“There is no practical use for isolated natural lignin–it is only used to study the structure and properties of that component of wood. In any case, the extraction process is far too tedious and expensive.”
– John M. Harkin, Chemist – from “Lignin and Its Uses,” July 1969 Research Note, Forest Products Laboratory, US Forest Service
From a microbial ecology perspective, biomass from the forest floor must be decomposed, and turned over. Lignin, a major structural component of trees and other plants, tends to resist that process.
‘Recalcitrant” is the word Dr. Bill Mohn, a professor in the department of microbiology and immunology uses to describe the chemically complex and irregular byproduct of one of British Columbia’s biggest industries. Cellulose has made an easy transition from its standby use in pulp and paper to other products, but lignin has been enduringly difficult to degrade and transform into useful products.
Lignin-based fuel pellets
As a result, lignin has typically been stockpiled, or burnt as-is, as a low-grade fuel. Up to 70 million tonnes have been disposed of in this way on an annual basis. Internationally, the market for pelletized lignin has been catching fire. Supporters say that used as biofuel, lignin pellets will reduce greenhouse gas emissions and use up wood products that would have otherwise been wasted. Critics contest the environmental claims, and maintain such projects could promote wasteful logging.
Over the past 11 years, Mohn along with LSI colleagues Dr. Lindsay Eltis and Dr. Steven Hallam have worked on an ambitious program to harness microorganisms to break down lignin into sustainable chemicals, biofuels, and building materials.
“It takes a microbial community to degrade it,” says Mohn. “One of the things we’ve been looking at is bacteria that can degrade lignin. I’m coming to the conclusion that in nature, bacteria alone probably do not extensively degrade lignin. We’re not finding much evidence for it. It really looks like fungi are the main degraders of intact lignin, with bacteria acting as scavengers.”
Nature’s biorefinery at work
Walking in the forest, what would nature’s biorefinery look like?
“What happens is fungi will break down the lignin – not because they want to use it, but so that they can get out the more-easily digestible cellulose and hemicellulose,” says Mohn.” They’ll only partly degrade the lignin and just leave these degradation products. You can imagine you’re eating a steak and you go after the nice, tender parts, and leave the bone and gristle. That’s what fungi are doing with wood.
“One thing you will often see are bracket fungi on trees and logs,” he adds. “These are the fruiting bodies that are equivalent to mushrooms. That means there’s a whole hyphal (branching filaments) network, growing throughout the log, where the fungi have already extensively degraded the wood, so now they’re producing fruiting bodies and a ton of spores. You might also see either brown rot, or white rot, where a log has been reduced to this this very fragile, dark brown or white, cracked state that you can tear apart with your fingers–that’s the work of two different fungal processes. In both cases, the cellulose is gone, and they’ve partly degraded the lignin to get at it, leaving behind this sort of brittle skeleton.”
Unlike cellulose, and hemicellulose, which are large molecules made mainly of sugars, lignin is a large molecule made out of aromatic monomers. “There are lots of reasons to think that bacteria basically scavenges the aromatic residual material from lignin, after the fungi have broken it down,” says Mohn. “We’ve done experiments with synthetic C-13 labeled lignin, where we see that if you incubate that in a soil microcosm, it is rapidly assimilated mainly by bacteria, and not fungi. In this model system, the fungi are not bothering with lignin breakdown products.
“So, from our microbial ecology point of view, the fungi are the first degraders, and leave the residual lignin for the bacteria to scavenge. Bacteria have all kinds of pathways for this range of aromatic material that the fungi leave behind. That’s now the focus in our lab–looking at those catabolic pathways to degrade aromatic compounds.
Focusing on bacterial pathways
From a biorefinery perspective, what really seems to look promising is to use chemical catalysts to initiate breakdown of lignin, says Mohn, who counts his current project as the third focusing on lignins.
Previously, researchers looked for fungal enzymes to break down lignin. So far, that has not been very practical. “What seems to work best are chemical pretreatments–oxidative catalytic fractionation, for example–followed by a bacterial biocatalyst that can transform the mishmash of aromatic material from lignin breakdown. What you want to do, is funnel that into one or a few valuable chemicals. We want to develop bio-catalysts that can do that for us,” adds Mohn.
Collaborators including UBC Forestry’s Dr. Scott Renneckar, do the chemical treatment of lignin.“They tell us what comes out of it, and we look for bacteria that can use those chemicals,” adds Mohn. Gara Dexter, a PhD student in the Mohn lab, has discovered a pathway for the degradation of acetovanillone, one of the main aromatic compounds that comes from oxidative catalytic fractionation of lignin–the separation of the polymeric sugar and lignin fractions in the presence of a catalyst that promotes cleavage of the lignin into aromatic monomers.
Established techniques identify new pathway
“This pathway allows the organism to grow on acetovanillone, and it merges with another pathway for vanillin and vanillate,” says Mohn. No one had previously shown how bacteria degrade acetovanillone. Dexter used selective enrichment, a low-tech approach, to find acetovanillone degraders. She made bacterial medium with acetovanillone as the only growth substrate, and inoculated it with a bit of compost, which contains a rich microbial community. After growing and transferring the enrichment cultures a few times, she isolated bacteria that grow on acetovanillone. She then used high-tech approaches, genomics, transcriptomics and bioinformatics, to identify the genes, the enzymes, and the pathway for acetovanillone degradation.
“That’s all worked really well, so we have this new pathway, and we’re working on the paper right now. We’ve done this in the past for many other pathways, especially for aromatic compounds, so that’s kind of a recurring theme in my research going back 20 years.
“After the necessary pathways are found, biocatalysts need to be developed, where you put these enzymes together into a cell that’s optimized to do what you want,” says Mohn. “Right now, lignin is mostly burnt as a waste product – essentially, a low-grade fuel. Whereas, you know, it could be converted into some really expensive chemicals.”
Looking to the future, Mohn hopes approaches emerging from LSI labs will enable industry to extract the most valuable range of products that it’s possible to obtain from lignin. This could encourage repurposing of shuttered pulp mills in communities hard hit by shifts in the appetite for traditional forest products – or new biorefineries that use chemical and biological catalysts to generate chemical products from biomass, rather than petroleum.
“On Earth Day, I’ll be hoping that biorefineries will be one solution to our many critically urgent environmental challenges,” concludes Mohn.
This work has received support from Genome Canada, Genome BC, and NSERC.