Thayer School of Engineering & Biofuels: The Future of Cellulosic Ethanol Technology

In 1908, Henry Ford labeled ethanol the fuel of the future.  Ford designed his model-T to run on a combination of gasoline and alcohol, but cars soon traversed the nation on gasoline alone (1).  A hundred years later, environmentally-conscious scientists and policy-makers are turning back to ethanol and other biofuels to find solutions to America’s costly dependence on oil.  In fact, the United States Department of Energy’s deadline for replacing thirty percent of current gasoline consumption with biofuels is 2030 (2).

Meanwhile professors at Dartmouth’s Thayer School of Engineering are helping to define the future of energy for our nation.  Their research reflects these contemporary concerns.  Thayer School researchers, in conjunction with Lebanon’s Mascoma Corporation, recently reported a method of metabolic engineering of a thermophilic bacterium that produces a high yield of ethanol in the Proceedings of the National Academy of Sciences last November (3).

Biofuels, Basically

Biofuels are the product of biomass, the term given to organic renewable material.  Biomass includes crops, wood, manure, and some degradable garbage.  It can be divided into two main groups: agriculture and waste.  Whereas agricultural biomass may be used for either food or fuel, waste biomass is only used for the production of biofuels (4).

Creating biofuels is a complex process.  Before reaching the consumer as fuel, biomass must undergo a series of transformations, each with sub-steps. Once harvested, biomass is prepared as feedstock, converted to intermediate products and converted once again to a final, energetic fuel.  These energy products are then distributed accordingly (4).  The integration of farming, engineering, technology, and economics determines the success of the process.

The main conversion techniques of biomass vary.  The process of gasification occurs in a gasifier whose heat, steam, and controlled amount of oxygen decompose biomass into gaseous hydrogen, carbon monoxide, carbon dioxide and other compounds.  Pyrolysis, another technique, is gasification without the presence of oxygen (5).  The method of starch and sugar fermentation relies on enzymes to decompose glucose-containing material in the presence of oxygen (6).  Biomass containing lignin, cellulose, and/or hemicellulose may also undergo this starch and sugar fermentation after being pretreated to break into component sugars within lignocellulosic biomass fermentation.  In transesterification an alcohol catalyst bonds to fatty acids found in greases, oils, and fat to reduce the viscosity thereby producing a combustible form (4).  Landfill gas collection captures naturally produced methane and carbon dioxide at waste disposal sites with a series of wells and vacuums (7).  Multiple engineered techniques are used within each conversion method (4).

Additionally, anaerobic digestion may be used for the conversion of biomass.  During anaerobic digestion, bacteria in the absence of oxygen digest biomass and release gas (4).  Psychrophilic, mesophilic, or thermophilic bacteria that can work at low, medium, and high temperatures, respectively, are used for this process.  The work conducted by Mascoma Corporation cofounder and Thayer School of Engineering professor Lee Lynd et al. focuses on a particular technique using anaerobic digestions of thermophilic bacteria.

More on Mascoma
Established in 2005 by professors Lee Lynd and Charles Wyman of Thayer School, Mascoma Corporation develops cellulosic techniques for the conversion of biomass to ethanol.  Professor Lynd cites the creation of Mascoma as a fusion of “science and technology from the bottom-up” with “a top-down goal of worldly contribution” (8).  Mascoma’s solutions call for “a complete rethinking of the way in which we fuel our economy” (9).
Currently, Mascoma has 115 employees, over half of whom have Ph.D.s.   Receiving its funding mainly from state and national grants, Mascoma dedicates roughly 70 percent of its efforts toward research.  Although corporate headquarters are in Boston, MA, the Research and Development Lab is located in Lebanon, NH, which allows for conjunctive work with Dartmouth (9).

Mascoma strives to execute a “strategy of technology discovery, development, and deployment” (10).  By creating a network with research institutions and innovative corporations, Mascoma hopes to establish a collaborative effort.  Its work with Dartmouth on thermophilic bacteria echoes these goals.

The Study
Within the PNAS study, researchers from Thayer School of Engineering and Biological Science Department as well as Mascoma Corporation engineered Thermoanaerobacterium saccharolyticum to produce ethanol at a high yield.  As a thermophilic saccharolytic anaerobe, T. saccharolyticum normally produces organic acids and ethanol; however, within this study knockouts of the genes acetate kinase (ack-), phosphate acetyltransferase (pta-), and L-lactate dehydrogenase (L-ldh-) led to a strain, ALK2, which produces ethanol as the only detectable organic product.  These genes were selected because of their involvement in organic acid formation.  Ethanol fermentation in ALK2 differs from other microbes with homoethanol fermentation, because while using pyruvate:ferredoxin oxioreductase the electrons are transferred along a new pathway, from ferredoxin to NAD(P).  Furthermore, although previously developed mesophilic strains show a preference toward glucose consumption, ALK2 uses xylose and glucose simultaneously.  When compared to the wild type, ALK2 showed slight differences that were easily accounted for based on the techniques used.  At 37 g/liter, this engineered strain’s maximum ethanol titer is the highest reported amount for a thermophilic anaerobe (11).

Before developing ALK2, the fermentation products in xylose-grown cultures of knockout mutants of T. saccharolyticum with L-ldh-, ack- pta-, and ack- pta- L-ldh- strain ALK1 genotypes were analyzed.  Knockout plasmids pSGD9 and pSGD8E were used to target ack- pta- and L-ldh-, respectively.  All of these mutants yielded an increase in ethanol with strain ALK1 yielding ethanol as its only product.  Mutant L-ldh- did not produce lactic acid and ack- pta- produced less hydrogen and did not produce acetic acid (11).
ALK1 cultivated in continuous culture for approximately 3000 hours with progressively higher feed xylose concentrations produces strain ALK2.  As previously mentioned, this strain exhibited a greater capacity for xylose consumption and produced a mean ethanol yield of 0.47 g of ethanol/g xylose.  This yield did not decrease in continuous culture without antibiotic selection for over hundreds of generations (11).

Previously engineered methods for creating biofuels using anaerobic digestion had revolved around mesophilic bacteria.  Although these methods increase the ethanol yield, they rely upon “costly cellulose enzymes” (3).  The genetically engineered thermophilic bacteria can produce ethanol without the addition of enzymes, thereby reducing costs (10).  These cost-effective efforts improve the chance of establishing a cellulosic biofuels industry.
However, much work remains before the strain ALK2 can be incorporated industrially. Because these strains can withstand higher concentrations of ethanol before ceasing production, one objective is to reduce the difference between this maximum tolerated concentration and the maximum concentration of ethanol produced.  Compared to the work done on other organisms, this goal is realistic (11).

The Defining Challenge of Our Time
Professor Lynd labeled the attainment of energy as “the defining challenge of our time” (8).  Although biofuels provide an economically viable alternative to gasoline, the success of contemporary sustainable efforts relies on more than alternative fuels alone.  Lynd asserts that “a sustainable world involves multiple complementary changes” (8).  Thayer School has consistently supported the efforts of Lynd and others by choosing to emphasize energy and the environment.  “[Thayer] is willing to let things start small,” Lynd explains, “the institution understands it is not possible to always operate on a huge scale” (8).

A blend of technology and public policy are required to make the necessary systemic alterations for a sustainable world.  Although the process will take time, work like that of Mascoma Corporation and Professor Lynd provides the base for these alterations.  Multilateral integration will be necessary if we are to one day come to fulfill Ford’s prophecy and live sustainably.

References
1. K. Addison, Ethanol Fuel: Journey to Forever (2008).  Available at http://journeytoforever.org/ethanol.html (1 Nov 2008).
2. U.S. Department of Energy, Biofuels Initiative (2007). Available at
http://www1.eere.energy.gov/biomass/biofuels­_initiative.html (9 Nov 2008).
3. S. Knapp, Dartmouth Researchers Advance Cellulosic Ethanol Production (2008). Available at
http://www.dartmouth.edu/~news/releases/2008/09/08.html (1 Nov 2008).
4. U.S. Environmental Protection Agency, Biomass Conversion: Emerging Technologies, Feedstocks, and Products (EPA Publication 600-R-07-144, 2007; http://www.epa.gov/sustainability/pdfs/Biomass%20Conversion.pdf).
5. U.S. Department of Energy, Biomass Gasification (2008).  Available at http://www1.eere.energy.gov/hydrogenandfuelcells/production/biomass_gasification.html (4 Jan 2009).
6. Oregon Department of Energy, Biofuel Technologies (2006). Available at http://www.oregon.gov/energy/renew/biomass/biofuels.shmtl (4 Jan 2009).
7. U.S. Environmental Protection Agency, Landfill Methane Outreach Program (2009). Available at http://epa.gov/lmop/overview.htm (4 Jan 2009).
8. L. Lynd, personal interview, 31 October 2008.
9. L. Lynd, “Biofuel Production” (2008). Presentation Delivered at DUJS Paper Party, 12 November 2008.
10. Mascoma Corporation (2008).  Available at http://www.mascoma.com/index.html (27 Oct. 2008).
11. A. J. Shaw, K. Podkaminer, S. Desai, J. Bardsley, S. Rogers et al, Proc. Nat. Acad. Sci.U.S.A. 105, 13769-13774, (2008).