2.2.10 Leaching experiment
To assess the amount of desorbed enzymes from the surface of the support, 5 mg of the immobilized derivatives (60 mg/g) was incubated in a solution containing NaCl (1M). The concentration of protein content in the supernatant was evaluated by using both the Bradford’s method and enzyme activity assay.
2.2.11 Biodiesel synthesis
In a typical experiment, the reaction was carried out in a 5 ml screw-capped vial including 130 mg of waste cooking oil and anhydrous methanol at various oil-to-methanol molar ratios (1:3, 1:4, 1:5, 1:6, and 1:7). The mixtures were incubated with the immobilized lipase at different temperatures (35, 40, 45, 50 and 55°C). Methanolysis reactions were performed with different amounts of t-butanol, water adsorbent (for RML and CALB) and water (for TLL). To analyze the impact of the solvent on methanolysis, the amount of t-butanol was varied (5, 20, 35, 50 and 65 wt. %) by the substrate weight. Reactions were performed for 72 h under constant agitation of 500 rpm. Aliquots of the reaction medium was taken at different times, mixed with methyl laurate (as an internal standard) and analyzed by GC.
2.2.12 GC analysis
The FAME yield in the reaction mixture was analyzed using gas chromatography (Thermo-Quest-Finnigon) equipped with a flame-ionization detector (FID) connected to Zebron capillary column (30 m × 0.25 mm i.d.; Phenomenex, USA.) Nitrogen was used as the carrier gas at a constant flow of 0.8 ml/min. The sample was weighed and mixed with 1000 µL of 0.8 mg/mL methyl laurate in n-hexane as an internal standard. Then, 0.5 µL of the diluted sample was injected into the GC. The column temperature was held at 150 oC for 1min, raised to 210°C at 25°C /min, and then increased to 240°C at 10°C /min and kept for 8 min. The injector and detector temperatures were set at 150 and 300°C, respectively. Eq. (1) was used to calculate biodiesel yield:
$$C=\frac{\sum A-{A}_{IS}}{{A}_{IS}}\times \frac{{C}_{IS}\times {V}_{IS}}{m}\times 100\%$$
1
In this equation, (∑A) is the total sub-peak area, (\({A}_{IS}\)) is the internal standard sub-peak, (\({C}_{IS}\)) the internal standard concentration in (mg/mL), (\({V}_{IS}\)) volume of the internal standard (mL), and (m) sample mass (mg).
2.2.13 Experimental Design
Optimization of the transesterification process of waste cooking oil was investigated using a 5-level, 5-factor Response Surface Central Composite Design (RSCCD) of Design Expert Software version 7.0.0 (State Ease Inc. Minneapolis, USA). The study required 45 experiments, consisting of 32 factorial points, 10 axial points, and 3 repetitive tests in central points. In addition, there were five independent identified variables, contained reaction temperature (35–55°C), enzyme amounts (5–25 w/w %), as well as t-butanol concentration (5, 20, 35, 50, and 65 wt %), water for TLL (0–40%) and water absorbance for RML and CALB (20–60%).The 3 stepwise methanol feeding was applied (oil: methanol ratio 1:3, 1:4, 1:5, 1:6and 1:7). The level of each independent variable was considered based on previous researches (Ashjari et al. 2020b)(Yang et al. 2005). In addition, the independent variables were encoded into low (-1) and high (+ 1) levels. Although, the axial points are coded as -2 (-α) and + 2 (+α). In this paper, the value of α, the distance from the center, is fixed at 2, which makes the rotating design.
2.2.14 Statistical analysis
The experimental data obtained from central composite design (CCD) were analyzed by response surface methodology. A quadratic equation (Eq. 2), which containing all the terms of interaction, was used to calculate the predicted values:
$$Y={}_{0}+\sum _{i=1}^{5}{}_{i}{X}_{i}+\sum _{i=1}^{5}{}_{ii}{X}_{i}^{2}+{\sum }_{i=1}^{3}\sum _{j=i+1}^{5}{}_{ij}{X}_{i}{X}_{j}+{\sum }_{i=1}^{5}{}_{\text{i}\text{i}\text{i}}{X}_{i}^{3}$$
2
Where (Y) is the yield of biodiesel from waste cooking oil, β0 the offset term, (βi) represents linear effect, (βii) represents squared effect, (βij) represents interaction effect, (Xi) represents the i-independent variable, and (Xj) represents the j-independent variable. The data were analyzed using the Design Expert program and the coefficients were calculated by F-test. All statistical steps including analysis of variance (ANOVA), regression analysis and plotting of contour plot were used to establish the optimum conditions to obtain high efficiency of methyl ester.