Effect of bacterial contamination on mixotrophic growth of Chlorella minutissima for biofuel production

Mixotrophic growth of algae has attracted attention due to the very high lipid production achieved with this platform. However, given the presence of organic substrates, contamination with heterotrophic organisms could dramatically affect productivity in these cultures. Experiments were carried out in which Chlorella minutissima were cultivated on glucose, glycerol, and acetate under mixotrophic conditions. Axenic algae cultures were grown under varying carbon loadings to provide baseline biofuel production levels and results were comparable to existing literature (50-120 mg/L/day of biomass productivity when grown on 1% glucose, glycerol, and acetate). The same experiments were repeated with controlled bacterial “contamination” by adding Escherichia coli. Due to the mixotrophic environment, E. coli were expected to compete with and displace algal biomass, particularly at high substrate concentrations. Large increases (~200%) in overall biomass productivity were observed in the co-cultures at 1% substrate concentration compared to the axenic algae cultures. Interestingly, the lipid content of the co-cultures did not decline compared to axenic algae, in fact, they nearly doubled at 1% substrate concentration based on GC-MS analysis of fatty acids. Quantitative PCR of 16S rDNA revealed that the algal fraction constituted 92-95% of total biomass at the highest substrate level and that total algal biomass increased by 200-500% in the co-culture compared to the axenic algae culture suggesting a symbiotic relationship. The high oil content in the co-cultures appeared to result from nitrogen limitation that occurred due to very high biomass accumulation.