Fall 2020

Cover Story

Three key players in ongoing nitrogen fixation research at CALS — from left, agronomy professor Natalia de Leon, horticulture faculty associate Claudia Calderón, and bacteriology researcher Valentina Infante — are pictured (standing six feet apart) in an experimental corn plot at West Madison Agricultural Research Station. Photo by Michael P. King


In 1909, the German chemist Fritz Haber sparked an agricultural revolution. Using enormous pressures and high temperatures, he had learned how to efficiently transform nitrogen, so abundant in the air, into ammonia. Artificial fertilizer was born.

By converting atmospheric nitrogen into a form plants can use — a process known as “fixation” — Haber helped farmers make their fields vastly more productive. He was lauded for conjuring “bread from air.” Today, artificial fertilizers and their fixed nitrogen feed half the world’s population.

At the time of Haber’s invention, UW scientists were establishing the university as an epicenter of nitrogen-fixation research. But instead of high-pressure tanks of superheated natural gas, they turned to nature’s chemical factories: plants. Scientists had recently discovered that legumes — peas, beans, alfalfa — partner with bacteria to fix nitrogen in their roots. This discovery set off a decades-long race to understand this vital alchemy and harness it to feed a growing population.

For more than a century, CALS scientists have played the parts of bacterial purveyors and biochemical sleuths, of mutant wranglers and slime whisperers, to unlock the secrets of nitrogen fixation. Today, UW remains a leader in understanding, applying, and expanding the potential for nitrogen fixation to improve agriculture worldwide.

Future Crop

As you follow the curving road of the West Madison Agricultural Research Station toward the experimental plots, the corn catches your gaze. It looms several feet above the familiar, Mid-western corn planted nearby. Even though it’s October, this corn remains green while the other varieties have turned to yellow husks.

On closer inspection, the corn sports dense arrays of thick, red roots on its stalks, set at regular intervals above the ground. Clinging to these roots is a viscous, slime-like gel. This gel is the reason the corn is here in Madison, thousands of miles from its native Mexico.

Gel exudes from the aerial roots of a special nitrogen-fixing corn variety growing at West Madison Agricultural Research Station. Photo by Michael P. King

“These aerial roots produce an insane amount of gel,” says Jean-Michel Ané, a professor of agronomy and bacteriology who has studied the special corn for years. “What we found is that gel is a good environment for nitrogen-fixing bacteria.”

In 2018, Ané and his partners at the University of California, Davis and Mars, Inc. announced that special varieties of corn from central Mexico can use this gel to fix nitrogen. The corn can acquire 30% to 80% of its nitrogen from bacteria in the gel. It was the first time that a grain crop — not a legume — was discovered that could acquire a significant amount of nitrogen from the air by partnering with bacteria.

At the West Madison research station, Ané and professor of agronomy Natalia de Leon MS’00, PhD’02 are testing dozens of varieties of this tropical corn to funnel into breeding programs. Their goal is to cross this nitrogen-fixation trait into elite varieties of corn adapted to different regions around the world. If high-yielding varieties of corn could produce even a fraction of their own nitrogen, it would have global benefits.

In wealthy countries, farmers can afford to apply large amounts of fertilizers to maximize their crop yields. But excess nutrients spill into waterways and promote the growth of algae that deplete oxygen in the water and kill animal life. As the Mississippi River funnels nitrogen from Midwestern corn fields every summer, it creates a low-oxygen “dead zone” in the Gulf of Mexico more than 7,000 square miles across.

In developing countries, access to fertilizers can be limited by either supply or price. The lack of fertilizer suppresses yield and hobbles profits. And the world spends about 1% of its total energy use — and creates an equal proportion of its greenhouse gases — just to produce nitrogen fertilizers using Haber’s original method.

“The benefits of nitrogen fixation [in corn] for developed countries, it’s really to develop an agriculture that’s more sustainable and less damaging to the environment,” says Ané. “And for developing countries, it’s a matter of increasing yield, because when they cannot have access to fertilizers, [a lack of] nitrogen is limiting their yield.”

Close-up of the spacing of aerial roots on the stalk of a special nitrogen-fixing corn variety growing at West Madison Agricultural Research Station. Photo by Michael P. King

Ané’s corn seed came from CIMMYT, a globally renowned plant breeding nonprofit based in Mexico that is dedicated to improving corn and wheat. CIMMYT and organizations like it source thousands of unique, local varieties of crops that can contribute new traits, such as pest or drought resistance, to high-yielding lines. Ané’s goal is to make his research — and any new seeds — widely available to improve agriculture broadly.

“I want to make sure everything I do is publicly accessible,” he says.

At the start of his career, Ané couldn’t have foreseen working with this unique corn. Like many nitrogen-fixation researchers over the decades, Ané hoped to engineer non-legumes to partner with nitrogen-fixing bacteria — the field’s holy grail. And he’s continued probing the genes that could make that possible, with some promise.

But in other ways, his tenure on campus has followed a well-worn path. With one foot in bacteriology and another in agronomy, and his work with agronomist de Leon and others, Ané exemplifies perhaps the defining feature of nitrogen-fixation research at UW–Madison: cooperation.

Fixated on Nitrogen Fixation

“In this long history of nitrogen fixation here, there have been strong collaborations,” says Gary Roberts, professor emeritus of bacteriology, as he sits in the light-filled atrium of UW’s Microbial Sciences Building.

He would know. As one in a long line of researchers at CALS who probed the legume-bacteria partnership at the heart of nitrogen fixation, Roberts himself had strong collaborations, especially with biochemists.

The century-long story of nitrogen-fixation research at UW–Madison and CALS includes a large cast of characters, many of whom are shown here in a rough chronology of their contributions. Photos courtesy of University of Wisconsin– Madison Archives and Gary Roberts and by Jeff Miller, Bryce Richter, and Michael P. King.

That has been a common link. In CALS, the close association between the bacteriology and biochemistry departments — down the block from one another — drove the research forward faster than either department could have accomplished alone.

Much of that work began with Perry Wilson BS’28, PhD’32. Hired as an assistant professor in the Department of Agricultural Bacteriology in 1932, Wilson became an early pioneer in the biochemistry of nitrogen fixation, in part because of access to stable external grants before such funding was common. In his greenhouse attached to King Hall, Wilson investigated how different gases affect nitrogen fixation. He showed that carbon dioxide provides critical support for photosynthesis and that hydrogen gas inhibits the process.

Wilson went on to train Bob Burris MS’38, PhD’40, who earned a faculty appointment in biochemistry. By using a stable isotope of nitrogen to follow the process, Burris established that the Rhizobia bacteria behind fixation turned nitrogen in the air into ammonia — precisely the product of Haber’s industrial reaction that so electrified the world. Burris went on to isolate and probe the proteins at the heart of the bacteria’s nitrogen-fixing reactions.

“Almost every important biochemical advance [in nitrogen fixation] for 20 years, Burris’s lab either did it first or second,” says Roberts.

Those proteins that Burris studied collectively make up the heart of nitrogen fixation: nitrogenase. The enzyme used by bacteria to convert atmospheric nitrogen into ammonia, nitrogenase is the only known example in the biological world where inert nitrogen gas is converted into a form that plants can use. It’s a mercurial enzyme, requiring strict conditions and enormous sums of energy to function — a feature that led to winding but productive research careers.

Next door to Wilson’s lab, Winston Brill set up shop as a professor of bacteriology in 1968. Working in the evenings next to Wilson’s student, Bob Fisher MS’67, PhD’69, Brill pitched in by helping to isolate a mutant strain of bacteria that couldn’t fix nitrogen, which helped Fisher explore the biochemistry of the process.

“That’s how I sneaked up on the field,” says Brill, now a retired creativity consultant living in Washington state. “From then on, I think almost ​​all of my research had to do with nitrogen fixation.”

Mutant bacteria became the Brill lab’s go-to method for piecing together the genes required for nitrogen fixation. With CALS bacteriologist Vinod Shah, Brill identified the active site of nitrogenase — where the alchemy takes place. Some of his other collaborations included faculty from the agronomy and entomology departments.

At one point, Brill’s lab isolated a mutant of Azotobacter, another nitrogen-fixing group, that excreted ammonia. They decided to test whether the mutant could provide enough nitrogen to help plants grow and settled on corn for their experiment.

The corn grew taller in the lab. “Under sterile conditions, it looked pretty neat. But we knew from working with Rhizobia that working in the soil is very different from working in a sterile flask,” says Brill. In field conditions, the growth gains vanished, which Brill says they expected.

Brill even looked at indigenous corn varieties, spotting one from Ecuador that looked to be incorporating fixed nitrogen. But when they didn’t see any improvement in growth in a lab experiment, they dropped the project. When Ané published his findings about nitrogen-fixing corn, Brill was floored, and he quickly offered his congratulations.

UW faculty members attend the groundbreaking of the Bacteriology Building for the College of Agriculture on July 27, 1953. Pictured are E. B. Fred, Elizabeth McCoy, H. L. Russell, E. M. Foster, O. N. Allen, I. L. Baldwin, W. D. Stoval, R. K. Froker, W. D. Frost, W. C. Frazier, K. B. Raper, C. A. Elvehjem, J. B. Wilson, P. W. Wilson, S. G. Knight, W. B. Sarles, and Noble Clark. Photo courtesy of University of Wisconsin-Madison Archives

Brill trained both Jo Handelsman PhD’84, now the director of the Wisconsin Institute for Discovery (WID), and Roberts, who eventually took over the lab once Brill went to work for the biotech company Agracetus west of Madison, where he continued to study nitrogen-fixing bacterial mutants.

With Brill, Handelsman studied the physiology of different strains of Rhizobia bacteria, their ability to help plant roots form nitrogen-fixing nodules, and their interaction with other bacteria present in alfalfa seeds. As a professor of plant pathology at CALS, former chair of the bacteriology department, and now at WID, Handelsman has continued to study the microbial communities surrounding legume roots.

When Roberts secured his faculty appointment, he collaborated with biochemist Paul Ludden PhD’77 to study the active site of nitrogenase. They worked out how the metal-rich enzyme core was put together. And they studied how bacteria regulate nitrogen fixation, an energy-intensive process. “It’s so slow, and so expensive, that you don’t do it unless you really, really need to,” says Roberts.

Burris and Brill long ran a Monday journal club on nitrogen fixation that attracted campus researchers from bacteriology, biochemistry, horticulture, botany, and agronomy, furthering the kinds of collaborations that spurred the research along in CALS.

“[The seminar] was one of the highlights of my postdoctoral experience because we had expertise in all of these different aspects of symbiosis on campus,” says CALS Dean Kate VandenBosch, who trained in nitrogen fixation at UW–Madison as a postdoctoral researcher in the 1980s with Elden Newcomb. “It got me out of my silo and pushed me to understand other aspects of the science that had not been part of my training.”

With Newcomb, a professor of botany, VandenBosch researched the enzymes found within the nodules that nitrogen-fixing bacteria form on plant roots. They pioneered new ways to see specific proteins using high-powered microscopy, which provided a greater understanding of how the plant cooperates with bacteria to establish the partnership.

“Nitrogen fixation is a field that was very intellectually stimulating and brought a lot of people into the field for a variety of reasons,” says VandenBosch. “It’s the interplay of the compatibility interactions between Rhizobium and the plant and how they influence each other’s development to create this stable, beneficial interaction that is of economic significance.”

But with much of the key biochemistry and bacteriology worked out, and practical advances in agriculture hard to come by, much of the nitrogen fixation work shifted to investigating how plant genes govern the partnership between bacteria and host plant. As the 20th century closed, emphasis on plant genomic analysis increased, and research on nitrogen fixation and its potential to improve agriculture moved to other institutions.

That is, until Ané joined the university in 2004. The inclusion of nitrogen-fixing corn has further reinvigorated the line of research, enticed new researchers to the field, and reignited hopes for developing practical applications. A century ago, even before Perry Wilson began his work, that same spirit of optimism and practicality permeated the UW’s approach to nitrogen fixation.

Research Rooted in Helping Farmers

As knowledge of legumes’ useful partnership with microbes spread around the country at the turn of the 20th century, so did the need for the particular bacteria that make the partnership possible. While these microbes are widespread where beans or alfalfa have long been grown, they can be absent in other soils. Enter the inoculating culture.

In 1916, CALS scientists started growing cultures of nitrogen-fixing bacteria and supplying them, at cost, to farmers around the state. Photo courtesy of Department of Bacteriology

At the end of the 19th century, companies began bottling and selling nitrogen-fixing bacteria to farmers to spread on their fields or coat their seeds. But poor understanding of microbial growth, confusion over the appropriate strains, and shoddy quality control often combined to make the effectiveness of these cultures suspect.

So in 1916, CALS scientists started growing cultures and supplying them, at cost, to farmers around the state. By 1925, UW was supplying more than 100,000 cultures a year, principally to alfalfa farmers. Private companies, weary of competing with the university, entreated the state legislature to halt the university’s practice.

But a December 1924 article in The Capital Times reported that the university had persuaded legislators that their efforts to distribute high-quality cultures benefited the state’s farmers. “There were a lot of farmers that were pretty insistent that [the university] stay in it because they were confident in the UW product,” recalls Burris in an oral history.

By 1930, the U.S. Department of Agriculture valued all the country’s cultures, commercial and not-for-profit, at $1 million, equivalent to $15 million in 2020. Today, seed companies continue to provide inoculated seed to ensure robust crops of soybeans, alfalfa, and other legumes.

E.B. Fred led the charge to perfect and distribute inoculating cultures. As one of the first bacteriology professors on campus and dean of the College of Agriculture at the time (and eventually university president), Fred set the tone for the university’s century-long commitment to nitrogen fixation research.

In addition to his work advancing inoculating cultures, Fred turned his attention to organizing the ballooning field. He partnered with Ira Baldwin PhD’26, his successor as College of Agriculture dean, and bacteriologist Elizabeth McCoy BS’25, MS’26, PhD’29 to publish a mighty tome — 343 pages long — organizing all existing knowledge of nitrogen fixation in 1934, starting with the use of legume crop rotation in classical antiquity and ending with the latest advances in bacteriology and biochemistry.

Around the same time, Ethel Allen MS’30 trained with Fred, Baldwin, and Wilson as a research fellow in nitrogen fixation. Later, in the 1940s, she and her husband, O. N. Allen, joined the bacteriology department, where they published dozens of articles on nitrogen fixation. After her husband’s death in 1976, Ethel completed their magnum opus, an 830-page encyclopedic description of legumes and their partnership with nitrogen-fixing bacteria, published in 1981.

As women working in male-dominated fields, McCoy and Ethel Allen rarely received the attention that Fred did as future university president. Ethel spent much of her career working in her husband’s lab without pay, while McCoy received belittling news coverage despite achieving full professorship in 1942. But by their own accounts, both women felt welcome and accepted by their colleagues at UW. And McCoy advanced much of the early nitrogen-fixation bacteriology at UW. “[McCoy] just had an amazingly broad spread of information in all kinds of areas,” Burris recalls in an oral history. “She was a bacteriologist’s bacteriologist.” (For more about McCoy, see The Sweeping Landscape of Her Work in the spring 2020 issue of Grow and UW’s Elizabeth McCoy Was a Pioneer of 20th Century Microbiology.)

Fred went on to train Wilson, who continued UW’s line of nitrogen fixation research forward to today.

A Corn (and Sorghum) Collaboration

Out at the West Madison research fields, Natalia de Leon is in her second year supervising the breeding program with the nitrogen-fixing corn varieties from CIMMYT. Hints that cereal crops such as corn might be able to incorporate fixed nitrogen had swirled around her during her scientific career. But Ané’s confirmation that indigenous varieties exhibited robust fixation opened her eyes to new possibilities.

Vânia C. S. Pankievicz, an assistant scientist in the Department of Bacteriology, inspects nitrogen-fixing sorghum plants growing in a climate-controlled room at the Biotron on the UW–Madison campus. Photo by Michael P. King

“I was shocked when I saw the numbers showing the amount of nitrogen that they were able to incorporate when the right conditions were provided,” says de Leon. “If this is something that can be consistently done, it could have a big impact.”

That impact could be even greater. Since demonstrating nitrogen fixation in corn, Ané has since spotted it in sorghum as well. A specialty crop in North America, sorghum is a foundational grain in the tropical world.

“These sorghum accessions also get a large amount of nitrogen from the air by the same mechanisms; and so, at this point, it’s not unique to corn,” says Ané, who recently received a $5.8 million grant from the Department of Energy to study sorghum nitrogen fixation along with Ophelia Venturelli, professor of biochemistry; Sushmita Roy, professor of biostatistics and medical informatics in the UW School of Medicine and Public Health; and Wilfred Vermerris, professor of microbiology and cell science at the University of Florida.

For the corn project, de Leon worked with horticulture faculty associate Claudia Calderón MS’09, PhD’10, researcher Valentina Infante, professor of agronomy Shawn Kaeppler BS’87, and other breeders to help Ané select new lines to test and to develop the breeding program at West Madison. In these early stages, the researchers are linking physical characteristics such as aerial roots to nitrogen fixation so they can track the trait during crosses.

For de Leon, this collaboration has generated fresh ideas for her existing projects and provided new opportunities to pursue. Producing the gel that fixes nitrogen requires wet conditions, which gives the staff in de Leon’s lab a new trait to study as they focus on environmental responses in corn. For this project, the group is concentrating heavily on corn germplasm from outside North America. If the breeding program could imbue these lines with the nitrogen-fixation trait, it would have a tremendous benefit for farmers in developing countries, says de Leon.

But perhaps the defining feature is the collaboration itself. “It goes all the way from very foundational biology to the very practical, like how do we find varieties of corn that express these traits the best, and how can we deploy them to places where it’d have a real impact on farmers’ fields,” says de Leon.

That means bringing bacteriologists and agronomists together. “We’re each learning to speak a different language,” says de Leon. “That has made it so much more enjoyable and meaningful.”

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