Scenedesmus is a genus of microalgae employed for several industrial uses. Industrial cultivations are performed in open ponds or in closed photobioreactors (PBRs). In the last years, a novel type of PBR based on immobilized microalgae has been developed termed porous substrate photobioreactors (PSBR) to achieve significant higher biomass density during cultivation in comparison to classical PBRs. This work presents a study of the growth of Scenedesmus vacuolatus in a Twin Layer System PSBR at different light intensities (600 mu mol photons m(-2) s(-1) or 1000 mu mol photons m(-2) s(-1)), different types and concentrations of the nitrogen sources (nitrate or urea), and at two CO2 levels in the gas phase (2% or 0.04% v/v). The microalgal growth was followed by monitoring the attached biomass density as dry weight, the specific growth rate and pigment accumulation. The highest productivity (29 g m(-2) d(-1)) was observed at a light intensity of 600 mu mol photons m(-2) s(-1) and 2% CO2. The types and concentrations of nitrogen sources did not influence the biomass productivity. Instead, the higher light intensity of 1000 mu mol photons m(-2) s(-1) and an ambient CO2 concentration (0.04%) resulted in a significant decrease of productivity to 18 and 10-12 g m(-2) d(-1), respectively. When compared to the performance of similar cultivation systems (15-30 g m(-2) d(-1)), these results indicate that the Twin Layer cultivation System is a competitive technique for intensified microalgal cultivation in terms of productivity and, at the same time, biomass density.