SUPPLEMENTARY MATERIAL
Mass Cultivation of Various Algal Species and their Evaluation as a Potential Candidate for Lipid
Production
Nadia Sharif, Neelma Munir* Faiza Saleem, Farheen Aslam and Shagufta Naz
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
[email protected]
Microalgae have been proposed as a promising source for biodiesel production. Focusing on algal
strains for biodiesel production, efforts should be made to search new strains. Experiments were carried
out to investigate the effects of growth parameters (nutrients, pH, light, aeration and temperature) and
the lipid percentage of 8 algal strains (Chlorella sp., Cladophora sp., Hydrodictylium sp., Oedogonium
sp., Oscillatoria sp., Spirogyra sp., Stigeocolonium sp., Ulothrix sp.). Results show that 6.5 – 7.5 is the
optimum pH for the growth of all algal species. Temperature showed a greater variation (25o- 40oC).
Ulothrix sp. gave more biomass productivity and is the most suitable strain for biodiesel production
due to higher oil percentage (62%). Least biomass production was observed for Stigeocolonium sp. and
least lipid content was obtained from Hydrodictylium sp. It was observed that among these 8 algal
strains for biodiesel production Ulothrix and Chlorella are the most promising algae species.
Key Words: Biodiesel, Fabrication, Microalgae, Nutrient; pH
[email protected]
Experimental
Study aimed at isolating and identifying algae as potential feedstock for the production of biodiesel. Unicellular
as well as multicellular algae were collected from the freshwater and brackish types of environments. In
Laboratory samples were centrifuged and concentrated to increase the biomass density and were stored at 4°C.
23 algae strains were identified morphologically and microscopically among these 8 strains were selected on the
basis of availability of facilities in laboratory for cultivation and characterization. All used chemical were of
Sigma and Merck Company. The collected algal samples were screened, isolated and characterized for growth
rate and oil production. Pure culturing of algae was made by agar plate and serial dilution methods. Microalgae
samples were spread on the plates containing BB and BG agar medium and the plates were incubated for 15-25
days with 16:8 hours light/dark photoperiod. After incubation the cultures were subjected for continuous
subculture till the pure culture is obtained (Jayaramareddy et al., 2014). For the identification of algae we
observed the characteristics described by the by Graham et al. (2009) . Cultures and experiments were
monitored trough microscopic observations.
Cultivation and Harvesting: Growth experiments were carried in Plastic containers and jars. Cultivation of
cultures was optimized in three different media - Bold Basal Medium (BBM), Blue green Medium (BGM) and
Chalkey’s medium. The mediums were sterilized in an autoclave for 20 min at 121 oC in order to prevent any
contamination. 0.1 gram fresh inoculum was inoculated in 1L media and was cultures for 2 weeks. For mixing
orbital shaker (OS 5 KIKA - WERKE) was used and the flasks were kept over the shaker at 300 rpm. Aeration
was supplied through aerating pumps (CE – SB – 348A0). Cultures were placed indoor where constant sunlight
was given also culture was monitored in the florescent light (500 lux). Growth was monitored at different
temperatures, growth media and pH (5.5 – 9). Cultivation was done in the range of 20 o- 45 oC temperature.
Cultures were allowed to grow till they were considered to have reached the end of the exponential phase / start
of the stationary phase before harvesting. This was determined by morphological means by following the
cultures in light microscope on weekly basis. Biomass was then gently centrifuged at 3000 rpm for 15 min
(Chen et al., 2011) to remove extracellular water and air-dried for 12 h with forced air at room temperature. Airdried biomass was oven-dried at 60ºC for 3hrs and weighed. The dried biomass was used as feedstock for oil
extraction.
Oil Extraction: Microwave extraction was performed by using domestic microwave oven (Orient TM). Oil was
extracted by adding 50 ml of chloroform/methanol (2:1) in 0.5 g dried algal sample. The sample was then heated
in microwave for 120 sec at 800 power level. The vessel content is filtered through filter paper and the lipid
extract evaporated to dryness. The percentage lipid content was calculated as determined by (Putri et al., 2011).
References
Chen, MTang, HMa, HHolland, TCNg, K and Salley, SO. 2011. Effect of nutrients on growth and lipid
accumulation in the green algae Dunaliella tertiolecta. Bioresource technology, 102: 1649-1655.
Graham, LGraham, J and Wilcox, L. 2009. Algae, 720, 2nd ed. Pearson Education, San Francisco, CA. pp. 720.
Jayaramareddy, DKrishnappa, R and Thimmappa, GS. 2014. Microalgae Cultivation in Different pH,
Temperature and Media for Lipid Production. International Journal of Life Sciences, 8: 13-17.
[email protected]
Putri, EVDin, MFMAhmed, ZJamaluddin, H and Chelliapan, S. 2011. Investigation of microalgae for high lipid
content using palm oil mill effluent (POME) as carbon source. International Conference on
Environment and Industrial Innovation, 12: 85-89.
FIGURES
Figure S1. Algae sampling A, axenic culture B, Hydrodictyon sp. C, Cldophora sp. D, Chlorella
sp. E, Oedogonium sp F.
[email protected]
Figure S2. Effect of different pH ranges on the growth of different algal species.
Figure S3. Effect of different temperatures on the growth of different algal species.
[email protected]
Figure S4. Optimum temperature and pH media of selected algal species.
Figure S5. Lipid Percentage of selected algal species
[email protected]
Table S1. Morphological Characteristics of algal Samples
*Corresponding author. [email protected]