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The Future, Unzipped

Biochemist John Ralph and his colleagues have pioneered a technology that could revolutionize how industry produces biofuels and other value-added goods

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.”