## 3 Results and Discussion  
### Experiment 1: Effect of Light Stress and Nutrient Starvation  
High-throughput cultivations are based on utilising microtiter plates or micro bioreactor based systems, where it is possible to grow simultaneously many microbial species and strains under different conditions [36; 37; 55; 56; 57; 58]. Such systems, as, for example, Bioscreen C, MTPS-Duetz or microbioreactors have not been evaluated for screening phototrophic microalgae because they often have limited light transparency which does not allow optimal lighting during cultivation. For this reason, we used a traditional shake flask system combined with the high-throughput FTIR spectroscopy to assess qualitatively how nutrient and light treatments affect the metabolism and composition of _C. vulgaris_ biomass over time. FTIR detects the IR radiation, which is absorbed by specific molecular bonds, generating a spectrum that can be considered a chemical fingerprint of the biomass. The characteristic absorbance peaks of the different bonds, and the corresponding molecular components and spectral regions are detailed in Table 1 and Figure 2. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Chemical Components** & **Wavenumber (cm\({}^{-1}\))** & **Peak Assignment** & **Reference** \\ \hline \multirow{3}{*}{Polysaccharides (Red area on Figure 2)} & 3500–3200 & O-H stretching & [59] \\  & 1200–1000 & C-O and C-O-C stretching & \\  & 1080 & C-O-H bending in starch & [12; 60] \\  & 1155 & C-O stretching in starch & \\ \hline \multirow{6}{*}{Lipids (Blue area on Figure 2)} & 3010 & \multicolumn{2}{c}{=C-H stretching} \\  & 2955 & \multicolumn{2}{c}{-C-H (CH3) stretching} \\ \cline{1-1}  & 2925 & \multicolumn{2}{c}{\textgreater{}CH2 of acyl chain} & \\ \cline{1-1}  & 2855 & \multicolumn{2}{c}{-C-H (CH2) stretching} & \\ \cline{1-1}  & 1745 & \multicolumn{2}{c}{-C = O stretching in esters} & \\ \cline{1-1}  & 1465 & \multicolumn{2}{c}{-C-H (CH2, CH3) bending} & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Peak assignments of the FTIR-HTS spectra in the algal cells in experiment 1 (main chemical components are marked on Figure 2). Figure 2 shows the spectral emission of cultures grown under different nutrient conditions using data from the last time point at a high light intensity and is an example of how the treatments generate different chemical fingerprints. The spectrum shows a marked difference in the starch peaks at the wavelengths of 1080 and 1155 cm\({}^{-1}\), with nitrogen starvation giving the highest signal, whereas the other polysaccharide region is homogeneous between treatments, thus we consider starch accumulation as a storage compound to be the main driver for differences in polysaccharide region, being starch the most abundant carbohydrate in _C. vulgaris_[12]. In the lipid region, there is again a strong signal for the nitrogen deprived culture which has a specific pattern and a higher peak at 1745 cm\({}^{-1}\) related to ester bond stretching in acyl glycerides. Proteins have a higher signal at control and P- conditions though the peaks are similar for all treatments. Finally, the signals at 885 and 1265 cm\({}^{-1}\) have been attributed to polyphosphates which are known to be accumulated in microalgae, including _C. vulgaris_, as inorganic IP3 granules [61; 62]. As confirmed by further principal component analysis (PCA), the areas with higher variability are the lipid and polysaccharide regions. Their changes over time are detailed in Figure 3. The PCA results have shown significant chemical changes in the algal cells grown under different nutrient conditions, although no changes were detected for the different light intensities. Generally, among all tested conditions, nitrogen and sulphur starvation showed the most pronounced effect on the algal cellular chemical profile, while control and phosphorus did not show any significant effect.