This Small Business Innovation Research (SBIR) Phase I project helps unlock the potential of forests to provide sustainable, carbon-neutral raw material for much of the nation?s energy needs. Tethys will search for enzymes specific for ether bonds between lignin and the hardwood hemicellulose, xylan. A fluorogenic model of xylan-lignin ether bonds will be synthesized that fluoresces when the xylan-lignin ether bond is broken. It will be used to bioprospect for xylan lignin etherase (XLE) activity in culture collections and sites of hardwood decay in Maine forests. Putative positives will be tested for autofluorescence, XLE solubility, and XLE cofactor requirement. Results of this project will advance understanding of etherase enzymatic mechanisms, increase knowledge about the chemistry of sugar-lignin ether bonds, and contribute to the understanding of wood decay.
The broader/commercial impacts of this research are two-fold. First, development of a novel, environmentally friendly process for pulp products becomes possible. The new process will also create new feedstocks for biofuels and platform chemicals. Second, the development of the new method means that (i) wood can be used to meet a significant portion of America?s energy needs; (ii) corn slated for ethanol production can be directed to food products; (iii) energy and industrial chemicals used in pulp and paper mills will be reduced and (iv) America?s pulp and paper industry (and the rural towns where mills are located) will receive a much needed economic boost from reduced costs and increased revenues from valuable new products.
This Small Business Innovation Research (SBIR) Phase I project was undertaken to help unlock the potential of forests to provide sustainable, carbon-neutral raw material for much of the nation’s energy needs. Most of a tree's mass is composed of three large molecules, cellulose, hemicellulose and lignin. Two of those molecules (cellulose and hemicellulose) are composed of useful sugars. The third component, lignin, is chemically very different and unlike sugars, provides significant heat energy when burned. However, the three molecules are chemically bound together to create a strong matrix. Some of those chemical bonds are difficult to break, and the current practice of using of harsh chemicals to break them creates environmental hazards and costs. Most molecules and chemical bonds found in nature can be broken down by enzymes. Enzymes are specific and environmentally benign molecules made by living organisms. Tethys Research LLC searched for enzymes that specifically cleave ether bonds (a type of strong chemical bond common in wood) between lignin and the hardwood hemicellulose, xylan. A chemical resembling one type of xylan-lignin ether bonds was synthesized. Unlike natural compounds containing ether bonds in hardwoods, this chemical fluoresces when the ether bond is broken. The chemical was used to search for sources of an enzyme activity that can break this ether bond between lignin and xylan. The search encompassed microorganisms already isolated and in existing culture collections and microorganisms gathered from sites of hardwood decay in Maine forests. From culture collections, 12 bacterial and 28 fungal strains were chosen. 30 samples were taken from Maine sites of hardwood decay. All of these microbes were grown under conditions in which only those microbes that could break the ether bond could grow. Twelve microbes were able to grow by breaking the ether bond. Most of those microbes able to generate fluorescence from the substrate were fungi. Many fungi have non-enzymatic mechanisms for breaking down wood. These methods are indiscriminate and not useful for industry. Most of fungal strains chosen from culture collections had never been screened for these mechanisms. As a consequence, fungi that appeared to be positive may not be truly positive. Work to eliminate all false positives due to non-enzymatic mechanisms is continuing, but fungal positives were not considered good candidates for further testing. All putative positives were tested to see if some of the fluorescence seen in the cultures came from a source other than splitting the ether bond. Additionally, they were tested to see if the enzyme activity was freely soluble in the medium or if it was attached to the outside of cells. Finally, they were tested to determine whether the enzyme activity required outside energy to break the ether bond. The need for an energy source or the attachment of the enzyme activity to a cell changes the way the enzyme and its parent microbe must be handled in the laboratory and used in industry. One candidate isolated from a Maine forest, E518, was partially characterized. This novel microorganism appears to make a freely soluble enzyme activity that does not require an additional energy source. It appears to be an excellent candidate for further development. Intellectual Merit: Results of this project will advance understanding of etherase enzymatic mechanisms, increase knowledge about the chemistry of sugar-lignin ether bonds, and contribute to the understanding of both wood structure and wood decay. The broader/commercial impacts of this research are two-fold. First, for pulp mills, development of a novel, environmentally friendly process for pulp products (ie. paper) becomes possible. The new process will also create new feedstocks for biofuels and platform chemicals. Second, for biorefineries, the development of the new method means that (i) wood can be used to meet a significant portion of America’s energy needs; (ii) corn slated for ethanol production can be directed to food products; (iii) energy and industrial chemicals used in pulp and paper mills will be reduced and (iv) America’s pulp and paper industry (and the rural towns where mills are located) will receive a much needed economic boost from reduced costs and increased revenues from valuable new products.
