# Valuable Nutri from _Ulva rigida_: Modulation by Seasonal and Cultivation Factors
## 3 Results and Discussion  
### Crude Protein  
The collected data reflected significant seasonal variations of protein content common to all tested samples (Figure 1). The maximum levels were obtained during autumn/winter (17.6 \(\pm\) 1.2 to 25.8 \(\pm\) 0.7% DW), whereas the lowest were observed in spring, particularly in May (7.6\(-\)11.8% DW). Similar trends were found by [PERSON] et al. [31], who reported slightly higher protein levels during fall/winter seasons corresponding to 30% DW for _Ulva_ spp. cultivated in a land-based intensive culture system in Pacific Northwest, albeit without statistical differences [31]. Furthermore, some studies have reported higher protein levels in farmed macroalgae in comparison to their wild counterparts, as aquaculture effluent is richer in nutrients, especially dissolved nitrogen, than seawater [32,33]. Indeed, this fact could in part justify that the maximum value found in our study was slightly higher than those reported for several specimens of wild _Ulva_ from Northwest Europe (25.8 vs. 23.6% DW) [34, 35, 36]. The annual protein content fluctuations observed in our samples were comparable to those assessed by [PERSON] et al. [34] for wild _U. rigida_ collected at the Brittany coast in France. Interestingly, [PERSON] et al. [26] reported the opposite trend for protein variation of wild _U. rigida_ collected at the west coast of the Cape Peninsula in South Africa, with maximum protein contents being registered during March and April. Still, although these results might seem discrepant, in both cases, the minimum levels of protein were found in seasons characterized by intense sunlight and high temperatures. In fact, the exposure to light saturating conditions is known to reduce nitrogen uptake in macroalgae tissue, resulting in decreased protein levels [37]. In contrast, the lower durability of the days and temperatures during the autumn/winter months seem to promote the uptake of nitrogen and the consequent synthesis of protein. However, one must not forget that other factors might affect the levels of protein in seaweeds. In this respect, [PERSON] et al. [38] registered a positive correlation with nitrogen content and a negative correlation with water temperature and salinity, in _Gracilaria cervicornis_ and _Sargassum vulgarte_ from Brazil [39]. In fact, the water temperature and salinity of tanks from different conditions during our study showed a decreasing tendency of these parameters from November to January and February, respectively (Supplementary Materials Figure S1). This might be partly associated with the high protein levels obtained in this period. However, further data would be needed in order to assess the influence of such abiotic factors. Our results also indicate that protein content in the green macroalgae grown under an IMTA regime could be changed by setting flow rate (Fr) and stocking density (Sd), or both parameters. Between these two, flow rate was the most relevant factor and macroalgae submitted to high flow rate (H\({}_{\text{Fr}}\)) showed in general higher protein content than those  
Figure 1: Seasonal variation of protein content in _U. rigida_ produced by IMTA under different conditions of flow rate, high (\(\Circle\)) and low (\(\blacktriangle\)); and of stocking density, high (full lines) and low (dotted lines). Standard deviations are represented as bars (n = 3, in triplicate). cultivated under low flow rate (L\({}_{\text{Fr}}\)) conditions. In more detail, the combination of H\({}_{\text{Fr}}\) and high stocking density (H\({}_{\text{Sd}}\)) yielded the best results, whereas the opposite was achieved for L\({}_{\text{Fr}}\) with low stocking density (L\({}_{\text{Sd}}\)) conditions. Still, significant differences were found among samples cultivated under fixed Fr and variable Sd.