Development of direct transesterification (DT) method for accurate quantification of microalgal lipid content
4.3 Results and discussion
4.3.1 Selection of best combination of biomass type and transesterification method Eight different transesterification methods were screened for three types of biomass
temperature was kept at 250°C with a split ratio of 1:20 and the oven temperature was kept at 100°C (5 min) followed by ramping at a rate of 5°C per min till 250°C followed by 15 min hold. The detector temperature was kept at 280°C and the injection volume of 1 µL was used for analysis. FAME mix C14-C22 (Supelco, USA) was used as the standard for GC- FID.
4.2.6 Fourier transform infrared spectrophotometer (FTIR) Analysis
The infrared spectra were analyzed by Fourier transform infrared spectrophotometer (IR Affinity-1 Shimadzu) from 500 to 4000 cm−1 to analyze the functional group of the diesel and biodiesel using KBr pellets.
4.2.7 Statistical analysis
Statistical analyses of the results obtained were performed using the software Minitab version 16.1.1 (Lead Technologies Inc.,). All the experiments were conducted in triplicate and the data were expressed as mean ± standard error. The significant difference in the various biomass types used and significant difference among the methods M1-M8 for each type of biomass were analyzed through one-way analysis of variance by evaluating their p-values.
yield. In case of oven dried biomass the reduction in FAME yield may be due to the oxidation of fatty acids (Oehrl et al., 2001). For instance, reduction in total lipid content was reported when algal biomass was dried at temperature greater than 60°C in an atmosphere with oxygen/air (Widjaja et al., 2009). Oxidation of fatty acids results in formation of ketone bodies or aldehydes resulting in overall reduction of fatty acid content (Widjaja et al., 2009).
Oehrl et al. (2001) demonstrated that unsaturated fatty acids are prone to oxidation than saturated fatty acids. In the present study, GC-FID profiling of the total fatty acids in Chlorella sp. FC2 IITG revealed the abundance of unsaturated fatty acids (>75% w/w, of total FAME) which might have undergone oxidation in the course of oven drying.
Fig. 4.2 FAME yield (%, w/w DCW) of the Chlorella sp. FC2 IITG obtained from eight different transesterification methods (M1-M8) and three different types of biomass. The significant difference among the biomass types is represented as ‘*’ and the significant difference among the methods M1-M8 for each type of biomass are represented as ‘a’
obtained from one way analysis of variance (corresponding p-values for * = 0; ** = 0.022;
a = 0)
Wet biomass showed reduced FAME yield than any other type of biomass due to high water content of about 80% (v/w) (Komers et al. 2001). Presence of water in the wet algal biomass interferes with the acid/alkali catalyst reducing the overall catalyst availability and transesterification efficiency. Fatty acids are saponified by the alkali catalyst in presence of water which in turn results in the formation of viscous froth and hinders effective transesterification (Griffiths et al., 2010; Kusdiana and Saka, 2004). In case of transesterification with acid catalysts, the fatty acids are protonated by the acid followed by the formation of tetrahedral intermediate with alcohol and finally the proton migrates to form FAME (Lotero et al. 2005). In the presence of water, acid catalyst binds to water leading to a reversible acid catalyst deactivation and the water molecules masks the acid by forming proton clusters around them (Lotero et al. 2005) and characteristic decrease of FAME yield in presence of water was also reported (Liu et al., 2006). Depending upon the type of biomass, the degree of transesterification varied significantly among the eight different methods. Maximum FAME yield was obtained in the two stage DT method M7 for lyophilized biomass in comparison to all other methods. M5 showed maximum FAME yield in cases of both wet and oven dried biomass when compared with other evaluated methods (Fig. 4.2).
Conventional Bligh and Dyer lipid extraction-transesterification method (M1) resulted in lowest FAME yield (7.87%, w/w in lyophilized biomass) in all types of biomass. The commonly used Bligh and Dyer method underestimated the FAME yield which may be attributed to inadequate extraction and unoptimized solvent ratios (Griffiths et al., 2010;
Laurens et al., 2012). Three different catalysts (NaOH, HCl and H2SO4) were used to determine the individual effect on the single stage DT (M2-M4). Very less FAME yield was obtained in case of NaOH and HCl as catalysts (M2 and M3) in comparison to conventional extraction transesterification which may be due to incomplete extraction and
transesterification in the single stage DT methods M2 and M3. When CH3ONa was used as catalyst in single stage transesterification, no FAME yield was detected in case of Nannochloropsis sp. and reported that two stages (CH3ONa /BF3) are necessary to obtain higher transesterification efficiency (Laurens et al., 2012). However, when H2SO4 was used as single catalyst in M4, a 2.4 fold increase in FAME yield was obtained in comparison to conventional method M1 and single stage DT methods M2 and M3 applied to lyophilized biomass. The two stage DT methods M5 and M7 with base and acid catalyzed treatments for first and second stage respectively showed higher FAME yield. Interestingly, when catalysts order was reversed in M6 and M8, the process resulted in lower FAME yield. In a recent study, it has been reported that the alkaline hydrolysis of algal biomass in the first stage of DT prior to methylation might have lead towards higher FAME yield (Griffiths et al., 2010). DT is effective transesterification method that can bypass the oil extraction step by merging it into transesterification step (Cheng et al., 2011) with subsequent reduction in the production cost of biodiesel from algal feedstock. The sequential two stages DT method M7 with lyophilized algal biomass was selected for further optimization using CCD on the basis of highest FAME yield obtained.