Integrated multitrophic level aquaculture (IMTA) in land-based recirculating aquaculture systems (RAS) has been proposed as an effective nitrogen bioremediation strategy. It can improve the utilization of added nitrogen through further biomass production, which increases the sustainability and efficiency of the farming system. Few studies have evaluated how co-farming in cold-water land-based marine recirculating aquaculture systems affects the nitrogen utilization; here, we quantify the effects of co-farming sea lettuce (Ulva fenestrata) with Atlantic wolffish (Anarhichas lupus) and rainbow trout (Oncorhynchus mykiss) in such a system. Nitrogen budgets were used to evaluate the efficiency of the system by summarizing all processes, both inputs and outputs. In alternating four-week periods for four months, the recirculating system held either only fish or fish together with sea lettuce. Fish biomass held 90–91% of the system nitrogen, while 9–10% was available as dissolved inorganic nitrogen. The nitrogen uptake by the sea lettuce biomass was quantified to 1.0–1.5% of the total nitrogen input. Extrapolations based on these results show that a wet weight ratio of sea lettuce:fish of 0.7 would be optimal for remediation of all added nitrogen.

Introducing novel and sustainable feed ingredients is essential to improve the environmental impact of fish aquaculture. Some species of seaweed, such as the green alga sea lettuce (Ulva fenestrata), constitute a promising novel ingredient due to their suitable amino acid profile and favourable growth traits. However, the effects of farmed Ulva in feed for cold-water salmonids have not yet been studied. Here we show that rainbow trout (Oncorhynchus mykiss) growth was improved by including Ulva meal in the feed while maintaining intestinal health and fish welfare. The study lasted for 12 weeks, and experimental diets included a control feed, and two feeds with 3% and 15% Ulva meal, respectively. The growth rate was highest for fish given the 15% Ulva diet during the first 6 weeks and the 3% Ulva diet for the last 6 weeks. This was likely due to an increased feed intake (FI) and improved palatability resulting from the inclusion of Ulva. Analysis of intestinal health parameters, primary barrier function and histomorphology, revealed no impaired barrier or inflammatory responses to any of the diets. Similarly, other primary and secondary stress indicators including plasma cortisol levels and oxidative stress markers in the liver and muscle tissues, were unaffected by dietary treatments. Interestingly, the fillet colour became more yellow with increased Ulva inclusion, which could affect the acceptance of the product to customers. Nevertheless, the results demonstrate that sea-farmed Ulva can contribute to improving the sustainability of future aquafeeds for rainbow trout.

To investigate the NO3- tolerance of Candidatus Scalindua sp., a continuous reactor was gradually exposed to increasing NO3- concentrations up to 3,200 mg N L-1. High NH4+ and NO2- removal efficiencies were maintained up to 2,600 mg N L-1, above which performance declined and Ca. Scalindua relative abundance decreased to 0.8%. After one year of recovery, removal efficiencies exceeded 97%, whereas Ca. Scalindua relative abundance only reached 6.5%. EC50 values for NH4+ and NO2- were both 3,000 mg N L-1. We demonstrated that our enriched Ca. Scalindua population tolerated NO3- up to 2,600 mg N L-1, far exceeding the levels typically encountered in most human-derived wastewaters.

Monitoring stress in captive fish is crucial for their welfare, but continuous physiological measures in unrestrained animals are challenging. Rainbow trout (Oncorhynchus mykiss) exhibit divergent personalities, ranging from bold to shy, which correlate with cortisol-mediated stress responses. To determine whether personality affects the sympathetic nervous system, heart rate was measured during three potentially stressful events as a proxy for sympathetic nervous system responses. Firstly, trout were classified as bold or shy, using a novel object test. Subsequently, trout were implanted with biologgers to record heart rate in vivo at rest during and after the behavioral tests. Following recovery, the fish underwent a second novel object test, a confinement test, a pair-wise contest, and a final novel object test to explore the degree of boldness over the experimental period, which remained consistent. Heart rate was relatively higher in both bold and shy animals during the confinement test and the pair-wise contest compared with the novel object test, which indicated that heart rate monitoring was a valid gauge of the valence of the experience. Heart rate responses did not differ between bold and shy trout, indicating that behavioral phenotype did not influence the autonomic stress response. Thus, heart rate is a reliable indicator of stress without the need to account for intra-specific behavioral variations.

Integrated multi-trophic aquaculture (IMTA), with fed and extractive species in recirculating aquaculture systems (RAS), has been identified as a promising and sustainable farming system with the potential to increase nutrient utilization. Additionally, co-farming has been shown to improve the nutrient content, growth of the farmed organisms, and water quality parameters in the RAS. However, there is a knowledge gap on how co-farming can interact with health and welfare of the co-farmed species. In this study it was shown that co-farming rainbow trout, Oncorhynchus mykiss, with sea lettuce, Ulva fenestrata, in a salt-water RAS had beneficial effects on both organisms and improved the water quality. Throughout the experiment, the sea lettuce grew, and its crude protein content increased by 25-30 % during the four week co-farming periods. Within the RAS, sea lettuce also contributed to locally increasing the pH. Since no systemwide effects were observed on pH or dissolved nitrogen concentrations, it is suggested that a larger macroalgae biomass should be used in relation to the total water volume and fish biomass. It was observed within the co-farming experiment that rainbow trout showed an improvement in observational welfare indicators such as less snout wounds, along with reduced plasma K+ concentrations. There were no negative effects on the remaining physiological and welfare indicators. This study demonstrates that co-farming sea lettuce with rainbow trout can benefit the RAS and sea lettuce without compromising fish health and welfare.

The Atlantic wolffish (Anarhichas lupus) is a cold-water species with the potential to diversify aquaculture in Northern countries. Few studies have investigated the stress physiology of Atlantic wolffish, and the current knowledge on stress in wolffish species is largely derived from the closely related spotted wolffish (Anarhichas minor). In the current study, Atlantic wolffish were exposed to handling stress mimicking common husbandry conditions in aquaculture such as repeated air exposure and net-chasing. Samples were taken prior to stress exposure (pre-stress; control) as well as 5- and 24-h post-stress. A series of primary and secondary acute stress response parameters were assessed: plasma cortisol, glucose and lactate levels, hematological indices (hemoglobin, Hb; hematocrit, Hct; mean corpuscular hemoglobin concentration, MCHC), and osmoregulatory capacity through plasma osmolality and gill Na+/K+ATPase (NKA) activity. Other secondary stress responses with implications for fish health and welfare are intestinal integrity and transport functions. These parameters were assessed using the Ussing chamber technique. The cortisol peak values were low in comparison to other fish species studied after acute handling stress and occurred as late as 24 h post-stress, suggesting that Atlantic wolffish is a slow and low cortisol responder. Plasma glucose remained stable, whereas lactate concentrations significantly decreased between 5 and 24 h after stress. There was no effect on pH, Hb, or Hct, although a significant increase in MCHC was found after 5 h and 24 h, originating from a small increase in Hb. This result suggests a minor increase in Hb synthesis after stress exposure. The intestinal integrity and transport functions as well as gill NKA-activity remained unchanged after stress exposure. In conclusion, Atlantic wolffish appears to exhibit a relatively moderate stress response, characterized by a slow and low primary stress response and minimal secondary effects following husbandry-related acute stress. These findings contribute to the understanding of the species’ potential for development as a candidate for marine, cold-water aquaculture.

The anammox process using marine anammox bacteria is a promising nitrogen removal process for recirculating aquaculture system wastewater. Marine anammox bacteria are typically found in oxygen-deficient zones and coastal areas under low phosphate concentrations. The optimal phosphate concentration for marine anammox bacteria remains unknown because most laboratory studies on these bacteria have been conducted under high phosphate concentrations. Therefore, the present study investigated the long-term effects of varying phosphate concentrations on the marine anammox bacteria, Candidatus Scalindua sp., to identify the optimal range of phosphate. Anammox activity and average growth rates were evaluated under seven phosphate concentrations (0, 0.23, 0.46, 0.68, 1.14, 6.15 [control], and 15.48‍ ‍mg P L-1) over a period of 70 days. After 50 days of reactor operation, reactor performance under phosphate concentrations ranging from 0.23 to 6.15‍ ‍mg P L-1 stabilized at 70% of total nitrogen removal efficiency, indicating the successful establishment of the anammox process. Conversely, anammox reactor performance under conditions without phosphate addition (0‍ ‍mg P L-1) and the highest phosphate concentration (15.48‍ ‍mg P L-1) did not reach 70% of total nitrogen removal efficiency, indicating a suboptimal phosphate concentration for normal anammox activity. Average growth rates calculated from total biomass samples varied from 0.0006 to 0.0012 h-1. These results indicate that Ca. Scalindua need to be kept at phosphate concentrations between 0.23 and 6.15‍ ‍mg P L-1 for optimal functioning in wastewater treatment ecosystems.

A nutritional bottleneck in salmonid aquaculture is the procurement of marine-derived compounds, such as essential amino and fatty acids, including omega-3 fatty acids, lysine, and methionine. Therefore, insects containing these compounds are highly promising as feed ingredients. The present study evaluates larvae of a "marine" insect (Coelopa frigida, the bristly-legged seaweed fly larvae, SWFL) reared on brown algae side streams as a feed ingredient for rainbow trout (Oncorhynchus mykiss). SWFL contained, on a dry matter basis, 60% crude protein (CP), 3.5% lysine, and 1.5% methionine, as well as 17% lipids, including 4% eicosapentaenoic acid and docosahexaenoic acid. Four isoenergetic (ca. 23 MJ kg-1 gross energy) and isoproteic (ca. 45% CP) feeds were evaluated in a 10-week feeding trial. The diets included a control containing 25% fish meal, a commercial reference, and two diets substituting 40% fish meal with either SWFL or partially defatted black soldier fly larvae (BSFL) meal (Hermetia illucens). SWFL-fed fish displayed higher specific growth rates (SGR) compared to BSFL-fed fish and similar SGR compared to control and reference diet-fed fish. Feed intake in SWFL-fed fish was higher than for the control and BSFL diets and similar to the reference diet. The feed conversion ratio of fish fed the BSFL and SWFL diets was higher compared to the control, suggesting lower bioavailability of both insect meals compared to fish meals. No difference in intestinal health parameters was noted between the insect diets and the control diet, indicating good intestinal health across all treatments. However, changes in electrogenic intestinal transport were observed between the SWFL and BSFL diets, illustrating the heterogeneous effect of different insect products. Overall, SWFL meal is a promising alternative marine feed ingredient, compatible with circular production systems, as it can be efficiently cultivated using marine side streams.

The Atlantic wolffish (Anarhichas lupus) is a cold-water fish with potential for aquaculture diversification. To unveil the mechanisms underlying the compromised growth in Atlantic wolffish when reared at higher temperatures, we investigated the relationship between temperature, growth rate, aerobic capacity, stress biomarkers, and gut barrier function. Juveniles acclimated to 10°C were maintained at 10°C (control) or exposed to 15°C for either 24 h (acute exposure) or 50 days (chronic exposure). Fish exposed to 15°C exhibited reduced growth, higher standard, and maximum metabolic rates compared to those at 10°C. In the chronically exposed group at 15°C, metabolic rates were lower than those of acutely exposed fish. The absolute aerobic scope exhibited no significant variation in temperatures; however, the factorial scope showed a notable reduction at 15°C in both acute and chronic exposed groups, aligning with a correlated decrease in individual growth rates. Chronic warming led to increased plasma glucose levels, indicating energy mobilization, but cortisol levels were unaffected. Furthermore, chronic warming resulted in reduced intestinal barrier function, as evidenced by increased ion permeability and a negative potential in the serosa layer. We conclude that warming elevates metabolic rates while reducing intestinal barrier function, thus increasing energy expenditure, collectively, limiting energy available for growth at this temperature from increased allostatic load. Thus, juvenile wolffish maintaining their aerobic scope under thermal stress experience slower growth. This research provides insights for improving the welfare and resilience of wolffish in aquaculture at elevated temperatures and understanding their response to increased environmental temperatures.

The objective of the present study was to investigate the optimal dietary protein requirement and the effect of varying protein levels on the growth and health of juvenile, wild-caught Atlantic wolffish, Anarhichas lupus, a promising candidate for cold-water aquaculture diversification. Six iso-energetic (ca. 18.3 MJ kg-1), fish meal-based experimental diets were formulated with crude protein levels ranging from 35% to 60%, with graded increments of 5% in a 12-week feeding trial in a recirculating aquaculture system (RAS). Weight gain, specific growth rate (SGR), and condition factor (K) were evaluated in response to dietary protein levels. Liver, muscle, and blood parameters were assessed for possible changes in protein and lipid metabolism and welfare. Overall growth was highly variable throughout the experiment on all diets, as expected for a wild population. The feed with highest in protein (60%) inclusion resulted in the highest growth rates, with an average weight gain of 37.4% ± 33.8% and an SGR of 0.31% ± 0.2% day-1. This was closely followed by feeds with 55% and 50% protein inclusion with an average weight gain of 22.9% ± 34.8% and 28.5% ± 38.3%, respectively, and an SGR of 0.18% ± 0.3% day-1 and 0.22% ± 0.3% day-1, respectively. Fish fed the high protein diets generally had increased hepatic lipid deposition (17%-18%) and reduced free fatty acid levels (3.1-6.8 μmol L-1) in the plasma relative to fish that were fed the lower protein diets (35%-45%). No effects of diet were found on plasma protein levels or muscle protein content. Furthermore, stress parameters such as plasma cortisol and glucose levels were unaffected by diet, as were plasma ghrelin levels. Overall, these results suggest that a high protein inclusion in the diet for Atlantic wolffish is required to sustain growth with a minimum protein level of 50%.

Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH4+) is biologically converted into nitrate (NO3−) via nitrite (NO2−) by nitrifying bacteria. As a result, NO3− usually accumulates in RAS and must subsequently be removed through denitrification in full RAS, or by regular water exchanges in partial RAS. The marine anammox bacteria Candidatus Scalindua can directly convert toxic NH4+ and NO2− into harmless nitrogen gas (N2) and has previously been identified as a promising alternative to the complex denitrification process or unsustainable frequent water exchanges in marine RAS. In this study, we evaluated the impact of high NO3− levels typically encountered in RAS on the performance and abundance of Ca. Scalindua in a laboratory-scale bioreactor. The bacterial composition of the granules, including the relative abundance of key nitrogen-cycling taxa, was analyzed along with the functional profile (i.e., NH4+ and NO2− removal efficiencies). For this purpose, a bioreactor was inoculated and fed a synthetic feed, enriched in NH4+, NO2−, minerals and trace elements until stabilization (Phase 1, 52 days). NO3− concentrations were then gradually increased to 400 mg·L−1 NO3−-N (Phase 2, 52 days), after which the reactor was followed for another 262 days (Phase 3). The reactor maintained high removal efficiencies; 88.0 ± 8.6% for NH4+ and 97.4 ± 1.7% for NO2− in Phase 2, and 95.0 ± 6.5% for NH4+ and 98.6 ± 2.7% for NO2− in Phase 3. The relative abundance of Ca. Scalindua decreased from 22.7% to 10.2% by the end of Phase 3. This was likely due to slower growth of Ca. Scalindua compared to heterotrophic bacteria present in the granule, which could use NO3− as a nitrogen source. Fluorescence in situ hybridization confirmed the presence of a stable population of Ca. Scalindua, which maintained high and stable NH4+ and NO2− removal efficiencies. These findings support the potential of Ca. Scalindua as an alternative filtering technology in marine RAS. Future studies should investigate pilot-scale applications under real-world conditions.

Habitat fragmentation may cut off anadromous salmonids from parts of their potential native habitat and separate previously connected populations. Understanding the consequences of this is vital for fish management and prioritization of restoration activities. Here, we show that there is a significant difference in the body morphology, physiological stress response, and aspects contributing to aerobic capacity between juvenile anadromous brown trout, Salmo trutta, collected at a downstream site and an upstream site, separated by 2 km and several challenging stream sections, in a small unfragmented stream system in western Sweden. Following a standardized stress test, there were significant differences between fish from the upstream and downstream sites (plasma cortisol concentration, plasma osmolality, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin concentration). Plasma glucose concentration did not significantly differ between fish from the two sites. Fish from the upstream site had larger spleen mass, although there was no evidence of differences in ventricle mass or proportion of compact ventricular myocardium. These physiological differences indicate local variation in stress response and highlight the importance of considering local trait variation in river management. If a section of the river becomes fragmented or degraded, and there are differences in the juveniles in different parts of the river, the consequence for the population might be larger than the proportional loss of habitat.

Haemoglobin concentration ([Hb]) assessment in fish blood has become a routine parameter to measure the health and welfare status of the animals. The original method (haemoglobincyanide method, best known as the Drabkin method) for measuring Hb in human and animals is not well suited for work outside of a laboratory setting. It is relatively time consuming, contains hazardous cyanide elements, and requires specific laboratory material. As an alternative to the Drabkin method, portable analysers have been developed for human blood, but they need to be first validated for fish blood before being used in experiments. In this study, the performance of the new HemoCue Hb 801 portable haemoglobin analyser was compared to the validated Drabkin method to determine [Hb] in three fish species. Hb readings between the two methods were not different for any of the species tested (rainbow trout, Onchorynchus mykiss, Atlantic wolffish, Anarhichas lupus, and Nile tilapia, Oreochromis niloticus). Therefore, this new portable device can be readily used to measure Hb in fish blood. Unlike the previous model from HemoCue, the Hb 201+, this device does not need an incubation time or a correction factor, representing a major gain of time and precision.

Due to the growth of aquaculture and the finite supply of fishmeal and oil, alternative marine protein and lipid sources are highly sought after. Particularly promising is the use of side streams from the fish processing industry, allowing for the recovery and retention of otherwise lost nutrients in the food production chain. The aim of the present study was to evaluate the potential of three fish processing side streams as fish feed ingredients. The side streams originated from different stages of the production chain, were used without further processing, and included sprat trimmings (heads, frames, viscera), marinated herring (fillets), and mackerel in tomato sauce (fillets and sauce). The three side streams contained moderate levels of protein (28-32% dry matter) and high levels of lipid (34-43%). The sprat trimmings included ca. 29% ash and 1.5% phosphorous, which may add value due to the high level of essential minerals but needs to be considered in feed formulations. Three diets were formulated to include 50% of each side stream replacing all fishmeal and ca. 80% of the fish oil of the control diet, which contained 35% fishmeal and 10% fish oil. The diets were evaluated in a 12-week feeding trial using rainbow trout (Oncorhynchus mykiss). Fish fed the sprat diet displayed the highest feed intake and growth and showed no negative effects on the intestinal health. The mackerel side stream displayed a good digestibility but resulted in lower growth rates compared to the sprat trimmings. Fish fed the herring diet displayed the lowest performance regarding growth, feed intake, and digestibility. They further exhibited a reduction in nutrient uptake in both proximal and distal intestines, likely contributing to the observed lower digestibility and growth, and a reduction in plasma ghrelin levels. As part of a circular approach to increase marine lipid and protein production for fish feed, the tested sprat and mackerel side streams are promising raw materials; however, additional studies using more commercial-like feed formulations are encouraged.

Assessing the nutritional status and identifying major causes of mortality in larvae experiencing varying degrees of starvation are crucial for establishing appropriate feeding protocols and enhancing the welfare of hatchery-reared fish. The black rockfish Sebastes schlegelii is an important species in aquaculture and stock enhancement efforts in China, Japan, and Korea. This study aimed to identify optimal diagnostic morphometric indicators of starvation in newly hatched (0-6 days post-hatch, DPH) and postlarval stages (27-37 DPH) of this valuable fish species through histological analyses. Our findings revealed that certain morphometric parameters, including body length, the ratios of eye diameter to head height, body height to body length, and abdomen height to body height, exhibit sensitivity to starvation during both larval and postlarval stages. Particularly, the ratios of body height to body length and abdomen height to body height emerged as the most sensitive morphometric indicators of starvation. Histological examinations of the digestive system revealed rapid alterations in the morphology of hepatic parenchymal cells, accompanied by a significant decrease in the number of lipid cells in the liver during episodes of food deprivation. Starvation induced cellular degeneration in the digestive organs, manifested by reduced heights of epithelial cells and mucosal layers in the intestine, oesophagus, and stomach, along with degeneration and separation of muscle fibers. Among these variables, the height of the intestinal submucosa and muscle layer emerged as the most sensitive indicators reflecting nutritional conditions in newly hatched larvae. In contrast, the height of intestinal striated borders and mucosal folds proved to be the most sensitive indicators in the postlarval stage. Furthermore, the height of intestinal epithelial cells and the number of lipid vacuoles in enterocytes exhibited high sensitivity to food deprivation in both newly hatched larvae and postlarvae. These findings underscore the varying resilience of fish to starvation during different developmental phases and highlight the utility of morphological sensitivity characteristics as reliable diagnostic indices for assessing nutritional status in relation to starvation or suboptimal feeding during the early developmental stages of black rockfish in hatchery-reared processes.

Empirical evidence has demonstrated that fish experience pain, and so to ensure their good welfare, it is vital that we can recognize and assess pain. A range of general, behavioral, and physiologic indicators can be used when assessing pain in fish. Many of these can be used at the tank side and are termed operational welfare indicators, whereas some require further computer or laboratory analysis. Behavioral indicators are valid and have been shown to profoundly differ between nonpainful and painful treatments in fish. However, these are not universal, and species-specific differences exist in behavioral responses to pain.

Recirculating aquaculture systems (RAS) are promising candidates for the sustainable development of the aquaculture industry. A current limitation of RAS is the production and potential accumulation of nitrogenous wastes, ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−), which could affect fish health and welfare. In a previous experiment, we have demonstrated that the marine anammox bacteria Candidatus Scalindua was a promising candidate to treat the wastewater (WW) of marine, cold-water RAS. However, the activity of the bacteria was negatively impacted after a direct exposure to RAS WW. In the current study, we have further investigated the potential of Ca. Scalindua to treat marine RAS WW in a three-phase experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2− and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2−, respectively, were achieved. In the second phase (116 days), we gradually increased the exposure of Ca. Scalindua to nitrogen-enriched RAS WW over a period of about 80 days. In the last phase (79 days), we investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Our results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2− throughout the experiment. Despite a slight decrease in relative abundance (from 21.4% to 16.7%), Ca. Scalindua remained the dominant species in the granules throughout the whole experiment. We conclude that Ca. Scalindua can be successfully used to treat marine RAS WW, without the addition of TE, once given enough time to acclimate to its new substrate. Future studies need to determine the specific needs for optimal RAS WW treatment by Ca. Scalindua at pilot scale.

Species invasions are a global problem of increasing concern, especially in highly connected aquatic environments. Despite this, salinity conditions can pose physiological barriers to their spread, and understanding them is important for management. In Scandinavia's largest cargo port, the invasive round goby (Neogobius melanostomus) is established across a steep salinity gradient. We used 12,937 SNPs to identify the genetic origin and diversity of three sites along the salinity gradient and round goby from western, central and northern Baltic Sea, as well as north European rivers. Fish from two sites from the extreme ends of the gradient were also acclimated to freshwater and seawater, and tested for respiratory and osmoregulatory physiology. Fish from the high-salinity environment in the outer port showed higher genetic diversity, and closer relatedness to the other regions, compared to fish from lower salinity upstream the river. Fish from the high-salinity site also had higher maximum metabolic rate, fewer blood cells and lower blood Ca2+. Despite these genotypic and phenotypic differences, salinity acclimation affected fish from both sites in the same way: seawater increased the blood osmolality and Na+ levels, and freshwater increased the levels of the stress hormone cortisol. Our results show genotypic and phenotypic differences over short spatial scales across this steep salinity gradient. These patterns of the physiologically robust round goby are likely driven by multiple introductions into the high-salinity site, and a process of sorting, likely based on behaviour or selection, along the gradient. This euryhaline fish risks spreading from this area, and seascape genomics and phenotypic characterization can inform management strategies even within an area as small as a coastal harbour inlet.

The overall goal of this study was to provide solutions to innovative microalgae-based technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second "stress" phase using the RAS wastewater to enhance the production of high-value metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and β-carotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values.