Virginia iGEM 2007

Harvesting Cellulose and Light to Power Butanol Biosynthesis

Motivation

Economic, environmental and geopolitical factors have played a significant role in driving forward current research in biofuel production. Plant biomass, including fast-growing feedstock plants such as grasses and agricultural waste products such as corn stalks, can be converted to liquid transportation fuel by microbial metabolic pathways. Although the concept of deriving fuel from biomass was first proposed in the 1970s, only recent technological advances promise to exploit this abundant, renewable and sustainable source of hydrocarbons that can be domestically produced (Stephanopoulos, 2007).

There are many potential biologically-produced fuels including methane, ethanol, butanol and hydrogen. Subsidized ethanol, fermented by yeast, is currently blended into gasoline fuel in the US. DuPont and BP have recently announced that they will collaborate in retrofitting a bioethanol production facility into a biobutanol plant this year in the UK (McCormick, 2006). Other companies are attempting to develop microorganisms capable of producing hydrogen gas for fuel, which may be a long time coming due to the many engineering challenges involved.

Butanol

Although there are many options, butanol fuel has key advantages. Butanol has an energy density that approaches gasoline, unlike ethanol. Additionally, butanol at atmospheric conditions is a liquid, unlike hydrogen, and is relatively easy to handle. It is less hydroscopic than ethanol, it can be transported through the existing pipeline infrastructure, and it easily blends with diesel or gasoline; it has a higher octane rating than gasoline, so that it can be used by any internal combustion engine made for burning gasoline; and it has a very low vapor pressure so little butanol will escape into the atmosphere (McCormick, 2006; Buckley and Wall, 2006). Butanol has a relatively low latent heat of vaporization and, therefore, will burn better in colder weather than ethanol, which has a much higher heat of vaporization.

Butanol fuel is renewable because it can be produced biologically via fermentation; solvent-producing species of Clostridium such as Clostridia acetobutylicum manufacture butanol naturally (Weizmann and Rosenfeld, 1937). The problem with the bioproduction of butanol is that it is not economically viable in the currently petroleum-dominated market, although with development it could be. The root of the problem lies in the high cost of substrate and the butanol toxicity to the fermenting microorganisms, inhibiting growth and further butanol production (Bowles and Ellefson, 1985). Therefore, major goals in butanol bioproduction include finding a way to grow the fermenting microorganisms on inexpensive feedstock and developing butanol-tolerant organisms. This will be the focus of the remainder of the paper. Other areas of research not discussed in this paper include bioreactor and process design and operation considerations (e.g., extracting butanol from the bioreactor as it is produced).

Energy from Biomass

Humans use approximately 2.0(10)17 Btu/yr. The amount of solar energy that is stored in biomass via photosynthesis is about 2.0(10)18 Btu/yr., only a fraction of the total amount of solar energy received at the Earth’s surface, which is around 2.5(10)21 Btu/yr. (Demain et al., 2005). Biomass contains primarily cellulose and hemicellulose in addition to a variety of other polysaccharides for structural or storage functions (Schwarz, 2001). Almost all of the biomass produced is mineralized again by certain microorganisms, which are capable of hydrolyzing complex polysaccharides with the aid of multi-enzyme systems (Schwarz, 2001).

Butanol-producing Clostridia have limited ability to degrade biomass and therefore cannot take full advantage of biomass as a plenteous substrate. A complete cellulase system was discovered recently in a marine bacterium, Saccharophagus degradans (Andrykovitch and Marx, 1988, Ekborg et al., 2005, Taylor et al., 2006). This microorganism is the only known marine bacterium to be able to metabolize every major polysaccharide found in plant cell walls including cellulose, hemicelluloses (xylans, arabinans, glucans, and β-mannans) and pectin (Taylor et al., 2006). This cellulase system from Saccharophagus degradans has the potential to unlock the largely untapped renewable energy in biomass.

Energy from Sunlight

Although this presents a natural mechanism for the human extraction of energy from biomass, there remains a vast amount of direct solar energy that could potentially be harvested by other biological means. Proteorhodopsin, a membrane-bound, light-driven proton pump, was discovered in 2000 through genomic analyses of uncultured marine bacteria (DeLong, 2000). The finding indicates that light-driven energy production other than photosynthesis may occur commonly in the world’s oceans. In addition to utilizing efficient cellulase systems, integration of proteorhodopsin in a butanol-producing and butanol-tolerant microorganism may drive down the cost of substrate even more by putting to use the free energy radiated by the sun. In this way, butanol can be biosynthesized by microorganisms that have the ability to harvest solar energy directly via proteorhodopsin and indirectly by metabolizing complex polysaccharides from fast-growing feedstock plants and agricultural waste. This paper presents a synthetic biology approach to the metabolic pathway design for butanol bioproduction from plant biomass and sunlight.

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Team Members

From left to right: Amy Schell (BME 2008), George McArthur (ChE 2008), Kevin Hershey (ChE 2008), Ranjan Khan (BME and Neuroscience 2009), Emre Ruhi (Biology 2009)


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