## 4 Discussion  
This study demonstrates the possibility of successfully growing the strain _H. pluvialis_ CCAP 34/1D in urea as the principal NS, reaching a high biomass yield and using a low-cost renewable source of nitrogen (Figure 1). The stressors used in our study had a significant effect on carotenoid accumulation, with a minimal negative impact upon the cell number obtained during vegetative growth (Figure 2). Namely, all the treatments, barring FeNO\({}_{3}\), increased the astaxanthin accumulated and all, except NH\({}_{4}\)Cl, showed a further increase in the cell number following exposure to stressors (Table 1). Previous reports showed that the best performance of _H. pluvialis_ in terms of growth rate, cell density, and long vegetative stage, without premature encysting, is usually achieved supplying NaNO\({}_{3}\) and/or KNO\({}_{3}\) among possible NS [32; 33], with results differing among the strains and the nitrogen concentrations. In this study, cells grown in urea had a significantly greater growth rate (\(\mu\) = 0.4) (\(p\) < 0.0001) and maximum cell density (4.98 \(\times\) 10\({}^{5}\) cells/mL, \(p\) < 0.0046) when compared to cultures grown in any other source of nitrogen tested. Studies investigating growth in different NS are few and have reported conflicting results. For instance, in [PERSON] et al. [34] when comparing different NS including urea, a rate of 0.47 d\({}^{-1}\) was achieved in NaNO\({}_{3}\) (a maximum cell density of 1.4 \(\times\) 10\({}^{5}\) cells/mL) while 0.44 d\({}^{-1}\) was in urea (a maximum cell density of 4.5 \(\times\) 10\({}^{4}\) cells/mL), data that was considerably lower than the highest cell density reached in our study. Additionally, [PERSON] et al. [35] reached the maximum growth rate (0.26 d\({}^{-1}\)) in NaNO\({}_{3}\) and found out that their strain did not grow under urea as sole NS. Many studies comparing these different NS, found _H. pluvialis_ growing optimally in sodium nitrate, which is three and eight times more expensive than  
Figure 3: The letters indicate significant differences in the ratios between different nitrogen treatments during either the green or red growth phases (\(p\) < 0.05), while the differences between the green and the red phase in each treatment are denoted by \({}^{\star}\) (\(p\) < 0.05). ammonium and urea, respectively [36]. In another study, [PERSON] et al. [37] calculated that NaNO\({}_{3}\) accounts for 83.3% of the total chemical cost of all those used to make BG11 medium. Urea (CO(NH\({}_{2}\))\({}_{2}\)) is an organic compound, thus it provides additional C, which is important for the green-red transition and the accumulation of astaxanthin. As a low-cost N-source, urea could enhance the economics of the whole process. The replacement of sodium nitrate with urea has been attempted for other species cultivation, e.g., _Spirulina platensis_ with the same purpose of making its cultivation more cost-effective by reducing the input cost without sacrificing the production efficiency [38, 39]. Upon improving the green phase of cultivation in terms of biomass productivity, the astaxanthin induction was achieved by applying a mix of stressors at moderate intensity and assessed through a previous screening (Supplementary Materials 2), where the aim was to find the compromise between the highest cell concentration and the highest carotenoids accumulation. This was attained using modified JM (without the addition of extra NS in the form of NaNO\({}_{3}\) but keeping the original recipe containing Ca(NO\({}_{3}\))\({}_{2}\) and (NH\({}_{4}\))6 Mo7O24.4H2O) with the addition of 0.25% (\(w/v\)) sodium acetate and exposed to 250 umol photons m\({}^{-2}\) s\({}^{-1}\) for 7 days. Previous studies on the nutrition of _H. pluvialis_ have shown that acetate appears to be an important carbon source, enhancing both growth and carotenogenesis [40, 41]. This was confirmed in our study and was used in the astaxanthin induction medium. [PERSON] et al.