Immobilization of microalgae on chitosan-based carriers represents a promising approach for producing value-added metabolites, biocapturing nutrients, and removing organic pollutants from wastewater. Although chitosan is non-toxic, biocompatible, and biodegradable, its commercial application is limited by high cost. To address this limitation, we propose the use of polysaccharide-rich biomass waste as an additive. This study investigates novel chitosan-based composite carriers containing three types of additives: spent biomass from cultured cells of the medicinal plant Ajuga turkestanica remained after bioactive compound extraction, apple pomace, and mushroom mycelium, the latter two being widespread food industry byproducts. These composites were prepared via cryopolymerization at varying chitosan-to-biomass ratios (1:3, 1:1, 3:1). The immobilization efficiency for the model cultures Lobosphaera sp. IPPAS C-2047 and Chlorococcum sp. increased with higher proportions of plant biomass and declined with elevated mycelium or pomace content. Notably, all carriers, except those containing 75

The generalized response of the cyanobacterium Nostoc sp. PCC 7120 to and its recovery from phosphorus (P) starvation stress differ drastically under diazotrophic and non-diazotrophic growth modes. In nitrogen (N) -replete medium, Nostoc sp. PCC 7120 cells were resilient even to prolonged P starvation when its growth was supported by mobilization of cellular reserves of P (polyphosphate) and glycogen on the background of accumulation of nitrogen and carbon reserves (mainly cyanophycin). The P-starving cells quickly recovered upon re-feeding with inorganic phosphate (Pi). Under diazotrophic conditions, P starvation essentially diminished the fixation of dinitrogen. As a result, most of the vegetative cells comprising the trichomes of the cyanobacterium died and decomposed while other cells retained their structural integrity but did not divide. In turn, the latter fell into two categories: some of them showed signs of nutrient starvation; while the other became dormant but did not display the signs of starvation. They resembled neither akinete nor chlorotic cells but were similar to arthrospores lacking a thickened sheath. Re-feeding with Pi triggered a quick resuscitation of the dormant vegetative cells manifested by mobilization of their internal reserves, resumption of the cell growth and division. These processes took place faster than the formation of heterocytes with well-developed envelope (thus, nitrogenase activity recovered by the 7th day after re-feeding of the cells with Pi). The results provide a deeper insight into the mechanisms of stress tolerance in Nostoc sp. PCC 7120 and modulation of the cyanobacterial productivity in natural ecosystems and artificial cultivation facilities by nitrogen and P availability.

Despite the long research history on the genus Coelastrella, its species diversity and biotechnological potential have not been fully explored. For the first time, cluster analysis of morphological characteristics was done in the representatives of the said genus. The results obtained have shown that morphological similarity does not necessarily indicate a molecular genetic relationship. It the light of it, the taxonomic status of species can reliably be determined using specific DNA region, such as 18S-ITS1-5.8S-ITS2. The V4 and V9 regions of gene 18S rRNA are relatively conservative fragments which are not suitable for species identification. The ITS2 can be used as a "short barcode". Among the advanced machine methods for delimitation species, the most effective algorithm for distinguishing Coelastrella species was the Generalized Mixed Yule Coalescent (GMYC) method. This paper represented for the first time our comprehensive review of the works devoted to the analysis of the biotechnological potential of representatives of the genus Coelastrella and shows that fatty acid composition of the three main chemogroups within the studied genus differs. In the future, this may form the basis for predicting the composition of the fatty acid profile of new strains, which is important while searching for organisms with specified biotechnological properties. In conclusion, an integrative approach was employed to describe Coelastrella affinis sp. nov., a new species of the genus Coelastrella with high biotechnological potential. Also, a new description of C. thermophila var. astaxanthina comb. nov. was proposed.

Microalgae are the richest source of natural carotenoids—accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis—gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Microalgal biotechnology is a promising solution for bioremoval of nutrients, especially phosphorus (P). Microalgae can accumulate large quantities of P and the P-enriched algal biomass can be used as bio-fertilizer. Immobilization of microalgae on cheap, biocompatible, and biodegradable chitosan-based carriers facilitates the biomass harvesting, increases the stress resilience of their cells and improves the economic viability of the technology. Here, we demonstrated that immobilization of the green microalga Lobosphaera sp. IPPAS C-2047 on a chitosan-based carrier increased the bioremoval of P from the cultivation media. This effect was accompanied by the build-up of P-rich deposits in the cell wall and an expansion of the vacuolar compartment harboring the bulk P of the cell. An increase in the abundance of P-rich intracellular inclusions as compared to the cells of suspended culture was revealed by ultrastructure analysis and confirmed by energy-dispersive spectroscopy (EDS). The immobilization of the Lobosphaera sp. cells resulted in reorganization of their cell wall, increase in its electron density and thickness, probably due to the penetration of the chitosan nanoparticles into cells. This hypothesis was supported by the pattern of the changes in elemental composition of the Lobosphaera sp. cell wall which revealed, in addition to the anions (phosphate, nitrate, sulfate and chloride) adsorbed to the positively charged nanoparticles of chitosan, a characteristic presence of cationic counterions (potassium, sodium, magnesium, calcium). The presence of the putative chitosan particles was also noted in the vacuoles. Overall, the rearrangements in the cell of the Lobosphaera sp. triggered by its immobilization on chitosan seem to be among the factors increasing its cell quota for P accumulation and hence the removal efficiency for this nutrient as compared to the suspended cell.

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5′-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.

Photogeneration of hydrogen in microalgae is thought to be among the mechanisms increasing their resilience to stresses including those caused by nutrient deprivation by re-routing the flow of electrons and reducing power in the cell. Metabolism of phosphorus (P), an essential nutrient, and its reserve forms such as polyphosphate (PolyP), is affected by and plays a role in the responses to diverse stresses, too. However, the potential interplay of the capability of photogeneration of hydrogen and turnover of phosphorus-rich inclusions in stressed microalgae cells so far escaped the attention of researchers. Here, we present a quantitative ultrastructural view of the turnover of P-rich inclusions in the model microalga Chlamydomonas reinhardtii strains, the parent strain CC-425 and its hydEF-1 mutant lacking hydrogenase activity as a function of sulfur and oxygen availability in the medium. In addition to the electron microscopy cell image analysis of the studied strains, we followed the elemental composition of the inclusions in different (sub) compartments of the cells obtained with energy dispersive X-ray spectroscopy. The stress caused by sulfur deprivation and subsequent transition of the microalgae culture to anaerobic conditions declined the size of phosphorus-containing inclusions but increased their number in the parent strain. Overall, the accumulation of the phosphorus-rich inclusions in hydEF-1 mutant was much lower than in the fully functional parent strain regardless of the cultivation conditions. We believe that impaired hydrogenase activity and correspondingly reduced sink of electrons and reducing power in the mutant strain indirectly affects the turnover of P and its reserves in the cell. These effects were manifested by the changes in the abundance, morphology, and elemental composition of the P-containing inclusions. We hypothesized that the sulfur-deprivation stress increased the initiation of the biosynthesis of PolyP chains, but their elongation and hence the formation of large PolyP-containing inclusions was hindered by anaerobiosis.

Phosphorus (P) is an essential macronutrient central to the exchange and storage of energy and information in the cell. Due to its limited bioavailability, P often becomes a limiting nutrient in aquatic and terrestrial ecosystems hence the studies of responses to stress caused by P starvation cyanobacteria, the primary producers, are of considerable interest. Indeed, the availability of P is among main factors limiting diazotrophy in cyanobacteria. To gain a deeper understanding of the effect of P starvation on cyanobacteria in non-diazotrophic conditions, we studied a model system of two near—isogenic strains, Nostoc sp. PCC 7120 and Nostoc sp. PCC 7118 differ in their ability to form heterocysts. Specifically, we investigated the differences in the responses of these strains to P starvation by comparing their growth kinetics, photosynthetic pigment content, ultrastructural rearrangements of vegetative cells, and the expression profile of key genes of phosphorus metabolism. The tolerance of PCC 7120 to P starvation was higher than that of PCC 7118, which manifested itself in a higher growth rate, less profound ultrastructural changes (in particular, phycobilisomes as well as polyphosphate reserves were retained in the cells of PCC 7120). At the same time, the accumulation of cyanophycin, a depot of nitrogen and energy, increased several-fold in the cells of both strains during P starvation, but this increase was larger in PCC 7118 cells. Assumably, the increased resilience of the PCC 7120 to P starvation stems from its higher ability to accumulate intracellular reserves of P in the form of polyphosphates. Our findings suggest that the phenotypic differences between the strains Nostoc sp. PCC 7118 and Nostoc sp. PCC 7120 are not limited to the different ability to form heterocysts. A deeper understanding of the drivers of stress response phenotypic diversity in near-isogenic strains would require a comparative analysis of their whole-genome sequences.

The productivity of microalgal cultures and their resilience to unfavorable conditions is largely determined by the availability of mineral nutrients, particularly nitrogen. Nitrogen starvation is a strong stressor that induces a broad range of responses in microalgae at the cell and cell population (culture) levels. These responses such as lipogenesis and secondary carotenogenesis are widely used in biotechnology to obtain valuable secondary metabolites of microalgae. It was believed that microalgal cells lack specific structures that function as long-term nitrogen depot, but recent studies assigned this role to microcrystalline inclusions constituted by nitrogenous bases (most often guanine). It is also known that purine microcrystals are biophotonic structures widespread in nature. These structures modify the intensity and spectral composition of radiation in illuminated cells and tissues of living organisms. In this regard, we studied (i) the dynamics of the formation of guanine crystals in the cells of green carotenogenic microalgae from the genus Coelastrella (C. thermophila NAMSU CM1/23 and C. rubescens IPPAS C-2066) depending on the availability of nitrogen in the medium and (ii) the effect of the presence of these crystals on cell resilience to high light intensity. Nitrogen-rich crystal inclusions were accumulated in cells when the cultures reached the stationary growth phase in a medium with ample nitrate nitrogen. During rapid growth, these inclusions were hardly detectable in the cells and disappeared during cultivation in the absence of nitrogen. Optical polarization microscopy and Raman microspectroscopy demonstrated that these nitrogen-rich inclusions are birefringent microcrystals composed of guanine. C. thermophila cells harboring abundant guanine crystals showed resistance to short-term (10–15 min) exposure to high-intensity light (600 µmol PAR quanta/m2/s). The results obtained are discussed in the context of the multifaceted role of guanine crystals in the stress tolerance of microalgae.

Coal mining endangers the environment by contaminating of soil, surface, and ground water with coal mine drainage water (CMW) polluted by heavy metals. Microalgal cultures, hyper-accumulators of heavy metals, represent a promising solution for CMW biotreatment. A bottleneck of this approach is the availability of microalgal strains that combine a large capacity for heavy metal biocapture with a high resilience to their toxic effects. Biotopes contaminated with heavy metals are frequently inhabited by microalgae evolved to be resilient to heavy metal toxicity. Therefore, the autochthonous (locally isolated) microalgal strains are a priori considered to be superior for biotreatment of heavy metal-polluted waste streams. Still, strains from biocollections combine a high pollutant resilience with other biotechnologically important traits such as high productivity, high CO2 sequestration rate etc. Moreover, the strains available “off-the-shelf” would enable rapid development of bioprocesses. Here, we compared the efficiency of CMW biotreatment with autochthonous (isolated from the coal mine drainage sump) and allochthonous microalgae (from a geographically distant phosphate-polluted site). Both autochthonous strains and allochthonous strains turned to be interchangeable under our experimental conditions. Still, the autochthonous strains showed a higher capacity for sequestration of iron, zinc, and manganese, the specific pollutants of the studied CMW. It can be important when the duration of unattended exploitation of the CMW treatment facility is a priority or spikes of the heavy metal concentration in CMW are expected. Therefore, the “off-the-shelf” strains can be a plausible solution for rapid development of CMW treatment technologies from scratch (although screening for acute toxicity of CMW is imperative). On the other hand, locally isolated strains can offer distinct advantages and should be always considered if sufficient time and other resources are available for the development of microalgae-based process for CMW treatment.

Apart from their functions in the nervous system of animals, neurotransmitters operate as regulatory agents and signals in diverse kingdoms of life. Some neurotransmitters have recently been revealed to exert specific effects on microalgae, predominantly functioning as algal growth stimulators. This article presents new data on the effects of such neurotransmitters as serotonin, norepinephrine, dopamine, histamine, and acetylcholine on the fatty acid and pigment composition of the green microalga Scenedesmus quadricauda (Turp.) Breb. K-1149. It was established that acetylcholine and, to a lesser extent, histamine increased the total fatty acid content of S. quadricauda cells, whereas serotonin and dopamine decreased the fatty acid content. Acetylcholine, histamine, and norepinephrine elevated the percentage of polyunsaturated fatty acids; in contrast, serotonin and dopamine increased the share of saturated fatty acids. Acetylcholine and, to a lesser extent, norepinephrine increased the total chlorophyll content per gram of dry weight in S. quadricauda, while histamine decreased the chlorophyll content. Histamine also increased the chlorophyll a/chlorophyll b and carotenoid/chlorophyll ratios, which were decreased by dopamine. The data obtained are of biotechnological and ecological interest. The stimulation of fatty acid accumulation and the increase in the percentage of polyunsaturated species was caused by the neurotransmitters acetylcholine and histamine at low (1–10 μM) concentrations, which potentially enables facilitating the biotechnological production of health-promoting preparations for therapeutic and cosmetic purposes. However, other neurotransmitters (dopamine and serotonin) tested increased the relative content of saturated fatty acids; therefore, they apparently can be used to stimulate biofuel production, since saturated fatty acid-rich lipids are advantageous raw materials for biodiesel production. The impact of neurotransmitters on microalgal fatty acid composition and photosystem components may be considered in terms of ongoing chemical interaction between microalgae and other aquatic ecosystem components that are known to produce neurotransmitters.

Green microalgae, including those from the genus Lobosphaera, are exploited in various fields of biotechnology to obtain valuable fatty acids (e.g., arachidonic acid (C20:4, ARA)) for the production of infant formulae, food and feed additives. In nature, microalgae frequently exist in naturally immobilized state (as biofilms) with a limited cell division rate and increased stress resilience. In the fields of biotechnology, immobilization of microalgae on artificial cell carriers simplifies biomass harvesting and increases culture robustness and productivity. The choice of a suitable cell carrier is central to biotechnology involving immobilized cultures. Cell carriers based on the natural amine-containing polymer chitosan and synthetic polyethylenimine (PEI) are promising candidates for immobilization of phototrophic microorganisms. This is the first report on the effects of immobilization on PEI and chitosan on the accumulation and composition of polyunsaturated fatty acids, including ARA, in Lobosphaera sp. IPPAS C-2047. Immobilization on PEI increased the ARA percentage in the total fatty acids and ARA accumulation by 72% and 81% compared to the suspended cells cultured in complete or nitrogen-deprived medium 14 days, respectively. Immobilization of Lobosphaera sp. on the chitosan-based carrier reduced the ARA percentage but increased oleic and α-linoleic acid percentages. The mechanisms of the effects of immobilization on the fatty acid profiles of the microalgae are discussed.

The effect of nitrogen starvation and, for the first time, low temperature, as well as their simultaneous effect, on the physiology and ultrastructure of cells of microalgae of the genus Lobosphaera (Chlorophyta, Trebouxiophyceae) was studied. Nitrogen deficiency in both strains led to a decrease in the content of chlorophyll by three times and an increase in the proportion of carotenoids by two times. A decrease in the content of both chlorophyll and carotenoids was observed at +10°C. The simultaneous effect of two factors resulted in a threefold decrease in the chlorophyll content in NAMSU 924/2 and a sixfold decrease in NAMSU (CALU) 1497; the proportion of carotenoids in both strains decreased by 1.5–2 times. Data on ultrastructural changes in cells of microalgae of the genus Lobosphaera under the influence of stress factors have been obtained. A similar nature of the response in both strains to stress conditions was noted. Nitrogen deficiency led to the accumulation of numerous lipid droplets in the cytoplasm of cells along the cell wall. Long-term incubation on a nitrogen-free medium led to the filling of the entire volume of cells with lipid droplets, disassembly of the membrane system of chloroplasts, that reduction in sizeand being located between densely lying lipid droplets. At low temperatures, the number of thylakoids decreased, while the interthylakoid space and the size of chloroplasts increased. With simultaneous exposure to nitrogen starvation and low temperature, numerous lipid droplets accumulated, the number of thylakoids decreased, the interthylakoid space and the size of the chloroplast increased, which was noted under separate exposure to stress factors. The pyrenoid in both strains did not undergo significant changes in all cases.

Microalgae are naturally adapted to the fluctuating availability of phosphorus (P) to opportunistically uptake large amounts of inorganic phosphate (P-i) and safely store it in the cell as polyphosphate. Hence, many microalgal species are remarkably resilient to high concentrations of external P-i. Here, we report on an exception from this pattern comprised by a failure of the high P-i-resilience in strain Micractinium simplicissimum IPPAS C-2056 normally coping with very high P-i concentrations. This phenomenon occurred after the abrupt re-supplementation of P-i to the M. simplicissimum culture pre-starved of P. This was the case even if P-i was re-supplemented in a concentration far below the level toxic to the P-sufficient culture. We hypothesize that this effect can be mediated by a rapid formation of the potentially toxic short-chain polyphosphate following the mass influx of P-i into the P-starved cell. A possible reason for this is that the preceding P starvation impairs the capacity of the cell to convert the newly absorbed P-i into a "safe" storage form of long-chain polyphosphate. We believe that the findings of this study can help to avoid sudden culture crashes, and they are also of potential significance for the development of algae-based technologies for the efficient bioremoval of P from P-rich waste streams.

Due to their valuable properties, nanoparticles are in demand in industry, medicine, and ecology. Traditional physical-chemical synthesis methods of nanoparticles are energy-consuming and/or are carried out with the participation of toxic substances that limit their further use, e.g., in medicine. In this regard, attention is drawn to the “green” synthesis of nanoparticles including with the participation of microalgae cells. The ability of cells of the green microalgae Micractinium simplicissimum IPPAS C-2056 to form ultra-small Fe (1–4 nm) and P (3–9 nm) nanoparticles, Mn (10–60 nm) nanoparticles as well as mixed (P–Fe, P‒Mn, Fe–Mn, Fe–P–Ca–Mg) nanoparticles, which vary in composition, localized in the intercellular matrix, on the surface, and in the cell wall is shown by transmission electron microscopy combined with energy-dispersive X-ray spectroscopy. The obtained data evidence a high potential of M. simplicissimum IPPAS C2056 for the green synthesis of nanoparticles containing Mn, Fe, and P. The determinants of this ability and prospects for using microalgae to produce nanoparticles with valuable properties are discussed.

Broad application of CuO nanoparticles (CuO-NP) for industrial and household purposes leads to a continuous increase in their discharge to, and, hence, ever-increasing environmental hazards for aquatic ecosystems. Microalgae-based technologies hold promise for bioremediation of diverse hazardous micropollutants (HMP), including NP, from wastewater. In this study, we tested the ability of the green microalga Desmodesmus sp. to accumulate CuO-NP or their components. We also assessed the tolerance of this microalga to the environmentally relevant concentrations of CuO-NP. Using scanning electron microscopy, we demonstrated that the average size of CuO-NP was 50–100 nm, and their purity was confirmed with elemental composition analysis. Tests of the colloidal suspensions of CuO-NP showed that the hydrodynamic diameter of CuO-NP and their aggregates was below 100 nm. Flow cytometry analysis showed that CuO-NP at a concentration of 100 µg L−1 slightly inhibited the viability of microalgae cells and led to an increase in their oxidative stress. The assessment of the condition of photosystem II showed that CuO-NP exert a multifaceted effect on the photosynthetic apparatus of Desmodesmus sp., depending on the concentration of and the exposure to the CuO-NP. Desmodesmus sp. turned to be relatively tolerant to CuO-NP. In addition, the ICP-MS method revealed increased bioaccumulation of copper by microalgae cells in the experimental groups. The outcomes of this study indicate that the Desmodesmus sp. has a significant potential for bioremoval of the copper-based nanostructured HMP from an aquatic environment.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.