Chemistry lessons from bacteria may improve biofuel production

Thursday, June 9th, 2016

If you’re made of carbon, precious few things are as important to life as death.

A dead tree may represent a literal windfall of the building blocks necessary for making new plants and animals and the energy to sustain them.

“The recycling of plant carbon is fundamental to the function of our ecosystems,” says Cameron Currie, professor of bacteriology at the University of Wisconsin–Madison. “We get food, water, air, energy — almost everything — through those ecosystem services. It’s how our planet operates.”

But the component parts of a dead tree were carefully assembled in the first place, and don’t just fall apart for easy recycling.

In the case of cellulose — a key structural component in plant cell walls and the most abundant organic compound in life on land — a world of specialized microbes handles this careful deconstruction. Much of that work is done by fungi growing on decaying plants, but bacteria in the soil, in the guts of animals like cows and working alongside insects, get the job done, too.


Researchers tried to grow different strains of Streptomyces bacteria on dead plant material (filter paper). Successful cellulose processing strains tapped special genes to produce enzymes that break down cellulose. Photo: Currie Lab/UW-Madison.

A new analysis of a group of bacteria called Streptomyces reveals the way some strains of the microbe developed advanced abilities to tear up cellulose, and points out more efficient ways we might mimic those abilities to make fuel from otherwise unusable plant material.

Streptomyces were long thought to be prominent contributors at work in breaking down cellulose, and to be equally active in the cause across hundreds or thousands of strains of the bacteria.

“That assumption — which is based on a very good, old study of one type of Streptomyces — is not right on the mark,” says Brian Fox, a UW–Madison biochemistry professor and co-author of the study published today in the journal PLOS Biology. “What we see now is that there’s a relatively small group of types of Streptomyces that is far more effective at breaking down cellulose, and a much larger group that is far less effective.”

The UW–Madison researchers measured the relative abilities of more than 200 types of Streptomyces bacteria by growing them on simple sugar and on a good source of cellulose: filter paper (which is made of dead trees).

They were able to collect the genomes of more than 120 of those strains, and identify the genes — and the ways key genes were expressed — that set strong cellulose degraders apart from poor ones.

“The strains that aren’t good at degrading cellulose mostly express the same genes whether we grow them on glucose or on plant material,” says Gina Lewin, a bacteriology graduate student and study co-author. “The strains that are really good at degrading cellulose totally change their gene expression when we grow them on plant material.”

The successful Streptomyces strains — which were typically those found living in communities with insects — ramp up production of certain enzymes, the proteins that do the cleaving and dissolving and picking apart of cellulose.


Counter to long-held belief about the bacteria Streptomyces, seen growing here in a petri dish, the ability to break down a stubborn molecule in plant cell walls called cellulose may be limited to just a few gifted strains. Photo: Adam Book/UW-Madison

“There are families — six or eight or maybe 10 of these enzymes — that all of the active Streptomyces have,” Fox says. “And this paper shows that the most abundant one of them has to be there or the whole thing collapses.”

It’s the particular combinations of enzymes that makes the research useful to scientists working on biofuels.

Biofuels are typically made from the sugars easily extracted from the same parts of plants we eat.

“We eat the kernels off the corn cob,” Currie says. “But most of the energy in that corn plant is in the part which is not digestible to us. It’s not in the cob. It’s in the green parts, like the stalk and the leaves.”

Evolving microbes like Streptomyces have been sharpening the way they make use of those parts of plants almost as long as the plants themselves have been growing on land. That’s hundreds of millions of years. On the other hand, the Department of Energy’s UW–Madison-based Great Lakes Bioenergy Research Center, which funded the Streptomyces study, was established in 2007.

“The natural world is responding to the same kind of things that humans are,” Fox says. “We need to get food. We need to get energy. And different types of organisms are achieving their needs in different ways. It’s worth looking at how they do it.”

The new study identifies important enzymes, and new groups of enzymes, produced when Streptomyces flex particular genes. If they represent an improvement over current industrial processes, the microbes’ tricks could make for a great boon to bioenergy production.

“For a cellulosic biofuel plant, enzymes are one of the most expensive parts of making biofuels,” Lewin says. “So, if you can identify enzymes that work even just slightly better, that could mean a difference of millions of dollars in costs and cheaper energy.”

Banner photo: The ability to break down the cellulose in plant material is rare in Streptomyces bacteria, except in strains that live alongside insects — honeybees, leaf-cutter ants (above), some beetles —that eat or make use of the woody parts of plants. Photo: Don Parsons/UW-Madison.

This story was originally published on the UW-Madison News site.

UW expert at White House summit

Thursday, May 12th, 2016

On Friday, May 13, the White House Office of Science and Technology Policy will host an event on microbiomes – communities of microorganisms that live on and in people, plants, soil, oceans and the atmosphere. Microbiomes maintain healthy function of these ecosystems, influencing diverse features of the planet – human health, climate change and food security.

During the event, the administration will announce steps to advance the understanding of microbiome behavior and enable protection and restoration of healthy microbiome function, including by investigating fundamental principles that govern microbiomes across diverse ecosystems, and developing new tools to study microbiomes.

Lending expertise to the summit will be UW-Madison bacteriology Professor Timothy Donohue. Donohue directs the Department of Energy-supported Great Lakes Bioenergy Research Center and is an expert on biofuels and bio-based renewable energy technologies. Donohue can talk about microbiome research in general and, more specifically, how it might inform strategies for devising alternative fuel technologies. He can be reached at (608) 770-1283.

Researchers collaborate to break down lignin and advance biofuels

Thursday, January 14th, 2016

To tackle what many consider the next frontier in biofuels research, the Great Lakes Bioenergy Research Center (GLBRC) recently joined forces with the Joint BioEnergy Institute (JBEI) in Emeryville, California. The focus of their collaboration? Lignin, a glue-like compound in the cell wall of most living plants that gives them their sturdiness.

While lignin accounts for up to one third of plant carbon, it’s also the most difficult part to break down and remains a formidable obstacle to accessing the valuable sugars contained within biomass. As a result, the biofuels and paper industries mostly treat it as a waste product, isolating it to be burned or discarded.

GLBRC lignin Kate Helmich

Kate Helmich analyzes bacterial enzymes called LigF and LigE that break particular chemical bonds within larger lignin molecules.

But GLBRC and JBEI researchers hope to change all that by bringing their collective expertise to the challenge of unraveling lignin’s strong and unique molecular bonds. With two new studies already complete, the payoff for their effort could be significant.

“If we can convert lignin from an undesirable byproduct into a starting material for advanced biofuels and other lucrative chemicals,” says Tim Donohue, GLBRC director and UW–Madison professor of bacteriology, “we would dramatically change the economics of tomorrow’s biorefineries.”

The researchers began by studying bacterial enzymes that cleave specific chemical bonds inside larger lignin molecules with the idea of creating a single new enzyme to break down lignin. That turned out to be a difficult task, however, because lignin molecules are similar to snowflakes: they have a multitude of spatial configurations, even within a single plant, and each of them requires a different approach to deconstruction.

“Making a single enzyme would be like trying to make a glove that’s designed for your left hand fit on your right hand,” explains Kate Helmich, co-first author of one of the studies and a recent PhD graduate of UW–Madison’s biochemistry department. “Our two hands are different configurations of the same fingers, and lignin is like a chain of many different hands. Degrading that entire chain would require an enzyme, or glove, that can attach to both the left and the right hands within it.”

Nature has instead evolved multiple, highly specialized bacterial enzymes that work in concert to break down lignin, each of them cleaving different sets of chemical bonds in the complex molecule.

Though a single lignin-cracking “master hybrid enzyme” remains elusive, the study informs future enzyme engineering efforts and sets the stage for more GLBRC and JBEI collaborations to convert lignin and other parts of plant biomass into biofuels and valuable chemicals.

GLBRC’s Helmich and Daniel Gall share first authorship, ​with JBEI’s Jose Henrique Pereira, on a paper recently published in the Journal of Biological Chemistry; and former GLBRC researcher and Rice University professor George Phillips, Jr. is senior author. In a sister study led by JBEI (currently slated for publication), the team reports new insights into bacterial enzymes that could someday help produce aromatic compounds of great interest to the chemical industry from lignin.

“It is exciting to see that our collaboration has resulted in the simultaneous publication of these two important papers,” Donohue says. “Collaboration is at the center of everything we do at GLBRC. We look forward to doing more of this mutually beneficial work with JBEI and other academic and industrial partners to advance the growing biofuels industry.”

GLBRC is one of three Department of Energy-funded Bioenergy Research Centers created to make transformational breakthroughs that will form the foundation of new cellulosic biofuels technology. For more information on the GLBRC, visit

This story was originally published on the Great Lakes Bioenergy Research Center website.

The future, unzipped: Pioneering a technology that could revolutionize how industry produces biofuels

Thursday, January 7th, 2016

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.

John Ralph

John Ralph standing before a nuclear magnetic resonance (NMR) spectrometer, a sophisticated tool of his trade. Banner photo: Lignin is a touch organic compound that gives plants like this poplar tree their structure – and it’s one of the biggest obstacles in breaking down plants to produce biofuel. Photos: Matthew Wisniewski/WEI.

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.

Pile of lignin

A pile of lignin that has been extracted from corn stover using various pretreatment techniques. Photo: Muyang Li/MSU.

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.

Continue reading this story in the Fall 2015 issue of Grow magazine.

Six-year study suggests perennial crop yields can compete with corn stover

Monday, December 28th, 2015

A six-year Great Lakes Bioenergy Research Center (GLBRC) study on the viability of different bioenergy feedstocks recently demonstrated that perennial cropping systems such as switchgrass, giant miscanthus, poplar, native grasses, and prairie can yield as much biomass as corn stover.

The study is significant for beginning to address one of the biofuel industry’s biggest questions: can environmentally beneficial crops produce enough biomass to make their conversion to ethanol efficient and economical?

students with miscanthus

Undergraduate students examine giant miscanthus, the subject of a recent GLBRC publication comparing the yield and total growth of perennial crops to that of corn over the course of six years.

Since 2008, University of Wisconsin–Madison research scientists Gregg Sanford and Gary Oates, and their colleagues at Michigan State University, have cultivated more than 80 acres of crops with the potential to become feedstocks for so-called “second-generation” biofuels – i.e., biofuels derived from non-food crops or the non-food portion of plants – at UW’s Arlington Field Station and MSU’s Kellogg Biological Station.

“We understand annual systems really well, but little research has been done on the yield of perennial cropping systems as they get established and begin to produce, or after farmland has been converted to a perennial system,” says Oates.

To find out basic information about how well certain crops produce biomass, Sanford and Oates, also affiliated with the Department of Agronomy, tested the crops across two criteria: diversity of species, and whether a crop grows perennially (continuously year-after-year) or annually (needing to be replanted each year).

Highly productive corn stover has thus far been the main feedstock for second-generation biofuels. And yet perennial cropping systems, which are better equipped to build soil quality, reduce runoff, and minimize greenhouse gas release into the atmosphere, confer more environmental benefits.

Corn, when grain is included, proved to be most productive over the first six-year period of the study at the Wisconsin site, but giant miscanthus, switchgrass, poplar, and native grasses were not far behind. While the soil is not as fertile at the MSU site, there, miscanthus actually produced the same amount of biomass as corn (grain included) in the experiment, with poplar and switchgrass within range.

“All of this means that, at large scales and on various soils, these crops are competitive with corn, the current dominant feedstock for ethanol,” Sanford says.

Gregg Sanford in field

Study lead author Gregg Sanford, describes the more than 40 acres of agricultural systems examined at UW-Madison’s Arlington Agricultural Research Station in Arlington, WI to visitors.

Data from the project also suggests improvements will come fast for these perennial crops.

“Corn has a huge head start from a historical standpoint – it’s been bred by farmers and seed companies in the U.S. for more than a century,” Oates adds, “but advances in our understanding of chemistry, genetics, and biology means these other crops can catch up quickly.”

If there are limitations to the success of the trial thus far, it’s that it takes time for some perennial crops to produce enough biomass for harvest.

“In our case, the ramp-up of native prairie grass and switchgrass took almost two years,” says Sanford. “That’s a long time for farmers to wait for income.”

“One bad year can also have a big impact,” Sanford adds, noting the failure of the miscanthus crop at Arlington in the first year of the study. “If not for an unusually cold winter, it would have been more competitive with corn in Wisconsin,” he continues. “Looking forward, putting financial protections in place that are similar to those for commodity producers could help insure farmers when this happens.”

Now in the midst of the study’s eighth year, Sanford says the study will continue for the foreseeable future.

“We know that perennial systems can prevent negative impacts such as soil erosion and nitrate leaching and that they also provide habitat for native species that provide beneficial ecosystem services,” Sanford says, “but there are still a lot of questions we want to answer about soil processes and properties, questions that take many years to answer.”

The GLBRC is one of three Department of Energy Bioenergy Research Centers created to make transformational breakthroughs and build the foundation of new cellulosic biofuels technology. For more information on the GLBRC, visit or visit us on twitter @glbioenergy.

This story was originally published on the Great Lakes Bioenergy Research Center website.

Engineered softwood could transform pulp, paper and biofuel industries

Wednesday, April 22nd, 2015

Scientists have demonstrated the potential for softwoods to process more easily into pulp and paper if engineered to incorporate a key feature of hardwoods. The finding, published in this week’s Proceedings of the National Academy of Sciences, could improve the economics of the pulp, paper and biofuels industries and reduce those industries’ environmental impact.

“What we’ve shown is that it’s possible to pair some of the most economically desirable traits of each wood type,” says John Ralph, the Great Lakes Bioenergy Research Center’s (GLBRC) plants leader and a University of Wisconsin-Madison professor of biochemistry.

According to Ralph, altering what once was the hard and fast distinction between softwoods and hardwoods — which process into largely separate product streams — could create opportunities for the multi-billion dollar industries that process biomass for profit.


John Ralph. Banner photo: Softwood pine tracheary elements (TEs) from cells engineered to produce hardwood-type lignin. Photograph courtesy of Lloyd Donaldson, Scion, New Zealand and Matt Wisniewski, GLBRC, WI.

Like most plants, hardwood trees such as birch or poplar contain lignin, the notoriously hard-to-process “glue” that lends plant tissues their structure and sturdiness. Lignin is derived from binding molecules called G- and S-monomers, with S-monomers producing a simpler and more easily degradable lignin. As hardwoods contain both G- and S-monomers, they have traditionally been prized for their relatively easy processing into pulp or paper.

Softwoods such as pine or spruce, on the other hand, derive their lignin from G-monomers only, producing a lignin that is much harder to degrade and which renders softwoods more difficult to process. Their industrial advantage, however, is their long fibers, which are particularly well suited for use in making strong paper products such as shipping containers and grocery bags. In addition, the sugar found within softwoods converts more easily and in higher volume to ethanol, making softwoods a potentially superior feedstock for biofuels.

Ralph and a team of collaborators, including first author Armin Wagner from Scion, one of New Zealand’s Crown Research Institutes, and GLBRC’s Fachuang Lu, used a model called the “tracheary element” (TE) system to prove that it’s possible to engineer conventionally long-fibered softwoods to contain the easier-to-process lignin found in hardwoods.

The TE system induces suspension-cultured cells to make secondary cell walls representative of those found in real wood fibers. In this study, the researchers transformed cells from softwood pine within the TE system by introducing genes for two key enzymes known to produce lignin in flowering plants, showing that the resulting softwood was capable of making and incorporating the S-monomers needed to produce a hardwood-type lignin in its cell wall.

Next, the researchers will attempt to use the same approaches to engineer actual softwood plants to produce S-monomers and S/G lignins. The transition from model to plant is highly anticipated.

“If we could implement this in real plantation softwoods, we could decrease the intensity of pre-treatment processes and increase yields across a variety of industries,” Ralph says. “But there’s a tangible environmental benefit as well: processing biomass faster and more efficiently cuts out a significant amount of waste and energy.”

The research was funded partially by GLBRC, one of three Department of Energy Bioenergy Research Centers created to make transformational breakthroughs that will form the foundation of new cellulosic biofuels technology.

Using cover crops to replenish soil carbon

Wednesday, April 15th, 2015

Traversing the landscape of the Upper Midwest, there is a high likelihood you’ll see corn fields. Lots and lots of corn fields.  Here, leftover stalks are most often plowed under the earth in late autumn, where they can replenish the ground, becoming soil organic matter.

Soil organic matter is made up of partly decomposed plants, soil animals, and microbes. It’s mostly carbon, but it also includes nitrogen and other nutrients that bind together to encourage plants to take in water, while preventing both runoff and drought.


A field at the University of Wisconsin Arlington Agricultural Research Station.

These days, corn stalks have become more and more valuable for use as a biofuels feedstock. Farmers are now able to use leftover corn stover to produce energy and extra money. But that added value also poses a problem: if there’s no plant matter going back into the soil, how does carbon get replenished?

At the University of Wisconsin Arlington Agricultural Research Station about 30 minutes north of Madison, Anna Cates is studying how to replenish, or fix, carbon into the soil by planting other crops along with the corn.

“If you leave nothing, you have no carbon inputs in the ground, and the nutrient levels become so low that they can’t be beneficial to the crops.” says Cates, a University of Wisconsin–Madison graduate student in agronomy and a researcher at the Great Lakes Bioenergy Research Center (GLBRC).

Working under the tutelage of Randy Jackson, a UW–Madison agronomy professor and grassland ecology expert, Cates is replacing the stover that might otherwise be left behind with two different cover crops.

In one plot, a hardy grass called winter rye is planted after the corn harvest and allowed to grow until early May. Throughout the winter, it fixes carbon into the soil, before being harvested just before corn planting season.

“The great thing about winter rye is that it is so resilient in cold conditions,” says Cates. “It can be planted in November and grow enough in a month to survive the winter, so a good crop can be grown before it’s time to sow the corn. We’re trying to optimize the system so that both crops are successful, all while maintaining a soil balance.”


Anna Cates

To find out if the plan works, Cates will measure carbon in corn and cover crop biomass. She’s also going to gauge soil respiration, which is the carbon breathed out by the tiny little microbes living below the surface when they decompose plant roots and leaves. A system with lower soil respiration produces more plant carbon, and thus may be storing more of the nutrient in the soil.

In another plot, bluegrass, is cultivated in between rows of corn. In this trial, a carpet of bluegrass grows around the plant year-round.

“The trick is for the grass to replenish the soil throughout the year without inhibiting corn growth,” says Cates. “Because it’s a perennial, bluegrass should be really good at maintaining soil carbon levels, keeping microbial communities healthy, and attracting beneficial insects that keep the pest populations down.”

After each season, Cates compares soil carbon measurements from the bluegrass and winter rye plots to a conventionally sown corn field.

Now into its second season, the experiment will take time to germinate, so to speak. That said, Cates has already seen data from 2014 demonstrating that the rye cover crop appears give a boost to soil carbon. The grass plot measurements will begin in earnestness in 2015.

“In the end, it’s a simple math problem,” says Cates. “We add up how much plant matter is produced and subtract the carbon dioxide and the harvest. If we end up building more carbon than we take out, it’s a victory for the soil, but also for the biofuels industry.”

This story was originally published on the Great Lakes Bioenergy Research Center website.

David Duncan: Finding quirk and charisma in microbes

Tuesday, April 14th, 2015

David Duncan loves to think about dirt, and a quick glance at his family tree could lead one to believe he comes by it naturally. His grandfather was an agricultural extension agent and his handful of uncles includes two agronomists and an expert on fungi.

But Duncan, a University of Wisconsin–Madison doctoral student in agronomy and a Great Lakes Bioenergy Research Center researcher, asserts that what really pulled him into agronomy was his mother.

“My interest in biology was agricultural from the get-go and I think it’s because my mom always loved the idea of having a great big garden,” Duncan says. “Early on, I learned from her that growing things was a good pursuit, something worth doing.”


David Duncan in his lab at the Wisconsin Energy Institute.

Duncan spent his childhood in central Wyoming, an arid region where it’s hard to grow much of anything. He recalls tending some strawberries and rhubarb with his mom, but remembers even better the summers he spent digging around in the decomposing pile of grass clippings out by the shed.

“It was sort of an early experiment in microbial ecology,” Duncan says. “Unfortunately, it ended when the hornets set up a nest there.”

Boy scouts and soccer eventually displaced the compost pile, but Duncan ventured back to biology as an undergraduate at Stanford University where he studied plant biology and biotechnology and became interested in understanding the ecology of sustainable cropping systems.

Duncan graduated from Stanford in 2006 and joined UW­–Madison’s College of Agricultural and Life Sciences master’s program in agroecology in 2007.

In 2010, he became a doctoral student in agronomy where his study of cropping systems brought him right back to the microbial ecology he enjoyed as a boy.

Today, when Duncan looks at dirt, he sees habitat for the millions of different species of microbes residing there.

“Microbes are incredibly charismatic if you have the right mindset,” says Duncan. “They’re quirky. They do things that don’t make sense at the human scale. For example, there are microbes that split up metabolic processes among multiple different organisms … kind of like you eating something and then your partner being the one to actually digest it.”

Microbes, the oldest life form on earth, are single-cell organisms so tiny that billions can live in a thimbleful of soil. But Duncan, whose research focuses on sustainable biofuel feedstocks, is searching agricultural fields for something even smaller – a single microbial gene called nitrous oxide reductase (nosZ) that’s responsible for making the loss of agricultural nitrogen less harmful to the environment.

When farmers apply nitrogen fertilizer to crops, the fertilizer is eventually transformed into nitrate. Nitrate is a simple form of nitrogen that’s easy for plants to use, but can also be lost in run-off or end up in the soil. Nitrogen loss is bad for farmers because they have to pay for the fertilizer, but even worse is what what nitrogen turns into when you lose it: nitrates in your drinking water and nitrous oxide in the atmosphere.

Nitrous oxide is a significant ozone-depleting chemical and a highly potent greenhouse gas, and agricultural use of fertilizers plays a significant role in its production. In fact, nitrogen loss from agricultural soil accounts for about 75% of total U.S. nitrous oxide emissions.

Duncan is comparing cropping systems including corn, switchgrass, and native prairie grass in an effort to understand how the microbial community as well as system variables such as crop type, temperature, moisture, and nitrogen availability contribute to nitrous oxide emissions. And the nosZ gene is a central focus of his research.

Some microbes have a group of genes that allows them to “breathe” nitrate. When microbes with an active nosZ gene breathe nitrate, it’s chemically converted to the harmless nitrogen gas that comprises 70% of our atmosphere. For microbes lacking an active nosZ gene, however, the nitrate conversion process produces nitrous oxide.


A soil sample

“If the nosZ gene does it job,” Duncan says, “we don’t see nitrous oxide emissions. But if nosZ is absent, or not working, then we see a lot of nitrous oxide emissions.”

Duncan would like to use the prevalence of nosZ to predict whether the nitrogen conversion process in a particular soil will be environmentally harmful. But it’s not that simple ­– sometimes, active microbes with the nosZ conversion ability don’t convert nitrogen at all.

For the nosZ conversion of nitrogen to take place, a number of conditions have to be met: (1) The microbe has to have the nosZ gene (many don’t); (2) The microbe has to be “awake” (many are inactive in a state resembling hibernation); and (3) The nosZ gene has to be “turned on” (in scientific terms, “expressed”).

Duncan’s work looks closely at both the microbial community and the environmental variables in order to understand the conditions that best foster the presence and wakefulness of nosZ microbes as well as the “turning on” of the nosZ gene.

“It’s important to have both research components in place,” Duncan explains, “because variables such as temperature, moisture, and nitrogen availability determine whether the microbes with the preferred conversion ability are awake and ready to render excess nitrogen harmless.”

While many of Duncan’s research hours are spent in the field — sampling soils and documenting nitrous oxide emissions — far more of his time is spent analyzing vast digital datasets of microbial genetic data, looking at the abundance and types of nosZ present in agricultural soils.

Making sense of the data generated by sequencing microbial DNA is complicated, in part because most sequencing technologies have been developed to analyze human DNA. Any two people will have DNA sequences that are about 99.9% identical. In sharp contrast, just one gram of soil can contain somewhere between one and ten million different species of microbes, and some of these species are nothing alike.

Duncan, however, appears completely undaunted by the complexity.

“I’m happiest when I’m doing data analysis,” Duncan explains. “There’s something extremely rewarding about uncovering patterns in your data, particularly when they’re patterns you can fit into a biologically coherent narrative.”

Duncan’s careful analysis of local soil is helping create a coherent narrative for biofuel cropping systems. His work is a starting point for understanding the complex relationships among energy crops, microbial communities, environmental variables, and nitrogen loss.

“Currently, we plant crops and measure nitrous oxide emissions, which is laborious,” Duncan says. “But we’re working to get to the point where we could instead use soil analysis, including nosZ content, to predict potential emissions.”

Short-term, Duncan believes his research can help both farmers and scientists evaluate potential bioenergy crops. Long-term, Duncan hopes his work will help scientists predict the environmental effects of different biofuel cropping systems in changing climates across the world.

This story was originally published on the Great Lakes Bioenergy Research Center website.

Daniel Amador-Noguez engineers better bacteria for biofuels

Tuesday, March 3rd, 2015

In high school,” Daniel Amador-Noguez recalls, “I took a science class where one of the lectures was all about the future – where the research was taking us, what were some potential future discoveries – and I thought, ‘oh, all this sounds really cool, so how come it hasn’t happened yet?’”

Seventeen years later, it’s not at all difficult to square that eager young man with the energetic scientist Amador-Noguez has become. An assistant professor of bacteriology at the University of Wisconsin–Madison and researcher at the Great Lakes Bioenergy Research Center (GLBRC), Amador-Noguez emanates anticipation, for next week’s result, the future of his research, and the future of biofuels.


David Stevenson (left), research specialist, and Daniel Amador-Noguez examine a plate of microbes in the Microbial Sciences Building. Photos by Matthew Wisniewski.

“It’s difficult to come up with a biological process that isn’t affected by bacteria,” he says. “They are virtually everywhere, not just in the environment but also inside our bodies. And if we can improve our understanding of microbes we can do a lot of enormously important things, including improving biofuels.”

Amador-Noguez’s lab focuses on understanding metabolism in biofuel-producing bacteria with the goal of engineering microbes that can more efficiently convert plant biomass to energy. It’s exciting research, but it’s also the kind of data-driven work that Amador-Noguez hungered for from a very early age.

Growing up in Pachuca, a small city in central Mexico, in the 1980s Amador-Noguez struggled to find information on biology, the subject that interested him most. Science education in Mexico at that time was not strong, the Internet was not yet in widespread use, and bookstores were scarce, so Amador-Noguez found himself reading and re-reading the same encyclopedia periodical his mother had arranged to be sent to the house.

“I must have read that encyclopedia three or four times by the time I was nine or ten,” he says. “My favorite volume had a section on genetic mutations with all this classic imagery of flies, where you have all these different flies with three eyes or four wings. It’s a bit creepy but for a kid that’s really interesting!”

After high school, Amador-Noguez headed to the Monterrey Institute of Technology in northern Mexico, where he had his first real exposure to science, including laboratory research. Since he hadn’t been able to find a good biology program in Mexico, he majored in chemistry.

After graduating from Monterrey in 2001, Amador-Noguez pursued a doctorate in molecular genetics at Baylor College of Medicine in Houston, Texas. While studying the molecular mechanisms of aging in the long-lived Ames Dwarf mice and Little mice, Amador-Noguez discovered that liver metabolism played a key role in the mice’s longevity. His research has focused on metabolism ever since.

He left Baylor with doctorate in hand in 2007 and headed to Joshua Rabinowitz’s lab at Princeton University to do a postdoctoral fellowship in cellular metabolism. He began using mass spectrometry, an analytical chemistry technique, to study the metabolism of bacteria related to biofuel production, and there his interest in biofuels was born.

After five years at Princeton, Amador-Noguez joined UW–Madison and GLBRC in the fall of 2013. To study bacteria in his lab, he now uses metabolomics, a relatively new, systems-level approach to understanding the behavior of metabolites and the regulation of metabolism in microorganisms.

Amador-Noguez uses analytical tools such as mass spectrometry and liquid chromatography, which enable him to identify and measure most of the metabolites inside bacteria, as well as determine the bacteria’s metabolic flux, or which of the bacteria’s metabolic pathways are the most active (or inactive) as the cell converts nutrients into the energy and building blocks it needs to grow.


The Amador-Noguez Lab

Metabolic flux, the extent to which carbon is passing through one metabolic pathway versus another, is of particular importance to biofuel production. Often, the way to engineer bacteria that are more efficient at converting biomass into biofuels is to maximize flux through pathways known to be involved in biofuel production.

The linked nature of metabolic pathways also makes inhibition of any one pathway a significant issue in studying bacteria; if one pathway is not working, a cell will simply shut down the rest of its metabolism.

In one recent GLBRC project, Amador-Noguez and his team sought to understand how a particular toxin inhibits the conversion of plant sugars into biofuels. The toxin in question is produced from lignin – the woody backbone of plants – during the process of breaking down plant biomass into its sugar components such as glucose.

Amador-Noguez discovered that, in general, these “lignotoxins” are powerful inhibitors of the enzymes the cell needs to synthesize nucleotides, which are essential to the cell’s DNA.

Now that Amador-Noguez knows which enzymes are affected, he and other researchers can find a way to direct those particular enzymes to be more resistant to lignotoxins. Or he can break the regulatory connections between nucleotide biosynthesis pathways and fermentation pathways so that the bacteria would simply remain unaware that a particular biosynthetic pathway was not functioning properly, and would thus continue to produce biofuels.

Looking to the future, Amador-Noguez is excited about the possibility of developing new metabolomics tools that could help reveal how metabolism is regulated within entire bacterial communities, and not just in isolated bacterial species. He says that much remains unknown about how bacteria play nice with one another but that gaining an understanding of the community’s rules could yield powerful results.

“If we could understand how different bacteria interact with each other metabolically in these complex bacterial communities we could do a lot of things,” he explains. “We could understand what shapes the bacterial communities living inside of our bodies, which would have important ramifications for human health, and we could engineer each bacteria in those communities to specialize in doing one thing, which could make the process of biofuel production much more efficient.”

“Biofuels,” he adds, “are an important solution for many of our energy issues. We’re always going to need fuel and we’re always going to need chemicals. And the biological production of biofuels and high-value chemicals from plant biomass is one of the most promising strategies for the sustainable generation of these essential commodities.”

Like many scientists working today on energy-related research, Amador-Noguez remains motivated by the impact his day-to-day research could have on the national and global energy picture, as well as by his long-burning desire to make important advances happen.

“The fact that what we’re doing can contribute to solving big problems in society, that is very motivational,” he says.

This story was originally published on the Great Lakes Bioenergy Research Center website.

Bioenergy center’s research leads to 100th patent application

Wednesday, February 25th, 2015

The Great Lakes Bioenergy Research Center (GLBRC), one of three bioenergy research centers established in 2007 by the U.S. Department of Energy (DOE), recently celebrated the filing of its 100th patent application.

Led by the University of Wisconsin-Madison and major partner Michigan State University, GLBRC consists of more than 400 scientists, students and staff working to develop a robust and sustainable pipeline for producing biofuels and chemicals from the nonedible, or cellulosic, portion of plants. Each year the center brings in about $25 million in federal funding.

glbrc logoSince 2011, the center has reported 50 percent more inventions than expected for a university entity of its size and funding level. Its research has spurred startup companies and supports a range of industries: energy, automotive, and biochemical among them.

“Collaboration is the center’s lifeblood,” GLBRC director Tim Donohue says. “Across different research areas – sustainability, plants, deconstruction and conversion – GLBRC scientists are working with and learning from each other every single day. Our relationship with industry, government and other research institutions helps us produce knowledge that meets real-world needs. By spurring the development of cellulosic biorefineries that will produce the fuels and chemicals we need and use, we are driving the economy and addressing a critical societal need.”

Candace Wheeler, technical fellow at the General Motors Research and Development Center, says research from the DOE’s Bioenergy Research Centers is helping to meet the diverse energy needs of a growing fleet of GM vehicles.

“We look to Bioenergy Research Center researchers for new knowledge and scientific discoveries that will support the sustainable production of biofuels,” Wheeler says.

In recently developed GLBRC technology, UW-Madison’s John Ralph, Michigan State’s Curtis Wilkerson and the University of British Columbia’s Shawn Mansfield pooled their diverse expertise to engineer poplar trees whose plant walls contain a modified form of the polymer lignin to make them easier to degrade for conversion to fuel. The resulting technology, reported in Science last April, is of broad interest to the bioenergy, bioproducts and fiber industries.

“GLBRC is at the forefront of new concepts for lignin utilization,” says Jerry Gargulak, Business Development Manager at LignoTech USA. “The work being done to design modified poplar lignin with decreased complexity is spectacular. This technology has the potential to open up a completely new route to lignin-based chemicals and change the bio-based chemical industry in a new and exciting way.”

GLBRC technology has also laid the foundation for several startup companies. Hyrax Energy, a private company begun by UW-Madison researcher Ronald Raines, was the first company to emerge. Its technology converts biomass into fermentable sugars usable in biochemicals, bioproducts and drop-in fuels.

GlucanBio, which draws from technologies developed by GLBRC’s Jim Dumesic, focuses on de-risking and scaling up the use of a plant-derived solvent capable of dramatically reducing the cost of producing biofuels and bioproducts.

“What we’re seeing is that collaborative partnerships like GLBRC’s greatly increase the potential for transformative research and technology transfer,” Donohue says.

Several technology transfer organizations, including the Wisconsin Alumni Research Foundation, Michigan State University’s MSU Technologies and Texas A&M’s Office of Technology Commercialization, support the center’s intellectual property efforts and enable the licensing of its technologies.

The 100th patent “is an exciting milestone for GLBRC,” says Leigh Cagan, chief technology commercialization officer for WARF. “We’re proud to support the UW-Madison researchers and partners working to meet the greatest energy challenge of our time.”

For more information on GLBRC technologies, contact