Isolation and Characterization of Wild Fresh Water Microalgae
2.3 Results and Discussion
2.3.1 Preliminary analysis of biochemical characteristics of isolated strains .1 Morphological and molecular identification
2.2.7 Fatty acid methyl esters (FAME) analysis
Transesterification of lipids and FAME analysis: Transesterification of neutral lipids was carried out with methanol using a 2-step procedure reported by Mishra and Mohanty (2019). The resultant FAME were further analyzed for the profiles (or composition) using gas chromatograph–mass spectrometer (GC–MS) system. The GC-MS system comprised of a gas chromatograph (Clarus 680, Perkin Elmer, USA) employing Elite-5MS capillary column (dimensions 60 m × 0.25 mm × 0.25 μm with stationary phase of 5% diphenyl and 95% dimethylpolysiloxane) and a Clarus 600C mass spectrophotometer (Perkin Elmer, USA). The peaks were identified using data analysis software NIST-2008.
Fourier transform infrared (FTIR) spectrophotometer analysis: FTIR spectrophotometer (Shimadzu, IR-Affinity-1) was used to obtain infrared spectra of FAME in the range 500 to 4000 cm-1 for analysis of functional groups present in the algal biodiesel.
identification as of genus Tetradesmus. The identified strain SGM19 was green-colored, small single or colonial microalgae. Most of the colonies comprised of 2–4 oval-shaped cells. Both the isolated microalgae showed similarity in some of the morphological characteristics. Hence, genetic identification of both the strains was conducted using sequence analysis of 18S rRNA to attain a clear distinction between them.
At molecular level, the strains were further distinguished by relative sequence analysis of the ITS regions of ribosomal RNA. The sequencing of the 18S rRNA showed 921 and 702 bp long nucleotide sequences in the isolated microalgal strains. The pairwise alignment of the isolate sequences with the NCBI database sequences, demonstrated that strains were 98% identical to Scenedesmus sp. The sequences of the isolated microalgae were submitted to NCBI database. Accession numbers - MG760624.1 (Tetradesmus obliquus SGM19) and MG754459.1 (Tetradesmus obliquus SGM09).
The microalgal identification was also verified by phylogenetic tree analysis. For constructing the phylogenetic tree, other microalgal gene sequences from Genbank were compared with the sequence of the isolates by employing the use of Neighbor-Joining method in Mega 5.0 as described by Saitou and Nei (1987). The isolated microalgal strains were labeled by NCBI as Tetradesmus obliquus SGM19 and Tetradesmus obliquus SGM09. Fig. 2.1C depicts the Neighbour-Joining phylogenetic tree with the location of respective strains based on 18S rRNA sequence.
Fig. 2.1D depicts the FTIR spectrum of raw biomass in the wavenumber range of 500 – 4000 cm-1. The major peaks in the spectrum and their assignment are as follows:
1026 cm1 = P=O bond in phospholipids and nucleic acids; 1153, 1238, 1392 and 1454 cm-1 = bending and stretching of various bonds in polysaccharide, aliphatic groups and carbonate ions (C-O-C, C=O stretching and C-H bending); 1705–1460 cm-1 = proteins, 3000–2800 cm-1 = lipids; 3424 cm-1 = O-H stretching; 1650 cm-1 = ester groups.
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(C) (D)
Figure 2.1. Characterization of isolated microalgae T. obliquus SGM19. (A) light micrograph, (B) FE-SEM micrograph, (C) phylogenetic tree based on 18S rRNA sequences, (D) FTIR spectrum of raw biomass
2.3.1.2 Biomass and lipid content
The growth and biochemical compositions of the isolated microalgae are presented in Table 2.2. T. obliquus SGM19 exhibited a significant growth, displaying maximum specific growth rate of 0.177 ± 0.014 d-1. The sigmoidal growth curve (Fig. 2.2A) showed 1 d lag-phase and a gradual shift to stationary phase, which promotes production of storage substances. On 15th day from inoculation, the final biomass concentration attained was 2.3 ± 0.8 g/L.
Table 2.2. Growth and biochemical characteristics of the isolated microalgae
Parameters T. obliquus SGM19 T. obliquus SGM09
Specific growth rate, µ (d-1) 0.177±0.014 0.161±0.011
Biomass concentration (g/L) 2.3±0.8 1.51±0.62
Biomass productivity (g/L/d) 0.17±0.03 0.15±0.02
Chlorophyll a (µg/mL) 17.71±1.1 17.82±1.8
Chlorophyll b (µg/mL) 10.43±0.8 9.28±1.2
Total Chlorophyll (µg/mL) 28.14±1.9 24.49±1.5
Total Carotenoid (µg/mL) 7.91±0.5 8.45±0.43
Protein (% DCW) 19.7±2.1 19.2±1.6
Carbohydrate (% DCW) 21.5±1.2 19.6±1.2
Lipid (% DCW) 27.5±1.5 20.5±2.2
β-carotene (mg/g DCW) 0.61±0.1 0.55±0.13
Antioxidant activity (%) 69.1±6.3 63.8±5.4
The lipid content was 27.5 ± 1.5 % of DCW, and the maximum lipid productivity was found to be 41 mg/L/d. The microalgal strain with lipid content > 20% and lipid productivity of higher than 40 mg/L/d are considered as potential lipid producers for biofuels (Chen et al., 2017). Based on this criterion, T. obliquus SGM19 is worth of further investigation. Furthermore, with the Nile red staining, the microalgae displayed
lipid bodies as small red dots, indicating high content of intracellular lipid (Fig. 2.2B).
T. obliquus was found to be a promising biomass producer. Biomass and lipid production by the T. obliquus SGM19 strain varied proportionately. The highest lipid content noted in T. obliquus SGM19 was 27.5± 1.5% DCW.
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Figure 2.2. (A) growth profile in BG-11 medium, (B) Nile red stained micrograph
2.3.1.3 Protein and carbohydrate content
Microalgae are potential feedstock for food and pharmaceutical industry because of their high carbohydrate and protein content. With procedure outlined in section 2.2.5, protein content of T. obliquus SGM19 was determined as 19.7% dry biomass, while carbohydrate content was 21.5% dry biomass. Comparing these values with previous literature (Selvarajan et al., 2015), we find that protein content of T. obliquus SGM19 is at par with microalgal strains of other genres such as Chlorella, Dunaliella, and Chlamydomonas. However, carbohydrate content of T. obliquus SGM19 was relatively smaller than other species. Carbohydrate content of the biomass can be enhanced by full optimization of growth conditions supplementation of the medium with CO2 and other promoters. Chandra et al. (2016) reported an increase in carbohydrate content of biomass from 7.73% to 23.91% by sparging CO2 at 20% v/v. Similarly, Patnaik and Mallick (2015) reported that the carbohydrate content could be enhanced from 22.2 to 55.1%
DCW by supplementing growth medium with acetate and citrate.
2.3.1.4 Pigment composition analysis
Chl-a, Chl-b and total carotenoid contents of T. obliquus SGM19 were determined as 17.71, 10.43 and 7.91 µg/mL, respectively, which can be attributed to significant photosynthetic rate and biomass production. Among the two microalgal strains isolated by us, carotenoids/total chlorophyll and chlorophyll a/b ratio were higher in T. obliquus SGM09 which indicates active stress conditions caused by reduction in light-harvesting complex and PS II activity (Zhang et al., 2013). β-carotene yield in both the isolated strains, viz., T. obliquus SGM09 and T. obliquus SGM19 was estimated to be 0.55 and 0.61 mg/g DCW, respectively.
2.3.1.5 Antioxidant assay
The extract from microalgae was further fractionated with methanol, acetone (75%),
ethanol (70%), and water. For the DPPH method, the standard antioxidant showed highest antioxidant activity of 94.6%. The methanolic extract of T. obliquus SGM19 registered the highest activity of 69.1%, followed by methanolic extract of T. obliquus SGM09 (activity = 63.8%) and water extracts of T. obliquus SGM19 (activity = 63.5%). Other extracts of both microalgal strains showed activities below 60%. The antioxidant activities determined with FRAP assay showed similar trend as the DPPH scavenging method with the highest antioxidant activity of 92% for standard, followed by methanolic extract of T. obliquus SGM19 (72.2 %). The other extracts showed activities below 65%.
As the preliminary analysis of biochemical facets of the two isolated microalgal strains (viz. biomass productivity, lipid/carbohydrate/protein/pigment content and antioxidant activities, as described in preceding sections) indicated performance of both the strains to be more or less similar, thus, further studies on simultaneous production of lipids and β-carotene were carried out using T. obliquus SGM19.
2.3.2 Growth of T. obliquus SGM19 and associated product synthesis