Journal of Energy Bioscience 2024, Vol.15, No.5, 301-313 http://bioscipublisher.com/index.php/jeb 305 5 Biotechnological Strategies to Enhance Biohydrogen Production 5.1 Genetic Engineering of Algal Strains Genetic engineering of algal strains has emerged as a promising strategy to enhance biohydrogen production. Techniques such as metabolic pathway engineering and the modification of hydrogenase and nitrogenase enzymes are pivotal in this context. By targeting specific genes and pathways, researchers have been able to increase the efficiency of hydrogen production (Figure 2). For instance, the engineering of hydrogenases to improve their activity under oxygenic conditions has shown significant promise (Dubini and Ghirardi, 2014; Anwar et al., 2019; Khan and Fu, 2020). Additionally, the introduction of genetic modifications that enhance the photosynthetic efficiency and biomass accumulation in algae can further boost hydrogen yields (Mathews and Wang, 2009; Vargas et al., 2016). Several case studies have demonstrated the potential of genetically modified algae in enhancing biohydrogen production. For example, the D1 mutant strains of Chlamydomonas reinhardtii have shown increased hydrogen production under simulated outdoor conditions, with strain D239-40 being particularly effective (Oncel et al., 2015). Another study highlighted the use of a chloroplast-targeted genetic modification parameter, which significantly increased hydrogen production efficiency up to a certain threshold (Vargas et al., 2016). These case studies underscore the importance of genetic engineering in optimizing algal strains for biohydrogen production. Figure 2 Representation of the hydrogen photoproduction-related pathways in Chlamydomonas (Adopted from Dubini and Ghirardi, 2014) Image caption: Hydrogen production occurs in the chloroplast, where the photosynthetic chain and the hydrogenases are located (see text for more details). The respiratory chain is located in the mitochondrion, and there is an extensive communication between the two organelles that can impact the level of hydrogen production (adapted from Kruse et al. 2005). The circled numbers indicate where current genetic engineering efforts have impacted H2 photoproduction, as described in the text. The barriers overcome by these modifications are: (1) O2 sensitivity, addressed by PSII inactivation and/or increased O2 consumption; (2) proton gradient dissipation, addressed by the pgrl1 knockout mutation (decreased CEF); (3) photosynthetic efficiency, addressed by knockdown of light-harvesting antennae or truncating antenna proteins; (4) competition for electron, addressed by Rubisco mutagenesis; (5) low reductant flux and hydrogenase expression, addressed by impacting starch accumulation/degradation, FDX-HYD fusion, and overexpressing hydrogenase, respectively. It must be noted that, for clarity, not all the genetic engineering approaches mentioned in the text are represented in the figure (Adopted from Dubini and Ghirardi, 2014)
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