(129c) Douglas-fir Tannin Inhibition of Trichoderma Reesei Cellulase

Oleson, K., University of Washington
Sprenger, K., University of Washington
Pfaendtner, J., University of Washington
Schwartz, D. T., University of Washington
Forestry residues from timber harvest and forest thinnings are an abundant potential source of biomass for biofuels. This material is comprised of braches, broken or defective tree parts, tops, and trees not meeting grade specification. Biofuel production from forestry residues is currently being investigated at the pilot plant production level for processes based on pretreatment, saccharification, and fermentation. Finding ways to reduce the price of every step in the process will help make this technology reach commercial production.

Trees have evolved to contain a variety of different molecules that defend the tree from degradation. This broad class of molecules are often called “extractives”, and they can make up 5 - 25% of the dry mass of forestry residues1. The tannins in Douglas-fir bark are the most potent extractive inhibitor and can be abundant in bark rich feedstocks. Better understanding of how tannins cause inhibition can help us adapt biofuel processing to mitigate this inhibition.

In this study we will show how Douglas-fir tannins inhibit the cellulase Cellobiohydrolase I (Cel7a) from Trichoderma reesei. This cellulase can make up 55% of the enzyme mass in a cellulase cocktail2 and is noted for its ability to hydrolyze crystalline cellulose into cellobiose. Experimental saccharification results show that inhibition from Douglas-fir bark tannins can reduce cellobiohydrolase activity 20% for high concentration bark feedstocks. The binding of tannins onto Cel7a are being characterized through isothermal titration calorimetry and this information is paired with molecular dynamics simulations to identify how a Douglas-fir tannin binds to Cel7a. Tannin binding to both the carbohydrate binding module and the catalytic core is being investigated via molecular simulations.

As biofuel processing considers forest residues as a renewable feedstock, it is necessary to devise process strategies to remove or neutralize tannin inhibition, or create cellulases resistant to their inhibitory effects. Additional process steps or chemical additives could increase saccharification yields with the downside of additional processing costs, whereas inhibition resistant cellulases would make process modification unnecessary. This work provides molecular level information that can be used identify possible residues for mutagenesis to make cellulases tannin-resistant.

1. Oleson, K. R.; Schwartz, D. T., Extractives in Douglas-fir forestry residue and considerations for biofuel production. Phytochemistry Reviews 2016, 15, (5), 985-1008.

2. Evans, B. R.; Margalit, R.; Woodward, J., VERATRYL ALCOHOL OXIDASE ACTIVITY OF A CHEMICALLY-MODIFIED CELLULASE PROTEIN. Archives of Biochemistry and Biophysics 1994, 312, (2), 459-466.