Adapted from a UC Santa Barbara news release by Sonia Fernandez

It’s a tough job, but someone’s got to do it. In this case, the “job” is the breakdown of lignin, the structural molecule that gives plants strength and rigidity. One of the most abundant terrestrial polymers (large molecules made of repeating subunits called monomers) on Earth, lignin surrounds valuable plant fibers and other molecules that could be converted into biofuels and other commodity chemicals – if we could only get past that rigid plant cell wall.

Fortunately, the rather laborious process already occurs in the guts of large herbivores through the actions of anaerobic microbes that cows, goats, and sheep rely on to release the nutrients trapped behind the biopolymer. In a paper published in the journal Nature Microbiology, UC Santa Barbara chemical engineering and biological engineering professor Michelle O’Malley and collaborators prove that a group of anaerobic fungi – Neocallimastigomycetes – are up to the task. O’Malley is part of the Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) where she serves as the Deputy Director for Microbial and Enzyme Discovery. The mission of this group is to explore targeted ecosystems and discover novel microbes and enzymes that break down plant cell walls, and in particular the lignin within them.

“You can think of lignin as kind of a skeletal system for plants,” said O’Malley, whose research focuses on finding and accessing alternate sources of energy and chemicals from what would otherwise be considered plant waste. Additionally, she said, lignin has properties that make the plant resistant to physical degradation by enzymes and pathogens. “Lignin is really important – it provides that hardiness and structure, but it’s equally difficult to break down for the exact same reason.”

For decades it was thought that lignin could be degraded only in the presence of oxygen. “It takes time, and depends on certain chemical species – such as free oxygen radicals – that to the best of everyone’s knowledge could only be made with the help of oxygen,” O’Malley explained.

However, there have been hints all along that nature has more than one way of stripping away the lignin. In the industrial biomass world, to access the cellulose and hemicellulose behind the lignin, plant biomass typically has to undergo pre-treatment. But in the O’Malley Lab’s work with anaerobic microbes, pre-treatment has never been necessary.

“We’ve never had to extract the lignin out of there because the fungi we work with are just happy to extract the cellulose and hemicellulose on their own,” she said. “So the fact that these fungi could grow on non-pretreated plant biomass was always a feature that was unique and unusual, and we hypothesized that they must have a way of moving the lignin around.”

To find out for sure, the O’Malley Lab conducted experiments with members of the Neocallimastigomycetes group, based on genetic findings previously made by collaborators at the DOE Joint Genome Institute (JGI). Tom Lankiewicz, the study’s lead author, cultivated some of these fungi on poplar, sorghum and switchgrass biomass in an oxygen-free environment. The choice of these three types of biomass came from the various ways lignin presents itself in nature, from the flexible stems and leaves of the grasses to the more rigid wood of poplar. In addition, these plants are being eyed by DOE as renewable carbon sources to produce sustainable biofuels and bio-based products.

Then the team, along with collaborators at Great Lakes Bioenergy Research Center (GLBRC), tracked the progress of the fungi as they went to work on the tough fibers.

The researchers found that indeed, Neocallimastix californiae did break down the plants’ tough cell walls. Using nuclear magnetic resonance spectroscopy performed at JBEI, they could identify specific lignin bond breakages in the absence of oxygen.

“The nuclear magnetic resonance showed that sorghum biomass is favored by the anaerobic fungi, as compared to switchgrass and poplar,” said Yu Gao, a co-author and project scientist in the Plant Systems Biology group at JBEI. “We were excited to see almost complete breakdown of the key structural bonds between lignin monomers in the sorghum.”

“This is really a paradigm shift in terms of how people think about the fate of lignin in the absence of oxygen,” O’Malley said. “You could extend this to understand what happens to lignin in a compost pile, in an anaerobic digester, or in very deep environments where no oxygen is available. It pushes our understanding of what happens to biomass in these environments and alters our perception of what’s possible and the chemistry of what’s happening there.”

While this research proves that lignin can be broken down by fungi in oxygen-free environments, the next challenge for the researchers is to find out exactly how. Are there enzymes mediating this process? Is this a feature of anaerobes in general? Like with any intriguing research, each answer opens up more questions – questions that invite more research and fruitful collaborations.

Co-author Igor Grigoriev, a senior staff scientist at JGI, is looking forward to future work detailing the fungi’s lignin-digesting machinery and delving into the other interesting functions that these microbes have to offer. “At JGI, we’re very interested in engaging the research community into characterization of fungal genes of unknown function, especially those that are found across such a large group of fungi as Neocallimastigomycetes – the family that N. californiae belongs to. Genes that are maintained across large evolutionary distances are usually very important, because otherwise evolution efficiently eliminates what is not needed.”

JBEI and GLBRC are both DOE Bioenergy Research Centers. JBEI and JGI, a DOE Office of Science User Facility, are both managed by Lawrence Berkeley National Laboratory.

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