Materials and Methods

5.2.1 Materials

The de-oiled microalgal biomass was obtained from the previously isolated oleaginous microalgae Tetradesmus obliquus KMC24 after lipid extraction. The WCO was collected from the hostel of IIT Guwahati, Assam, India. All the chemicals and reagents were procured from HiMedia® and Sigma-Aldrich, India.

5.2.2 De-oiling of microalgal biomass

The T. obliquus KMC24 biomass (10 g) was de-oiled by extracting the lipid through the soxhlet extraction method employing n-hexane (200 mL) as a solvent [365]. Lipid, hexane,

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123 | P a g e and de-oiled biomass were recovered at the end of extraction. The de-oiled biomass was made solvent free through oven drying for carbon catalyst preparation.

5.2.3 Synthesis of DMB catalyst

The DMB catalyst was synthesized via a two-step process where carbonization of de- oiled microalgal biomass was followed by sulfonation of the carbonized biochar with sulphuric acid. In the first-step, 10g of de-oiled biomass was pyrolyzed in a tubular reactor at 400 °C- 700 °C at a heating rate of 4 °C min^-1 under a stream of Argon (100 mL min^-1) for 4 h to obtain biochar. The biochar yield was determined from the following equation:

𝐵𝑖𝑜𝑐ℎ𝑎𝑟 𝑦𝑖𝑒𝑙𝑑 =𝑚𝑎𝑠𝑠 𝑜𝑓 𝑏𝑖𝑜𝑐ℎ𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑡𝑎𝑘𝑒𝑛 × 100% (5.1) The biochar was ground to a fine powder using a mortar and pestle before sulfonation.

2g of biochar was sulfonated using (5, 10, 15, 20 mL) concentrated sulphuric acid for a reaction time of 2, 4, 6, and 8 h in a 500 mL glass beaker. The variable’s ranges were selected based on the previously reported literature [366,367]. The reaction mixture was heated at 110 °C on a hot plate and was occasionally mixed with a glass rod. After the sulfonation process, the mixture was cooled and placed in deionized water. Later, the carbon catalyst was repetitively washed with hot deionized water until the washing solution was neutral in pH and free of sulfate ions. The synthesized solid acid catalyst was oven dried (110 °C) overnight and was stored in a vacuum desiccator for further study.

5.2.4 Characterization

The structural analysis of the catalyst was determined by X-ray diffraction (XRD) technique employing X-ray diffractometer (TTRAX III 18 kW; Rigaku Corporation, Japan) with Cu Kα radiation (λ = 1.5406 Å). Diffractograms were obtained in the 2θ range of 10° to 90°. The morphology of the samples was determined by FESEM (Zeiss Sigma-300 Field Emission Scanning Electron Microscopy). Elemental components were determined by using energy dispersive spectroscopy (EDX) coupled with FESEM. The KBr pellet technique was employed to record the Fourier transform-infrared (FT-IR) spectra over a wavenumber range of 4000 to 400 cm^-1 by a spectrometer (Perkin Elmer Spectrum 2 FTIR). Raman spectra were recorded with a LabRAM HR Raman microscope (laser: 514 nm). Elemental analysis (C, H, N, S) of biochar, catalyst, and reused catalyst was carried out in a CHNS analyzer (Flash EA 1112 series, Thermo Finnigan, Italy). The BET (Brunauer-Emmett-Teller) surface area of the

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124 | P a g e samples was determined by N2 adsorption-desorption measurements at 77.3 K using a TriStar II 3020, Micromeritics instrument. The hydrophilic and hydrophobic properties of the catalyst were determined by a drop shape analyzer. The thermal stability of the samples was determined using TGA (STA7200, Hitachi, Japan) under N2 atmosphere. X-ray photoelectron spectroscopy (XPS) analysis on NEXSA; Thermo Scientific Instruments with a monochromatic Al Kα radiation was performed to analyze the chemical composition of the catalyst. The total acid density of the catalyst was determined by the titration method as reported in the literature [78]. The total acidity of the catalyst was also analyzed by temperature programmed desorption of ammonia (NH3-TPD, MicrotracBEL BELCAT II). At first, degassing of the catalyst was carried out under He flow at 300 °C for 1 h. The degassed catalyst was then cooled to 40 °C by He gas at a flow rate of 50 mL min^-1. After cooling, a gas mixture of 10% NH3/He was allowed to pass through the sample cell for 2 h. Subsequently, desorption of the physisorbed NH3 was conducted from ambient temperature to 800 °C at the heating rate of 10 °C min^-1 with a flow of He (30 mL min^-1).The desorbed NH3 was detected using a TCD detector.

5.2.5 Reaction study

Catalyst synthesis conditions such as pyrolysis temperature, sulfonation time, and the amount of H2SO4 added during the sulfonation reaction were optimized by evaluating the percentage of FFA conversion during the esterification reaction. Esterification reaction of a known amount of oleic acid with methanol was conducted in a round-bottomed flask equipped with a refluxing condenser at 80 ^oC reaction temperature, methanol to oleic acid molar ratio of 10:1, a reaction time of 6 h, and catalyst concentration of 5 wt. %. At the end of the esterification process, the catalyst was retrieved via centrifugation, and the unreacted methanol was evaporated. Finally, the liquid product was analyzed for acid value and FFA conversion using the previously reported equations [368]. The percentage FFA was calculated by multiplying the factor 0.503 with the acid value [369].

Further, the catalytic activity of the DMB catalyst prepared under optimized conditions was tested for transesterification of lipid derived from T. obliquus biomass (AO) and WCO.

The reaction parameters such as methanol/oil molar ratio, catalyst concentration, reaction temperature and time were optimized based on FAME yield in a round-bottomed flask equipped with a refluxing condenser. The variable’s ranges of the reaction parameters were selected based on the previously reported literature [74]. At the end of the reaction, the product was shifted to an Eppendorf tube, where the catalyst was recovered through centrifugation.

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125 | P a g e Subsequently, hexane and warm distilled water were incorporated into the reaction mixture and vortexed. Finally, the mixture was centrifuged to separate the phases based on their density.

The FAME was separated from the upper phase, and unreacted methanol was evaporated. The top layer consisting of hexane and FAME was collected. The hexane was vaporized, and the FAME yield (%) was determined using the following equation [74]:

𝐹𝐴𝑀𝐸 𝑦𝑖𝑒𝑙𝑑 (%) = 𝐹𝐴𝑀𝐸 𝑂𝑏𝑡𝑎𝑖𝑛𝑒𝑑 (𝑔)

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑜𝑖𝑙 (𝑔)× 100 (5.2) The catalytic efficiency of the DMB catalyst was compared with the conventional acid- base catalyst. A two-step, acid-base (HCl-NaOH) transesterification reaction of AO and WCO was carried out under the optimized reaction conditions.

5.2.6 Catalyst reusability study

The stability of the DMB catalyst was evaluated by reusing it for consecutive cycles at the optimized reaction conditions. The catalyst recovered through centrifugation at the end of the experiment was rinsed with methanol, dried at 80 °C for 6 h, and was reused for the next experiment.

5.2.7 Analysis of FAME and biodiesel properties

The FAME composition and biodiesel properties were analysed using the protocol mentioned in the methodology section of chapter 3.