*3.2. Process Optimization*/*Characterization*

#### 3.2.1. Characteristics of Feedstock

The density and the specific gravity of feedstock were 0.918 ± 0.050 g/cm<sup>3</sup> and 0.934 ± 0.051 respectively, while the acid value was calculated to be 1.68 ± 0.12 mg/KOHg, the peroxide value was 15.80 ± 0.32 mEq/Kg, iodine value was 96.20 ± 1.00 and the saponification value of waste cooking oil feedstock was 182.0 ± 1.0 mg KOH/g.

#### 3.2.2. Composition Analysis of Produced Biodiesel by GC-MS

The product (biodiesel) was analysed by GC-MS. Major fatty acid methyl esters consisted of methyl esters of palmitic acid (16:0), lenoleic acid (18:2), stearic acid (18:0), gonodic acid (20:1) and arachidic acid (20:0) were identified by NIST library of GC-MS. Noshadi et al. [32] reported waste cooking oil with myristic acid, palmitoleic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. Uzun et al. [33] used waste oils for biodiesel production with a following fatty acid content of palmitic acid, stearic acid, oleic acid, linoleic acid and docasanoic acid. Comparable results have also been reported by [34–36], who found that the variations in fatty acid content might be due to presence of di fferent edible oils and their varying amounts in cooking oils.

#### 3.2.3. FTIR Monitoring of Biodiesel Production

Conversion of triglycerides into the biodiesel was confirmed by the FTIR spectral comparison of feedstock oil with the synthesized biodiesel. Characteristic peaks of waste cooking oil were observed at 723, 1700–1800 and 2800–3000 cm<sup>−</sup>1, which corresponds to –CH2 rocking, C=O starching and symmetric C-H stretching vibrations, respectively. While in the case of biodiesel, adsorption peak at 1437 cm<sup>−</sup><sup>1</sup> corresponding to methyl ester and peak appearing at 1196 cm<sup>−</sup><sup>1</sup> for C-O ester bond are the characteristic biodiesel adsorption peaks. The absence of 1380 cm<sup>−</sup><sup>1</sup> peak in biodiesel that represents the O–CH2 bonds in glycerol site of triglycerides further proves the formation of methyl esters (Figure 7). The results obtained in the present work are comparable to previous research [37–39].

**Figure 7.** FTIR of (**a**) WCO and (**b**) WCO based biodiesel.

## 3.2.4. Optimized Reaction Parameters

Table 1 describes the optimum reaction conditions for the optimized yield of waste cooking oil-based biodiesel. By carrying out 50 experiments on various conditions of selected parameters, it has been observed that Fe3O4\_PDA\_Lipase catalysed the transesterification of feedstock oil giving a maximum yield of 92%, under the given set of reaction conditions/parameters viz., biocatalyst concentration (10%), CH3OH to oil molar ratio (6:1), reaction time (30 h), reaction temperature (37 ◦C) and water content (0.6%). While biocatalyst concentration (1%), methanol to oil molar ratio (3:1), reaction time (12 h), reaction temperature (20 ◦C) and water content (1%) gave the lowest response (41.5%).

Stoichiometrically, methanol reacts with triglycerides in 3:1 molar ratio, but excess amount of methanol was provided to increase the rate of forward reaction because transesterification is equilibrium limited reaction [40]. Moreover, the viscosity of the oils is high, which hinders the mass transfer. The addition of excess methanol lessens the reaction mixture viscosity, so the rate of reaction is increased by the improved mass transfer [41]. However, excess of methanol can lead to the emulsification of glycerol, which can recombine with the esters to reduce the biodiesel yield. In addition, excess amount of methanol can deactivate the lipase by changing its globular structure [42,43]. In the current study, a 6:1 methanol to oil ratio was observed to be the optimum CH3OH concentration for the biocatalytic transesterification of waste cooking oil.

Water presence influences the activity of the immobilized enzyme and stability of the free enzyme. Water/oil interface may also be required for better catalytic activity of lipase. Furthermore, the enzyme deactivation due to small chain alcohols can be prevented if the lipase is dispersed in water [43]. However, in the presence of higher content of water hydrolysis may compete with the methanolysis [44]. Therefore, water content is required to be adjusted with minimal content as per experimental requirements. Highest triglyceride conversion into biodiesel was obtained while using 0.6% H2O content in present research. Temperature significantly effects the enzymatic transesterification. The activity of lipase increases with temperature till the optimum point, i.e., 37 ◦C, and a further increase in temperature can lead to denaturisation of lipase.

Similar conditions have also been investigated by other researchers. Ying and Dong obtained the maximum biodiesel yield using immobilized lipase with 31.3% nano-biocatalyst, 38 ◦C temperature and 4.7:1 methanol to oil ratio [27]. Thangaraj et al. reported the maximum biodiesel yield of 89% at an optimum condition i.e., 1:3 methanol/oil ratio, 12 h of reaction period and 45 ◦C temp., using the immobilized NS81006 lipase as the catalyst [45]. Iso et al. reported the highest enzyme activity at 0.3% water content using immobilized lipase [46]. Mumtaz et al., using enzymatic transesterification, reported 95.9% biodiesel yield with 0.75% NOVOZYME-435, 6:1 methanol to oil ratio and 60 h of reaction at 32.50 ◦C [47]. Arumugam and Ponnusami used lipase immobilized on activated carbon for biodiesel production; optimum parameters were 1:9 oil to methanol ratio, 10% water content and 30 ◦C temperature, achieving a yield of 94.5% biodiesel with these conditions [41]. Xia also used RSM to optimize the enzymatic transesterification process, the optimum conditions reported are 4:1 methanol to oil ratio, 6.8% of biocatalyst and 42.2 ◦C reaction temperature [26]. Andrade et al. used nano-biocatalyst prepared by immobilization of lipase on magnetic nano-particles and achieved the highest biodiesel yield at 4:1 methanol/oil raio, 20% enzyme conc., 37 ◦C temp., and 12 h of time period [25]. Andrade et al. attained 95.5% methyl ester yield at 8:1 molar ratio of alcohol to oil, 8% biocatalyst amount and 45 ◦C temperature [48].

**Table 1.** Optimum reaction conditions for biodiesel production from Fe3O4-PDA-Liapase catalysed transesterification.

