Understanding a Toxic Necessity

Understanding a Toxic Necessity

Jannell Bazurto, assistant professor of Plant and Microbial Biology at the University of Minnesota, is pursuing a better understanding of formaldehyde, a chemical that is carcinogenic, toxic, and produced by all living things.

By Reed Grumann

If you dissected a pig in high school biology, you might remember a sharp, acrid smell permeating the classroom and the teacher’s warning about a carcinogenic chemical called formaldehyde. Though often labeled a killer chemical, every organism on Earth, including humans, produces small amounts of formaldehyde. In very small quantities, it’s manageable. Produce or consume too much, and formaldehyde will kill otherwise healthy cells by attacking critical proteins and DNA.

While most organisms can neutralize small amounts of formaldehyde, researchers are just beginning to understand the mechanisms involved in formaldehyde regulation. Jannell Bazurto, a member of the BioTechnology Institute and an assistant professor of Plant and Microbial Biology, looks for clues in Methylobacterium, a type of bacteria that produces and neutralizes formaldehyde levels that would kill most other microbes.

As their name implies, methylobacteria have a unique ability to metabolize or breakdown, single-carbon molecules like methane and methanol. The process helps the cell maintain enough energy to survive, but also generates formaldehyde as an intermediate step. Fortunately, the bacteria are also uniquely equipped to handle sudden changes in concentrations of the toxin.

A key mechanism of Methylobacterium is an enzyme that converts formaldehyde into less harmful chemicals. While the enzyme is sufficient at normal levels of formaldehyde concentration, it’s not enough to handle exceptionally higher formaldehyde levels. To identify the function of two additional proteins suspected of playing a role in regulating formaldehyde levels, a group of researchers at the University of Idaho removed them from bacterial cells. And just like a cake without sugar and eggs, something was off. “If you don’t have [the two proteins] … you can see [the cells] accidentally overproduce formaldehyde, and they end up secreting it in the growth medium,” says Bazurto.

It’s still unclear exactly how these two proteins keep formaldehyde levels low in methylobacterium, but they aren’t alone in their efforts. Dozens of genes express proteins as formaldehyde levels change—a strong indicator of their importance in regulating the toxin. The challenge for Bazurto is knowing which of these genes, and the proteins they encode, actually play a role in formaldehyde metabolism. By manipulating each gene and looking at the results, Bazurto hopes to crack the code and establish which genes impact formaldehyde metabolism and their role in the process.

Once understood, these metabolic pathways could be hardwired into other microbes (like E. coli) through genetic engineering. Modified E. coli could consume methanol, neutralize formaldehyde, or produce marketable chemicals like biofuels and organic acids. In some facilities, formaldehyde is produced in large quantities as a building block for other chemicals. Wastewater remediation at these facilities would greatly benefit from bacteria genetically modified to directly consume formaldehyde and withstand toxic concentrations.

Throughout its industrial lifecycle, formaldehyde has the potential to creep into our air and water, putting humans at risk of exposure. The relationship between excessive formaldehyde exposure and human health issues—cancer, respiratory issues, and skin irritation—has been well established, yet we still know very little about how humans (and other organisms) sense and handle exposure to formaldehyde. As the search for practical applications in biotechnology, medicine, and environmental remediation continues, Bazurto remains fascinated by the basic science and “scenario where we actually know how to resolve formaldehyde toxicity itself.”

Reed Grumann is a writing intern in the Science Communication Lab, majoring in microbiology and political science. He can be reached at gruma017@umn.edu.

Image courtesy of Janelle Buzurto. Timecourse of Methylobacterium chemotaxing toward a capillary tube that has a formaldehyde plug in it. (Zero and five minutes). Cells seen faintly in the background at zero minutes begin to move toward the plug by the five minute point, forming a halo around the end of the tube.


Clean Energy from Beer Waste?

Clean Energy from Beer Waste?

MnDRIVE-funded researcher harvests natural gas from brewery wastewater

By Nick Minor

From industry pioneers like St. Paul’s Summit Brewery to small-town brewpubs, Minnesota’s craft beer industry has become a point of pride for local beer enthusiasts. But for every pint that flows through the tap, 3 to 10 pints of wastewater–high in carbohydrates, acids, and alcohol–end up in the municipal waste stream to be treated by the city. Breweries pay a premium to remove and treat this wastewater. Still, that same nutrient-rich content provides an ideal food source for hungry microbes capable of turning the waste into energy at the brewery.

With funding from the University of Minnesota MnDRIVE Environment Initiative, researchers led by Professor Paige Novak set out to treat this brewery wastewater while achieving two additional benefits: reducing the load on municipal water treatment systems and producing energy to help fuel brewery operations. 

Kuang Zhu, a recent PhD graduate from Novak’s lab, designed a 2-stage process to treat the brewery wastewater. In the first stage, microbes (called acetogens) feed on the wastewater. Housed in an airtight, oxygen-free compartment, they digest the carbohydrates in the wastewater, producing hydrogen and acetate as byproducts. These byproducts are then siphoned into a second oxygen-free compartment where microbes (called methanogens) consume the acetate, and produce methane, a significant component of natural gas. One of the team’s original innovations was to house the microbes in beads made of a carbohydrate derived from brown algae. This keeps the active microbes in the reactors where they can do their work simply and with little energy expenditure. Hydrogen collected from the first stage, and methane collected from the second can be used to generate power for the brewery while the treated water, significantly cleaner, flows to the local municipal wastewater treatment facility.

Scaling up this research from the lab to the brewery, presented its own set of challenges. Partnering with Fulton Brewing in Minneapolis, the team embarked on “months and months of troubleshooting,” Novak recalls. There were constant tweaks, leaks, and spills; parts to replace, cross-contamination, and even a small wastewater “explosion,” all within a wastewater storage room that averaged a steamy 85 degrees Fahrenheit. 

But this process, slow and tedious though it may be, is a critical part of science. Novak credits the demonstration grant from MnDRIVE, a state-funded initiative that aims to connect basic research with real-world impacts, for making this possible. “It’s such a different kind of trial and error and troubleshooting process,” explains Novak, “and there are so few funding sources for that kind of work. Going through this demonstration project has been invaluable because we figured out all those things we need to pay attention to when we do this again.” 

“Plus,” Novak adds, “it’s been really fun.”

One part of that fun is a public art exhibit that was stimulated by a requirement of MnDRIVE grants to have a public outreach component. After meeting local artist Aaron Dysart at a conference, Novak knew his data-driven approach to art would be a perfect fit for her project and for making the required outreach component much more visible. Dysart plans to create a disco ball suspended outside the Fulton taproom. Not just any disco ball, Dysart’s installation will spin at a rate proportional to the gas produced by the bioreactors. It will project color sequences linked to the ratio of hydrogen and methane. Mounted sideways, Dysart’s disco ball project will be a stream of data bubbling up from the wall of the brewery’s outdoor beer garden.

Of course, getting the bioreactor design into the real world won’t just involve science and engineering. It also requires understanding how the bioreactor fits into the marketplace. Again, thanks to MnDRIVE funding, Novak was able to partner with the Carlson School of Business to conduct some preliminary market research. “They analyzed the technology and the market,” Novak says, “and evaluated different food and beverage industries that would be a good match for our technology.” To Novak’s surprise, Carlson’s research showed that breweries weren’t the only market for the bioreactor. “Breweries don’t typically spend enough money [for this to make a big difference for them].” Instead, potato chip makers and candy manufacturers, both of which generate high concentration wastewaters, could benefit more from the team’s design. Furthermore, the business school team clearly showed that before the technology will be accepted by industries, the design must be plug and play and ready to work out of the box, regardless of the nature of the waste stream.

This goal is now much closer to reality thanks to progress made through the demonstration grant. One day, Novak hopes high concentration industrial wastewater treatment will be as simple as “getting your beads, dumping them in, and watching them go to work no matter what.” MnDRIVE funding not only helped move the technology forward, it also allowed the team to identify industries most likely to benefit from the research and provided an opportunity for consumers to learn about wastewater treatment through a public art installation.            

Nick Minor is an alumnus of the Science Communications Lab, pursuing a master’s degree in zoology and physiology at the University of Wyoming. He can be reached at minor092@umn.edu.


Stopping PFAS in Its Tracks

Stopping PFAS in Its Tracks

UMN researchers Matt Simcik and William Arnold trap harmful chemicals before they can pass through the environment to our drinking water

by Caroline Frischmon

When you turn on the faucet, you probably trust the water in your glass will be safe to drink. For Minnesotans living in the eastern Twin Cities, this trust evaporated when toxic PFAS chemicals (or per and poly-fluoroalkyl substances) infiltrated their groundwater. PFAS are found in many products, ranging from nonstick cookware and food packaging to waterproof clothing. Despite their ubiquity, scientists suspect high concentrations of the chemicals lead to cancer, obesity, and other health problems. 3M formerly manufactured PFAS at its Cottage Grove facility, which caused the east metro contamination. Now the chemicals are threatening drinking water for Minnesotans across the state.

“This is an issue everywhere,” says Matt Simcik, a University of Minnesota Environmental Health Science professor who has studied PFAS for nearly 20 years. He explains that contamination is widespread because landfills throughout the state can also leak the chemicals.

When we throw away PFAS-coated products like raincoats and upholstery, the compounds leach into the water that passes through the landfill. Operators either pump this water (called landfill leachate) to a settling pond or truck it to a wastewater treatment facility. Neither option effectively filters PFAS before discharging the contaminated leachate into the environment. Plants absorb some of the released PFAS, but the rest can percolate into groundwater. With the help of a MnDRIVE Environment seed grant, Simcik and his colleague William Arnold in the Department of Civil Engineering, are developing a solution to prevent the release of landfill PFAS.

Known as forever chemicals, PFAS can persist indefinitely in the environment. We don’t have an effective way to break them down, so Simcik plans to trap the compounds instead. He and Arnold previously developed a groundwater treatment technology that uses a coagulant or clumping agent. These big molecules bind to PFAS to make the chemicals stick together and become entangled in the soil — where they can’t travel to our faucets. While it has shown great promise in the laboratory, they are now field testing this method. Depositing the coagulant within a landfill could immobilize the chemicals in layers of waste even as the leachate flows through the site. Simcik’s plan would effectively turn landfills into PFAS storage containers, and prevent contamination from spreading further in the environment.

While this sounds promising, applying the groundwater technique to landfills is more complicated than simply identifying the best location to bury the coagulant. Groundwater is relatively clean, but landfill leachate can pick up contaminants other than PFAS as it passes through layers of waste, which reduces the coagulant’s effectiveness. Before testing the treatment method in a landfill, the MnDRIVE project will investigate how to maintain performance even as the leachate composition varies.

Simcik must also confirm that the treatment is long-lasting. If the chemicals can leak out of their trap, the coagulation method would just delay the problem rather than solve it. The lab will monitor the longevity of the coagulants, but Simcik anticipates the solution will be long term.
Forever chemicals require a lasting solution because they can endure many years in the environment. 3M phased out production of the two most prevalent types of PFAS (called PFOS and PFOA) in the early 2000s after they were detected in animal bloodstreams worldwide. Over a decade later, the chemicals still linger in the environment near the 3M facility. After 3M phased out PFOS and PFOA, other manufacturers introduced new PFAS chemicals as replacements without proof they were any less toxic.

Ongoing research on the substitute PFAS compounds now points to similar health hazards as the originals, so companies may someday end up replacing these “replacement” chemicals. As this toxic cycle repeats itself, Simcik hopes to at least keep PFAS, both old and new, locked away in landfills and out of our bloodstreams. “Hopefully, we can prevent future contamination. That’s our goal,” he says.

Caroline Frischmon is a writing intern in the Science Communications Lab, majoring in Bioproducts and Biosystems Engineering. She can be reached at frisc109@umn.edu

Signal and Noise        

Signal and Noise        

Enzyme-based coatings developed at the University of Minnesota help protect port infrastructure by disrupting the signals underwater bacteria use to communicate.

By Nick Minor and Kristal Leebrick

In any seaport or freshwater marina around the world, just beneath the surface, and you’ll find an ongoing battle between the boats, docks, bridges—anything made of steel—and a cast of aquatic bacteria in search of a submerged surface to call home. The biocorrosion created by these bacterial hitchhikers is especially dire in cold climates where winter brings the added wear and tear of scraping ice. And Duluth-Superior Harbor is ground zero, as aquatic bacteria corrode nearly 50,000 pounds of steel there each year.

Two University of Minnesota scientists—Randall Hicks, a microbial ecologist in Duluth, and Mikael Elias, a biochemist in the Twin Cities—have developed an enzyme coating they believe could rewrite the story of biocorrosion in Duluth and around the world. Their work shows extraordinary promise in helping prevent biocorrosion in seaports and could have the added bonus of being environmentally friendly.

The scientists’ collaboration began in late 2016, after Elias read about Hicks’s and postdoctoral associate Simon Huang’s work on testing anti-biocorrosion coatings in Gateway, published by the University of Minnesota BioTechnology Institute. The timing was auspicious for Elias. He and his students had recently engineered an enzyme that breaks down the chemical signals bacteria use to coordinate and build things like biofilm, a matrix of proteins and carbohydrates that can lead to biocorrosion. The interruption of those signals is like overlaying an impenetrable static onto construction workers’ walkie-talkies. Without the ability to communicate, the bacteria can’t coordinate enough to build anything.

Communication-disrupting enzymes are well-known and widely available, yet their potential ability to prevent bio-induced corrosion was unknown. In addition, in order to prevent biocorrosion in the Duluth-Superior Harbor, an enzyme needs to be hardy enough to withstand organic solvents of paints, temperature shifts that can kill most plants and animals, and endure scrapes from massive winter ice flows. This is where Elias’ specialty—protein engineering—came into play. Elias refined the enzyme to such an extent that it is now “so stable that we can dilute them into paint, a very harsh treatment for a protein,” says Elias, “and they still remain active.”

After reading about Hicks’s and Huang’s work, Elias reached out and asked if Hicks could squeeze one more coating into his tests. From there, the duo started with a two-month, proof-of-concept test in the lab, made possible through funding from the University’s MnDRIVE Environment initiative, which supports promising research on environmental remediation. In this short-term test, the enzyme, which was suspended in a durable acrylic, outperformed every other coating Hicks had been examining. But the real test lay ahead. After presenting the enzyme coating to companies like PPG, BASF, and Ecolab, Elias and Hicks heard the same message over and over: The companies needed to know if it would remain effective for years, not just two months.

Elias and Hicks received a much larger “demonstration grant” from MnDRIVE, which supported two years of testing, including testing in the Duluth-Superior Harbor. The work exceeded all expectations: over those two years, the enzyme coating was more effective at preventing biocorrosion than any other available coatings and it appears to do no harm to the environment as it kills nothing outright. Currently, 85 percent of the market for anti-biocorrosion coatings is dominated by toxic copper oxide paints. As with nearly every other coating available, copper oxide paints work by brute force, killing the organisms responsible for biocorrosion. Copper oxide’s toxicity to biocorrosive organisms also means it’s toxic to other living things.

Copper oxide paints were technically banned by multiple U.S. states. “But, because there is no alternative,” explains Elias, “the ban is constantly being pushed back.” Copper oxide, a heavy metal and potent environmental toxin, has been accumulating in portside ecosystems around the world for decades.

“The alternative that we’re working on,” says Elias, “is ecological because it’s a protein. A protein, by definition, is biodegradable. It’s amino acids.” The enzyme’s approach—disrupting the communication between bacteria that get biocorrosion started—is utterly novel.

The enzyme coating could rewrite the story of biocorrosion in Duluth and enable additional infrastructure protections to take effect. The aquatic ecosystem around the Duluth-Superior Harbor, along with similar portside ecosystems around the world, could start to recover from decades of copper pollution.

Based on their initial work, the team received funding from the Minnesota Sea Grant and Minnesota Aquatic Invasive Species Research Center-LCCMR to study the coatings’ ability to inhibit biofouling and the adhesion of aquatic invasive species to underwater surfaces.

“This may just be another arrow in the quill of possible coatings that could be used,” Hicks explains cautiously, “but potential applications are certainly way beyond Lake Superior. The market could be potentially unlimited.”



Also see Battling Biocorrosion in Duluth-Superior Harbor

The Fight for Safer Food

The Fight for Safer Food

To confront the threat of persistent foodborne pathogens, Steve Bowden turns to novel techniques to understand and take down those threats.

By Kyle Wong

Nearly one in six Americans suffer from foodborne illnesses each year, according to the Centers for Disease Control and Prevention (CDC). Scientists like the University of Minnesota’s Steve Bowden are working to change that.

Standard food-safety processes like pasteurization, freezing, and fermentation kill foodborne pathogenic bacteria, but these pathogens evolve to survive. Through mutation and the acquisition of new genes, pathogens can cause outbreaks from unfamiliar sources such as fresh produce, spices, and peanut butter. Bowden, an assistant professor in the Department of Food Science and Nutrition and a member of the BioTechnology Institute, is developing strategies to eliminate new and persistent pathogens.

One common offender is Salmonella, a bacterial species that infects millions of Americans each year, leading to thousands of hospitalizations and hundreds of deaths. Bowden studies how Salmonella responds to stresses in the food environments where it thrives. He seeks to identify common genetic traits that enable Salmonella to survive and grow under varied conditions. Ultimately, he hopes to develop procedures that eliminate harmful bacteria from the food system. “We want to understand why certain outbreaks occur in specific types of food and if there is a correlation with the genes found in those specific types of bacteria,” says Bowden.

To target specific foodborne pathogens, Bowden’s lab engineers a type of virus called a bacteriophage that infects and then kills bacteria. But identifying phages that work is only half the battle. Because pathogens are so diverse, effective treatment requires the right combination of phages to remove all of the pathogen targets and ensure food safety. To make things more complicated, the food matrix can also affect the phage’s efficacy. One cocktail of phages might succeed on one food but fail to remove the same pathogen in another.

Nonetheless, the Food and Drug Administration (FDA) has approved some phage cocktails. They are entirely harmless to humans and assist in the control of foodborne pathogenic bacteria during food processing. Their potential use as a safe, natural method to improve food safety is garnering interest in the food industry. Still, further research is required to enhance their efficacy and improve manufacturing methods.

Despite these challenges, Bowden’s longstanding fascination with molecular biology and microbiology keeps him motivated: “I was surprised by how many foodborne illnesses there are. I want to make use of genome sequencing to try and control these pathogens. What’s interesting about molecular biology is how we can apply it to make the world safer.”

Bowden’s drive to understand foodborne pathogens couldn’t have come at a better time. Addressing global threats to food safety, such as climate change and antibiotic resistance, requires broad thinking and a flexible approach. Bowden brings a global perspective to his work. After earning his Ph.D. in biochemistry at the University of Cambridge, he worked as a postdoc in the United Kingdom and Japan. “I feel lucky to have been in so many labs and learn different perspectives,” he says. “It’s helped broaden my appreciation for different ways to do research.”

The experience helped prepare Bowden for his research at the University of Minnesota. “The drive to understand a problem and develop techniques to study that problem has kept me in academia,” he says. “I’m excited to see what direction it takes.”

Kyle Wong is a writing intern in the University of Minnesota Science Communications Lab, majoring in Microbiology. He can be reached at wong0511@umn.edu

Making the Best of the Rest

Making the Best of the Rest

MnDRIVE initiative helps Second Harvest Heartland turn inedible food into useful products

By Mary Hoff

Every day, Second Harvest Heartland gathers more than 100 tons of food from donors across Minnesota and western Wisconsin and redistributes it to food shelves and others who serve people in need. In the process, the food bank—the second largest in the U.S.—ends up with some 3 tons of bad cabbage, spoiled milk, too-old-to-eat cereal and other “unfit for consumption” bits and pieces left over from this process.

And pays $200,000 per year to have it hauled away.

Now, Second Harvest is looking to turn this waste into a “third harvest” with the help of a MnDRIVE demonstration grant, University of Minnesota agricultural engineers, and an invention that began as a way to reduce problems with pig poop.

It all began when Bob Branham, director of produce strategy for the food bank, began looking for a way to reduce the need to spend money that could be going to feed people on disposing of inedible organics.

“I’m paying people to take away high-value waste,” he recalls thinking. “Why shouldn’t I keep that waste to the benefit of Second Harvest Heartland?”

Branham reached out to the University of Minnesota where, coincidentally, associate professor of bioproducts and biosystems engineering Bo Hu had recently, with the help of a MnDRIVE seed grant, developed an anaerobic digester system for turning another type of organic material—pig manure—into useful materials. Hu and Timothy LaPara, professor of civil, environmental, and geo-engineering, applied for and received a MnDRIVE demonstration grant proposal to apply the concept to meet Second Harvest needs.

With support from MnDRIVE and the help of undergraduate and grad students, Hu and LaPara designed a two-stage system capable of transforming Second Harvest’s highly variable organic waste stream into heat, fertilizer, and a valuable soil amendment.

Anaerobic digestion has some similarities to traditional composting but is miles beyond it in both technical sophistication and value of output. Whereas composting takes place with organic materials exposed to air and produces a soil amendment, anaerobic digestion relies on bacteria that break down materials in the absence of oxygen and produces a gas that can be burned to produce heat.

“Digestion of food waste is actually a very sexy idea right now,” Hu says, noting that some large cities are banning food waste from landfills, and sustainability advocates are pushing to reduce the greenhouse gas contributions of waste while deriving useful products.

To make the process suitable for application at Second Harvest, Hu and his team refined it to work with a variable waste stream, at a relatively small scale, with a minimal need for water, and with a bio-electrochemical system that removes adverse odors. They also did an economic analysis to determine whether a digester would make dollars-and-cents sense for Second Harvest, which operates on a tight budget and aims to put every extra penny into helping allay hunger and reduce food waste.

The project penciled out, so Hu and team built an experimental digester to refine the process. Among other things, they looked at strategies to reduce odor and corrosiveness of the gas it produces, explored how the mix of digesting microbes might be tweaked over time to meet seasonal changes, and identified ways to automate the process.

Now, with many of the bugs worked out of the system, the team is installing a pilot digester at its Brooklyn Park facility to test its performance with the mix of waste the food bank produces. Once the pilot confirms the functionality of the system, Hu will advise Second Harvest on installing a full-scale facility.

Along with the benefits the system will reap from what is currently treated as a liability, Hu envisions a fourth harvest from the project as well: Inspiration and motivation for other food banks, as well as other businesses that manage organic waste.

Hu says MnDRIVE has been “very vital” in making this research possible because federal grants are increasingly hard to come by.

“It’s actually really good seed money to obtain federal support,” he says. “We are doing applied research, but we are also gaining fundamental knowledge at the same time that will help us [pursue funding from] federal agencies like USDA or the National Science Foundation.”

For his part, Branham is delighted to have the opportunity to do an even better job of making the most of the resources his organization stewards. “This is a whole new validation that waste and renewable energy can be great partners,” he says. “I wish there wasn’t organic waste, but there is—it just happens for a variety of reasons and that’s not going to go away. So let’s find a better use for it to fight food insecurity.”