Genomic Science Program

Biohydrogen Production

Hydrogen is a promising energy carrier of the future: It can be derived from a variety of energy sources and used in fuel cells with high efficiency; “combustion” of hydrogen produces only water as a by-product, making it a nonpolluting, carbon-free energy alternative. The most common industrial methods for producing hydrogen include steam reformation of natural gas, coal gasification, and splitting water with electricity typically generated from fossil fuels. These energy-intensive industrial processes release carbon dioxide and other greenhouse gases and pollutants as by-products. Some microorganisms produce hydrogen naturally, and biotechnologies based on these microbial systems could lead to the development of clean, renewable sources of hydrogen. In a recent report on the hydrogen economy, however, the National Research Council (NRC) noted that “substantial, fundamental research needs to be undertaken before photobiological methods for large-scale hydrogen production are considered” (Hydrogen Economy 2004).

Several reviews have examined the potential of biological hydrogen production (Madamwar, Garg, and Shah 2000; Ghirardi et al. 2000; Melis and Happe 2001; Tamagnini et al. 2002; Levin, Pitt, and Love 2004; Nath and Das 2004; Prince and Kheshgi 2005). Although microorganisms produce hydrogen by different mechanisms, the step can be represented by the simple chemical reaction 2H⁺ + 2e^– <--> H₂. This reaction is known to be catalyzed by either nitrogenase or hydrogenase enzymes. Although alternative biological hydrogen production-pathways exist, each with its own set of advantages and disadvantages, the following discussion on biohydrogen production will focus on challenges that must be overcome to improve one type of biological hydrogen production known as biophotolysis.

Biophotolysis of Water

Under certain conditions, green algae and cyanobacteria can use water-splitting photosynthetic processes to generate molecular hydrogen (H₂) rather than fix carbon, the normal function of oxygenic photosynthesis (see sidebar, Photosynthetic Production of Hydrogen from Water). Bidirectional hydrogenases in these organisms use electrons from the photosynthetic electron-transport chain to reduce protons to yield H₂. Biophotolysis holds potential for the scale of hydrogen production necessary to meet future energy demand. This approach to hydrogen production is promising because the source of electrons or reducing power required to generate hydrogen is water—a clean, renewable, carbon-free substrate available in virtually inexhaustible quantities. Another advantage of biophotolysis is the more efficient conversion of solar energy to hydrogen. Reengineering microbial systems for the direct production of hydrogen from water eliminates inefficiencies associated with carbon fixation and biomass formation. Theoretically, the maximal energetic efficiency for direct biophotolysis is about 40% (Prince and Kheshgi 2005) compared with a maximum of about 1% for hydrogen production from biomass (Hydrogen Economy 2004). Recognizing the important potential of biophotolysis, NRC has recommended that DOE “refocus its biobased program on more fundamental research on photosynthetic microbial systems to produce hydrogen from water at high rate and efficiency” (Hydrogen Economy 2004).

Biohydrogen Research Targets

Engineering Oxygen-Tolerant, Efficient Hydrogenases. Hydrogenases known to tolerate oxygen generally are not very efficient hydrogen producers. During biophotolytic hydrogen production, oxygen is released from the water-splitting reaction; thus, engineering hydrogenases with sufficient activity and oxygen tolerance will be needed. Engineered hydrogenases then could be used in bioinspired nanostructures that maintain optimal conditions for hydrogen production.

Designing Microorganisms Optimized for Hydrogen Production. Photosynthetic microbes that have been genetically modified to produce hydrogen at high rates and efficiency from the biophotolysis of water could be grown in extensive farms of sealed enclosures (photobioreactors). Hydrogen would be harvested for use in energy applications, with oxygen released as a by-product.

Gaps in Scientific Understanding

Understanding biophotolysis well enough to model hydrogenase structure and function, regulatory and metabolic networks, and eventually entire organisms will stimulate the kind of biotechnological innovation needed to engineer the ideal organism to use in hydrogen bioreactors or the ideal enzyme-catalyst to use in bioinspired nanostructures for hydrogen production. But achieving this level of understanding will require basic research that investigates a greater range of hydrogen-producing enzymes and organisms, mechanisms of hydrogenase assembly, oxygen sensitivity of hydrogenase, electron-transfer rate limitations, and regulatory and metabolic processes that influence hydrogen production. Some specific issues relevant to these basic research needs follow.

• What is the extent of natural diversity among hydrogenases and hydrogen-producing organisms? A vast majority of organisms that contain hydrogenases have not been identified and probably cannot be cultured in the laboratory using current procedures. Studying hydrogenase enzymes involved in nonbiophotolytic pathways could provide structural or functional insights to guide the engineering of biophotolytic systems.
• How are hydrogenases assembled, and how are metals incorporated into the active site? Two major types of hydrogenases are defined by their biologically unique metallocenters: Nickel-iron (NiFe) and iron only (Fe). NiFe hydrogenases are found in many bacteria and some cyanobacteria. Fe hydrogenases are found in some bacteria and green algae. In green algae, hydrogenases are bidirectional (capable of catalyzing hydrogen oxidation or proton reduction to produce H₂); in cyanobacteria, hydrogenases are either bidirectional or they uptake enzymes. Although turnover is much higher for Fe hydrogenases, NiFe hydrogenases are more oxygen tolerant. The metallocenters of both NiFe and Fe hydrogenases form complexes with such unusual inorganic cofactors as carbon monoxide (CO) or cyanide (CN). Little is known about the assembly of an active hydrogenase, and several genes may be involved in the synthesis of cofactors required for activity. A better understanding of hydrogenase assembly will enable the engineering of enzymes with improved function.
• How do we overcome the oxygen-sensitivity problem of hydrogenases? The bidirectional Fe hydrogenases that catalyze the hydrogen-evolution reaction in biophotolytic systems are highly sensitive to oxygen, a product of the water-splitting reaction in the first step of the photosynthetic pathway. Oxygen sensitivity also makes hydrogenase isolation from cells and its subsequent analysis a challenge that will be met by new technologies.
• What are the potential electron-transfer rate limitations associated with each step of the biophotolytic hydrogen production pathway? Key factors that can impact the partitioning of electrons between hydrogenase and competing pathways include the buildup of a pH gradient across the photosynthetic membrane and variations in the concentrations of critical electron-transport carriers. Understanding how electron fluxes in an organism are regulated will aid the development of mechanisms for directing more electrons towards proton reduction and hydrogen production.
• What are the regulatory and metabolic pathways that influence H₂ production? A thorough examination of hydrogen metabolism in green algae and several different strains of cyanobacteria from diverse habitats will provide new insights into how hydrogen production pathways are controlled. By understanding how an organism sustains and regulates hydrogen-production, we will be able to determine which metabolic pathways contribute, how eliminating hydrogen-consuming reactions affects hydrogen metabolism and other cellular processes, and how organisms can be adapted to increase hydrogen yields.

Scientific and Technological Capabilities Required to Achieve Goals

Key capabilities needed to address many of the gaps in current understanding of biophotolytic hydrogen production include developing microbial hosts to produce hydrogenase enzymes, screening large numbers of enzymes for desired functionalities, large-scale molecular profiling to provide a global view of hydrogen production, in vivo visualization of hydrogenase structure and activity, modeling of regulatory and metabolic networks, and metabolic engineering (see Table: Roadmap for Development of Biophotolyic Hydrogen Technologies, and Table: Biophotolytic Hydrogen Production Challenges, Scale, and Complexity). Specific needs include the following:

• Suites of microbial hosts to produce hydrogenases from many different organisms. Potentially thousands of enzymes from many different organisms will need to be produced and analyzed. Other requirements include methods for producing eukaryotic enzymes in simpler prokaryotic systems, designing host organisms that can provide the intracellular environment required for proper protein assembly and folding, and screening the proteins produced from these host organisms.
• Methods to produce large numbers of enzymes to screen for desired hydrogenase properties. With so much variability among natural hydrogenases and engineered variants, developing high-throughput capabilities for producing large numbers (perhaps hundreds of thousands to millions) of enzymes to screen for O₂ tolerance, H₂ production, spectroscopic examination, and structural analysis could accelerate the discovery of enzymes best suited for biotechnological applications.
• Molecular profiling to provide a global view of cellular activity during hydrogen production. Improvements in computational capabilities and large-scale molecular profiling techniques (transcriptomics, proteomics, metabolomics, measurements of metal abundance) are needed to obtain a global view of microbial hydrogen production. Systems-level analyses could guide experimental investigations by defining gene regulatory networks controlling the expression of genes involved in hydrogen production or cofactor synthesis. Pathways activated or deactivated during hydrogen production could be identified for multiple organisms under varying conditions.
• Methods to perform in vivo visualization and characterization of molecular machines. Although crystal structures of some hydrogenases have been determined, this information provides only snapshots of enzyme structure. Advanced techniques for visualizing the different stages of hydrogenase assembly or monitoring hydrogenase activity in living cells will be critical to building predictive models that can be used to engineer hydrogenases optimized for biotechnological applications.
• Support and techniques for systems-level studies to model and simulate regulatory and metabolic networks. Studying hydrogenase function within the context of a network maintained by living cells is essential to understanding how this process is influenced by different pathways and environmental conditions. Traditional in vitro biochemical methods that study hydrogenase activity one enzyme at a time in the laboratory do not provide sufficient information to understand enzymatic activities in living cells. Tools for monitoring hydrogenase activity in vivo and integrating diverse sets of experiment data are needed to build in silico models of a biophotolytic organism under hydrogen producing conditions.
• Metabolic engineering. Metabolic engineering involves genetically modifying microorganisms to target and manipulate enzymatic, regulatory, or transport pathways that impact a particular microbial process such as hydrogen production. Models could guide metabolic engineering, for example, by identifying control points for manipulating the flow of electrons to hydrogenase or by predicting how cellular activity and hydrogen yields may be impacted by a variety of conditions. These conditions include the elimination of a particular metabolic pathway or the buildup of a pH gradient across the photosynthetic membrane.

Roadmap for Development of Biophotolytic Hydrogen Technologies

Total Processes That Must Be Optimized, with a Number of Challenges for Each

Biophotolytic Hydrogen Production Challenges, Scale, and Complexity

Text adapted from Genomics:GTL Roadmap: Systems Biology for Energy and Environment, U.S. Department of Energy Office of Science, August 2005. DOE/SC-0090.