Friday, August 12, 2011

A Case for BioHydrogen

Our energy dilemma

The hydrocarbon economy is faltering as oil reserves dwindle worldwide (Hirsch, 2008).  Commodity prices have begun to fluctuate drastically due to the uncertain cost of petroleum, which resulted in food riots around the world in 2008.  With a steadily decreasing energy supply and the demands on energy systems continually growing, the planet is in dire economic, geopolitical, and environmental straits.  In order to halt the advance of climate change, prevent ecological collapse, rescue the global economy, and ensure our energy security, humanity must find a way to harness currently available (non-fossilized) energy.  The largest source of energy on Earth, excluding the future potential for thermonuclear fusion reactors, is the sun.  Human civilization consumes 15 TW annually while approximately 80,000 TW of solar energy fall on the Earth’s surface each year (Makarieva et al., 2008).  For hundreds of millions of years, this solar flux has been the driving force for life on Earth.  It is estimated that photosynthesis absorbs and distributes seven times more energy to the biosphere (~100 TW) than is consumed anthropogenically each year (Makarieva et al., 2008).  There are many promising technologies in development that will cheaply harness solar output, like next generation photovoltaics, advanced wind turbines, tidal power systems, wave energy generators, solar thermal collectors, biofuels, solar fuels, electrofuels, etc.  In the end, some combination of these solutions, coupled with improved energy and transportation infrastructure, will be needed to resolve our energy dilemma.  In this article, I focus on cyanobacteria and their potential for hydrogen production, which, if properly developed, could provide sustainable fuel for transportation and industry in a post-petroleum world. 

BioHydrogen Airship of the Future?

Cyanobacteria and the rise of oxygenic photosynthesis

Approximately four billion years ago, after the formation of Earth, the first signs of life began to appear, scratching out a lithotrophic existence in an oxygen-starved atmosphere (Towe, 1996; Cleaves et al., 2008).  Anoxygenic photosynthesis probably developed very early in evolution (Pierson and Olson, 1989), harnessing the power of the sun, but the real metabolic revolution didn’t occur for another billion or so years, when photosynthetic eubacteria evolved the ability to split water into electrons, protons and gaseous oxygen (Nisbet et al., 2007).  This new metabolic mode, termed oxygenic photosynthesis, effectively gave living organisms access to an endless supply of electrons from water and became such a prolific process that it filled Earth’s atmosphere with oxygen, fundamentally transforming the biogeochemistry of the planet (Wille et al., 2007).  Moving electrons from chemically stable water molecules to high-energy carbon-carbon bonds spanned an immense reduction-oxidation (redox) gap, powered by a reliable stellar wind of solar electromagnetic radiation, which ultimately increased the size and scope of life and made the development of structurally complex eukaryotic (multicellular) organisms possible (Blankenship and Hartman, 1998).  Almost all ecosystems on Earth today are directly or indirectly dependent on oxygenic photosynthesis.

The subjects of this Earth-transforming story, and the only organisms known to have evolved oxygenic photosynthesis, are the cyanobacteria (Nisbet et al., 2007).  For a billion years, the electrons in water sat untouched by photosynthetic life because it was energetically unfeasible to oxidize water and reduce NADP+ (an electron carrier needed for the reduction of inorganic carbon) in a single step.  In an evolutionary leap, cyanobacteria pioneered the coupling of two photosynthetic reaction centers, P680 and P700 (named for the wavelengths of light they optimally absorb), referred to as photosystem II (PSII) and photosystem I (PSI), respectively (Allen and Martin, 2007; Mimuro et al., 2008).  Thus, the redox problem was resolved by first pumping low-energy electrons up the redox gradient to a temporary reservoir (the quinone pool/electron transfer chain), from which the second photosystem (PSI) could pull out excited electrons in order to transfer them to an even higher energy state, sufficient for the reduction of NADP+ to NADPH + H+.



The rising concentration of oxygen in the atmosphere presented new challenges to early life on Earth.  Most species at the time were obligate anaerobes or microaerophiles, and only those organisms that had the ability to deal with highly reactive oxygen radicals and persist in an oxygen-enriched atmosphere would inherit the majority of Earth’s habitats (Brioukhanov and Netrusov, 2007; Bendall et al., 2008).  Cyanobacteria, in addition to overcoming the general toxicity of oxygen (a byproduct of their new metabolism), had to find a way to acquire nitrogen under aerobic conditions (fixing atmospheric nitrogen gas into biologically-available ammonia).  The molybdenum-iron nitrogenase enzyme is highly conserved among diazotrophs (nitrogen-fixers) and is very sensitive to oxygen, which destroys the activity of the enzyme.  Cyanobacteria got around this issue by separating oxygenic photosynthesis and nitrogen fixation, either spatially or temporally (Tsygankov, 2007).  Some cyanobacteria only activate nitrogen fixation under dark anaerobic conditions, when PSII is unable to evolve oxygen.  Other cyanobacteria, like filamentous Nostoc punctiforme, form a specialized cell type for nitrogen fixation, called a heterocyst.  Heterocysts deactivate their PSII complexes, grow thickened cell walls, and exhibit higher intracellular respiration rates, which keep oxygen levels very low (Cardona, 2009).  Vegetative cells provide the heterocysts with carbohydrates, while the heterocysts provide the vegetative cells with fixed nitrogen (Cardona, 2009).  

Nostoc punctiforme

Hydrogen metabolism in cyanobacteria

Nitrogenase is responsible for the following reaction: N2 + 8 H+ + 8 e + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi (Tikhonovich and Provorov, 2007).  This process is very energy-intensive, requiring 8 moles of ATP for every mole of ammonia produced.  In addition to ammonia, the nitrogenase enzyme also produces a molecule of hydrogen gas for every molecule of gaseous nitrogen that it fixes.  Hydrogen production via nitrogenase is relatively inefficient, due to the large amounts of ATP required, but it can still be used to produce measurable amounts molecular hydrogen.  One impediment to producing hydrogen in this way is the presence of uptake-hydrogenases (Schütz et al., 2004), which oxidize nitrogenase-produced hydrogen in order to minimize the energy loss from N-fixation by reclaiming ATP via the oxyhydrogen reaction, removing oxygen from the interior of the cell and providing reducing equivalents for other cellular processes (Tamagnini et al., 2007).  Concordantly, uptake-hydrogenase enzyme activity has been shown to correlate with nitrogenase activity (Schütz et al., 2004).  Therefore, it is not surprising that a Nostoc uptake-hydrogenase knockout mutant exhibits a significantly higher hydrogen output than the wild-type (Lindberg et al., 2004).

Nitrogenase
Bidirectional hydrogenases, as their name implies, are capable of reversible catalysis of hydrogen formation or consumption (2H+ + 2e- <=> H2).  Unlike the uptake-hydrogenases, the activity of these enzymes is independent of nitrogenase activity (Schütz et al., 2004).  These enzymes seem to act as putative escape valves for the excess reducing power that could build up during metabolism, but their exact function is still under debate (Tamagnini et al., 2007).  Cyanobacterial bidirectional hydrogenases have a nickel-iron active site.  These nickel-iron hydrogenases are more aerotolerant, but less productive than the iron-iron hydrogenases found in other anaerobic eubacteria and in eukaryotic green algae (Ghirardi et al., 2007).  Both nickel-iron and iron-iron hydrogenases require maturation proteins in order to attain catalytic activity, but it has also been suggested that certain [Fe-Fe] hydrogenases do not require a maturation process (Asada et al., 2000; King et al., 2006).  These maturation proteins are involved with the insertion of metal clusters into the active site of the hydrogenases (Fontecilla-Camps et al., 2009).  Hydrogenases are much more efficient hydrogen producers than nitrogenases, and hold great promise for future biotechnological applications (Tamagnini et al., 2007).

The overall physiology of heterocystous cyanobacteria is highly complex.  Advances in genomics, transcriptomics, proteomics and metabolomics are helping scientists develop a holistic view of hydrogen metabolism (Cardona, 2009).  In terms of the obstacles facing photobiological hydrogen production, they can be broken down into a few basic issues (Tamagnini et al., 2007).  The first main hurdle is that the most prolific hydrogen-producing enzymes, the [Fe-Fe] hydrogenases, are highly oxygen sensitive.  For a photobioreactor to be cost-effective, it should produce hydrogen under atmospheric conditions.  Some investigations have looked into finding or engineering oxygen-tolerant hydrogenases (Ghirardi et al., 2007).  The difficulty with oxygen-tolerant hydrogenases is that their hydrogenase activity has so far been inversely proportional to their level of oxygen tolerance (as seen in [Ni-Fe] hydrogenases).  In heterocystous cyanobacteria, this obstacle has been overcome by creating an anoxic microenvironment inside the heterocyst.  Another complication arises due to the competition of different metabolic pathways for electrons from PSI.  Most of the reducing power generated via oxygenic photosynthesis is diverted towards carbon and nitrogen fixation in Nostoc punctiforme.  The goal is to better insulate energy-yielding pathways from competing metabolic processes (Agapakis et al., 2010).  Dr. Matthias Rögner, from Ruhr-Universität in Bochum, estimated at a recent conference in Sigtuna, Sweden, that under optimal growth conditions ~75% of all electrons coming from PSI could potentially be diverted to hydrogen production without negatively impacting the organism.  While metabolic engineering can be vastly complex, scientists can theoretically simplify this problem by catching the electrons at their source (PSI).  PSI hands its electrons off to a ferredoxin, which then shuttles the electrons away to various pathways (Tamagnini et al., 2007).  One idea is to physically link a ferredoxin ligand to a hydrogenase, in order to compete with the native ferredoxins and snatch electrons away from alternate pathways and transfer them directly to the hydrogenase (Agapakis et al., 2010).

The ideal hydrogen-producing organism

Nostoc punctiforme was isolated from a mutualistic association with a cycad (Costa and Lindblad, 2002).  Nostoc, when living symbiotically, has a higher heterocyst frequency than it does as a free-living organism, and will devote much of its metabolism to the production and secretion of specific nitrogen-rich metabolites that are beneficial to its plant host (Enderlin and Meeks, 1983).  Since Nostoc has evolved to mass-produce a specific metabolite when living symbiotically, it seems almost pre-programmed for fuel production.  Heterocystous N-fixing cyanobacteria have minimal nutritional requirements, high photosynthetic efficiencies and can create anoxic microenvironments inside specialized cells that allow anaerobic processes to occur under aerobic conditions.  All of these attributes are conducive to affordable bioreactor design for hydrogen production.  If Nostoc hydrogen metabolism can be effectively engineered, using novel synthetic biology tools, the door to developing successful photobiological hydrogen-yielding technologies is opened. 

Nostoc living symbiotically in plant tissue.

Synthetic biology: an emerging field

An astounding result of recent genomic sequencing projects is that the length of a genome does not predict the morphological or physiological complexity of an organism.  For example, length of the human genome is similar to that of the fruit fly (Mukherji and van Oudenaarden, 2009).  Instead, it has been found that biological modularity can help explain the diversity of form and function in the natural world (Mukherji and van Oudenaarden, 2009).  A limited subset of predictable biological “parts” can be assembled in various ways to produce molecular “devices”, which can be arranged into “systems” to carry out different functions.  In this light, one can view a living cell as a combination of co-regulated genetic circuits, working in tandem.  Another surprising discovery made recently shows the inherent resiliency of biological circuitry to rewiring.  Isalan et al. (2008), in order to test the limits of perturbing regulatory networks in biological systems, found that randomly rewiring Escherichia coli transcriptional networks by synthetically altering transcription factor/promoter pairings only resulted in faulty growth in 5% of cases.  This level of tolerance to random restructuring of biological networks is highly conducive to the adaptation and evolvability of living systems by allowing large-scale alterations to be made to an organisms genome without significantly impeding its growth (Isalan et al., 2008; Mukherji and van Oudenaarden, 2009).  Biological modularity and network resiliency provide powerful mechanisms for the rapid evolution of novel or optimized processes by reshuffling pre-existing genes/proteins (Isalan et al., 2008; Mukherji and van Oudenaarden, 2009; Picataggio, 2009). 

Transcriptase enzymes moving along the DNA strand, from a recent synthetic biology comic book published in Nature.
As our understanding of biology grows in breadth and in depth, we come closer to the goal of being able to rationally design biological systems.  Thanks to the enormous accumulation of whole-system biological data and the discovery of the modular nature of genetic and enzymatic elements during the past few decades, coupled with advances in in silico data analysis/modeling and rapid in vitro DNA synthesis technology, a new field, called synthetic biology, has emerged (Picataggio, 2009).  Synthetic biology allows for the rational manipulation of microbial phenotypes by combining systems biology, bioinformatics, protein design and engineering.  With a comprehensive understanding of the molecular regulation of gene expression and protein function, biologists can begin to assemble a toolbox of reliable promoters, repressors, activators, ribosomal binding sites, reporters, signaling devices and enzymes, that can be used to design metabolic circuits in a cellular chassis - an autonomous self-replicating framework, or superstructure, that acts as the platform for synthetic circuitry (Picataggio, 2009).  Standardization of genetic tools will streamline process engineering and expand the potential to quickly develop microbial systems for the production of renewable fuels and high-value molecules (Picataggio, 2009).

Synthetic biologists have already succeeded in characterizing thousands of biological parts with defined functions and performance parameters, which can be accessed openly at the wiki site http://partsregistry.org/ maintained by the Massachusetts Institute of Technology, but they are not yet capable of engineering whole biological systems with the same precision and reliability that, say, electrical engineers are accustomed to.  One of the main challenges is to identify the subset of genes that are absolutely necessary for the survival of a minimal genome - the smallest number of genes that allows for the replication of an organism in a particular environment (Cho et al., 1999).  Until researchers can build a living cell from the ground up, they won’t totally understand the limits of metabolic engineering.  The immense potential for engineering crucial synthetic metabolic circuits has already been demonstrated by the work of Dr. Jay Keasling, who produced an important precursor to the anti-malarial drug artemisinin in E. coli (Hale et al., 2007).  Keasling’s work has cut the cost of artemisinin by ten fold and will provide many people in the third world with access to crucial malaria treatments, literally saving millions of lives. 

Dr. Jay Keasling

E. coli is an ideal cellular chassis for molecular reprocessing of low-value substrates into high-value products, but in order to use synthetic biology to tackle humanity’s energy needs, it is important that we move away from heterotrophic organisms and focus on developing a photosynthetic chassis that can harness the power of the sun.  Approaching solar fuel production via direct synthesis from sunlight avoids the drawbacks of traditional fermentation-based methods whose biomass feedstocks often compete directly with food crops (Tenenbaum, 2008).  Researchers at UC Davis have recently developed efficient synthetically-derived fuel (isobutyraldehyde, which can be converted to isobutanol) from cyanobacteria (Atsumi et al., 2009).  This result is encouraging, and suggests that the field of photosynthetic fuels will continue to grow.  In the absence of a minimal genome, heterocystous cyanobacteria seem to be ideal chassis for hydrogen production.  


The development of synthetic biology tools for cyanobacteria

The creation of standard biological parts (promoters, ribosomal binding sites, repressors, activators, etc.) for cyanobacteria is simplified by the work already completed in E. coli and by the current open-source nature of synthetic biology resources, fostered by organizations like the BioBricks Foundation (http://bbf.openwetware.org/) and iGEM (http://2009.igem.org). With our current understanding of transcriptional and translational regulation in cyanobacteria, we can re-design genetic regulatory elements and codon-optimised genes from distantly related organisms in silico and synthesize these constructs in vitro for expression in cyanobacteria.  A team in France has developed codon-optimized (for expression in E. coli, Synechocystis, Nostoc and Anabaena) synthetic [Fe-Fe] hydrogenase genes from Chlamydomonas reinhardtii and Clostridium acetobutylicum that have been linked to a synthetic ferredoxin ligands derived from a chlamydomonal ferredoxin (Jaramillo, A., 2009, École Polytechnique, Palaiseau, France, privileged information).  Recently, researchers in the Department of Photochemistry and Molecular Science at Uppsala University have characterized Ptrc promoters, derived from the lacUV5 promoter, ribosomal binding sites and an expression vectors (pPMQAK1 and pPMQAC1) that are broadly functional in E. coli and in cyanobacteria (Brosius et al., 1985; Huang et al., 2010; Gibbons, 2010).  With these tools, it is now possible to express prolific [Fe-Fe] hydrogenases and their respective maturation proteins in cyanobacteria.  It is only a matter of time before viable hydrogen-yielding systems are in place. 

Synthetica, a novel branch to the tree of life.

Conclusion

In this post, I wanted to provide a glimpse of recent developments in photobiological hydrogen production (a field that I'm familiar with), but this is just one small piece of the puzzle.  There are many teams of brilliant researchers around the globe chipping away at their own corners of sustainable energy.  The future green economy will have to take a multi-faceted approach to energy, incorporating dozens of the most successful technologies, alongside behavioral and policy reform (i.e. less consumption, governmental regulation of greenhouse gasses, more localized food production, ecosystem preservation/restoration etc.).  My point was to inspire hope, despite all the doom and gloom surrounding climate change, by illuminating a subset of the work and ideas of our best and brightest, who toil day and night to build a better tomorrow.  While we cannot ignore the perils that lie ahead, we must maintain a certain degree of hope in order to keep our heads above water and look towards a better horizon.  Fear not, fellow humans - for every Senator James Inhofe, there are a hundred thousand scientists, social activists, community horticulturists and conservationists.


Citations

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