| Algae offer the potential to simultaneously produce biofuels and treat wastewater but significant barriers to commercialization remain. Wastewater often contains organic material in addition to a diverse microbial community. Under high organic loadings, heterotroph microorganisms may out-compete algae for resources. To gain insight into algal-microbial interactions we studied a co-culture system in which we utilized the model green alga Chlorella minutissima and the model bacterium Escherichia coli. We attempted to isolate the effects of the bacteria on culture growth rates, lipid production, starch production, organic substrate uptake, and substrate utilization efficiency under a variety of autotrophic and mixotrophic conditions. The results suggested a symbiotic relationship between the two organisms particularly at high substrate levels. Notably, C. minutissima growth and substrate uptake increased significantly in the presence of E. coli whereas C. minutissima only had modest effects on E. coli growth. These results contrasted with our initial hypothesis that E. coli would occupy a greater fraction of biomass under increasing substrate loads. Moreover, co-cultures exhibited high lipid content under high substrate conditions based on the results of the sulfo-phospho-vanillin lipid assay. This surprising result led to further investigation of lipids by other analysis methods.;Several methods have been employed for algal lipid analysis, however, shortcomings of these methods led to the development of a novel microplate assay to measure neutral lipids. Neutral lipids, particularly triacylglycerols, are considered an ideal biodiesel feedstock. Nile red dye has been used extensively to visualize and measure neutral lipid bodies in a variety of cell types including algae. Efforts to use cell staining for absolute lipid quantitation have thus far proved elusive, however, due to variable dye penetration into different cell types. To avoid this challenge, we used Nile red dye to stain lipids after extraction from algae. Algal pigments were found to completely quench the Nile red fluorescence signal, however, we overcame this challenge by using bleach to destroy the algal pigments. The result was an assay that measured neutral lipids -- particularly triacylglycerol and to a much lesser extent, sterol lipids. The assay result was independent of the algae strain tested, in contrast to cell staining procedures. Other advantages included a lack of sensitivity to acyl chain saturation and good reproducibility.;This assay was used in conjunction with gas chromatography of fatty acid methyl esters and thin layer chromatography of neutral and polar lipids to more thoroughly characterize the lipids of co-cultures. We observed very high levels of neutral lipids, specifically triacylglyerols, in high-substrate co-cultures. Moreover, the fatty acid profiles in those same co-cultures had lower levels of saturation than their axenic counterparts which should lead to higher quality biodiesel with better oxidative stability. These results suggest that co-culturing can enhance both the quantity and quality of biofuel from algae but additional research is required to determine the extent of this phenomenon among species. |
