Transesterification process

SCG lipids extracted from an instant coffee ground sample (ICG2) through the New Holland Extractions Ltd. pilot plant (Section 3.2.4) were used in all transesterification experiments, and the FFA content of the oil was found to be 29.91 ± 0.51 % w/w according to the method described in Section 3.2.6. A two-step transesterification process was selected as the most appropriate for the conversion of oil to FAMEs based on previous studies that investigated biodiesel synthesis from SCG oils with high FFA content (Al-Hamamre et al., 2012; Caetano et al., 2012; Haile, 2014; Vardon et al., 2013). In this method, the FFAs were initially converted to esters with methanol in an acid-catalyzed pretreatment step, and thereafter, when the FFA content of the oil had been reduced to a suitable level, a base-catalyzed transesterification step was conducted. Methanol was used in both steps and was preferred over ethanol as it is the smallest molecular weight alcohol, and can potentially increase the reaction speed (Leung and Guo, 2006; Ma and Hanna, 1999). Prior to esterification, the ICG2 oil samples were subjected to heating at 100 °C for 5 hours in an oven to remove any residual water traces.

3.2.7.1 Acid-catalyzed pretreatment

In the first part of the transesterification process, ICG2 coffee oil which had been previously homogenized by preheating at 55 °C in a water bath (Fisherbrand 60301) for 30 minutes, and methanol were mixed in the presence of sulfuric acid in a 250 ml conical glass flask located on a magnetic hotplate stirrer (Fisherbrand ARE) at temperatures of 50 °C and 60 °C.

Methanol and oil are immiscible and therefore the stirring speed was kept

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constant at 600 rpm so as to ensure efficient mixing, as suggested by previous researchers (Caetano et al., 2012; Chai et al., 2014; Haile, 2014).

The mixture was stirred for 4 hours after the start of the reaction, a duration that was found to be sufficient in previous studies (Al-Hamamre et al., 2012;

Vardon et al., 2013), while the % w/w FFA content of the mixture was measured at 2, 3 and 4 hours through titration (Section 3.2.6).

In order to determine the desired experimental molar ratios of methanol-to-FFA and weight percentages of catalyst to methanol-to-FFA, the density of the oil and the average molecular weight of fatty acids present in the oil had to be measured.

The density of methanol is 0.791 g/ml, while its molecular weight is 32.04 g/mol (Sadeghi and Azizpour, 2011), and the density of sulfuric acid is 1.83 g/ml (Shitov et al., 2009).

The density of the oil was calculated by adding quantities of 1 ml into a 10 ml volumetric cylinder, measuring the weight on a precision balance and then drawing a graph of mass against volume. Thereafter, a straight best fit line was drawn and the ICG2 oil density was found from its gradient (Appendix C).

The average molecular weight of fatty acids present in the ICG2 oil was calculated based on the fatty acid profile of SCG oil from the same source (ICG1) determined through gas chromatography, where the molecular weights of the individual fatty acids present in the oil were calculated by summing the appropriate number of carbon, hydrogen and oxygen atoms (Appendix D).

The transesterification and gas chromatography method used at New Holland Extraction Ltd. to determine the fatty acid profile of the ICG1 oil can be found in Appendix D. The average molecular weight (g/mol) of a single fatty acid in this oil was calculated as per Equation 3.12:

𝐹𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 = ^{∑ 𝑓𝑖}

∑( ^𝑓𝑖

𝑀𝑊𝑖)= 273.54 (Equation 3.12) Where fi corresponds to the % w/w fraction of each fatty acid in ICG1 oil and MWi to the molecular weight of an individual fatty acid. The molecular weight of a triglyceride in ICG2 oil was measured by Equation 3.13:

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𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 = 3 𝑥 𝑀𝑊_𝑓+ 38.049 = 858.68 (Equation 3.13)

Where MWf is the fatty acid average molecular weight calculated by Equation 3.12, and 38.049 the weight of the glycerol backbone (Shrestha and Gerpen, 2010).

Oil samples that had been pre-treated at the aforementioned conditions were left to settle for 24 hours in a separating funnel and the bottom layer was separated from the top layer of unreacted methanol and water with a separation funnel. The resulting sample was then subjected to rotary evaporation (Section 3.2.3.4) to remove any residual methanol and water. Any oil loss relative to the initial samples weight was measured according to Equation 3.14:

% 𝑤/𝑤 Oil loss = ^{𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙−𝑊𝑝𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑}

𝑊_{𝑖𝑛𝑖𝑡𝑖𝑎𝑙} × 100 (Equation 3.14) Where Winitial and Wpretreated correspond to the initial and pre-treated oil respectively.

3.2.7.2 Base-catalyzed transesterification and FAMEs purification

In this step of the process, pre-treated oil with FFA content below 1.5 % w/w was mixed with methanol in the presence of potassium hydroxide. All the alkali-catalyzed experiments were conducted in a 250 ml conical flask located on a magnetic hotplate stirrer (Fisherbrand ARE), at a temperature of 60 °C (measured with a thermometer) for 4 hours at varying methanol-to-pretreated oil molar ratios and catalyst-to-pretreated oil weight percentages, while the mixture was constantly stirred at 600 rpm with a magnetic stirrer.

In order to determine the desired experimental methanol-to-oil molar ratios and catalyst to oil weight percentages, the density and molecular weight of oil previously subjected to acid-catalyzed esterification were determined. The density of the pre-treated oil was measured with the method used for the determination of the crude oil density (Appendix C). The molecular weight of the pre-treated oil was calculated based on the assumption that it contained approximately 70 % w/w triglycerides, assuming that the majority of

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triglycerides have not yet reacted with methanol to yield FAMEs, ~1.5 % w/w FFAs and ~28.5 % FAMEs due to the esterification process. The average molecular weight of FFAs and triglycerides were previously determined (Equation 3.12, Equation 3.13), while the average MW of FAMEs was found to be 287.54 g/mol by replacing in the FFA molecule the hydrogen atom of the – OH group with a methyl group (-CH3). Therefore, the average MW of the pre-treated oil was measured as per Equation 3.15.

𝑃𝑟𝑒𝑡 − 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑜𝑖𝑙 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑀𝑊 = (0.285 𝑥 𝑀𝑊_𝑓) + (0.7 𝑥 𝑀𝑊_𝑡) +

(0.15 𝑥 𝑀𝑊_{𝑓𝑎𝑚𝑒}) = 724.05 (Equation 3.15)

Where MWf, MWt and MWfame correspond to the molecular weights of FFAs, triglycerides and FAMEs found in the pre-treated oil respectively.

The reaction product was allowed to settle in a separation funnel and after 24 hours the upper biodiesel and unreacted methanol phase was separated from the lower glycerol phase. Unreacted methanol was removed from the FAMEs by rotary evaporation (Section 3.2.3.4) and the resulting ester phase was successively washed with warm (55 °C) distilled water until neutral pH. The pH was measured constantly measured by a Hannah, HI991001 pH meter.

The washing process also served as a way to remove residual catalyst, glycerol, methanol and soap from the coffee biodiesel. Thereafter, the FAMEs were subjected to thermal heating at 100 °C for 5 hours and subsequently a further drying step of drying with sodium sulfate to remove residual water was carried out. Finally, a filtration process with cellulose membranes (Whatman, 4-7μm) was performed to remove solid traces. The % w/w FAME reaction yield relative to pre-treated ICG2 oil was calculated according to Equation 3.16.

% 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 = ^{𝑤𝐹𝐴𝑀𝐸}

𝑤𝑝𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑜𝑖𝑙 × 100 (Equation 3.16) Where WFAME and Wpretreated oil represent the mass of biodiesel and pre-treated ICG2 oil respectively.

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