# Development of an Organic Culture Medium for Autotrophic Production of _Chlorella vulgaris_ Biomass
## 3 Results and Discussion
### Screening of Organic Substrates for Autotrophic Production of C. vulgaris  
A preliminary trial was carried out at laboratory scale (2 L bubble column reactors) using the four OS and the synthetic culture medium at a normalized N concentration of 2 mmol L\({}^{-1}\) as control (Figure 1). The results obtained revealed that _C. vulgaris_ grew best in Bioscape and EcoMix4, reaching a final biomass concentration of \(\sim\)0.6 g L\({}^{-1}\), similar to that obtained with the synthetic growth medium (control; \(p\geq 0.05\)). On the other hand, significantly slower growth performance was observed in cultures grown with Nutrimais and Nutriverde (\(p<0.05\)), which reached about half of the final biomass concentration (\(\sim 0.31\) g L\({}^{-1}\)) obtained with the other OS. Although Nutriverde and Nutrimais \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Element** & **Bioscape** & **Nutrimais** & **Nutriverde** & **EcoMix4** & **Synthetic Medium** & **Local Water** \\ \hline N (mmol L\({}^{-1}\)) & 1113.9 & 11.5 & 3.6 & 63.8 & 2.0 & \textless{}LOD \\ P (\(\mu\)mol L\({}^{-1}\)) & 864.2 & 167.3 & 135.4 & 566.4 & 180 & \textless{}LOD \\ Fe (\(\mu\)mol L\({}^{-1}\)) & 101.4 & 59.1 & 34.6 & 66.8 & 20 & 15.4 \\ K (\(\mu\)mol L\({}^{-1}\)) & 15,095.7 & 6581.5 & 3492.7 & 38,320.2 & 0 & 114.3 \\ Mg (\(\mu\)mol L\({}^{-1}\)) & 2950.1 & 349.8 & 231.0 & 282.1 & 0.04 & \textless{}15.5 \\ Zn (\(\mu\)mol L\({}^{-1}\)) & 1.0 & 0.8 & 0.3 & 0.7 & 0.02 & \textless{}LOD \\ Cu (\(\mu\)mol L\({}^{-1}\)) & 0.4 & 0.6 & 0.3 & 0.6 & 0.002 & \textless{}LOD \\ Co (\(\mu\)mol L\({}^{-1}\)) & \textless{LOD} & \textless{LOD} & \textless{LOD} & \textless{LOD} & 0 & 0.4 \\ Mo (\(\mu\)mol L\({}^{-1}\)) & \textless{LOD} & \textless{LOD} & \textless{LOD} & \textless{LOD} & 0.02 & 0.4 \\ Mn (\(\mu\)mol L\({}^{-1}\)) & 28.6 & 0.9 & 0.5 & 0.1 & 0.02 & \textless{}LOD \\ \hline \end{tabular} \(\text{LOD}=\text{limit of detection}\).  
\end{table} 
Table 1: Elemental composition of different organic substrates solubilized in water (300 g L\({}^{-1}\)) at 60 \({}^{\circ}\)C, and synthetic culture medium (Guillard’s F/2). The synthetic medium values are presented in the final concentration used for autotrophic growth. The local groundwater used for microalgae cultivation was also analysed. displayed an N:P ratio closer to the optimum, the higher volume of both culture media needed to reach an N content of 2 mmol L\({}^{-1}\) augmented its colour and, therefore, the light attenuation of the final medium. As a result, the photosynthetic capacity of cultures in this specific medium might have been compromised. Alternatively, these culture media might lack some micronutrients essential for microalgal growth or contained compounds that inhibited cell division. Higher biomass productivities were obtained using EcoMix4 (\(0.038\pm 0.003\) g L\({}^{-1}\) d\({}^{-1}\)), Bioscape (\(0.033\pm 0.005\) g L\({}^{-1}\) d\({}^{-1}\)), and synthetic (\(0.037\pm 0.001\) g L\({}^{-1}\) d\({}^{-1}\)) culture media without significant differences among them (\(p\geq 0.05\); Table 2). _C. vulgaris_ grown with Nutrimais displayed a productivity of \(0.017\pm 0.002\) g L\({}^{-1}\) d\({}^{-1}\), significantly lower than that achieved in the aforementioned media, but significantly higher than those obtained with Nutriverde (\(0.009\pm 0.002\) g L\({}^{-1}\) d\({}^{-1}\); \(p<0.05\)). Similarly, cultures grown with EcoMix4 (\(0.122\pm 0.009\) d\({}^{-1}\)), Bioscape (\(0.111\pm 0.011\) d\({}^{-1}\)), and synthetic (\(0.123\pm 0.001\) d\({}^{-1}\)) culture media displayed higher specific growth rates (\(\mu\)), but identical among them (\(p\geq 0.05\)), followed by Nutrimais (\(0.075\pm 0.004\) d\({}^{-1}\)), and Nutriverde with the lowest \(\mu\) (\(0.036\pm 0.008\) d\({}^{-1}\); \(p<0.05\)). Regarding reagents' cost, synthetic and Bioscape were the most competitive media with 1.32 and 1.56 \(\upmu\)kg of biomass produced. Nutrimais and EcoMix4 followed with a cost of 14.23 and 21.24 \(\upmu\)kg of biomass produced.