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Aquaculture is one of the most resource-efficient and sustainable ways to produce animal protein. The Food and Agriculture Organization predicts that cultivated aquatic species will provide around 53% of the world's seafood supply by 2030. Further growth of intensive farmed aquatic species may be limited by a shortage of feed resources. The aquaculture sector therefore needs to intensify its search for alternative ingredients based on renewable natural resources. A significant increase in production will require an accelerated transition in technology and production systems, better use of natural available resources, development of high-quality alternative feed resources and exploitation of available space. The present review discusses the urgent need to identify appropriate alternative ingredients for a sustainable future salmonid production. We describe and evaluate the most promising marine ingredients, including low-trophic species (mesopelagic fish, zooplankton, polychaetes, macroalgae and crustaceans), novel microbial ingredients (bacteria, yeast and microalgae), insects (black soldier fly, yellow meal worm and crickets), animal by-products (poultry meal, meat and bone meal, blood meal and hydrolysed feather meal) and by-products from other commercial productions (trimmings and blood). Furthermore, we discuss the available volumes and need for new processing technologies and refining methods to ensure commercial production of nutritionally healthy ingredients. The essential production steps and considerations for future development of sustainable and safe seafood production are also discussed. publishedVersion

The following policy recommendations aim to help the EU aquaculture sector to apply a circular-economy approach in order to participate in the green transition set by the European Green Deal: Define circularity in aquaculture Define a common methodology to measure circularity in aquaculture Increase circularity in aquaculture production by increasing circularity in feed production and by valorising aquaculture wastes (effluent and sludge) Encourage sectorial and cross-sectorial co-governance

In chapter 1 we introduce microalgae, photosynthetic microorganisms with potential to replace commodities (such as food, feed, chemicals and fuels). Production costs are still high, reason why microalgae are still only economically feasible for niche markets. We suggest to borrow the concept of plant domestication to select industrial microalgae cells. Two approaches can be successfully used to domesticate microalgae: adaptive laboratory evolution (ALE) and fluorescence assisted cell sorting (FACS). ALE takes advantage of the natural adaptability of microorganisms to different environments, while FACS actually select cells with specific phenotypes. This thesis aimed to select cells of Chlorococcum littorale with improved phenotypes, assuming that these cells could establish new populations with increased industrial performance. In Chapter 2 we wanted to know what happened during time to biomass and lipid productivities of Chlorococcum littorale repeatedly subjected to N-starvation. We tested 2 different cycles of N-starvation, short (6 days) and long (12 days). Short cycles didn’t affect lipid productivity, highlighting the potential of C. littorale to be produced in semi-continuous cultivation. Repeated cycles of N-starvation could have caused adaptations of the strain. Hence, we also discussed the implications of using repeated N-starvation for adaptive laboratory evolution (ALE) experiments. Chapter 3 introduces a method to detect and to select microalgae cells with increased lipid content. The method requires only the fluorescence dye Bodipy505/515 dissolved in ethanol, and the method was designed to maintain cellular viability so the cells could be used to produce new inoculum. In chapter 4 we evaluated a question that emerged while deciding which criteria to use to sort lipid-rich cells: does cellular size affects lipid productivity of C. littorale? We hypothesized that cells with different diameters have different division rates, which could affect lipid productivity. Therefore, we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of Chlorococcum littorale (comparing populations before and after sorting, based on different diameters). Results showed that the size of vegetative cells doesn’t affect the lipid productivity of C. littorale. In chapter 5 we present a strategy to sort cells of C. littorale with increased TAG productivity using the method developed at chapter 3. Both the original and the sorted population with the highest lipid productivity (namely, S5) were compared under simulated Dutch summer conditions. The results confirmed our data from experiments done under continuous light: S5 showed a double TAG productivity. Our results showed also that the selected phenotype was stable (1.5 year after sorting) and with potential to be used under industrial conditions. In chapter 6 we extrapolated our results (indoor and outdoor) to other climate conditions. We ran simulations changing the light conditions to four different locations worldwide (the Netherlands, Norway, Brazil and Spain) to estimate both biomass and TAG productivities. Results indicated that biomass yields were reduced at locations with higher light intensities (Brazil/Spain) when compared with locations with lower light intensities (Norway/Netherlands). Hence, the choice of location should not be based on light intensity, but on how stable irradiation is. Chapter 7 is the general discussion of the thesis, demonstrating that both ALE and FACS are effective approaches to select industrial microalgae cells. We also present our view on how ALE and FACS could further improve microalgae strains for industry.