## 4 Conclusions  
Medium optimization through surface response methodology revealed increments of 44.9-51.2% in _Chlorococcus amblystomatis_-specific growth rates and 36.6-40.8% in biomass production under heterotrophy and photoautotrophy, respectively. Heterotrophic cultivation registered a 5.5-fold increase in biomass production compared to photoautotrophic cultivation at the laboratory scale, supporting the two-stage approach for further industrial \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Pigments (mg\(\cdot\)g\({}^{-1}\))**} & \multicolumn{2}{c}{**Hetetrotrophy**} & \multicolumn{2}{c}{**Photoautotrophy**} \\ \cline{2-5}  & **Basal** & **Optimized** & **Basal** & **Optimized** \\ \hline Neoxanthin & 0.52 \(\pm\) 0.05 \({}^{\text{c}}\) & 0.79 \(\pm\) 0.06 \({}^{\text{c}}\) & 1.46 \(\pm\) 0.12 \({}^{\text{b}}\) & 3.66 \(\pm\) 0.33 \({}^{\text{a}}\) \\ Violaxanthin & 0.02 \(\pm\) 0.00 \({}^{\text{c}}\) & 0.06 \(\pm\) 0.01 \({}^{\text{c}}\) & 0.51 \(\pm\) 0.03 \({}^{\text{b}}\) & 0.75 \(\pm\) 0.06 \({}^{\text{a}}\) \\ Lutein & 1.23 \(\pm\) 0.10 \({}^{\text{c}}\) & 1.60 \(\pm\) 0.11 \({}^{\text{c}}\) & 4.32 \(\pm\) 0.15 \({}^{\text{b}}\) & 5.27 \(\pm\) 0.37 \({}^{\text{a}}\) \\ \(\beta\)-carotene & 0.81 \(\pm\) 0.16 \({}^{\text{b}}\) & 4.15 \(\pm\) 0.22 \({}^{\text{a}}\) & 5.37 \(\pm\) 0.44 \({}^{\text{a}}\) & 5.84 \(\pm\) 0.98 \({}^{\text{a}}\) \\ Chlorophyll \(a\) and \(b\) & 8.53 \(\pm\) 0.35 \({}^{\text{c}}\) & 14.59 \(\pm\) 0.74 \({}^{\text{b}}\) & 12.88 \(\pm\) 0.61 \({}^{\text{b}}\) & 29.32 \(\pm\) 0.39 \({}^{\text{a}}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 10: Pigment profile in mg\(\cdot\)g\({}^{-1}\) of _Chlorococcus amblystomatis_ biomass grown with hetero- and photoautotrophic basal and optimized heterotrophic media. Different letters indicate significant differences between media. Values are given as mean \(\pm\) standard deviation (\(n=3\)). high-quality biomass production with a reduced scale-up time. Moreover, optimizing the cultivation medium towards biomass production has shown significant differences in the biochemical composition of the resultant biomass in comparison to the biomass obtained from the basal media cultivation. The optimized biomass revealed an interesting composition that is rich in protein (61.49-73.45% of dry weight (DW)), registering higher content than those reported for the biomass obtained from the basal media (33.49-56.67% of DW) in heterotrophy and photoautotrophy, respectively, representing an alternative protein source. _C. amblystomatis_ was also demonstrated to be a promising source of polyunsaturated fatty acids (51.64-72.82% of total fatty acids) and might be a natural food colorant due to its high carotenoids content (1.60-5.27 mg\(\cdot\)g\({}^{-1}\) of DW of lutein and 4.15-5.84 mg\(\cdot\)g\({}^{-1}\) of DW of \(\beta\)-carotene) in heterotrophic and photoautotrophic optimized biomasses, respectively, being a sustainable alternative as a source of nutrients to address the increasing world food and feed demands. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13042089/s1](https://www.mdpi.com/article/10.3390/app13042089/s1)]([https://www.mdpi.com/article/10.3390/app13042089/s1](https://www.mdpi.com/article/10.3390/app13042089/s1)), Table SI: Calibration curves of pigments standards; Table S2: Experimental layout of the Plackett-Burman design and results of heterotrophic _Chlorococcus amblystomatis_-specific growth rates (\(\mu\)). Conceptualization, N.C., H.P., J.L.S., L.G., J.V. ; methodology, N.C., H.P., P.S.C.S., M.M.C., G.E.S., I.G., M.T., J.L.S., L.G., J.V. ; validation, N.C., H.P., P.S.C.S., A.B., H.C., J.I.S., L.G., J.V. ; formal analysis, N.C., H.P., P.S.C.S., J.L.S., L.G., J.V. ; writing--original draft preparation, N.C., H.P., G.E.S., I.G., M.T., J.L.S., L.G., J.V. ; writing--review and editing, N.C., H.P., P.S.C.S., M.M.C., G.E.S., I.G., M.T., A.B., H.C., J.L.S., L.G., J.V. ; supervision, J.L.S., L.G., J.V. ; funding acquisition, J.L.S., L.G., J.V.