Activation of stress reactions in the dinophyte microalga Prorocentrum cordatum as a consequence of the toxic effect of ZnO nanoparticles and zinc sulfate.

According to the results of the experimental study, the main regularities of changes in morphological, structural-functional and fluorescent indices of P. cordatum were established when zinc oxide nanoparticles ZnO NPs (0.3-6.4 mg L-1) and Zn in form of salt (0.09-0.4 mg L-1) were added to the medium. The studied pollutants have cytotoxic (growth inhibition, development of oxidative stress, destruction of cytoplasmic organelles, disorganization of mitochondria) and genotoxic (changes in the morphology of nuclei, chromatin condensation) effects on microalgae, affecting almost all aspects of cell functioning. Despite the similar mechanism of action of zinc sulfate and ZnO NPs on P. cordatum cells, the negative effect of ZnO NPs is also due to the inhibition of photosynthetic activity of cells (significant decrease in the maximum quantum yield of photosynthesis and electron transport rate), reduction of chlorophyll concentration from 3.5 to 1.8 pg cell-1, as well as mechanical effect on cells: deformation and damage of cell membranes, aggregation of NPs on the cell surface. Apoptosis-like signs of cell death upon exposure to zinc sulfate and ZnO NPs were identified by flow cytometry and laser scanning confocal microscopy methods: changes in cell morphology, cytoplasm retraction, development of oxidative stress, deformation of nuclei, and disorganization of mitochondria. It was shown that the first signs of cell apoptosis appear at 0.02 mg L-1 Zn and 0.6 mg L-1 ZnO NPs after 72 h of exposure. At higher concentrations of pollutants, a dose-dependent decrease in algal enzymatic activity (up to 5 times relative to control) and mitochondrial membrane potential (up to 4 times relative to control), and an increase in the production of reactive oxygen species (up to 4-5 times relative to control) were observed. The results of the presented study contribute to the disclosure of fundamental mechanisms of toxic effects of pollutants and prediction of ways of phototrophic microorganisms reaction to this impact.

Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate.

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25-200 µg L-1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 µg L-1) and lethal (200 µg L-1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 µg L-1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 µg L-1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

The comprehensive effect of copper oxide nanoparticles on the physiology of the diatom microalga Thalassiosira weissflogii.

The high rate of production and use of copper oxide nanoparticles (CuO NPs) results in its accumulation in the environment. However, the effect of large quantities of CuO NPs on aquatic ecosystems is not fully known. In aquatic ecosystems, phytoplankton is the primary producer of organic matter and the basis of all the trophic interactions; accordingly, the potential effect of CuO NPs on the microalgae community is of great concern. This study established the main patterns of changes in morphological, structural, functional, fluorescent and cytometric parameters in the marine diatom Thalassiosira weissflogii after adding CuO NPs to the medium at concentrations of 250-2500µgL-1 . As shown, the investigated pollutant has cytotoxic, genotoxic and mechanical effect on the microalga covering almost all the aspects of cell functioning. A two-fold decrease in the culture abundance relative to the control is observed at the toxicant content of 550µgL-1 in the medium. At CuO NPs content above 750µgL-1 , a pronounced inhibition of the alga growth is recorded, as well as a decrease in the efficiency of its photosynthetic apparatus, a disturbance of membrane integrity, an increase in cell volume, a rise in abundance of dead/inactive cells in the culture, enlargement and deformation of nuclei, an increase in reactive oxygen species production, and depolarisation of the mitochondrial membrane. Our results show that high CuO NPs concentrations in water can cause serious disruptions in phytoplankton functioning and in equilibrium of aquatic ecosystems in general.

Responses of Prorocentrum cordatum (Ostenfeld) Dodge, 1975 (Dinoflagellata) to copper nanoparticles and copper ions effect.

In the present study, changes were determined in morphological, structural-functional, and fluorescent parameters of Prorocentrum cordatum with the addition of CuO nanoparticles (NPs) and copper ions (CuSO4). A stimulating effect of low Cu2+ concentrations (30 µg L-1) on algal growth characteristics was observed. Higher Cu2+ concentration of 60-600 µg L-1 and CuO NPs concentration of 100-520 µg L-1 inhibited algal growth. Ionic copper is more toxic to P. cordatum than NPs. After 72 h of algae cultivation in the medium supplemented with CuSO4 and CuO NPs, EC50 values (calculated based on cell abundance) were of 60 and 300 µg L-1 (in terms of copper ions), respectively. Reduction in algal growth rate is due to disruption in cell cycle, changes in nuclear morphology, chromatin dispersion, and DNA damage. The studied pollutants slightly affected the efficiency of P. cordatum photosynthetic apparatus. Addition of the pollutants resulted in an increased production of reactive oxygen species (ROS). At a concentration of Cu2+ of 120 µg L-1 and a concentration of CuO NPs 0-300 µg L-1 of CuO NPs increase in ROS production is short-term with a decrease at later stages of the experiment. This is probably due to the activation of antioxidant mechanisms in cells and an increase in the concentration of carotenoids (peridinin) in cells. The high values of ROS production persisted throughout the experiment at sublethal copper concentrations (400-600 µg L-1 of CuSO4 and 520 µg L-1 of CuO NPs). Sublethal concentrations of pollutants caused restructuring of cell membranes in P. cordatum. Shedding of cell membranes (ecdysis) and formation of immobile stages (temporary or resting cysts) were recorded. The pronounced mechanical impact of NPs on the cell surface was observed such as-deformation and damage of a cell wall, its "wrinkling" and shrinkage, and adsorption of NP aggregates.

Growth and B-Phycoerythrin Production of Red Microalga Porphyridium purpureum (Porphyridiales, Rhodophyta) under Different Carbon Supply.

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.

Differential responses of Pleurochrysis sp. (Haptophyta) to the effect of copper and light intensity.

The effect of light, copper ions, copper oxide nanoparticles on the change in the structural, functional, cytometric, fluorescent parameters of coccolithophore Pleurochrysis sp. was investigated. The culture Pleurochrysis sp. was represented by two cell forms: (1) covered with coccoliths; and (2) not covered, the ratio of which depends from growth conditions. An increase in light from 20 to 650µEm-2 s-1 led to a decrease in the concentration of cells covered with coccoliths from 90 to 35%. With an increase in light, the decrease in the values of variable chlorophyll a fluorescence was observed, a decrease in the chlorophyll concentration was noted, and an increase in cell volumes and their granularity due to coccoliths 'overproduction' was recorded. A tolerance of Pleurochrysis sp. to the effect of copper was registered, both in the ionic form and in the form of a nanopowder. This is probably due to the morphological (presence of coccoliths) and physiological (ligand production) peculiarities of species. Copper did not affect the ratio of cells covered with coccoliths; its value was about 85%. Growth inhibition, a 2-fold decrease in the intracellular chlorophyll content, a decrease in F v /F m , and a pronounced cell coagulation were recorded at the maximum Cu2+ concentration (625µgL-1 ). The mechanical effect was registered of CuO nanoparticles on the surface of Pleurochrysis sp. coccosphere, which results in the emergence of destroyed and deformed coccoliths. A hypothesis is proposed considering the protective function of coccoliths acting as a barrier when the cells are exposed to nanoparticles and copper ions.

Productivity and morphometric parameters of the microalga Dunaliella salina IBSS-2 under pilot cultivation in continental mid-latitude climate in spring.

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.