## 5 Conclusions  
This study highlights the advantages of a modified JM medium where urea is the primary NS, not only for the enhanced growth of _H. pluvialis_ but also for the low-cost sustainable source of nitrogen which could be economically employed at the industrial level. It also scans a broader image, including an innovative method to induce astaxanthin by a combination of abiotic factors acting as stressors of moderate intensity without the loss of biomass. The combination of the following factors, including nitrogen limitation, the increase of light, and sodium acetate addition, was found to be the best. Different nitrogen sources showed a different mosaic of pigments that converged in the astaxanthin accumulation after exposure to the induction conditions. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12031261/s1](https://www.mdpi.com/article/10.3390/app12031261/s1)]([https://www.mdpi.com/article/10.3390/app12031261/s1](https://www.mdpi.com/article/10.3390/app12031261/s1)), Figure S1: Growth rate of cultures in each different medium 3N-BBM+V+V, BG11, MWC, JM at 6, 6, 5, 7, 7, 75, 8 pHs; Figure S2: _H. pluvialis_ 34/1D grown in different N/P ratios: Control with NaNO3 2.6/1; 1/1; 5/1; 10/1; Figure S3: Effect of sodium acetate 0%, 0.25%; 0.5%; 1%; 1%; 1.5%; 1.75%; 2% on carotenoids accumulation (OD\({}_{480}\)) and cell density (OD\({}_{750}\)) under ML and HL; Figure S4: _H. pluvialis_ 34/1D calibration curve used for cell quantification; Figure S5: Representative HPLC chromatograms of _H. pluvialis_ green (A) and red (B) phases of growth, when cultivated under FeNO\({}_{3}\), NaNO\({}_{3}\), NH\({}_{4}\)Cl, or Urea as the principal nitrogen source in Jaworski's Media; Table S1: Biochemical table reporting the mean value (% DW) \(\pm\) S.D. within samples (\(n=3\)); Table S2: Ordinary one-way ANOVA and [PERSON]'s multiple comparison test, with a single pooled variance were performed for _H. pluvialis_; Table S3: The relative change of _H. pluvialis_ CCAP 34/1D cell number based on the number of cells/mL at the beginning and end of the 7 day induction process; Table S4: Quantitative pigment composition (in mg/g DW), obtained by HPLC analysis, of _H. pluvialis_ cells at the end of the green phase, in cultures previously supplied with equimolar concentrations of nitrogen from different sources; Table S5: For cathaxanthin, a _t_-test was used to determine if there is a significant difference between the means of two groups, NaNO\({}_{3}\) and FeNO\({}_{3}\); Table S6: Quantitative pigment composition (in mg/g DW), obtained by HPLC analysis, of _H. pluvialis_ cells at the end of the red phases stationary phase, in cultures previously supplied with equimolar concentrations of nitrogen from different sources; Table S7: Pigments variation between the green and the red phase; Table S8: Ordinary two-way ANOVA and Sidak's multiple comparison test, with individual variances computed for each comparison, were performed; Table S9: Carotenoids/Chlorophylls ratio analysis between the green and the red phase. **Author Contributions:** [PERSON], [PERSON] and [PERSON] conceived and planned the project, and designed the experiments. [PERSON] conducted the experiments, and generated, collated, and processed the data. [PERSON], [PERSON], [PERSON] and [PERSON] interpreted the results. [PERSON] wrote the manuscript. [PERSON], [PERSON], [PERSON] and [PERSON] reviewed and edited the manuscript. [PERSON], [PERSON] and [PERSON] supervised the project and acquired the funding. All authors have read and agreed to the published version of the manuscript. **Funding:** The project leading to this application has received funding from the Erasmus+/KA1 2017 with a personal scholarship for [PERSON]. The authors acknowledge the financial support from the ABACUS project, grant agreement No. 745668, funded by the BBI-JU under the European Union's Horizon 2020 R&I programme.