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Abstract As a result of human activities, surface waters worldwide are experiencing increasing levels of eutrophication, leading to more frequent occurrences of microalgae, including harmful algal blooms. We aimed to investigate the impact of decomposing camelina straw on mixed phytoplankton communities from eutrophic water bodies, comparing it to the effects of barley straw. The research was carried out in 15 aquaria (five of each type – containing no straw, camelina straw, and barley straw). The experiment lasted eight weeks, and the water used in the aquaria was sourced from an eutrophic reservoir. Our research revealed that the camelina straw had the most significant inhibitory effect on the growth of specific groups and species of phytoplankton (especially chrysophytes, potentially toxic cyanobacteria, and dinoflagellates). This inhibition was achieved by releasing polyphenols, primarily gallic and caffeic acids and flavonoids. Simultaneously, polyphenols promoted the growth of filamentous green algae. Our findings present novel data on the vulnerability of freshwater species and taxonomic groups of algae to the effects of camelina and barley straw exposure.

The recognition and use of algae in the very trend-driven cosmetic industry is progressively increasing. Up to now, the main focus was on large seaweeds and a limited number of microalgae. However, motile microalgae, flagellates, remain underscored in this aspect, although some of them are utilized commercially. Flagellates from different taxonomic groups occupy various habitats and contain bioactive high-value multifunctional compounds, some of which are novel. Moreover, they may simultaneously produce different substances, which together with the development of downstream processing technologies, makes them a promising source for modern biotechnology. The present review covers data on 411 strains, 251 species from 110 genera from 6 phyla, and is oriented generally towards less explored flagellates. It demonstrates their great potential as bearers of interesting novel compounds that can be beneficially applied in modern cosmetics. Safety aspects of both sources and products are also discussed. Considering the gaps in the knowledge, the necessity to expand the research on both well-known and yet unexplored microalgae is shown, encouraging the development of upstreaming processes, including phycoprospecting. Last but not least, this paper outlines the role of living culture collections and of using good taxonomic expertise before running the biochemical tests, cultivation, and bioengineering experiments.

This paper presents the first data on the biodiversity of lithophytic algae from Bulgarian megaliths obtained after the application of the direct sampling method, subsequent cultivation, and processing by light microscopy. A rich algal flora was found: 90 species and 1 variety of 65 genera from Cyanoprokaryota/Cyanobacteria (29 species, 13 genera), Chlorophyta (40 species and 1 variety, 38 genera), Streptophyta (5 species, 1 genus), and Ochrophyta (16 species, 13 genera). Among them were the globally rare Pseudodictyochloris multinucleata (Chlorophyta), found for the first time in such lowland and warm habitats, and Scotiella tuberculata (Chlorophyta), for which this is the first finding in the country. Three of the recorded species are conservationally important. The low floristic similarity between the sites (0–33%) shows the diversity of the algal flora, with no common species found for all the megaliths studied. The most widespread were the strongly adaptive and competitive Stichococcus bacillaris, Apatococcus lobatus, and Chloroidium ellipsoidium (Chlorophyta). The correlations estimated between the species number and substrate temperature (18.1–49.6 °C) suggest the prospect of future research related to the impact of global warming. In addition, the study points to the safety aspects as it revealed species from nine potentially toxin-producing cyanoprokaryotic genera that could be harmful to visitors’ health.

This study aims at improving the existing algal-based wastewater treatment technologies by overcoming some of the major drawbacks of these systems such as large required land area, culture contamination, and energy-intensive algal harvesting. The experiments were carried out in an open photo-sequencing batch reactor at a laboratory-scale for nearly 2 months. A specific strain ACUS00207 of the aeroterrestrial green microalga Klebsormidium nitens (Kützing) Lokhorst was used. The strain is native to Bulgaria and belongs to a species that has never been used before in suspended growth systems for wastewater treatment for phosphorus removal. The culture of K. nitens showed promising results: phosphorus removal rates ranging from 0.4 to 1 mg total phosphorus L−1 d−1, efficient settling properties, and resistance to culture contamination with native microalgae. On the basis of the observed phosphorus removal mechanism of biologically mediated chemical precipitation/phosphorus precipitation, an innovative working mode of the sequencing batch reactor is suggested for reducing the hydraulic retention time and the required land area. HIGHLIGHTS Klebsormidium nitens used for the first time in suspended growth wastewater treatment.; Phosphorus removal rates ranging from 0.4 to 1 mg total phosphorus L−1 d−1.; Efficient settling properties and resistance to culture contamination.; Potential for reduction of the hydraulic retention time and the required land area.;

Cyanotoxins (CTs) are a large and diverse group of toxins produced by the peculiar photosynthetic prokaryotes of the domain Cyanoprokaryota. Toxin-producing aquatic cyanoprokaryotes can develop in mass, causing “water blooms” or “cyanoblooms,” which may lead to environmental disaster—water poisoning, extinction of aquatic life, and even to human death. CT studies on single cells and cells in culture are an important stage of toxicological studies with increasing impact for their further use for scientific and clinical purposes, and for policies of environmental protection. The higher cost of animal use and continuous resistance to the use of animals for scientific and toxicological studies lead to a progressive increase of cell lines use. This review aims to present (1) the important results of the effects of CT on human and animal cell lines, (2) the methods and concentrations used to obtain these results, (3) the studied cell lines and their tissues of origin, and (4) the intracellular targets of CT. CTs reviewed are presented in alphabetical order as follows: aeruginosins, anatoxins, BMAA (β-N-methylamino-L-alanine), cylindrospermopsins, depsipeptides, lipopolysaccharides, lyngbyatoxins, microcystins, nodularins, cyanobacterial retinoids, and saxitoxins. The presence of all these data in a review allows in one look to advance the research on CT using cell cultures by facilitating the selection of the most appropriate methods, conditions, and cell lines for future toxicological, pharmacological, and physiological studies.

Nowadays, the flourishing development of modern cosmetics, and of “green cosmetics” especially, leads to rapid product innovation, with the increasing use of novel natural ingredients from unusual sources. A peculiar group of slime molds that have long been controversially classified as plants, fungi, or animals represents such an uncommon resource. In this regard, it is strange that these fascinating low-eukaryotic organisms are conspicuously absent from the current reviews of natural cosmetic sources and have no industrial cosmetics utilization. Chemical analyses have confirmed that the slime molds produce a plethora of novel or rare secondary metabolites of interest for cosmetics (127 substances), many of which exhibit biological activity. Interestingly, novel compounds were isolated from 72% of the 53 checked species. At the same time, the number of studied species, from a total of more than 900 currently recognized, is strikingly low (0.06). Such great unexplored biodiversity leaves a space wide open for new discoveries, presenting the slime molds as a reservoir of new biologically active substances that may provide valuable natural ingredients (pigments, lipids, aromatic substances, etc.) for application in modern cosmetics. Therefore, the current review aims to provoke a stronger interest in this neglected aspect, outlining the knowledge that has been obtained so far and indicating some challenges and perspectives for the future.

This paper presents results from the first application of molecular-genetic analysis combined with conventional light microscopy (LM) for identification of cylindrospermopsin (CYN) producers in field phytoplankton samples from Bulgaria. In total 68 cyanoprokaryotes were found by LM, out of which 18 were considered potential CYN-producers according to the literature. They occurred in different abundance (0-40%) in five waterbodies. The presence of CYN-producers was sought by application of the primer pair cynsulfF/cylnamR specific for the sulfotransferase gene (cyrJ) considered the best genetic marker for detection of CYN-toxigenic strains. Although CYN was detected by enzyme-linked immunosorbent assay (ELISA) in two waterbodies (lake Vaya and reservoir Mandra), we did not find cyanoprokaryotes with cyrJ gene. Therefore, Raphidiopsis raciborskii, despite being identified by LM in both waterbodies where CYN occurred, was not the producer of this toxin and belonged to the European nontoxic population of the species. The same was true for the less widely spread Raphidiopsis mediterranea and Chrysosporum bergii (both found in small amounts only in lake Vaya). Our results on CYN occurrence without the assignment of its producer in the collected samples suggest that: 1) CYN remains stable in water after disappearance of the toxigenic species, or, more probably, 2) besides the already known species, there are others that are associated with the production of CYN. Because revealing of novel CYN producers is of ultimate importance for health risk assessment and water management, we propose more analyses to be performed in future studies.

The paper presents the first proof of planktonic cyanoprokaryote genus Cuspidothrix as an anatoxin A (ATX) producer in Bulgarian wetlands. The results from polymerase chain reaction (PCR) obtained from two summer sampling campaigns in 26 selected lakes and reservoirs demonstrated presence of the anaC gene, responsible for ATX production in 21 strains of the genus. They were found in three waterbodies sampled in 2018 (coastal lake Vaya, coastal reservoir Poroy, inland reservoir Sinyata Reka) and in four waterbodies sampled in 2019 (inland reservoirs Duvanli, Koprinka, Plachidol 2, Sinyata Reka). The detected genetic diversity generally corresponds to the observations conducted by conventional light microscopy, by which we distinguished three species of Cuspidothrix (Cuspidothrix issatschenkoi, Cuspidothrix elenkinii and Cuspidothrix tropicalis, the latter considered alien in the country). Eleven strains showed high similarity to two sequences of C. issatschenkoi available from the National Centre for Biotechnology Information (NCBI). Ten other strains assembled in a group, which—in lack of available from NCBI genetic sequences—were presumed related to C. tropicalis and C. elenkinii after comparison with the results from light microscopy. Cuspidothrix strains found in Bulgarian waterbodies showed high genetic similarity to those isolated and sequenced from Asia (Japan, China) and Northern Europe (Norway, Finland).

Microalgae are subject of extensive scientific and commercial interest in terms of both fundamental ecology and biotechnological potential. Here, we describe five Bulgarian aeroterrestrial strains of the Eustigmatos/Vischeria group, which have shown potential as valuable carotenoid producers. The strains are stored in the algal collection of Sofia University (ACUS). The main cytological and morphological diagnostic features of the studied eustigmatophycean stramenopile algae remained stable during long-term cultivation, showing that immediate light microscopy (LM) identification in cultures is reliable. We used a polyphasic approach for taxonomical identification, combining conventional microscopy of culture material with molecular-genetic methods (based on rbcL gene sequencing). This approach led to almost similar taxonomic results, supporting the need for unification of the genera Eustigmatos and Vischeria (which are presently separated on the basis of morphological features). Based on the obtained results, we speculate that there could be new eustigmatophyceaen aeroterrestrial taxa. This, however, needs to be confirmed in more refined molecular-genetic studies with a view to exploiting the full biotechnological potential of the aeroterrestrial Eustigmatos/Vischeria strains described here.

Carotenoids have received particular attention both for their importance in algal systematics and hydrobiology and for their health benefits for humans, along with other applications in various industries. Here, we provide new data on the carotenoid content of five Vischeria/Eustigmatos strains isolated from aeroterrestrial habitats in Bulgaria and kept in the Algal Collection of Sofia University (ACUS). The obtained pigment pattern with nine carotenoids and generalization of literature data allowed us to update the knowledge on the chemotaxonomic characteristics of Eustigmatophyceae with a total of 47 pigments reported for the group, out of which 37 are carotenoids. The important photosynthetic pigment lutein, considered also a high-value product with extensive applications in feed, food, nutraceutical and pharmaceutical industries, was proved as a novel carotenoid for the group. The results also confirmed the presence of luteoxanthin, which is rarely reported in algae. All strains had a high content of the commercially valuable health-promoting xanthophyll astaxanthin. There were also different amounts of beta-carotene, violaxanthin, vaucheriaxanthin, canthaxanthin, zeaxanthin and antheraxanthin. Based on the differences in the pigment composition of the members of the ecological groups of aquatic and aeroterrestrial species, we propose the existence of two main pigment types in the class Eustigmatophyceae, with the aquatic type further divided into freshwater and marine pigment subtypes.

Recently, scientists are focused on the discovery of alternative sources of high-value products such as proteins, lipids and carbohydrates. Microalgal cells have great potential for accumulation of bioactive components important in food, feed, pharmaceutical and cosmetics industry, as sources of biofuel, for waste water remediation, etc. The main advantages of microalgae as producers of bioactive components are fast biomass growth; short reproduction cycle; resistance to harsh conditions and contaminations; smaller area for growing in comparison with agriculture crops, etc. However, the production of high-value components from microalgae biomass is still associated with high costs, so efforts are focused on cost reduction. This study evaluated the possibilities for production of low-cost high-value products from two microalgal strains, Chlorela vulgaris CAUP H1993 and Desmodesmus communis CAUP H522. Microalgal biomass was grown under simple low-cost media supplemented with urea as nitrogen source for extraction of crude protein. To completely utilize the harvested biomass, we applied two consecutive steps of the extraction procedure for protein and lipids accumulated in the exhausted biomass. The media supplemented with urea was promising, especially for growing C. vulgaris H1993 CAUP. The results showed that after two consecutive extraction steps, it is possible to obtain 8–9.0% crude protein and 21–24% lipids from the exhausted dry biomass of D. communis and C. vulgaris, respectively.

This review provides data on the possibilities for commercial use of algae of the green streptophyte genus Klebsormidium. It is one of the basal algal progenitors of land plants and shows many mechanisms and features which allow its adaptation to different environmental stresses associated with land colonization. This has led to formation of structures and compounds with countless possible applications in biotechnology and nanotechnology, starting from food to pharmaceuticals and biofuels. Therefore, the present work aims to provide: (1) a review on this yet neglected topic for the first time; (2) reliable identification using the polyphasic taxonomic approach of three local Klebsormidium strains isolated from soils of two protected areas in Bulgaria (Kresnendko Defile and National park Rila). It also outlines the cultivation advantages of Klebsormidium broad tolerance to harsh conditions and its reproductive features (easy disintegration and possibility to produce resting stages). The possibility to use inoculum of akinetes in soil algalization as biofertilizers is proposed. The results from standard microscopical identification and molecular data (ITS and rbcL, used separately and in combination), combined in the applied polyphasic approach led to distinguishing between geographically distinct strains collected at different altitudes. After identification of two strains (ACUS 00001 and ACUS 00014) as belonging to Klebsormidium dissectum and of ACUS 00207 as belonging to Klebsormidium klebsii, the similarity of the latter taxon with K. nitens is discussed.

Today’s global problems and challenges have given rise to a new field of interest – bioeconomics. It is strongly related to phycoprospecting, or searching for specific algal strains of commercial importance. There has been growing interest in the small algal class Eustigmatophyceae in recent years. These microscopic stramenopilic algae, which have all the advantages of microalgal cultivation, have proved to be promising commercial sources of valuable compounds (e.g. carotenoids, unsaturated fatty acids, amino acids) in aquaculture, agriculture, biofuels production, medicine, pharmaceutics, cosmetics, wastewater treatment, environmental control, etc. The present review shows the main genera and strains of commercial importance, outlines their main fields of application and some gaps in our knowledge in this aspect. Today, the great promising bioeconomical potential of these algae has generally been recognized, but in the present state of its infancy, it is far from being fully exploited.

Microscopic prokaryotic and eukaryotic algae (microalgae), which can be effectively grown in mass cultures, are gaining increasing interest in cosmetics. Up to now, the main attention was on aquatic algae, while species from aeroterrestrial and extreme environments remained underestimated. In these habitats, algae accumulate high amounts of some chemical substances or develop specific compounds, which cause them to thrive in inimical conditions. Among such biologically active molecules is a large family of lipids, which are significant constituents in living organisms and valuable ingredients in cosmetic formulations. Therefore, natural sources of lipids are increasingly in demand in the modern cosmetic industry and its innovative technologies. Among novelties in skin care products is the use of lipid nanoparticles as carriers of dermatologically active ingredients, which enhance their penetration and release in the skin strata. This review is an attempt to comprehensively cover the available literature on the high-value lipids from microalgae, which inhabit aeroterrestrial and extreme habitats (AEM). Data on different compounds of 87 species, subspecies and varieties from 53 genera (represented by more than 141 strains) from five phyla are provided and, despite some gaps in the current knowledge, demonstrate the promising potential of AEM as sources of valuable lipids for novel skin care products.

Bulgaria, situated on the Balkan Peninsula, is rich in small and shallow, natural and man-made non-lotic waterbodies, which are threatened by blooms of Cyanoprokaryota/Cyanobacteria. Although cyanotoxins in Bulgarian surface waters are receiving increased attention, there is no information on microviridins and their producers. This paper presents results from a phytoplankton study, conducted in August 2019 in three lakes (Durankulak, Vaya, Uzungeren) and five reservoirs (Duvanli, Mandra, Poroy, Sinyata Reka, Zhrebchevo) in which a molecular-genetic analysis (PCR based on the precursor mdnA gene and subsequent translation to amino acid alignments), combined with conventional light microscopy and an HPLC analysis of marker pigments, were applied for the identification of potential microviridin producers. The results provide evidence that ten strains of the genus Microcystis, and of its most widespread species M. aeruginosa in particular, are potentially toxigenic in respect to microviridins. The mdnA sequences were obtained from all studied waterbodies and their translation to amino-acid alignments revealed the presence of five microviridin variants (types B/C, Izancya, CBJ55500.1 (Microcystis 199), and MC19, as well as a variant, which was very close to type A). This study adds to the general understanding of the microviridin occurrence, producers, and sequence diversity.

Cyanoprokaryotes (=cyanobacteria, blue-green algae) are the most ancient oxygen-producing phototrophic microorganisms, spread all over the Globe, which form the important basis of different food chains in aquatic and terrestrial habitats. However, due to strong anthropogenic pressure during the last decades they are also responsible for causing nuisance algal blooms in different water bodies with deleterious effects on the mankind and ecosystems mainly due to production of toxic substances (cyanotoxins). Amongst them are the microcystins, nodularins, lyngbyatoxins and aplysiatoxins, known as tumor-promotors with increase of exposure routes through which humans and animals can be placed at risk (Meriluoto et al. 2017). However, the investigations on the relations between the occurrence and development of such diseases with the cyanotoxins and their producers are extremely scarce at a global scale (Yu and Chen 1994, Ueno et al. 1996, Fleming et al. 2002, Svircev et al. 2009, Drobac et al. 2011, Labine et al. 2015). During the last 15 years cyanoblooms and microcystins, nodularins and saxitoxins were detected in 16 different Bulgarian freshwater bodies, including some drinking-water reservoirs (Stoyneva-Gärtner et al. 2017). Amongst the detected toxins some new forms were recognized by their characteristic spectra (Pavlova 2007, Pavlova et al. 2007), and, more recently, a new potential producer of lyngbyatoxin was found in the Black Sea (Stoyneva et al. 2015). The poster shows a pilot assessment of the spread of cancer distribution and mortality vs. spread of cyanoblooms and cyanotoxins in Bulgaria. The pilot assessment is made on the basis of comparison of the general regions of spread of cyanotoxins in Bulgarian water bodies and toxin-producing cyanospecies during the period 2000-2017 (Stoyneva-Gärtner et al. 2017) with the spread of cancer in Bulgaria (e.g. Valerianova et al. 2015). The comparison shows general conformities between the spread of the “most dangerous” water bodies and main regions of cancer diseases in the country. The results obtained served as a basis for a new project proposal which aims at a deepening of the studies for improvement of prevention of cancer in the country.

The rising interest in harmful cyanoprokaryote blooms promotes an increase of phycological and ecological research on potentially toxic species and their hazardous substances. The present study aimed to identify the main microcystin (MC) producers and their contribution to the phytoplankton of shallow waterbodies in Bulgaria, applying different methods. The sampling was performed in August 2019 in nine lakes and reservoirs, two of which (reservoirs Kriva Reka and Izvornik 2) were studied for the first time. The high contribution of cyanoprokaryotes to the total species composition and phytoplankton abundance was proved by light microscopic (LM) observations and HPLC analysis of marker pigments. The LM identification of potential MC-producers was supported by PCR amplification of mcyE and mcyB genes. The MCs amounts, detected by HPLC-DAD, varied by sites with a range from undetectable concentrations to 0.46 µg L−1 with only one recorded variant, namely MC-LR. It was found only in the reservoirs Mandra and Durankulak, while toxigenic MC-strains were obtained by PCR from five more waterbodies. Both LM and PCR demonstrated that the MC-producers were Microcystis aeruginosa and M. wesenbergii, despite their occurrence in low amounts (

With the increasing demand for natural and safe products in cosmetics, algae with their diverse and valuable bioactive compounds are gaining vital importance. Until now, cosmetics have focused mainly on the use of freshwater and marine algae. However, algae are not restricted to aquatic habitats. They are found in essentially every type of aeroterrestrial and extreme environment on the Earth. There, they have to cope with harsh ecological conditions and have developed special strategies to thrive in these inimical habitats. Although not thoroughly studied, their adaptations include protective biochemical compounds which can find their application or are already used in the field of cosmetics. With proper cultivation techniques, algae from these habitats can provide novel sources of high-value functional products for the cosmetics industry, which have the advantage of being obtained in eco-friendly and cost-effective processes. However, it has to be considered that a few aeroterrestrial and extremophilic algae can be toxin producers, and in order to ensure conformity to the safe quality standards, all new ingredients must be properly tested. The aim of the present review is to unveil the hidden and underestimated potential of the enigmatic algae of aeroterrestrial and extreme habitats for the rapidly developing modern cosmetic industries.

The paper presents results from the first application of polyphasic approach in studies of field samples from Bulgaria. This approach, which combined the conventional light microscopy (LM) and molecular-genetic methods (based on PCR amplified fragments of microcystin synthetase gene mcyE), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus Microcystis. During the molecular identification of toxin-producing strains by use of HEPF × HEPR pair of primers, we obtained 57 sequences, 56 of which formed 28 strains of Microcystis, spread in six clusters of the phylogenetic tree. By LM, seven Microcystis morphospecies were identified (M. aeruginosa, M. botrys, M. flos-aquae, M. natans, M. novacekii, M. smithii, and M. wesenbergii). They showed significant morphological variability and contributed from Microcystis is needed, proved the presence of toxigenic strains in M. aeruginosa and M. wesenbergii, and suppose their existence in M. natans. Our results demonstrated also that genetic sequencing, and the use of HEPF × HEPR pair in particular, can efficiently serve in water quality monitoring for identifying the potential risk from microcystins, even in cases of low amounts of Microcystis in the water.