In this study, marine microalgae Chlorella pyrenoidosa produced 186 ml H-2/l under nitrogen deprivation in natural seawater, and adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to medium reduced the total volume of hydrogen production by 85%. This suggested water was the main electron donor for hydrogenase. An active starch accumulation was observed during the first two days in nitrogen deprivation. But the starch content in cell decreased only by 7% at the end of the hydrogen evolution stage. This was shown the absence of a large contribution of starch to the hydrogen production by C. pyrenoidosa in nitrogen deprivation. Different from the hydrogen production by Chlamydomonas reinhardtii under sulfur deprivation condition, the concentration of acetate in the medium decreased not only at the stage of oxygen consumption but also during the stage of hydrogen evolution by C. pyrenoidosa. Thus, acetate is involved not only in the establishment of anaerobiosis but also plays an important role in the production of hydrogen by C. pyrenoidosa as an exogenous electron donor. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.