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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

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

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