1 Introduction

The rising demand for sustainable and renewable energy sources has intensified research efforts into alternative energy technologies. Among these, biohydrogen production has emerged as a promising candidate, offering a clean, efficient, and environmentally friendly energy carrier. Biohydrogen, produced through biological processes, is particularly attractive due to its potential for large-scale production using diverse biomass feedstocks. This process not only provides a renewable energy source but also contributes to the reduction of greenhouse gas emissions, making it an integral part of future energy solutions (Saravanan et al., 2021; Lin, 2024).

Biohydrogen production leverages the natural metabolic processes of microorganisms to convert organic substrates into hydrogen gas. This biological approach is advantageous over traditional methods, such as steam reforming of hydrocarbons, as it operates under milder conditions, has lower energy requirements, and can utilize a wide range of renewable organic materials (Aslam et al., 2018). Various microorganisms, including algae, cyanobacteria, and anaerobic bacteria, have been studied for their hydrogen-producing capabilities. Among these, anaerobic bacteria are particularly notable for their efficiency in converting complex organic compounds into hydrogen under oxygen-free conditions (Fuess et al., 2019).

Anaerobic bacteria play a critical role in biohydrogen production due to their ability to thrive in oxygen-depleted environments and efficiently degrade organic matter (Bengelsdorf et al., 2018). These bacteria, through fermentation and anaerobic digestion processes, are capable of producing significant amounts of hydrogen. Their metabolic pathways are diverse and can be optimized for enhanced hydrogen yield through genetic engineering (El-Dalatony et al., 2020). The study of these bacteria not only aids in understanding the fundamental mechanisms of biohydrogen production but also provides opportunities to engineer strains with improved hydrogen production capacities, paving the way for industrial-scale applications (Jia et al., 2021).

This study will delve into the metabolic mechanisms of anaerobic microorganisms involved in biohydrogen production, analyzing strategies to enhance hydrogen yield through genetic engineering. It will also evaluate the potential applications of these genetically modified microorganisms in advancing sustainable energy development. By providing a comprehensive overview of the metabolic pathways of anaerobic bacteria in biohydrogen production, this research offers new insights into the fundamental scientific principles of bioenergy conversion, thereby promoting innovation and advancement in biotechnology for clean energy.

2 Overview of Anaerobic Bacteria in Biohydrogen Production

2.1 Types of anaerobic bacteria involved

Anaerobic bacteria are pivotal in the biological production of hydrogen due to their ability to thrive in environments devoid of oxygen, where they efficiently metabolize organic substrates into hydrogen. A variety of anaerobic bacterial species have been identified as efficient producers of biohydrogen. These bacteria are categorized based on their metabolic capabilities and the specific pathways they utilize to generate hydrogen.

2.1.1 Clostridium species

Clostridium species are among the most extensively studied anaerobic bacteria for biohydrogen production. These Gram-positive, spore-forming bacteria are known for their high hydrogen yield during the fermentation of carbohydrates. Clostridium butyricum and Clostridium acetobutylicum utilize the butyrate and acetate pathways to produce hydrogen. The process involves the fermentation of glucose and other sugars into organic acids and hydrogen gas, with the production of acetate being particularly beneficial for hydrogen generation (Pason et al., 2020).

2.1.2 Enterobacter species

Enterobacter species, such as Enterobacter cloacae, are facultative anaerobes that have also shown significant potential in biohydrogen production. Unlike Clostridium species, Enterobacter species can grow in both aerobic and anaerobic conditions, although hydrogen production occurs under anaerobic conditions. These bacteria utilize the formate hydrogen lyase (FHL) pathway, where formate is cleaved into hydrogen and carbon dioxide. This pathway is less energy-intensive compared to other metabolic routes, making Enterobacter species attractive candidates for biohydrogen production (Nizzy et al., 2020).

2.1.3 Other significant species

In addition to Clostridium and Enterobacter species, other anaerobic bacteria have also been identified as efficient hydrogen producers. These include Thermotoga maritima and Caldicellulosiruptor saccharolyticus, both of which are thermophilic bacteria that thrive at high temperatures. These species utilize unique pathways, such as the ferredoxin-dependent hydrogenase pathway, which allows for efficient electron transfer during the breakdown of complex carbohydrates into hydrogen. The high temperature stability of these bacteria makes them particularly suited for industrial-scale biohydrogen production (Saidi et al., 2018; Ben Gaida et al., 2022).

2.2 Natural metabolic pathways for hydrogen production

The metabolic pathways employed by anaerobic bacteria for hydrogen production are diverse and reflect the adaptability of these organisms to different environmental conditions (Figure 1). The primary pathways include:

Ferredoxin-Dependent Hydrogenase Pathway: This pathway is commonly used by Clostridium species, where electrons generated during the oxidation of pyruvate are transferred to ferredoxin. The reduced ferredoxin then donates electrons to hydrogenase, leading to the production of hydrogen (Buckel, 2021).

Formate Hydrogen Lyase (FHL) Pathway: Enterobacter species primarily use this pathway, where formate is directly split into hydrogen and carbon dioxide by the formate hydrogen lyase enzyme complex. This pathway is highly efficient in generating hydrogen under anaerobic conditions (Nizzy et al., 2020).

Butyrate and Acetate Pathways: In Clostridium species, these pathways involve the fermentation of sugars into butyrate and acetate, with hydrogen being produced as a byproduct. The acetate pathway is particularly important due to its higher hydrogen yield (Amin et al., 2019).

These pathways highlight the versatility and efficiency of anaerobic bacteria in biohydrogen production, making them crucial players in the development of sustainable energy solutions.

3 Metabolic Pathways for Biohydrogen Production

3.1 Fermentative pathways

The production of biohydrogen by anaerobic bacteria is governed by various metabolic pathways, each with distinct biochemical mechanisms and efficiencies. Fermentative pathways are central to the production of biohydrogen in anaerobic bacteria. These pathways involve the breakdown of organic substrates, such as carbohydrates, into simpler molecules, resulting in the generation of hydrogen gas.

3.1.1 Acidogenic fermentation

Acidogenic fermentation is a crucial pathway in the production of biohydrogen, where organic substrates are converted into volatile fatty acids (VFAs), carbon dioxide, and hydrogen. During this process, complex organic compounds are first hydrolyzed into simpler sugars, which are then fermented by acidogenic bacteria. The main products of acidogenic fermentation include acetic acid, butyric acid, and lactic acid, with acetic acid being particularly important for biohydrogen production due to its high hydrogen yield (Gu et al., 2020). This pathway is often coupled with other fermentative processes to enhance hydrogen production (Buckel, 2021).

3.1.2 Butyrate-type fermentation

Butyrate-type fermentation is another significant pathway in biohydrogen production, characterized by the production of butyric acid alongside hydrogen and carbon dioxide. This pathway is often favored by Clostridium species and operates efficiently under anaerobic conditions. Although butyrate fermentation yields less hydrogen per mole of substrate compared to acetic acid pathways, it remains a key route for biohydrogen production due to its robustness and compatibility with various substrates (Pason et al., 2020). The pathway's ability to process complex organic materials makes it suitable for industrial-scale hydrogen production from biomass (Tang et al., 2021).

3.2 Hydrogenases and their role

Hydrogenases are enzymes that play a pivotal role in the biohydrogen production process by catalyzing the reversible oxidation of molecular hydrogen. These enzymes are classified into three major types based on their metal cofactors: [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-only hydrogenases.

[NiFe]-hydrogenases are commonly found in both bacteria and archaea and are known for their robustness in various environmental conditions. They play a crucial role in the metabolism of hydrogen under both anaerobic and microaerophilic conditions (Lee et al., 2019).

[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 CRISPR-Cas9, researchers can knock out genes that divert electrons away from hydrogen production or introduce mutations that enhance the activity of hydrogenases. This precision editing is essential for fine-tuning bacterial metabolic pathways to maximize hydrogen output (Jia et al., 2021).

4.1.2 Gene knockout and overexpression

Gene knockout and overexpression are classic genetic engineering techniques that have been extensively used to manipulate the metabolic pathways of anaerobic bacteria. Gene knockout involves the deletion or inactivation of specific genes to eliminate undesirable pathways or reduce the production of competing by-products. Conversely, gene overexpression increases the activity of key enzymes involved in hydrogen production by introducing multiple copies of a gene or using strong promoters to drive higher expression levels. For example, overexpression of hydrogenase genes can significantly boost hydrogen production, while knocking out pathways that lead to the formation of lactate or ethanol can redirect metabolic flux toward hydrogen synthesis (Sekoai and Daramola, 2018).

4.1.3 Synthetic biology approaches

Synthetic biology combines principles of engineering with molecular biology to design and construct new biological parts, devices, and systems. In the context of biohydrogen production, synthetic biology approaches involve the construction of artificial metabolic pathways, the design of synthetic promoters for controlled gene expression, and the creation of engineered bacterial strains with optimized metabolic networks. These approaches enable the assembly of novel pathways that do not exist naturally, thereby expanding the capabilities of anaerobic bacteria to produce hydrogen from a wider range of substrates and under various environmental conditions (Kracke et al., 2018).

4.2 Case studies of genetically modified anaerobic bacteria

The application of genetic engineering to anaerobic bacteria has led to several successful modifications that have significantly improved biohydrogen production.

4.2.1 Enhanced hydrogen production

One notable example is the modification of Clostridium acetobutylicum to overexpress [FeFe]-hydrogenases, resulting in a substantial increase in hydrogen yield. By knocking out competing pathways and enhancing the expression of hydrogenase genes, researchers were able to redirect the metabolic flux toward hydrogen production, achieving higher yields than wild-type strains (Yu et al., 2019). Similarly, Enterobacter cloacae has been engineered to improve its tolerance to oxygen, allowing for more robust hydrogen production under less stringent anaerobic conditions (Lee et al., 2019).

4.2.2 Improved pathway efficiencies

Another case study involves the use of CRISPR-Cas9 to edit Thermotoga maritima, a thermophilic bacterium known for its ability to produce hydrogen at high temperatures. By knocking out specific genes involved in competing metabolic pathways, researchers enhanced the efficiency of the butyrate and acetate pathways, leading to improved hydrogen production under industrial conditions (Saidi et al., 2018). Additionally, synthetic biology techniques have been applied to construct artificial operons that combine multiple hydrogen production pathways into a single, highly efficient system, further optimizing the metabolic processes involved (Kracke et al., 2018).

4.3 Challenges and limitations

Despite the successes achieved through genetic engineering, several challenges and limitations remain. One significant challenge is the complexity of metabolic networks in anaerobic bacteria. The introduction of genetic modifications can lead to unintended effects, such as the accumulation of toxic intermediates or the disruption of cellular homeostasis, which can negatively impact overall hydrogen production.

Another limitation is the difficulty in achieving stable expression of engineered pathways, especially under industrial conditions where factors such as temperature, pH, and substrate availability can vary. Additionally, the 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 acetobutylicum has 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.

For instance, co-culturing Clostridium thermocellum, a cellulose-degrading bacterium, with hydrogen-producing bacteria such as Thermotoga maritima has been shown to improve hydrogen yield from lignocellulosic biomass (Figure 2) (Ben Gaida et al., 2022). The cellulose is first broken down into simpler sugars by C. thermocellum, which are then utilized by T. maritima to produce hydrogen. This cooperative interaction not only enhances the efficiency of substrate utilization but also stabilizes the production process under varying environmental conditions.

Microbial consortia can also be engineered to optimize the metabolic environment for hydrogen production. By selecting and cultivating strains that complement each other’s metabolic activities, researchers can create highly efficient consortia that outcompete single-species cultures in terms of hydrogen yield and stability.

5.4 Environmental and process conditions optimization

Optimizing environmental and process conditions is critical for maximizing the efficiency of biohydrogen production. Factors such as pH, temperature, substrate concentration, and hydraulic retention time (HRT) can significantly influence the metabolic activity of hydrogen-producing bacteria.

Maintaining an optimal pH, typically in the range of 5.5 to 6.0, is crucial for ensuring the activity of hydrogenases and preventing the accumulation of inhibitory by-products. Similarly, controlling the temperature within the optimal range for the specific bacterial species being used can enhance metabolic rates and hydrogen yield (Zhu et al., 2021).

Substrate concentration and the type of feedstock also play a pivotal role in hydrogen production. Using pretreated or hydrolyzed substrates can improve the availability of fermentable sugars, leading to higher hydrogen yields. Additionally, optimizing HRT in bioreactors ensures that the microbial community has sufficient time to degrade the substrate and produce hydrogen, while also preventing washout of the bacteria (Tang et al., 2021).

Advanced bioprocessing techniques, such as fed-batch and continuous-flow systems, can also be employed to optimize hydrogen production. These systems allow for the controlled addition of substrates and the removal of inhibitory by-products, thereby maintaining optimal conditions for sustained hydrogen production (Saidi et al., 2018).

The optimization of metabolic pathways, the application of directed evolution, the development of co-culture systems, and the fine-tuning of environmental and process conditions are all crucial strategies for enhancing biohydrogen production. By integrating these approaches, it is possible to significantly improve the efficiency and yield of hydrogen production in anaerobic bacterial systems, paving the way for more sustainable and economically viable biohydrogen technologies.

6 Recent Advances and Breakthroughs

In recent years, significant advancements have been made in the field of biohydrogen production through the metabolic engineering of anaerobic bacteria. These breakthroughs have been driven by novel genetic modifications, advances in synthetic biology, and the integration of omics technologies.

6.1 Novel genetic modifications and their impact

The advent of advanced genetic engineering techniques has enabled the development of anaerobic bacteria strains with enhanced capabilities for biohydrogen production. One of the most impactful strategies has been the targeted modification of key genes involved in hydrogen production pathways. For instance, the introduction of mutations in the [FeFe]-hydrogenase gene in Clostridium acetobutylicum has led to strains with significantly higher hydrogen yields due to improved enzyme activity and stability (Kracke et al., 2018).

Another notable genetic modification involves the knockout of competing pathways that divert electron flow away from hydrogen production. By inactivating genes responsible for lactate and ethanol formation, researchers have successfully redirected metabolic flux toward hydrogen synthesis, thereby enhancing overall hydrogen yield (Sekoai and Daramola, 2018).

6.2 Advances in synthetic biology

Synthetic biology has played a pivotal role in advancing biohydrogen production by enabling the design and construction of novel metabolic pathways and regulatory circuits. Recent developments in this field have focused on creating synthetic operons that combine multiple genes involved in hydrogen production into a single, highly efficient system. These synthetic operons allow for coordinated expression of the entire pathway, resulting in enhanced hydrogen yields under various environmental conditions (Kracke et al., 2018).

Additionally, synthetic biology has facilitated the creation of novel biosensors and regulatory elements that can dynamically control gene expression in response to environmental cues. For example, researchers have developed synthetic promoters that are activated by specific metabolic intermediates, ensuring that hydrogen production is maximized only when the conditions are optimal (Jia et al., 2021).

6.3 Integration of omics technologies

The integration of omics technologies—genomics, proteomics, and metabolomics—has revolutionized the study of biohydrogen production by providing comprehensive insights into the complex networks of genes, proteins, and metabolites involved in the process. These technologies have enabled researchers to identify key genetic and metabolic bottlenecks that limit hydrogen production and to develop targeted strategies to overcome these challenges.

Genomics: Advanced genomic techniques have been used to sequence the genomes of hydrogen-producing bacteria, allowing for the identification of genes involved in hydrogen metabolism and the discovery of novel hydrogenases with unique properties. This has provided a foundation for the rational design of engineered strains with improved hydrogen production capabilities (Tang et al., 2021).

Proteomics: Proteomic analyses have revealed the expression levels and post-translational modifications of hydrogenases and other enzymes involved in hydrogen production. This information is crucial for understanding how these enzymes are regulated and how their activity can be enhanced through genetic or environmental manipulation (Lee et al., 2019).

Metabolomics: Metabolomic profiling has provided insights into the flux of metabolites through hydrogen production pathways, identifying key intermediates that can be targeted for optimization. This has led to the development of engineered strains with improved metabolic efficiency and higher hydrogen yields (Zhu et al., 2021).

6.4 Case studies highlighting significant progress

Recent advances in genetic engineering, synthetic biology, and omics technologies have significantly enhanced our ability to engineer anaerobic bacteria for improved biohydrogen production. These breakthroughs are paving the way for more sustainable and economically viable hydrogen production technologies, with the potential to contribute significantly to the global transition to renewable energy sources.

Enhanced Hydrogen Production in Clostridium acetobutylicum: Through a combination of CRISPR-Cas9-based gene editing and synthetic biology, researchers developed a strain of C. acetobutylicum that overexpresses a synthetic operon for [FeFe]-hydrogenase, leading to a threefold increase in hydrogen yield compared to wild-type strains (Yu et al., 2019).

Optimization of Thermotoga maritima for Industrial Hydrogen Production: By integrating genomic and metabolomic data, scientists were able to engineer T. maritima to produce hydrogen more efficiently at high temperatures, making it a viable candidate for industrial-scale hydrogen production from lignocellulosic biomass (Ben Gaida et al., 2022).

Directed Evolution of Enterobacter cloacae for Oxygen Tolerance: Using directed evolution, a strain of E. cloacae was developed that exhibits enhanced hydrogen production under less stringent anaerobic conditions, thanks to the evolution of a more robust hydrogenase enzyme (Lee et al., 2019).

These case studies underscore the potential of advanced genetic and synthetic biology techniques to overcome the limitations of traditional biohydrogen production methods and to create more efficient, scalable systems.

7 Challenges and Future Directions

The field of biohydrogen production through the genetic engineering of anaerobic bacteria holds great promise but also faces significant challenges. These challenges span technical, economic, and environmental domains, each presenting obstacles that must be addressed to fully realize the potential of biohydrogen as a sustainable energy source.

7.1 Technical challenges in genetic engineering

One of the primary technical challenges in the genetic engineering of anaerobic bacteria for biohydrogen production is the complexity of metabolic networks. Genetic modifications often lead to unintended consequences, such as the accumulation of toxic intermediates or the disruption of cellular homeostasis, which can reduce the efficiency of hydrogen production. Additionally, the stability of engineered traits over time and under industrial conditions remains a significant hurdle. Maintaining the functionality of genetically modified pathways in the face of fluctuating environmental factors, such as pH, temperature, and substrate availability, is crucial for the success of biohydrogen production on a commercial scale (Tang et al., 2021).

Another challenge is the difficulty in achieving high levels of gene expression without triggering metabolic burden or toxic effects. Overexpression of hydrogenase genes or other key enzymes can lead to the depletion of essential cellular resources, negatively impacting overall cell growth and viability. Balancing the need for high enzyme activity with the health of the host organism is a delicate task that requires careful optimization of genetic constructs and regulatory elements (Jia et al., 2021).

7.2 Economic and Scalability Considerations

The economic viability and scalability of biohydrogen production are major considerations that will determine the feasibility of this technology in real-world applications. One of the significant economic challenges is the cost of genetic engineering itself, which includes the development and optimization of engineered strains, as well as the necessary infrastructure for large-scale production. The use of specialized bioreactors, stringent anaerobic conditions, and precise control of environmental variables can drive up costs, making it difficult for biohydrogen to compete with more established methods of hydrogen production, such as steam methane reforming or electrolysis (Saidi et al., 2018).

Scalability is another key issue. While small-scale laboratory studies have demonstrated the potential of genetically engineered bacteria for hydrogen production, scaling up these processes to an industrial level introduces numerous challenges. These include maintaining consistent performance across large bioreactors, preventing contamination, and managing the logistics of substrate supply and waste disposal. The development of cost-effective, scalable bioprocesses is essential for the widespread adoption of biohydrogen production technologies.

7.3 Potential environmental impacts and biosafety concerns

The release of genetically modified organisms (GMOs) into the environment poses potential risks that must be carefully managed. While the use of anaerobic bacteria for biohydrogen production is typically conducted in controlled bioreactors, there is still a risk of accidental release. Such events could lead to unintended ecological consequences, including the horizontal transfer of engineered genes to native microbial populations, which could disrupt local ecosystems (Ben Gaida et al., 2022).

Biosafety concerns also extend to the production process itself. The metabolic by-products of hydrogen production, such as volatile fatty acids and gases like methane, must be carefully managed to prevent environmental pollution. Additionally, the use of antibiotics or other selective agents in the cultivation of genetically engineered strains raises concerns about the potential spread of antibiotic resistance genes.

7.4 Future research priorities and potential areas of innovation

To address the challenges outlined above, future research in biohydrogen production should focus on several key areas:

1) Advanced Metabolic Engineering: Developing more sophisticated genetic tools and strategies to finely tune metabolic pathways will be crucial. This includes the use of synthetic biology to create more robust and flexible genetic constructs that can adapt to varying environmental conditions without compromising hydrogen production efficiency (Kracke et al., 2018).

Cost-Reduction Strategies: Research should focus on reducing the costs associated with genetic engineering and large-scale production. This could involve the development of low-cost substrates, the use of waste materials as feedstocks, or the improvement of bioprocessing technologies to increase yields and reduce operational expenses (Yu et al., 2019).

Biosafety and Environmental Impact Assessment: Rigorous assessment of the biosafety and environmental impact of genetically modified strains is essential. This includes the development of containment strategies, such as the use of synthetic auxotrophy (requiring a nutrient not found in the environment) to prevent the survival of GMOs outside of the bioreactor. Additionally, the impact of metabolic by-products on the environment should be minimized through innovative waste management solutions (Tang et al., 2021).

Integration with Renewable Energy Systems: There is significant potential to integrate biohydrogen production with other renewable energy systems, such as solar or wind power. This could involve using excess renewable energy to power biohydrogen production processes or integrating biohydrogen with other biofuel production systems to create hybrid energy solutions (Lee et al., 2019).

Exploration of New Microbial Hosts: Expanding the range of microbial species used for biohydrogen production could lead to the discovery of new, more efficient pathways and enzymes. Extremophiles, for example, may offer unique metabolic capabilities that can be harnessed for biohydrogen production under conditions that are challenging for conventional organisms (Ben Gaida et al., 2022).

While significant challenges remain, the future of biohydrogen production through genetic engineering is bright. By addressing technical, economic, and environmental concerns, and by pursuing innovative research avenues, biohydrogen could play a crucial role in the global transition to sustainable energy.

8 Concluding Remarks

The exploration of metabolic pathways and genetic engineering in anaerobic bacteria for biohydrogen production has revealed significant advancements and highlighted ongoing challenges. As the global demand for renewable energy sources continues to increase, biohydrogen production has emerged as a promising avenue for sustainable energy generation. This review has provided a comprehensive analysis of the metabolic pathways involved in biohydrogen production, including acidogenic and butyrate-type fermentations, and the critical role of hydrogenases in these processes. Genetic engineering techniques, such as CRISPR-Cas9, gene knockout, and synthetic biology approaches, have been instrumental in enhancing hydrogen yields by optimizing these pathways.

The integration of omics technologies has provided deeper insights into the regulatory networks governing biohydrogen production and has facilitated the identification of new targets for genetic engineering. Additionally, the development of co-culture systems and microbial consortia, which leverage synergistic interactions between different microbial species, has further enhanced hydrogen production from complex substrates. Despite these advances, challenges such as the stability of engineered traits, economic scalability, and biosafety concerns remain significant hurdles to the widespread adoption of biohydrogen technology.

Genetic engineering has undeniably played a pivotal role in advancing biohydrogen production, enabling the optimization of metabolic pathways and the creation of engineered strains with enhanced capabilities. The precise modification of hydrogen-producing bacterial genomes has opened new possibilities for improving efficiency, yield, and process stability. As the field progresses, the integration of synthetic biology and omics technologies will further expand the potential of genetic engineering. The design of synthetic metabolic pathways, the development of dynamic regulatory circuits, and the use of directed evolution to refine enzyme function are all promising areas of innovation. However, the successful commercialization of biohydrogen technology will require a holistic approach that addresses not only the technical challenges but also the economic and environmental aspects. Ensuring the biosafety of genetically modified organisms and minimizing the environmental impact of biohydrogen production are critical considerations that must be prioritized.

In conclusion, the future of biohydrogen production is bright, with genetic engineering at the forefront of this exciting field. By continuing to innovate and address the challenges, biohydrogen has the potential to become a key player in the global transition to sustainable energy.

Acknowledgments

The author expresses gratitude to the two anonymous peer reviewers for their feedback.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Amin M., Ghasemian M., Bina B., Taheri E., and Fatehizadeh A., 2019, Biohydrogen production as a clean fuel by acid and alkaline pretreated mixed culture during glucose fermentation, Health Scope, 8(2): e12903.

https://doi.org/10.5812/JHEALTHSCOPE.12903

Aslam M., Ahmad R., Yasin M., Khan A., Shahid M., Hossain S., Khan Z., Jamil F., Rafiq S., Bilad M., Kim J., and Kumar G., 2018, Anaerobic membrane bioreactors for biohydrogen production: Recent developments, challenges and perspectives, Bioresource Technology, 269: 452-464.

https://doi.org/10.1016/j.biortech.2018.08.050

Ben Gaida L., Gannoun H., Casalot L., Davidson S., and Liebgott P., 2022, Biohydrogen production by Thermotoga maritima from a simplified medium exclusively composed of onion and natural seawater, Comptes Rendus. Chimie., 25(S2): 129-143.

https://doi.org/10.5802/crchim.136

Bengelsdorf F., Beck M., Erz C., Hoffmeister S., Karl M., Riegler P., Wirth S., Poehlein A., Weuster-Botz D., and Dürre P., 2018, Bacterial anaerobic synthesis gas (Syngas) and CO₂+H₂ fermentation, Advances in Applied Microbiology, 103: 143-221.

https://doi.org/10.1016/bs.aambs.2018.01.002

Buckel W., 2021, Energy conservation in fermentations of anaerobic bacteria, Frontiers in Microbiology, 12: 703525.

https://doi.org/10.3389/fmicb.2021.703525

El-Dalatony M., Zheng Y., Ji M., Li X., and Salama E., 2020, Metabolic pathways for microalgal biohydrogen production: current progress and future prospectives, Bioresource Technology, 318: 124253.

https://doi.org/10.1016/j.biortech.2020.124253

Fuess L., Zaiat M., and Nascimento C., 2019, Novel insights on the versatility of biohydrogen production from sugarcane vinasse via thermophilic dark fermentation: Impacts of pH-driven operating strategies on acidogenesis metabolite profiles, Bioresource Technology, 286: 121379.

https://doi.org/10.1016/j.biortech.2019.121379

Gu X., Wang Y., Li H., Li J., and Wang S., 2020, Characteristics of biohydrogen production and performance of hydrogen-producing acetogen by increasing normal molasses wastewater proportion in anaerobic baffled reactor, Archaea, 2020(1): 8885662.

https://doi.org/10.1155/2020/8885662

Jia D., He M., Tian Y., Shen S., Zhu X., Wang Y., Zhuang Y., Jiang W., and Gu Y., 2021, Metabolic engineering of gas-fermenting clostridium ljungdahlii for efficient co-production of isopropanol, 3-hydroxybutyrate, and ethanol, ACS Synthetic Biology, 10: 2628-2638.

https://doi.org/10.1021/acssynbio.1c00235

Kracke F., Lai B., Yu S., and Krömer J., 2018, Balancing cellular redox metabolism in microbial electrosynthesis and electro fermentation - a chance for metabolic engineering, Metabolic Engineering, 45: 109-120.

https://doi.org/10.1016/j.ymben.2017.12.003

Lee S., Kim M., Kang S., and Lee H., 2019, Biohydrogen production of obligate anaerobic archaeon Thermococcus onnurineus NA1 under oxic conditions via overexpression of frhAGB-encoding hydrogenase genes, Biotechnology for Biofuels, 12: 1-8.

https://doi.org/10.1186/s13068-019-1365-3

Lin J., 2024, Sustainable Development strategy of bioenergy and global energy transformation, Journal of Energy Bioscience, 15(1): 10-19.

Mumtha C., Subashri D., and Mahalingam P., 2022, Biohydrogen producing facultative anaerobic bacteria from different anaerobic sludge, Journal of Pure and Applied Microbiology, 16(3): 1861-1872.

https://doi.org/10.22207/jpam.16.3.32

Nizzy A., Kannan S., and Anand S., 2020, Identification of hydrogen gas producing anaerobic bacteria isolated from sago industrial effluent, Current Microbiology, 77: 2544-2553.

https://doi.org/10.1007/s00284-020-02092-2

Pason P., Tachaapaikoon C., Panichnumsin P., Ketbot P., Waeonukul R., Kosugi A., and Ratanakhanokchai K., 2020, One-step biohydrogen production from cassava pulp using novel enrichment of anaerobic thermophilic bacteria community, Biocatalysis and Agricultural Biotechnology, 27: 101658.

https://doi.org/10.1016/j.bcab.2020.101658

Saidi R., Liebgott P., Gannoun H., Gaida L., Miladi B., Hamdi M., Bouallagui H., and Auria R., 2018, Biohydrogen production from hyperthermophilic anaerobic digestion of fruit and vegetable wastes in seawater: simplification of the culture medium of Thermotoga maritima, Waste management, 71: 474-484.

https://doi.org/10.1016/j.wasman.2017.09.042

Saravanan A., Kumar P., Khoo K., Show P., Carolin C., Jackulin C., Jeevanantham S., Karishma S., Show K., Lee D., and Chang J., 2021, Biohydrogen from organic wastes as a clean and environment-friendly energy source: Production pathways, feedstock types, and future prospects, Bioresource Technology, 342: 126021.

https://doi.org/10.1016/j.biortech.2021.126021

Sekoai P., and Daramola M., 2018, Effect of metal ions on dark fermentative biohydrogen production using suspended and immobilized cells of mixed bacteria, Chemical Engineering Communications, 205: 1011-1022.

https://doi.org/10.1080/00986445.2018.1428958

Tang T., Chen Y., Liu M., Zhang Y., and Zhilong Y., 2021, Biohydrogen production, sludge granulation, and microbial community in an anaerobic inner cycle biohydrogen production (AICHP) reactor at different hydraulic retention times, International Journal of Hydrogen Energy, 46(59): 30300-30309.

https://doi.org/10.1016/j.ijhydene.2021.06.186

Yu Y., Zhu X., Xu H., and Zhang X., 2019, Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli, Metabolic Engineering, 56: 181-189.

https://doi.org/10.1016/j.ymben.2019.10.002

Zhang L., Li J., Ban Q., He J., and Jha A.K., 2012, Metabolic pathways of hydrogen production in fermentative acidogenic microflora, Journal of Microbiology and Biotechnology, 22(5): 668-673.

https://doi.org/10.4014/jmb.1110.10076

Zhu S., Yang X., Zhang Z., Zhang H., Li Y., Zhang Y., and Zhang Q., 2021, Tolerance of photo-fermentative biohydrogen production system amended with biochar and nanoscale zero-valent iron to acidic environment, Bioresource Technology, 338: 125512.

https://doi.org/10.1016/j.biortech.2021.125512