Date of Award


Document Type


Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering and Earth Science


Walker, Terry H

Committee Member

Bridges , William C

Committee Member

Ladner , David


Biodiesel production using Chlorella protothecoides microalgae species has become an attractive topic due to the intense requirements of renewable energy in past decades. While, the expensive capital cost of the carbon and nitrogen substrates for algae growth is becoming a restrictive problem in this filed. In this study, heterotrophic cultivation of Chlorella protothecoides in the dark was conducted in 500mL shake flasks. Chlorella protothecoides growth in mixed substrate of brewer fermentation and biodiesel crude glycerol by-products containing a relatively high concentration of carbon and nitrogen was discussed and compared with that in microalgae basal medium which was supplied with 30 g/L pure glucose and 4 g/L yeast extract as its carbon and nitrogen sources. An old Chlorella protothecoides which was stored for six month and a new C. protothecoides strains were inoculated in three different batches supplying with two tested medium. The results of biomass accumulation (g/L), lipid concentration (g/L), total organic carbon (g/L), total nitrogen (g/L) and accumulated biomass and lipid productivities (g/L/day) were discussed and compared in this study. For the heterotrophic experiments, the biomass concentration of the old and new C. protothecoides strains in basal medium with supplement of pure glucose and yeast extract (BM-GY) were 14.47 g/L and 11.43 g/L on the sixth day, respectively. Using mixed by-product substrate, the biomass concentration on day 6 reached 14.07 g/L in the old strains batch and 12.73 g/L in the new strains batch. There were no significant differences of lipid content between BM-GY group and mixed waste group, while, the new strains achieved higher lipid contents than those of the old strains. Approximately 81.5 wt% of total organic carbon and 65.1 wt% of total nitrogen in the mixed waste were removed during the cultivation period. The accumulated lipid productivities achieved in BM-GY medium were 2.07 g/L/day of the old strains and 1.61 g/L/day of the new strains. In the mixed waste groups, lipid productivitiy of the old strains was 2.12 g/L/day and 1.81 g/L/day of the new strains. Based on the data analysis, optical density was a reasonable indicator to predict biomass accumulation in further studies and there was a linear relationship between the optical density and cell concentration. In summary, this study showed the potential usage of industrial waste stream such as brewer fermentation waste and crude glycerol for C. protothecoides fermentation to produce biofuel. These alternative sources of microalgae substrates could lower the capital cost as well as meet the goal of environmental friendly technologies within the biofuel industry.