of 55-60 mass. % and 35-40 wt. % of the amount of carotenoids, respectively, which is 2.4-2.6% of dry weight, with a productivity of 3.6-3.8 mg per day of carotenoids per 1 liter of culture, capable of bioabsorbing phosphates from the medium at a rate of 2.3 -2.6 mg⋅l ^-1 day ^-1 .

Cultivation conditions providing the maximum level (yield) of useful property (product): any stressor that reduces the cell division rate (illumination of 460-480 μmol of photons of PHAR m ^-2 s ^-1 , excluding nitrogen from the composition of the culture medium).

Genotyping.

DNA isolation.

5-10 mg of microalgae culture biomass was selected for DNA isolation. DNA is isolated by phenol-chloroform extraction. Before isolation, the samples are frozen three times at -4 ° C, followed by thawing. This is necessary for the destruction of the strong cell walls of algae. Samples were incubated for one hour in 300 μl of TE buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA) containing 10 mg / ml lysozyme at 37 ° C. Then add 2% sodium dodecyl sulfate and incubate for one hour at 40 ° C with vigorous stirring. Then add 1 M NaCl and leave overnight on ice to salting out the proteins. Then carry out the procedure of phenol-chloroform extraction. The purity of DNA samples was evaluated by electrophoresis in a 1.5% agarose gel. The resulting DNA samples were stored in TE buffer at -4 ° C.

Phylogenetic analysis.

Phylogenetic relationships of the IPPAS C-2045 strain and related strains are evaluated by the similarity of the partial sequence of the 18s pRNA gene. Presented phylogenetic tree obtained by the NJ method (Fig. 4). Phylogenetic analysis was performed in the BioNJ program. Visualization was carried out in the program TreeDyn 198.3. The percentage of replicative trees into which taxa were combined in the bootstrap test are shown next to the branches.

As a result of the phylogenetic analysis, the species affiliation of the studied isolate was established. The isolate was identified as Bracteacoccus aggregatus (syn. Cohaerens) and received the IPPAS identifier C-2045; after depositing in the Russian Collection of Microalgae at the establishment of the Institute of Plant Physiology K.A. Timiryazev Russian Academy of Sciences (IPPAS) he was assigned the identifier IPPAS S-2045.

Phosphate Absorption Kinetics

The kinetics of phosphate uptake by a microalgae culture of Bracteacoccus aggregatus (syn. Cohaerens) strain IPPAS C-2045 is evaluated by the residual content of orthophosphate in the medium by ion chromatography. The initial density of the culture - 0.5 g s.v.⋅l ^-1 illuminance - 60-80 micromol photons m ^-2 PAR ⋅s ^-1. When orthophosphate is added to a final concentration of 400 µmol⋅l ^-1 to the culture of IPPAS C-2045 strain, previously incubated for 10 days on BG-11 medium that does not contain a phosphorus source, fast absorption of orthophosphate is observed. As a result, the ability of the microalgae strain IPPAS S-2045 to bioabsorb phosphates from the medium is 2.3–2.6 mg⋅l ^-1 ⋅ days ^-1 in terms of orthophosphate.

The present invention is illustrated by specific examples of execution, which are not the only possible, but clearly demonstrates the possibility of achieving the desired technical result.

For cultivation used a liquid nutrient medium BG-11 [Stanier, R. et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Microbiology and Molecular Biology Reviews. 1971, 35 (2): 171-205], prepared from stock solutions.

Environment BG-11 of the following composition:

K ₂ HPO ₄ - 0.04 g / l, NaNO ₃ - 0.3 g / l, MgSO ₄ ⋅ 7H ₂ 0 - 0.075 g / l,

CaCl ₂ ⋅ 2H ₂ 0 - 0.036 g / l, citric acid - 0.006 g / l,

FeSO ₄ ⋅7H ₂ 0 - 0.006 g / l, Na ₂ CO ₃ - 0.2 g / l, EDTA - 0.001 g / l,

FeSO ₄ ⋅7H ₂ 0 solution (7.45 g / l) + EDTA (5.57 g / l) - 1 ml / l,

trace element solution (Н ₃ BO ₃ - 2.86 g / l, MnCl ₂ ⋅4Н ₂ 0 - 1.86 g / l, ZnSO ₄ ⋅7H ₂ 0 - 0.22 g / l, CuSO ₄ ⋅5H ₂ 0 - 0.08 g / l, Na ₂ MoO ₄ ⋅ 7H ₂ 0 - 0.39 g / l, Co (NO ₃ ) ₂ ⋅ 6H ₂ 0 - 0.05 g / l) - 1 ml / l,

pH at the beginning of cultivation - 7.4-7.7

pH at the end of cultivation 6.9-7.2

air sparging rate 0.8-1.2 l / min

The strain was cultured at a temperature of 23-25 ° C and round-the-clock illumination (illumination of 60-80 μmol of photons PHAR m ^-2 ⋅ s ^-1 , type of lamp: LED, 4700 K).

Cultivation was carried out for 10-14 days.

To induce carotenogenesis and transition to the “red” stage of cultivation, cells at the exponential growth phase are washed twice with nitrogen-free medium BG-11 ₀ [Rippka, R. et al. Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Journal of General Microbiology. 1979, 111, 1-61), are resuspended in 300-400 ml of this medium and incubated under illumination of 460-480 μmol of photons PHAR m ^-2 s ^-1 for 7-10 days.

After that, the biomass is separated from the medium by centrifugation with an acceleration of at least 3000 g for 4-5 minutes.

The analysis of carotenoids is carried out immediately after separation of the biomass, or after 30 days, keeping the samples at -80 ° C. For pigment analysis, spectrophotometric and chromatographic methods are used [K. Chekanov et al. Accumulation of Astaxanthin by a New Haematococcus pluvialis Strain BM1 from the White Sea Coastal Rocks (Russia). Marine Drugs. 2014, 12, 4504-4520].

Productivity under optimal cultivation conditions:

the accumulation of biomass (dry weight, mg / l per day): 100-200;

yield of a useful product: the amount of carotenoids is estimated at 2.4-2.6% dry weight, carotenoid productivity 3.6-3.8 mg per day with 1 liter of culture, the simultaneous accumulation of astaxanthin and β-carotene (55-60 wt. % and 35-40 wt.%, respectively, of the sum of carotenoids). The results are shown in FIG. 2 and 3.

Thus, the microalgae strain Bracteacoccus aggregatus (syn.cohaerens) IPPAS C-2045 shows a high ability to simultaneously accumulate astaxanthin and provitamin A (β-carotene), which is not found in known analogues. The accumulation of astoxanthin and β-carotene is 55-60 mass. % and 35-40 wt. %, respectively, of the amount of carotenoids at the "red" stage of cultivation, which is 2 times higher than the same indicator for the known prototype. The amount of carotenoids is 2.4-2.6% of dry weight, which is 2.5 times higher compared to the prototype. The strain Bracteacoccus aggregatus (syn.cohaerens) IPPAS C-2045 maintains high productivity (100-200 mg of dry biomass and 3.6-3.8 mg of carotenoids per day with 1 liter of culture) and the simultaneous ability to bioabsorb phosphates from the medium at a rate of 2 , 3-2.6 mg⋅l ^-1 ⋅ days ^-1 , which exceeds the level of known analogues that are not capable of the simultaneous accumulation of astaxanthin and β-carotene.

The microalgae strain Bracteacoccus aggregatus (syn.cohaerens) IPPAS C-2045 successfully passed the preliminary test and the stage of test cultivation in experimental and semi-industrial photobioreactors up to 50 l.

Claims (1)

The microalgae strain Bracteacoccus aggregatus, deposited in the Russian Collection of Microalgae at the establishment of the Institute of Plant Physiology named after K.A. Timiryazev Russian Academy of Sciences "with the assigned identifier IPPAS S-2045, - producer of a mixture of natural bioantioxidant astaxanthin and provitamin A.

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