Gustavo B. Leite, Patrick C. Hallenbeck*

Departement de Microbiologie et Immunologie, Universite de Montreal, Montreal, Quebec, Canada
Corresponding author email: patrick. hallenbeck@umontreal. ca

OUTLINE

Introduction 389

Engineering Cyanobacteria 392

Strains, Tools and Methods 392

Cyanobacteria as a Production System for Biofuels: Current Status 393

Hydrogen 393

Hydrogen Bioproduction 394

Hydrogen-Evolving Enzymes 394

Hydrogen Bioproduction 395

Ethanol 398

Ethylene 398

Microbial Production of Ethylene 399

Bioproduction of Ethylene Using efe 399

Isoprene 400

Butyraldehyde and Butanol 401

Photosynthetic Production of Aliphatic Alcohols and Alkanes 402

Conclusion and Outlook 403

References 403

INTRODUCTION

Paleontological and geochemical data as well as molecular analysis of the plastid genome point to a single prokaryote as the origin of several groups of organisms scattered throughout the tree of life, including the entire kingdom of Plantae (Knoll, 2008; Yoon, 2004). A cyano- bacterial ancestor is believed to be the only organism ever to couple together two photosystems, harvesting electrons from water to produce energy-rich molecules such as adenosine triphosphate (ATP) and reduced nico­tinamide adenine dinucleotide phosphate (NADPH) (Knoll, 2008) (Figure 22.1). These molecules provide the necessary chemical energy, protons and electrons for cellular reactions and the synthesis of other molecules, most importantly powering CO2 fixation through the Calvin-Benson-Bassham cycle. This event is thought to have happened between the mid-Archean and early Pro­terozoic eras (2000—3000 millions of years ago). The
atmosphere was poor in oxygen and rich in CO2, and the oceans were rich in salts and minerals; perfect conditions for the first algal blooms. The invention of oxygenic photosynthesis conferred a great advantage to this ancient cyanobacterium, starting widespread speciation and changing the composition of the atmo­sphere through the oxidation of water into protons and molecular oxygen (Figure 22.1). This was probably the first universally relevant instance of primary production and established a food chain by transforming inorganic nutrients into organic molecules that could be used by heterotrophic organisms (Knoll, 2008). The role of primary producers, so important in fully establishing life on earth, is still equally important today, when cyano­bacteria are thought to be responsible for 25% of all carbon dioxide fixation and together with eukaryotic microalgae sustain most of oceanic life, fixing CO2 and carrying out important steps in various biogeochemical nutrient cycles (Field et al., 1998).

Bioenergy Research: Advances and Applications http://dx. doi. org/10.1016/B978-0-444-59561-4.00022-X

Humanity is totally dependent on photosynthesis for food and fuel. As well as a source of organic carbon, mankind relies on photosynthesis as energy source, through the use of fossil fuels, ancient photosynthetic products stored and cooked under pressure for millions of years, the burning of readily available biomass, or more recently through the use of biofuel crops as a new source of liquid fuels. Sugarcane or corn ethanol and biodiesel have been produced from crops for more than 40 years, with a greatly increased role the last two decades. These first-generation biofuels are presently being produced at large scale, with worldwide produc­tion of ethanol and biodiesel of 50 billion and 9 billion liters, respectively, in 2007. Even though these seem like significant quantities, biofuels still represent a miniscule fraction of the world’s primary energy use; in 2011, 161 tons per day of renewable liquid biofuels were produced, whereas 12 million tons per day of crude oil were consumed (BP, 2012).

Humans have been constantly perfecting agricultural technology since the dawn of civilization, and with the green revolution, food crop yields have shown consider­able increases decade after decade, although this progress is now stagnating in many food producing areas (Ray et al., 2012). At any rate, given the enormous demand for energy and the predicted increase in the world’s population to 9 billion by 2050, it is evident that there is not enough arable land to satisfy both nutritional and energy demands through food and fuel crops. Of course, in addition to renewable energy derived through photosynthesis, other sources of
sustainable energy exist: solar, wind, geothermal, hydro­electric, etc., but together these energy sources cannot supply the quantity and types of energy demanded worldwide since electricity is not suitable for all applica­tions. Modern society is built around liquid and gaseous fuels, which are very efficient energy carriers suitable for a variety of applications, in particular mobile power. Liquid biofuels are essentially photosynthetically derived compounds, at present sustainably produced through the cultivation of energy crops, but as discussed above, this directly competes with the production of food crops.

A possible and promising alternative for sustainable energy production system is intimately related to crude oil formation over the previous millions of years. Before the appearance of vascular land plants on earth, ancestral cyanobacteria were already occupying a large variety of environments and now, after a long period of evolution, cyanobacteria and the microalgae formed through endosymbiosis of cyanobacteria, can be isolated from virtually any natural water sample, from extremely fresh water to hypersaline lakes, from snow in the Arctic Circle to hot or relatively dry environments. The richness of this speciation over billions of years can be appreciated through the variety of morphological forms that are found. These organisms show themselves to be a promising system for the production of hydrocarbons and other desirable products. Cultivation can be carried out using nonarable land; seawater and wastewater have been shown to support growth, bioremediating effluents while fixing atmospheric carbon dioxide into
possible commercial products. The rather simple nutri­tion requirements of these organisms highlight the capa­bility of their metabolism to produce all the molecules needed for cellular growth. Their pathways frequently contain metabolites with commercial interest that can be readily used or easily processed into a final product (Figure 22.2).

Although cyanobacteria and eukaryotic algae share these attributes, cyanobacteria have the additional advantage of being relatively easily manipulated genet­ically. Thus, using cyanobacteria, if a desired product is not naturally produced, genetic engineering techniques allow the insertion of genes or even entire pathways to make novel products, either high-value compounds or

commodity chemicals such as biofuels. Of course any molecule that is produced using cyanobacteria could be produced in other microorganisms, especially fermentation workhorses such as Saccharomyces cerevi — siae or Escherichia coli, but these are heterotrophs requiring carbon compounds previously fixed through photosynthesis, i. e. agriculturally produced.

Thus, the cyanobacteria are uniquely positioned to carry out CO2 fixation driven by solar energy capture while at the same time being amenable of genetic engi­neering to produce a wide variety of liquid and gaseous biofuels. In this chapter the current achievements on research toward the production of biofuels and crude oil substitutes using cyanobacteria as a model organism are reviewed. As will be seen, although much has already been achieved in terms of engineering toward the production of biofuels, in most cases productivity is the greatest bottleneck, although some steps in down­stream processing also present many challenges. Thus, at present, use of a cyanobacterial system for commer­cial production of biofuels at cost-effective levels still faces significant hurdles.