## 3 Results and Discussion
### Biodiesel Production by Enzymatic Transesterification (First Stage)  
For a better understanding of the results, Table 4 summarizes the nomenclature employed to distinguish experiments in the first stage. Figure 2 shows the biodiesel production and yield for all experiments considering the effect of reaction time and enzyme concentration. An interesting trend was observed for E1, E4 and E7 experiments as well as E2, E5 and E8 experiments. For reaction time of 8 h and 12 h, higher biodiesel yield was reached in the following order of enzyme concentration 14%, 18% and 16%. For reaction time of 16 h, higher production of esters was observed in the following \begin{table}
\begin{tabular}{c c} \hline \hline
**Properties** & **Result** \\ \hline Density (g/mL) & 0.9572 \(\pm\) 0.00014 \\ Moisture (mass fraction) & 0.0600 \(\pm\) 0.016 \\ Acidity index (mg KOH/g) & 2.5624 \(\pm\) 0.3945 \\ pH & 5.7 \(\pm\) 0 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Physicochemical characterization of WFO. Values are mean +/- standard deviation (n = 2). \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**References**} & \multicolumn{2}{c}{**Fatty Acid Composition (\%)**} \\ \cline{2-3}  & **Palmitic Acid (C16:0)** & **Oleic Acid (C18:1)** \\ \hline This work & 30.35 & 18.84 \\ [PERSON] et al. [43] & 6.1 & 64.2 \\ [PERSON] et al. [44] & 34.80 & 53.30 \\ [PERSON] et al. [45] & 17.82 & 40.98 \\ [PERSON] et al. [46] & 60.1 & 27.20 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Fatty acids composition of WFO reported in literature. order of enzyme concentration 16%, 14% and 18%. According to these results, longer reaction times affected transesterification process when using catalyst concentration of 14 and 18% that may be attributed to the formation of undesired products such as dimers or the reaction of impurities from WFO with reagents. Also, a slight recombination of esters and glycerol to monoglyceries may occur [47]. Lipase activity may decrease over time as a result of the accumulation of substances inhibiting the enzyme or the substrate is being used up [48]. The low biodiesel production in the E4 experiment leads to the lower consumption of WFO in comparison to experiments E1 and E7. Hence, substrate availability is high enough not to inhibit enzyme activity over time. The mechanism governing enzymatic transesterification of WFO is shown in Figure 3.  
The E1 experiment reached the highest production with 437.863 g (91.49% biodiesel yield), followed by the E7 experiment with a production of 419.827 g (87.72% biodiesel yield), which can be explained by the effect of catalyst concentration on biodiesel production. As is well known, enzyme concentration is an essential parameter for improving transesterification process [49]. The lower biodiesel production for experiments with higher enzyme concentration (16% and 18%) is supported by other works with similar trends [50]. These results suggested an excess of enzyme over required owing the limitations in liquid volume for reaction, hence, liquid phase was insufficient to suspend the solid catalyst causing external mass transfer resistance [51]. The excess solid enzyme available in the reactive mixture can limit glycerol separation due to the noninteraction between methanol and oil [52]. The existence of a minimum at concentration of 16% in the correlation of biodiesel yield and enzyme concentration may be explained by the formation of dimers with lower activity through the union of two molecules of lipase in the regions of their active centers, which is a common phenomenon when using _Thermonyces languinosus_[53]. The complexity of the substrate and the state of reuse of the oil allows to infer the difficulty of the lipase for the transesterification reaction, generating a possible inhibition in the active site of the enzyme at concentration of 16% or in its allosteric center [54].