Photosynthesis is the most efficient natural mechanism for transduction of energy from the primary energy source, the sun. By developing a way to transfer this energy production pathway to a scalable and industrial host we can replicate natural systems to generate clean renewable energy. By engineering a photosynthetic pathway into Escherichia coli from algae naturally expressing it, we can produce a clean hydrogen fuel source from sunlight and water. This year the Macquarie Australia iGEM team focused on the hydrogenase component from the photosynthetic pathway, enabling the production of hydrogen gas for use as a future energy source.

Hydrogen as a source of fuel are gaining popularity in the current energy market due to the absence of polluting emissions and high energy yield. As hydrogen fuel cells generate energy via the redox reaction of hydrogen with atmospheric oxygen, the only byproduct from this process is water while the generated energy can provide: motive power to vehicles, such as cars, boats, aircrafts, and rockets; as well as electrical power for manufacture and potentially households. At 120 MJ kg−1, hydrogen has a higher energy content compared to petroleum, as automotive gasoline, producing 46.4 MJ kg−1, and as automotive diesel producing 45.6 MJ kg−1 (Lakatos et al., 2014). This indicates the energetic value to be gained by widespread adoption of hydrogen fuels cells when compared to the current dominant market products.

Hydrogen fuel cells require pure hydrogen, and since this is not naturally available, in large quantities, it must be manufactured via techniques which are energy intensive. Currently, the hydrogen generation industry produces over 50 million tons of hydrogen per year, sourcing over 95% of its product from fossil fuels. Unfortunately, this production process is not free of pollutants. These processes include steam reforming of natural gas (48%) or partial oxidation of hydrocarbons (30%) and coal (18%), as shown in figure 1 (Koumi Ngoh and Njomo, 2012). All these production processes contribute to the resource depletion and the increase in the atmospheric concentration of the two main greenhouse gases, carbon dioxide and methane. The most advanced process of hydrogen production is by the electrolysis of pure alkaline water but has not yet been made economically viable due to dependence on electrical power and to date only addresses 4% of the world’s demand (Koumi Ngoh and Njomo, 2012). As such, the environmental costs involved in hydrogen fuel production negate a large portion of the environmental benefits seen in hydrogen fuel use.

To make progress towards economically viable and environmentally sustainable Hydrogen production, the Macquarie Australia iGEM team have successfully transformed E. coli with a hydrogenase gene cluster capable of converting glucose to hydrogen gas (fig. 2). This was achieved with our main biobrick submission, the Hydrogen Gas Producing Gene Cluster.

Currently three hydrogenases are known: [Fe] hydrogenase, found mostly in archaea (Salomone-Stagni et al., 2010); [NiFe], endogenous to E. coli (Kim Jaoon et al., 2010); and [FeFe] hydrogenase from Chlamydomonas reinhardtii (Mulder et al., 2011) (fig. 3). We have successfully incorporated the [FeFe] hydrogenase from Chlamydomonas reinhardtii into DH5α E. coli. The complex we have incorporated consists of the hydrogenase enzyme (Hyd1), ferredoxin, ferredoxin-NADPH-reductase (FNR) and the maturation enzymes (HydEF and HydG). Together these enzymes work cohesively to produce our desired hydrogen gas product whilst avoiding the detrimental emission present in current production processes. A similar experiment was carried out by Agapakis et al. (2010) producing hydrogen via co-expression of an [FeFe] hydrogenase with ferredoxin and pyruvate-ferredoxin oxidoreductase (PFOR) in E. coli. Through testing combinations of hydrogenases, ferredoxins and PFOR from different sources they found the highest levels of hydrogen production were seen with the PFOR from Desulfovibrio africanus co-expressed with the hydrogenase and ferredoxin from Clostridium acetobutylicum. However, this model limited the theoretical yield to two moles hydrogen per one mole of glucose. They did not co-express the C. reinhardtii hydrogenase with the C. reinhardtii FNR as we have. Theoretical yield for hydrogen gas production from glucose is twelve molecules of hydrogen per glucose, however natural and genetically-modified microorganisms to date cannot produce hydrogen with a yield of more than four (Zhang, 2015).

Our other Biobrick parts, which we have submitted, include the three components of the Hydrogen Gas Producing Gene Cluster. They are the HydEFG biobrick, encoding the two enzymes involved in the maturation of the hydrogenase (HydEF and HydG); FerHyd which includes the genes encoding ferredoxin, FNR and Hyd1; and the fourth part is HydG which had not previously been submitted.

We integrated what we learned from consulting the public and industry into a business plan and prototype for fuelling cars at home. By doing this, we have designed a potential future platform for bacteria transformed with the Hydrogen Gas Producing Gene Cluster. More broadly these bacteria could represent an alternate, clean, renewable source for electrical power generation. We hope that the progress we have made will lead to the further exploration of the potential of our Hydrogen Gas Producing Gene Cluster.

AGAPAKIS, C. M., DUCAT, D. C., BOYLE, P. M., WINTERMUTE, E. H., WAY, J. C. & SILVER, P. A. 2010. Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering, 4:3.

KIM JAOON, Y., JO, B. & CHA, H. 2010. Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli. Microbial Cell Factories , 9:54.

KOUMI NGOH, S. & NJOMO, D. 2012. An overview of hydrogen gas production from solar energy. Renewable and Sustainable Energy Reviews , 16, 6782-6792. LAKATOS, G., DEAK, Z., VASS, I., RETFALVI, T., ROZGONYI, S., RAKHELY, G., ORDOG, V., KONDOROSI, V. & MAROTI, G. 2014. Bacterial symbionts enhance photo-fermentative hydrogen evolution of Chlamydomonas algae. Green Chem., 16, 4716-4727.

MULDER, D. W., SHEPARD, E. M., MEUSER, J. E., JOSHI, N., KING, P. W., POSEWITZ, M. C., BRODERICK, J. B. & PETERS, J. W. 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19, 1038-1052.

SALOMONE-STAGNI, M., STELLATO, F., WHALEY, C. M., VOGT, S., MORANTE, S., SHIMA, S., RAUCHFUSS, T. B. & MEYER-KLAUCKE, W. 2010. The iron-site structure of [Fe]-hydrogenase and model systems: an X-ray absorption near edge spectroscopy study. Dalton Transactions, 39, 3057-3064.

ZHANG, Y. H. P. 2015. Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnology advances, 33, 1467-1483.