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Lawrence Berkeley National Laboratory
Addressing the Need for Alternative Transportation Fuels: The Joint BioEnergy Institute
Lawrence Berkeley National Laboratory
LBNL Paper LBNL-3258E
American Chemical Society Journal of Chemical Biology, 3, 1, 17-20, 2008
Addressing the Need for Alternative Transportation Fuels: The Joint BioEnergy
Harvey W. Blanch†,‡,§, Paul D. Adams†,§,¶, Katherine M. Andrews-Cramer†,¿, Wolf B. Frommer†,§,**, Blake A.
Simmons†,††, and Jay D. Keasling†,‡,§,¶,* †Joint BioEnergy Institute, ‡Department of Chemical Engineering,
University of California, Berkeley California 94720, §Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720, ¶Department of Bioengineering, University of California, Berkeley, California
94720, ¿Sandia National Laboratories, Albuquerque, New Mexico 87185, **Department of Plant Biology, Carnegie
Institute for Science, Stanford, California 94305, and ††Sandia National Laboratories, Livermore, California 94551
Today, carbon-rich fossil fuels, primarily oil, coal, and natural gas, provide 85% of the energy consumed in
the U.S. As world demand increases, oil reserves may become rapidly depleted (1). Fossil fuel use increases
CO2 emissions and raises the risk of global warming. The high energy content of liquid hydrocarbon fuels
makes them the preferred energy source for all modes of transportation. In the U.S. alone, transportation
consumes ›13.8 million barrels of oil per day and generates "0.5 gigatons of carbon per year (2). This
release of greenhouse gases has spurred research into alternative, nonfossil energy sources. Among the
options (nuclear, concentrated solar thermal, geothermal, hydroelectric, wind, solar, and biomass), only
biomass has the potential to provide a high-energy-content transportation fuel. Biomass is a renewable
resource that can be converted into carbon-neutral transporation fuels.
Currently, biofuels such as ethanol are produced largely from grains, but there is a large, untapped
resource (estimated at more than a billion tons per year) of plant biomass that could be utilized as a
renewable, domestic source of liquid fuels. Well-established processes convert the starch content of the grain
into sugars that can be fermented to ethanol. The energy efficiency of starch-based biofuels is however not
optimal, while plant cell walls (lignocellulose) represent a huge untapped source of energy (3). Plant-
derived biomass contains cellulose, which is more difficult to convert to sugars; hemicellulose, which
contains a diversity of carbohydrates that have to be efficiently degraded by microorganisms to fuels; and
lignin, which is recalcitrant to degradation and prevents cost-effective fermentation. The development of
cost-effective and energy-efficient processes to transform lignocellulosic biomass into fuels is hampered by
significant roadblocks, including the lack of specifically developed energy crops, the difficulty in separating bio-
mass components, low activity of enzymes used to deconstruct biomass, and the inhibitory effect of fuels and
processing byproducts on organisms responsible for producing fuels from biomass monomers.
The Joint BioEnergy Institute (JBEI) is a U.S. Department of Energy (DOE) Bioenergy Research Center
that will address these roadblocks in biofuels production. JBEI draws on the expertise and capabilities of
three national laboratories (Lawrence Berkeley National Laboratory (LBNL), Sandia National Laboratories
(SNL), and Lawrence Livermore National Laboratory (LLNL)), two leading U.S. universities (University of
California campuses at Berkeley (UCB) and Davis (UCD)), and a foundation (Carnegie Institute for Science,
Stanford) to develop the scientific and technological base needed to convert the energy stored in lignocellulose
into transportation fuels and commodity chemicals. Established scientists from the participating
organizations are leading teams of researchers to solve the key scientific problems and develop the tools and
infrastructure that will enable other researchers and companies to rapidly develop new biofuels and scale
production to meet U.S. transportation needs and to develop and rapidly transition new technologies to the
JBEI’s biomass-to-biofuels research approach is based in three interrelated scientific divisions and a
technologies division. The Feedstocks Division will develop improved plant energy crops to serve as the raw
materials for biofuels. The Deconstruction Division will investigate the conversion of this lignocellulosic plant
material to sugar and aromatics. The Fuels Synthesis Division will create microbes that can efficiently convert
sugar and aromatics into ethanol and other biofuels. JBEI’s cross-cutting Technologies Division will develop and
optimize a set of enabling technologies including high-throughput, chipbased, and ’omics platforms; tools for
synthetic biology; multi-scale imaging facilities; and integrated data analysis to support and integrate JBEI’s
Figure 1. Overview of JBEI structure and research pipeline, courtesy of LBNL Creative Services Office.
Energy Feedstocks: Understanding Recalcitrant Biomass.
Atmospheric carbon dioxide is fixed by plants into carbohydrates through photosynthesis. Sugar cane
and beet roots store large amounts of simple sugars, corn grain and wheat kernels store carbon as starch, and
cellulose and hemicellulose are found in the leaves of all agricultural crops, as well as in the leaves and wood of
trees. Most ethanol for fuel use today is produced from corn grain, and the technology for breaking down starch
into simple sugars is well developed. Lignocellulosic biomass, such as wood, forest product residues, grasses,
agricultural residues, and specialty energy crops, can provide much larger amounts of biomass for production
of transportation fuels. However, lignocellulosic biomass is resistant to breakdown; plants have evolved
complex means to employ cellulose and hemicellulose as structural materials that are very resistant to
microbial attack. Lignin, a polyphenolic material, serves to strengthen the cellulosic material to form the
plant cell wall, which provides resistance to pests and pathogens. The crystalline cellulose core of cell walls
is very resistant to chemical and biological breakdown, and the complex structures of the cell wall also
contribute to its recalcitrance. Research in JBEI’s Feedstocks Division is directed at overcoming the
recalcitrance of lignocellulosic plant matter so that it can be more easily deconstructed.
The main objectives of the Feedstocks Division are to elucidate the mechanisms involved in
synthesis of plant cell wall constituents using high-throughput functional genomics and glycomics and to
expand knowledge of lignin polymerization to allow the development of plants with novel types of lignin
with equivalent biological function but with improved susceptibility to enzymatic and chemical
Deconstruction: Converting Lignocellulosic Biomass to Sugars.
The most direct approach to overcoming the recalcitrance of biomass relies on pretreatment by
mechanical or chemical methods. Pretreatment aims to decrease the crystallinity of cellulose and increase
the accessibility of the bio-mass for subsequent hydrolysis. Biomass pretreatment by dilute acid hydrolyzes
the hemicellulose component, whereas treatment with alkali removes part of the lignin. These approaches,
however, are not economically optimal. Other pretreatment approaches include steam and alkaline
explosive decompression and hydrothermolysis. All of these pretreatment methods expose the cellulose
fibers and make them more accessible to cellulase enzymes, which can then hydrolyze the cellulose to
fermentable sugars. Enzymatic hydrolysis does not produce byproducts and thus offers the possibility of
improving the costs of biofuels production.
Cellulolytic microorganisms (fungi and bacteria) produce enzymes that act synergistically to
hydrolyze plant cell wall materials. Presently, our understanding of the fundamental mechanisms of enzymatic
cellulose degradation is limited. Fungi produce three types of cellulolytic enzymes. Random-acting
endoglucanases produce free ends from cellulose fibrils that can be degraded by exoglucanases, which produce
the glucose dimer cellobiose. The third type of enzyme, $-glucosidase, hydrolyzes the released cellobiose to produce
glucose. Some bacteria employ a molecular “machine”, the cellulosome, to break down cellulose.
Hemicellulose is degraded by a class of enzymes known as hemicellulases, which are multidomain
enzymes containing structurally discrete catalytic and noncatalytic domains. Hemicellulases from different
organisms are classified generally as either glycoside hydrolases or carbohydrate esterases, which hydrolyze
acetate or ferulic acid side groups of the hemicellulose polymer, respectively. Compared to the research and
development effort in the scientific community to understand and optimize cellulase enzymes, very little is
known about the exact mechanisms of hemicellulases, and methods to engineer these enzymes are nascent.
Lignin, the third major component of biomass, is the component most resistant to enzymatic attack. Lignases
generally consist of a family of enzymes including phenol oxidase (laccase), peroxidases (lignin peroxidase),
and manganese peroxidase.
Thus, the major objectives of JBEI’s Deconstruction Division are i) to improve pretreatment methods
with broad applicability to a range of feedstocks, ii) to explore new sources of lignocellulolytic enzymes from
natural environments, relying on high-throughput protein production and directed evolution using on-chip
technologies, iii) to examine microbial communities for new sources of cellulolytic and lignolytic enzymes,
and iv) to develop lignin models and lignase assays that enable the creation of modified ligninases for enhanced
degradation and conversion of modified lignin.
Fuel Synthesis: Capturing the Energy Content of Sugars.
Sugars derived from starch-based biomass such as corn are readily fermented to ethanol, because they
are present in nearly pure solutions. In contrast, lignocellulose deconstruction results in both five- and six-carbon
sugars together with a number of inhibitory compounds, including organic acids, furan derivatives, phenolics,
and inorganics. Hardwoods and agricultural residues contain 5–25% pentose sugars, primarily xylose and
These are not fermented to ethanol by the most commonly used yeast, Saccharomyces cerevisiae.
Anaerobic bacteria ferment pentose sugars but are typically inhibited by low concentrations of ethanol and other
byproducts from deconstruction. Filamentous fungi are able to tolerate these inhibitors but grow and produce ethanol
too slowly to be commercially attractive.
The challenge in the biofuels production division of JBEI is thus to convert all of the monomer sugars
(hexoses and pentoses) released from depolymerization of lignocellulosic biomass into transportation fuels and
other chemicals. Accomplishing this objective will first rely on developing and improving fuel production systems
in selected model microorganisms: the bacterium Escherichia coli, the yeast S. cerevisiae, and the thermoacidophilic
archaeon Sulfolobus solfataricus. JBEI is initially employing E. coli and S. cerevisiae strains that have been
previously engineered to produce ethanol from five- and six-carbon sugars. All three hosts will be engineered to
improve their tolerance to byproducts formed during biomass processing and to high concentrations of ethanol and
other fuel products.
JBEI will develop biochemical synthesis pathways for production of a range of other candidate fuel
molecules and chemicals that are currently based on petroleum feedstocks. We will construct and validate
these pathways in E. coli and then, when functional, introduce them into S. cerevisiae and S. solfataricus.
JBEI is initially targeting five existing or proposed fuel molecules: ethanol, butanol, isopentanol, hexadecane,
and geranyl decanoate ester. Unlike ethanol, these potential fuel molecules may be used to power jet and
diesel engines and can be distributed via existing infrastructure that is used to distribute petroleum-based
Technologies for Biofuels Research and Production.
Successfully meeting JBEI goals is critically dependent on the application and development of
advanced technologies deployed in the context of a research environment that is fully integrated through
state-of-the-art information systems and informatics methods. The JBEI Technologies Division will develop
and implement technologies that can be applied to the research in each of the scientific and engineering
divisions. Using high-throughput protein expression, purification, and screening, JBEI researchers will
generate thousands of gene clones per year, fully characterize the plant cell wall synthesis machinery by
synthesis and tagging to identify functional complexes, and perform functional analysis of tens of
thousands of wild-type and engineered lignocellulose-degrading enzymes.
In functional genomics, JBEI researchers will characterize the transcript and protein profiles of
natural and engineered organisms including plants, undertake metabolite and flux profiling of modified
organisms to optimize fuel production, and perform high-throughput glycomics. In the area of synthetic
biology, JBEI will develop platform hosts for the production of enzymes and fuels and create parts and
devices for the construction of new fuel-generating organisms and improved plants. Finally, in high-
throughput imaging, JBEI researchers will develop new and improved technologies for visualizing cell
walls. The technologies developed within JBEI will be of general use for a wide variety of biological
applications and will benefit the biofuels research com-munity, GTL, and other DOE initiatives.
A Single Facility Integrates JBEI’s Research.
JBEI is designed to be a dynamic organization with all research teams working together at a single
location. This colocation of researchers will enable scientists to share their ideas, develop technologies that
will benefit all scientific divisions, and ad-dress cellulosic biomass problems at a systems level. JBEI is
positioned to take advantage of the significant capabilities of its partners and other institutions and
companies in the San Francisco Bay Area. JBEI’s close working relationships with its industry partners
will ensure that JBEI creates the fundamental knowledge and scalable technologies to solve real-world
problems in commercial-scale biofuels production.
Conclusions and Perspectives.
The challenges in converting lignocellulosic feedstocks into transportation fuels are significant.
The integrated approach taken by JBEI’s researchers will realize basic science and engineering
developments to meet these challenges. The potential payoffs from this research are significant: renewable,
carbon-neutral transportation fuels; lessening the impact of global warming; and reducing our reliance on
foreign oil while improving trade balances.
National Research Council (2006) Trends in Oil Sup-ply and Demand, the Potential for Peaking of Conven-
tional Oil Production and Possible Mitigation Op-tions, National Academies Press, Washington, DC.
U.S. Department of Energy (2005) Emission of Green-house Gases in the United States, www.eia.doe.gov/
U.S. Department of Energy and U.S. Department of Ag-riculture (2005) Biomass as Feedstock for a Bioen-
ergy and Bioproducts Industry: The Technical Feasibil-ity of a Billion-Ton Annual Supply, www.osti.gov/ bridge.
This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under Contract No. DE-AC02-05CH11231.
This work was supported by the Assistant Secretary for Energy Efficiency and Renewable Energy,
Office of Building Technology, State, and Community Programs, of the U.S. Department of Energy under
Contract No. DE-AC02-05CH11231.