*2.7. Experimental Design*

Process of biodiesel production using Fe3O4\_PDA\_Lipase was optimized using five factor CCRSM (central composite response surface methodology) provided by Design Expert version 10.0.7 (State-Ease Inc., Minneapolis, MN, USA). The five independent variables chosen for the investigation of the optimization process with their ranges are A (methanol/oil ratio 3:1 to 9:1), B (biocatalyst concentration 1 to 10%), C (reaction temperature 20 to 50 ◦C), D (reaction time 12 to 48 h) and E (water content 0.2 to 1%). Fifty reactions were carried out as per experimental design.

A reaction mixture containing a specified amount of oil, nano-biocatalyst, methanol and water were allowed to react in a conical flask placed in shaking incubator, while reaction parameters were set in accordance to the RSM (response surface methodology) design. After completion of reaction, the glycerol was separated from biodiesel. Crude biodiesel was purified, and remaining methanol was recovered by using a rotary evaporator under the conditions of reduced pressure. GC-FID, having a highly-polar BPX-70 capillary column (30 m × 0.25 mm), was utilized for the analysis of products. The oven temperature was kept at 100 ◦C for 30 s, then raised to 250 ◦C at a rate of 10 ◦C/min, while the temperature of the detector was set at 270 ◦C. 500 ppm of sample was dissolved in hexane containing methyl heptadecanoate as an internal standard, and 1 μL of this mixture was injected in column [26,27]. The percentage conversion of biodiesel was calculated by given formula [28]

$$\text{FAME} \left( \text{Biodiesel} \right) \%= \frac{\sum \text{A}\_{\text{ME}} - \text{A}\_{\text{IS}}}{\text{A}\_{\text{IS}}} \times \frac{\text{C}\_{\text{IS}} \times \text{V}}{\text{M}} \times 100\%$$

where - AME denotes the sum of peak areas of FAME's peaks, AIS is the peak area of the internal standard peak (methyl heptadecanoate), CIS is the concentration of the internal standard, V is the volume of the internal standard and M is the mass of biodiesel.

#### *2.8. Recovery and Recycling of Nano-Biocatalyst*

The Fe3O4\_PDA\_Lipase was recovered by magnetic decantation from both the biodiesel and glycerol layers. Separated Fe3O4\_PDA\_Lipase was then washed and dried at ambient temperature for reuse.
