Acetone-butanol-ethanol (ABE) fermentation from wastewater microalgae by Clostridium saccharoperbutylacetonicum N1-4 is a novel bioprocess that utilizes waste substrate to generate valuable solvents. Butanol, the most abundant product resulting from ABE fermentation, is an environmentally safe and high performing fuel that can be utilized as a drop-in-fuel; however, high operational costs and low ABE yield present challenge in scale-up of the process. The utilization of algae as a substrate requires pretreatment prior to fermentation to increase the bioavailability of the algal fermentable sugars and to improve the conditions of the pre-fermentation medium. The purpose of this thesis was to optimize wastewater microalgae pretreatment through (1) the optimization of microalgae saccharification, and (2) the use of cheese whey as co-substrate and supplement. Optimal conditions for sugar liberation from wastewater algae through acid hydrolysis were determined for subsequent fermentation to acetone, butanol, and ethanol (ABE). Acid concentration, retention time, and temperature were evaluated to define optimal hydrolysis conditions by assessing sugar and ABE concentrations, and the associated costs. Additionally, the effect of cheese whey as a supplement and substrate was determined for acetone, butanol, and ethanol (ABE) fermentation from wastewater microalgae. Three media constituents, potassium phosphate, magnesium sulfate, and ferrous sulfate, were evaluated to assess their need as supplements in the medium to be inoculated, when 50 g/L of cheese whey was present. The optimization of wastewater microalgae pretreatment results in increasing ABE production and decreasing process costs.