AbstractThis study evaluates the feasibility of advanced biofilm microalgae cultivation in a twin layer (TL) system for nutrient removal (N and P) as the tertiary treatment in small wastewater treatment plants (WWTPs) located in sensitive areas. Furthermore, the potential valorisation of microalgae biomass as a component of bio-based fertilizers is assessed. Scenedesmus sp. was chosen among 33 microalgae strains for inoculation of TL due to its high growth rate and its nutrient uptake capacity. The tests carried out in the prototype were markedly efficient for total soluble and ammoniacal nitrogen removal (up to 66 and 94%, respectively). In terms of potential valorisation of microalgae, the nutrient content was 5.5% N (over 40% protein), 8.8% P2O5 and 1.5% K2O, high enzymatic activity, very low levels of heavy metals and no detectable pathogen presence. However, in the formulation of solid-state bio-based fertilizers, the microalgae proportions in blends of over 2% of microalgae led to negative effects on ryegrass (Lolium perenne L. ssp.) and barley (Hordeum vulgare ssp.). The obtained results demonstrate that TL represents a promising technology, which allows efficient tertiary treatment of urban wastewater and the production of high-quality bio-based fertilizer.