Aims: 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. Methods and ResultsA 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. Significance and Impact of the Study: 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.