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Emerging enzymes: Synbiomics integrates pulp mill data into biocatalyst toolkit

Enzymes are being used by the pulp and paper industry to reduce energy and chemicals by breaking down or degrading the polymeric components of woody biomass (Figure 1, above) into smaller, simpler molecules.

But there are other kinds of enzymes that can modify or add chemical groups to cellulose, hemicellulose or lignin, so that they can be used in higher-value applications.

This is the premise of Synbiomics, a large-scale research project supported by Genome Canada, Ontario Genomics and Genome BC led by Dr. Emma Master of the University of Toronto and Dr. Harry Brumer from the University of British Columbia.

This is a four-year, $10-million project with research conducted at five universities (Concordia, Queen’s, Toronto, UBC in Canada, and Aalto University in Finland) and industry partners Canfor, DuPont, EcoSynthetix, Rayonier, InnoTech, IGPC, Millar Western, West Fraser and Fortress Advanced Bioproducts.

The power of enzymes

Nature is smarter than us. Whereas we use brute force with high mechanical stresses at elevated temperatures, or harsh chemicals at high pressures, to convert wood into fibres and biochemicals, nature uses gentle chemicals such as enzymes under ambient conditions. Could we do better by learning from nature?

Enzymes are nature’s catalysts that are used to speed up chemical reactions. These catalysts are proteins made by microbes to break down wood into smaller chemical components. Familiar examples would be the bacteria in a termite’s gut or brown rot fungi that attack cellulose, leaving the darker materials behind.

Synbiomics focuses on enzyme discovery from micro-organisms that can modify and upgrade the key components of wood to create bio-polymers and materials of higher value.

Years ago, these observations suggested that the enzymes within these fungi or bacteria could be used to naturally break down wood to pulp, and reduce the cost of chemicals and energy.

Over 40 years ago, various enzymes began to be used commercially in the pulp and paper industry. Cellulases are being used to improve drainage by breaking down dissolved and colloidal material, to lower refining energy, and in the production of micro-fibrillated cellulose (MFC) and cellulose nano-crystals (CNC).

Enzyme use in pulp and paper

Xylanases are used in combination with cellulases in deinking to improve recycling and to reduce chemical use. They can also cleave hemicelluloses, which can reduce bleaching process chemicals by up to 30 per cent.

Lipases are used to control pitch in pulping by converting tri-glycerides to fatty acids, which are less likely to form deposits. Amylase is used to lower the viscosity of starch, esterase to break down stickies, and pectinase to reduce cationic demand of peroxide-brightened mechanical pulp.

The global market for enzymes in the pulp and paper industry has been growing to over $250 million per year.

In contrast to previous technologies that have focused on the deconstruction of wood into simpler molecules such as sugars for conversion to fuels and chemicals, Synbiomics focuses on enzyme discovery from micro-organisms that can modify and upgrade the key components of wood to create bio-polymers and materials of higher value.

Segregating hemicellulose and lignin

In kraft pulping, cellulose is the coveted product, while most of the lignin and hemicellulose is burned in a boiler to recover pulping chemicals and to generate energy (Figure 2).

Wastewater generated throughout the mill gets treated in an aerobic activated sludge process where residual woody biomass gets converted to CO2 or ends up in biosludge.

Increasingly mills are treating wastewater in an anaerobic digester to convert the biodegradable components into methane for energy (Figure 2). As shown in figure 2, if hemicellulose and lignin are segregated, enzymes could be used on these streams to increase their value.

Figure 2. Biomass fractionation in a forest biorefinery. Lightning bolts indicate stages in the process where energy can be recovered. Photo: Synbiomics

There are many challenges in this area of research but the greatest one is the complexity of the chemistry.

Diverse families of fungi and bacteria have devised their own unique chemistries to feed on lignocellulosic materials such as wood.

The vastness of the different mechanisms can be overwhelming and confusing, but the opportunity is that there are special enzymes yet undiscovered that may impart special properties (e.g., tailored miscibility, enhanced reactivity) to cellulose, hemicellulose or lignin.

By establishing enzyme screens that incorporate commercial conditions from the start, Synbiomics aims to identify enzyme and microbial technologies that can create new bio-based products.

Finding enzymes with special properties

Nature modifies and disassembles lignocellulosic material using enzymes and non-catalytic proteins that gently nudge and pry things loose.

The analogy would be the tools that a watchmaker uses to repair a fine mechanical pocket watch. By contrast, in chemical and mechanical pulping, we are using a sledgehammer after which we pick through the shattered pieces.

To mimic nature’s processes, we need genetics. Each organism has a blueprint or instructions to make proteins (enzymes) for specific purposes in its DNA.

Using sophisticated tools, it is possible to identify the strand of DNA that has the code for a known enzyme. The next step is to look for similar DNA strands that code related enzymes that could have special properties, such as new substrate preference or higher stability, using tools such as phylogenetic analysis and sequence similarity networks to analyze DNA for proteins of similar function.

To make a sufficient quantity of this enzyme, the DNA blueprint is incorporated into a host organism and sent to a bioreactor.

This is done through recombinant DNA by grafting the DNA strand into a production host, such as fungi (Aspergillus niger – black mould) or bacteria (E. coli), which are selected to produce the enzyme.

Cellulose, hemicellulose and lignin are then exposed to this enzyme to test for activity. Through this process, which can also include iterative improvement of the production host, a number of new enzymes are being discovered.

Figure 3. Protein families studied by Synbiomics (green) to upgrade lignocellulosic fractions (yellow) to make products (blue) that will be used by project partners to make coatings, adhesives and plastics. Photo: Synbiomics

Matching enzymes to lignocellulosic material

Synbiomics is uncovering enzymes that introduce new and desirable chemical functional groups to main lignocellulose fractions.

Synbiomics’ is examining monooxygenases for the ability to tailor cellulose surfaces, -transaminases and carbohydrate oxidases to upgrade hemicellulose, and test dye-decolourizing peroxidases, laccases and o-demethylases as surface modifiers of lignin to increase reactivity (Figure 3).

They are also looking at non-catalytic proteins from fungi, including hydrophobins and loosenins, which, as their names suggest, are predicted to alter physical properties (e.g., interfacial behaviour, porosity) of cellulosic materials.

Recent progress includes direct conversion of underused hemicelluloses into difunctional crosslinking molecules, and activation of industrial lignins through enzyme-mediated grafting.

Technoeconomic assessments are performed with project partners to evaluate impact and product costs.

Creating a database

Ultimately, their goal is to create a biocatalyst toolkit that will be captured in a gold-standard database called CLAE (Characterized Lignocellulosic-Active Enzymes Database at clae.fungalgenomics.ca), which will include genetic information, enzyme activity and operational conditions.

Synbiomics is integrating operational data of anaerobic bioreactors from partnering pulp mills with corresponding microbial community sequence data to provide more process stability and maximize energy recovery. Multivariate analysis is used to track the performance of full-scale anaerobic bioreactors, bringing immediate benefit to project partners.

Now entering its fourth year, the Synbiomics team is focusing on market assessment and demonstration of the most promising pathways.

Equipped with new DNA sequence analysis tools, protein production hosts and a database of biocatalysts geared towards bio-based materials engineering, the Synbiomics team is building a community of practice within Canada to support the development of emerging bio-based economies.

For more information, readers are welcome to contact Emma Master at Emma.Master@utoronto.ca.

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J. David McDonald is president of JDMcD Consulting Inc., an adjunct professor at McMaster University and a PAPTAC Fellow.

This article originally appeared in the Fall 2020 edition of Pulp & Paper Canada. Access the digital edition here.