As spring warms up Wisconsin, humans aren't the only ones tending their gardens.

At the University of Wisconsin-Madison Department of Bacteriology, colonies of leaf-cutter ants cultivate thriving communities of fungi and bacteria using freshly cut plant material.

While these fungus gardens are a source of food and shelter for the ants, for researchers, they are potential models for better biofuel production. "We are interested in the whole fungus garden community, because a lot of plant biomass goes in and is converted to energy for the ants," says Frank Aylward, a bacteriology graduate student and researcher with the Great Lakes Bioenergy Research Center.

Aylward is the lead author of a study identifying new fungal enzymes that could help break down cellulosic-or non-food-biomass for processing to fuel. His work appears on the cover of the June 15 issue of the journal Applied and Environmental Microbiology.

"All the enzymes that we found are similar to known enzymes, but they are completely new; no one had identified or characterized them until now, " Aylward says.

Building on Aylward's previous study of these gardens, the researchers relied on genome sequencing provided by the U.S. Department of Energy Joint Genome Institute (JGI) and support from Roche Applied Science's 10 Gigabase Grant Program to understand the unique roles of fungi and bacteria. In addition to sequencing the genome of Leucoagaricus gongylophorous, the fungus cultivated by leaf-cutting ants, the researchers looked at the genomes of entire, living garden communities.

"We really tried as thoroughly as possible to characterize the biomass degrading enzymes produced," Aylward says. "Identifying all these new enzymes really opens the door to technological applications, because we could potentially mix and match them with others that we already know about to achieve even better biomass degradation."

Illustration of the leaf-cutter ant Atta cephalotes in its fungus garden habitat. The variation in texture between the top and bottom strata represents the different stages of biomass degradation occurring in each layer of the fungus garden. Image credit: Cara Gibson

In a symbiotic relationship, L. gongylophorous provides food for the leaf-cutter ant Atta cephalotes by developing fruiting bodies rich in fats, amino acids and other nutrients. To fuel production of these fruiting bodies, the fungus needs sugar, which comes in the form of long cellulose molecules packed inside the leaf clippings the ants deliver. To get at the sugars, the fungus produces enzymes that break the cellulose apart into glucose subunits.

After sequencing the L. gongylophorous genome, the researchers noticed that the fungus seemed to be doing the lion's share of cellulose degradation with its specialized enzymes. However, they also realized that it was by no means working alone: in fact, the gardens are also home to a diversity of bacteria that may help boost the fungus's productivity.

"We think there could potentially be a division of labor between the fungus and bacteria," says Garret Suen, co-author of the study and a UW-Madison assistant professor of bacteriology and Wisconsin Energy Institute researcher.

The researchers have a few leads in their investigation of the mysterious role of bacteria in leaf-cutter ant communities, which they are pursuing in collaboration with JGI. In addition to providing nitrogen and key vitamins, the bacteria appear to help the fungus access energy-rich cellulose by breaking apart other plant polymers that encase it, such as hemicellulose.

Accessing and deconstructing cellulose is also the goal of GLBRC researchers, who want to ferment the stored sugars to ethanol and other advanced biofuels. Enzymes such as those of the leaf-cutting ants' fungus specialize in breaking down leaves, but understanding how they work in the context of the ant community could help researchers create similar methods for processing cellulosic biofuel feedstocks, such as corn stalks and grasses.

The researchers are discovering, however, that both the beauty and the challenge of the leaf-cutter ant garden lie in its complexity. A peek into UW-Madison's resident colony in the Microbial Sciences Building reveals a metropolis of brown insects bustling around the pale, pitted surface of the fungus garden, many with leaf sections held aloft. The strong resemblance to a small city drives home the point that energy production in such a meticulously coordinated system would be difficult to replicate in a lab or a bio-refinery.

"In an industrial setting, you need a system that's reproducible, sustainable, controlled — and that produces a consistent level of ethanol," Suen says.

A potential alternative to re-creating these natural processes is to extract, replicate and purify biomass-degrading enzymes synthetically. New enzymes could be added to known combinations and tested for their ability to break down biofuel feedstocks. However, this process can be time-consuming and costly.

To put their findings in perspective, the researchers plan to study other insects in addition to ants, including certain species of termites and beetles, which also act as gardeners in fungal communities. They hope that a better understanding of these complex systems will help them share their biomass-degrading secrets with bioenergy researchers.

"It's difficult to think that we can actually find a process that improves on nature," says Aylward, "so it probably makes sense to learn from it."