Already biodiesel Volkswagens are driving around Berkeley, California, reeking of French fries from recovered veggie oil — a low-tech response to President Bush's plea to break the nation's oil addiction.

The Berkeley Biodiesel Collective sponsors veggie-powered car shows.

In nearby Walnut Creek the approach is a bit more sophisticated. Work is underway at the Department of Energy Joint Genome Institute (DOE JGI) to bring the technology of DNA sequencing to bear on the effort to make alternative fuels cheaper, easier to produce, and easier on the environment.

Ethanol from cornstarch and biodiesel from various oil-producing plants currently account for only two percent of the U.S. fuel market. According to DOE, ethanol can replace 20 percent of the fossil fuel consumed for transportation by 2030 — but reaching this goal will require a different approach, namely the conversion of biomass to ethanol.

Biomass is any plant-derived material; it's comprised of three main compounds: cellulose, hemicellulose, and lignin. During biomass conversion, cellulose from plants is broken down, chemically or enzymatically, into simple sugars that in turn are fermented by bacteria or yeast into ethanol.

"Lignocellulosic materials such as wood chips, crop residue, and various grasses have high energy content, but the sugars are less readily available than those in cornstarch," says DOE JGI director Eddy Rubin. "By directing DNA sequencing and the tools of molecular biology, we can survey the vast catalog of microbial biodiversity to improve the processes of converting these feedstocks to usable fuel."

The right place for the job

DOE JGI, launched in 1997 to speed the completion of the Human Genome Project, now unites the expertise of five national laboratories — Lawrence Berkeley, Lawrence Livermore, Los Alamos, Oak Ridge, and Pacific Northwest — along with the Stanford Human Genome Center. Even before the sequences of chromosomes 5, 16, and 19 were published by DOE JGI researchers in the journal Nature in 2004, the institute had set its sights on targets that factor prominently in DOE's clean-energy, carbon-cycle management, and environmental clean-up missions.

	In Walnut Creek, DOE JGI combines university and national laboratory expertise in genetic research.

The effort has involved sequencing the genomes of dozens of microbes, including organisms that convert ethanol more efficiently and tolerate higher levels of ethanol, and those involved in degrading stubborn plant polymers. Most recently, DOE JGI led an international effort to decode the first tree, the fast-growing poplar.

Partnerships with hundreds of academic institutions have steadily driven the growth of JGI's bioenergy portfolio. In November, DOE JGI was selected to participate in the consortium of laboratories that will sequence corn, the leading U.S. ethanol fuel crop. In January, DOE put in place a memorandum of understanding with the U.S. Department of Agriculture to establish a framework for coordinating plant and microbial genome sequencing relevant to the agencies.

Bean power

The first fruit of the relationship, one catalyzed by DOE JGI's new Laboratory Science Program (LSP), is the soybean, Glycine max, the world's most valuable legume crop. Soybean is of particular interest to DOE because it's the principal source of the renewable alternative fuel, biodiesel. Biodiesel has the highest energy content of any alternative fuel and is significantly more environmentally friendly than comparable petroleum-based fuels, since it degrades rapidly in the environment.

But there is room for improvement, says Gerald Tuskan, an Oak Ridge National Laboratory geneticist with more than 15 years of biofuel research experience. Tuskan was recently tapped as leader of the LSP, a program geared to foster large-scale strategic sequencing projects that involve several national laboratories and are aligned with future funding opportunities in DOE's biology programs. A second goal is to provide small-scale sequencing that meets the needs of individual investigators at the DOE national laboratories.

"In illuminating soybean biology through sequencing, assembling, and annotating its genome, we can develop a customized biomass production platform for combining oil seed production for biodiesel with the enhanced vegetative growth for converting to ethanol — and as a result, doubling the energy output of the crop," Tuskan says. "Ultimately we'd like to modify soybeans so that they would not only produce and store higher levels of oil in the seed, but throughout the entire plant. The molecular and cellular machinery for doing this is native in soybean. We just need to find a way of regulating its development and expression in leaf tissue."

Bug power

Another approach is exploring and modifying the microbial players in the biodiesel equation. DOE JGI is pursuing this line of attack in silico — in the computer — through the Integrated Microbial Genomes (IMG) data management system. IMG is the result of collaboration between DOE JGI and Berkeley Lab's Biological Data Management and Technology Center (BDMTC).

Computer programs calculate how desirable traits from different organisms can be combined.

"IMG provides access to comparative information about organisms, genes, and their metabolic pathways," says DOE JGI biologist Athanasios "Thanos" Lykidis. "So you can find organisms that have certain pathways, identify another organism lacking that capability, then use one as a donor for the other."

One scenario is to modify the triacylglycerol biosynthetic pathway for optimized biodiesel generation. Currently, the most common form of biodiesel is fatty-acid methyl esters (FAAE), produced through the reaction of triacylglycerols (TAGs) with methanol in the presence of a catalyst; triacylglycerol is the major lipid reserve in plants and animals.

"The main source of TAG in the U.S. is soybean oil and yellow grease," says Lykidis. "In both cases the production cost of biodiesel is two to three times higher than the petroleum-based product — so it isn't a competitive alternative to petroleum-derived fuel. However, metabolic engineering presents an opportunity to dramatically lower the costs associated with biodiesel production."

Says Lykidis, "TAGs and FAAEs serve as storage compounds for energy and carbon, and their occurrence is widespread in plants. But their occurrence in bacteria is limited." He and his colleagues are identifying and characterizing genes involved in TAG and FAAE biosynthesis. They will then seek to generate microorganisms and plants with enhanced TAG and FAAE production.

Poplar possibilities and termite travails

In the nearer term, sequencing may help harvest biomass more efficiently. DOE JGI's Dan Rokhsar and Jerry Tuskan were among the investigators who led the effort to sequence the genome of the poplar (Populus).

	With genetic modifications, the poplar may become a highly efficient source of biomass.

"We now have the candidate genes that will help us domesticate poplar for biomass and reduce the cost from $50 to about $20 per ton," says Tuskan. "Lignocellulosic biomass is inherently resistant to deconstruction. If we could gain a better understanding of cell-wall biosynthesis, then we could devise strategies for better cell-wall deconstruction."  

Tuskan adds that "even greater gains in domestication are coming from changing hormones that control the distribution of carbon; the enzymatic pathways that shape the ratios of cellulose, lignin, and the hemicellulose in the stem; and the transcription factors that regulate branching. These domestication steps, along with developing microbial-based bioreactors — communities that produce and tolerate high levels of ethanol — are some of the low-hanging fruits of technology."

Yet another target of DOE JGI's bioenergy efforts is the remarkable termite, a creature that is capable of cranking out two liters of hydrogen from fermenting just one sheet of paper, making it one of the planet's most efficient bioreactors. Termites accomplish this by exploiting the metabolic capabilities of about 200 different species of microbes that inhabit their hindguts.

"Termites have spread throughout the world and play a critical role in recycling wooden biomass," Rubin says. "They are so successful in eating our houses from underneath us that they cause more than $1 billion in damage in the United States annually."

Because less than one percent of all microorganisms in the biosphere can be grown in the lab, DOE JGI researchers Phil Hugenholtz and Falk Warnecke have had to tackle these critters on their own turf — in the jungle of Costa Rica. Through the emerging strategy of metagenomics — which means isolating, sequencing, and characterizing DNA extracted directly from an organism's actual habitat — they are obtaining a profile of the "bugs" inside the bug.

It's the microorganisms in a termite's guts that allow it to convert wood to usable energy.

Collaborators in this venture, undertaken by the DOE JGI Community Sequencing Program (CSP), include Jared Leadbetter and colleagues from Caltech; Diversa, a San Diego-based biotechnology company; and InBio, Costa Rica's National Biodiversity Institute.

Termites eat wood, but they can't extract energy from the complex lignocellulose polymers within it. These polymers are broken down into simple sugars by the fermenting bacteria in the termite's gut, which use enzymes that produce hydrogen as a byproduct.

"It's not as if we are going to put termites in our tank," Hugenholtz says. "But if we can harness the termite microbe enzymes that breakdown lignocellulose and make hydrogen, we may end up with a commercially viable process."

Additional information

• More about the DOE JGI Laboratory Science Program
• Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply, a joint study sponsored by the U.S. Department of Energy and the U.S. Department of Agriculture, was published in April 2005 (pdf)