Hibberdia magna (Chrysophyceae): a promising freshwater fucoxanthin and polyunsaturated fatty acid producer.

BACKGROUND: Algae are prominent producers of carotenoids and polyunsaturated fatty acids which are greatly prized in the food and pharmaceutic industry. Fucoxanthin represents a notable high-value carotenoid produced exclusively by algae. Its benefits range far beyond just antioxidant activity and include cancer prevention, anti-diabetes, anti-obesity, and many other positive effects. Accordingly, large-scale microalgae cultivation to produce fucoxanthin and polyunsaturated fatty acids is still under intensive development in the commercial and academic sectors. Industrially exploitable strains are predominantly derived from marine species while comparable freshwater fucoxanthin producers have yet to be explored. RESULTS: In this study, we searched for freshwater fucoxanthin producers among photoautotrophic flagellates including members of the class Chrysophyceae. The initial screening turned our attention to the chrysophyte alga Hibberdia magna. We performed a comprehensive cultivation experiments using a temperature × light cross-gradient to assess the impact of these conditions on the target compounds productivity. Here we present the observations that H. magna simultaneously produces fucoxanthin (max. 1.2% dry biomass) and polyunsaturated fatty acids (max. ~ 9.9% dry biomass) and is accessible to routine cultivation in lab-scale conditions. The highest biomass yields were 3.73 g L-1 accompanied by maximal volumetric productivity of 0.54 g L-1 d-1 which are comparable values to marine microalgae fucoxanthin producers in phototrophic mode. H. magna demonstrated different optimal conditions for biomass, fucoxanthin, and fatty acid accumulation. While maximal fucoxanthin productivities were obtained in dim light and moderate temperatures (23 °C× 80 µmol m-2 s-1), the highest PUFA and overall biomass productivities were found in low temperature and high light (17-20 °C × 320-480 µmol m-2 s-1). Thus, a smart biotechnology setup should be designed to fully utilize H. magna biotechnological potential. CONCLUSIONS: Our research brings pioneer insight into the biotechnology potential of freshwater autotrophic flagellates and highlights their ability to produce high-value compounds. Freshwater fucoxanthin-producing species are of special importance as the use of sea-water-based media may increase cultivation costs and prohibits inland microalgae production.

Comparison of various approaches to detect algal culture contamination: a case study of Chlorella sp. contamination in a Phaeodactylum tricornutum culture.

Microalgal contamination in algal culture is a serious problem hampering the cultivation process, which can result in considerable economic and time losses. With the field of microalgal biotechnology on the rise, development of new tools for monitoring the cultures is of high importance. Here we present a case study of the detection of fast-growing green algae Chlorella vulgaris (as contaminant) in a diatom Phaeodactylum tricornutum culture using various approaches. We prepared mixed cultures of C. vulgaris and P. tricornutum in different cell-to-cell ratios in the range from 1:103 to 1:107. We compared the sensitivity among microscopy, cultivation-based technique, PCR, and qPCR. The detection of C. vulgaris contamination using light microscopy failed in samples containing cell ratios <1:105. Our results confirmed PCR/qPCR to provide the most reliable and sensitive results, with detection sensitivity close to 75 cells/mL. The method was similarly sensitive in a pure C. vulgaris culture as well as in a mixed culture containing 107-times more P. tricornutum cells. A next-generation sequencing analysis revealed a positive discrimination of C. vulgaris during DNA extraction. The method of cultivation media exchange from sea water to fresh water, preferred by the Chlorella contaminant, demonstrated a presence of the contaminant with a sensitivity comparable to PCR approaches, albeit with a much longer detection time. The results suggest that a qPCR/PCR-based approach is the best choice for an early warning in the commercial culturing of microalgae. This method can be conveniently complemented with the substitution-cultivation method to test the proliferating potential of the contaminant. KEY POINTS: • PCR-based protocol developed for detection of Chlorella cells. • Synergy of various approaches shows deeper insight into a presence of contaminants. • Positive/negative discrimination occurs during DNA extraction in mixed cultures. • Newly developed assays ready to use as in diagnostics of contamination.

Control of cytokinin and auxin homeostasis in cyanobacteria and algae.

BACKGROUND AND AIMS: The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [3H]trans-zeatin (transZ) and auxin ([3H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. METHODS: Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [3H]transZ and [3H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. KEY RESULTS: The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [3H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. CONCLUSIONS: Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

The microalga Parachlorella kessleri--a novel highly efficient lipid producer.

The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (1-10% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or fivefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using fivefold or 10-fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5- or 10-fold) were identified as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufficient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale-up solar open thin-layer photobioreactor were described.

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris.

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l(-1) day(-1). We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l(-1) day(-1), biomass density was 5.7 g l(-1) dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: II. The effect of aluminium, nickel and copper.

The effect of the toxic metal ions, aluminium (Al3+), nickel (Ni2+), and copper (Cu2+), on both the actin and tubulin cytoskeleton of the green alga Spirogyra decimina was studied. Batch cultured cells were grown for different time intervals at concentrations of 10, 15, 40 and 100 microM of aluminium as AlCl3, nickel as NiCl2 and copper as CuSO(4).5H2O. The impact of copper on the morphology of both MTs and AFs was much more prominent than the other two metals. A rapid irreversible depolymerization of cytoskeletal structures occurred, whereas in the presence of aluminium or nickel, changes in the cytoskeleton were slight and reversible to some extent. Nickel changed the orientation of cortical MTs, which turned from a transverse to a skewed or longitudinal direction. Aluminium caused slight depolymerization of the cytoskeleton, which reverted spontaneously to the normal cytoskeletal state (in AlCl3 free nutrient solution). Copper exerted a strong effect on both the MT and AF cytoskeleton, which fragmented and disorganized rapidly. The extent of cytoskeletal damage by copper was dosage and time dependent and AFs were slightly more sensitive than MTs.

Chemico-physical and pharmacodynamic properties of extracellular Dictyosphaerium chlorelloides biopolymer.

Microalgae occupy all territories and their products represent a rich source of phytochemicals for human being. Green microalga Dictyosphaerium chlorelloides was found to be a significant producer of the extracellular biopolymer. Dominant components of the biopolymer were found to be Gal (39 wt%) with its methyl derivatives (15 wt%), Rha (19 wt%) and Man (14 wt%). 2-OMe-Gal was found to be the major derivative while other sugars, namely 3-OMe-, 6-OMe- and 2,3-di-OMe-Gal, 3-OMe-Glc and 4-OMe-Xyl were in smaller amounts. NMR spectroscopy revealed complex structure with galactan backbone branched by sugars in furano and pyrano forms in alpha and beta configurations. NMR data of 2-OMe, 3-OMe, 2,3-OMe and 6-OMe galactoses afforded characteristic values for O-methyls in each position. Biopolymer antitussive effect was similar to that of centrally acting antitussive drugs, indicating its relatively good antitussive potential.

A gene transfer event suggests a long-term partnership between eustigmatophyte algae and a novel lineage of endosymbiotic bacteria.

Rickettsiales are obligate intracellular bacteria originally found in metazoans, but more recently recognized as widespread endosymbionts of various protists. One genus was detected also in several green algae, but reports on rickettsialean endosymbionts in other algal groups are lacking. Here we show that several distantly related eustigmatophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Trachydiscus minutus CCALA 838, revealed genomic features (size, GC content, number of genes) typical for other Rickettsiales, but some unusual aspects of the gene content were noted. Specifically, Phycorickettsia lacks genes for several components of the respiration chain, haem biosynthesis pathway, or c-di-GMP-based signalling. On the other hand, it uniquely harbours a six-gene operon of enigmatic function that we recently reported from plastid genomes of two distantly related eustigmatophytes and from various non-rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely related to the one from Phycorickettsia, suggesting a gene transfer event between the endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize an important role of the operon in the physiology of Phycorickettsia infection and a long-term eustigmatophyte-Phycorickettsia coexistence.

Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?

Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte.

Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri.

Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

ZOOSPOROGENESIS, MORPHOLOGY, ULTRASTRUCTURE, PIGMENT COMPOSITION, AND PHYLOGENETIC POSITION OF TRACHYDISCUS MINUTUS (EUSTIGMATOPHYCEAE, HETEROKONTOPHYTA)(1).

The traditional order Mischococcales (Xanthophyceae) is polyphyletic with some original members now classified in a separate class, Eustigmatophyceae. However, most mischococcalean species have not yet been studied in detail, raising the possibility that many of them still remain misplaced. We established an algal culture (strain CCALA 838) determined as one such species, Trachydiscus minutus (Bourr.) H. Ettl, and studied the morphology, ultrastructure, life cycle, pigment composition, and phylogeny using the 18S rRNA gene. We discovered a zoosporic part of the life cycle of this alga. Zoospore production was induced by darkness, suppressed by light, and was temperature dependent. The zoospores possessed one flagellum covered with mastigonemes and exhibited a basal swelling, but a stigma was missing. Ultrastructural investigations of vegetative cells revealed plastids lacking both a connection to the nuclear envelope and a girdle lamella. Moreover, we described biogenesis of oil bodies on the ultrastructural level. Photosynthetic pigments of T. minutus included as the major carotenoids violaxanthin and vaucheriaxanthin (ester); we detected no chl c. An 18S rRNA gene-based phylogenetic analysis placed T. minutus in a clade with species of the genus Pseudostaurastrum and with Goniochloris sculpta Geitler, which form a sister branch to initially studied Eustigmatophyceae. In summary, our results are inconsistent with classifying T. minutus as a xanthophycean and indicate that it is a member of a novel deep lineage of the class Eustigmatophyceae.