Modulation of the metabolite content of the unicellular rhodophyte Porphyridium purpureum using a 2-stage cultivation approach and chemical stressors.

There have been growing interests in microalgal biotechnology for the biorefining of bioactive compounds such as carotenoid pigments, ω-3 fatty acids, antioxidants or antimicrobials for sectoral applications in the pharmacology, nutraceutical and cosmetic fields. This study focused on the unicellular marine rhodophyte Porphyridium purpureum CCAP 1380/1 A, which was cultivated via a two-stage batch growth mode for 10 days using hydrogen peroxide (H2O2), the phytohormone methyl jasmonate (MJ) and three plant extracts (Passiflora incarnata, Panax ginseng and Valeriana officinalis). The microalgal biomass was then analysed for its protein, phycoerythtin, carbohydrate and pigment composition together with its pigment content and antioxidant activity. Of note, MJ increased the protein and phycoerythtin content (up to 225 µg BSA eq./mg DW and 15 mg/ml, respectively) while both the MJ and H2O2 treatments increased carotenoid pigment yields (ß-carotene and zeaxanthin, up to 5 and 4 mg/g, respectively). Carbohydrates were enhanced ∼10 fold by the Valeriana officinalis treatment (up 192 µg starch eq./mg). Overall, neutral lipids and antioxidants were mostly negatively affected by the plant extracts. The greatest antioxidant activity registered was obtained with the H2O2 treatment (15 µmol Trolox eq./g DW with TEAC assay). P. purpureum contains multiple valuable compounds of commercial interest. These results indicate that they can be favorably modulated using specific cultivation regimes and chemical enhancers, thereby facilitating the exploitation of the biomass by applying a suitable co-refinery pipeline.

Cold stress combined with salt or abscisic acid supplementation enhances lipogenesis and carotenogenesis in Phaeodactylum tricornutum (Bacillariophyceae).

Compounds from microalgae such as ω3-fatty acids or carotenoid are commercially exploited within the pharmacology, nutraceutical, or cosmetic sectors. The co-stimulation of several compounds of interest may improve the cost-effectiveness of microalgal biorefinery pipelines. This study focussed on Phaeodactylum tricornutum to investigate the effects on lipogenesis and carotenogenesis of combined stressors, here cold temperature and addition of NaCl salt or the phytohormone abscisic acid, using a two-stage cultivation strategy. Cold stress with NaCl or phytohormone addition increased the neutral lipid content of the biomass (20 to 35%). These treatments also enhanced the proportions of EPA (22% greater than control) in the fatty acid profile. Also, these treatments had a stimulatory effect on carotenogenesis, especially the combination of cold stress with NaCl addition, which returned the highest production of fucoxanthin (33% increase). The gene expression of diacylglycerol acyltransferase (DGAT) and the ω-3 desaturase precursor (PTD15) were enhanced 4- and 16-fold relative to the control, respectively. In addition, zeaxanthin epoxidase 3 (ZEP3), was downregulated at low temperature when combined with abscisic acid. These results highlight the benefits of applying a combination of low temperature and salinity stress, to simultaneously enhance the yields of the valuable metabolites EPA and fucoxanthin in Phaeodactylum tricornutum.

Influence of exogenous phytohormone supplementation on the pigment and fatty acid content of three marine diatoms.

Diatoms are ubiquitous photosynthetic microorganisms with great potential for biotechnological applications. However, their commercialisation is hampered by production costs, requiring hence optimisation of cultivation methods. Phytohormones are plant growth regulators which may be used to influence physiological processes in microalgae, including diatoms. In this study, the model species Phaeodactylum tricornutum (Phaeodactylaceae) and two Irish isolates of Stauroneis sp. (Stauroneidaceae) and Nitzschia sp. (Bacillariaceae) were grown with varying amounts of the phytohormones indoleacetic acid (IAA), gibberellic acid (GA3), methyl jasmonate (MJ), abscisic acid (ABA) or salicylic acid (SA), and their influence on pigment and fatty acid profiles was monitored. The application of GA3 (200 mg/l) stimulated the growth of P. tricornutum which accumulated 52% more dry biomass compared to the control and concomitantly returned the highest eicosapentaenoic acid (EPA) yield (0.6 mg/l). The highest fucoxanthin yield (0.18 mg/l) was obtained for P. tricornutum cultivated with GA3 (2 mg/l) supplementation. In Stauroneis sp., SA (1 mg/l) had the most positive effect on EPA, the content of which was enhanced up to 45.7 µg/mg (4.6% of total dry weight). The SA (1 mg/l) treatment also boosted carotenogenesis in Nitzschia sp., leading to 1.7- and 14-fold increases in fucoxanthin and ß-carotene compared to the control, respectively. Of note, MJ (0.5 mg/l) increased the EPA content of all diatom species compared to their controls. These results indicate that phytohormone-based treatments can be used to alter the pigment and lipid content of microalgae, which tend to respond in dose- and species-specific manners to individual compounds.Key points• Response to phytohormones was investigated in diatoms from distinct families.• MJ (0.5 mg/l) caused an increase in EPA cellular content in all three diatoms.• Phytohormones mostly caused dose-dependent and species-specific responses.

Comparative Response of Marine Microalgae to H2O2-Induced Oxidative Stress.

There have been growing interests in the biorefining of bioactive compounds from marine microalgae, including pigments, omega-3 fatty acids or antioxidants for use in the nutraceutical and cosmetic sectors. This study focused on the comparative responses of five marine microalgal species from different lineages, including the dinoflagellate Amphidinium carterae, chlorophyte Brachiomonas submarina, diatom Stauroneis sp., haptophyte Diacronema sp. and rhodophyte Rhodella violacea, to exposure during their batch growth to hydrogen peroxide (H2O2). A. carterae returned an enhanced signal with the DPPH assay (8.8 µmol Trolox eq/g DW) when exposed to H2O2, which was associated with reduced pigment yields and increased proportions in saturated C16 and C18 fatty acids. B. submarina showed enhanced antioxidant response upon exposure to H2O2 with the DPPH assay (10 µmol Trolox eq/g DW), a threefold decrease in lutein (from 2.3 to 0.8 mg/g) but a twofold increase in chlorophyll b (up to 30.0 mg/g). Stauroneis sp. showed a downward response for the antioxidant assays, but its pigment yields did not vary significantly from the control. Diacronema sp. showed reduced antioxidant response and fucoxanthin content (from 4.0 to 0.2 mg/g) when exposed to 0.5 mM H2O2. R. violacea exposed to H2O2 returned enhanced antioxidant activity and proportions of EPA but was not significantly impacted in terms of pigment content. Results indicate that H2O2 can be used to induce stress and initiate metabolic changes in microalgae. The responses were however species-specific, which would require further dosage optimisation to modulate the yields of specific metabolites in individual species.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.