Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae.

Microalgae, capable of accumulating large amounts of lipids, are of great value for biodiesel production. The high cost of such production stimulates the search for cultivation conditions that ensure their highest productivity. Reducing the content of nitrogen and phosphorus in the culture medium is widely used to change the content and productivity of lipids in microalgae. Achieving the right balance between maximum growth and maximum lipid content and productivity is the primary goal of many experimental works to ensure cost-effective biodiesel production from microalgae. The content of nitrogen and phosphorus in nutrient media for algal cultivation after converted to nitrogen (-N) and phosphorus (-P) lies in an extensive range: from 0.007 g L- 1 to 0.417 g L- 1 and from 0.0003 g L- 1 to 0.227 g L- 1 and N:P ratio from 0.12:1 to 823.33:1. When studying nutritional stress in microalgae, no single approach is used to determine the experimental concentrations of nitrogen and phosphorus. This precludes the possibility of correct interpretation of the data and may lead to erroneous conclusions. This work results from the systematisation of information on using nitrogen and phosphorus restriction to increase the lipid productivity of microalgae of different taxonomic and ecological groups to identify future research directions. The results of 301 experiments were included in the analysis using the principal components method. The investigation considered various divisions and classes: Cyanobacteria, Rhodophyta, Dinophyta, Haptophyta, Cryptophyta, Heterokontophyta/Ochrophyta (Bacillariophyceae, Eustigmatophyceae, Xanthophyceae), Chlorophyta, and also the ratio N:P, the time of the experiment, the light intensity during cultivation. Based on the concentrations of nitrogen and phosphorus existing in various nutrient media, a general scheme for designating the supply of nutrient media for nitrogen (as NO3- or NH4+, N g L- 1) and phosphorus (as РO4-, P g L- 1) has been proposed: replete -N (˃0.4 g L- 1), moderate -N (0.4-0.2), moderate N-limitation (0.19-0.1), strong N-limitation (˂0.1), without nitrogen (0), replete -Р (˃0.2), moderate -P (0.2-0.02), moderate P-limitation (0.019-0.01), strong P-limitation (˂0.01), without phosphorus (0).

Antioxidant Status and Biotechnological Potential of New Vischeria vischeri (Eustigmatophyceae) Soil Strains in Enrichment Cultures.

The functional state of enrichment cultures of the Eustigmatophycean strains Vischeria vischeri MZ-E3 and MZ-E4 after 25-day cultivation in the BBM medium was studied. The concentrations of chlorophyll a, total carotenoids, protein, vitamins A and E, fatty acid peroxidation product content, an antioxidant enzyme, and succinate dehydrogenase activity were measured. MZ-E3 succinate dehydrogenase activity was significantly higher by 2.21 times; the MZ-E4 strain had 2.94 times higher glutathione peroxidase activity. The MZ-E3 antioxidant activity index and the MZ-E3 unsaturation of fatty acids were 1.3 and 1.25 times higher than the MZ-E4. The retinol and α-tocopherol content of the MZ-E3 was 28.6% and 38.76% higher than MZ-E4. The main fatty acid profile differences were the 3.46-fold and 3.92-fold higher stearic and eicosapentaenoic acid content in the MZ-E4 biomass. MZ-E3 had higher antioxidant, energy, and metabolic and photosynthetic status than MZ-E4. The antioxidant status of the studied strains showed the dependence of the adaptive mechanisms of each, associated with differences in the ecological conditions of the biotopes from which they were isolated. These strains are promising for producing α-tocopherol and biomass enriched with omega-3 and omega-6 fatty acids.

A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool for Studying the Structure and Activity of the Nucleolus in Drosophila melanogaster.

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction.

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.

Phylogeny and fatty acid profiles of Aliinostoc vietnamicum sp. nov. (cyanobacteria) from the soils of Vietnam.

A new cyanobacterial species of Aliinostoc, A. vietnamicum sp. nov., is recorded in the tropical forest soil from the Cát Tiên National Park, Vietnam. The analysis is based on morphological characters, 16S rDNA phylogeny, ITS secondary structure, and fatty acid composition analysis. Aliinostoc vietnamicum differed from the other species of the genus by the size and shape of vegetative cells, size of akinetes and heterocytes, and presence of granular polyphosphate inclusions in vegetative cells. The evolutionary distance matrix based on the 16S rRNA gene shared 96.2-98.2% similarities with other Aliinostoc sequences. The phylogeny inferred by maximum likelihood and Bayesian inference placed A. vietnamicum in the Aliinostoc clade, within the Nostocaceae. For the first time, fatty acid composition analysis was obtained for a member of the genus Aliinostoc with cultivation time experiments. α-linolenic (27.54-37.75%), palmitic (13.87-22.65%), and stearic (10.08-20.27%) acids were the dominant fatty acids when cultured during the exponential growth phase, as well as during stationary. This is the first finding of a strain with such a high content of stearic acid among cyanobacteria with Nostoc-like morphology.

Optimization of Biomass Accumulation and Production of Phenolic Compounds in Callus Cultures of Rhodiola rosea L. Using Design of Experiments.

Rhodiola rosea L. is a valuable medicinal plant with adaptogenic, neuroprotective, antitumor, cardioprotective, and antidepressant effects. In this study, design of experiments methodology was employed to analyze and optimize the interacting effects of mineral compounds (concentration of NO3- and the ratio of NH4+ to K+) and two plant growth regulators [total 6-benzylaminopurine (BAP) and α-naphthylacetic acid (NAA) concentration and the ratio of BAP to NAA] on the growth and the production of total phenolic compounds (TPCs) in R. rosea calluses. The overall effect of the model was highly significant (p < 0.0001), indicating that NH4+, K+, NO3-, BAP, and NAA significantly affected growth. The best callus growth (703%) and the highest production of TPCs (75.17 mg/g) were achieved at an NH4+/K+ ratio of 0.33 and BAP/NAA of 0.33, provided that the concentration of plant growth regulators was 30 µM and that of NO3- was ≤40 mM. According to high-performance liquid chromatography analyses of aerial parts (leaves and stems), in vitro seedlings and callus cultures of R. rosea contain no detectable rosarin, rosavin, rosin, and cinnamyl alcohol. This is the first report on the creation of an experiment for the significant improvement of biomass accumulation and TPC production in callus cultures of R. rosea.

Diversity of Cyanobacteria and Algae During Primary Succession in Iron Ore Tailing Dumps.

The extraction of commercial minerals is often accompanied by the formation of large areas of quarry and dump technogenic ecosystems. This stimulates the search for measures to reduce their negative impact on the environment, as well as a detailed study of all the constituent elements of ecosystems that spontaneously or after reclamation form on them. Primary stages of syngenesis on the tailing dumps of iron ore mines in Kryvorizhzhia took place involving cyanobacteria and eukaryotic algae. The dynamics of the community structures of algae and cyanobacteria depends on the mineralogical composition of tailings, salinity conditions, pH, and content of particles of physical clay and humus. The assessment of the features of the dynamics of algae communities was carried out based on the ordination procedure because of the method of non-metric multidimensional scaling. The considered environmental variables were statistically significant predictors of community structure and could explain 47-90% of the variation in measurements. Diagnostic signs of the species composition of communities of algae and cyanobacteria tailing dumps were established by physical and chemical parameters. An increase in the content of physical clay particles in the substrate and a pH shift towards the alkaline side increased the species richness of cyanobacteria, while an increase in the humus content increased the total species diversity. Based on the specificity of the type of growth and the species composition of algae communities, when describing the primary successions, it is proposed to allocate the following stages of development of algae and cyanobacteria communities: dispersal aerophyton, stratose epilitophyton, algal crust, mixed moss and algal crust, and edaphone.

Influence of Light Conditions on Microalgae Growth and Content of Lipids, Carotenoids, and Fatty Acid Composition.

Microalgae are a valuable natural resource for a variety of value-added products. The growth of microalgae is determined by the impact of many factors, but, from the point of view of the implementation of autotrophic growth, light is of primary importance. This work presents an overview of the influence of light conditions on the growth of microalgae, the content of lipids, carotenoids, and the composition of fatty acids in their biomass, taking into account parameters such as the intensity, duration of lighting, and use of rays of different spectral composition. The optimal light intensity for the growth of microalgae lies in the following range: 26-400 µmol photons m-2 s-1. An increase in light intensity leads to an activation of lipid synthesis. For maximum lipid productivity, various microalgae species and strains need lighting of different intensities: from 60 to 700 µmol photons m-2 s-1. Strong light preferentially increases the triacylglyceride content. The intensity of lighting has a regulating effect on the synthesis of fatty acids, carotenoids, including ß-carotene, lutein and astaxanthin. In intense lighting conditions, saturated fatty acids usually accumulate, as well as monounsaturated ones, and the number of polyunsaturated fatty acids decreases. Red as well as blue LED lighting improves the biomass productivity of microalgae of various taxonomic groups. Changing the duration of the photoperiod, the use of pulsed light can stimulate microalgae growth, the production of lipids, and carotenoids. The simultaneous use of light and other stresses contributes to a stronger effect on the productivity of algae.

Lipid accumulation by Coelastrella multistriata (Scenedesmaceae, Sphaeropleales) during nitrogen and phosphorus starvation.

A novel freshwater strain of Coelastrella multistriata MZ-Ch23 was discovered in Tula region, Russia. The identification is based on morphological features, phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis places the novel strain in the "core" Coelastrella clade within the Chlorophyceae. This is the first record of Coelastrella multistriata in the algal flora of Russia. Cultivation experiments were carried out to evaluate growth dynamics of the newly identified strain and the impact of nitrogen and/or phosphorus depletion on the fatty acid profiles and lipid productivity. On the fully supplemented Bold's basal medium and under phosphorus-depleted conditions as well, the fatty acid profiles were dominated by α-linolenic acid (29.4-38.1% of total fatty acids). Depletion of either nitrogen or both nitrogen and phosphorus was associated with increased content of oleic acid (32.9-33.7%) and linoleic acid (11.9%). Prolongation of the growth to two months (instead of 25 days) resulted in increased content and diversity of very long-chain fatty acids including saturated species. The total very long-chain fatty acid content of 9.99% achieved in these experiments was 1.9-12.3-fold higher than in stress experiments. The highest variation was observed for oleic acid (3.4-33.7%). The novel strain showed the ability to accumulate lipids in amounts up to 639.8 mg L-1 under nitrogen and phosphorus starvation, which exceeds the previously obtained values for most Coelastrella strains. Thus, the newly identified MZ-Ch23 strain can be considered as a potential producer of omega-3 fatty acids on fully supplemented Bold's basal medium or as a source of biomass with high content of saturated and monounsaturated fatty acids after nitrogen and phosphorus starvation.

Differential Zn and Mn sensitivity of microalgae species from genera Bracteacoccus and Lobosphaera.

One of the most common pollutants in natural ecosystems is heavy metals. Algae are sensitive to the action of heavy metals. This allows to use algae to assess the toxicity of heavy metals, bioindication, and during phycoremediation. This study examines the effect of different Zn and Mn concentrations (1.0, 5.0, 25.0, 50.0, 500.0, 1000.0 mg L-1) on green algae Bracteacoccus minor and Lobosphaera incisa in a chronic bioassay. The results of this study showed that the toxic effect of Zn and Mn on B. minor and L. incisa begins to manifest itself at the lowest of the studied metal concentrations-1 mg L-1. The critical concentration of Zn, which leads to the complete death of B. minor and L. incisa, is 50.0 and 500.0 mg L-1, and Mn is 1000.0 mg L-1 and 500.0 mg L-1, respectively. It was found that principal component (PC) 1 accounts for 60.47% of the total variance and reflects changes associated with low concentrations of heavy metals (up to 5.0 mg L-1). PC2 accounts for 27.95% of the total variance. PC2 is mostly associated with high concentrations of ions of heavy metals. Thus, the effect of Zn and Mn concentrations up to 5 mg L-1and above 50 mg L-1on B. minor and L. incisa has a different character. At the same time, the response of the studied algae species to the action of Zn and Mn has individual differences. In general, B. minor is more resistant to Mn, while L. incisa is more resistant to Zn.

Biogeography of the cosmopolitan terrestrial diatom Hantzschia amphioxys sensu lato based on molecular and morphological data.

Until now, the reported diversity of representatives from the genus Hantzschia inhabiting soils from different parts of Eurasia was limited to the few species H. amphioxys, H. elongata and H. vivax and some of their infraspecific taxa. We have studied the morphology, ultrastructure and phylogeny of 25 soil diatom strains, which according to published description would be assigned to "H. amphioxys sensu lato" using 18S rDNA, 28S rDNA and rbcL. We show that strains are made up of seven different species of Hantzschia, including five new for science. Five strains were identified as H. abundans. This species has a slight curvature of the raphe near its external proximal ends. Four of the examined strains were represented by different populations of H. amphioxys and their morphological characteristics fully correspond with its isolectotype and epitype. The main specific features of this species include 21-25 striae in 10 µm, 6-11 fibulae in 10 µm, 40-50 areolae in 10 µm and internal proximal raphe endings bent in opposite directions. H. attractiva sp. nov., H. belgica sp. nov., H. parva sp. nov., H. pseudomongolica sp. nov. and H. stepposa sp. nov. were described based on differences in the shape of the valves, significant differences in dimensions, a lower number of striae and areolae in 10 µm and the degree and direction of deflection of the internal central raphe endings. Based on the study of the morphological variability and phylogeny of soil Hantzschia-species from different geographical locations we conclude that while some species such as H. amphioxys are truly cosmopolitan in their distributions, some sympatric populations of pseudocryptic taxa exist in the Holarctic.

A New Species of Freshwater Algae Nephrochlamys yushanlensis sp. nov. (Selenastraceae, Sphaeropleales) and Its Lipid Accumulation during Nitrogen and Phosphorus Starvation.

The new species Nephrochlamys yushanlensis sp. nov. is described from a freshwater plankton sample. A comparison of morphology, 18S rDNA gene and ITS2 sequences, and fatty acid profiles showed that the novel strain represents a new lineage within the genus Nephrochlamys. For the first time with a member of the Selenastraceae, experiments with phosphate and nitrate deprivation were conducted to evaluate changes in biomass, lipid and triacylglycerol (TAGs) accumulation, and composition of fatty acids. Biomass dry weight under simultaneous nitrogen and phosphorus depletion was 1.73 g · L-1 , which is significantly lower than the 2.41 g · L-1 observed in the control. All conditions of nutrient restriction significantly increased the lipid content in comparison with the control. The largest increase in the total lipid content, reaching 58.64% DW per cell at the end of cultivation, occurred with nitrogen deficiency. Significant increases in TAGs content (to 23.69% and 21.74%, respectively) occurred in phosphorus- and nitrogen-depleted conditions in comparison to the control (16.90%). Oleic (49.8-64.1%), palmitic (21.1-22.7%), and linoleic (8.6-10.3%) acids were the dominant fatty acids when cultured on standard BBM medium, as well as with the shortage of nutrients. Phosphorus deprivation as well as absence of both nitrogen and phosphorus led to the appearance of FAMEs α-linolenic (1.5-4.1%) and stearidonic (1.0-1.8%) acids. In general, FAME profiles revealed that the relative percentage of saturated and monounsaturated fatty acids increased (88.9% of total fatty acids) in nitrogen-depletion conditions, suggesting this strain may be suitable for biodiesel production.

Investigation of Chemical Constituents of Eranthis longistipitata (Ranunculaceae): Coumarins and Furochromones.

Aqueous-ethanol extracts (70%) from the leaves of Eranthis longistipitata Regel. (Ranunculaceae Juss.)-collected from natural populations of Kyrgyzstan-were studied by liquid chromatography with high-resolution mass spectrometry (LC-HRMS). There was no variation of the metabolic profiles among plants that were collected from different populations. More than 160 compounds were found in the leaves, of which 72 were identified to the class level and 58 to the individual-compound level. The class of flavonoids proved to be the most widely represented (19 compounds), including six aglycones [quercetin, kaempferol, aromadendrin, 6-methoxytaxifolin, phloretin, and (+)-catechin] and mono- and diglycosides (the other 13 compounds). In the analyzed samples of E. longistipitata, 14 fatty acid-related compounds were identified, but coumarins and furochromones that were found in E. longistipitata were the most interesting result; furochromones khelloside, khellin, visnagin, and cimifugin were found in E. longistipitata for the first time. Coumarins 5,7-dihydroxy-4-methylcoumarin, scoparone, fraxetin, and luvangetin and furochromones methoxsalen, 5-O-methylvisammioside, and visamminol-3'-O-glucoside were detected for the first time in the genus Eranthis Salisb. For all the above compounds, the structural formulas are given. Furthermore, detailed information (with structural formulas) is provided on the diversity of chromones and furochromones in other representatives of Eranthis. The presence of chromones in plants of the genus Eranthis confirms its closeness to the genus Actaea L. because chromones are synthesized by normal physiological processes only in these members of the Ranunculaceae family.

Immunohistochemical analysis of adiponectin in atherosclerotic lesions of human aorta.

BACKGROUND: Metabolic syndrome, a cluster of interrelated disorders including abdominal obesity, insulin resistance, dyslipidemia, and hypertension (HTN) plays an important role in development of atherosclerotic lesions in arterial wall. Dysregulation of adipose tissue hormones (adipokines) production is a possible link between abdominal obesity and other manifestations of metabolic syndrome. Adiponectin is a well-known adipokine which affects metabolism and inflammatory response. However, data on its role in atherogenesis are still controversial. The aim of this study is to investigate whether adiponectin is present in atherosclerotic lesions of human aorta. METHODS: Thirty-five autopsy segments from abdominal, thoracic aortas, and aortic arch of four men (mean age: 57 years) were fixed and stained for lipids [Oil Red O (ORO)], cells [hematoxylin-eosin (H&E)], and adiponectin [indirect immunoperoxidase assay (IPA) method]. Samples of both stable and unstable plaques were selected for analysis. Human adipose tissue, THP-1 monocytes/macrophages, and human endothelial hybrid cell line (EA.hy926) were chosen for detection of adiponectin messenger ribonucleic acid (mRNA) using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Adiponectin accumulations were found inside endothelial cells covering both stable and unstable atherosclerotic plaques. Focal depositions of adiponectin were also found in fibrous caps of stable lesions and atheromatous core of both stable and unstable plaques and also in adventitia. RT-PCR revealed mRNA expression of adiponectin gene in adipose tissue, but not in mononuclears and endothelial cells. CONCLUSION: Adiponectin is present in aortic plaques of humans, but is not synthesized in endothelial cells and mononuclears, at least in culture conditions. Detection of adiponectin in atherosclerotic lesions can serve as indirect evidence of possible participation of this adipokine in atherogenesis.

Fatty Acid Content and Profile of the Novel Strain of Coccomyxa elongata (Trebouxiophyceae, Chlorophyta) Cultivated at Reduced Nitrogen and Phosphorus Concentrations.

A novel freshwater strain of Coccomyxa elongata (MZ-Ch64) was isolated from the Zaporizhia region, Ukraine. The identification was based on the phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis placed this strain in the Coccomyxa group, within the class Trebouxiophyceae. The novel strain MZ-Ch64 formed a strongly supported lineage closest with C. elongata. The MZ-Ch64 strain differed from the morphological description of the species by the size of vegetative cells and absence of small mucilaginous caps at one end of the cell. A number of experiments with different concentrations of phosphate and nitrate were conducted to evaluate changes in the resulting fatty acid profiles and biomass productivity. The fatty acid profile and total fatty acids varied significantly under different nutrient deficiencies. The dominant fatty acid during cultivation on standard BBM medium, as well as in phosphorus-depleted conditions, was oleic acid (to 48.0%-54.6% of total fatty acids). Absence of nitrogen alone, and absence of both nitrogen and phosphorus, led to an increase of palmitic acid (to 24.7%-25.6%), cis-7-hexadecenoic acid (to 14.8%) and α-linolenic acid (to 9.1%-10.1%) in comparison with the control sample. The greatest variation was found for oleic acid (31.9%-54.6%). Thus, this strain can be considered as a potential producer of oleic acid or cis-7-hexadecenoic and α-linolenic acids for biotechnological applications.