## 2 Materials and Methods
### Experimental Design  
#### 2.3.1 Experiments at Laboratorial Scale  
Cultivation Assays  
Two experimental assays were conducted in laboratorial and controlled conditions. The experiments were conducted with specimens collected at _Mondego_ estuary in different seasons (late spring and late autumn) to assess the influence of seasonality in _Ulva_ spp. growth and ability to remove nutrients. Both assays were performed in a climate room, with an average temperature of 19 \({}^{\circ}\)C and a photoperiod cycle of 16 h day: 8 h night with constant light (1 Klux). The growth system was composed by six 10 L rectangular tanks with an aeration system mimicking the pilot-IMTA system settled in the local aquaculture. For experiment LU1 (performed in late spring), specimens of _Ulva_ spp. (density \(\approx\) 1.8 g/L) were cultivated (free in tank) in 8 L of fish effluent (pre-filtered with 38 \(\upmu\)m mesh) for 24 days. The experiment was performed in triplicate. Furthermore, three tanks with fish effluent and no algae addition were used as control. Effluent salinity registered an average value of 33.18 \(\pm\) 1.3 PSU and 7.88 \(\pm\) 0.33 for pH, during the experiment, and oxygen percentage was maintained above 75%. Effluent was changed every 6 days. During each renewal, physico-chemical parameters were registered, algae biomass was drained and weighed, and aliquots of effluent were taken from each tank (d6, d12, d18, d24) as well as from the recently collected effluent (d0, d6, d12, d18, d24), for nutrient analysis. For experiment LU2 (performed in late autumn), the cultivation assay was performed as described in LU1: _Ulva_ spp. was cultivated in three tanks (biomass density of 1 g/L in 8 L of effluent, 3 replicates) and fish effluent and no algae addition were used as control (3 replicates). _Ulva_ spp. was allowed to grow for 8 days. Effluent salinity registered an average value of 31.92 \(\pm\) 1.40 PSU and 7.6 \(\pm\) 0.5 for pH, during the experiment, and oxygen percentage was maintained above 75%. Effluent replacement was performed after 4 days. Aliquots of effluent were taken from each tank every day for nutrient analysis to assess the daily depletion of nutrient. In addition, during medium renewal (d0, d4 and d8), aliquots from the recently collected effluent were also collected, physico-chemical parameters were measured, and algae biomass was weighed. After each effluent renovation (in LU1 and LU2), the initial density of algal biomass was maintained and the remaining part of _Ulva_ spp. was dried at 40 \({}^{\circ}\)C, powdered using a ball mill mixer (MM400, Retsch, Dusseldorf, Germany) and stored until further analysis. Oxygen Production-Consumption  
Two stocks of _Ulva_ spp. (algal density 30 g/L. collected in late autumn in _Mondego_ estuary) were maintained separately, for 28 days, in seawater (32 \(\pm\) 3 PSU, volume 8 L) and in fish effluent (17 \(\pm\) 2 PSU, volume 8 L). Constant aeration and light (1 KLux) was assured, as well as, an average room temperature of 19 \({}^{\circ}\)C and a photoperiod cycle of 12 h day: 12 h night. Media renewal was performed every 4 days to avoid nutrient depletion. Oxygen production and consumption by _Ulva_ spp. grown in different media was assessed by periodical measurement of oxygen levels. For that, fifteen glass flasks (500 mL) were filled with autoclaved seawater, previously filtered (0.5 \(\upmu\)m porosity glass fiber filter), and the initial oxygen level was measured. Then, one gram of _Ulva_ spp. cultivated in seawater and one gram of _Ulva_ spp. cultivated in fish effluent were placed separately in autoclaved seawater (5 replicates per each variable). Autoclaved seawater without algae addition (5 replicates) was used as control. All replicates' containers were randomly distributed and sealed with Bemis(tm) Parafiltm(tm) to reduce gas exchanges. Then, oxygen level was measured every 4 h for a total period of 24 h in all flasks.