The Future, Unzipped

John Ralph PhD’82 talks with the easy, garrulous rhythms of his native New Zealand, and often seems amiably close to the edge of laughter.

So he was inclined toward amusement last year when he discovered that some portion of the Internet had misunderstood his latest research. Ralph—a CALS biochemist with joint appointments in biochemistry and biological systems engineering—had just unveiled a way to tweak the lignin that helps give plants their backbone. A kind of a natural plastic or binder, lignin gets in the way of some industrial processes, and Ralph’s team had cracked a complicated puzzle of genetics and chemistry to address the problem. They call it zip-lignin, because the modified lignin comes apart—roughly—like a zipper.

One writer at an influential publication called it “self-destructing” lignin. Not a bad turn of phrase—but not exactly accurate, either. For a geeky science story the news spread far, and by the time it had spread across the Internet, a random blogger could be found complaining about the dangers of walking through forests full of detonating trees.

Turning the misunderstanding into a teachable moment, Ralph went image surfing, and his standard KeyNote talk now contains a picture of a man puzzling over the shattered remains of a tree. “Oh noooo!” the caption reads. “I’ll be peacefully walking in a national park and these dang GM trees are going to be exploding all around me!”

That’s obviously a crazy scenario. But if the technology works as Ralph predicts, the potential changes to biofuels and paper production could rewrite the economics of these industries, and in the process lead to an entirely new natural chemical sector.

“When we talk to people in the biofuels industry, what we are hearing is that creating value from lignin could be game-changing,” says Timothy Donohue, a CALS professor of bacteriology and director of the UW–Madison-based Great Lakes Bioenergy Research Center, where Ralph has a lab. “It could be catalytic.”

After cellulose, lignin is the most abundant organic compound on the planet. Lignin surrounds and shapes our entire lives. Most of us have no idea—yet we are the constant beneficiaries of its strength and binding power.

When plants are growing, it’s the stiffening of the cell wall that creates their visible architecture. Carbohydrate polymers—primarily cellulose and hemicelluloses—and a small amount of protein make up a sort of scaffolding for the construction of plant cell walls. And lignin is the glue, surrounding and encasing this fibrous matrix with a durable and water-resistant polymer—almost like plastic. Some liken lignin to the resin in fiberglass.

Without lignin, the pine cannot soar into the sky, and the woody herb soon succumbs to rot. Found primarily in land plants, a form of lignin has been identified in seaweed, suggesting deep evolutionary origins as much as a billion years ago.

“Lignin is a funny thing,” says Ralph, who was first introduced to lignin chemistry as a young student during a holiday internship at New Zealand’s Forest Research Institute. “People who get into it for a little bit end up staying there the rest of their lives.”

The fascination is born, in part, from its unique chemistry. Enzymes, proteins that catalyze reactions, orchestrate the assembly of complex cell wall carbohydrates from building blocks like xylose and glucose. The types of enzymes present in cells therefore determine the composition of the wall.

Lignin is more enigmatic, says Ralph. Although its parts (called monomers) are assembled using enzymes, the polymerization of these parts into lignin does not require enzymes but instead relies on just the chemistry of the monomers and their radical coupling reactions. “It’s combinatorial, and so you make a polymer in which no two molecules are the same, perhaps anywhere in the whole plant,” says Ralph.

This flexible construction is at the heart of lignin’s toughness, but it’s also a major obstacle for the production of paper and biofuels. Both industries need the high-value carbohydrates, especially the cellulose fraction. And both have to peel away the lignin to get to the treasure inside. A combination of heat, pressure, and caustic soda is standard procedure for liberating cellulose to make paper; bleach removes the remaining lignin. In the biofuels industry, a heat and acid or alkaline treatment is often used to crack the lignin so that it is easier to produce the required simple sugars from cellulose. Leftover lignin is typically burned.

The economic cost of these treatments alone is significant, and lignin pretreatment is at the heart of many of the more egregious environmental costs of paper. On the biofuels side, lowering treatment costs to liberate carbohydrates from lignin could change the very economics of biofuels. In these large-scale, industrial processes, saving a percentage point or two is often worthwhile, but the Holy Grail is a quantum jump.

“Because it’s made this way”—Ralph jams his hands together, crazy-wise, fingers twisted together into a dramatic representation of lignin polymerization—“there is no chemistry or biology that takes it apart in an exquisite way,” he says. “We actually stepped back and thought: How would we like to design lignin? If we could introduce easily cleavable bonds into the backbone, we could break it like a hot knife through butter. How much can you actually mess with this chemistry before the tree falls down?”

Ralph’s team had their eureka moment more than 15 years ago, and have been trying to bring it to life ever since.

With a background in forage production and ruminant nutrition, John Grabber, an agronomist at the USDA–Agricultural Research Services’ Dairy Forage Research Center in Madison, got pulled into lignin chemistry through the barn door. On his family’s dairy farm he grew up with lignin stuck to his boots, though he never knew it. But during graduate school he became interested in how plants are digested by cows. Cell walls are potentially a great source of digestible carbohydrates—most plants contain anywhere from 30 to 90 percent of their mass in their cell walls—but it is entangled with lignin. “You quickly find out that lignin is the main barrier to feed digestion,” he says.

Grabber began working on a model system to understand plant lignification—for corn in particular—in 1989. After meeting at a conference, Grabber joined Ralph and plant physiologist Ronald Hatfield at the Dairy Forage Research Center back in 1992. There were many projects ongoing, but Grabber remained interested in trying to fully understand the structural characteristics of the lignin: how it’s made and how to modify it. In his model system they could make any kind of lignin they wanted to study, and see how the changes affected utilization.

Ralph and Hatfield advocated for the work, helping to find funding and offering their expertise. “If I had worked for somebody else I probably wouldn’t be doing this work,” says Grabber. “John and Ron gave me freedom and support to do it.”

Around the same time, Fachuang Lu joined Ralph’s lab seeking a Ph.D. His journey into lignin chemistry was not, at first, his idea. A native of mainland China, he’d enjoyed a successful undergraduate career in Beijing studying chemical engineering, then found himself assigned by the college to a master’s program in lignin chemistry. Lignin is an ingredient in the slurry of chemicals used in oil drilling, and that was his specialty. In 1989 Lu left Beijing for a teaching position at Guangxi University, but three years later he decided to continue his education. Though he’d never met Ralph, he was fascinated by the chemistry and applied to study in his lab.

As Ralph, Grabber, Hatfield and Lu continued to tinker with lignin chemistry, momentum began to build in the lab. Though lignin created a snowflake universe of different molecules, there were rules of assembly. A complex chemical pathway enabled lignin construction, with a mechanism that remained constant across different families of plants, but with many potential building blocks.

Ralph and his colleagues were the first to detail what was happening to lignin as the controlling genes of the biosynthetic pathway were turned on and off, a task ably completed by a slew of outstanding collaborators worldwide with expertise in biotechnological methods—but who lacked the diagnostic structural tools to determine what the plant was doing in response.

Ralph’s team quickly learned that lignification was somewhat flexible. “We figured that we could engineer lignin well beyond the previously held bounds,” says Ralph. As various pathways and chemical possibilities danced in their heads, it struck them: What if, during lignification, they could persuade the plant to slip in a few monomers that had easily broken chemical bonds? If they did it right, lignin would retain its structural value to the plant, but be easier to deal with chemically.

“In the course of our conversation we realized that if plants could do this, it could really revolutionize how readily you could make paper,” recalls Grabber. Says Ralph: “It’s almost impossible to tell which one of us actually verbalized it first—it is one of those great outcomes of the group dynamic.”

Lu’s particular genius was synthesizing the various complex chemicals needed, particularly a novel monomer-conjugate called coniferyl ferulate. It was the key to the zip-lignin—the teeth of the zipper. “He’s got to be one of the best in terms of making molecules,” says Grabber.

They were thrilled by such a revelation, but, in retrospect, they soon realized it was sort of an obvious idea—one suggested by the underlying chemistry and biochemistry of a pathway that was becoming increasingly well understood. Yet it was a discovery of huge potential value. They dropped into stealth mode and began to work on it. They finished important research and stuck it in drawers—signature research, the kind that, when finally published, would capture journal covers. And yet they sat on it, quietly chipping away for nearly a decade.

It helped that there was a flurry of controversy in the field—what Chemical & Engineering News called “the lignin war.” “Part of the reason we could sit on it was that, at the time, making these kinds of molecules was so far-fetched,” says Grabber. “Probably if we had talked about it, people would have laughed at us.”

But as the idea for zip-lignin grew in principle, it became stronger. Lu, Hatfield and colleague Jane Marita MS’97 PhD’01 found that balsa trees and a fiber crop known as kenaf produced very small amounts of coniferyl ferulate. But even as the idea seemed more and more feasible, Hatfield and Marita couldn’t isolate the gene needed to manufacture coniferyl ferulate because of its very low expression in these plants.

And they got stuck. “At the beginning we were thinking that this is just a fantastic idea, but we really didn’t have that much confidence,” says Lu. “Maybe John [Ralph] had more confidence than me.” So they just kept at it. “Every step you think, yes, we are closer, closer, closer.”

In 2008 Ralph moved his work from the Dairy Forage Research Center into UW labs, with research projects under the recently formed Great Lakes Bioenergy Research Center (GLBRC). The center, launched with a $125 million grant from the U.S. Department of Energy that has since been renewed, was just one manifestation of the money and intellectual heft infusing biofuels research—and for zip-lignin it was a lucky move.

During the center’s first full meeting, Curtis Wilkerson, a plant biologist at GLBRC partner Michigan State University, was sitting in the audience when Ralph took his turn at the podium.

Wilkerson is a cell wall specialist. Though lignin is a third of the wall’s carbon and is essential to the way plants conduct water, he confesses he’d never given it much thought. In a room full of cell wall specialists, Ralph would “likely be the only person talking about lignin,” he says. “It just split that way a long time ago. People like myself had very little exposure to what John was thinking.”

It was this kind of academic silo that a place like GLBRC was supposed to breach. Ralph talked about putting ester bonds into lignins and his team’s long search for the elusive enzyme. Wilkerson saw a solution. Due to recent technical advances, the price of determining all of the expressed enzymes in a plant had become more refined and much less expensive. He offered to use these recent developments to try to find the missing enzyme to enable zip-lignin.

From the previous work, Wilkerson knew essentially the size and shape of the puzzle piece he was looking for. He began, quite literally with Google, trolling through the scientific literature looking for a plant that made a lot of coniferyl ferulate. The Chinese medicinal “dong quai” or Chinese angelica (Angelica sinensis) soon emerged as a candidate. Its roots contained about 2 percent coniferyl ferulate.

The team used knowledge about the likely type of enzyme they were searching for and successfully identified the gene and its enzyme that could produce coniferyl ferulate. The whole search took less than six months.

Would you believe an essential tool for the genetic engineering of poplars is a hole punch? That’s the word from Shawn Mansfield, a molecular biochemist at the University of British Columbia, who took the zip-gene from the Angelica and made it work in poplar, a popular tree in the biomass and forest products industry.

Working from Wilkerson’s gene, the first job was figuring out how to tag the new protein so that it fluoresced during imaging. While not necessary to the function of the genetically modified plant, it essentially allows the scientists to check their work: see where the protein is, how much is there, and if it is behaving as a protein should.
Mansfield’s lab also had to find a way to turn the gene on at the right time and place. It could make all the coniferyl ferulate one wanted, but if it wasn’t made at the right time and tissue, there would be no zip-lignin.

After perfecting these finer points, the gene is inserted into a special bacterium—and then the hole punch finally comes into play. Disks punched from poplar leaves are mixed with bacteria that have been inoculated with a special chemical that stimulates the bacteria to share their DNA around. Then the leaf disks are put in a special growth medium. As many as 12 shoots might emerge off of a single disk, but the lab would select and nurture only one shoot from each disk.

In the end they had about 15 successful transgenic candidates that they grew in the greenhouse and then shipped off to Wilkerson and Ralph for further study. Final selection was made based on the amount of fluorescent yellow the trees gave off, and from a newly devised analytical method developed by Lu and Ralph that was particularly diagnostic for the incorporation of the zip monomer into the lignin polymer.

The team knew that genetically modified organisms are not popular or easily talked about—never mind the exploding trees. The idea of reworking a fundamental building block of the plant world will breed resistance.

Ralph argues that this is already part of nature’s vocabulary: they’ve found their building blocks within the plant kingdom, including mutants that do similar things. And now that they know what they are looking for, Steven Karlen, a member of Ralph’s group, is continuing to find more evidence that Mother Nature is doing it herself. “We managed to persuade plants to do this,” Ralph says. “Chances are that nature has already attempted it and you could actually get there by breeding.”

It’s no surprise that Mansfield, who created the final transgenic tree, argues that there is a role for this kind of technology. “We as scientists should be wise in advocating for the proper use of it,” he cautions. “I would never force it on anybody. I would never try to sway people to think that it is the end-all or be-all for everything.”
But given the growing human population and rising CO2 levels, something like zip-lignin has a definite use in reducing the carbon footprint by reducing processing energy and chemical loads. “That means there are less environmental pollutants that need to be cleaned up afterwards,” Mansfield says.

“Our ecological footprint can be much reduced using these kinds of transgenic trees,” he argues. “The caveat is that we need to be very smart about where and how we plant them.”

Not many things in the natural world can take apart lignin, but any homeowner with a deck knows that fungi are up to the task. A recent analysis of mushroom genomes suggests that fungi evolved this ability about 300 million years ago. This is about the end of the Carboniferous era, when earth’s coal production began to slow down. Coincidence? Perhaps not. Now that wood could rot, it probably slowed the burial of organic carbon via tree trunks and other lignin-rich plants.

Could the discovery of zip-lignin signal another transition, and hasten our move away from fossil fuels laid down in the Carboniferous?

Tim Donohue likes to think so. He likens biofuels now to the early oil industry, when oil was simply being turned into liquid fuel while the by-products were burned or dumped. It took a few decades for inventors to capitalize on this now valuable stream of raw materials to build the modern chemical industry.

“Lignin is about 25 to 30 percent of carbon in the plant. So if we’re going to catalyze an industry that makes clean energy and chemicals from plant biomass, figuring out what to do with the lignin is going to be key,” Donohue says.

People in the industry used to joke that you could do a lot of things with lignin except make money from it. But that may be changing. “The economics and profitability of the industry will be very different if lignin can be turned into valuable compounds,” says Donohue.

One of the early efforts to make use of lignin was in Rothschild, Wisconsin, at a company now known as Borregaard LignoTech. When processed properly, lignin has many uses, from the manufacture of vanilla flavor to additives for concrete. There is even a small amount of it in the battery of your car that allows it to keep recharging.

Jerry Gargulak is research manager at Borregaard LignoTech, and learned about zip-lignin recently in his capacity as a scientific advisor to the GLBRC. Despite its many uses, Gargulak and his colleagues dream about a time when lignin can replace carbon black in tires and be used to build carbon fibers and structural plastics.

Zip-lignin and the ideas behind it could bring this day closer. “It gives us a technology that might yield a more interesting lignin-derived starting material,” Gargulak says. “It could potentially lead to a lot of innovation downstream in lignin technology.” But he emphasizes, “There are a lot of i’s to be dotted and t’s to be crossed.”

This story is just beginning. Zip-lignin has a patent and has excited industrial interest that could be worth significant dollars. Ralph and his colleagues continue working to further refine the process, increasing the percentage of zippable bonds in poplar and also inserting the gene into more plants, such as corn and Brachypodium, both grasses.
And in the basement of the shiny new Wisconsin Energy Institute building, where the GLBRC is based, two massive new nuclear magnetic resonance (NMR) spectrometers work 24/7, providing a level of detail into lignin that Ralph has never had before.

“We spend a lot of time looking at these Rorschach test–like figures,” Ralph says of the information generated from the NMR. “The detail in them is unbelievable. These things have been revolutionizing what we do.”

Uganda: The Benefits of Biogas

Generating enthusiasm for a new kind of technology is key to its long-term success. Rebecca Larson, a CALS professor of biological systems engineering, has already accomplished that goal in Uganda, where students at an elementary school in Lweeza excitedly yell “Biogas! Biogas!” after learning about anaerobic digester systems.

Larson, a UW–Extension biowaste specialist and an expert in agricultural manure management, designs, installs and upgrades small-scale anaerobic digester (AD) systems in developing countries. Her projects are funded by the Wisconsin Energy Institute at UW–Madison and several other sources. Community education and outreach at schools and other installation sites are an important part of these efforts.

Children get excited by the “magic” in her work, she says. “It’s converting something with such a negative connotation as manure into something positive,” Larson notes. In an AD system, this magic is performed by bacteria that break down manure and other organic waste in the absence of oxygen.

The resulting biogas, a form of energy composed of methane and carbon dioxide, can be used directly for cooking, lighting, or heating a building, or it can fuel an engine generator to produce electricity.

Larson’s collaborators in Uganda include Sarah Stefanos and Aleia McCord, graduate students at the Nelson Institute for Environmental Studies who joined forces with fellow students at Makarere University in Kampala to start a company called Waste 2 Energy Ltd.
Along with another company, Green Heat Uganda, which has built a total of 42 digesters, Waste 2 Energy has helped install four AD systems since 2011.

“Most of these digesters are locally built underground dome systems at schools and orphanages,” Larson explains. Lweeza’s elementary school is a perfect example.

The AD systems use food waste, human waste from pit latrines and everything in between. The biogas generated by the digester is run through a pipeline to a kitchen stove where the children’s meals are prepared. Compared to traditional charcoal cooking, the AD systems greatly reduce the school’s greenhouse gas emissions.

Larson and her team are now focusing on enhancing the efficiency and environmental benefits of these systems. Their goals are to improve the digester’s management of human waste, reduce its water needs, increase the amount of energy it produces and generate cheap fertilizer to boost food crop yields.

“Our overall goal is to create a closed-loop and low-cost sustainability package that addresses multiple local user needs,” Larson says.

The beauty of the project is that all these needs can be met by simply adding two new components to the existing systems: heating elements and a solid-liquid separator.

To help visualize the impact of the fertilizer, Larson set up demonstration plots that compare crop yields with and without it. Down the road, a generator could be added to the system to provide electricity in a country where only 9 percent of the population currently has access.

As a next step, Larson hopes to replicate the project’s success in Bolivia. She is finalizing local design plans with Horacio Aguirre-Villegas, her postdoctoral fellow in biological systems engineering, and their collaborators at the Universidad Amazonica de Pando in Cobija.

Class Act: Energizing the Classroom

When biochemistry senior Hong-En Chen first got involved with a student organization called Energy Hub, she knew she could bring something special to the table.

As the daughter of a preschool teacher, she’d interacted a lot with young children throughout her own childhood and adolescence. While in high school she worked as a teacher and tutor in music, math and reading in both English and Mandarin at the Einstein School in Madison, a private preschool and after-school enrichment center for elementary school students.

Based on her experience, she saw an important niche for Energy Hub: The group could go out to local elementary schools and hold after-school classes about energy.

“When kids are young, they’re like sponges. They absorb a lot of information and are enthusiastic learners,” notes Chen. “When we introduced concepts about energy use, conservation and sustainability, the kids impressed us not only by handling complex material, but also by applying ideas to their everyday lives.”

As outreach director of Energy Hub, Chen got other club members on board to pilot their project, working with second- to fifth-grade students at four Madison elementary schools. Based on that experience, they applied for and received a Wisconsin Idea Fellowship grant to further develop their curriculum during the 2012–2013 school year. They created a 10-week program that is going strong this year.

Hands-on activities are key, says Chen, whether using an educational science toy like Snap Circuits to teach the concepts behind powering lights and fans, or having students divide into the fantasy cities of Greenville and Coaltown to talk about how they, as residents, would use energy from various sources to get through a day. “It was a fun way to get them thinking about the costs and benefits of renewable versus nonrenewable energy sources,” Chen says.

Chen’s thinking a lot about that topic herself. She is researching compounds for solar energy conversion in chemistry professor Song Jin’s lab. And she is considering graduate programs in materials chemistry with an eye toward working in renewable energy research.

Learn more about Energy Hub at www.uwehub.org.

Looking for “Hotspots”

In their quest to make cellulosic biofuel a viable energy option, many researchers are looking to marginal lands—those unsuitable for growing food—as potential real estate for bioenergy crops.

But what do farmers think of that? Brad Barham, a CALS/UW-Extension professor of agricultural and applied economics and a researcher with the Great Lakes Bioenergy Research Center (GLBRC), took the logical next step and asked them.

Fewer than 30 percent were willing to grow nonedible cellulosic biofuel feedstocks—such as perennial grasses and short-rotation trees—on their marginal lands for a range of prices, Barham and his team found after analyzing responses from 300 farmers in southwestern Wisconsin.

“Previous work in the area of marginal lands for bioenergy has been based primarily on the landscape’s suitability, without much research on its economic viability,” says Barham, who sent out the survey in 2011. “What’s in play is how much farmers are willing to change their land-use behavior.”

Barham’s results are a testament to the complex reality of implementing commercial cellulosic biofuel systems. Despite the minority of positive responses, researchers found that there were some clusters—or “hotspots”—of farmers who showed favorable attitudes toward use of marginal land for bioenergy.

These hotspots could be a window of opportunity for bioenergy researchers since they indicate areas where feedstocks could be grown more continuously.

“People envision bioenergy crops being blanketed across the landscape,” says Barham, “but if it’s five percent of the crops being harvested from this farm here, and 10 percent from that farm there, it’s going to be too costly to collect and aggregate the biomass relative to the value of the energy you get from it.

“If we want concentrated bioenergy production, that means looking for hotspots where people have favorable attitudes toward crops that can improve the environmental effects associated with energy decisions,” Barham notes.

CALS agronomy professor Randy Jackson is also interested in the idea of bioenergy hotspots. Jackson, who co-leads the GLBRC’s area of research focusing on sustainability, says that just because lands are too wet, too rocky or too eroded to farm traditionally doesn’t mean they aren’t valuable.

“The first thing we can say about marginal lands is that ‘marginal’ is a relative term,” says Jackson. Such lands have a social as well as a biophysical definition. “This land is where the owners like to hunt, for example.”

The goal of GLBRC researchers like Barham and Jackson is to integrate the environmental impacts of different cropping systems with economic forces and social drivers.

The environmental benefits of cellulosic biofuel feedstocks such as perennial grasses are significant. In addition to providing a versatile starting material for ethanol and other advanced biofuels, grasses do not compete with food crops and require little or no fertilizer or pesticides. Unlike annual crops like corn, which must be replanted each year, perennials can remain in the soil for more than a decade, conferring important ecosystem services like erosion protection and wildlife habitat.

The ecosystem services, bioenergy potential and social values that influence how we utilize and define marginal land make it difficult to predict the outcomes of planting one type of crop versus another. To tackle that problem, Jackson is working with other UW–Madison experts who are developing computer-based simulation tools in projects funded by the GLBRC and a Sun Grant from the U.S. Department of Energy.

Jackson hopes that these modeling tools will help researchers pinpoint where farmer willingness hotspots overlap with regions that could benefit disproportionately from the ecosystem services that perennial bioenergy feedstocks have to offer.

“These models will include data layers for geography, crop yield, land use, carbon sequestration and farmer willingness to participate,” says Jackson. “There could be as many as 40 data layers feeding into these models so that you can see what would happen to each variable if, say, you were to plant the entire landscape with switchgrass.”

Will Dead Species Live Again?

Stanley A. Temple is the Beers-Bascom Professor Emeritus in Conservation in forest and wildlife ecology at CALS and a former chair of the conservation biology and sustainable development program at the Gaylord Nelson Institute for Environmental Studies. For 32 years Temple occupied the faculty position once held by Aldo Leopold, and while in that position he received every University of Wisconsin teaching award for which he was eligible. Since his retirement from academia in 2008 he has been a Senior Fellow of the nonprofit Aldo Leopold Foundation. He and his 75 graduate students have worked on conservation problems in 21 different countries and have helped save some of the world’s rarest and most endangered species. Last spring Temple gave a TED talk at a special event devoted to de-extinction, a concept that has captured the imagination of scientists and the general public alike.

What is “de-extinction”?
De-extinction is a recent term that involves bringing back an extinct species using DNA that’s been recovered from preserved material. There are two ways that it can be accomplished: one would be cloning to produce a copy of an extinct individual’s genome. The second way is through genetic engineering to re-create a close approximation of what the extinct species’ genome might have once been. The reality is that it’s no longer science fiction. We’re getting close to being able to revive extinct species from recovered DNA.

This must make for some unusual scientific partnerships.
It’s an interesting synthetic endeavor that matches the biotechnologists in the laboratory with conservationists in the field. The biotech crowd will be responsible for recovering DNA from an extinct species and through either cloning or engineering turning that DNA into individuals. But once they’ve done that, the next step involves people like myself who know how to recover endangered species by taking a small number of individuals and turning them into a viable population and getting them back into the wild.

What opportunities might this technology present to conservation efforts?
On the plus side, obviously, it would be exciting to bring back a species that human beings drove to extinction. But even if we weren’t able to do that, the technology presents an appealing opportunity to recover DNA from preserved specimens of an endangered species and use it to enhance the genetic diversity of the surviving population.

Can you please elaborate on that?
Conservationists have recovered many endangered species from very low population levels and saved them from extinction. The problem is, they’re often genetically depauperate, or lacking in genetic diversity. If we can recover some of the lost genes from preserved specimens collected before the population crashed, we might greatly improve the species’ prospects for long-term survival.

How would a conservation biologist go about actually applying this?
De-extinction is still an unproven concept, but it’s likely that sometime in the coming decades it will happen. Once they have revived individuals of an extinct species in the lab, then conservation biologists could try to recover the species by captive breeding and reintroducing the species to the wild. But conservation biologists get concerned about some of the details: Which species are going to be revived? Are they the right species? Are they the species that have the best chances for long-term survival in the world today? Are they species that might actually enhance the ecological health of the ecosystem that they were once part of, like the wolves reintroduced to the Yellowstone ecosystem? These are all questions of setting priorities for which species to actually revive.

How would you recommend setting priorities?
As a conservation biologist I would certainly look first at recently extinct species that were affected by a threat we’ve now overcome. Not only are those the ones for which we’re likely to have good quality DNA, but their ecological niche in the wild hasn’t been vacant for very long. And as a result, the ecological community that they were once part of has not readjusted itself to their absence, and might once again easily accommodate the species in its midst. On the other hand, if you’re dealing with a species that’s been extinct for a very long period of time—centuries or even millennia—the ecosystem that they were part of has moved on, and a species like that, once back in the system, could essentially be the equivalent of an invasive species. It might disrupt the system and threaten extant species.

How would you like to see this development proceed?
Considering the timeline that we probably have years or even decades to do this right—I and other individuals and groups that are thoughtful and somewhat skeptical about this would like to see a very broad discussion of the implications. We would like to see a lot of input in deciding the priorities about which species to bring back. We would not like to see this done in secret, which, unfortunately, is where this seems to be heading. This very expensive work is not receiving government funding and doesn’t have any sort of public oversight. Hence, privately funded biotech labs seem to be focusing on reviving spectacular extinct species, like mammoths and other Ice Age animals, rather than species that have a real chance of surviving in today’s world.

What would be an important takeaway point for the general public?
De-extinction doesn’t mean we can ignore the significance of extinction—to think, “Oh well, we can let species go extinct because we can always save some DNA and bring them back later.” This would just be an open door for activities that have been constrained by concerns for biodiversity and basically give the green light to go ahead and precipitate extinctions of species that are already with us.

Biofuel for Teens

As students in Craig Kohn’s class at Waterford Union High School can tell you, you don’t need a grant or Ph.D. to do scientific research. A question and some curiosity are all that’s needed—along with a sturdy pair of gloves.

Kohn BS’08, who earned degrees in biology and agricultural education at CALS, teaches a class called Biotechnology and Biofuels in which students hunt for bacteria that naturally secrete enzymes called cellulases. Cellulases are named for their ability to break down cellulose, the sugar polymer in plant cell walls that gives stems and leaves their structure.

“Cellulases are important for bioenergy because they are necessary to turn cellulose into a fermentable product that can be made into ethanol and other biofuels,” says Kohn.

To find those cellulase-producing bacteria, Kohn sends students out to collect samples from the compost heaps and animal pens behind their school in a quest known as “bioprospecting.”

Back in the classroom, students drop the samples into test tubes filled with media solution and a strip of filter paper. If cellulases are present, the cellulose-based paper will disintegrate as the enzymes do their work.

That process of discovery excites students. “You see this light in their eyes when they realize that they are participating in science directly, and that their work could lead to actual breakthroughs and results,” Kohn says.

Kohn developed the activity as a participant in “Research Experience for Teachers,” a program at the UW’s Great Lakes Bioenergy Research Center (GLBRC). For his project he shadowed Cameron Currie, a CALS professor of bacteriology and a GLBRC researcher who uses genomic and ecological approaches to study biomass-degrading microbes.

“Teachers are not only learning about current science—they are embedded in the lab,” says John Greenler, GLBRC’s director of education and outreach. “When teachers have that primary experience, they are in a better position to engage their students because they ‘get it.’”

Connor Williams, a high school senior who helped develop the bioprospecting lab with Kohn through his participation in the National FFA Organization (formerly Future Farmers of America), says his favorite element is the hands-on, independent work.

“I learned that answers to biofuel challenges literally can be found right in our backyards,” Williams says. “You just need to know where to look.”

Where Are We Now?

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.

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.

Cash Crop Biomass

WISCONSIN FARMERS have been growing biomass for generations, says Kevin Shinners. They just have a different name for it.

“Biomass is really just poor-quality forage,” says the CALS agricultural engineer. “We allow it to get very mature and it’s really high in fiber, so it doesn’t make very good animal feed, but it
makes great biomass.”

And Wisconsin farmers have a leg up in the business of producing biomass, says Shinners, a specialist in forage systems who branched out into bioenergy crops about 10 years ago.

“We have all of the tools to harvest and handle and process it. And an added advantage is that when we take biomass off the field, we have new places to put our dairy manure,” he says. “When you take corn stover off the field, you’re
removing nutrients that you need for next year’s crop. A Wisconsin farmer can apply manure, while an Illinois farmer may have to go out and buy fertilizer.”

Wisconsin also is rich in off-farm resources. The state’s custom harvesters are expert at chopping stalks and grass, and biomass could fit nicely into their schedule. After they finish chopping corn silage in September, crews could move on to corn stover or switchgrass in October and November, spreading fixed costs over more acres and keeping employees working longer.

In fact, under some business models, farmers might job out most of their biomass crop production. If the crop is a perennial, such as switchgrass, the farmer may spend more time in front of the computer and on the phone than out in the field. “Once the crop is established, he’ll manage fertilization and weed control through an agronomic service, cutting and removal through a custom harvester and marketing through a biomass aggregator,” Shinners says.

But even though Wisconsin farmers may be very much at home with the types of crops involved and the mechanics of producing them, they’ll be on less familiar ground when it comes to marketing, Shinners notes.

“If you’re a cash crop farmer, you’re used to marketing your corn and beans through multiple paths, selling some out of the field, storing some, selling futures, to optimize what you earn on an annual basis,” he says. “For biomass, you’ll have to change your mindset.

“If a firm builds a large cellulosic biorefinery here, it will need an absolute dedicated supply,” Shinners says. “If half the people in the area decided not to produce biomass one year, that plant would be a dinosaur.” Meaning that a critical mass of local farmers must be willing to lock into a long-term production contract.

The economics of biomass are driven by the fact that, pound for pound, the stuff isn’t worth as much as other crops. Profit margins may be slim, so farmers will need to produce as efficiently as possible.

That’s where Shinners comes in. His research centers on streamlining the harvest and handling a variety of biomass crops, including such perennials as switchgrass and reed canarygrass, and annuals such as sorghum. But his biggest push has been in corn stover—the stalks and leaves and cobs left when the kernels are removed—simply because there’s so much of it.

“There are some 90 million acres of corn being grown in the United States this year, and with the prices we’re seeing, there’s going to be more and more of it grown. If you’re really interested in biomass, it’s right there at our doorstep,” he reasons.

Since profit-minded crop producers aim to make as few trips across the field as possible, Shinners’ first efforts focused on harvesting both corn grain and corn stover in one pass. Essentially, he grafted a forage harvester to the back of a combine and hitched a wagon behind to catch the chopped stover.

This impressive 50-foot train of machinery worked, he says, but handling two crops at the same time slowed down the grain harvest, putting both yield and quality at risk. “That’s even more of an issue these days, when we have seen corn go over $7 per bushel,” he says. “As corn grain increases in value, everything that slows the combine down has a much greater economic cost.”

Shinners is focusing now on a system in which the combine harvests grain and leaves the stover behind in a long, neat row. “A custom harvester could come in behind and chop these windrows and store them for the farmer.”

Since buyers will need year-round deliveries, storing biomass crop until it’s needed is part of the equation. Shinners thinks the best approach is one that dairy farmers use for forage—seal it from the air in long plastic bags or covered bunkers and let it ferment. “We know this from dairying: You can open up a silo bag from two years ago and it’s still good quality,” he says.

That fermented biomass could be good enough to eat—by livestock, at least—which may offer farmers a way to take advantage of the bioenergy market without having to wait for a biomass refinery to be built nearby.

“If we apply amendments like lime right before we store corn stover, the feed value can increase substantially,” says Shinners. “So instead of waiting for somebody to develop a biorefinery in Wisconsin to convert stover to ethanol, why not divert some of the grain normally used to feed cattle toward ethanol production and use the stover to replace the corn as animal feed?”

From Field to Fuel

Where are we in terms of moving toward the “green gas” of the future?

The Great Lakes Bioenergy Research Center, led by CALS with Michigan State University as a major partner, has over the past four years been conducting basic research to convert non-edible plants such as grasses and trees to ethanol and other advanced biofuels. Here we present an overview of research progress.

 

O Bioneers

It wasn’t exactly panning for gold, but a lesson in “bioprospecting,” as it’s called, had students scour the campus looking for something just as valuable: invisible forms of life that could one day be key in developing a sustainable alternative to oil.

“Instead of going out and looking for precious metals, we’re looking for precious microbes,” says John Greenler, director of education and outreach at the Great Lakes Bioenergy Research Center and lead instructor of the university’s first bioenergy course for freshmen, held this past fall semester.

“Out in the environment there are a lot of microorganisms that are really good at breaking down fibrous plant material,” Greenler notes—a vexing but essential step in producing biofuel.

“Before I took this class I was only a little curious with the concept of bioenergy. Now I feel involved with bioenergy research and the possibility of using it to solve many environmental, political, and economic problems.” -Michael Polkoff

“We’re hoping to figure out how those microbes do that and then utilize that process to make biofuels—essentially, capture energy for our transportation needs the same way the microbes capture energy as a source of food,” Greenler says.

“Bioenergy: Sustainability, Opportunities and Challenges” debuted as a First-Year Interest Group (FIG) program open to 20 freshmen, and it was snapped up quickly during registration. As the bioprospecting lab shows, the course was designed to have students work on real-world problems researchers face in a new and rapidly growing field.

That includes the frustrations. Student Michael Polkoff reports that the prospecting material chosen by his group—pond scum—came up negative for microbes that produce cellulose-busting enzymes.

“While the results are depressing for the work we put into this—especially going barefoot into a freezing, sludgy drainage pond—it’s part of doing scientific research,” says Polkoff. “Sometimes you get results, other times you don’t. More importantly, we learned how research is done.”

The course has galvanized Polkoff’s interest in bioenergy. “Before I took this class I was only a little curious with the concept of bioenergy,” he says. “Now I feel involved with bioenergy research and the possibility of using it to solve many environmental, political, and economic problems.”

The course is offered through a partnership between the Great Lakes Bioenergy Research Center and the Wisconsin Bioenergy Initiative. Students visit the UW campus labs of some of the nation’s foremost researchers, and one field trip took them to CALS’ Arlington Research Station to study bioenergy field plots.

The FIG program, which clusters three courses linked by a common theme—the bioenergy course was paired with introductory chemistry and environmental studies—targets low income, minority, “first in family to college students,” says Greenler. “Overall, about 30 percent of students in the FIG program are minorities.”

Catch up with…Jack Newman

AT AMYRIS BIOTECHNOLOGIES, JACK NEWMAN PhD’01 is focused on some of the world’s biggest problems. He co-founded the company in 2003 with the goal of creating a more cost-effective treatment for malaria, and after some notable successes toward that end, he has turned his eye on developing new renewable sources of energy. As senior vice president of research, Newman talks about the unique blend of science, idealism and business savvy that underlies the company’s lofty goals.

You’ve had some enormous successes in your career. Where do ideas this big come from?

I have always enjoyed studying science, but I was inspired to seek a career that played an important role in world stewardship by where I grew up. Cape Cod is one of the most naturally beautiful places in the world. To me, it represents a microcosm of what’s happening to all of our planet’s beautiful places—an environment fighting a difficult battle with pollution.

How did you go from there to starting Amyris?

I had been working in bioremediation—using microbes to clean up environmental waste—but there was a gulf between the technology and the marketplace that was fairly impossible to overcome. All the technology in the world really does no good without a viable business model, a way to take that cool new technology out of the lab and apply it into the impactful realm of the marketplace.

While doing a postdoc at the University of California-Berkley I met a few like-minded scientists, Kinkead Reiling and Neil Renninger, that wanted to solve real-world problems. We kind of stumbled into a real-world problem that our science could address. Strains of malaria were developing that were resistant to chloroquine, the reigning malarial treatment. To treat these new, increasingly life-threatening strains, medicine was turning to artemisinin, a pricey compound from wormwood plants that takes 14 months to produce. So we rallied around the idea of synthesizing an affordable alternative as a starting mission for our new company. With help from a grant from the Gates Foundation, Amyris met that goal, and we expect that this product will begin wide distribution within the next few years. Our process will be a stable second source to the plant-derived artemisinin for life-saving drugs.

How did you synthesize it?

The technology is based on isoprenoids, which are chemical compounds produced by a plant that was cultivated to replace sandalwood as an essential oil when it became expensive and scarce. That’s where the company name comes from—amyris is an oil derived from that plant.

What can we expect next?

Well, we thought about ways to deploy the same technology we used to solve the artemisinin problem. Of the countless possible applications, our new goal was use synthetic biology to engineer a biofuel with more energy and less carbon emissions that could replace the fuel in the engines we use now. We figured out how to do it on paper, then at small scale in the lab. Now we are producing it by the barrel and scaling up in California and Brazil. Within two to three years, we expect this fuel to reduce greenhouse gas emissions by 60 to 80 percent for every gallon of petroleum it replaces.