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1. The Effect of Temperature and Aeration on Chlorella Cell Growth and Oil Production Thi Yi Aung and Celia Dourado Leao Biology Department, Skyline College, San Bruno CA Abstract Concern about global petroleum supplies and global warming has lead to the search for an alternate renewable fuel source. Biofuels, obtained from living organisms, are a promising prospect because they do not add CO2 to the atmosphere. An additional advantage of microalgae-based biofuels is they do not require the cultivatable land of traditional oil-producing plants such as corn or soybean. The unicellular Chlorella algae can accumulate large amounts of lipid. The purpose of this project is to determine the optimum combination of temperature and aeration to maximize cell yield and oil production. Chlorella sp. was grown at 5°C to 45°C in 100-mL aliquots in 250-mL Erlenmeyer flasks with and without aeration for 25 days. Algal cells were counted using a hemacytometer. Intracellular oil was determined using Sudan IV staining. Maximum oil:cell yield is obtained at 25°C . The effect of aeration is being determined. These data will help identify the most efficient photobioreactor-design system for cultivating microalgae for biofuel. • Materials and Methods • Chlorella sp. algae (Niles Biological, Inc. ) were grown under different conditions (Table 1). • Pond water was a 1 dilution of Alga-Gro medium (Carolina Biological Supply Company, 15-3751.) • Cells were counted using a hemacytometer and their intracellular lipid content was determined by Sudan IV staining (5) (Figure 1). • 1.38 x 106 algae cells were placed in 250-mL Erlenmeyer flasks containing 100 mL pond water (Figure 2). • Flasks were incubated for 30 days as shown in Table 1. A 35 Watt incandescent lamp was provided for 11 hrs to simulate sunlight when artificial sunlight was required. An air stone from a compressed air supply was used when aeration was supplied. A magnetic stir bar was used when stirring was required. • Results • Chlorella grew best at 20-25°C; especially with aeration (Figure 2). • The growth rate at 20-25°C was 45% higher than that of 35°C. • Aeration at 20-25°C not only hastened the growth rate, but also resulted in more lipid content per cell (Figure 3). • The most lipid (average 4.0 m/cell, 85% of the cell) was produced at 20-25°C with aeration (Figure 4). • Providing artificial light for 11 hr/day hastened the growth rate but caused a decreased in lipid content to 2.3 m/cell, 60% of the cell. • Algae incubated with stirring clumped. Figure 4. Maximum cell yield and lipid content is obtained at 25°C with aeration. Stat= stationary. • Discussion & Conclusion • Chlorella cells incubated at 45°C died out after 20 days. • Bacterial contaminants are a problem when Chlorella is incubated at 35°C. If Chlorella cells are to grown in desert heat, sterilization will be required. • Although artificial light did increased the growth rate, the lipid content of such cells was low. • The best condition for cell growth and maximum lipid per cell is 25°C with aeration. Aim To determine the optimum combination of temperature and aeration to maximize algae cell yield and oil production. . • Background • Fossil fuels are a nonrenewable resource. • Burning of fossil fuels release a significant amount of CO2 into the air. • Usage of biodiesel, compared to petroleum diesel, showed a 78.5% decrease in carbon dioxide emission (1). • The most common source of biofuels is vegetable oils: corn and soy. • Studies have indicated that a net energy loss from • terrestrial oil seed crops due to the need for large tracts of land, irrigation water, and fertilizer (2). • Microalgae produce 30 times the amount oil per unit area of land (3). • Stresses, such as aeration and lack of nutrient, may increase oil content of algae. • Temperature could also affect the rate of algae growth (3). • The current downside to algae based oil is the need for an efficient photobioreactor designs (4). Literature Cited National Biodiesel Board. October 2009. Benefits of Biodiesel. Patzek, T. and D. Pimentel. 2005. “Is Ethanol from Veggies a Waste of Fossil Energy Sources.” Natural Resources Research 163: 84-85. National Renewable Energy Laboratory. 1998. Close-out Report. A Look Back at the U.S. Department of Energy’s Aquatic Species Program—Biodiesel from Algae. NREL/TP-580-24190. Vasudevan, P. T. and M. Briggs. 2008. “Biodiesel Production— Current State of the Art and Challenges.” Journal of Industrial Microbiology and Biotechnology 35: 421–430. K. Burdon, J. Stokes, and C. Kimbrough. “Studies of the Common Aerobic Spore-Forming Bacilli.” Journal of Bacteriology 43(6): 717-724, 1941. Figure 3. Algal growth within 29 days. Acknowledgements We are grateful to our mentor, Dr. Christine Case, for her advice throughout this project. Her great advice and her enthusiasm about biology made this project an extremely enjoyable learning experience for us. We would also like to thank Patricia Carter, Skyline College Biology lab technician, for her support. Last but not least, we would like to thank Stephen Fredricks, Skyline College MESA director for providing the opportunity for us to attend SACNAS National Conference. Figure 1. Algae cells. (A) Chlorella before lipid staining. (B) Chlorella after lipid staining. Figure 2. Algae cultures. (A) Chlorella at 25°C aerated. (B) Chlorella at 25°C stirred. (C) Chlorella at 25°C stationary.