RU2849068C1 - Micractinium simplicissimum microalgae strain - producer of lutein and beta-carotene, capable of bioaccumulating carbon dioxide when cultivated in salt water - Google Patents

Abstract

FIELD: photobiotechnology.

SUBSTANCE: Micractinium simplicissimum NAMSU-MS2022 is proposed, which is a producer of a mixture of lutein and β-carotene, resistant to brackish environments and capable of bio-removal of inorganic carbon from the culture medium. The strain is deposited in the Russian Collection of Microalgae at the K.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned identifier IPPAS C-2082.

EFFECT: strain has a biomass productivity of 0.35–0.37 g dry weight⋅ l^–1⋅ day(^{–1) ,}a high level of carotenoid accumulation of 13.3–13.7 mg⋅g^–1, in particular lutein 63–65% by mass and β-carotene 20–24% by mass, capable of absorbing CO₂ at a high rate from the growth medium, characterised by carbon dioxide removal at a rate of 0.30–0.32 l^–1⋅day^–1, and is capable of maintaining these indicators at a wide range of cultivation medium salinity values up to 18 ‰.

1 cl, 4 dwg, 6 tbl, 2 ex

Description

Scope of application

The invention relates to photobiotechnology and represents a new strain of the microalgae Micractinium simplicissimum , designed for the simultaneous production of lutein and β-carotene (provitamin A), capable of bioremoval of carbon dioxide when cultivated in brackish water.

State of the art

Microalgae cultivation is inexpensive and can be cost-effective, as they exhibit autotrophic growth, using _CO2 and sunlight as an energy source to convert them into biomass enriched with valuable compounds through photosynthesis. Microalgae cultivation in coastal wetlands unsuitable for agriculture can yield valuable raw materials for the production of biofertilizers for agriculture, as well as feed for the fisheries and livestock industries, while increasing carbon sequestration potential and promoting bioremediation of these areas.

Microalgae capable of growth and cell division under extreme conditions, such as extreme pH, temperature, or salinity, possess certain biotechnological advantages, such as accumulating protective metabolites that allow them to grow and thrive in extreme conditions. For example, microalgae can be used as a natural source of β-carotene, lutein, and other carotenoids. The number of microalgae species that can be sustainably produced on an industrial scale is limited.

The use of photoautotrophic microorganisms, namely microalgae and cyanobacteria, as raw materials for the production of a wide range of useful bioproducts has become widespread in recent decades. Currently, one of the most promising methods of biological conversion of CO ₂ with the concomitant production of a number of biologically active substances, antioxidant pigments and raw materials for biofuel production is considered to be the use of photobiotechnologies based on the cultivation of photoautotrophic microorganisms (cyanobacteria and eukaryotic microalgae). At the same time, the successful implementation and economic efficiency of these photobiotechnologies depend on the solution of a number of issues in the field of photophysiology and biochemistry of microalgae [Melnicki MR et al. Feedback-controlled LED photobioreactor for photophysiological studies of cyanobacteria // Bioresource Technology, 2013, 134 (127-133); Masojidek J. et al. A two-stage solar photobioreactor for the cultivation of microalgae based on solar concentrators // Journal of Applied Phycology, 2009, 21 (55-63)]. The use of microalgae in various photobiotechnologies is relevant as a means of reducing the risk to the environment due to the increasing concentration of climate-active (greenhouse) gases (CO ₂ ) in the atmosphere. In addition, the use of microalgae allows providing the industry with renewable raw materials for the production of environmentally friendly fuel, feed additives, biofertilizers, and valuable dietary supplements. The use of microalgae has a number of key advantages, such as the rapid growth of microalgae cultures (phototrophic microorganisms are significantly (several thousand times) superior to higher plants in the efficiency of biomass accumulation and lipid biosynthesis per unit area occupied by the culture), as well as the possibility of deploying biomass production on areas unsuitable for agriculture (non-arable lands). This allows for the conservation of cultivated land for food production, which is in short supply worldwide. Additional advantages of using microalgae include the " _CO2 -neutrality" of fuel produced from microalgae biomass—burning such fuel does not increase the overall _CO2 concentration in the atmosphere—as well as the ability to produce biologically and pharmacologically active compounds, including those with antioxidant, anti-inflammatory, and antitumor activity (arachidonic acid, astaxanthin, lutein, β-carotene, etc.).

Various strains of microalgae are known to produce carotenoids under various cultivation conditions. For example, a strain of microalgae is knownHaematococcus pluvialisVM1 (patent RU 2573944), deposited in the Russian Collection of Microalgae at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned IPPAS identifier H-2018, is intended for the production of natural astaxanthin, capable of high levels of astaxanthin accumulation and providing high productivity when grown in a medium containing sodium chloride at the level of brackish water. StrainHaematococcus pluvialisVM1 demonstrates intensive growth of the culture at increased salinity of the environment up to 25-27 ‰, the accumulation of astaxanthin in the culture is 3-6% of the dry biomass weight of the microalgae, that is, the yield of the useful product is 30-60 mg⋅g^-1biomass per day with the accumulation of biomass of 100-300 mg d.v. l^-1per day. In addition, a strain of green microalgae is knownDunaliella salinaTeod. IPPAS B-2026 (patent RU 2788527), maintaining a consistently high growth rate in open-type cultivators during industrial cultivation under naturally changing temperature and light conditions, maintaining commercially significant crop productivity in terms of biomass (7 g d.v. ⋅m^-2⋅day^-1) and demonstrating a carotenoid content of at least 700-750 mg⋅m by the end of the “green” stage of cultivation^-2A strain of microalgae has been described.Bracteacoccus aggregatus(syn.cohaerens) IPPAS C-2045 (patent RU 2710131) which demonstrates a high capacity for the simultaneous accumulation of astaxanthin and provitamin A (β-carotene). The accumulation of astaxanthin and β-carotene is 55-60 wt. % and 35-40 wt. %, respectively, of the total carotenoids; the total carotenoids are 2.4-2.6% of the dry biomass weight, while this strain maintains high productivity (100-200 mg of dry biomass and 3.6-3.8 mg of carotenoids per day from 1 l of culture) and the capacity for the simultaneous bioremoval of phosphates from the medium at a rate of 2.3-2.6 mg⋅l^-1⋅day^-1. Also known is a strain of microalgaeCoelastrellasp. K1 (patent RU 2703420), which demonstrates a high capacity for the simultaneous accumulation of astaxanthin and β-carotene, amounting to 45-50 wt. % and 45-50 wt. %, respectively (from the total carotenoids, the total carotenoids constitute 1.3-1.7% of the dry biomass weight), and also maintains high productivity (100-200 mg of dry biomass and 2.1-2.3 mg of carotenoids per day from 1 l of culture) and the ability to bioremove phosphates from the medium at a rate of 2.3-2.6 mg⋅l^-1⋅day^-1A strain of cyanobacteria has been described.Synechococcussp. IPPAS B-2053 (patent RU 2752609), which has a high accumulation level of mycosporine-like amino acids (3.7-3.9 wt%) and maintains high productivity during cultivation. A salt-tolerant microalgae is knownDunaliella bardawilMasazir CCAP 19/38 (patent EA017565B1), designed for the production and processing of biomass as an additive to feed for poultry, livestock, fish and sericulture, which can also be used in pharmacology and cosmetology.D. bardawilMasazir is a unique microorganism capable of synthesizing a number of beneficial compounds, such as β-carotene (provitamin A), glycerol, and unsaturated fatty acids, in water bodies with extremely high salt (NaCl) concentrations. Under certain conditions, this strain is capable of hypersynthesizing β-carotene, with its content reaching 9% of the microalgae's dry biomass weight. The microalgae is also known to be tolerant to high salinity, high temperatures, and strong light.Chlorella aromataMEM-A25 (patent CN107541466A) is a producer of aromatic compounds such as benzothiazoles and others, and is also capable of synthesizing neutral lipids, unsaturated fatty acids, sterols, pigments, proteins, polysaccharides, and other substances. The strain was isolated from brine from the saline-alkaline regions of Hainan, China. It was archived at the China Center for Type Culture Collection on January 10, 2017, under the identifier CCTCC NO: M2016793.

Despite the wide diversity of microalgae capable of producing carotenoids, they exhibit varying productivity under different cultivation conditions. Furthermore, not all strains are capable of maintaining productivity when grown in brackish environments and effectively absorbing _CO2 from the medium at a rate suitable for industrial use. Therefore, the search for new promising strains capable of producing carotenoids under unfavorable conditions (such as elevated chloride salinity levels of 13-16‰) is a pressing issue.

The closest strain to the claimed invention is the microalgae strain Desmodesmus subspicartus Dia 5 (patent RU 2835138), deposited in the Microalgae Collection of the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences under IPPAS C-2072. It produces carotenoids, particularly lutein and beta-carotene. This strain has the ability to accumulate high levels of both lutein and beta-carotene, while its biomass has good organoleptic properties. The strain is undemanding regarding cultivation conditions, including a low NaCl concentration in the culture medium. The well-known microalgae strain Desmodesmus subspicartus Dia 5 exhibits high levels of accumulation of a mixture of natural carotenoids (approximately 4.5 mg per g of dry biomass), primarily lutein (approximately 64%) and β-carotene (more than 50% at the growth stage, decreasing to 22% at the plateau stage). However, it exhibits low productivity of total carotenoids and a low rate of carbon bioremoval. Furthermore, this strain is unable to maintain its accumulation of the natural carotenoid mixture—lutein and β-carotene—when cultivated in brackish water.

The technical problem solved by the claimed invention is the production of a microalgae strain that eliminates the shortcomings of known analogues.

Disclosure of invention

The technical result of the invention is the production of a new strain of microalgae with a biomass productivity of 0.35-0.37 g dry matter ⋅L ^-1 ⋅day ^-1 , a high level of accumulation of carotenoids (13.3-13.7 mg⋅g ^-1 ), in particular lutein (63-65 wt.%) and β-carotene (20-24 wt.%), and capable of absorbing CO ₂ at a high rate from the growing medium, characterized by the bioremoval of carbon dioxide at a rate of 0.30-0.32 L ⋅L ^-1 ⋅day ^-1 , and capable of maintaining these indicators at a salinity of the cultivation medium in a wide range of values up to 18 ‰.

At higher salinity of the cultivation environment (up to 24 ‰), the growth of a new strain of microalgae with lower productivity values is also possible.

The technical result is achieved by obtaining the microalgae strain Micractinium simplicissimum NAMSU-MS2022, isolated from a natural seawater sample from the shelf zone of the Kara Sea and deposited in the Russian Collection of Microalgae at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned IPPAS identifier C-2082. The resulting microalgae strain is capable of high levels of simultaneous accumulation of a mixture of lutein and β-carotene, ensures high productivity during cultivation, exhibits the ability to significantly absorb _CO2 from the cultivation medium, and maintains these indicators at a salinity of at least 16‰.

In particular, the obtained strain is characterized by a high ability to simultaneously accumulate lutein and β-carotene (62-64 wt.% and 23-25 wt.%, respectively, of the total carotenoids), while the total carotenoids are estimated at no less than 13.5 mg⋅g ^-1 of dry biomass weight, the culture has high productivity in biomass accumulation (no less than 0.35-0.37 gl ^-1 ⋅day ^-1 ), the ability to bioremove carbon dioxide at a rate of 0.30-0.32 l⋅l ^-1 ⋅day ^-1 , and this strain is also able to maintain productivity indicators at a salinity of the environment of 15-18 ‰.

To maintain the viability of the strain, BG-11 nutrient medium [Stanier, R., et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Microbiology and Molecular Biology Reviews . 1971, 35(2): 171-205] is used, solidified by adding 1.5 agar-agar to it. Cells are seeded on the solid medium using the exhaustive streak method on Petri dishes and incubated under constant illumination with an intensity of 50-80 μmol quanta⋅m ^-2 ⋅s ^-1 using LEDs (color temperature of light - 4700 K) on light racks at a temperature of 23-25 °C.

For culturing the strain, BG-11 nutrient medium is used. The inoculum is added to the medium to a final chlorophyll concentration of 5-7 μg⋅ml ^-1. Cultivation is carried out in a photobioreactor under constant illumination with an intensity of 80-100 μmol quanta⋅m ^-2 ⋅s ^-1 using LEDs (light color temperature - 4700 K), at a temperature of 23-25 °C and bubbling the medium with a gas-air mixture containing 1.6-2.4% CO ₂ at a rate of 0.4-0.6 l min ^-1 . The pH of the medium at the beginning of cultivation is 7.0-7.4, at the end of cultivation (12 days) - 7.8-8.2. To ensure maximum productivity and achieve technical results, the Micractinium simplicissimum IPPAS C-2082 algae culture is preferably grown under elevated illumination of 210-230 μmol quanta ^m-2 s ^-1 in the presence of seawater salts in the cultivation medium (salinity of the medium 15-18 ‰). The cultivation duration is at least 12 days at a temperature of 23-25°C and a culture density change of 0.3 to 1.5 g dry matter/L .

As a result, 0.35-0.37 g⋅l ^-1 ⋅day ^-1 of biomass (by dry weight) is obtained per day, the sum of carotenoids is 13.3-13.7 mg⋅g ^-1 of dry biomass weight, in which the proportion of lutein is 62-64 wt.% and β-carotene 23-25 wt.%, while the rate of carbon dioxide absorption is 0.30-0.32 l⋅l ^-1 ⋅day ^-1 .

Compared to its closest analogue, the proposed microalgae strain exhibits higher productivity in total carotenoids and is characterized by a significantly higher rate of carbon bioremoval. Furthermore, the proposed strain can maintain its performance characteristics at the same level when cultivated in brackish water. Key advantages of this strain include its potential for industrial cultivation for the bioremoval of greenhouse gases (climate-sensitive gases)—carbon dioxide—in coastal and arid regions where brackish water is readily available but fresh water is scarce.

Brief description of the drawings

Fig. 1 shows cells of microalgae Micractinium simplicissimum IPPAS C-2082 under vegetative cultivation conditions - bright-field microscopy.

Fig. 2 shows the ultrastructure of the cell of the microalga Micractinium simplicissimum IPPAS C-2082 - electron microscopy.

Fig. 3 shows a graph of the accumulation of dry biomass of Micractinium simplicissimum IPPAS C-2082 when cultivated on BG-11 medium with 1.5-2 ‰ NaCl.

Fig. 4 shows a graph of the accumulation of dry biomass of Micractinium simplicissimum IPPAS C-2082 during cultivation on BG-11 medium with 15-18 ‰ NaCl.

Implementation of the invention

The microalgae strain Micractinium simplicissimum NAMSU-MS2022 was isolated from a natural seawater sample on the Kara Sea shelf and deposited in the Russian Microalgae Collection at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned IPPAS identifier C-2082. It was selected through screening for its ability to grow on a medium containing mineral salts from seawater and to accumulate carotenoids (a mixture of lutein and β-carotene).

Isolation method: from an enrichment culture obtained by incubating the original water sample on BG-11 medium, followed by plating the resulting strain on BG-11 mineral medium with the addition of 1 g/l of the antibiotic ampicillin and subculture of cells from single colonies with dilution on solid BG-11 medium (containing 1.5% agar).

Morphological features.

Growth pattern on storage medium: homogeneous, aggregation is possible at later stages.

Cytological description.

Cell shape, size: Micractinium simplicissimum strain IPPAS C-2082 is represented by immobile single spherical coccoid cells (4-6 µm in diameter).

The cell wall is unremarkable, the surface layer is felt-like.

The plastidome of vegetative cells is represented by one large bilobed chloroplast (Chl), surrounded by two membranes and having a well-developed thylakoid system in the stroma.

The pyrenoid is single, large, and two-shelled. The reserve inclusions are primarily starch grains (SG), which are typically deposited around the pyrenoid as a non-insulating, fragmented starch sheath, as well as as interthylakoid grains located diffusely within the stroma.

The nucleus is single, large, central. One nucleolus.

Reproduction: vegetative (mitotic division with the formation of two daughter cells); asexual: autosporangia with 16-32 fully formed daughter vegetative cells (autospores). Flagellates have not been found.

Physiological properties of the strain.

Optimal cultivation conditions:

For cultivation, use 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 are autoclavable.

Medium 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 O - 0.036 g/l, citric acid - 0.006 g/l,

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

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

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

pH at the beginning of cultivation is 7.0-7.4

pH at the end of cultivation (after 12 days) - 7.8-8.2

_CO2 content in the gas-air mixture (GAM) - 1.6-2.4%, bubbling rate 0.4-0.6 l⋅min ^-1

temperature 23-25°C

24-hour lighting, illumination 80 μmol quanta⋅m ^-2 ⋅s ^-1

lamp type: LED, 4700 K

culture density - 0.3-1.5 g dry matter/l

Biotechnological characteristics of the strain.

The microalgae strain Micractinium simplicissimum NAMSU-MS2022, deposited in the Russian Collection of Microalgae at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned IPPAS identifier C-2082, produces a mixture of natural lutein and β-carotene (provitamin A) in a ratio of 62-64 wt.% and 23-25 wt.% of the total carotenoids, respectively, the total carotenoids being 13.3-13.7 mg⋅g ^-1 of dry biomass weight, with a productivity of 4.8-5.0 mg per day from 1 l of culture for the total carotenoids, capable of bioremoving carbon dioxide from the environment at a rate of 0.30-0.32 l⋅l ^-1 ⋅day ^-1 .

Cultivation conditions that ensure the maximum level (yield) of the useful property (product): the presence of seawater salts in the cultivation medium (salinity of the medium 15-18 ‰), illumination 210-230 μmol quanta⋅m ^-2 ⋅s ^-1 .

Productivity under optimal cultivation conditions for biomass accumulation (dry biomass weight, g⋅l ^-1 ⋅day ^-1 ): 0.35-0.37.

Yield of useful product: productivity of carotenoids 4.8-5.0 mg per day from 1 liter of culture, simultaneous accumulation of lutein and β-carotene (62-64 wt.% and 23-25 wt.%, respectively, of the total carotenoids).

Genotyping.

DNA extraction.

For DNA extraction, 5-10 mg of microalgae culture biomass is collected. DNA extraction is performed using phenol-chloroform extraction. Before extraction, the samples are frozen three times at -4°C and then thawed. This is necessary to destroy the strong cell walls of the algae. The samples are incubated for 1 hour in 300 μl TE buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA) containing 10 mg ml ^-1 lysozyme at 37°C. Then 2% sodium dodecyl sulfate is added and the sample is incubated for 1 hour at 40°C with vigorous stirring. Next, 1 M NaCl is added and the sample is left on ice overnight to salt out the proteins. This is followed by phenol-chloroform extraction. The purity of the DNA samples is assessed by electrophoresis in a 1.5% agarose gel. The resulting DNA samples are stored in TE buffer at -4°C. A fragment of the ITS1-5.8S rRNA-ITS ribosomal gene cluster and the gene encoding the large subunit of the RubisCO enzyme ( rbcL ) are then sequenced.

As a result of the phylogenetic analysis of the obtained sequences, carried out according to the previously described method [Characterization of a new halotolerant artic strain of a carotenogenic microalga Halochlorella rubescens NAMSU sbb-20 / A. Zaytseva, D. Bakhareva, P. Zaytsev, E. Lobakova // Russian Journal of Plant Physiology . - 2023. - Vol. 70, no. 3.], the species affiliation of the studied isolate was established. The isolate was identified as Micractinium simplicissimum and received the identifier NAMSU-MS2022; after deposition in the Russian Collection of Microalgae at the K.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS), it was assigned the identifier IPPAS C-2082.

During testing, the microalgae strain Micractinium simplicissimum IPPAS C-2082 demonstrated a high capacity for the simultaneous accumulation of lutein and β-carotene in biomass, with a total carotenoid content of 13.3-13.7 mg g ^-1 of dry biomass weight. This is unmatched by known analogues and 10% higher than that of the known prototype. Lutein and β-carotene accumulation amount to 63-66% and 23-25%, respectively, of total carotenoids. The strain Micractinium simplicissimum IPPAS C-2082, when cultivated on a medium at a salinity of 15-18 ‰, maintains high productivity: 0.35-0.37 g⋅L ^-1 ⋅day ^-1 for the accumulation of dry biomass and 4.8-5.0 mg for the accumulation of total carotenoids per day from 1 L of culture and simultaneously the ability to bioremove carbon dioxide from the medium at a rate of 0.30-0.32 L⋅L ^-1 ⋅day ^-1 , which significantly exceeds the level of known analogs capable of growth on brackish media.

The microalgae strain Micractinium simplicissimum IPPAS C-2082 has successfully passed preliminary testing and the trial cultivation stage in experimental and semi-industrial photobioreactors.

The advantage of this strain is the possibility of its use for industrial cultivation for the purpose of carbon dioxide bioremoval in coastal and arid regions where brackish water is relatively easily accessible.

Example 1

For the cultivation of the microalgae strain Micractinium simplicissimum IPPAS C-2082, the classical BG-11 medium of the following composition was used:

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 O - 0.036 g/l, citric acid - 0.006 g/l,

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

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

microelement solution (H ₃ BO ₃ - 2.86 g/l, MnCl ₂ ⋅4H ₂ O - 1.86 g/l, ZnSO ₄ ⋅7H ₂ O - 0.22 g/l, CuSO ₄ ⋅5H ₂ O - 0.08 g/l, Na ₂ MoO ₄ ⋅7H ₂ O - 0.39 g/l, Co(NO ₃ ) ₂ ⋅6H ₂ O - 0.05 g/l) - 1 ml/l, to which additional salt solutions were added, namely the Uralmed sea salt solution containing NaCl, (NH ₄ ) ₂ SO ₄ , NaNO ₃ , MgSO ₄ salts, to a final salinity of 1.5-2 ‰.

The pH level at the beginning of cultivation was set at 7.2.

The culture density at the beginning of cultivation was 2 million cells/ml.

The cultivation was carried out in 1.6 L glass bubbling columns in a photobioreactor at a temperature of 23 to 25°C using a mixture of atmospheric air and _CO2 for bubbling, the _CO2 content was 1.6-2.4%, and the bubbling rate was 0.4-0.6 L⋅min ^-1 .

LED lamps (4700 K) were used for lighting; lighting was provided around the clock, and illumination was set from 210 to 230 μmol quanta⋅m ^-2 ⋅s ^-1 .

Cultivation time is 12 days (exponential phase of biomass accumulation from days 4 to 7).

The culture density at the end of cultivation was 40-60 million cells/ml.

As the conducted studies have shown, the productivity of the strain in terms of biomass accumulation (dry weight) was 1.3±0.15 g⋅l ^-1 , with an average daily growth rate of 0.35 g⋅l ^-1 ⋅day ^-1 .

To determine absolute carbon content, a 50 ml aliquot of the cell suspension was collected, the cells were pelleted by centrifugation at 5000 g for 10 min, the supernatant was discarded, the cell pellet was freeze-dried in a LabCONCO FreeZone freeze dryer, and its mass was measured. The percentage carbon content of the freeze-dried biomass was determined using an Elementar MicroCube elemental analyzer.

The rate of carbon bioremoval by the microalgae culture was determined using the following formula:

Where - average rate of CO fixation₂,- dry weight content (g/l) at a given timet, - dry weight content (g/l) at sowing (at the timet ₀),ω _C- the proportion of carbon in the dry weight of cells (g/g),V- volume of cell suspension (l),- the molar mass of carbon dioxide (g/mol), and - molar mass of carbon (g/mol). The rate of carbon dioxide bioremoval was calculated taking into account the mass fraction of carbon in CO₂.

The rate of carbon bioremoval and productivity parameters of the microalgae culture Micractinium simplicissimum IPPAS C-2082 grown for 12 days at a salinity of the cultivation medium of 1.5-2 ‰ are presented in Table 1:

Parameter Unit of measurement Salinity of the cultivation medium 1.5-2 ‰ Biomass accumulation g/l 1.3±0.15 Biomass productivity g/l/day 0.35±0.03 Total carotenoid content in biomass Mass.% 1.33±0.11 mg/g 13.48±0.85 mg/l/day. 4.91±0.25 Lutein content in biomass mg/g 8.50±0.77 Lutein productivity mg/l/day. 3.20±0.15 β-carotene content mg/g 2.7±0.16 β-carotene productivity mg/l/day. 0.68±0.05 Carbon bioremoval rate g _CO2 /l/day 0.58±0.09 l _CO2 /l/day 0.30±0.05 g C/l/day 0.14±0.01

As can be seen from Table 1, the yield of the useful product, carotenoids, was 13.48 mg/g at a salinity of 1.5-2‰, and the rate of carbon bioremoval was 0.30±0.05 l _CO2 ⋅l ^-1 ⋅day ^-1 or 0.14±0.01 g C⋅l ^-1 ⋅day ^-1 in terms of pure carbon.

The analysis of the pigment composition of cells was carried out as follows.

Pigments were extracted with 100% acetone. 500 μl of 100% acetone were added to the precipitated cells. For complete pigment extraction, the cells were disrupted with a hand-held glass-Teflon homogenizer. The mixture was then centrifuged for 5 min at 10,000 rpm. The supernatant was poured into a clean tube, and another 500 μl of 100% acetone was added to the sediment. The procedure was repeated until the sediment was completely decolorized, and the supernatants for each sample were pooled. The concentration of chlorophylls and total carotenoids were determined spectrophotometrically using an Agilent Cary 300 Bio spectrophotometer. Separation and analysis of individual pigments were performed by high-performance liquid chromatography on a Waters Sunfire C18 reversed-phase column (4.6 × 150 mm, 5 μm grain). To separate the pigment mixture, a mixture of 82% acetonitrile, 11% methanol and 7% water was used as solution A, and pure ethyl acetate was used as solution B. The gradient elution program was: 0-10 min: 10% solution B, 10-15 min: 15% solution B; 15-20 min: 45% solution B, 20-25 min: 0% solution B. The solvent feed rate was 1 ml/min.

The HPLC system consisted of a Shimadzu LC-20A pump with an FCV-20ALVP module for creating a solvent gradient on the low-pressure side, an SPD-M20A detector, and a CTO-20AC thermostat.

The composition of pigments contained in the biomass of the microalga Micractinium simplicissimum IPPAS C-2082, grown for 12 days at a salinity of the cultivation medium of 1.5-2 ‰, is presented in Table 2:

Pigments (mg/g d.v.) Salinity of the cultivation medium 1.5-2 ‰ Chlorophyll a 37.81 Chlorophyll b 14.55 Chlorophyll ( a + b ) 52.36 Carotenoids 10.11

An analysis of the carotenoid composition of the algae showed that the Micractinium simplicissimum IPPAS C-2082 strain produces various carotenoids, including lutein, β-carotene, neoxanthin, violaxanthin, and zeaxanthin. Among the carotenoids produced by the strain, lutein (approximately 65±5.2% at a salinity of 1.5-2‰) and β-carotene (65±5.2% at a salinity of 1.5-2‰) are the most abundant.

The composition of carotenoids contained in the biomass of the microalga Micractinium simplicissimum IPPAS C-2082 grown for 12 days at a salinity of the cultivation medium of 1.5-2 ‰ is presented in Table 3.

Carotenoids (% of total carotenoids) Salinity of the cultivation medium 1.5-2 ‰ Neoxanthin 4.7±0.28 Violaxanthin 4.6±0.15 Lutein 65±5.2 Zeaxanthin 0.1±0.05 β-carotene 65±5.2 Unidentified carotenoids 3.5±0.09

Example 2

For cultivating the microalgae strain Micractinium simplicissimum IPPAS C-2082, the BG-11 medium and the conditions specified in Example 1 were used, with the exception that the Uralmed sea salt solution containing the salts NaCl, (NH ₄ ) ₂ SO ₄ , NaNO ₃ , MgSO ₄ were added to a final salinity of the medium of 15-18 ‰.

As the conducted studies have shown, the productivity of the strain in terms of biomass accumulation (dry weight) was 1.4±0.15 g⋅l ^-1 at a salinity of 15-18 ‰, with an average daily growth rate of 0.37 g⋅l ^-1 ⋅day ^-1 .

The absolute carbon content and the rate of its bioremoval, as well as the bioremoval of carbon dioxide by the microalgae culture, were determined as described in Example 1.

The rate of carbon bioremoval and productivity parameters of the microalgae culture Micractinium simplicissimum IPPAS C-2082 grown for 12 days at a salinity of the cultivation medium of 15-18 ‰ are presented in Table 4:

Parameter Unit of measurement Salinity of the cultivation medium 15-18 ‰ Biomass accumulation g/l 1.4±0.15 Biomass productivity g/l/day 0.37±0.05 Total carotenoid content in biomass % 1.37±0.24 mg/g 13.52±0.65 mg/l/day 4.95±0.38 Lutein content in biomass mg/g 8.55±0.41 Lutein productivity mg/l/day 3.22±0.19 β-carotene content mg/g 3.17±0.19 β-carotene productivity mg/l/day 0.79±0.06 Carbon bioremoval rate g _CO2 /l/day 0.62±0.03 l _CO2 /l/day 0.32±0.03 g C/l/day 0.17±0.02

As can be seen from Table 4, the yield of the useful product, carotenoids, was 13.52±0.65 mg/g at a salinity of 15-18‰, and the rate of carbon bioremoval was 0.32±0.03 l CO2 _⋅l ^-1 ⋅day ^-1 or 0.17±0.02 g C⋅l ^-1 ⋅day ^-1 in terms of pure carbon.

The analysis of the pigment composition of the cells was carried out as described in Example 1.

The composition of pigments contained in the biomass of the microalga Micractinium simplicissimum IPPAS C-2082, grown for 12 days at a salinity of the cultivation medium of 15-18 ‰, is presented in Table 5:

Pigments (mg/g d.v.) Salinity of the cultivation medium 15-18 ‰ Chlorophyll a 32.16±1.99 Chlorophyll b 11.38±0.75 Chlorophyll ( a + b ) 43.54±5.1 Carotenoids 13.5±2.1

The analysis of the carotenoid composition of the algae showed, as in Example 1, that the Micractinium simplicissimum IPPAS C-2082 strain produces various carotenoids, such as lutein, β-carotene, neoxanthin, violaxanthin, and zeaxanthin. Among the carotenoids produced by the strain, the highest accumulation levels are for lutein (61±2.9% at a salinity of 15-18‰) and β-carotene (24.1±1.9% at a salinity of 15-18‰).

The composition of carotenoids contained in the biomass of the microalga Micractinium simplicissimum IPPAS C-2082 grown for 12 days at a salinity of the cultivation medium of 15-18 ‰ is presented in Table 6.

Carotenoids (% of total carotenoids)
Salinity of the cultivation medium 15-18 ‰ Neoxanthin 4.1±0.3 Violaxanthin 3.8±0.3 Lutein 61±2.9 Zeaxanthin 0.2±0.01 β-carotene 24.1±1.9 Unidentified carotenoids 4.8±0.4

Thus, the conducted study showed that the microalgae strain Micractinium simplicissimum IPPAS C-2082, when grown on media with a salinity of 15-18 ‰, has a high level of accumulation of a mixture of natural carotenoids (about 13.5 mg/g dry biomass), among which the most significant shares are lutein (61±2.9%) and β-carotene (24.1±1.9%), while the rate of carbon bioremoval is 0.32±0.03 l CO ₂ ⋅l ^-1 ⋅day ^-1 or 0.17±0.02 g C⋅l ^-1 ⋅day ^-1 in terms of pure carbon.

As a result, a strain of microalgae, Micractinium simplicissimum NAMSU-MS2022, was obtained. It is deposited in the Russian Collection of Microalgae at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) and has been assigned the IPPAS identifier C-2082. This strain exhibits a high level of accumulation of a mixture of lutein and β-carotene, maintains high productivity when grown in brackish environments, and has the ability to simultaneously bioremove carbon dioxide from the environment.

Claims (1)

The microalgae strain Micractinium simplicissimum NAMSU-MS2022, deposited in the Russian Collection of Microalgae at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPPAS) with the assigned IPPAS identifier C-2082, is a producer of a mixture of lutein and β-carotene, resistant to brackish environments and capable of bioremoval of inorganic carbon from the cultivation medium.