## Materials and Methods
### Biodiesel Production by Enzymatic Transesterification  
Figure 1 depicts the experimental methodology followed to determine optimum conditions for biodiesel production from WFO. The experiments were repeated in duplicate (n = 2) because of the limitations in the amount of feedstock as it comes from different used oils collected from local restaurants in Cucuta, which depended on frying conditions and their fatty acid profiles change significantly affecting the properties of the mixture. In the first stage, the effect of enzyme concentration and reaction time was evaluated while fixing temperature and pH. To this end, substrate must be present in excess amount to ensure that reaction rate is independent of WFO concentration [28]. The amount of WFO was fixed in 500 mL based on the results of previous researches with the same value for such operating parameter [8; 29]. The methanol to oil ratio was 3:1 because of the high conversion performance reached by [PERSON] et al. [8].  
The typical reaction time for enzymatic transesterifications of various oils ranged from 4 h to 72 h [30], hence, reaction times of 8, 12 and 16 h were selected for WFO as feedstock. It was considered different concentrations (14%, 16% and 18%) of lipase XX 25 split, which is a combination of lipase from _Candida rugosa_ and _Thermomyces languinosus_. Such enzyme was selected in this work owing its ability to efficiently hydrolyze edible oils [31]. The lipase combination system can provide higher hydrolysis yields because of the strong affinity to certain groups towards the ester synthesis such as unsaturated acids and long-chain fatty acids [32; 33]. Studies reported that mixture of two or more lipases contributes to increase ester yield in biodiesel, which is explained by the different specificities of each lipases [34]. _Candida rugosa_ is able to catalyze all the ester bonds of triglycerides to obtain fatty acids and glycerol, while, _Thermomyces languinosus_ is stable and able to maintain activity at 50-60 \({}^{\circ}\)C [35].  
The reaction took place in four-input reactors, in which the feedstock, methanol and enzyme were mixed. This was achieved by adding the enzyme to the oil after establishing the reaction temperature (38 \({}^{\circ}\)C) as well as pH = 7.0 and mixing for 15 min. Then, methanol was divided into three equal parts and one by one was added to the reaction at intervals of 15 min as suggested by [PERSON] et al. [29]. After reaction time, process temperature was increased to 60 \({}^{\circ}\)C for 15 min in order to inhibit the enzyme and thus end the reaction mechanism. The mixture was centrifuged at 5000 rpm to separated glycerol from biodiesel. The separated mixture containing biodiesel was washed thoroughly with distilled water at 60 \({}^{\circ}\)C and subjected to decantation. Finally, biodiesel was heated at 130 \({}^{\circ}\)C to remove unreacted methanol and water droplets [29].  
After selecting the optimum conditions for reaction time and enzyme concentration, the effect of pH (6, 7 and 8) was assessed in the second stage with the temperature set in 38 \({}^{\circ}\)C. These pH values were selected according to previous works reporting the effect of pH on transesterification performance [36]. The final stage is related to determining optimum temperature condition for the biodiesel production. Temperature influences reaction rate and yield of esters [37], hence, it was important to evaluate such parameter on transesterification at 35, 38 and 40 \({}^{\circ}\)C.