Date of Award


Document Type


Degree Name

Master of Science (MS)

Legacy Department

Biosystems Engineering


Walker, Terry H

Committee Member

Drapcho , Caye M

Committee Member

Sharp , Julia L


Chlorella protothecoides is a microalga that can grow both photo-autotrophically and/or heterotrophically under different culture and environmental conditions. In this study both the heterotrophic growth and mixotrophic growth have been conducted. The heterotrophic experiments were conducted completely in the dark while the mixotrophic experiments had the dark cycles with periodic light exposure. The aim of the study was to independently understand the effect of each mode on biomass and lipid yields.
For the heterotrophic experiments, glycerol was used as an external organic carbon source while yeast extract was used as the nitrogen source. The carbon and nitrogen source were added to a defined culture medium. Three different grades of glycerol were evaluated for their effect on the biomass and lipid yields in the heterotrophic experiments, with the 65% crude glycerol proving best giving an average biomass concentration of 22.13 ± 0.17 g/L and average lipid concentration of 9.75 ± 0.02 g/L at the end of an eight-day fed-batch fermentation. The average biomass concentrations did not increase after the eighth day of fermentation. The pH was maintained at a constant value of 6.8 and temperature at 280C. As the experiments were carried out in fed-batch mode, addition of the culture medium was done every 24 hours to
maintain the carbon and nitrogen sources at 30g/L and 4g/L respectively till the eighth day. Yeast extract was found to be a good nitrogen source, as it also provides vitamins, amino acids and important growth factors as oppose to some other sources like ammonia and urea (Shi et al., 2000; Gonzalez-Bashan et al., 2000; Illman et al., 2000; Chen et al.,
The mixotrophic experiments were aimed to expose the algae to alternating light and dark cycles to enhance biomass accumulation during light cycle and lipid accumulation during dark cycles. The light cycle help to assimilate CO2 and produce energy via photosynthesis, which comprises the catabolic reaction, while the switch to the
dark cycle allows anabolic reactions where accumulation of lipids and production of other compounds occur. Here, the algae were exposed to light for 8 hours and dark for 16 hours each day for eight days. The 65% crude glycerol was supplemented as the external carbon source to be utilized by the algae during the dark cycles while yeast extract was
used as the nitrogen source. Here the average maximum biomass concentration of 28.95 ± 0.26 g/L and the average lipid concentration of 13.14 ± 0.01 g/L were obtained which were found to be higher than the heterotrophic results. With intermittent light exposure, the lipid yields were found to increase from a maximum of 0.44 ± 0.004 gram lipid/gram
biomass for heterotrophic experiments to 0.46 ± 0.004 gram lipid/gram biomass for mixotrophic experiments. The mixotrophic experiments also provided an increase in the average maximum overall biomass concentration from 22.13 ± 0.17 g/L in heterotrophic to 28.95 ± 0.26 g/L in mixotrophic experiments.