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Genetics researcher Dana Wohlbach displaying a common industrial yeast, which she is infusing with a yeast gene found in bark beetles to more efficiently help convert an abundant plant sugar to biofuel. Photo by Matt Wisniewski

TIM DONOHUE HAS SPENT THE LAST FOUR YEARS BUILDING A PIPELINE—but not the kind that springs to mind when we think of fuel.

The professor of bacteriology heads the CALS-led Great Lakes Bioenergy Research Center (GLBRC), founded with $142 million from the U.S. Department of Energy and a groundbreaking charge—to create the next generation of biofuels by harnessing renewable energy from the nonfood plants that are so plentiful all around us: grasses, trees and crop residues.

“We need to create liquid transportation fuels that are more cost-effective, more sustainable and won’t compromise the Earth or our quality of life,” says Donohue. “We’re in the middle of developing ways to generate these new fuels that are essential for powering our daily lives.”

With Michigan State University (MSU) as UW–Madison’s major partner, Donohue has assembled a team that now includes more than 400 researchers and staff and an additional nine member institutions. The effort spans two countries, 11 states and more than 60 individual lab and field facilities.

That’s a lot of brainpower. But the magnitude of the effort is commensurate with the task at hand, Donohue notes.

“We need to be considering everything from roots in the ground to what’s coming out of the nozzle,” Donohue says. “Without such a holistic approach, we won’t be able to demonstrate that this technology is feasible or see the weak spots where we can make improvements.”

What GLBRC has built is a research pipeline, a process that considers all factors that go into developing and implementing cellulosic biofuels—from creating sustainable agricultural landscapes and building better bioenergy crops to innovations in plant biomass processing and converting plant sugars into fuels.

While the promise of creating sustainable plant-based fuels isn’t new, the level of public investment needed to tackle this challenge has only recently emerged. According to the International Energy Agency, the United States leads world spending on biofuels public research, development and demonstration projects, investing $189 million in 2010 alone.

“By relying on fossils fuels, we’re living on energy that arrived on Earth many millions of years ago,” says Steve Slater, GLBRC’s scientific programs manager. “In order to reach a sustainable energy economy, we need to learn to live on the energy that arrives from the sun today. There’s a lot of that solar energy held within plant biomass, if we can figure out how to sustainably convert it to liquid fuels.”

Four years into its five-year grant, GLBRC has made some significant breakthroughs along the research pipeline. Here are some major points of interest.

Biochemistry professor John Ralph and his team are working to create Zip-Lignin, a new technology to break apart one of the plant cell wall’s toughest compounds. Photo by Matt Wisniewski

First Stop: PLANTS

At agricultural research stations in Wisconsin and Michigan, GLBRC researchers tend to tall stands of such biofuel crops as switchgrass and miscanthus, measuring above-ground traits like crop yield and digging down in the dirt to monitor soil microbes and water movement. Sophisticated instruments measure greenhouse gases such as carbon dioxide and nitrous oxide. Researchers count birds and insects to measure biodiversity and use satellite data to capture a watershed-level view of land use patterns.

It’s a lot of information, but each measurement plays a role in determining how these crop contenders would fare as large-scale bioenergy crops.

The leaves and stalks of these potential bioenergy plants are comprised of large quantities of cellulose, the most abundant organic compound on the planet. Cellulose is a polysaccharide, a long chain of tightly linked sugar subunits that must be broken down into simple sugars before they can be processed into biofuel. That alone is difficult—but to make the process even harder, much of a plant’s cellulose is locked within cell walls that form a tough, protective barrier. Breaking past the walls, using enzymes or chemicals to do so, is one of the biggest challenges in creating economically viable cellulosic biofuels.

Plant cell wall structures have evolved over time to fight off pests and disease. The more scientists understand about how the walls are created, the easier it will be to break them apart. DNA sequencing capacity provided by the Department of Energy (DOE) Joint Genome Institute allows plant breeders access to genetic and genomic data that provide clues about how those cell wall layers are built.

While determining the best genetic traits for bioenergy crops is a long-range goal, GLBRC plant researchers already have made important headway when it comes to tackling lignin, one of the toughest compounds that make up plant cell walls. Researchers hope to take it apart to get at the cellulose locked inside and convert small pieces of lignin into valuable co-products. CALS biochemistry professor John Ralph and his team have identified a gene that would allow easily breakable bonds to be incorporated into plant cell walls. They’re calling this new technology Zip-Lignin™ for its ability to break apart—or unzip—the lignin within. By getting lignin out of the way, biomass processing could be completed at lower temperatures. And lower temperatures mean lower overall costs.

And on another track, GLBRC researchers at MSU have located an enzyme that creates a plant oil with unique biodiesel-like properties. Now they’re working to encourage plants to produce more of that oil, which could be used directly as a “drop-in” or ready-to-use diesel replacement.

Second Stop: PROCESSING

Just as people need to chew food to better access and digest the nutrients inside, mechanical and chemical pretreatment of plants disrupts the cell walls and allows access to the sugars within. Using ammonia, heat and pressure, a pretreatment method known as AFEX (ammonia fiber expansion) blasts open cell walls, allowing enzymes easier access to the sugar polymers inside. Enzymes then break polymers apart into simple sugars for conversion to biofuel.

AFEX technology, developed and patented by GLBRC researcher Bruce Dale, an MSU professor of chemical engineering and materials science, has moved one step closer to commercialization. Dale and his team have partnered with MBI, a subsidiary of the Michigan State University Foundation, and in June received a $4.3 million DOE grant to scale up the technology as part of a one-ton-per-day cellulosic ethanol plant.

“We’ve come up with a less costly way of doing AFEX that we think is ready to commercialize,” Dale says. Improvements to the AFEX pretreatment process have also reduced the need for costly enzymes by a factor of three. And if the team can push the technology further, accomplishing another three- or fourfold reduction, enzyme cost would no longer be a limiting factor in biofuel production.

A technician prepares biomass for a new pretreatment method known as AFEX, which blasts open plant cell walls. Photo courtesy of MBI

As Dale’s team tinkered with different approaches to implementing this technology, they found that a modified approach to AFEX actually changed cellulose into a slightly different form that is five times easier for enzymes to break apart.

“We can understand in a much deeper way now how the AFEX process works, how it operates to produce digestible biomass,” Dale says. “Because we know that, we can do a much more rational job of picking the enzyme cocktails.”

Third Stop: ENZYMES

In the GLBRC’s early days, CALS bacteriology professor Cameron Currie’s work with leaf-cutter ants shed light on how these remarkable insects actually grow food—in one of the world’s oldest instances of farming—by tending leaves that provide nutrients for a strain of fungi that is the ant’s dietary staple. Along the way, Currie discovered something else: the ant’s nests are home to a number of previously unknown microbes whose enzymes may help break down the leaves. Currie recognized this property as a potential asset in the attempt to break down cellulose for biofuel.

That research has given Currie insight into the way cellulose-degrading microbes like bacteria or fungi work. One thing he’s seen so far is that microbes rarely go it alone. “Microbes in nature do not occur in isolation,” says Currie. “They do not break down plant biomass in a pure culture. In many systems, like the ant system, you have increased success and ability to compete with other organisms through beneficial symbiotic associations.”

He predicts that within many of these symbiotic systems, combinations of microbial organisms are each producing different enzymes, and that these enzymes each play a part in the efficient breakdown of plant biomass.

With a selection of contenders sitting in cold storage, Currie has begun collaborating with Brian Fox, a CALS biochemist, to understand how different types of enzymes function. Currie and Fox have formed a tightly knit duo, in effect merging their labs and working off of each other’s expertise.

“This collaboration has made a major impact on the work we’re doing,” Currie says. “It’s allowed us to really pick apart these systems and the microbes within them.”

Connecting with new colleagues is just one way that research centers like the GLBRC are tackling big science. And access to next-generation DNA sequencing tools speeds up the discovery process. Since starting at the GLBRC, Currie has worked with the Joint Genome Institute to generate dozens of bacterial genomes for comparative analysis.

“Sequencing is an area that is changing science in so many different ways,” says Currie. “It really helps us understand not only the evolution of a community of microbes that break down plant biomass, but also the evolution of the process of enzyme production.”

Currie and Fox are working to deliver newly characterized enzymes to their colleague Jonathan Walton, an MSU plant biologist who uses a robot to create enzyme cocktails that release sugars from plant biomass. This robotics platform, GENPLAT, runs through an impressive 96 tests at once, allowing Walton and his team to quickly evaluate new combinations of enzymes on different types of bioenergy crops.

Fourth Stop: CONVERSION

Once biomass has been pretreated and the sugars released, GLBRC scientists work with bugs like yeast and E. coli to optimize the way they churn through sugars and ferment them to produce fuels.

Even though S. cerevisiae, an industrial yeast that has been used by brewers for centuries, is great at chewing through glucose, it hasn’t had much of an appetite for the five-carbon sugars like xylose—the second most abundant plant sugar—that make up a good part of the plant cell wall. That is, until now.

“Strains of yeast that are currently used for biofuel production can only convert xylose to ethanol very slowly and inefficiently,” says CALS genetics researcher Dana Wohlbach. “But the more sugars a yeast can consume, the better, since more sugar consumption means more ethanol.”

Biochemistry professor Brian Fox (here with researcher Lai Bergeman ) is working closely with bacteriology professor Cameron Currie to understand which enzyme combinations will best release sugars from plant biomass. Photo by Matt Wisniewski

Researchers have identified a species of yeast found in bark beetles that is able to efficiently use xylose. After engineering that species’ xylose-friendly genes into an industrial yeast, researchers found that the industrial yeast, too, could use xylose along with other sugars—a development that could significantly increase the amount and speed with which biomass sugars can be converted to biofuels.

Although encouraging bacteria and yeast to act as miniature biofuel factories shows incredible promise, GLBRC is putting a few other bets on the table.

“Ethanol will probably continue to have a place in the automotive industry in the U.S. and around the world for decades,” says Tim Donohue, “but it is never going to be an acceptable biofuel for diesels or aviation or the shipping industry.”

Donohue is eager to expand the Center’s suite of fuels so that if an airline or shipping company comes knocking, they’ll find options to help them meet ambitious industry goals for reducing petroleum use. (The airline industry, for example, has committed to achieving carbon-neutral growth by 2020, as stated by the International Air Transport Association.) These industries are demanding ready-to-use fuels that can be “dropped in” to existing infrastructure such as engines, gas tanks and pipelines.

GLBRC’s Ron Raines and James Dumesic are working hard to meet this tall order. Using an ionic solvent, CALS biochemist Raines can convert raw plant biomass first into fuel precursors and next into a potential drop-in fuel called DMF (for 2,5-dimethylfuran). In another chemical approach, Dumesic, a UW professor of chemical and biological engineering, has used a series of catalytic reactions to create hydrocarbons, which are the basis for petroleum fuels.

Fifth Stop: SUSTAINABILITY

Biofuels generated by GLBRC get a reality check courtesy of UW mechanical engineer David Rothamer and the UW–Madison Engine Lab, where ethanol and other fuel precursors can be burned in engines to measure data on emissions and fuel performance.

“We understand how important it is to evaluate the feasibility of our technologies before we can call them a success, before we can decide that we’ve accomplished even the smallest goal,” says Donohue. “If I were flying in a commercial jet burning our biofuels, I would want us to be confident about fuel performance at 30,000 feet.”

How can GLBRC researchers be sure that successful fuel production at the lab bench can be scaled up to meet the needs of a state, a region or a country?

One way is to look at the fuel from every angle—counting the inputs related to growing, transporting and converting plant material into fuel. By using robust modeling software, GLBRC researchers are examining the feasibility of potential fuels or technologies not just for scalability, but also for sustainability.

Much more than a buzzword, “sustainable” means that trade-offs—social, environmental and economic factors—have been measured, modeled and validated against actual “boots on the ground data” measured at agricultural research stations and on Midwestern farms, says Randy Jackson, a grassland ecologist and CALS professor of agronomy who co-leads GLBRC’s sustainability research group.

GLBRC research on bioenergy cropping systems, for example, has shown that such crops lead to everything from a reduced need for insecticide (due to an increase in beneficial insects) to increased bird and grassland diversity. “It’s really exciting that these systems offer the opportunity to actually improve both landscape management and ecosystem services, or benefits, that we get from the land,” says Jackson.

But the bottom line has to make sense to farmers, reports Scott Swinton, an MSU professor of agricultural, food and resource economics who has conducted studies exploring what it would take for farmers to transition their fields away from corn and soybeans. Farmers need to know that there’s a solid market for dedicated energy crops such as switchgrass or miscanthus, and that moving away from what they know would help them pay the bills, Swinton says.

Looking Forward

Close to a year from now, GLBRC researchers will be wrapping up their first five-year funding cycle and awaiting word from DOE about a second round. They plan to close out the year with a set of promising technologies for further pursuit and recommendations based on which crops have shaken out as biofuel feedstock winners.

Instruments positioned near biofuel crops at the Arlington Agricultural Research Station measure plant utilization of solar radiation, weather conditions, and temperature and moisture at different depths in the soil. Photo by Beth Skogen

So far it’s clear that deep-rooted perennials are great at sequestering carbon, a big benefit for reducing greenhouse gases and therefore combating climate change. And regardless of the particular source of biomass—corn stover, switchgrass, miscanthus or poplar—bioenergy crops will need to be productive if farmers are expected to make an investment in this budding industry. If clean water, erosion control and biodiversity are important to consumers, agricultural landscapes will need to be designed with these values in mind.

A big part of doing biofuels right may simply mean being aware of the trade-offs.

Randy Jackson frequently gets the question “What biofuel crop is best?” And he usually answers, “It depends on where you are in the landscape.” If you want the landscape to improve water quality in your area, for example, you might need perennial biomass crops on the bottomlands.

After four years of collecting data and building models, Jackson is eager to roll the sustainability work up into scenarios, simulations and flexible decision-support tools so that farmers and other rural community members will have the ability to evaluate how their local landscape could be utilized for a combination of food, fiber and fuel.

At some time in the near future, the Center’s findings and results likely will become—and should become—a point of societal discourse involving a wide range of stakeholders, including the general public. “We’re committed to providing reliable, useful, relevant and thorough information to inform that discourse,” says Jackson.

Entering the home stretch of this first phase, all projects are focused on making existing processes faster, cheaper and more sustainable.
“We’ve created this pipeline and developed technology at the core of our mission, and we’ve achieved it in a little more than three years,” says Tim Donohue. “But we’re far from finished.”