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Microalgae are a natural source of high-value fatty acids and could address the reduced availability of fish oil from pelagic fisheries and the increasing needs from the aquaculture sector and the ω-3 market (Chauton et al., 2015). Still, their exploitation on an industrial scale is scarce, mainly due to the lack of highly performant strains. Classical breeding constitutes a source of genetic diversification in microalgae, generating progeny strains with unique genetic heritage and bypassing the controversial issue of genetically modified organisms (GMOs). In the framework of the H2020 NewTechAqua project, our goal is to generate improved microalgae strains via classical breeding for application as aquafeed in European aquaculture. The heterothallic diatom species Seminavis robusta is particularly suitable for controlled breeding programs based on pedigree selection (Chepurnov et al., 2012). Eight wild-type strains of S. robusta were purchased from the BCCM/DCG collection (PAE laboratory, UGENT, Belgium) and crossbred according to their sexual compatibility (De Decker et al., 2018). Single cells of 66 F1 progeny strains from eleven breeding pairs successfully developed into monoclonal cultures. All the 74 strains were cultured for twelve days under the same growth conditions in 6-well plates. Sixteen bright field and fluorescence CY5 images per well were taken every three days using the Cytation™ 3 plate reader and imager from BioTek, and processed using Gen5 and ImageJ softwares. Cell density over time was estimated using the percentage of surface showing CY5 fluorescence over the total surface. Sixteen F1 progeny strains aroused biotechnological interest as they showed faster growth compared to their wild-type parents. Further studies will address fatty acid composition and quantification for all the strains. The most performant F1 strains will undergo crossbreeding experiments in an iterative manner, using a pedigree-based approach. Our findings will ultimately contribute to achieving a more sustainable, resilient, and cost-effective European aquaculture., ABS2022 organized by ABO

Microalgae are a natural source of high-value fatty acids and could address the reduced availability of fish oil from pelagic fisheries and the increasing needs from the aquaculture sector and the ω-3 market (Chauton et al., 2015). Still, their exploitation on industrial scale is scarce, mainly due to the lack of highly performant strains. Classical breeding constitutes a source of genetic diversification in microalgae, generating progeny strains with unique genetic heritage and bypassing the controversial issue of genetically modified organisms (GMOs). In the framework of the H2020 NewTechAqua project, our goal is to generate improved microalgae strains via classical breeding for application as aquafeed in European aquaculture. The heterothallic diatom species Seminavis robusta is particularly suitable for controlled breeding programs based on pedigree selection (Chepurnov et al., 2012). Eight wild-type strains of S. robusta were purchased from the BCCM/DCG collection (PAE laboratory, UGENT, Belgium) and crossbred according to their sexual compatibility (De Decker et al., 2018). Single cells of 66 F1 progeny strains from eleven breeding pairs successfully developed into monoclonal cultures. All the 74 strains were cultured for twelve days under the same growth conditions in 6-well plates. Sixteen bright field and fluorescence CY5 images per well were taken every three days using the Cytation™ 3 plate reader and imager from BioTek, and processed using Gen5 and ImageJ softwares. Cell density over time was estimated using the percentage of surface showing CY5 fluorescence over the total surface. Above 30% of the F1 strains aroused biotechnological interest as they showed faster growth compared to their wild-type parents. Further studies will address fatty acid composition for all the strains. The most performant F1 strains will undergo crossbreeding experiments in an iterative manner, using a pedigree-based approach. Our findings will ultimately contribute to achieve a more sustainable, resilient and cost-effective European aquaculture., ISMS-180

Assessment of growth and production of metabolites of interest from a given microalgae on a laboratory scale is crucial to evaluate its potential applicability on larger scales into the biotechnology sector. Our work within the H2020 NewTechAqua project focuses on fatty acid production for use of microalgae as aquafeed in the aquaculture industry. Growth studies of microalgae comprise the estimation of cell density over time, for which several methods have been described in the literature. While indirect methods are undoubtedly less time-consuming, their relevance needs to be evaluated case by case for the species and the experimental conditions used. Our study focused on the benthic diatom Nitzschia palea, a species of interest for biotechnology (Abdel-Hamid et al., 2013). Three native strains of N. palea from the Canaries were cultured for 26 days in triplicate flasks under the same culture conditions. Growth was monitored using cell counting as direct method, and optical density (OD) and basal fluorescence (Fo) as indirect methods. The three strains showed their maximum growth rates between days 4 and 7 (0.21 – 0.29 day-1), and entered the stationary phase at day 17. The relevance of OD and Fo was demonstrated during the exponential phase only (days 4 – 17), as they both positively correlated with cell density in a linear regression model (R2 > 0.89, n = 45). Preliminary results on the fatty acid (FA) composition indicated that the major FAs produced by N. palea were palmitic acid (18 – 41 %) and palmitoleic acid (12 – 25 %), in accordance with a previous study (Touliabah et al., 2020). As for the ω-3 FAs, DHA was poorly produced (≤ 3%), while EPA constituted a major FA for two of the strains (21% and 13%). This study showed that OD and Fo were suitable alternatives to cell counting for cell density estimation, at least during the exponential phase of growth, allowing faster execution of the daily laboratory work. More accurate studies on the fatty acid production should be developed in the future to have a more comprehensive view of the biotechnological value of these strains., ISMS-285

Microalgae of the genus Gambierdiscus are producers of precursors of ciguatoxins (CTXs), which bioaccumulate in the marine trophic chain and are responsible for ciguatera fish poisoning in humans. Ciguatera is a foodborne illness typically known as a tropical disease, but ciguatera cases are being increasingly reported in the North-Eastern Atlantic. The appearance of fish contaminated with CTXs has become more and more common in areas such as the Canary Islands, but the mechanisms underlying the bioaccumulation and biotransformation of the CTXs in this region are still poorly discerned (Soliño & Costa, 2020). The study presented here is part of the European project “Ciguarisk” and has the objective to assess cell growth and toxin production of two Gambierdiscus strains from the Atlantic region selected for fish feeding experiments, i.e., G. excentricus IRTA-SMM-17-429 and G. carolineanus BEA1923. Cell growth was performed in culture flasks under the same culture conditions, using a semi-continuous batch approach. The culture method is aimed to harvest the highest biomass production possible for use in dietary exposure experiments in fish. Two culture media (L1 and F2) were evaluated for their influence on the growth of the strains. Both media were suitable for culturing Gambierdiscus, although some slight differences were observed in growth rates and cell behaviour. G. excentricus IRTA-SMM-17-429 presented slower growth with respect to G. carolineanus BEA1923 in all conditions tested. Ciguatoxins were extracted from fresh biomass samples and pre-purified using liquid-liquid partitioning. CTX-like toxicity of the strains was screened using the neuroblastoma neuro-2a assay (Caillaud et al., 2010). Preliminary data suggest that G. excentricus IRTA-SMM-17-429 is more toxic than G. carolineanus BEA1923. These results presented in this study are in accordance with previous studies on other strains of G. carolinianus and G. excentricus (Pisapia et al., 2017). Further studies will focus on culture optimization and scale-up, quantitation of the toxic content, and micro-encapsulation of the biomass for the feeding experiments. The results obtained from this project will ultimately help to a better understanding of the bioaccumulation and biotransformation of CTXs in the food chain in the Atlantic region.

Nowadays, the availability of fish oil from pelagic fisheries has been declining, and the demand of high-value fatty acids from the aquaculture sector and the ω-3 market has been rapidly increasing. Microalgae are a natural source of ω-3 PUFAs, since they constitute the primary producers of all the eicosapentaenoic and docosahexaenoic acids in marine food webs. Still, their exploitation on industrial scale is scarce, mainly due to the lack of highly performant strains. Classical breeding constitutes an incredible source of genetic diversification in microalgae, bypassing the controversial issue of genetic modified organisms (GMOs), and a novel approach for strain improvement in biotechnological processes. After an extensive bibliographic research, we chose to work with heterothallic diatoms in the framework of the H2020 NewTechAqua project. For these species, gametogenesis only occurs when two sexually-compatible strains get physical contact while they both are in the adequate phase of their life cycle. Therefore, they are particularly suitable for controlled manipulation of sexual encounters and isolation of progeny strains with unique genetic heritage and, potentially, improved biotechnological traits. Our study screened for sexual activity of 35 strains belonging to eight diatom species, mixing them in all pairwise combinations. Crossbreeding experiments worked for the species Seminavis robusta only. Single cells of fourty-three sexual descendants were isolated from three breeding pairs and successfully developed into new monoclonal cultures. Further studies will address the potential of the recombined strains for biotechnological applications. Our findings will ultimately contribute to achieve a more sustainable, resilient and cost-effective European aquaculture.

Several methods for monitoring the growth of microalgae in the laboratory have been described in the literature. Direct measurements, such as cell counting or dry biomass, are considered the most reliable methods. Indirect methods generally rely on specific parameters that can be measured as a consequence of phototrophic activity. While indirect methods are undoubtedly less time-consuming, their relevance needs to be evaluated case by case for the species and the experimental conditions used. Our study focused on the relevance of optical density (OD) and basal fluorescence (Fo) measurements for batch cultures of the benthic diatom Nitzschia palea. Three Canarian strains of N. palea (BEA0255B, BEA0256B and BEA0392B) were cultured in triplicate 1 L Erlenmeyer flasks with 270 mL FDMed medium (surface-tovolume ratio: 0.5) for 26 days at an initial cell density of 1.3 x 105 cells mL-1 ± 27% (RSD). The three strains showed their maximum growth rate (μmax) between days 4 and 7 (0.21 day-1 ± 0.023 (SD) for BEA0255B, 0.23 day-1 ± 0.058 (SD) for BEA0256B and 0.42 day-1 ± 0.065 (SD) for BEA0392B), and entered the stationary phase at day 17, maintaining an average concentration of 1.5 x 106 cells mL-1± 11% (RSD). Comparison between cell density, OD, and Fo was performed using the data of each replicate of the three strains in the same analysis. The relevance of OD and Fo measurements was demonstrated during the exponential phase (days 4 – 17), as they both positively correlated with cell density in a linear regression model (R2 > 0.89, n = 45). No linear correlation was found during the stationary phase (days 17 – 26, R2 < 0.46, n = 45 ). Further studies will address the study of photosynthetic efficiency, biomass weight, and composition throughout the growth phases of N. palea, with the aim of determining the optimal harvesting time for biotechnological applications., Poster presentation in YouTube: https://www.youtube.com/watch?v=4S5HLHo_0Lk&t=70s&ab_channel=FrancescoPisapia

Direct measurements such as cell density, are considered the most reliable methods for growth monitoring of microalgae. In comparison, indirect methods are undoubtedly less time-consuming, but their relevance needs to be evaluated case by case for the species and the experimental conditions used. Our study focused on the relevance of optical density (OD) and basal fluorescence (FO) measurements for batch cultures of the benthic diatom Nitzschia palea. Three Canarian strains of N. palea were cultured in triplicate 1 L Erlenmeyer flasks with 270 mL FDMed medium for 26 days with 1.3 x 105 cells mL-1 ± 27% (RSD). The three strains showed their maximum growth rate between days 4 and 7 (BEA0255B: 0.21 day-1 ± 0.023 (SD), BEA0256B: 0.23 day-1 ± 0.058 (SD), BEA0392B: 0.42 day-1 ± 0.065 (SD)), and entered the stationary phase at day 17, maintaining an average concentration of 1.5 x 106 cells mL-1± 11% (RSD). Comparison between cell density, OD and FO was performed using the data of each replicate of the three strains in the same analysis. The relevance of OD and FO measurements was demonstrated during the exponential phase, as they both positively correlated with cell density in a linear regression model (R2 > 0.89, n = 45). No linear correlation was found during the stationary phase (R2 < 0.46, n = 45). Further studies will address the study of photosynthetic efficiency, biomass weight and composition throughout the growth phases of N. palea, with the aim of determining the optimal harvesting time for biotechnology., YAS2021 organized by EABA

Seaweeds are a source of macro and microelements, amino acids, vitamins, polysaccharides and hormones, such as auxins, auxin-like compounds and cytokinins (Craigie, 2011). Seaweeds extracts (SE) have long been used in agriculture as soil amendment for their beneficial properties on plants, because they stimulate both plant growth and production (Crouch & van Staden, 1992; Arthur et. al., 2003). They also increase fruit weight, fresh dry mass of root, leaf area, yield per plant, the chlorophyll content and minerals (Sivasankari et al., 2006; Rayorath et al., 2008; Roussos et al., 2009). It has also been shown that SE may help the plants to counteract abiotic and biotic stresses. Among abiotic stresses, Ashraf & Foolad (2007) demonstrated that SE are involved in overcoming plant stress conditions such as drought and salinaty. Several studies have shown antibiotic, antiviral and antifungal activities of SE against a number of plant pathogens such as Penicillium spp. and Fusarium oxysporum (Khallil et al., 2015) and Aspergillus sp. (Kosanić et al., 2015). Seaweeds extracts are also rich in several bioactive compounds, such as polysaccharides that are well known to be elicitors of plant defence responses. Simultaneously to the growing consumption of agricultural products, such as strawberry, there was an increase in the use of chemical fertilizers to improve plant yield, and of synthetic pesticides to control fungal plant pathogens. The continuous use of chemicals during the years has affected human and animal health and the ecosystem. Strawberry is one of the most consumed berries and its high nutritional value and composition have stimulated its consumption increase. A number of fungal pathogens can affect strawberry plants such as Colletotrichum spp., and several species of the soil borne pathogens, such as Rhizoctonia, Fusarium and Pythium causing the so-called black root rot complex (Manici et al., 2005). Among these pathogens, Botrytis cinerea, the agent of grey mold, cause several losses especially during shelf life. Alternative approaches to chemical treatments are necessary to preserve the quality of strawberries during the shelf life in order to protect health safety and limit the development of the fungus. In this study, the antifungal activity of cationic polysaccharides extracted from two macroalgae, Ecklonia sp. (Ochrophyta) and Jania sp. (Rhodophyta) was investigated against B. cinerea in vitro and in vivo on strawberry. Polysaccharides were extracted by selective precipitation with 2% (w/v) N-Cetylpyridinium bromide monohydrate (Cetavlon) (Diaz et al., 2011). In the in vitro assay, fungal colony portions were treated for 6 hours by immersion in three polysaccharides aqueous concentrations, 1.65, 0.82 and 0.41 mg/ml for Ecklonia sp. and 0.18, 0.09 and 0.045 mg/ml for Jania sp. The treated colony portions were inoculated in agarized medium and daily growth was measured for a week. Ecklonia sp. polysaccharides at 0.18 mg/ml and 0.09 mg/ml significantly inhibited B. cinerea growth by 21.0% and 22.8%, respectively, two days after treatment. Jania sp. polysaccharides did never inhibit fungal colony growth. For in vivo experiments, strawberry ripe fruits cv. Cristal were immerged before or after harvesting in polysaccharide aqueous solutions of Ecklonia sp. at the concentrations of 0.82 and 0.41 mg/ml, and of Jania sp., at 0.09 and 0.045 mg/ml. A spore suspension of B. cinerea (1 × 105 spores/ml) was inoculated by spraying fruits 24 hours after treatment. Disease symptoms over the total area inoculated of fruit were evaluated as percentage of infected area. The pre-harvest treatment with Jania sp. showed to reduce disease symptoms by 100% at 0.09 mg/ml and by 50% at 0.045 mg/ml and with Ecklonia sp. by 16.7% (0.82 mg/ml) and 11.11% (0.41 mg/ml). Post-harvest treatment did never inhibit disease symptoms. This study showed that SE could be considered for further investigation in control strategy against B. cinerea

Doctorado en Ciencias del Mar. Programa de Biotecnología

[EN]Two cryopreservation methods, controlled cooling and encapsulation/vitrification, were studied in order to find an appropriate protocol to maintain microalgae cultures by exposing them to ultra-low temperatures (cryogenics). This study has shown that the most efficient cryopreserving method is the use of cryoprotectants, being Glycerol and DMSO the best options for this procedure, and dismissing the encapsulation/vitrification method due to the low effectiveness, which results in a low post-thaw viability rate and a higher demanding of labour and consumables. [ES]Dos métodos de criopreservación, enfriamiento controlado y encapsulación/vitrificación, han sido estudiados con el objetivo de encontrar un protocolo adecuado para conservar los cultivos de microalgas mediante su exposición a muy bajas temperaturas (criogenización). Este estudio ha demostrado que el método de criopreservación más eficiente es el de enfriamiento controlado combinado con el empleo de crioprotectores, siendo el Glicerol y DMSO los crioprotectores más adecuados para este procedimiento. A su vez se descarta el método de encapsulación/vitrificación debido a su baja efectividad, que se traduce en una baja tasa de viabilidad tras el proceso de descongelación y supone un método con una mayor demanda de tiempo de trabajo y de material.

Programa de doctorado: Gestión de recursos vivos marinos y medioambiente. Bienio 2001-2003. [ES]Las diatomeas son un grupo muy diverso de algas unicelulares y uno de los productores de materia orgánica más importantes de los ambientes acuáticos terrestres. Colonizan todo tipo de ambientes húmedos, en aguas dulces, salobres y marinas. Gran parte de ellas vive en un tipo de hábitat determinado con propiedades fisicoquímicas específicas. Algunas especies de este grupo son muy sensibles a los cambios ambientales, mientras otras son muy tolerantes ya sea en cortos o largos periodos de tiempo por lo que están consideradas como unos buenos bioindicadores.