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Hydrogen is a renewable fuel that can be biologically produced by green algae in closed anaerobic photobioreactors with light and organic carbon as energy sources. This research aimed to investigate the influence of sulfur concentration and light intensity on hydrogen and ethanol production, as well as on nutrient removal by Chlamydomonas reinhardtii (CC425) in batch cultures. The strain was cultivated in two phases: in the first step, the cultures were maintained under aerobic conditions to obtain biomass; in the second step, the biomass was transferred to closed anaerobic photobioreactors for gas generation under continuous illumination. A factorial design was accomplished to improve the biomass production in the first step, with light variation, pH, and glucose addition. Afterward, light intensity and sulfur concentration were varied to test hydrogen production in the second step. The best hydrogen production occurred in photobioreactors without sulfur addition (average increase of 7 times in the production) and under higher light intensity the productivity was 37% higher than lower light intensity (39.64 ± 2.44 µmol H2 L−1 h−1). There was an effect of sulfur concentration in the ethanol production and under higher light intensity the production was higher (203.20 ± 31.49 mg L−1). Furthermore, under conditions with the presence of sulfur, there was greater removal of ammoniacal nitrogen (5.3%), phosphate (85.0%), COD (9.1%) and acetic acid (97.2%). This research demonstrates the efficient production of hydrogen by C. reinhardtii and it shows that the process can be associated with ethanol production and nutrient removal. [ABSTRACT FROM AUTHOR]