## 2 Results and Discussion  
### FFA Production from Enzymatic Hydrolysis of USCO: Screening the Lipase Sources  
In the first step, FFA production from the enzymatic hydrolysis of USCO using a conventional process (mechanical stirring and heating by immersion in a temperature-controlled water bath), in the absence of emulsifier and buffer agents, was investigated. As shown in Figure 1, a linear profile of enzymatic hydrolysis for USCO catalyzed by CRL, a known non-specific lipase, until an almost complete conversion into FFA and glycerol can be observed. As expected, the full hydrolysis reaction was achieved after a 180 min reaction time. On the other hand, hydrolysis reactions catalyzed from lipases from _Aspergillus niger_ (ANL) and _Rhizopus javanicus_ (RJL), two non-commercial _sn_-1,3-specific lipases, gave a maximum hydrolysis percentage of only 8% after a 360 min reaction time. CALA, a commercial _sn_-2-specific lipase, exhibited maximum hydrolysis at 32% after a 360 min reaction time, thus indicating an almost complete hydrolysis of the ester bonds at the middle position of the glycerol backbone (_sn_-2 position). Thus, a mixture of FFA and diacylglycerols (DAG) are the main products obtained in this process. The hydrolysis percentage is around 45% after a 300 min reaction time, using a _sn_-1,3 specific lipase from _Thermomyces lanuginos_ (Eversa(r) Transform 2.0--ET2.0) as a biocatalyst. In this case, the maximum hydrolysis percentage was 66.6%, with the expected preferential formation of 2-monoacylglycerolscerol (2-MAG) [49]. Interestingly, the high hydrolysis percentages of refined soybean and waste cooking oils catalyzed by a _sn_-1,3 specific lipase from _Thermomyces lanuginos_ in its immobilized form (Lipozyme TL IM) have been reported on in other studies, albeit under complex experimental arrangements (ultrasound irradiation), and with a process that requires FFA extraction steps using organic solvents [50; 51]. Thus, a reaction system using a combination of an _sn_-2 specific lipase (CALA) and an _sn_-1,3 specific lipase (ET2.0) was performed under the same experimental conditions to obtain a complete USCO conversion into FFA using an active and stable low-cost lipase preparation (ET2.0). Previous reports have demonstrated the efficient use of a combination of microbial lipases (enzymatic cocktails) with different specificities, including the combination of _sn_-1,3 specific lipases, as potential biocatalysts for producing FFA mixtures from the hydrolysis of different vegetable oils [52; 53]. However, the maximum hydrolysis percentage observedwas 78% after a 660 min reaction time. The objective of this study was to select a lipase preparation or a specific combination that provides full conversion of the waste oil into FFA, the target raw material, for producing esters. Therefore, further tests were conducted using only CRL as a biocatalyst. Comparative Performance of Soluble or Immobilized Lipase ET2.0 on MAE Production via Esterification in Solvent-Free Systems  
The catalytic performance of immobilized lipase ET2.0 in the production of monoalkyl esters (MAE) via the direct esterification of FFA from USCO and fused oil was investigated and compared with that of its soluble form. In this study, the influence of water removal strategies on the reaction when using molecular sieves and/or esterification reactions conducted in open systems was also evaluated and compared with a reaction system performed in the absence of any water removal strategies (esterification reaction in a closed reactor in the absence of molecular sieves). According to Figure 2, the FFA conversion percentage was only 30.2% after a 210 min reaction time using soluble lipase as biocatalyst, corresponding to a productivity of 0.8 \(\mu\)molFFA\(\cdot\)min\({}^{-1}\cdot\)mg\({}_{\text{prot}}\)\({}^{-1}\) (Table 1). By contrast, immobilized lipase provided better catalytic performance in the reaction, based on FFA conversion percentages and productivity values.