Tag Archives: Bacteriology

Gut Dwellers

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Fermentation Fest

Fermentation Fest, sponsored by the Wormfarm Institute, is an annual celebration of “live culture” held in Sauk County. These scenes are from the 2017 fest, where Federico Rey gave a presentation on the human gut microbiome. Photos by Katrin Talbot MS’85

The second floor gallery of the Wormfarm Institute in Reedsburg, Wisconsin, is a far cry from the funky glassware and biosafety protocols of a working microbiology lab. One corner contains an improv kitchenette, circa 1987. Sprout — the mascot offspring of the Jolly Green Giant — waves improbably from behind a cubicle wall across the room; the child-size plastic statue is missing a hand. A cool draft of autumn rain and small-town traffic noise flows through an open window.

Assistant professor of bacteriology Federico Rey chats with his Wormfarm hosts as more than three dozen attendees of the annual Fermentation Fest assemble into a casual arc of folding chairs and a couple of vintage couches. These are people who already understand the idea of microbes as friends. Makers of coleslaw and kimchi, kombucha and beer — they are motivated by microbes and have paid to hear Rey’s summary of the state of current human microbiome research.

Across the life sciences, the microbiome is the buzziest of buzzwords, invoking a symphony of hope, hyperbole, and high expectations. Rey shares in the overall enthusiasm, but he is careful about the speculative details. Yes, the microbiome might even match our frothiest expectations. And no, he can’t cure your diabetes or make you leaner, faster, or smarter. He can’t even tell you if your microbiome is healthy. Not today.

Because nobody can.

In front of a large wall hanging of textile orange circles representing the bubbles of fermentation, he begins where he has to begin, very near the beginning. “Microbes are the most abundant form of life on this planet,” he says, his thick Argentinian accent backlit by a docent’s enthusiasm. “They can live in places where we cannot imagine life.”

“Microbes outnumber us by many orders of magnitude. They power almost everything on the earth,” he says. They convert as much carbon dioxide into organic compounds as plants do and emit more methane than the oil and gas industry. “Literally, there is no place on earth where there are no microbes,” he continues.”It is impossible to get rid of them.” And despite our germophobia, they’re mostly good company: “A very small fraction of microbes are pathogens. Most of them are commensal — they don’t do good or bad — or they are beneficial for humans.” These last ones, the microbes that fuel our fantasies of easy cures and everlasting health, truly capture Rey’s interest.

Every surface on our bodies is colonized by some kind of microbe, and microbiologists have identified thousands of species of bacteria that can inhabit the human gut. Each of us, in turn, has a collection of between 100 and 200 different bacteria strains, comingled with other life forms from fungi and protists to viruses and archaea. These enteric ecosystems — different for every single human, even more unique than a fingerprint — each contain 100 times the genetic information of our own cells. They both supplement and interact with our bodily blueprints.

Deciphering who’s who is not even half the problem. How exactly do our bodies gather these microbes? What shapes the resulting ecosystem? How do humans and microbes interact? In 2017 alone, Rey published new research about microbiome effects in diabetes, Alzheimer’s, and the cycling of the nutrient choline (which may positively affect fetal brain development but also can lead to heart disease later). “Every single disease or health condition scientists look at, they find a microbiome connection,” he says.

And yet there is no single definition of a healthy microbiota. And what is healthy for you may not be healthy for me.

Research Revolution

Federico Rey arrived in the United States in 1999 with advanced biological questions on his mind and little idea that microbes could hold the answer. As a research fellow at the Henry Ford Health Sciences Center in Detroit, he focused on hypertension and vascular disease. But when he moved to the University of Iowa for his Ph.D. in 2001, he met Rhodopseudomonas palustris during a lab rotation. These extravagantly versatile bacteria are known for their ability to use four different modes of metabolism to scavenge energy, nitrogen, and carbon from a variety of sources — with or without oxygen. Intrigued by the diversity and adaptability of microbes, Rey says he fell in love with the bacterium.

R. palustris was an obvious stepping-stone towards biofuels. But in 2005, Rey saw a talk by Jeff Gordon, a pioneer in the study of human-microbiome interactions. Gordon began examining the development of the mammalian gut in the 1980s. Eventually he realized that microbes were essential to the process, and he set out to untangle this complex relationship using early sequencing techniques and transgenic and germ-free mice.

Rey joined Gordon’s lab at Washington University in St. Louis as a postdoc in 2006 just as microbiome science was gaining momentum. Tools for reading the genetic code were getting faster, cheaper, and more versatile. Computers used to crunch burgeoning data sets were growing in power as new statistical methods were increasing in sophistication.

One of the earliest hints of the power of microbes was that transplanting the microbiome from one mouse to another could also transfer basic metabolic conditions, such as obesity. It was in Gordon’s lab that Rey first met CALS professor of biochemistry Alan Attie — through the microbes of his mice. In Attie’s efforts to unravel the many mysteries of diabetes, his lab sent Gordon microbiome samples from genetically distinct mice that had been placed on a high-fat or a chow (i.e., grain-based) diet. It was known that both diet and genetics had a significant effect on the metabolic health of these mice. Gordon helped to show that the microbes played a role as well.

Rey took the project with him when he was hired by CALS in 2013, and he’s been collaborating with Attie since. It’s a task of daunting complexity: integrate two genetically complex systems that play a role in metabolic disease. Neither is close to being perfectly understood, yet they interact with each other, coalescing in each individual.

Hundreds of mammalian genes are already understood as part of the metabolic pathways that go awry on the way to diabetes. The human microbiome, meanwhile, produces thousands of chemicals that act within the genetic framework of humans. We’ve long understood how these microbes help us break down the complex compounds in the plant-based foods we eat, providing about 10 percent more energy. More recently, we’ve learned how these microbial bioreactors produce molecules called short-chain fatty acids — acetic acid, propionic acid, butyrate, and other products of fermentation that signal our bodies in health-promoting ways.

Our bodies sense these molecules, helping regulate things like gastrointestinal motility. Less well understood are the thousands of chemicals that train our immune system and help regulate everything from kidney function to brain chemistry.

“This is one of the most important aspects of the microbiome that is revolutionizing biology,” Rey says. But unraveling the interplay between host genetics and metabolism is anything but easy.

Julia Kreznar inspects a germ-free mouse while performing cage maintenance. Sterile germ-free mouse facilities on the UW– Madison campus provide a controlled model in which to study the interactions of microbial communities within mammals and how they impact anatomy, physiology, behavior, and susceptibility to disease. Photo by Michael P. King

In the Rey lab, senior scientist Bob Kerby holds two sealed test tubes (left) containing a pure strain of bacterium from a human fecal sample. The cloudy sample has been incubated for 24 hours; the clear sample has just been prepared. The Petri dish holds the bacterial colony. Photos by Michael P. King

In work published in Cell Reports in 2017, Rey’s student Julia Kreznar used eight different mouse strains and microbial transplants to help unravel this tangled web. The study found measurable differences in the microbiome established in the different strains. These microbes, in turn, influenced the likelihood that the mice would succumb to metabolic disease. The work also demonstrated a novel link between the gut microbiome and insulin secretion.

“We can show that microbiota affected pancreatic islet physiology and function,” Kreznar says. It’s a promising step, but it’s also an indirect interaction. Identifying the mediators of these microbe-host interactions is really challenging.

“It’s the dawn of a new field,” Attie says. “We have 3 or 4 pounds of organisms that are producing so many molecules, and we don’t know the least of them.”

“I’m feeling daunted,” he admits. “We had this idea that we would potentially connect the dots genetically. It is enormously complex, and it’s hard. It’s harder than we even thought it was going to be.”

A Big Genetic Black Box

Back in Reedsburg, Rey knows the probiotic question is coming, so he makes a preemptive strike: “When I talk about microbiota, I’m not talking about probiotics. The probiotics that you can get at Walgreens are basically microbes from dairy, microbes that can live in milk. Microbes that are not adapted to live in our intestines.”

If the microbiome has taught him anything, it’s that generalizations are tricky. “If they work for you, you should continue,” he emphasizes, trying to claim middle ground. Because of course, probiotics are microbes, and there actually is a lot of evidence that some may provide immune benefits.

But you won’t get a better microbiota by eating probiotics, and if you don’t eat your yogurt on Saturday, by Sunday those yogurt microbes are pretty much gone. They just pass through. “The effect is good as long as you keep eating them. And that is the perfect model for a company, right?” he says, with a tone of innocent mischief.

But if you look at probiotics through the lens of the microbiota, you have to acknowledge this essential truth: It’s been a landmark decade, but we still don’t have tools that are sophisticated enough to measure the microbiota in sufficient detail. We still don’t have an adequate biological understanding of what makes a healthy microbiota. And we barely understand the complex dance these microbes do with our body. Add in the fact that probiotics are dietary supplements, and thus not well regulated.

“You have to be careful. You have to do your research,” Rey says. “There are many different strains of probiotics, and there are big differences between different strains.” This is further complicated by the difficulty of even properly identifying bacteria, which can evolve rapidly. “There is a big difference between George Clooney the actor and somebody named George Clooney who lives in Atlanta. They have the same name, but they are completely different people,” he explains. “There are thousands of strains of Lactobacillus rhamnosus. Some may have an effect. Many of them probably don’t.”

In fact, it is our ability to identify these subtle differences in microbes that sparked the current revolution in microbiome science. First came 16S ribosomal RNA. Essential to the construction of proteins, 16S changes very slowly, which allowed scientists to, finally, reliably identify the members in a microbial community.

We talk about DNA as the book of life. At first, just reading a few pages was a chore. Then we developed machines to read more pages, faster. Then we learned how to read the sequels: DNA makes RNA, which makes proteins and enzymes responsible for carrying out cellular processes, including the making and breaking of sugar for energy. The technology responsible for reading these interrelated genetic codes is called next-generation sequencing, and it has powered this first golden age of microbiome research. The challenge now is sifting through that data to find biological meaning. Postdoc Lindsay Traeger PhD’15 is one of Rey’s primary number crunchers. “I’m attracted to very broad questions that we can throw a data hammer at and see what falls out,” she says.

Right now Traeger is focused on the next stage of collaboration with Attie and several other UW–Madison faculty including Joshua Coon (biomolecular chemistry), Karl Broman (biostatistics and medical informatics), and Brian Yandell (statistics). “We know that the gut microbiome is influenced by diet,” Traeger says. “But there is also this genetic component, which is a black box.”

Using genetically distinct mouse strains is advantageous when you’re trying to model a particular disease. But if you’re trying to tease out broader biological principles, using a single strain of mice could lead to bias. That’s why the labs are using a special breed called Diversity Outbred, a strategic genetic mash-up of common lab strains and some wild strains.

In one hand, Traeger has the genetic code of each individual mouse. And in the other, she has the genetic code of the microbiome of each mouse. Using advanced statistics, she’s searching for patterns that suggest some molecular matchmaking.

A germ-free mouse in its sterile quarters on the UW–Madison campus. Photo by Michael P. King

“I’m trying to identify how the host is selecting for or deselecting for the presence and abundance of certain microbial functions. Because the microbes are interacting somehow with the host.” One gene of interest is responsible for creating the important immune protein TNF-alpha, which plays roles in cancer and autoimmune disease. Early returns suggest that the TNF gene is also involved in sensing and responding to bacteria that have flagella.

Of course, the TNF gene is only one of about 23,000 genes, while the genes associated with bacterial flagella are just a few out of potentially hundreds of thousands. With numbers that big, there’s a lot of noise to filter out. And lots of distractions. “It’s a little hard to focus,” she laughs. “I think I could just spiral off. [Rey] keeps me thinking about the biology.” Rey’s endless creative energy helps. “He’ll just bust into the office and say, ‘I have this idea!’”

The immensity of the black box also keeps the work exciting. “We do think we’re going to find some interesting examples of interaction of host and microbiome,” she says.

Two T-Bones a Day

The microbiome is a dynamic force. Change the diets of lab mice, and overnight the communities that live in their gut change dramatically. So why care what microbes are in your gut if you can switch it up that quickly?

Rey points to his classic Argentinian upbringing as an example. “I grew up eating a T-bone for lunch and a T-bone for dinner,” he says. “Twenty-five years. And I miss it very much,” he quips, evoking another laugh from his audience. “But if I became vegetarian long term, I would definitely select for different microbial communities. And I would likely have new microbes colonize me.”

But even while your gut community is adaptable, the microbes in your gut can also have long-term consequences. That T-bone? “There are components in meat that microbes love and that cause problems,” Rey warns. “But you might not have them.” Which could mean several things: You’ve never been exposed to them, or you’ve been exposed but they didn’t take. Or maybe they’re there, but other microbes keep them in check. All of those are open biological questions, which now makes nutrition even more complicated.

In 2011, Stanley Hazen of the Cleveland Clinic published a paper linking microbes to the breakdown of lipid phosphatidylcholine (lecithin) into several choline-related compounds, particularly TMAO (trimethylamine N-oxide), a chemical already found to be a strong predictor of heart disease risk. Specifically, microbes metabolized choline into trimethylamine (TMA), which is then converted in the liver to TMAO.

While diet is a big part of this risk — eggs, milk, liver, red meat, poultry, shellfish, and fish are major dietary sources — the combination of microbial and host biology leading to TMAO accumulation intrigued Kymberleigh Romano PhD’17, who decided to dig deeper for her doctoral work with Rey and was co-mentored by Daniel AmadorNoguez, an assistant professor in bacteriology with expertise in metabolomics. Despite years of lab experience, she’d never worked with lab animals before, but she knew the time had come. “A lot of the phenotypes we study exist only in the context of a host-microbe interaction,” she says. “A test tube is never going to develop heart disease.”

First she needed microbes. Harvard researcher Emily Balskus had identified the genes involved in microbial conversion of choline to TMA, and Romano began looking for them in human-associated microbes and constructing experimental mixes of microbes. As she tinkered she found that, in mice at least, you need a TMA producer present in the intestinal microbiota to see TMA accumulation. And as you add more TMA-producing species, less choline is left for the host.

Even though she’d narrowed down the difference to a single organism in her custom microbiota, it still wasn’t enough. One bacterium contains anywhere from 3,000 to 5,000 genes — that’s a lot of variables. Fortunately, her collaborator from Harvard had identified a genetically tractable choline consumer and knocked out the TMA production gene. “Now the only difference in my communities was a single gene.”

Cardiovascular risk aside, choline is an underappreciated nutrient contributing to the process of epigenetic regulation of gene expression, and those without enough of it are more likely to suffer metabolic disease. In mice and rats, there is even a two-day window during pregnancy where lack of choline can impact fetal brain development. “Biology is never simple,” says Romano. “If it’s simple, you’re missing something.”

Eat Your Vegetables

Talking about poop makes people laugh, and as Rey wraps up in Reedsburg, the crowd has stayed engaged, surviving even his brief foray into 16S sequencing.

His advice for microbial health is folksy and charming. “Spouses share more microbes than people that don’t live together,” Rey tells the crowd. “We have found that spouses who get along together share more microbes than spouses that don’t. There is a lot of exchange going on there.” (In fact, if you’ve had to take antibiotics, he suggests family time will do more to restore your microbes than probiotics.)

And, noting that the most diverse microbiomes are found in places like the Amazon, he says being exposed to dirt is probably a good thing. “Get your hands dirty working in your garden,” he says. “I think that’s a health habit that we have lost over the years.”

Still, stubborn ideas persist among those gathered in the room. About a dozen times people bring up their pet microbiome theories for validation: probiotics, kombucha, fermented food, raw food, red wine vinegar, minimal vegetable washing, fecal transplants.

“The microbiome has come to mean anything you want it to mean,” Rey says disarmingly, for another laugh.

But “I don’t know” is his most honest answer. It’s a conundrum: The microbiome is hot in part because of some stunning findings. Most remarkable is the use of fecal transplants to cure drug-resistant Clostridium difficile infections, with cure rates running above 90 percent in some studies.

That extraordinary outcome certainly got the attention of both the medical community and the fad diet community. And even as it validates the power of the microbiome, that outcome actually runs against the grain of all the variation Rey is trying to figure out. “My lab is very interested in understanding the consequences of our interpersonal differences,” he explains.

“I can sequence your microbes, and I would not be able to tell you what vegetables to eat,” he says. “Maybe in 5 or 10 years personalized nutrition will be a reality, but it is not today. The one recommendation we can give right now is try to think about feeding your microbes. Because we cannot tell you what will be the best for your microbiota.”

In other words, eat your vegetables. Let’s say you eat pizza, with regular flour dough and cheese. Your body can digest every single ingredient of that pizza. By the time it reaches your large intestine, where most of your microbes live, your body has absorbed everything of nutritional value.

“You’re not sharing any of your food with your microbes,” he explains. “That’s one of the things we are doing with our Westernized diets: we are starving our microbes.”

“Maybe broccoli is the best for your microbiota whereas cabbage is the best for my microbiota,” he concludes. “But in general, if you eat a diverse diet that contains plant polysaccharides, eventually you are going to help the good guys

Shaping the Future of Farming

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Thirty-five years ago, when CALS bacteriologist Winston Brill and his colleagues set out to exploit science’s newfound ability to manipulate genes to confer new traits on crop plants, the technology was, literally, a shot in the dark.

Working in a facility in Middleton, just west of Madison, Brill and his team blasted plant cells using a gene gun—a device that fired microscopic gold beads laden with DNA.

The idea was to introduce foreign genes that could confer new abilities on the plants that would ultimately be grown from the altered cells. First as Cetus of Madison, Inc., later as Agracetus and still later as a research and development outpost of Monsanto Company, the Middleton lab was, by all accounts, a hub of plant biotechnology innovation.

“Agracetus was the first in the world to engineer soybean, first in the world to engineer cotton, first in the world to field-test a genetically engineered plant,” recalls Brill, who was recruited by Cetus to establish the lab in the early 1980s. “Thus, the Madison area and the UW influence led to historically important events.”

In December 2016, the $10 million,100,000-square-foot facility—a warren of labs, greenhouses and growth chambers—was donated to UW–Madison by Monsanto to become the Wisconsin Crop Innovation Center (WCIC).

The hope, according to agronomy professor Shawn Kaeppler BS’87—now WCIC’s director—is that the center will add to its string of plant biotechnology achievements as one of just a few public facilities in the country dedicated to plant transformation, where genetically modified plant cells are taken from tissue culture and regenerated into large numbers of complete fertile plants.

The advent of the WCIC “is an unprecedented opportunity to add capabilities and capacity we couldn’t afford otherwise,” says Kaeppler, an expert on corn. Its acquisition by UW–Madison, he and others note, comes at an opportune time as powerful new techniques in synthetic biology are poised to make the development of plants with new or improved traits much more than a shot in the dark with a gene gun.

WCIC will function very much like a core facility, providing cell culture, phenotyping and plant transformation services for researchers at UW– Madison and other universities. It is also coming online at a time when the need for such resources is acute.

“There is a recognized need nationally,” explains agronomy professor Heidi Kaeppler BS’87, an expert in plant transformation who is serving as WCIC’s transformation technology director. “There are just a few public facilities around the U.S. and demand is outpacing the abilities of those facilities. It is a bottleneck.”

For researchers like bacteriology and agronomy professor Jean-Michel Ané, a member of the WCIC scientific advisory board, the new center means he will be able to devote more time to exploring such things as the genetic interplay that occurs when plants and bacteria collude to draw nutrients from the air through the act of nitrogen fixation.

Nitrogen-fixing plants such as soybean, alfalfa and clover are staples of modern agriculture. They are essential to the crop rotation practices that prevent exhaustion of soil from crops such as corn. Ané and many other scientists have long dreamed of engineering the ability to fix nitrogen into plants like corn to transcend the need for expensive and environmentally harmful chemical fertilizers.

However, engineering complex traits such as nitrogen fixation in plants that don’t have that innate ability is a monumental scientific and technological undertaking. To begin with, there are two organisms—the plant and a bacterium—working cooperatively. Each has its own genome, and many different genes from each organism are in play to accommodate the act of drawing life-sustaining nutrients from the air.

To confer that trait on corn, for example, is an exercise far more complicated than tinkering with one or a few genes, notes Ané. “The goal is to create maize that has this association. However, modifying a single gene will not be sufficient,” he says. “We modify many genes at a time. There is a lot of trial and error. We need to try many combinations.”

Those combinations come about in the lab as scientists alter individual plant cells by adding or subtracting genes of interest. Today, scientists can harness new techniques such as CRISPR– Cas9—a fast, cheap and accurate genome editing tool—and potent new cloning technologies that allow scientists to easily assemble multiple DNA fragments and their assorted genes into novel sequences.

Even with potent new tools like CRISPR–Cas9, engineering plants is a big, difficult task. A gene needs to be dropped in the right place on the genome and be in association with the right “promoters,” segments of DNA that initiate gene transcription, the first step toward expressing a new gene in an organism. Once plant cells are genetically altered, they must be transformed into large numbers of actual plants for further testing in the lab and, ultimately, the field. It is essential to know, for example, that the new genetic construct is stable, that the new genes are passed from generation to generation, and what effects they may have on plant growth or yield.

The promise of WCIC, Ané believes, will be the opportunity to work through all of those steps more efficiently and cost-effectively, and carry projects from the lab to the field much faster.

“We can focus on really doing science instead of growing plants,” Ané says. “We can now make genetic constructs very quickly. Within a month we can make hundreds of constructs. The limiting aspect is plant transformation. However, the scale of transformation we can do at WCIC allows us to think seriously about applying synthetic biology to plants.”

To begin with, WCIC is providing plant transformation services for corn, soybean and sorghum, big commercially important crop species. But Shawn Kaeppler envisions WCIC playing a role, as well, with crop plants that have not yet risen to the top of commercial research agendas.

To date, commercial interest has focused primarily on just a handful of traits—insect and herbicide resistance—in a handful of widely planted crops. Uncharted territory, Kaeppler says, exists in the full range of crop plants and their many different traits.

A ready example is switchgrass, a native perennial that is under the microscope at the Great Lakes Bioenergy Research Center (GLBRC), a U.S. Department of Energy- funded multi-institutional research center headquartered on the UW–Madison campus. The grass is seen as a potential feedstock for converting its biomass to liquid fuel. However, efficient conversion of plant materials to energy remains a challenge, and plant genetics will play a big role in refining the traits that will make that possible.

“WCIC will help lead us to the next generation of crop breeding and plant genetics,” explains Kate VandenBosch, the dean of CALS, referencing, broadly, the genetic makeup of the crop plants in play. “Scientific agencies at the federal level have invested a lot in understanding genomes, but we still have a lot of work to do to understand how those genes function.”

Indeed, genetic sequencing technologies have advanced to the point where new plant genomes are sequenced with increasing regularity. The genomes of crop plants like watermelon, cucumber, potato, soybean, wheat, corn and many others have been sequenced, but as VandenBosch notes, exploring those sequences to identify the genes that govern plant traits is an unexplored frontier.

Shawn Kaeppler’s own research, for example, is a window to both the complexity and opportunity that lurk in the genomes of plants. One of his interests is the complex of genes—involving anywhere from tens to hundreds of genes—that governs the root architecture of corn. Knowing more about the combination of genes that directs the plant to send shoots into the soil, it might one day be possible to engineer a plant that can send its roots deeper into the earth, providing farmers with a hedge against drought.

“Fifty to 70 percent of all maize genes are expressed in roots,” Kaeppler says. “Some control processes in all parts of a plant, and some specifically control root development and response to environmental stimuli.”

A gene of interest for Kaeppler and his team is one that influences root angle. “Altering root angle even five to 10 degrees can dramatically increase the rate that roots get deep in the soil,” as well as how much root biomass a plant lays down at depth, he explains.

Identifying those candidate genes and mutations of those genes means they can be selected and manipulated in the laboratory to generate plants with different root structures. At WCIC, those plants can be grown in quantity, their new qualities studied and, if promising, tested in the field. The goal, of course, is to provide a practical outcome that is useful to growers.

In plant science, numbers matter. The more plants you can grow to test a new genetic combination, the better, as there are so many variables in play.

“In many aspects of science, doing things on a large scale is critical,” says biochemistry professor Rick Amasino, an expert on flowering in plants. “To have WCIC in our capability is great. Large-scale transformation opens up a lot of possibilities.”

Amasino, who is also a member of WCIC’s scientific advisory board, views the center as an important new national resource. Individual labs, he explains, do not have the same capacity.

“This has the potential to be on a scale greater than any other university’s,” Amasino says. “Individual labs can’t generate the hundreds or thousands of transgenic plants needed to fully test certain hypotheses. Labs around the country and, hopefully, around the world can now do experiments they couldn’t otherwise do. There are so many opportunities out there.”

A Facility With Deep CALS Roots

The name is new, but the Wisconsin Crop Innovation Center (WCIC) holds a prominent place in the young history of agricultural biotechnology. The facility also has long and deep ties to CALS researchers and alumni.

Originally known as Cetus of Madison, Inc., the Middleton facility—owned by
the Cetus Corporation of Emeryville, California—opened in 1981 under the direction of CALS bacteriology professor Winston Brill. The Wisconsin Alumni Research Foundation (WARF) played a key funding role in the early days of the company.

Cetus of Madison, Inc. initially focused on evaluating and testing a wide variety of natural rhizobia species to better understand their role in nitrogen fixation and nodulation in legumes, with the hope of someday enabling maize to have that capacity.

As interest in biotechnology grew in the early 1980s, the facility’s focus changed to inventing and innovating ways to introduce genes into plants. In 1984, Cetus Corp. sold half of its interest in Cetus of Madison, Inc. to the WR Grace Co.—and thus the company name “Agracetus” was born.

Great discoveries followed. An electric “gene gun” and transformation methods developed at Agracetus revolutionized the plant transformation process. Many plant species were subsequently transformed, including tobacco, peanut, sunflower, soybean, maize, cotton, cranberry, canola, poplar, wheat and rice. CALS researchers Kenneth Raffa, Brent McCown PhD’69 and Elden Stang, as well as WCIC associate director Michael Petersen BS’87 (then still an undergraduate) and Richard Heinzen MS’74, collaborated with Agracetus scientists during that period. But that wasn’t the only significant research taking place. Other studies critical to agricultural improvement focused on cotton fiber quality, transformation process improvements, polymerase chain reaction (PCR) method development, insect and disease resistance and herbicide tolerance. A number of CALS faculty, including Michael Sussman, Richard Amasino and Andrew Bent, were highly involved in consulting with Agracetus in many of these areas.

In 1990, WR Grace Co. acquired full ownership of Agracetus. During the early 1990s, Agracetus ventured into research in DNA vaccines—using an improved “gene gun”—and contracted plant transformation services to others within the industry, including, most notably, the Monsanto Company. Collaborating with biological systems engineering professor Richard Straub PhD’80 (now CALS senior associate dean) and other CALS researchers, the company also worked on producing industrial enzymes in plants.

After successfully generating plants that eventually became commercial products
for Monsanto, including Roundup Ready Soybeans and Bollgard Cotton, the facility was acquired by Monsanto in 1996.

Over the next 20 years, Monsanto used the facility as its primary site for soybean and cotton transformation. Other R&D at the site included corn, canola, wheat, rice and alfalfa transformation, gene expression, molecular testing and seed chipping/genotyping.

The site was considered a “center of excellence” for Monsanto due to its highly innovative employees, high throughput transformation capabilities and ability to consistently perform above and beyond expectations.

In July of 2016, Monsanto relocated a number of remote functions back to its St. Louis headquarters in the interest of business consolidation. In the hope that the Middleton facility would continue to work toward the betterment of agriculture, Monsanto the following December donated it to longtime collaborator the University of Wisconsin– Madison, along with University Research Park.

Not surprisingly, given the long history of CALS involvement, agronomy professor Shawn Kaeppler BS’87 was chosen to serve as facility director.

Safer Native Foods

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At the edge of a remote Alaskan peninsula, 30 miles north of the Arctic Circle, lies the city of Kotzebue. Snow-covered in winter and starless for weeks in sum- mer, Kotzebue is home to roughly 3,300 people, most of whom are native Iñupiat Eskimos.

People there consume a diet rich in animals found in the region, including caribou, seal and whale. Following Native tradition, foods often are fermented or consumed raw.

But they sometimes are contaminated with one of the most poisonous known toxins: botulinum toxin, produced by a bacterium called Clostridium botuli- num. In fact, Alaska has one of the highest rates of food-borne botulism in the U.S., most likely because of those traditional foods. Botulism can cause paraly- sis, respiratory failure and death, so traditional foods are not allowed to be served in state-run facilities like nursing homes.

A group called the Seal Oil Task Force, comprising Native organizations like the Maniilaq Association along with state government partners, has formed to try to change that. They want Native elders to continue enjoying foods they have known their whole lives.

Which is how CALS bacteriology professor Eric Johnson, one of the world’s foremost experts on Clostridium, came to find himself on a boat in Kotzebue last summer, traveling to a Native process- ing facility where seal oil is produced.

Seal oil is to many Alaska Natives what soy sauce is to some Asian cultures: a staple of their diets, Johnson explains. It is also especially prone to botulinum contamination. The task force contacted Johnson in 2015 to see if he could help.

“Many of the foods they absolutely cherish can result in botulism,” Johnson says. “They want to inte- grate food safety into traditional Native foods.”

The catch is that any new processing methods can- not alter the final product or significantly stray from traditional production. For instance, heating the oil would kill the bacteria, but it also changes the taste.

Johnson is working with the task force to deter- mine how the bacteria are contaminating traditional food products. This has involved rendering seal oil back in his campus lab, testing for toxin as the blubber stripped from hunted seals emulsifies at ambient tem- perature into the nutrient-rich, yellow-hued delicacy.

In Kotzebue, seal oil is produced by cutting fresh blubber into pieces, placing it in a covered vat, and stirring—twice a day—until the fat eventually gives way to oil.

Johnson has a theory that Clostridium, found naturally in soil, may colonize minuscule pockets of water present in the fat as it breaks down. He wants to develop a method to prevent the bacteria from contaminating the oil, or a method to neutralize the toxin.

In the process, Johnson is learning more about Alaska Native culture and believes his work could have even greater reach. “It could have an impact on cultures elsewhere,” Johnson says.

Partnering for safety: Bacteriologist Eric Johnson (right) chatting with a colleague in Kotzebue.
Photo credit: Eric Johnson

Gut Reactions

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GARRET SUEN, an assistant professor in the Department of Bacteriology and an Alfred Toepfer Faculty Fellow, focuses on microbiomes and how microbes convert biomass into nutrients. “Microbiome” has become a more common word in the public consciousness in recent years. While the definition of microbiome remains somewhat up for debate, Suen defines it as the totality of the microbes that make up a community living within a particular environment— whether that’s an ocean, the tip of a pinky finger or—in Suen’s case—a cow rumen.

Through his studies of the microbiome of the cow rumen, Suen is working to understand the evolution and ecology of microbial communities and how those communities change in response to the host, the animal’s diet and other influences. He wants to use the microbes and their activities to improve the health of the animals, benefit farmers and even produce biofuels. Suen’s research has also led him to the microbiomes of other herbivores, including sloths and pandas.

Why are you looking at the microbiome of the cow rumen?

I’m very interested in helping Wisconsin farmers improve milk production in their cows. I’m trying to understand the interaction between the host cow and the microbes it has inside its rumen, and I want to know how we might go about altering that interaction so that we can improve milk production efficiency. There are a lot of farms in Wisconsin with small herds—100 or 200 head. Especially for these farmers, milk production efficiency is really important.

What role do microbes play in milk production?

Well, cows are strict herbivores. They only eat plant biomass, and without microbes they would not be able to digest that biomass. The microbes break down the plant polysaccharides found in the plant cell wall—things like cellulose—and they convert that into simple sugars like glucose, which is then fermented into fatty acids that the animal uses as its source of energy. It is those fatty acids that are also the building blocks of milk fat. So if we can better understand that process and which microbes do it best, we can improve milk production and make the animals more efficient in how they use the biomass they consume.

Why is understanding the relationship between microbes and milk production important?

Beyond the benefits to cows and farmers, making milk production more efficient will help feed the expanding population. It’s a better option than increasing the number of farms and land usage. Also, if we can use microbes to change milk composition, we could help cows produce milk with different fats or sugars. Studies have shown that human breast milk is healthier for babies in terms of promoting immune development, and we know that the types of sugars found in human milk are different from those in cow’s milk. So can we learn from that? Could we find ways to use microbes to make cow’s milk more like human breast milk? Changing milk composition could also affect the quality of downstream products such as yogurts and cheese.

How does your work with cow microbiomes relate to biofuel production?

Let’s take corn as an example of a crop we can use to make biofuels. The corn kernel is just one small part of the plant. The rest of the plant, called stover, is usually either silaged or burned. But there’s a lot of carbon in the stover that’s being wasted. So we want to know if we could take that carbon, break it down into simple sugars and have microbes ferment them into new fuels like ethanol. Cows are highly optimized to do that first part because we domesticated cows. We pushed cows to be as efficient as possible to produce as much milk as possible, and optimized the microbes at the same time.

So we’re very interested in taking some of the individual microbes from the cow rumen, bringing them into the lab and seeing what types of products they can produce. One of the microbes we study actually produces ethanol directly from cellulose. We view the rumen as a place where we might be able to identify novel enzymes that could be part of a larger industrial production facility producing next generation biofuels. We’re learning from nature, as I like to call it.

Another animal you study is the panda. Why are you interested in the gut microbiomes of panda bears?

In captivity, giant pandas get very painful episodes, called mucoidal episodes, during which they produce abnormal poop known as mucoids. Normally panda poop looks like chewed bamboo. Their system is inefficient at extracting energy from the food that they’re consuming, so bamboo moves very quickly through the gastrointestinal tract. But once or twice a year, they stop eating completely and produce these mucoids, poop that looks like their gut lining—the gooey, mucosal layer of the gastrointestinal tract.

But why would pandas shed their gastrointestinal tract lining? To answer that question, we worked with Ashli Brown Johnson, an associate professor at Mississippi State University, to look at the microbiota in the mucoids and compare them to regular poop of two giant pandas at the Memphis Zoo. We found that they’re very different from each other. So we came up with the hypothesis that maybe what’s happening is that pandas are eating these rough pieces of bamboo, which are actually causing physical abrasions to the gastrointestinal tract. The pandas then have an inflammatory response to the abrasions that results in the sloughing off of the internal gastrointestinal tract layer, producing mucoids.

Why is helping these pandas important?

The key thing is that these mucoidal episodes usually coincide with the gestation period of a panda. If the pandas are trying to get pregnant but not eating, how hard will it be to get pregnant? How hard will it be to carry a fetus to term—especially when you should be eating more to support the developing fetus? We don’t know why these episodes coincide with gestation, but anything to help pandas breed is important. Successful breeding of pandas is difficult and a big problem.

Are you studying other animals with interesting gut microbiomes?

We’re working with Hannah Carrey in the School of Veterinary Medicine to study what happens to microbes in ground squirrels during hibernation. When animals prepare to hibernate, they pack on weight, and while hibernating, they drop their internal core body temperature to around the temperature inside your refrigerator. We’d like to know what’s happening in that system. Understanding the activity of the microbiomes before and during hibernation can give us insight into host metabolism and diseases such as diabetes and obesity.

We also recently published a paper on sloths, which are on the complete opposite end of the spectrum from pandas. Pandas are eating all the time and are inefficient at getting energy from their food. Sloths eat much less than what you would predict for their body size. Physiologically it makes sense because they have much fewer energetic needs, but the three-toed sloth poops only once a week. That made me wonder what is going on from a digestive perspective! What we’ve found in sloths is completely different from anything we’ve seen in terms of microbial composition, so we want to figure out what’s so different about them. Animals that eat too much or too little for their body size are very interesting in terms of their gut microbiomes.

Garret Suen using an anaerobic chamber to study ruminal bacteria.
Photo by Matt Wisniewski/UW–Madison WEI

Antibiotics Off the Beaten Path

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As more antibiotic-resistant “superbugs” emerge, it’s clear that we desperately need new antimicrobial drugs. Yet, over the past couple of decades, antibiotic discovery has largely been stagnant.

“The reality is there’s almost no new antibiotics that are developed. And that’s because pharmaceutical companies have decreased their investment—in part because of the rediscovery issue,” explains bacteriology professor Cameron Currie.

The “rediscovery issue” refers to the fact that soil has historically been the prime source of new antibiotics—but it seems to be tapped out. When scientists screen soil microbes for new antibiotics, they keep finding the same compounds over and over again.

Currie is part of a team that is looking elsewhere.

Currie and his colleagues have been focusing their efforts on microbes that are associated with insects, plants and marine life from all around the United States, funded by a $16 million grant from the National Institutes of Health that was awarded in 2014.

“One of the major hurdles is finding new compounds, and that’s where we’re really excelling,” says Currie, a co-principal investigator on the grant. His partner is David Andes in the UW–Madison School of Medicine and Public Health.

At the front end, the work involves some good old-fashioned bioprospecting. Currie’s group, which is in charge of the terrestrial sphere, has gathered more than 2,000 flies, aphids, caterpillars, bees, ants and other insects, as well as mushrooms and plants, from locales near and far, including Alaska, Hawaii and Wisconsin’s Devil’s Lake.

Back at the lab, things get high-tech pretty quickly. Microbes are isolated from the samples and tested for antimicrobial activity. Promising strains undergo genetic sequencing that allows Currie’s group to determine how likely they are to produce novel antibiotic compounds. From there, other scientists involved in the grant go on to test the most promising compounds in a mouse model of infection. This approach has already yielded some exciting drug candidates.

“We have 9,000 strains to screen, and we have already found some new compounds that are effective at combating infections in mice and have low toxicity,” says Currie.

With so many samples to process, Currie’s group adopted bar code technology to help them keep track. They have a bar code reader—like you’d find in a grocery store— connected to a lab computer that they use to scan petri dishes, look up samples and add new data. For each microbial strain they’ve isolated, the database has photos of the “host” insect or plant, GPS coordinates for the collection site, assay results, genetic sequence and much more.

At this point, Currie feels confident that the project will pay off, and he’s eager to see one of the group’s compounds go into human clinical trials.

“If you find one new antibiotic that gets used in treatment, it’s a major success. You’re saving people’s lives,” Currie says.

To Market, to Market

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If you’re familiar with the College of Agricultural and Life Sciences (CALS), you no doubt know all about Stephen Babcock and his test that more than 100 years ago revolutionized the dairy industry by providing an inexpensive, easy way to determine the fat content of milk (thus preventing dishonest farmers from watering it down). What you might not know is that his great discovery went unpatented. The only money Babcock received for his invention was $5,000 as part of a Capper Award—given for distinguished service to agriculture—in 1930.

Just years before Babcock received that award, another entrepreneur was hard at work in his lab—and his discovery would break ground not only in science, but also in direct remuneration for the university.

In 1923, Harry Steenbock discovered that irradiating food increased its vitamin D content, thus treating rickets, a disease caused by vitamin D deficiency. After using $300 of his own money to patent his irradiation technique, Steenbock recognized the value of such patents to the university. He became influential in the formation in 1925 of the Wisconsin Alumni Research Foundation (WARF), a technology transfer office that patents UW–Madison innovations and returns the proceeds back to the university.

Discoveries have continued flowing from CALS, and WARF plays a vital role for researchers wanting to patent and license their ideas. But today’s innovators and entrepreneurs have some added help: a new program called Discovery to Product, or D2P for short.

Established in 2013, and co-funded by UW–Madison and WARF, D2P has two main goals: to bring ideas to market through the formation of startup companies, and to serve as an on-campus portal for entrepreneurs looking for help. Together, WARF and D2P form a solid support for researchers looking to move their ideas to market. That was the intent of then-UW provost Paul DeLuca and WARF managing director Carl Gulbrandsen in conceiving of the program.

“The idea of D2P is to make available a set of skills and expertise that was previously unavailable to coach people with entrepreneurial interests,” explains Leigh Cagan, WARF’s chief technology commercialization officer and a D2P board member. “There needed to be a function like that inside the university, and it would be hard for WARF to do that from the outside as a separate entity, which it is.”

D2P gained steam after its initial conception under former UW–Madison chancellor David Ward, and the arrival of Rebecca Blank as chancellor sealed the deal.

“Chancellor Blank, former secretary of the U.S. Department of Commerce, was interested in business and entrepreneurship. D2P really started to move forward when she was hired,” says Mark Cook, a CALS professor of animal sciences. Cook, who holds more than 40 patented technologies, launched the D2P plan and served as interim D2P director and board chair.

With the light green and operational funds from WARF and the University secured, D2P was on its way. But for the program to delve into one of its goals— helping entrepreneurs bring their ideas to market—additional funding was needed.

For that money, Cook and DeLuca put together a proposal for an economic development grant from the University of Wisconsin System. They were awarded $2.4 million, and the Igniter Fund was born. Because the grant was good only for two years, the search for projects to support with the new funds started right away.

By mid-2014, veteran entrepreneur John Biondi was on board as director, project proposals were coming in and D2P was in business. To date, 25 projects have gone through the Igniter program, which provides funding and guidance for projects at what Biondi calls the technical proof of concept stage. Much of the guidance comes from mentors-in-residence, experienced entrepreneurs that walk new innovators down the path to commercialization.

“For Igniter projects, they need to demonstrate that their innovation works, that they’re not just at an early idea stage,” explains Biondi. “Our commitment to those projects is to stay with them from initial engagement until one of three things happen: they become a startup company; they get licensed or we hand them over to WARF for licensing; or we determine this project might not be commercial after all.”

For projects that may not be destined for startup or that need some additional development before going to market, the collaboration between WARF and D2P becomes invaluable. WARF can patent and license discoveries that may not be a good fit for a startup company. They also provide money, called Accelerator funding, for projects that need some more proof of concept. Innovations that may not be ready for Igniter funds, but that are of potential interest to WARF, can apply for these funds to help them move through the earlier stages toward market.

“Some projects receive both Accelerator and Igniter funding,” says Cagan. “Some get funding from one and not the other. But we work together closely and the programs are being administered with a similar set of goals. We’re delighted by anything that helps grow entrepreneurial skills, companies and employment in this area.”

With support and funding from both WARF and D2P, entrepreneurship on campus is flourishing. While the first batch of Igniter funding has been allocated, Biondi is currently working to secure more funds for the future. In the meantime, he and others involved in the program make it clear that the other aspect of D2P—its mission to become a portal and resource for entrepreneurs on campus—is going strong.

“We want to be the go-to place where entrepreneurs come to ask questions on campus, the starting point for their quest down the entrepreneurial path,” says Biondi.

It’s a tall order, but it’s a goal that all those associated with D2P feel strongly about. Brian Fox, professor and chair of biochemistry at CALS and a D2P advisory board member, echoes Biondi’s thoughts.

“D2P was created to fill an important role on campus,” Fox says. “That is to serve as a hub, a knowledge base for all the types of entrepreneurship that might occur on campus and to provide expertise to help people think about moving from the lab to the market. That’s a key value of D2P.”

Over the past two years, D2P, in collaboration with WARF, has served as precisely that for the 25 Igniter projects and numerous other entrepreneurs looking for help, expertise and inspiration on their paths from innovation to market. The stories of these four CALS researchers serve to illustrate the program’s value.

Reducing Antibiotics in Food Animals

Animal sciences professor Mark Cook, in addition to helping establish D2P, has a long record of innovation and entrepreneurship. His latest endeavor, a product that has the potential to do away with antibiotics in animals used for food, could have huge implications for the animal industry. And as he explains it, the entire innovation was unintentional.

“It was kind of a mistake,” he says with a laugh. “We were trying to make an antibody”—a protein used by the immune system to neutralize pathogens—“that would cause gut inflammation in chickens and be a model for Crohn’s disease or inflammatory bowel disease.”

To do this, Cook’s team vaccinated hens so they would produce a particular antibody that could then be sprayed on feed of other chickens. That antibody is supposed to cause inflammation in the chickens that eat the food. The researchers’ model didn’t appear to work. Maybe they had to spark inflammation, give it a little push, they thought. So they infected the birds with a common protozoan disease called coccidia.

“Jordan Sand, who was doing this work, came to me with the results of that experiment and again said, ‘It didn’t work,’” explains Cook. “When I looked at the data, I saw it was just the opposite of what we expected. The antibody had protected the animals against coccidia, the main reason we feed antibiotics to poultry. We knew right away this was big.”

The possibilities of such an innovation—an antibiotic-free method for controlling disease—are huge as consumers demand antibiotic-free food and companies look for ways to accommodate those demands. With that potential in hand, things moved quickly for Cook and Sand. They filed patents through WARF, collaborated with faculty colleagues and conducted experiments to test other animals and determine the best treatment methods. More research was funded through the WARF Accelerator program, and it became clear that this technology could provide the basis for a startup company.

While Cook didn’t receive funds from D2P to bring the product to market, he and Sand used D2P’s consulting services throughout their work—and continue to do so. Between WARF funding and help from D2P, Cook says starting the current company, Ab E Discovery, has been dramatically different from his previous startup experiences.

“D2P is a game changer,” says Cook. “In other cases, there was no structure on campus to help. When you had a technology that wasn’t going to be licensed, you had to figure out where to get the money to start a company. There were no resources available, so you did what you could, through trial and error, and hoped. Now with WARF and D2P working together, there’s both technical de-risking and market de-risking.”

The combination of WARF and D2P has certainly paid off for Cook and Sand. They have a team and a CEO, and are now producing product. Interest in the product is immense, Cook says. He’d like to see the company grow and expand—and stay in Wisconsin.

“It’s been a dream of mine to make Wisconsin a centerpiece in this technology,” Cook says. “I’d like to see the structure strong here in Wisconsin, so that even when it’s taken over, it’ll be a Wisconsin company. That’s my hope.”

Better Corn for Biofuel

Corn is a common sight in Wisconsin and the upper Midwest, but it’s actually more of a tropical species. As the growing regions for corn move farther north, a corn hybrid has to flower and mature more quickly to produce crop within a shorter growing season. That flowering time is determined by the genetics of the corn hybrid.

Conversely, delayed flowering is beneficial for other uses of corn. For example, when flowering is delayed, corn can produce more biomass instead of food, and that biomass can then be used as raw material to make biofuel.

The genetics of different hybrids controls their flowering time and, therefore, how useful they are for given purposes or growing regions. Shawn Kaeppler, a professor of agronomy, is working to better understand those genes and how various hybrids can best fit a desired function. Much of his work is done in collaboration with fellow agronomy professor Natalia de Leon.

“We look across different populations and cross plants to produce progeny with different flowering times,” Kaeppler explains. “Then we use genetic mapping strategies to understand which genes are important for those traits.”

Throughout his work with plant genetics, Kaeppler has taken full advantage of resources for entreprenuers on campus. He has patents filed or pending, and he has also received Accelerator funds through WARF. For his project looking at the genetics behind flowering time, Kaeppler and graduate student Brett Burdo received Igniter funds from D2P as well. The Igniter program has proven invaluable for Kaeppler and Burdo as they try to place their innovation in the best position for success.

“I found the Igniter program very useful, to go through the process of understanding what it takes to get a product to market,” says Kaeppler. “It also includes funding for some of the steps in the research and for some of the time that’s spent. I can’t fund my graduate student off a federal grant to participate in something like this, so the Igniter funding allowed for correct portioning of funding.”

The end goal of Kaeppler’s project is to develop a transgenic plant as a research model and license the technology, not develop a startup company. His team is currently testing transgenic plants to work up a full package of information that interested companies would use to decide if they should license the technology. For Kaeppler, licensing is the best option since they can avoid trying to compete with big agricultural companies, and the technology will still get out to the market where it’s needed to create change.

“In this area of technology transfer, it is important not only to bring resources back to UW but also to participate in meeting the challenges the world is facing with increasing populations,” says Kaeppler. “Programs like D2P and WARF are critical at this point in time to see the potential of these discoveries realized.”

A Diet to Treat Disease

Around the world, about 60,000 people are estimated to have phenylketonuria, or PKU. Those with the inherited disorder are unable to process phenylalanine, a compound found in most foods. Treatment used to consist of a limited diet difficult to stomach. Then, about 13 years ago, nutritional sciences professor Denise Ney was approached to help improve that course of treatment.

Dietitians at UW–Madison’s Waisman Center wanted someone to research use of a protein isolated from cheese whey—called glycomacropeptide, or GMP—as a dietary option for people living with PKU. Ney took on the challenge, and with the help of a multidisciplinary team, a new diet composition for PKU patients was patented and licensed.

“Mine is not a typical story,” says Ney, who also serves as a D2P advisory board member. “Things happened quickly and I can’t tell you why, other than hard work, a good idea and the right group of people. We’ve had help from many people—including our statistician Murray Clayton, a professor of plant pathology and statistics, and the Center for Dairy Research—which helped with development of the foods and with sensory analysis.”

Being at the right place at the right time had a lot to do with her success thus far, Ney notes. “I’m not sure this could have happened many places in the world other than on this campus because we have all the needed components—the Waisman Center for care of patients with PKU, the Wisconsin Center for Dairy Research, the clinical research unit at University of Wisconsin Hospitals and Clinics, and faculty with expertise in nutritional sciences and food science,” she says.

Ney is currently wrapping up a major clinical trial of the food formulations, referred to as GMP medical foods, that she and her team developed. In addition to those efforts, the new diet has also shown surprising promise in two other, seemingly unrelated, areas: weight loss and osteoporosis prevention.

“My hypothesis, which has been borne out with the research, is that GMP will improve bone strength and help prevent fractures, which are complications of PKU,” explains Ney. “I have a comprehensive study where I do analysis of bone structure and biomechanical performance, and I also get information about body fat. I observed that all of the mice that were fed GMP, whether they had PKU or not, had less body fat and the bones were bigger and stronger.” Interestingly, the response was greater in female compared with male mice.

To support further research on this new aspect of the project, Ney received Accelerator funds from WARF for a second patent issued in 2015 titled “Use of GMP to Improve Women’s Health.” Ney and her team, including nutritional sciences professor Eric Yen, are excited about the possibilities of food products made with GMP that may help combat obesity and also promote bone health in women.

“There is a huge market for such products,” says Ney. “We go from a considerably small group of PKU patients who can benefit from this to a huge market of women if this pans out. It’s interesting, because I think I’m kind of an unexpected success, an illustration of the untapped potential we have here on campus.”

Fewer Antibiotics in Ethanol Plants

Bacteria and the antibiotics used to kill them can cause significant problems in everything from food sources to biofuel. In biofuel production plants, bacteria that produce lactic acid compete with the wanted microbes producing ethanol. At low levels, these bacteria decrease ethanol production. At high levels, they can produce so much lactic acid that it stops fermentation and ethanol production altogether.

The most obvious solution for stopping these lactic acid bacteria would be antibiotics. But as in other industries, antibiotics can cause problems. First, they can be expensive for ethanol producers to purchase and add to their workflow. The second issue is even more problematic.

“A by-product of the ethanol industry is feed,” explains James Steele, a professor of food science. “Most of the corn kernel goes toward ethanol and what remains goes to feed. And it’s excellent animal feed.”

But if antibiotics are introduced into the ethanol plant, that animal feed byproduct can’t truly be called antibioticfree. That’s a problem as more and more consumers demand antibiotic-free food sources. But Steele and his colleagues have a solution—a way to block the negative effects of lactic acid bacteria without adding antibiotics.

“We’ve taken the bacteria that produce lactic acid and re-engineered it to produce ethanol,” says Steele. “These new bacteria, then, compete with the lactic acid bacteria and increase ethanol production. Ethanol plants can avoid the use of antibiotics, eliminating that cost and increasing the value of their animal feed by-product.”

The bacteria that Steele and his team have genetically engineered can play an enormous role in reducing antibiotic use. But that benefit of their innovation didn’t immediately become their selling point. Rather, their marketing message was developed through help from D2P and the Igniter program.

“Learning through D2P completely changed how we position our product and how we interact with the industry,” says Steele. And through that work with D2P, Steele plans to later this year incorporate a company called Lactic Solutions. “D2P has helped us with the finance, the organization, the science, everything. Every aspect of starting a business has been dealt with.”

Steele and his collaborators are now working to refine their innovation and ideas for commercialization using Accelerator funds from WARF. Steele’s work, supported by both WARF and D2P, is a perfect example of how the entities are working together to successfully bring lab work to the market.

“There is no doubt in my mind that we would not be where we are today without D2P,” says Steele. “On top of that you add WARF, and the two together is what really makes it so special. There’s nothing else like it at other campuses.”

With such a strong partnership campaigning for and supporting entrepreneurship at UW–Madison, CALS’ strong history of innovation is poised to endure far into the future, continuing to bring innovations from campus to the world. And that is the embodiment of the Wisconsin Idea.

 

To Eat It—Or Not

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Food engineer Sundaram Gunasekaran, a professor of biological systems engineering, works with gold. But you won’t find the shiny yellow stuff in his lab; instead, the vials on his bench are mostly purple and red. Gunasekaran works with tiny pieces of gold—nanoparticles—that come in almost every color except gold. And those colors can tell a story.

Gunasekaran’s research focuses on food safety concerns, such as whether a food product was transported and stored properly or whether it has become contaminated. But how can a producer or consumer easily know a product’s history and whether it is
safe to eat? That’s where gold nanoparticles come in handy.

“The color of gold nanoparticles will change depending on the size and shape of the particle,” explains Gunasekaran. “At different temperatures, depending on how long you let the particles grow, they acquire different sizes and shapes. And that changes their colors.”

Gunasekaran’s lab is using those color changes for a difficult task—tracking the time and temperature history of a food product as it is packaged, transported and stored. Up to now similar sensors have given consumers some of this information, but they can miss such critical events as, for example, a short temperature spike that could be enough to kick-start the growth of a dangerous microorganism.

The sensors that Gunasekaran and his team are developing provide a more complete and accurate story. The new sensor can differentiate between food stored at high temperatures first and low temperatures second versus a product stored first at low temperatures and then at high temperatures. And that’s thanks to the properties of the gold nano-particles. The color of the first sample would be different than that of the second because of how and when the particles changed size and shape.

“We’re able to do this because the nanoparticle synthesis is affected by how the particles grow initially versus later,” explains Gunasekaran. “We call this the thermal history indicator, or THI.”

These gold nanoparticle sensors are being patented through the Wisconsin Alumni Research Foundation (WARF), and students in Gunasekaran’s lab won a UW–Madison Discovery to Product award. The student team also won a People’s Choice
award in the 2014 Agricultural Innovation Prize competition.

They are now working to further develop and optimize the system. Since different food products travel through different channels, some sensors will be best used to track long-distance travel over the course of a month, while others will track history for only a matter of hours. Some sensors will work best in frozen storage and others will be optimized for various room temperatures.

The goal of optimization is a simple-to-use biosensor customized for any given food product. Gunasekaran envisions the sensors—now being developed as self-adhesive dots or stickers—being used anywhere along the food production channel. Producers, packagers, transporters and even consumers could easily use the biosensors to understand the thermal history of their product, saving time and money and avoiding recalls and health issues.

“There are a number of ways to use this technology, and making a food product’s history easy to see is the key,” says Gunasekaran. “Food is being wasted because people are throwing it out according to an expiration date, or people are getting sick because they eat food that’s gone bad. Those things can be avoided by having a better product safety indicator.”

Give: Supporting Food Safety

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When Kikkoman wanted to establish a naturally brewed soy sauce plant in Walworth, Wisconsin—an operation that was to become the world’s largest—the company had a top-notch consultant at CALS to help them out.

That expert was professor Edwin “Mike” Foster, a noted bacteriologist who was the first director of the Food Research Institute (FRI) and the person responsible for FRI moving to UW–Madison from the University of Chicago in 1966.

“Mike was invaluable in offering guidance on how to address and validate regulatory issues related to the safety of soy sauce as Kikkoman went through the process of gaining FDA approval,” says FRI director Charles Czuprynski. Over the years UW–Madison has continued to play a role in testing potential new uses of sauce and products derived in the fermentation process, he notes.

Out of long-standing gratitude, the Kikkoman Foods Foundation has named a new scholarship fund in Foster’s honor. The “Kikkoman Scholarship in Honor of Dr. Edwin (Mike) Foster,” as it is called, will be awarded by the FRI each year “to a deserving undergraduate student with a demonstrated interest in food microbiology and food safety,” says Czuprynski. The award amount will be in the range of $1,000 to $1,200.

Czuprynski regards the Kikkoman plant as a remarkable Wisconsin success story—and a tribute to Kikkoman’s long-range leadership vision, supportive relationship with their workers and cooperation with local businesses and communities. “This scholarship is just one example of their generous support of UW–Madison and the UW System,” Czuprynski says.

The UW Foundation maintains more than 6,000 gift funds that provide critical resources for the educational and research activities of CALS.

Contributions to the Kikkoman Scholarship in Honor of Dr. Edwin (Mike) Foster fund are welcome at http://go.wisc.edu/08c3m5.

If you wish to establish your own scholarship fund, contact Sara Anderson at the University of Wisconsin Foundation, sara.anderson@supportuw.org, (608) 263-9537.

To make a more general contribution to scholarships at CALS, visit the Agricultural and Life Sciences Scholarship Fund at http://go.wisc.edu/3q63sr.

Microbes & Human Health

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Jim Steele used to be one of the skeptics. He’d be at a conference, listening to early research on the health benefits of probiotics. Steele scoffed at the small experiments. “We would literally try not to laugh in the audience, but we’d laugh pretty hard when we went out that night,” he admits.

But slowly the punch lines gave way to revelation. Steele, a professor in CALS’ Department of Food Science, conducts research on lactic acid bacteria, with a focus on Lactobacillus species. They’re important for human gut health, critical for the production of cheese and yogurts, and are the most common probiotic genus. He knew how incredibly useful they were, but still watched with a humbling disbelief as the data on the health potential of these microbes kept getting broader, deeper and more intriguing.

Our microbiota—what we call the totality of our bacterial companions—is ridiculously complex. Each human harbors a wildly diverse ecosystem of bacteria, both in the gut and elsewhere on the body. They have us completely outnumbered: where the typical body may contain a trillion human cells, your microbial complement is 10 trillion. They have 100 times more genes than you, a catalog of life potential called the microbiome. (The terms “microbiome” and “microbiota” are often used interchangeably in the popular press.)

While our initial, germaphobic impulse may be to freak out, most of these bacterial companions are friendly, even essential. On the most basic level they aid digestion. But they also train our immune system, regulate metabolism, and manufacture vital substances such as neurotransmitters. All of these things happen primarily in the gut. “In many ways the gut microbiota functions like an organ,” says Steele. “It’s extraordinarily important for human health,” with as much as 30 percent of the small molecules in the blood being of microbial origin.

Early research has suggested possible microbiota links to protecting against gastric cancers, asthma, numerous GI disorders, autoimmune disease, metabolic syndrome, depression and anxiety. And the pace of discovery seems to be accelerating; these headlines broke in just a few months last spring:
• Mouse studies suggested that the microbe Akkersmania muciniphila may be a critical factor in obesity;
• Kwashiorkor, a form of severe malnutrition that causes distended bellies in children, was linked to a stagnant microbiota;
• Risk of developing Type 2 diabetes was linked to an altered gut microbiota.

The catch: For all the alluring promise of microbes for human health—and it’s now clear they’re critically important—we have almost no idea how this complex system works.

The human gastrointestinal (GI) tract is a classic black box containing hundreds or thousands of species of bacteria (how many depends on how you define a species). There are viruses, fungi and protozoans. Add to that each person’s distinct DNA and their unique geographic, dietary and medical history—each of which can have short- and long-term effects on microbiota. This on-board ecosystem is as unique as your DNA.

Beyond these singularities, the action is microscopic and often molecular, and even depends on location in the GI tract. Most microbiota studies are done with fecal material. “Is that very informative of what’s going on in the ileum?” asks Steele, referring to the final section of the small intestine, which is thought to be the primary site where immunomodulation occurs. “From an ecosystem perspective, fecal material is many miles away from the ileum. Is it really reflective of the ileum community?”

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Protecting our Pollinators

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People and bees have a long shared history. Honeybees, natives of Europe, were carried to the United States by early settlers to provide honey and wax for candles. As agriculture spread, bees became increasingly important to farmers as pollinators, inadvertently fertilizing plants by moving pollen from male to female plant parts as they collected nectar and pollen for food. Today, more than two-thirds of the world’s crop plants—including many nuts, fruits and vegetables—depend on animal pollination, with bees carrying the bulk of that load.

It’s no surprise that beekeeping has become a big business in the farm-rich Midwest. Wisconsin is one of the top honey-producing states in the country, with more than 60,000 commercial hives. The 2012 state honey crop was valued at $8.87 million, a 31 percent increase over the previous year, likely due in part to the mild winter of 2011–2012.

But other numbers are more troubling. Nationwide, honeybee populations have dropped precipitously in the past decade even as demand for pollination-dependent crops has risen. The unexplained deaths have been attributed to colony collapse disorder (CCD), a mysterious condition in which bees abandon their hives and simply disappear, leaving behind queens, broods and untouched stores of honey and pollen. Annual overwintering losses now average around 30 percent of managed colonies, hitting 31.1 percent this past winter; a decade ago losses were around 15 percent. Native bee species are more challenging to document, but there is some evidence that they are declining as well.

Despite extensive research, CCD has not been linked to any specific trigger. Parasitic mites, fungal infections and other diseases, poor nutrition, pesticide exposure and even climate change all have been implicated, but attempts to elucidate the roles of individual factors have failed to yield conclusive or satisfying answers. Even less is known about native bees and the factors that influence their health.

Poised at the interface of ecology and economy, bees highlight the complexity of human interactions with natural systems. As reports of disappearing pollinators fill the news, researchers at CALS are investigating the many factors at play—biological, environmental, social—to figure out what is happening to our bees, the impacts of our choices as farmers and consumers, and where we can go from here.

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Ann Berres-Olivotti

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Ann Berres-Olivotti is a senior manager on the technical service team at Foremost Farms USA, where she primarily works in pharmaceutical lactose operations in the areas of quality assurance, process improvement and product functionality. “My favorite parts of the job are process improvements and educating end users regarding product capabilities,” she says. “Designing a practical process for a new product is one of the more exciting aspects of the job.”

Donald H. Burr

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Keeping the public safe from emergencies or outbreaks involving biological, chemical or radiological contamination of food is all in a day’s work for Donald Burr, who is a captain in the U.S. Public Health Service assigned to the FDA’s Center for Food Safety and Applied Nutrition. Burr helped design the FDA’s Food Emergency Response Network (FERN), which coordinates the response of food-testing laboratories at the local, state and federal levels following an emergency or outbreak. FERN was formed following the highly publicized anthrax attacks of 2001, and since then Burr has remained involved in the agency’s food defense and counter-terrorism activities. “It’s been gratifying to see the effectiveness of this program when there have been threats to our food supply,” Burr says.

While earning his doctorate at CALS, Burr worked at the UW–Madison Food Research Institute, where he helped develop animal models for infant botulism. After graduating he decided to pursue careers in both the U.S. Navy and the U.S. Public Health Service. “That allowed me to continue in the field of public health microbiology while at the same time serving my country,” he says.