## 4 Discussion
Such environmental features, as well as others such as salinity and nutrient availability, can influence the synthesis and inhibition of protein [67]. Apart from this, in AQUA1, the entrapment of algae in the aeration system affected the growth of the cultivated _Ulva_ spp., which also contributed to the decrease on protein content. Previous works reported an increment in protein content (20-35%) in cultivated _Ulva_ spp. [36, 66, 68, 46]. Alternatively, [PERSON] et al. registered protein contents (5.23-9.89% dry weight) similarly to those obtained in this study and observed no significant seasonal variation on protein contents [37]. A similar trend was registered for pigments contents. Photosynthetic parameters in algae have been shown to respond to environmental changes faster than the content of C and N [69]. The levels of chlorophyll \(a\) and \(b\) and carotenoids tended to decrease throughout the cultivation periods in AQUA1 and AQUA2, results that are in accordance with the reduction of oxygen production capability of _Ulva_ spp. cultivated, for 28 days, in fish effluent. Macroalgae productivity is known to change depending on the metabolic expression and adaptations to the environmental variability to which they are exposed [70]. In fact, ecotoxicological studies show that stress factors can inhibit respiration rates [71; 72; 73], and _Ulva_ species have been used as a quick response environmental bioindicator [74]. Stress factors, such as algae poor circulation in cultivation system (AQUA1) and consequent lower exposure to light, may contribute to the reduction on pigments levels. The content of chlorophyll can determine the potential photosynthesis [75] and therefore the production of oxygen [76]. Biotic and abiotic factors, such as temperature, salinity, irradiance, high nutrient supply, zoospore formation, growth, can influence photosynthetic response [51; 69; 77; 78; 79] and stimulate or inhibit the production of pigments. As for algae growth, flow rate and stocking density are two important parameters that can influence the protein content of cultivated macroalgae and photosynthesis response [36]. The combination of high flow rates and stocking density favours the nitrogen-uptake since macroalgae have access to higher loads of nutrients and are less exposed to light [36]. Therefore, the operation on continuous flow is a pivotal improvement to the IMTA-pilot system. A comparison between _Ulva_ spp. grown in an estuarine area and _Ulva_ spp. grown in a coastal area (more exposed to tidal currents, higher salinity and with less nutrient availability) was also conducted. In terms of growth, _Ulva_ spp. from coast required a longer period (7 days) to adapt to the cultivation medium (lower in salinity and higher in nutrient load comparing to seawater) than _Ulva_ spp. from estuary. After 7 days, SGRs and yields were within the same range. Enriched 815N signatures were found in estuarine _Ulva_ spp., as expected. Protein and pigments content throughout the cultivation assays did not differ statistically. NRE was also similar, although at the end of the cultivation period it was observed a higher reduction of [PO4\({}^{3-}\)] in tanks with estuarine _Ulva_ spp. and of [NH4\({}^{+}\)] in tanks with coastal _Ulva_ spp. A previous work reported differences regarding the growth performance of _Ulva_ species from seawater or brackish water habitats, denoting a better performance for _Ulva_ naturally occurring in brackish water [80]. Similar to this work, no significant differences for nutrient uptake were reported. In one's case, the origin of the algae (from the coast or the estuary) seems not to be a determining factor to ensure the successful production of algae in IMTA systems. For the trials performed, the pilot system implemented required low energy and low maintenance to properly work.