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The New Biofuel Boom

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With nationwide gasoline prices averaging $3.29 a gallon, research into alternative fuels — particularly biofuels — is a bigger venture than ever. Biofuel research has been around for several decades, but it stalled in the 1990s when the price of oil was low. Today, the field is booming again. In 2007, President Bush announced the goal of reducing US gasoline consumption by 20 percent in 10 years. To that end, government, industry, and academia are pouring research dollars and efforts into developing biofuels.

Large-scale biology is enjoying a key role in this work. "Any route to biofuels seems to go through genomics," notes Scott Baker, a fungal genome researcher from Pacific Northwest National Labs.

Biofuel funding has certainly been a boon to the genomics field. British Petroleum gave $500 million to establish the Energy Bioscience Institute, a collaboration including the company and the University of California, Berkeley; the University of Illinois at Urbana-Champaign; and Lawrence Berkeley National Laboratory. Last year, the US Department of Energy awarded $135 million to Oak Ridge National Lab and its regional academic partners; the state of Tennessee also kicked in $70 million to research cellulose-based fuels. These second-generation biofuels — which can be grown on agriculturally marginal land and require little fertilizer — are more resource-friendly than corn-based ethanol, which necessarily uses plants that could've been food. "You can't take food off the table to fill a gas tank," says Stephen Mayfield, a cell biologist at the Scripps Research Institute.

Much of the money is going toward studying cellulosic ethanol, derived from the breakdown and fermentation of a plant. Getting from field to fuel is a multi-step process that involves growing and harvesting the plant, using micro-organisms or enzymes to convert the plants' cell walls into sugars, and fermenting those into alcohol.

At each step of the way, genomics is poised to make the process a bit easier. Through sequencing and synthetic biology, scientists are engineering plants to contain more sugars in their cell wall, and to make those sugars more accessible during the conversion process. There's plenty of research into bacteria and fungi as well; these organisms might be made more efficient at converting cell walls to sugars or at turning sugar into a longer-chain alcohol, such as butanol or isopropanol, both of which are better fuels than ethanol. All of this is predicated upon having the genome of the organism and an understanding of the proteins, enzymes, and metabolic pathways involved to make that plant more sugar-dense or that bacteria more productive

The feedstocks

The first step toward a biofuel is to grow a plant or other photosynthetic organism. The feedstock provides the raw material — sugar — in its cell walls to contribute to the subsequent fermentation. With genome sequences in hand, scientists can engineer these organisms to be more energy-dense or easier to tend in the fields.

"About two years ago, we sequenced the [poplar] genome, so we have a catalog of all of the genes that are there. With modern molecular biology techniques, we can identify genes that affect cell wall chemistry or disease resistance or yield in a very efficient manner now," says Oak Ridge National Laboratory's Gerald Tuskan, who led the poplar sequencing effort. "It makes improvement and accelerated domestication a lot more feasible today than it was prior to having the genome sequenced."

Even without being engineered, poplar offers some advantages as a biofuel feedstock. It is a woody perennial that only needs to be planted once. It grows quickly and in temperate climates on marginal lands that do not easily support agriculture. Even so, Tuskan and his team have set their sights on improving the tree's conversion efficiency and its productivity. "As an undomesticated species, there's a lot of headroom for improvement," he says.

First, they want to make the sugars of the tree's cell walls easier to break down. "To state it in a technical term, we are trying to overcome recalcitrance. The cell wall is recalcitrant to enzymatic conversion or enzymatic breakdown, microbial conversion," Tuskan says. To overcome that, they will be changing the chemistry of the plant's cell walls to reduce the amount of lignin, which usually binds cell walls together to give them more mechanical strength. They also want to add more sugars to the cell wall so there is more energy to be harvested.

Another series of improvements is targeted at making poplar more productive and therefore more economical to grow. For instance, a shorter, thicker tree that doesn't put a lot of effort into growing far-reaching limbs would concentrate the energy that scientists hope to harvest. Also, Tuskan says, "We want to increase the ability of water use efficiency and nitrate, nutrient use efficiency and we want to, of course, convey disease and insect resistance into these materials."

Tuskan is also part of the Joint Genome Institute's effort to get another perennial, switchgrass, sequenced. Switchgrass has many of the same advantages as poplar — the ability to be grown on marginal lands, less of a need for fertilizer and chemical inputs, and a higher ethanol yield per unit of land than corn. To landowners, says Tuskan, switchgrass would represent an earlier return on investment than poplar since it can be harvested during its first year.

"We are trying to pull together people from government agencies, universities, and private industry and formulate … a five-year plan to outline how we would go about sequencing the switchgrass genome," he says.

Another potential source of biofuel is algae, but they don't quite fit into the generic scheme. They are both a feedstock and the conversion organism that produces a biodiesel. "We're taking sunlight and turning it directly into biofuel," says Stephen Mayfield, who studies Chlamydomonas, whose genome was published last September.

Algae already produce triglycerides, whose fatty acids can be turned into biodiesel. "Our goal is to understand [the biochemistry and genetics] so that we can get algae to grow fast for a while and, at some point, stop growing and switch all of their photosynthetic energy into storage lipids. If we can do that, we'll be well on our way to making biofuels [economical]," Mayfield says.

Before getting to that point, he will be studying Chlamydomonas' gene transcripts, expressed proteins, and metabolomics to understand how that algae made a particular lipid and how it can be engineered to produce more of it.

Baker, who studies fungi, is impressed by the potential of algae. "I think there's a consensus, maybe in the far-off future, [that algae] will be economically viable biofuels," he says.

Then there's synthetic biology, which Craig Venter has hailed as the replacement for the petrochemical industry. Researchers at his company, Synthetic Genomics, are taking a similar approach to biofuel research, but with a twist: they don't want to study one organism to optimize its potential as a source of biofuel or as a catalyst to break down feedstock. Instead, they plan to cobble together a new organism by combining "cassettes" of genes from many different sources.

"We are looking at many varieties and ultimately we could just borrow from different microbes and combine them as cassettes into the chassis of a synthetic genome and, in a sense, combine the best properties of the possible candidates we can think of — in terms of efficacy, in terms of speed, in terms of suitable conditions or function [or] resilience," says Synthetic Genomics President Ari Patrinos. "It's not a technology that's available today on a massive scale, but we are working very, very hard to get to that stage very quickly."

The destroyers

For the next step of developing a biofuel — breaking down the plant's cell walls and converting those sugars to alcohol — researchers are studying organisms that do this naturally. Termites are notorious for destroying the wooden foundation of houses and other buildings. To do that, they — or more precisely, the bacteria in their series of guts — break down the wood's cell walls. Another system is fungi, which are natural decomposers. Plants really don't want their cell walls to be easily disassembled, says Phil Hugenholtz at JGI. "If you can do that, though, you have an inexhaustible supply of sugar for making biofuels," he says.

Hugenholtz has taken a metagenomics approach to studying the bacteria contained in a termite's hindgut and how they break down wood. "The reason we are studying termites is because they are arguably the preeminent natural system for breaking down plant cell walls," he says. "If you can find out how they are doing it and sort of emulate it in the laboratory, then that's the plan."

From sequencing the bacteria from termites' hindgut, Hugenholtz has found 300 bacterial species, plus some archaea, with 600 different enzymes involved in breaking down cell walls. He and his team have found cellulases, xylases, and novel carbohydrate-binding proteins that they think hold the enzymes and substrate together so they act more efficiently. "We're basically at the beginning of piecing it all back together," he says.

Meanwhile, Baker at Pacific Northwest National Labs is studying filamentous fungi, which have not been as aggressively sequenced as bacteria. These fungi, he says, contain a large number of enzymatic pathways that break things down and could be harnessed in biorefineries. "Fungi are great because they degrade biomass; they have all the enzymes needed to degrade plant cell wall material," he says.

Baker and other fungal researchers are calling for a genome sequencing program for fungi. In a commentary coming out in Fungal Biology Reviews, he and his colleagues argue that filamentous fungi encode a lot of degradation pathways that could be harnessed in biofuel research. "They are very good at making enzymes and organic acids that can utilize all the kinds of sugars that are out there," he says. "There's a lot that we can learn about fermentation and a lot of improvements that can be made that will benefit the biofuels though the enzymes."

The to-do list

Beyond engineering organisms or creating new ones to yield a biofuel, there are other technological hurdles to meeting demand for renewable transportation fuel, including finding a biochemical pathway to a fuel that is more effective than ethanol and consolidating the breakdown and fermentation steps.

"Ethanol really sucks as a biofuel because of its light density," says JGI's Hugenholtz. It gets fewer miles to the gallon than gasoline, and because of its light density, it cannot replace jet fuel. Ethanol also attracts water, which rusts pipes. Longer-chain alcohols are better, Hugenholtz says, because they contain more carbons. These higher alcohols, such as butanol or isopropanol, have higher densities and octane ratings.

"To make butanol or pentanol or any of these higher forms of alcohol, we will actually have to modify and add pathways that don't currently exist," says Tuskan at JGI. "This is probably a five- to eight-enzyme step to go beyond ethanol production into isopropanol or butanol or some of these other alcohols."

If the steps between breaking down the cell walls and fermenting sugars can be combined, producing biofuel from cellulose will be less expensive. "The big trend now is to do everything in a single fermentation. Every time you add a unit process to making any sort of fermentative product, that's more capital costs. If you get everything in a single tank, then … you decrease your processing costs," says PNNL's Baker. "I think that ultimately we will get there, but it is going to take some interesting metabolic engineering of the organisms."

As for the technological hurdles, researchers are optimistic. "It's going to crack in the next couple of years. Something's going to give," says Hugenholtz.

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