Bioscience Evidence 2024, Vol.14, No.2, 81-92 http://bioscipublisher.com/index.php/be 86 potential for horizontal gene transfer in engineered strains raises concerns about the release of genetically modified organisms into the environment, necessitating strict containment measures (Tang et al., 2021). The economic feasibility of large-scale biohydrogen production using genetically modified bacteria remains a challenge. The cost of genetic engineering, coupled with the need for specialized bioreactors and fermentation conditions, can make it difficult to compete with traditional hydrogen production methods. While genetic engineering offers powerful tools to enhance biohydrogen production, overcoming these challenges will require continued research and innovation. Advances in synthetic biology, metabolic engineering, and bioprocess optimization will be essential for realizing the full potential of genetically modified anaerobic bacteria in sustainable hydrogen production. 5 Optimization Strategies for Enhanced Biohydrogen Production Optimization strategies are essential for maximizing the efficiency and yield of biohydrogen production by anaerobic bacteria. These strategies encompass a range of approaches, including metabolic engineering, directed evolution, co-culture systems, and environmental optimization. 5.1 Metabolic engineering for pathway optimization Metabolic engineering involves the deliberate modification of an organism's metabolic pathways to increase the yield of desired products, such as hydrogen. By manipulating key enzymes and regulatory elements within these pathways, researchers can enhance the efficiency of substrate conversion and redirect metabolic flux towards hydrogen production. One of the primary approaches in metabolic engineering is the overexpression of genes encoding hydrogenases and other critical enzymes involved in hydrogen production. For example, the overexpression of [FeFe]-hydrogenases in Clostridium acetobutylicumhas been shown to significantly increase hydrogen yield by enhancing the electron flow towards hydrogen production (Kracke et al., 2018). Additionally, knocking out genes responsible for the production of competing by-products, such as lactate or ethanol, can further improve the efficiency of hydrogen production pathways (Jia et al., 2021). Synthetic biology also plays a crucial role in pathway optimization by enabling the design and construction of novel metabolic circuits. For instance, synthetic promoters and operons can be engineered to control the expression of multiple genes simultaneously, allowing for precise regulation of metabolic pathways and improved hydrogen production (Kracke et al., 2018). 5.2 Directed evolution techniques Directed evolution is a powerful tool for optimizing enzymes and metabolic pathways by mimicking the process of natural selection in the laboratory. Through iterative cycles of mutation and selection, enzymes with enhanced activity, stability, or substrate specificity can be evolved, leading to improved biohydrogen production. In the context of biohydrogen production, directed evolution has been used to evolve hydrogenases with greater resistance to oxygen and higher catalytic efficiency. For example, by subjecting Enterobacter cloacae to directed evolution, researchers have developed strains with increased hydrogenase activity and enhanced tolerance to oxygen, resulting in more robust hydrogen production under industrial conditions (Lee et al., 2019). This approach can also be applied to other enzymes in the hydrogen production pathway, allowing for the fine-tuning of metabolic processes to maximize yield. 5.3 Co-culture systems and microbial consortia Co-culture systems and microbial consortia offer a promising strategy for enhancing biohydrogen production by leveraging the synergistic interactions between different microbial species. In these systems, multiple microorganisms work together to degrade complex substrates, transfer metabolites, and optimize the flow of electrons towards hydrogen production.
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