Bioscience Evidence 2024, Vol.14, No.2, 81-92 http://bioscipublisher.com/index.php/be 84 [FeFe]-hydrogenases are typically more active than [NiFe]-hydrogenases but are more sensitive to oxygen. They are primarily involved in hydrogen production during the fermentation processes in strict anaerobes like Clostridium species (Mumtha et al., 2022). [Fe]-only hydrogenases, also known as iron hydrogenases, are less common but play a significant role in some hydrogen-producing microorganisms. These enzymes facilitate the reduction of protons to produce hydrogen, particularly in hyperthermophilic conditions, as seen in Thermotoga species (Ben Gaida et al., 2022). 3.3 Pathway regulation and optimization The regulation of metabolic pathways for biohydrogen production involves intricate genetic and environmental controls to maximize hydrogen yield. Various strategies have been employed to optimize these pathways, including genetic engineering to overexpress hydrogenase genes or suppress competing metabolic pathways, such as those leading to the production of lactate or ethanol, which divert electrons away from hydrogen production. Additionally, environmental factors such as pH, temperature, and substrate concentration are crucial in optimizing the pathways. For instance, maintaining a slightly acidic pH (around 5.5-6.0) is optimal for many hydrogen-producing bacteria as it favors the activity of hydrogenases and the acidogenic fermentation pathway (Zhu et al., 2021). Advanced biotechnological approaches, such as CRISPR-Cas9-based gene editing, are also being explored to fine-tune the expression of key enzymes and enhance the efficiency of hydrogen production pathways. 3.4 Comparative analysis of different metabolic pathways A comparative analysis of the various metabolic pathways involved in biohydrogen production reveals distinct advantages and limitations: Acidogenic Fermentation: This pathway offers a high hydrogen yield and is highly efficient in terms of substrate conversion. However, it is sensitive to pH fluctuations and requires careful control of environmental conditions (Buckel, 2021). Butyrate-Type Fermentation: While this pathway has a lower hydrogen yield compared to acidogenic fermentation, it is more robust and can handle a wider range of substrates. It is particularly well-suited for the fermentation of lignocellulosic materials and other complex organic wastes (Pason et al., 2020). Hydrogenase Activity: The type of hydrogenase enzyme plays a critical role in determining the efficiency of hydrogen production. [FeFe]-hydrogenases are more active but less stable than [NiFe]-hydrogenases, which are more resilient in fluctuating environmental conditions (Lee et al., 2019). Overall, the choice of metabolic pathway depends on the specific application, substrate availability, and desired hydrogen yield. A combination of these pathways, optimized through genetic and environmental modifications, can potentially maximize biohydrogen production for industrial applications. 4 Genetic Engineering Approaches 4.1 Overview of genetic engineering techniques Genetic engineering has revolutionized the field of biohydrogen production by enabling the modification of anaerobic bacteria to enhance their metabolic pathways and improve hydrogen yield. The application of genetic engineering to anaerobic bacteria for biohydrogen production involves several sophisticated techniques. These techniques aim to modify bacterial genomes to optimize metabolic pathways, increase the efficiency of hydrogen production, and reduce the formation of by-products that compete with hydrogen synthesis. 4.1.1 CRISPR-Cas9 CRISPR-Cas9 is a powerful tool that has been widely adopted in genetic engineering due to its precision and efficiency in gene editing. This technology allows for the targeted modification of specific genes involved in biohydrogen production, such as those encoding hydrogenases or enzymes in fermentative pathways. By using
RkJQdWJsaXNoZXIy MjQ4ODYzMg==