BACKGROUND: Bacillus subtilis synthesizes surfactin, a powerful surface-active agent. It has interesting potential applications. However, due to its high cost of production, commercial use is impracticable. The downstream processing represents approximate to 60% of production costs and the culture medium approximate to 30%. Many reports focused, separately, on production of surfactin using by-products (reduced cost) or the purification using synthetic medium. Therefore, the aim of this work was to evaluate, for the first time, the impact of using a by-product as fermentation medium on the downstream processing based on membrane filtration. RESULTS: Membranes of PES-100-kDa efficiently retained surfactin micelles - the first step of ultrafiltration, whereas, the second step required membranes of 50 kDa to separate surfactin monomers from proteins. Ultrafiltration of crude biosurfactant was associated with fouling and/or concentration polarization resulting in lower purity than when synthetic medium was used. Further improvement in purity was achieved by partial removal of proteins before ultrafiltration by acid precipitation and extraction. The NMR and MALDI-TOFMS analyses identified 11 potential surfactin homologues composed of two amino acid sequences. CONCLUSION: Production of surfactin using cassava wastewater as a low-cost culture medium and its purification by the 2-step ultrafiltration process is feasible, nevertheless, the higher protein content of this medium compared with the synthetic one leads to a lower purity product; further increase in purity can be achieved by applying additional purification steps prior to ultrafiltration with subsequent increased process cost. (C) 2016 Society of Chemical Industry