The bioalcohol paradigm

The emergence of a globalized economy over recent decades has led to the expansive use of non-renewable resources, especially petroleum from crude-oil. According to the Energy Information Administration, the U.S. consumes more than 35 quadrillion Btu’s (3.70 10¹⁹ J) of liquid energy annually, which represents almost 40% of total energy usage.¹ Petroleum has consistently represented roughly 95% of all liquids since 1958, with the transportation sector consuming between 50% and 70% during the same period.² As of 2008, over 63% of refined petroleum was delivered to market as motor gasoline.³ Because the national – and global – demand for energy is expected to increase substantially over coming decades, the development and implementation of renewable technologies will be crucial for continued economic growth and sustainability.

Biofuels represent a particularly viable, broad-reaching alternative to many (if not all) fossil fuels. Unlike petroleum-based fuels, biofuels are inherently renewable, exhibit diversity, burn cleaner, and can be produced in purity using bottom-up processing strategies. In addition, many biofuels can be blended with gasoline or diesel? to create bridge fuels (i.e., E10 and B20) which could provide industry, still in its infancy, with enough time to adapt to the looming threat of peak oil. However, at the moment significant barriers to commercialization reinforce petroleum as the most attractive, cost-effective liquid fuel.⁴

Biofuels currently account for less than 5% of total liquid consumption which leaves considerable room to improve upon the current state of the art. Though several types of biofuels have been defined—including biodiesel, biogasoline, biogas, biomass, biowaste?, and biocrude?—industrial-scale production is currently limited to bioalcohol?s from fermentation, namely the short-chain alcohol ethanol (C₂H₆O). Ethanol (EtOH?), a first generation C₂ alcohol, differs from hydrocarbon gasoline, diesel, and jet fuel?, which consist of a range of carbon molecules; C₄-C₁₂, C₈-C₂₁, and C₅-C₁₆, respectively. Although ethanol can be used as a primary motor fuel or fuel additive, upon combustion it produces less energy and is incompatible with some distribution and storage infrastructure. Despite these disadvantages, ethanol has enjoyed success in Brazil as a primary fuel—used in over 90% of cars—and has recently grown popular in the U.S as a gasoline oxygenate since the phasing out of methyl tert-butyl ether (MTBE).

Alternative, advanced biofuels such as various butanols (C₄) and pentanols (C₅) are purported to be viable substitutes for gasoline in a growing body of literature.^{5, 6, 7, 8, 9} These higher alcohols are gaining in popularity over ethanol because they (i) have a higher energy content (ii) can be used in pure form, (iii) do not require modification to existing combustion car engines or distribution infrastructure, (iv) are less hygroscopic, (v) are less corrosive, and (vi) have lower vapor pressures.^{6, 9} Conveniently, yeast cells can be used to produce all of these alcohols.

The budding yeast Saccharomyces cerevisiae? is widely employed in industrial processes such as beer and wine fermentation, baking, preparation of bulk chemicals and polymer precursors, synthesis of drugs, and production of biofuels. In addition, yeast cells have been used to study a wide range of biological processes resulting in a vast collection of literature on topics like aging, DNA repair, mRNA transport, and the cell cycle.¹⁰ S. cerevisiae cells grow rapidly, have simple nutrient requirements, and are unusual in that they prefer to ferment glucose (sugar) to form alcohols rather than oxidize glucose, even in the presence of high levels of oxygen.¹⁰ Yeast cells are currently widely used in North America for production of ethanol from cornstarch and are being intensively investigated for use in production of advanced biofuels from cellulosic feedstocks?.¹¹

Glucose is metabolized in yeast cells through glycolysis to form an important intermediate chemical, pyruvate. Carbon flux may then proceed either through respiration to form carbon dioxide and water or via fermentation pathways to form carbon dioxide and ethanol, and long-chain higher alcohols. Under typical growth conditions, conversion of pyruvate to ethanol is the preferred pathway.

Pyruvate can also follow an alternative pathway to create branched-chain amino acids (BCAAs), the dominant precursors for branched-chain alcohol production in yeast cells. These amino acids - leucine, valine, and isoleucine – are catabolized to C₄ and C₅ alcohols via the Ehrlich pathway in three steps, two of which involve standard fermentation reactions.^{7, 8, 12} Thus, leucine forms 3-methyl-1-butanol (3MB – C₅H₁₂O), valine forms 2-methyl-1-propanol (2MP – C₄H₁₀O), and isoleucine is converted to 2-methyl-1-butanol (2MB – C₅H₁₂O).

Humans have enjoyed branched-chain alcohols as flavor compounds in beer and wine for centuries. Although significant efforts have been made to improve total alcohol titers in yeasts at the cellular level, results have repeatedly demonstrated that increased production leads to decreased cell viability.¹³ The current understanding is that ethanol stress in yeast results in metabolism inhibition, increased frequency of petite mutants (loss of mitochondrial function), loss of membrane transport functions, and impeded growth.¹⁴ Bioengineering efforts are underway to overcome these limitations to fermentation.

Of importance lately, concern over food shortage has shone a spotlight on biofuel feedstocks, leading to a worldwide shift in attitudes about possible carbon sources for biofuels. Consensus has settled on lignocellulosic biomass? as the most viable choice as a renewable carbon source for advanced biofuel production. Lignocellulosic biomass, which comprises cellulose, hemicellulose, and lignin, represents by far the most abundant source of bioenergy on Earth. However, lignin is highly recalcitrant and current methods used to break it down into useful sugars can introduce over 100 inhibitory compounds into fermentation feedstocks.¹⁵ As a corollary, fermentation has become more complex, and more intolerable to yeasts. Nonetheless, to date, only S. cerevisiae have demonstrated the ability to perform in toxic environments containing lignocellulosic hydrolysate feedstocks.¹⁶

References

1) Energy Information Administration, Annual Energy Review 2008 , Table 1.3 Primary Energy Consumption by Source, 1949-2008. 2009, U.S. Department of Energy, Washington, D.C.

2) Energy Information Administration, Annual Energy Review 2008, Table 5.13c Estimated Petroleum Consumption: Transportation Sector, 1949-2008. 2009, U.S. Department of Energy, Washington, D.C

3) O’Donnell, M. Master’s Thesis, University of Texas at Austin, 2009

4) Kerr, R. Science 2010, 329, 780-781

5) Peralta-Yahya, P.P.; Keasling J.D. Biotechnol. J., 2010, 5, 147-162.

6) Dürre, P. Biotechnol. J., 2007, 2, 1525-1534.

7) Hazelwood, L. A.; J.M. Daran, et al. Applied and Environmental Microbiology, 2008, 74, 2259-2266.

8) Dickinson, J. R.; L. Eshantha, et al. The Journal of Biological Chemistry, 2003, 278, 8024-8034.

9) Cann, A. F., Liao, J. C. Appl. Microbial. Biotechnol. 2010, 85, 893-9.

10) Nevoigt, E. Microbiol. Mol. Biol. Rev. 2008, 72, 379-412.

11) Fischer, C.R.; Klein-Marcuschamer, D.; Stephanopoulos, G. Metabolic Engineering, 2008, 10, 295-304.

12) Atsumi, S.; Hanai, T.; Liao, J. C. Nature, 2008, 451, 86-89.

13) Walker, G. M. Yeast: Physiology and Biotechnology, Ed.; Wiley: New York, 1999, 163-165.

14) Walker, G. M. Yeast: Physiology and Biotechnology, Ed.; Wiley: New York, 1999, 66-75.

15) Liu, Z. L.; Slininger, P. J.; Gorsich, Appl Biochem. Biotechnol., 2005, 124, 451-460.

16) Vertes, A. A.; Qureshi, N.; Blaschek, H.P.; Yukawa, H. Biomass to Biofuels: Strategies for Global Industries, Ed. Wiley: New York, 2010, 271.