## 3 Results and Discussion  
### Preliminary Screening Using Plackett-Burman Design  
The Plackett-Burman experimental layout was obtained with Minitab(r) v.20 software, and the respective results of the specific growth rates are described in Table S2 in Supplementary Materials. The results of the variance analysis are presented in Table 1.  
The nitrogen sources, NaH\({}_{2}\)PO\({}_{4}\cdot\)H\({}_{2}\)O + K\({}_{2}\)HPO\({}_{4}\), and CuSO\({}_{4}\cdot\)5H\({}_{2}\)O were the statistically significant factors (\(p<0.05\)) affecting the _C. amblystomatis_ heterotrophic-specific growth rate, suggesting that the remaining factors were not limiting culture growth. Moreover, since the categoric factor registered a negative effect, the lower-level factor of (NH\({}_{2}\))\({}_{2}\)CO was the most appropriate nitrogen source.  
The model registered a multiple correlation coefficient (\(R^{2}\)) of 0.951, indicating that more than 95% of the data in this screening could be explained by this model. Furthermore, an \(F\)-value of 7.70 and a \(p\)-value lower than 0.05 also support the significance of the model and the fit between the data and the model used.  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Factor** & **Low Level (\(-\))** & **High Level (\(+\))** & **Effect** & \(t\)**-Value** & \(p\)**-Value** \\ \hline Nitrogen source & (NH\({}_{2}\))\({}_{2}\)CO & (NH4)\({}_{2}\)SO\({}_{4}\) & \(-\)0.006 & \(-\)7.33 & 0.000 \\ \hline (NH\({}_{2}\))\({}_{2}\)CO & \multirow{2}{*}{20.00 mM} & \multirow{2}{*}{60.00 mM} & \multirow{2}{*}{\(-\)0.002} & \(-\)2.24 & 0.067 \\ (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & & & & & \\ \hline NaH\({}_{2}\)PO\({}_{4}\cdot\)H\({}_{2}\)O + & \multirow{2}{*}{10.00 mM} & \multirow{2}{*}{100.00 mM} & \multirow{2}{*}{\(-\)0.005} & \(-\)6.05 & 0.001 \\ K\({}_{2}\)HPO\({}_{4}\)\({}^{*}\) & & & & & \\ \hline CaCl\({}_{2}\cdot\)2H\({}_{2}\)O & 1.00 mM & 5.00 mM & 0.000 & 0.45 & 0.667 \\ \hline MgSO\({}_{4}\cdot\)7H\({}_{2}\)O & 1.00 mM & 10.00 mM & \(-\)0.002 & \(-\)2.43 & 0.051 \\ \hline FeSO\({}_{4}\cdot\)7H\({}_{2}\)O & 0.05 mM & 0.50 mM & 0.001 & 1.69 & 0.142 \\ \hline ZnSO\({}_{4}\cdot\)7H\({}_{2}\)O & 0.50 mM & 2.50 mM & 0.000 & \(-\)0.08 & 0.935 \\ \hline Cl\({}_{2}\)Co-6H\({}_{2}\)O & 0.00 mM & 0.04 mM & 0.000 & \(-\)0.52 & 0.621 \\ \hline Na\({}_{2}\)MoO\({}_{4}\cdot\)2H\({}_{2}\)O & 0.00 mM & 0.10 mM & 0.000 & \(-\)0.12 & 0.905 \\ \hline MnSO\({}_{4}\cdot\)H\({}_{2}\)O & 0.20 mM & 1.50 mM & 0.000 & 0.29 & 0.778 \\ \hline NiCl\({}_{2}\cdot\)6H\({}_{2}\)O & 0.00 mM & 0.02 mM & 0.001 & 1.37 & 0.221 \\ \hline CuSO\({}_{4}\cdot\)5H\({}_{2}\)O & 0.00 mM & 0.02 mM & \(-\)0.002 & \(-\)2.88 & 0.028 \\ \hline H\({}_{3}\)BO\({}_{3}\) & 0.50 mM & 1.50 mM & 0.000 & \(-\)0.44 & 0.673 \\ \hline Vitamins Mix ** & 0.5 dose & 3 doses & 0.000 & 0.22 & 0.835 \\ \hline Temperature & 26 \({}^{\circ}\)C & 30 \({}^{\circ}\)C & 0.000 & 0.55 & 0.601 \\ \hline Model: \(R^{2}=0.951;\)\(R_{\rm adj}^{2}=0.827;\)\(R_{\rm pred}^{2}=0.597;\)\(p\)-value = 0.010; \(F\)-value = 7.70; **/K ratio = 3.24. ** Heterotrophic vitamins mix: Thiamine-HCl (1.0 g L\({}^{-1}\)), d-Biotin (0.015 g L\({}^{-1}\)), cyanocobalamin (B12) (0.012 g L\({}^{-1}\)), calcium pantotheane (0.030 g L\({}^{-1}\)) and PABA (p-Aminobenzoic acid) (0.060 g L\({}^{-1}\)). Dose: 1.50 mL L\({}^{-1}\). \\ \hline \end{tabular}
\end{table}
Table 1: Statistical analysis of Plackett–Burman experiment for specific growth rates of heterotrophic cultures.