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Microalgae have been positioned as excellent models for producing new sources of energy (biofuels and biohydrogen). Some investigations in these biological models have been directed to know if the enzymes ferredoxin (FDX) and hydrogenase (HYD) are involved in the algae producing different concentrations of molecular hydrogen (H 2 ). To date, little is known about the concomitant transcriptional regulation of both enzymes during H 2 evolution in algae. In this research, we evaluated the relative expression of hdy and fdx genes during the evolution of H 2 in three microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) in N-deprived anaerobic cultures in the presence of Fe, and 12:24 and 24:24 h dark:light cycles. We also detected structural differences in the enzymes. The 3D modeling indicated that the 3D structure of HYD and FDX are conserved in most algal genera, and the results of our grouping according to the aa characteristics of the proteins showed two grouping trends: One, according to the algae's phylogenetic classification, and another one according to the species-specific enzyme's characteristics, and the grouping could perhaps be more influenced by the algae's ability to produce H 2 . The three microalgae species reached maximum H 2 accumulation values in 24h:24 h dark:light conditions in Fe-supplemented media (4.2 AE 0.12 mL L À1 in C. vulgaris, 3.9 AE 0.10 mL L À1 in S. obliquus, and 4.5 AE 0.10 mL L À1 in C. reinhardtii), and the highest global relative expression of hyd and fdx genes was reached during the first hour of exposure to light, which suggests concomitant expression of both enzymes at the beginning of H 2 production. The behavior of the expression of the hyd and fdx genes in these algal species proved to be similar between species. A better understanding of the concomitant regulation of both enzymes could lay the groundwork for the future use of both enzymes to improve H 2 yields in microalgae.

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