ABSTRACT This study aimed to evaluate third generation biodiesel production by microalgae Phormidium autumnale using cassava processing wastewater as a platform. Experiments were performed in a heterotrophic bubble column bioreactor. The study focused on the evaluation of the bioreactor (batch and fed-batch) of different operational modes and the analysis of biofuel quality. Results indicate that fed-batch cultivations improved system performance, elevating biomass and oil productions to 12.0 g L−1 and 1.19 g L−1, respectively. The composition of this oil is predominantly saturated (60 %) and monounsaturated (39 %), resulting in a biodiesel that complys with U.S., European and Brazilian standards. The technological route developed indicates potential for sustainable production of bulk oil and biodiesel, through the minimization of water and chemical demands required to support such a process.