6-07: Lignin distribution measurements via confocal laser scanning microscopy

The presence of lignin in a cellulosic matrix reduces the efficiency of hydrolyzing enzymes by acting as a physical barrier to heterogeneous catalysis and by presenting surface sites for nonproductive adsorption.  For a given substrate, incremental delignification steps will invariably increase the amount of monomeric sugar released by a constant enzyme dose.  However, two substrates that arrive at the same bulk lignin content using different treatments can show drastically different enzymatic hydrolysis efficiencies.  A speculation is proposed that differences in hydrolysis efficiencies for such samples are related to lignin distribution throughout the substrate matrix.  A suitable technique for measuring lignin distribution needs to be identified before any hypotheses related to the role of lignin distribution during enzymatic hydrolysis can be tested.   

Confocal laser scanning microscopy (CLSM) was evaluated as a tool for measuring lignin distribution.  Using a lignin specific fluorochrome the CLSM can analyze fibers and produce images of lignin distribution across the cell wall. Preliminary work was performed on a series of softwood samples with approximately equal bulk lignin contents (12-14%) produced via different chemical delignification techniques; kraft and kraft followed by oxygen, ozone, or chlorite.  After analyzing cell walls from each set, the standard error in the distribution measurement is approximately 10%.  Relating lignin distribution to enzymatic hydrolysis performance, there seems to be slight advantages of removing outside surface lignin (kraft v. kraft-ozone; 32% v. 42%) but larger gains occur if the treatment removes lignin from the bulk of the cell wall (kraft v. kraft-oxygen/kraft-chlorite; 32% v. ~60%).