**2. Genetic Modification**

Krishna et al. reported that sustained H2 production is achieved by altering the ratio between PSI and PSII [31]. In this work, a *C. reinhardtii* C3 mutant with a modified PSI/PSII ratio (0.33) produced H2 with a rate of 3 mL H2/L/d for 42 days. Chen et al. identified a *C. reinhardtii* mutant strain hpm91 lacking proton gradient regulation 5, with 30-fold H2 production yield compared to wild type (WT) [32]. Characterization of the hpm91 strain revealed an increased reactive-oxygen-species-scavenging capacity. This translates into an enhanced stability of PSII complex and increased H2 production yield. Steinbeck et al. investigated the capacity of *C. reinhardtii pgr5* and *pgr5 pgrl1* double mutant to produce H2 [33]. The *pgr* mutants showed four times higher maximal enhanced H2 production rate (7 mL/L/h) than the WT. Pinto et al. studied a *Chlamydomonas* mutant with reduced rubisco levels, activity, and stability [34]. This mutant was used to reduce carbon fixation by Calvin cycle activity, which is the main competitor for the reducing power required by the hydrogenase. In this work, the rubisco mutant presented 15 times higher H2 production than the WT. Eilenberg et al. studied the in vivo H2 production efficiency of a *C. reinhardtii* strain Fd-HydA containing ferredoxin fused to HydA. H2 production rate was 4.5 times higher than that of the native HydA in vivo [35]. Torzillo et al. showed that the in vivo H2 production of the *C. reinhardtii* mutant strain L159I-N230Y was up to 5-fold higher (16 nmoles H2/μgchl/h) than that of *C. reinhartii* CC 124 [36,37]. Batyrova et al. developed a genetically modified *C. reinhardtii* strain that activates photosynthesis in a cyclical manner. In this strain, the low O2 production benefits H2 production [38]. In comparison with the WT, this genetically modified strain presented higher H2 production levels. Kosourov et al. showed that a truncated light antenna *C. reinhardtii* mutant could produce six times more H2 compared to the WT strain [39]. Xu et al. introduced a catalase gene from *Synechococcus elongatus* PCC7942 and an *Escherichia coli* pyruvate oxidase gene, both driven by a HSP70A/RBCS2 promoter, into the chloroplast of *C. reinhardtii* [40]. Under low light, these microalgal cells consumed more O2 than WT, resulting in a lower O2 content and increased H2 production [40]. Kruse et al. used the *Chlamydomonas* strain Stm6, which has a modified respiratory metabolism and large starch reserves compared with the WT [41]. *Chlamydomonas* strain Stm6 presented 5–13 times increased H2 production rate (540 mL H2/Lculture) compared to the WT [41]. Later, Volgusheva et al. obtained similar results by using the *Chlamydomonas* Stm6 mutant [42]. They attained an anaerobic condition much faster in the Stm6 strain than in the WT. This was a result of the higher respiration rate and lower initial O2 production rate. H2 production was four times higher in the Stm6 strain compared to the WT. Oey and co-workers reported the knock-down of the LHCMB 1, 2, and 3 proteins in the *C. reinhardtii* strain Stm6Glc4 [43]. The produced *C. reinhardtii* mutant exhibited increased light-to-H2 and biomass conversion efficiencies of 180% and 165%, respectively. Wu et al. introduced a leghemoglobin gene (lba) into chloroplasts of *C. reinhardtii*. The genetically modified *Chlamydomonas* with lba gene consumed O2 faster than WT, thus improving H2 production [44]. Noone et al. introduced the clostridial hydrogenase gene into *C. reinhardtii* that contains insertionally inactivated hydrogenase genes. The presence of the more O2-tolerant clostridial hydrogenase led to more sustained H2 production [45].

Nowadays, the primary current challenge of such a process is the development of an oxygen-resistant hydrogenase. However, other bottlenecks may also be of significant importance, such as the oxygen sensitivity of hydrogenases. In this case, a number of other scientific and engineering issues are very likely to arise. They may include: (1) maximizing photosynthetic light-conversion efficiency (LCE); finding the proper redox potential balance in the organism to facilitate H2 production; (2) preventing the effect of the buildup of high relative H2 partial pressure restricting the process by feedback

inhibition; (3) addressing inefficient metabolic processes such as unneeded ATP generation during H2 production in microalgae; (4) examining issues associated with the generation of destructive, active-oxygen species; and (5) minimizing the production of alternative, carbon-containing products that drain usable reducing power from the system. Recently, an increased H2 output was attained by bioengineering photosynthesis [46].

In the following paragraphs, some of the most recent strategies used for sustained photobiological H2 production by microalgae are summarized.
