## 3 Results and Discussion
### Experiment 1: Effect of Light Stress and Nutrient Starvation
Moreover, lipids and carbohydrates were the most affected by the nitrogen and sulphur treatments, while changes in protein content and profile were not detected if not at the last time points, where cultures were also visibly stressed (data not shown). \begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{**Chemical Components**} & \multicolumn{1}{c}{**Wavenumber (cm\({}^{-1}\))**} & \multicolumn{1}{c}{**Peak Assignment**} & \multicolumn{1}{c}{**Reference**} \\ \hline Proteins & 1680–1630 & -C = O stretching, Amide I & [59] \\ (Green area on Figure 2) & 1530–1560 & C-N-H deformation, Amide II & \\ \hline Polyphosphate & 1265 & P = O stretching & [59] \\ (Grey area on Figure 2) & 885 & P-O-P stretching & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 2: Preprocessed FTIR-HTS spectra of _Chlorella vulgaris_ grown for 94 h on control medium (dark blue), nitrogen starvation (light blue), phosphorus starvation (green) and sulphur starvation (red) under light intensity 1800 μmol m\({}^{-2}\) s\({}^{-1}\) in experiment 1. Thus, in Figure 3A, we can observe how nitrogen and sulphur starvation influenced polysaccharide accumulation in algal cells. In these conditions the polysaccharides increased rapidly from 4 h to 16 h, then it stabilized until 32 h and, finally, it dropped slightly for cells grown under nitrogen starvation, while in sulphur starvation, we can observe a considerable decrease. Additionally, a significant increase over time in the lipids (Figure 3B) can be seen under nitrogen starvation, with the highest value indicated after 94 h. Suplhur starvation also slightly affected total lipid with the highest content at 16 h-94 h while control and phosphorous conditions were shown not to be affecting the total lipid content. Starch and lipid storage patterns in our study are in agreement with several examples existing in the literature that show how starch accumulation is a fast response to environmental changes, with an anabolic phase lasting 12-48 h followed by starch degradation and concomitant lipid synthesis [63; 64; 65]. This sequential accumulation is a mechanism that is activated when sub-optimal conditions are long-term, and the cells turn from starch to lipid synthesis, which is preferred as storage having higher energy density and stability [21; 66]. Among the treatments, phosphorous starvation did not have an effect on the biomass composition, although [PERSON] et al. in 2011 [18] reported it has a role in inducing starch accumulation. We theorise that this may be explained by the presence of polyphosphates, detected with FTIR spectroscopy, which may have been induced by high phosphorous content in the medium prior to starvation, through luxury uptake [61], whereas in the aforementioned study phosphorous in the growth medium was lower, possibly hindering IP3 uptake and resulting in stress. Nitrogen and sulphur starvation proved to both be promising triggers for polysaccharide accumulation and, despite sulphur being reported to induce up to 60% starch in the literature [18], nitrogen led to a faster and steadier accumulation pattern. It is also worthy  
Figure 3: FTIR-HTS spectra PCA score plots of (**A**) polysaccharide (1200–900 cm\({}^{-1}\)) and (**B**) lipid (3000–2800 cm\({}^{-1}\) and 1800–1700 cm\({}^{-1}\)) regions of _C. vulgaris_ biomass grown on different nutrient conditions (blue for control, red for nitrogen starvation, green for phosphorus starvation and orange for sulphur starvation) and under three light intensities in experiment 1.  
to note that, according to the literature, sulphur limitation is associated with lower cell mortality and better growth performance over a long period of time [18; 20]. However, since our work has focused on producing starch, which increased and should be harvested in a short time span, it was considered that the expected long term cell mortality and productivity loss in nitrogen starvation would not be deemed an issue for this specific purpose, and a faster and steadier accumulation would be preferable.