A Helpful Fungus Among Us

A Helpful Fungus Among Us

Mine wastewater bioremediation on Minnesota’s Iron Range

By Evan Whiting

When people think of fungi, they typically conjure images of mushrooms: portobellos, oysters, truffles, or shiitakes. But a mushroom—the fruiting body we see above ground—is merely the tip of the iceberg when it comes to fungi. Most fungi are comprised of a tangled network of small thread-like structures called hyphae. Others, like yeasts, are microscopic, consisting only of single cells.

In fact, there is a huge diversity of fungi out there—some experts estimate that there are over two million species of fungi living on Earth today. And they’re important members of modern ecosystems. Excellent scavengers and nutrient recyclers, many fungi also gather materials from their surroundings and can even capture and store environmental contaminants. Experts are actively working to understand the underlying mechanisms, but scientists, engineers, and industry professionals also see the potential for using fungi in bioremediation—the cleanup of environmental contaminants or pollutants using living organisms, usually microbes.

Dr. Cara Santelli, an associate professor of Earth & Environmental Sciences and a member of the University of Minnesota Biotechnology Institute, is an expert on bioremediation and the interface between microbes and minerals. Santelli and her colleagues investigate organisms with the potential for bioremediation of mining waste, including several species of fungi. One of her ongoing projects, funded by MnDRIVE Environment, involves a species of fungus native to northern Minnesota’s Iron Range, where high concentrations of metals in the wastewater from an inactive underground iron mine could, if left untreated, threaten ecosystems and natural resources on the surface.

Located near Lake Vermilion and the Boundary Waters Canoe Area Wilderness, the Soudan Iron Mine was once a prolific source of iron ore. In the 1960s, when mining operations shut down, the mine became a research and educational facility. But more than fifty years later, there are still lingering issues with toxic metals in the mine’s wastewater, which is currently treated with expensive ion-exchange filters. Luckily, there may be a cheaper, local, and ‘organic’ solution to this problem: bioremediation with a native species of fungus (Periconia sp.) that grows in the salty, metal-rich waters in the depths of the Soudan Mine. Periconia is capable of producing manganese oxide minerals that incorporate metal ions in their chemical structures, much like blocks in a microscopic game of Tetris.

Dr. Brandy Stewart, a postdoctoral researcher in the Santelli Lab, has been studying this fungal bioremediation system in detail. Stewart is an expert in biogeochemistry, or the interplay between chemicals, microbes, and their environment. With additional background in environmental consulting, she brings a wealth of knowledge and experience to this new project. Currently, Stewart studies the optimal growth conditions for the project’s focal fungus, as well as how efficiently it produces manganese oxide minerals using nearby metal ions from the surrounding brine.

The fungus needs a cocktail of nutrients to grow and remain healthy, but the actual wastewater from the mine might not be the ideal growth medium. Even for this mighty fungus, certain metals, like copper, can be toxic at higher concentrations, so Stewart has set out to facilitate the removal of these metals without hurting the fungus itself. She likens this to our own dietary choices: “If you ate nothing but doughnuts, you probably wouldn’t be very healthy, but if you ate well and got plenty of fruits and veggies most of the time, you could still have a doughnut every once in a while and be okay.”

Thankfully, the fungus seems to grow quite well in the suboptimal, mine-like conditions simulated in the laboratory, especially when grown in the presence of carpet fibers. These fibers may be acting as a sort of ‘scaffolding’ upon which the fungus can grow, or even as an additional food source for the fungus. Regardless, Stewart has found that the presence of carpet fibers dramatically improves the growth and productivity of the fungus, which creates a thick biofilm where the manganese oxide minerals form and accumulate . As revealed by X-ray fluorescence images, these minerals are often laden with captured metals, demonstrating the efficiency of this system for removing metals from wastewater.


Periconia sp

X-ray fluorescence imagery showing very similar groupings of manganese oxide minerals (left) and copper ions (right) along strands of Periconia sp. fungal hyphae growing in the lab. Courtesy of Brandy Stewart, UMN.

Although still in the experimental stages, Santelli and Stewart eventually hope to build larger bioreactors to scale up their fungal bioremediation system for applications in the Soudan Mine. These bioreactors may be able to help prolong the lifespans of the ion-exchange filters already in use—or potentially replace them, if they prove to be at least as effective at removing metals like copper, cobalt, and nickel from the mine’s wastewater. These metals are not just potential environmental contaminants; they’re also economically important for myriad purposes, including electronics and battery production. If enough of these metals could be efficiently captured in manganese oxides produced by fungi and later recovered, we could potentially capitalize on them.

And mining wastewater might be just the beginning, according to Santelli. There are large amounts of dissolved solids in many types of industrial wastewaters, so there is great potential for using fungi (and/or other microbes) for bioremediation of contaminants in wastewaters well beyond the Iron Range. With such a huge diversity of fungi out there, it’s only a matter of time until another helpful fungus among us becomes the next big hit in bioremediation.

Evan Whiting is a PhD Candidate in the Department of Earth & Environmental Sciences at the University of Minnesota and an affiliate writer in the University of Minnesota Science Communication Lab. He can be reached at whiti101@umn.edu.

Feature photo: Photomicrograph of Periconia sp. fungal hyphae. Courtesy of Brandy Stewart, UMN

Tapping the Talents of Enzymes

Tapping the Talents of Enzymes

BTI researchers are working to discover, understand, and improve our ability to enlist the help of molecules that catalyze life.

by Mary Hoff

Enzymes are the movers and shakers of the biomolecular world. A class of proteins found in every cell of every living thing, they bring together molecules that would otherwise be reluctant to interact so they can combine, exchange parts, or otherwise alter each other.

In nature, enzymes facilitate thousands of processes from photosynthesis to decomposition. Enzymes speed up chemical reactions thousands of times faster than the reactions would occur on their own. Over the millennia, humans have found ways apply this superpower to accomplish a variety of useful goals from making beer and cheese, to making our laundry whiter and brighter, to cranking out millions of copies of a sample of DNA to solve crimes or find long-lost relatives.

“And that’s just the beginning,” says Romas Kazlauskas, professor in the department of Biochemistry, Molecular Biology, and Biophysics. “Enzymes can also learn how to do different things. We’re trying to figure out which parts are important and why they’re important, then design something different to do a reaction that doesn’t occur naturally.” By identifying enzymes, improving understanding of how they work, and applying them to new tasks, Kazlauskas and BTI colleagues are taking advantage of the billions of years of evolution that produced these molecules to make medicines, degrade pollutants, speed industrial processes, and more.


Trash to Treasure Kazlauskas is working with an enzyme found in nature that degrades PET plastic. First synthesized in the 1940s, PET (a form of polyethylene) is widely used for beverage and food containers with some 70 million tons generated every year—and much of that ending up in landfills and the ocean. Current efforts to recycle the material can only produce lower-quality plastics.

“One idea is to break down to component parts and resynthesize fresh plastic that would be just as good as what you started with,” he says.

Sound like a job for an enzyme? Kazlauskas thought so, too. But the likelihood of finding a suitable one in nature was slim because PET didn’t exist for most of evolutionary history. Nevertheless, in a compost heap in Japan, researchers recently found an enzyme that bacteria use to break down the waxy part of plants that could also break down PET.

Kazlauskas, along with graduate student Colin Pierce and colleagues, is working on modifying that enzyme known as cutinase, to improve its ability to degrade PET. The goal, ultimately, is to be able to develop a commercially viable way to turn old PET into new plastics without experiencing the loss of quality. This would not only reduce the load of plastic waste, it would also reduce pressure to produce more plastic, often from fossil fuels.

Kazlauskas sees huge opportunities beyond PET for efforts to extend nature’s ability to degrade compounds. “There are a lot of cases where we start making stuff and putting it out in nature, and people say it doesn’t degrade,” he says. “We’re finding bugs that can degrade these forever chemicals.” he says. By advancing the ability to modify such natural enzymes to make them do the job even better, the hope is to avoid the buildup of undesirable, manufactured compounds in the environment and reduce the need to make more from raw materials.


Life on the Edge

Turning somersaults in a sulfurous hot spring or clinging tenaciously to life on a glacier, the organisms studied by McKnight Presidential Fellow Trinity Hamilton, associate professor in the department of Plant and Microbial Biology, are the stars in microscopic world’s version of “Survivor.” Having evolved within living things, most enzymes work best under fairly mild conditions with respect to things like temperature and acidity. But not all. In the hot springs of Yellowstone National Park, Hamilton is studying how microbes—and the enzymes they use to carry out the activities of daily life—can survive regular temperature fluctuations of 40 degrees Celsius (104 degrees Fahrenheit) or more. And, on the other end of the spectrum, she’s also looking at how other microorganisms that live on ice maintain their function at low temperatures in the absence of liquid water.

Hamilton studies, among other things, enzymes that facilitate photosynthesis and function well up to 72 degrees Celsius, and then suddenly quit. “We have no idea why,” she says. “It’s really hard to perform autopsies on microbes.”

The ability of bacterial enzymes to function at various temperatures is behind the process that makes it possible to sequence DNA using what is called the polymerase chain reaction (PCR) technique. The enzyme used for this was isolated from bacteria in a hot spring in Yellowstone National Park. A better understanding of how bacteria keep these proteins active under extreme conditions could open the door to advancing our ability to solve other human challenges like preserving food or thriving in the face of climate change.

Molecular Assembly Line 

For Michael Smanski, a McKnight land grant professor in the department of Biochemistry, Molecular Biology, and Biophysics, the focus is on bringing together enzymes from different sources to create an efficient work team that can produce a specific molecule.

Most recently he’s been focusing on serofendic acid, a molecule found in the blood of fetal calves. In the early 2000s, researchers in Japan discovered that serofendic acid could prevent the untimely death of neurons and potentially serve as a therapy for minimizing damage from Parkinson’s disease, strokes, and other neurological trauma. The problem? It occurs in such small quantities that it would take 1,000 calves to make a minute 3 milligrams of active substance—providing dismal prospects for practical use.

Biosynthesis and enzymes to the rescue. Using databases describing enzymes from different organisms and the type of work each does, Smanski and colleagues designed a molecular assembly line that could make serofendic acid from readily available feedstock chemicals. Then they introduced the corresponding genes into a bacterium, enabling it to make the desired product.

As any industrial engineer would know, populating an assembly line with ready workers is only part of the solution, however. For efficiency’s sake, it’s also important to have the right capacity at every step of an assembly process to ensure that partially made products don’t pile up, or a slow step doesn’t drag down everything else. To optimize production of serofendic acid, Smanski has been tweaking the activity levels of the various enzymes relative to each other.

Even as he’s working on a strategy to improve outcomes for individuals with brain disorders, Smanski is also discovering principles that can be applied more broadly to optimizing artificial biosynthetic processes.

Optimizing a process means testing multiple levels of expression for the specific gene that’s coding for each enzyme along the way. If the assembly line consists of 15 enzymes, and researchers want to test five levels of expression for each, that would require 5 15 power—more than 30 billion—individual trials. Using combinatorial mathematics, Smanski is devising a strategy for selecting from those millions of possibilities, a mere 100 or so that are most likely to succeed, making the optimization process far more manageable.

“As improvements in the technical aspects of genetic engineering make it easier to rewrite the genetic blueprints of living organisms, learning how to efficiently sift through all of the possibilities of what-to-write is becoming more important,” Smanski says. “We think that our work at the interface of combinatorial mathematics and genetic engineering will benefit any application that requires more than one or a few genes working together.”


Self-Assembling Scaffold

Bringing multiple enzymes together in an assembly line is far more easily said than done. The molecules involved in sequential steps must be physically located close enough to each other to be able to pass a product-in-the-making down the line. They also must be positioned correctly at their stations so they can accomplish their task.

Distinguished McKnight University Professor Claudia Schmidt-Dannert is on it. Rather than identifying enzymes to string together to accomplish a molecule-making task, she is working on the assembly line itself.

Her goal is to not only design a stable assembly line of enzymes able to make a useful product but to genetically modify a microorganism—say, the bacterium E. coli—so that it can produce it on its own. Such a self-assembling scaffold, she believes, could dramatically improve the function of molecular assembly lines, reducing the cost of producing pharmaceuticals and other useful products.

“If we stabilize enzymes, we can make reactions more efficient,” she says.

Schmidt-Dannert and colleagues recently received a patent for a self-assembling, modular scaffold that co-locates enzymes in a way that enhances their ability to efficiently produce desired molecules. Her current focus is on developing scaffolds as materials that can correctly self-assemble into 3-dimensional architectures to organize and support enzyme functions. This would allow the manufacture of a broad range of chemicals including pharmaceutically active molecules—a process that can be extremely difficult using conventional chemical synthesis methods. 

She’s also applying her strategy to making molecular assembly lines more economical by including enzymes that recycle required co-factors, which are molecules or metals that assist enzymes in assembling a molecular product.

“You need to recycle the [co-factors]. Otherwise, the process is too expensive,” she says. “A scaffold helps make that possible.”


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.


BTI-NAIST Exchange Marks 15 Years

BTI-NAIST Exchange Marks 15 Years

Tim Montgomery

Following a visit to Minnesota by three Japanese graduate students from the Nara Institute of Science and Technology (NAIST), a group of four Minnesota graduate students from the BioTechnology Institute (BTI) visited Japan in mid-October. Chris Flynn, Grayson Wawrzyn, Jessica Eichmiller and Maria Rebolleda-Gomez were graciously hosted by their NAIST counterparts on a 3-week trip that completed the 15th exchange in a program organized by former BTI Director, Ken Valentas in 1996.

The 15th BTI-NAIST exchange featured a symposium on progress in microbial biotechnology, enzyme engineering and systems biology – and a five-year renewal of the agreement that created the program.

Since its conception, the exchange has successfully connected graduate students from one institution to research groups in the other based on common interests with the intent of learning new skills and techniques. Students from the host laboratory also become cultural mentors for the visitors. Lasting professional and personal bonds are forged in the process, sometimes resulting in collaborative research initiatives.

“I think that my favorite part of Japan,” commented Grayson Wawrzyn, “was learning to be part of a culture so strikingly different from our own.”

Wawrzyn, a graduate student researcher in the lab of Claudia Schmidt-Dannert, was assigned to Takashi Hashimoto’s laboratory and worked with one of his students to help characterize some of the enzymes involved in nicotine biosynthesis in tobacco plants.

Other members of the BTI group participated in equally compelling genomic research projects. Eichmiller studied novel intracellular proline transporters and tested the stress tolerance of mutant yeast strains in the lab of Hiroshi Takagi. Rebolleda-Gomez was introduced to systems biology in the study of bacterial genomics while in the lab of Hirotada Mori. And Flynn learned how cells repair damaged DNA while in the Maki lab.

For Japanese lab members who are required to present their lab work in English each week, working with the exchange group from BTI was an opportunity to practice speaking scientific English.

Living and working together, lab groups also had fun together. Several of the Japanese labs had their own baseball teams, and the last week of the exchange featured ‘lab Olympics day’ where Japanese lab members dressed in super hero outfits competed for fun in a series of relay races.

In addition to experiencing traditional Japanese foods like sushi and okinomiyaki, BTI exchange members also experienced the cultural environment in trips to local shrines around Nara and the old hilltop estates in Arishiyama near Kyoto. The highlight of their cultural experience was a 4-day holiday break that brought most of the group to Tokyo before members went their separate ways. Wawrzyn and Rebolleda-Gomez explored Tokyo further while Eichmiller visited a Japanese friend and Flynn and his wife toured a world heritage shrine and marveled at the beauty of the ponds and cascading waterfalls of Chuzenji in Nikko.

“The hospitality of our hosts was superb,” concluded Flynn. “Everyone was super friendly.”

Added Eichmiller, “an unexpected benefit of the trip to Japan is that I can better relate to my Japanese colleagues at the University.”

A Rewarding Experience in Japan

A Rewarding Experience in Japan

by Tim Montgomery

Janice Frias, Katherine Volzing, Chad Satori and Josh Ochocki visited Japan this past November as part of the BioTechnology Institute’s ongoing exchange program with the Nara Institute of Science and Technology (NAIST). They travelled to Japan with returning exchange students from NAIST whom they had previously hosted at BTI.

Exciting cultural experiences complemented the students’ lab work at NAIST, beginning with an informal welcome party where dried squid “candy” was served. The BTI group stayed on-campus in guest houses, were chaperoned to various tourist and cultural attractions and experienced new food and pastimes – from a hearty noodle meal of hiroshimayaki to a traditional foot bath where fish nibbled the dirt particles off their toes.

Katherine Volzing visited the ginza, a high style shopping district in Tokyo, and was invited to breakfast with a family in the Tsukiji Fish Market where they served her raw tuna on a stick.

“It looked yucky,” she confessed, “but it was the best thing I ever ate.”

Cultural excursions included time spent at the Todai-ji Temple in Nara; the Kiyomizu Temple, Sanjeusangen-do Temple Garden and Ginkaku-ji Gardens in Kyoto; the Floating Torii at Miyajima; Himeji Castle, and aboard the 200 mph Shinkansen or “bullet train” while in transit. But the exchange group from BTI also accomplished quite a bit in their lab work.

“Professor Takagi said we were the best working group ever,” exclaimed Chad Satori proudly. Satori was excited to work with cell transfections and binding assays in the Sato lab in a change of pace from his mostly analytical work under Edgar Arriaga at BTI.

Janice Frias, who has worked to synthesize biohydrocarbons in the Wackett lab at BTI, was assigned to the lab of NAIST exchange coordinator Hiroshi Takagi, Professor of Cell Biotechnology specializing in applied microbiology and protein engineering. Frias assisted in Takagi’s work with stress tolerance in yeast as an element in improving industrial fermentation in the production of bioethanol.

Frias, Satori and the other members of the exchange group from BTI each found their assignments while at NAIST to be rewarding. Distefano lab member Josh Ochocki was introduced to the work of Professor Kinichi Nakashima exploring neuron stem cells and how they develop into different types of brain cells. And Katherine Volzing found her experience in the lab of Ko Kato examining differentiation in gene expression in stem cells extracted from mice to be a change of pace from her statistical and computational modeling in the Kaznessis lab at BTI. All were impressed with the professionalism as well as the aggressive English requirements of their Japanese counterparts.

“They’re required to put together and present their lab work and plate results in English each week,” explained Janice Frias in amazement.

“The labs were impeccably clean,” concluded Chad Satori. “And everyone was very professional and kind.”


BioTechnology Institute

The BioTechnology Institute is the central University of Minnesota vehicle for coordinated research in the biological, chemical, and engineering aspects of biotechnology. more >>

Department of Bioproducts and Biosystems Engineering

The Department of Bioproducts and Biosystems Engineering was formed on July 1, 2006, from the merger of the former departments of “Bio-based Products” and “Biosystems and Agricultural Engineering.” The department focus is on discovery, development and application of renewable resources and sustainable technologies to meet society’s needs while enhancing the environment in Minnesota and beyond. more >>

Department of Biochemistry, Molecular Biology
and Biophysics – Microbial Biochemistry and Biotechnology Division

The department has equally strong interests in understanding the molecular mechanisms of metabolic diseases and cancer, developing novel strategies in biocatalysis and biotechnology, and advancing knowledge through structural biology and molecular biophysics. more >>

Department of Chemistry

The Department of Chemistry has graduate faculty specializing in interdisciplinary areas of chemical biology that include: bioanalytical and biomaterials chemistry, computational chemistry, design and synthesis, enzyme chemistry, structure and spectroscopy, and nucleic acids. more >>

Department of Chemical Engineering
and Materials Science

The Department of Chemical Engineering and Materials Science has a graduate focus on biotechnology and bioengineering research. more >>

Department of Medicinal Chemistry

The College of Pharmacy has a Medicinal Chemistry department and graduate program that is one of the top-rated in the country. This department consists of a diverse group of faculty members, graduate students and postdoctoral-fellows working at the interface of chemistry and biology and is home to the editorial office of the Journal of Medicinal Chemistry. more >>