OBJECTIVE Aim of the paper was to assess the feasibility of producing hydrogen as a biofuel by photofermentation of fermented water lettuce (Pistia stratiotes L.) waste biomass, after a nitrogen-stripping treatment. RESULTS A natural (42OL) and an engineered strain (CGA676, with low-ammonium sensitivity) of Rhodopseudomonas palustris were used for producing hydrogen. The stripping procedure was highly effective for ammonium removal, with an acceptable selectivity (91% of ammonium was removed; only 14% of total organic acids were lost). Both strains were able to produce hydrogen only in the nitrogen-stripped substrate. The natural strain R. palustris 42OL showed a higher Biochemical Hydrogen Potential (1224 ml l-1 vs 720 ml l-1 ; 50·0 mol m-3 vs 29·4 mol m-3 ), but at a lower rate (5·6 ml l-1  h-1 vs 7·3 ml l-1  h-1 ; 0·23 mol m-3  h-1 vs 0·29 mol m-3  h-1 ) than strain CGA676. CONCLUSIONS Water lettuce waste biomass can be used for biofuel production, after hydrolization, fermentation and nitrogen stripping. CONCLUSIONS The investigation on novel, low cost and sustainable biomasses as feedstocks for biofuel production is a priority. Aquatic plants do not compete for arable land. Moreover, water lettuce is a floating and invasive weed, thus its biomass must be harvested when detrimental, and can now be biotransformed in clean hydrogen.