## 3 Results and Discussion
### Biodiesel Production by Enzymatic Transesterification (First Stage)
#### 3.2.1 Physicochemical Characterization of Biodiesel  
Table 5 reports the results for characterization analysis of the biodiesel obtained in each experiment. The densities of the biodiesel was quantified with an average value of 0.92 g/mL, which is close to the density of the biodiesel produced by alkaline transesterification using WFO as reported by [PERSON] et al. [8]; these numbers were higher than the range required by ASTM 6751, EN (European standards) 14214 and NTC 5444 (0.86-0.90 g/mL). The high density value of diesel could be due to the high density of the oil used [29]. It was found a difference between biodiesel density in experiment E4 and experiment E6 suggesting that biodiesel with lower density is produced during experiments with less reaction time because there are still unreacted amounts of methanol inferring in the density. Moisture content obtained for each experiment were different with values between 0.11 and 0.5%, a range greater than the maximum value required by standards 0.05% (500 mg/Kg). Although the moisture content was expected to be lower owing the final drying stage at 130 \({}^{\circ}\)C, the repetitive washing of separated mixture and the low efficiency of drying contributed to reach higher moisture than allowed for biodiesel. Also, the remaining ternary mixture methanol-water-glycerol after separation stages may interfere water evaporation [59]. The acidity index reported values between 2.0667 and 4.6129 mg KOH/g, where lowest results were reached during experiments E8 and E1. All these values were significantly higher than those allowed by NTC 5444, EN 14214 and ASTM 6751, which may be attributed to the unknown handling of raw material in the frying and storage process and impurities may affect this property [9]. The flash point is very important in the handling and storage of fuels; therefore, to reduce the risk of fire, it is required high flash point of substances [60]. The flash points obtained in each experiment (from 227.5 to 257.5 \({}^{\circ}\)C) are considered favorable because they are significantly higher than the minimum value required by the standards, specifically 120 \({}^{\circ}\)C for standards EN 14214 and NTC 5444, and 130 \({}^{\circ}\)C for ASTM 6751. The kinematic viscosity showed higher values than the established by standards of 3.5-5 mm\({}^{2}\)/s for EN 14214, and 1.9-6 mm\({}^{2}\)/s for ASTM 6751 and NTC 5444. This is an undesired result for the biodiesel, since viscosity exceeding the limits lead to poor fuel atomization and incomplete combustion [60]. Viscosity is related to the presence of unreacted triglycerides, showing the performance of transesterification [27]. In this sense, it was expected that experiments with higher viscosity results  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Exp. ** & **Density (g/mL)** & \begin{tabular}{c} **Moisture** \\ **Content (\%)** \\ \end{tabular}  & \ \begin{tabular}{c} **Acidity Index (mg** \\ **KOH/g Sample)** \\ \end{tabular}  & \begin{tabular}{c} **Flash Point (\({}^{\circ}\)C)** \\ **Viscosity (mm\({}^{2}\)/s)** \\ \end{tabular}  \\ \hline E1 & 0.922 \(\pm\) 0.0041 & 0.279 \(\pm\) 0.01 & 2.84 \(\pm\) 0.28 & 252.5 \(\pm\) 5 & 10.53 \(\pm\) 0.04 \\ E2 & 0.925 \(\pm\) 0.0042 & 0.381 \(\pm\) 0  
were E1 (10.53 mm\({}^{2}\)/s) and E4 (10.06 mm\({}^{2}\)/s) due to the short reaction times to conduct enzymatic transesterification. For longer reaction times, it was observed that experiments E6 (8.66 mm\({}^{2}\)/s) and E9 (7.08 mm\({}^{2}\)/s) reached lower viscosity at same enzyme concentration. For reaction times of 8 h and 16 h, kinetic viscosity decreased when increasing enzyme concentration similar to the trend reported by [PERSON] et al. [52] for enzyme concentration range of 6-9 wt%. To overcome the high viscosity of biodiesel, additives such as Diethyl Ether/DDE and the commercial additive Viscoplex 10-330 CFI can be used according to the results of [PERSON] et al. [61] for reduced viscosity of blends. Also, [PERSON] et al.