Microalgae are photosynthetic microorganisms that convert solar energy into chemical energy, which can be redirected physiologically to produce hydrogen (H2). Photosynthetic H2 production by microalgae has many advantages, such as CO2 sequestration; bioremediation of wastewater when it is used as culture medium to grow algal biomass; microalgae growth is not seasonal; cultures can be installed in nonarable lands; and depending on the microalgae species, valuable byproducts can be obtained [1].
H2 production by microalgae is classified into 2 categories: direct photolysis, when H2 is produced from electrons that arise from the water-splitting process of photosynthesis, and indirect photolysis, when H2 is produced from electrons that are derived from the carbohydrate catabolism [2]. In both processes, [Fe]-hydrogenase reduces the electrons to molecular H2 gas. However, hydrogenase expression and its activity are suppressed by oxygen (O2) that is produced by photosynthesis [3]. Thus, during direct photolysis, in addition to generating H2, microalgae produce O2 which in turn inhibits [Fe]-hydrogenase and thus, H2 production.
To address this incompatibility in simultaneous H2 and O2 production, some researchers have focused on identifying or improving genetically engineering microalgae with O2-torelant hydrogenases in which O2 is restricted from gaining access to the catalytic site [4]. Conversely, indirect photolysis is effected when O2 and H2 production are separated by time: oxygenic photosynthesis activity is decreased or inhibited, whereas mitochondrial respiration remains unchanged, consuming the O2 present. Under such conditions, there is a net consumption of O2 by the cells, establishing the anaerobic conditions required to express the O2-sensitive hydrogenase and to carry out H2 production. To alter the photosynthesis:respiration ratio in microalgae, researchers have proposed several strategies, such as the deprivation of sulfur nutrients that reversibly inhibits the PSII [5]; mutants that contain a truncated light-harvesting antenna to increase H2 production [6]; mutants with low sulfate permease activity, which limits sulfates from supplying the chloroplast, resulting in a sulfate-deprived cell phenotype [7]; and starch-over accumulating mutants when H2 production has improved [8]. Nevertheless, none of these methods has achieved commercial maturity.
Thus, simple and economical methods that foster a condition, known as “anaerobic oxygenic photosynthesis” must be developed, in which photosynthetic O2 is consumed as soon as it is released, creating conditions in which anaerobic H2 metabolism occurs [9]. Such O2 consumption would improve the H2 production because both processes—photosynthesis and carbohydrate catabolism—will donate electrons to the hydrogenase. To demonstrate this concept, reducing agents are used to decreasing the generated O2 by photosynthesis and to induce the anaerobic H2 production in microalgae, when photosynthesis and mitochondrial respiration occur simultaneously.
Various agents reduce dissolved O2 in culture media. Sodium sulfite is an inorganic salt with antioxidant and preservative properties, it is oxidized and converted into sodium sulfate and thus scavenge O2. Sulfites affect hydrogenase formation in cultures of Scenedesmus but not its activity [10]. Also, sodium sulfite and sodium bisulfite prevent the accumulation of photosynthetic O2 in Phaeodactylum tricornutum cultures [11]. Sodium thiosulfate is a reducing reagent that scavenges reactive O2 species (ROS) and protects Chlorella sp. and Chlorella sorokiniana cells [12] and [13]. Hydroquinone is a phenol that reacts with molecular O2 (auto-oxidation) in alkali solutions [14] and removes O2 from aqueous medium [15]. The rate of auto-oxidation is pH-dependent, occurring very rapidly at alkaline pH to yield a brown solution but very slowly in acidic medium. Its ecotoxicity is high (