Development of sustainable technologies for production of the red microalga Porphyridium purpureum is an important objective to reduce the production costs and expand its incorporation into food, feed, and nutraceutical products. The aim of this study was to evaluate the production potential of P. purpureum under different cultivation modes and to optimize its pilot–scale cultivation in open ponds under mid-latitude climate conditions (spring–summer and autumn). Batch and semicontinuous photoautotrophic cultivation, as well as batch mixotrophic cultivation (with glycerol), were compared in terms of biomass and B–phycoerythrin (B–PE) productivity. The highest biomass productivity (22.3 g m–2 day–1) was obtained under mixotrophic cultivation in summer, which was 2–3 times higher than in photoautotrophic modes. However, mixotrophic growth markedly reduced the B–PE content in the biomass (down to 2.85

This study investigated the effect of sodium bicarbonate (NaHCO3) supplementation on the growth and morphological characteristics of the red microalga Porphyridium purpureum. The NaHCO3 concentration in the nutrient medium was found to significantly influence both culture productivity and morphometric parameters. A linear relationship was established between production characteristics (biomass yield and productivity) and NaHCO3 concentration within the tested range, resulting in a twofold increase in these parameters with increasing sodium bicarbonate concentration from 0 to 2 g L- 1. Morphological analysis revealed improved culture conditions at high NaHCO3 concentrations (2 g L- 1), evidenced by a prevalence of medium-sized cells and a low proportion of cells with cytoplasmic retraction or in aggregates during the stationary phase. In contrast, cultures with low or no bicarbonate supplementation showed signs of stress, including a sharp increase in cell aggregation and the presence of autospores. A positive correlation was identified between the proportion of cells with cytoplasmic retraction and aggregate formation, while a negative correlation was observed for autospore abundance relative to NaHCO3 content. Prolonged cultivation led to an increase in average cell diameter with higher bicarbonate concentrations. The results demonstrate that NaHCO3 is an effective carbon source for enhancing P. purpureum biomass production, and that specific morphological changes are reliable indicators of the culture's physiological state in response to growth conditions.

The aim was to evaluate the stability of pigments of the phycobiliprotein group extracted from the biomass of the Spirulina (Arthrospira) platensis cyanobacterium and the Porphyridium purpureum red microalgae. Water extracts of phycobiliproteins were obtained following a double freezing of the raw biomass of Arthrospira platensis and Porphyridium purpureum. An extraction was carried out with a phosphate buffer (0.05 M, pH = 7) in the cold (5 degrees C) for 24 hours. To the extracts obtained, 96% ethanol was added until its concentration in the solution was 20%. The hydroalcoholic extracts of phycobiliproteins were stored for three months. Pigment concentrations were monitored by an optical method. The allophycocyanin pigment demonstrated the highest storage stability. The highest degradation rate of phycobiliproteins was observed during their storage in the light at room temperature. The degra- dation rate of pigments under these conditions was 9- and 80-fold higher (for B-phycoerythrin and C-phycocyanin, respectively) than similar indices during their storage in the dark and in the cold. C-phycocyanin was the least stable, compared to other studied phycobiliproteins. Its degradation rate under all storage options was 5- to 10-fold higher than that of B-phycoerythrin under similar conditions. An essential conservation requirement for C-phycocyanin and beta-phycoerythrin in hydroalcoholic solutions was the absence of light. For C-phycocyanin, a low temperature was necessary as well. Storage of B-phycoerythrin in the dark at room temperature is acceptable. These conditions can ensure the conservation of up to 86% of pigments in hydroalcoholic solutions for 25-30 days.

Porphyridium purpureum is a promising microalga species due to the content of various valuable compounds. In this study, specific irradiance parameter, representing the amount of light energy per unit of microalgae biomass, was introduced. The growth characteristics and pigments and protein accumulation of P. purpureum culture were investigated under semi-continuous mode. Varying dilution rate and surface irradiance resulted in a specific irradiance of 0.2-6.7 W g-1. Using mathematical modeling, we determined the patterns of changes in biomass, pigments, protein content and productivity of P. purpureum culture depending on specific irradiance. The content of target compounds was maximized under the lowest level of specific irradiance (0.2-1.2 W g-1), while the highest productivity of this components was reached under 1.2-1.7 W g-1. Overall, lower irradiance levels were favorable for P. purpureum cultivation based on the energy consumption and production characteristics of this species.

Red microalga Porphyridium purpureum (Bory) Drew is a well-known object of biotechnology due to its unique ability to synthesize a wide range of biologically active compounds. Enough minerals in an accessible form in a medium are a prerequisite for maintaining a high growth rate of P. purpureum. Carbon is the main element of microalgal biomass and is a component of all organic compounds. The work aimed to study the morphological features of cells and the accumulation and production of B-phycoerythrin and total protein in P. purpureum biomass in different ways of supplying CO2 into the culture. In Variant 1, CO2 was directly injected into a gas–air mixture (2–3 percent v/v) used for culture bubbling via capillary. In Variant 2, the air was supplied to the culture through the aquarium sparger. Variant 3 was like the first one but without the additional introduction of carbon dioxide. The application of the method for sparging atmospheric air led to a significant increase in both the productivity of the P. purpureum and the rate of protein and B-phycoerythrin synthesis in comparison with growing it using the air without spraying (two-and-a-half times, five times, and more than eight times, respectively). Moreover, there were significant changes in the morphological structure of P. purpureum cells, which were visualized both by microscopy and by changes in the color of the culture. Based on the experimental data obtained, the variants for the carbon supply experiment were ranked as follows: Variant 1 is better than Variant 2 and Variant 3. The use of atomization as a technological method made it possible to speed up the transfer of carbon dioxide from the air to the medium, which helped to keep the growth rate of P. purpureum biomass and B-phycoerythrin accumulation high.

The red microalga Porphyridium purpureum (Bory de Saint-Vincent, 1797) Drew et Ross, 1965 is of great interest to researchers as a source of various biologically valuable substances, with their content in cells being determined by cultivation conditions. Phycobiliproteins concentration in P. purpureum cells depends directly on nitrogen concentration in the culture medium and cell irradiance. Semi-continuous cultivation allows maintaining these parameters at a level given. The aim of the work was to study P. purpureum culture growth and B-phycoerythrin (B-PE) accumulation and production at low irradiance, with minimal rates of pigment photodestruction. P. purpureum semi-continuous (quasi-continuous) cultivation was carried out at a specific flow rate of 0.1 and 0.2 day−1 and mean surface irradiance of 5 and 25 W·m−2. P. purpureum culture productivity increased by 1.6–17 times both with a rise in surface irradiance 5 to 25 W·m−2 and an increase in the medium specific flow rate 0.1 to 0.2 day−1. Maximum productivity values for the experimental conditions (0.21 g·L−1·day−1) were recorded at 25 W·m−2 and 20 % medium specific flow rate, but those were 1.5–2 times lower than the precalculated ones. In P. purpureum cells, protein and B-PE concentrations decreased both with an increase in surface irradiance (by 15–20 %) and with a rise in a specific flow rate (by 1.5 times) for all the variants. The shifts in protein and B-PE concentration in P. purpureum culture had a unidirectional character as well; those mainly corresponded to the shift in the culture density. P. purpureum B-PE productivity increased by 1.5–1.9 times with a rise in surface irradiance 5 to 25 W·m−2. Maximum B-PE productivity (13 mg·L−1·day−1) was recorded for the variants of the experiment with a surface irradiance of 25 W·m−2 (0.1 and 0.2 day−1). An increase in specific irradiance of P. purpureum cells 7 to 26 W·g−1 resulted in a rise in biomass productivity by 2.6 times; in B-PE productivity, by 1.8 times; and in protein productivity, by 1.7 times. In the experiment, irradiance was the factor determining the production characteristics of P. purpureum culture, and it was confirmed by the data obtained.

In this study, we aimed to investigate the taxonomy and various characteristics of Dunaliella salina IBSS-2 strain and describe its cultivation potential in mid-latitude climate during springtime. In addition, our analysis confirmed the essentiality of combining morphological, physiological, and other characteristics when identifying new species and strains of the genus Dunaliella, along with the molecular marker (internal transcribed spacer (ITS) of rDNA gene). The pilot cultivation of microalgae during the springtime in the south of Russia demonstrated that the climatic conditions of this region allow D. salina cultivation for biomass accumulation during this season, highlighting light and temperature conditions as the main factors determining the growth rate of D. salina. A two-fold increase in daily insolation and, consequently, in temperature in April resulted in a more than three-fold increase in productivity of D. salina culture. The maximum productivity of D. salina both in April and May was comparable and reached 2 g m-2 day-1, and the total yield for 8-10 days was about 14.5-16 g m-2. The additional CO2 supply into the D. salina culture did not show any significant effect on its growth rate; however, it contributed to maintaining the diversity of morphometric characteristics over a longer period of time. Changes in the morphological and morphometric characteristics of algal cells, including size reduction, were observed during the batch cultivation. Thus, the production potential of the green carotenogenic microalga D. salina was determined in the springtime, which allows expanding the seasonal interval of its cultivation in temperate latitudes.

The research deals with theoretical calculation of the maximal growth rate of green halophilic microalga Dunaliella salina in the conditions of natural illumination at South regions of Russia (Sevastopol city as an example). The calculation is based on the concept that microalgae growth rate is defined as the difference between gross productivity and endogenous biomass expenditure rate. Gross productivity is a function of photosynthetic active radiation (PAR), absorption coefficient and energy utilization efficiency. For maximal productivity calculation it is suggested that all incident of the pond surface light energy is absorbed by microalgae culture. As far as photobiosynthesis efficiency value depends on illuminance in a complex way, we used average value 5.58%. The rate of endogenous biomass expenditure was determined based on the value of night losses, which for D. salina was about 5%. Computations showed that for D. salina maximum biomass gain makes 26 g DW/(m(2) . day). It is shown that the maximum observed productivity of D. salina in the conditions of natural light in the southern regions of Russia cannot exceed 26 g DW/(m(2) . day).

The red pigment B-phycoerythrin (B-PE) belongs to the group of phycobiliproteins (PBPs). It is a part of the light-harvesting pigment complex of the red microalga Porphyridium purpureum (Bory) Drew et Ross, and its amount in cells is determined by the level of irradiation and nitrogen supply. B-PE is a valuable natural pigment whose biotechnological potential is used in nutraceuticals, pharmaceuticals, food and cosmetic industries, and in biomedical research and clinical diagnostics. The dynamics of cell number and the content of photosynthetic pigments are quite often assessed in experiments with P. purpureum. The culture’s density is low in the majority of the experiments, although it is known that it can reach 10 g/L of dry matter. The aim of the study was to investigate the features of the accumulation and production of PBPs in a dense culture of P. purpureum under varying illumination. The molecular-genetic analysis confirmed the taxonomic affiliation of P. purpureum culture. Algae were grown in flat plate photobioreactors with an average illumination of 5, 10, and 15 klx. The dynamics of culture density, the content of B-PE, and production characteristics were used as indicators. It was shown that an increase in the microalgae culture density corresponding to the calculated nitrogen concentration in the nutrient medium was obtained only under illumination of 5 klx. Illumination increased to 10, and 15 klx caused a decrease in both the maximum and average productivities of the culture by 2.5–3.5 times. It was experimentally shown that the highest content of B-PE in the cells and culture of P. purpureum (5.5% of dry matter and 74 mg/L, respectively) was observed under illumination of 5 klx. The pattern of the change in B-PE content in P. purpureum culture was determined depending on the specific illumination of microalgae cells: the pigment concentration hyperbolically decreased with increasing specific illumination. Thus, the level of illumination of P. purpureum microalgae cells had a significant effect on the growth characteristics of the culture (growth rate, B-PE synthesis rate and yield): a lower level of surface illumination was preferred for P. purpureum cultivation. The approach proposed in the study allows one to reduce material costs during the cultivation of P. purpureum while maintaining a high growth rate of the culture.

The productivity characteristics of green halophilic algae Dunaliella salina during its pilot cultivation were compared between two nutrient media and under different illumination. Cultivation was carried out in ponds located in the greenhouse during summer season. Two main factors that determine rates of growth and pigments synthesis, as well as the pigments ratio in D. salina grown outdoors in summer, are light and temperature conditions. The mean productivity of D. salina culture grown in two nutrient media, differing in nutrients concentration, was the same, and the mean carotenoid accumulation rate was 30% higher when using Ben-Amotz medium (52 mg center dot m(-2)center dot day(-1)) than Johnson medium. D. salina cell illumination was decreased by pond shading or by doubling their depth. Both these methods allowed reducing the level of the stress impact on algal culture under extreme light and temperature conditions. Shading of the ponds resulted in an increase in biomass and carotenoid productivity by 30% and 50%. However, a two-fold increase in ponds depth proved to be a more effective technological method, which allowed for an increase in D. salina areal productivity both in biomass and carotenoids by a factor of 3.5-3.8. In conclusion, less concentrated medium and increasing culture depth may be recommended as the optimization factors for D. salina outdoor cultivation.

The microalga Dunaliella salina was studied at the main stages of transition from laboratory to pilot-scale cultivation: strain selection, nutrient medium selection, estimation of influence of physicochemical factors on accumulation of carotenoids and evaluation of the selected strain growing technology at pilot conditions. Dunaliella salina strain IBSS-2 was recognized as promising for commercial cultivation due to the combination of high-production characteristics, environmental stress resistance, and relative easiness of transition to the carotenogenesis stage. The influence of stress factors on the D. salina culture productivity in two nutrient media was estimated. It was shown that light effect combined with nutrients deficiency is the key factor for β-carotene accumulation. The influence of increased irradiance caused the increase of carotenoid content in D. salina cells up to 8%, and increasing irradiance and salinity resulted in carotenoid productivity going up 1.5 times. Testing of D. salina pilot cultivation system demonstrated that productivity at the first cultivation stage was about 6 g m −2 day −1 in both batch and semicontinuous mode. During pilot D. salina cultivation in Crimea, the culture transition to the carotenogenesis stage was achieved both in summer and autumn. The concentration of carotenoids in the ponds was 200 and 600 mg m −2 with a carotenoid/chlorophyll a ( Car/Chl a ) ratio of 7 and 4.5 in summer and autumn, respectively. The possibility to use natural population of D. salina cells from salterns as inoculum and brine as a nutrient medium base was demonstrated. The study results suggest that the proposed approach can be recommended for D. salina commercial cultivation.

The investigation of cyanobacteria Spirulina (Arthrospira) platensis growth in the chicken manure extract (MCE) was carried out. The content of nitrogen and phosphorus in 1 liter of nutrient medium prepared on the basis of chicken manure extract is calculated. It is experimentally shown that the extraction of chicken manure in a concentration of 5-15% causes serious violations of the pigments biosynthesis processes. In this case, the protein and photosynthetic pigments contents in the cells of the S. platensis is significantly reduced by the 6th days and leads to the culture death. Increasing the volume fraction of the MCE in the nutrient medium has a significant impact on the pigments contents and total protein in the cyanobacteria biomass. Optimum concentrations of extract (20-30%), which provide a high rate of spirulina growth and the accumulation of biologically active substances in the obtained biomass, were determined. It was shown that 20-30% of MCE concentration allows to increase culture yield by 10-20% compared to control sample and to obtain the S. platensis biomass containing significant amounts of biologically active substances. It was found that the content of protein, chlorophyll a and C-phycocyanin in the S. platensis cells increased 2.5-6 times at the linear stage of culture growth at the increase of the volume fraction of MCE in the nutrient medium from 5 to 30%. S. platensis biomass, produced from such cultivation process, meets the criteria of high-quality biomass contents of protein, chlorophyll a, carotenoids and C-phycocyanin (51, 1.1, 0.4 and 5.5% correspondingly). The approach proposed in the paper allows to reduce material costs for the traditional mineral media preparation for microalgae cultivation.

Productional and biochemical characteristics of green alga Dunaliella salina (Dunal) Teodoresco were studied during pilot cultivation at the south-west of Crimean Peninsula, Russia. Two-phase cultivation was carried out in ponds located in the greenhouse module. In the "green" phase, biomass concentration reached 1.2 g/l, with maximum cell density of 1.69.10(6) cell/ml and the maximum productivity of 0.08 g/(1-day) (7 g/(m(2).clay)). Twofold culture dilution was applied for carotenogenesis induction. The maximum cell density in the "red" phase was 0.84.10(6) cell/ml, while biomass reached 0.95 g/l. Mean net carotenoid accumulation rate at the second stage (1-6 day) was 0.65 mg/(1.day). Carotenoids content in culture doubled compared to initial values and reached about 20 mg/l or 800 mg/m(2). Carotenoids/chlorophyll a ratio of 11.6 was observed by the end of the "red" phase. Night biomass loss was higher in the "red" phase (up to 9.5% of biomass) compared to the "green" phase (up to 5.6%). Optimal duration of both the first and the second cultivation stages was found to be 10-12 days for the studied weather conditions, which enables production of 3 g of carotenoids from 1 m(2) (100 l) of starter culture. In conclusion, Crimea is a prospective region for D. salina algobiotechnology.

The calculation of limit productivity for microalgae cultures during cultivation in the autumn period in the southern regions of Russia is carried out. The maximum observed productivity of Spirulina platensis and Dunaliella salina in semi-industrial conditions in autumn (Sevastopol, october-november 2018) was estimated. It is noted that the growth rate is limited by temperature conditions. The possibility of obtaining additional biomass of the studied species during the off-season is shown.

Aim. In this work, we set out to study the effect of surface irradiance on the growth rate and the pigment ratio of D. salina, as well as to test a technology for semi‐industrial cultiva‐ tion of D. salina aimed at obtaining its biomass enriched with β‐carotene.Methods. D. salina was cultivated under semi‐industrial conditions in a greenhouse module of the A. O. Kovalevsky Institute of Marine Biological Research. Square tanks (1 × 1 m) lined with polyethylene film and laid on a flat ground surface were used as propagators. The culture layer had a thickness of 10 cm and a volume of 100 litres. Pre‐grown D. salina was diluted using fresh medium without salts – sources of nitrogen and phosphorus. Dunaliella was cultivated under natural light with continuous stirring.Results. We determined the range of surface irradiance, which can be considered optimal for intensive cultivation of D. salina in terms of energy and mineral costs. Across this range, the average growth rate of the culture under experimental conditions amounted to 0.23–0.27 g DW/(l∙day), whereas the ratio of Car / Chl a increased by a factor of 1.5‐ 2, which indicates changes in the pigment composition of D. salina. It is experimentally shown that the content of carotenoids in the tanks increased by 1.3 times amounting to 600 mg per 1 m2 at a Car/Chl a ratio of 4.5 at the second stage of D. salina cultivation.Conclusions. Carotenoid accumulation during semi‐industrial cultivation of D. salina occures due to two factors: in‐ creased natural irradiance and temperature without an additional increase in salinity and blowing of carbon dioxide, which reduces the costs of its industrial cultivation. This two‐stage cultivation method can be used to develop a technology for obtaining Dunaliella biomass enriched with β‐carotene in the southern regions of the Russian Federation.

The work is focused on modeling of chlorophyll and carotenoids content dynamics in the in cells of the unicellular algae D. salina , living in salt water, at carotenogenesis induction phase. A mathematical model of pigments content in microalgae cells, which experience excess of light energy and the limit of nutrient medium mineral components, is proposed. The model is based on assumption, that observed rate of variation in pigment concentration is an algebraic sum of the rates of synthesis, photodestruction and partial recovery of photo-oxidized pigments. The rate of secondary carotenoids synthesis does not depend on external conditions and is determined by the quantity of key enzyme complex and its turnover rate. The rate of secondary carotenoids and chlorophyll photodestruction depends on the effective light intensity and is proportional to the amount of absorbed photosynthetically active radiation energy. The verification of the derived equations was conducted in the course of D. salina cultivation at the carotenogenesis stage. The specific rate of chlorophyll a photodestruction was determined, which resulted in 0.12 days –1 . The secondary carotenoids concentration increases up to the maximum value, which is determined by the ratio of synthesis and photodestruction specific rates, as well as the maximum culture density. Under conditions of natural light in the Sevastopol region, the maximum concentration of carotenoids was 18.33 mg/l or 0.73 g/m 2 .

The paper presents studies of morphological and morphometrical characteristics of green halophilic carotenogenic microalga Dunaliella salina (Dunal) Teodoresco, 1905, from the south-west region of Crimean Peninsula. D. salina was cultivated in two-phase mode under conditions of natural illumination at the premises of A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (IBSS), Sevastopol, Russia. The maximum D. salina cell density was 1.69·106 cell/ml in the “green” phase and 0.84·106 cell/ml in the “red” growth phase. The maximum productivity by cell number reached 0.15 ∙ 106 cell/(ml·day)in the “green” phase while it was lower by 73% in the “red” phase (0.04∙106 cell/(ml·day). Along with the maximum productivity, linear growth stage in the first phase was characterized by a maximum fraction of small (up to 500 μm3 in volume) cells (about 15-29%) and a decrease in cell volume by 40-45% as compared with initial value. The mean of D. salina cell volume in the “red” phase was 30% higher than in the “green” phase. At the same time, the large cell fraction in the “red” phase was consistently high (15-35%). The patterns of change in morphological and morphometrical cell parameters were in accordance to stage and conditions of growth. Thus, cell elongation was noted in the stage of linear growth, while under unfavorable conditions at growth-declining stage cells became more round-shaped, with orange and tile-red coloration and granulation of cell content. It was shown that morphological and morphometric cell parameters can serve as additional criteria for assessment of physiological condition in D. salina culture. The experiment demonstrated the prospects for two-stage D. salina cultivation in Crimea.

Недостаток углерода может являться основным лимитирующим фактором при выращивании микроводорослей, поэтому подбор оптимального способа подачи углерода в культуральную среду для конкретной культуры и фотобиореактора является определяющим для их интенсивного культивирования. Культуру микроводоросли Dunaliella salina выращивали в лабораторных фотобиореакторах плоскопараллельного типа при круглосуточном искусственном освещении 15 кЛк. Подачу воздуха осуществляли аквариумным компрессором со скоростью 0.8 л·л-1·мин-1 культуры в минуту. В первом варианте барботаж осуществляли через стеклянную трубку, во втором — через специальный распылитель воздуха. Экспериментально показана возможность интенсивного выращивания культуры D. salina на распыляемом атмосферном воздухе с максимальной продуктивностью 0.34 г сухой биомассы с 1 л в сутки. Проведена сравнительная оценка затрат для выращивания культуры зелёной микроводоросли D. salina на распыляемом атмосферном воздухе и с дополнительным введением СО2 в газовоздушную смесь.