Sampling, isolation and identification of the suitable microalgal strain for biodiesel production
3.3 Results and discussion
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 the fatty acid composition analysis using GC-FID and the total lipid was quantified in terms of FAME using calibration curve shown in Fig. 3.2.
Fig. 3.2 Standard correlation graph for the estimation of total lipid as fatty acid methyl esters assayed in gas chromatograph with standard FAME mix C14-C22
FC12, FC13 and FC14. To obtain a pure culture conventional serial dilution, plating and streaking method was used. The collected strains were stored as slants and as glycerol stocks (20% w/v, glycerol) at -80°C. Further axenic culture was screened for high lipid accumulation under photoautotrophic condition.
3.3.2 Screening and selection of neutral lipid accumulating microalgal strains The main aim of screening is to identify the “Oleaginous” algal strain which can accumulate substantial amount of neutral lipids. The neutral lipid content of the organism was quantified by Nile-red method detailed in section 3.2.5.2. Among the ten microalgal strains screened, five strains showed neutral lipid accumulation as shown in Fig. 3.3 only in BG11 growth media. Other five strains did not show any neutral lipid accumulation even after incubation for a period of over 30 days under photoautotrophic conditions. As observed from the fluorescence intensity of neutral lipid Nile-red complex FC6 was found to be the best neutral lipid accumulating organism (Fig. 3.3). The neutral lipid content in FC6 culture was found to be 16.30% (w/w, DCW) at the end of 30 days of cultivation followed by FC8 with 4.0% (w/w, DCW) as shown in Fig. 3.3.
Fig. 3.3 Screening and selection of high lipid accumulating indigenous microalgal strains in BG11 media. The neutral lipid content was measured using Nile-red method.
Further, neutral lipid accumulation was confirmed by Nile-red neutral lipid complex under confocal microscope analysis of microalgal cell (Fig. 3.4). With the maximum total lipid content, FC6 was identified as the best neutral lipid accumulating strain among the screened indigenous strains. Thus, the strain was chosen for further detailed identification and media screening experiments.
Fig. 3.4 Confocal imaging of strain FC6 under higher magnification (A) superimposed image of bright field cells, auto-fluorescence of cells stained with Nile red and fluorescence from Nile red-neutral lipid complex; (B) Cells showing auto-fluorescence in red color and Nile red-neutral lipid complex fluorescence as golden yellow color. The images were obtained using confocal microscope with Olympus software.
3.3.3 Selection of the suitable growth medium for selected indigenous microalgal strain
The growth medium for selected FC6 strain was screened by growing it in six different medium compositions. The best media for the growth were screened on the basis of dynamic profile of its growth for 16 days (Fig. 3.5). Amongst all medium composition used, BG11 medium supported the maximum growth with a biomass concentration of 0.69 g L-1 (Fig. 3.5). This may be attributed to the availability of high concentration of sodium nitrate in the BG11 medium supporting maximum growth of the strain.
Less growth was observed in case of Watanabe media followed by Beijerinck media, Bold Basal media, Algae broth media and Diatom medium (0.47 g L-1, 0.37 g L-1, 0.31 g L-1, 0.16 g L-1 and 0.12 g L-1and respectively). The reduced growth in Bold Basal Media, Beijerinck media and Diatom media may be attributed to the lower concentration of nitrate source 0.25 g L-1, 0.15 g L-1 and 0.02g L-1 respectively. Thus, BG11 media was chosen as the best growth supporting medium for the selected strain and further medium optimization was performed to enhance the biomass titer.
Fig. 3.5 Selection of best growth media for the growth of indigenous microalgal strain FC6.
Six different media compositions were used as detailed in section 3.2.2 (Table 3.1)
3.3.4 Morphometric and molecular identification of the organism
The colonies on BG11 agar plate were green in color, spherical convex in shape and shiny with regular edges. Observation under light microscope showed that the cells are spherical in shape, green in color, non-motile and measured about 4-6 µm in diameter (Fig.
3.6 A). FESEM analysis of the cells showed the absence of flagella and spikes over their surface (Fig. 3.6 B). The partial 18S rDNA sequence of length 1074 base pairs was obtained through sequencing and submitted to the Genbank (Accession No.: JX453208). BLAST analysis showed that the isolate is novel in terms of its 18S rDNA sequence and belongs to
the genus Chlorella sp. with a maximum similarity of 97%. Phylogenetic analysis of the strain and 19 other organisms under the order Chlorellales with 97% similarity showed that the strain belongs to Chlorella sorokiniana.
Fig. 3.6 Morphometric identification of the strain FC6: (A) Cells under phase contrast microscope and (B) Field effect scanning electron microscopic image of the cell obtained at 2.0KV EHT and 5 KX magnification
Thus based on the morphometric analysis and molecular analysis, the isolated strain was designated as Chlorella sorokiniana FC6 IITG (Fig. 3.7). The strain Chlorella sorokiniana FC6 IITG is referred as FC6 in this thesis.
3.3.5 Fatty acid profile of the stored lipid obtained from Chlorella sorokiniana FC6 IITG
In order to evaluate the suitability of selected microalgal stain for biodiesel production, fatty acid composition of stored lipid was analyzed. The FAME composition of FC6 was analyzed using gas chromatography. Palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) were the three major fractions that constitute the majority of total fatty acid compositions. These fatty acids were also found to be abundant in other Chlorella sp.
reported in the literature. In the present study, the selected strain was able to produce fatty acids with 85% contributions from saturated (C16:0) and unsaturated (C18:2, C18:2) fatty acids, which are considered to be the key elements for suitable quality biodiesel (Liu et al.,
2011). Hence, Chlorella sorokiniana FC6 IITG can be a potential candidate for good-quality biodiesel production.
Fig. 3.7 Molecular identification of FC6. Phylogenetic tree based on 18S rDNA sequences of the strain and genus within the order Chlorellales. The tree was constructed using neighbor-joining method with Jukes-cantor model. Bootstrap test (1000 replicates in %) is shown next to the branches and taxon name starts with the gene accession number. The isolated strain reported in this study is marked with (●).