3. Broadly biodieselis composed ofCarbon - 77%,Hydrogen -12%,Oxygen - 11% andtraces of Nitrogenand Sulfur(Tomasevic et al,2003) Advantages –Renewable,biodegradable,less emissions,can be producedfrom cooking oil(Nantiyakul,2007) Fuel suitable to beused in compressionignition dieselengines mainlycomprising of fattyacid monoalkylesters derived frombiological sources ofoils and fats likevegetable oils,animal fats andmicroalgal oils (CEN,2008)

5. Table 1: Comparison of the fuel properties of Biodiesel from Microalgae and Diesel (Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China, 2004)

6. Compare transesterification efficiency using homogeneous, immobilized enzyme and heterogeneous catalyst Compare oil extraction from biomass using ultrasonic waves and microwave heating Production of oil from Chlorella vulgaris and study of its growth dynamics

8. Pure microbial culture of Chlorella vulgaris was procured from TISTR Pure microbial culture of Chlorella vulgaris was procured from TISTR Culture grown on Nutrient agar slants Working culture prepared by inoculating in Bold’s basal medium (BBM) Working culture prepared by inoculating in Bold’s basal medium (BBM) Growth conditions: Temperature – 25 0 C, RPM – 100, pH – 6.7, Air – 1 L/min, CO 2 - 0 ml/min, 10 ml/min and 20 ml/min Growth conditions: Temperature – 25 0 C, RPM – 100, pH – 6.7, Air – 1 L/min, CO 2 - 0 ml/min, 10 ml/min and 20 ml/min Two table lamps with 14 W cool white bulbs were fixed 20 cm away from the bioreactor to provide illumination Two table lamps with 14 W cool white bulbs were fixed 20 cm away from the bioreactor to provide illumination Optical Density was checked every day once by UV Spectrophotometer at 682 nm Optical Density was checked every day once by UV Spectrophotometer at 682 nm

9. C.Vulgaris grown in BBM for 15 days C.Vulgaris grown in BBM for 20 days Grown in N-deficient BBM for 10 days Grown in N-deficient BBM for 15 days Grown in N-deficient BBM for 10 days Grown in N-deficient BBM for 15 days Centrifuged at 5000 rpm for 15 minutes and then thick cell mass slurry freeze-dried at -48 deg C overnight Centrifuged at 5000 rpm for 15 minutes and then thick cell mass slurry freeze-dried at -48 deg C overnight

10. Figure1: Bioreactor set-up along with external illumination Figure2: Bioreactor vessels containing microalgae C. vulgaris during growth and incubation for oil accumulation phases

11. Figure3: Freeze dried microalgal biomass

12. Microwave Heating Ultrasonication Figure4: Microwave oven Figure5: Ultrasonic processor

13. Freeze dried Chlorella Vulgaris Microwave @ 450 W 2,4,6,8,10 mins Microwave @ 450 W 2,4,6,8,10 mins Microwave @ 900 W 2,4,6,8,10 mins Microwave @ 900 W 2,4,6,8,10 mins Ultrasonication @ Half wave cycle 2,4,6,8, 10 mins Ultrasonication @ Half wave cycle 2,4,6,8, 10 mins Ultrasonication @ Full wave cycle 2,4,6,8, 10 mins Ultrasonication @ Full wave cycle 2,4,6,8, 10 mins Oil from Chlorella vulgaris biomass

14. Density at 15 0 C Kinematic viscosity at 40 0 C Acid value Fatty acid composition

15. Chemical homogeneous Catalyst Potassium hydroxide (KOH) Biodiesel Immobilized enzyme catalyst Novozyme 435 Chemical Heterogeneous catalyst CaO/Al 2 O 3 Recycled Enzyme and Heterogeneous catalyst : Used catalyst mixed with fresh catalyst in a ratio of 1:1

16. Table 2: Standard method of biodiesel testing according to Department of Energy Business, B.E.2548 (2005)

18. End of exponential phase of growth - 20 days after inoculation Optimum level of CO 2 concentration in delivered air - 20 ml/min The biomass production after 15 day growth was 0.63 mg of biomass/ml of culture and that after 20 day growth period was 0.91 mg of biomass/ml of culture. Figure 6: Growth curves (based on optical density) of Chlorella vulgaris under standard fermentation conditions and external supply of carbon dioxide mixed in air at various concentrations

19. Effect of growth period and oil accumulation incubation period Average oil extraction after incubation in normal media 15 day – 26 % 20 day – 29 % Average oil extraction after incubation in normal media and N-deficient media 15 day growth and 10 day oil accumulation – 37 % 20 day growth and 10 day oil accumulation – 37.4 % 15 day growth and 15 day oil accumulation – 44.2 % 20 day growth and 15 day oil accumulation – 45 % Figure 7: Oil production for different oil accumulation incubation period More incubation in N-deficient medium More accumulation of oil

20. 2 4 6 8 10 Optimum 8 Optimum 8 Figure 8: Oil extraction efficiency after different treatment time for microwave heating and ultrasonication applied on dry biomass after growth in normal medium and incubation in N-deficient medium for oil accumulation Effect of treatment time

21. Figure 9: Oil extraction efficiency by microwave heating at 450 W and 900 W power levels for biomass obtained after different periods of growth and oil accumulation Effect of treatment method – Microwave heating

22. Figure 10: Oil extraction efficiency by ultrasonication treatment at half cycle and full cycle wave modes for biomass obtained after different periods of growth and oil accumulation Effect of treatment method – Ultrasonication

23. Effect of treatment method 41.18% 41.07% 0.388 kWh/min 0.432 kWh/min 0.487 kWh/min 0.789 kWh/min Ultrasonication Half cycle Ultrasonication Full cycle Microwave 450 W Microwave 900 W Microwave Treatment more economic in terms of power consumption

24. Liquid at room temperature Yellowish in color with a tinge of green, no odor Appearance and consistency of normal vegtable oil Acid value=0.08 mgKOH/g Limit – 0.5 mgKOH/g max. Viscosity = 32.28 cSt Limit – 3.5-5.0 cSt Density = 914 kg/m 3 Limit – 860-900 kg/m 3

25. Table 3: Comparison of the different methods of transesterification on basis of catalyst used, reaction conditions and the resultant FAME % obtained

26. Algal oil Density - 914 kg/m 3 Viscosity – 32.28 cSt Acid value – 0.08 mgKOH/g Algal Biodiesel Density – 852 kg/m 3 Viscosity – 4.5 cSt Acid value – 0.08 mgKOH/g All the properties were within specifications of the Department of Energy Business B.E. 2548 (2005) All the properties were within specifications of the Department of Energy Business B.E. 2548 (2005) Highest methyl ester content obtained was 93.44 % which is less than the specified limit of 96.5% for B-100. Biodiesel obtained from transesterification of the algal oil can thus only be used by blending with normal petroleum diesel in lieu of its slightly less FAME content

27. Optimum concentration of CO 2 in air delivered - 20 ml/min Optimum time period of growth - 20 days Biomass production (mg of biomass/ml of culture) - 0.63 mg/ml (15 day growth) and 0.91 mg/ml (20 day growth). Main factor influencing oil extraction - Treatment time. No effect of different power and wave cycle modes on oil extraction. Oil extraction increased with increasing processing time. Optimal time for treatment - 8 mins. The average oil extraction by microwave heating - 41.07 % and ultrasonication - 41.18 %. On basis of power consumption microwave heating is much economical compared to ultrasonication. Major fatty acid in the oil Linoleic acid (69.1 wt. %) and the average molecular weight of the oil - 874.86 g/mole. Density of the oil at 15 0 C was 914 kg/m 3, Kinematic Viscosity at 40 0 C was 32.28 cSt and acid value was 0.08 mg KOH/g. Homogeneous catalyst gave maximum FAME conversion Biodiesel obtained from transesterification of the algal oil can be used only by blending with normal petroleum diesel in lieu of its slightly less FAME content Recycled catalysts mixed with fresh catalysts can give considerable amount of FAME conversion which add to the commercial viability of those catalysts. Density of oil was reduced to 852-854 kg/m 3 from 914 kg/m 3. Viscosity of oil was reduced to about 4.5 cSt from 32.28 cSt. Both the properties were in range of DOEB specifications

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