## 4 Discussion
[42] tested different concentrations of sodium acetate, observing that 0.25% (\(w/v\)) acetate led to carotenoids accumulation and biomass increase, while higher concentrations (>0.5% \(w/v\)) caused growth inhibition, including a reduction in the chlorophyll \(a\) content and cell number decrease, whereas, [PERSON] et al. [34] recorded that the addition of sodium acetate (0.15% \(w/v\)) did not produce any increase in cell numbers but induced cyst formation. [PERSON] et al. [43] exposed the cultures to 10 mM (0.08% \(w/v\)) sodium acetate under solar irradiation, obtaining astaxanthin yields more than two-fold compared to the control, after 6 days. Moreso, in other studies, the induction process in mixotrophy led to a decrease in cell density, and in the present study, induction conditions did not stop algal growth. This result can be explained by two factors: (i) the resuspension of the 14 days old cultures in the fresh NaNO\({}_{3}\) depleted buffered medium with the application of a moderate intensity stressor, which could have contributed to further growth; and (ii) the moderate increase of irradiance and the addition of an organic carbon source at 0.25% \(w/v\), did not reduce the cell density, elongating the growth curve and concomitantly enhancing the degree of cell encystment. Finding moderately stressful conditions is, indeed, essential to guarantee cell survival, e.g., the limitation of nitrogen instead of depletion, or exposure to a moderate increase in light [13]. The presence of nitrogen is essential for the cell division rate and for the accumulation of secondary carotenoids, and in addition, the synthesis of astaxanthin requires nitrogen for continuous protein synthesis in order to support its hyper-accumulation [44]. In the JM original recipe, the nitrogen from NaNO\({}_{3}\) accounts for 941 \(\upmu\)M, and in this study, it was eliminated from the induction medium. Meanwhile, the additional sources of nitrogen contained within the original JM recipe, including Ca(NO\({}_{3}\))\({}_{2}\)\(\times\) 4H\({}_{2}\)O (84 \(\upmu\)M N) and (NH\({}_{4}\))6 Mo7O24.4H2O (0.8 \(\upmu\)M N) were retained and their presence was considered enough to maintain background levels of cell metabolism and growth. When an NS (NaNO\({}_{3}\) in our case) remained available for the cultures, chlorophylls, and total carotenoid concentrations remained at constant levels. However, under nitrogen deficiency, a rapid chlorophyll decline and carotenoid increase per cell occurred--this was avoided in this study due to the medium refresh without the total lack of nitrogen source. As shown in the Supplementary Materials 3, the protein amount remained nearly constant, thanks to the presence of nitrogen in the medium (Table S1 in Supplementary Materials 3). Other authors found a decrease of proteins and a consequent decrease in photosynthetic activity [45, 46]. The increase in the carbohydrate content is an indicator of the carotenogenesis induction, being the initial precursors in the carotenoids pathway (Table S1 in Supplementary Materials 3) [47]. The carotenoids detected by the HPLC analysis varied from the primary carotenoids commonly found in _H. pluvialis_, such as lutein and \(\beta\)-carotene, to the less frequent antharaxanthin and canthaxanthin [48]. The significant increase of the carotenoid/chlorophyll ratio between green and red phases of cells grown with urea and NaNO\({}_{3}\) was mainly caused by a concomitant reduction of chlorophylls after the induction and a concomitant increase in the carotenoids value (Figure 3). On the other hand, it was observed that cultures grown in FeNO\({}_{3}\) and NH\({}_{4}\)Cl, started to encryst before the induction process, from day 10 and 12, respectively, which was not observed in the cultures grown with either urea or NaNO\({}_{3}\). The presence of astaxanthin in the green phase of the FeNO\({}_{3}\) and NH\({}_{4}\)Cl treatments confirmed the presence of red cysts already formed before the induction process, as observed under the microscope.