ESR-measurements show a high number of radicals in the laccase treated samples compared to untreated control fibers (Fig. 2). Drying of laccase treated fibers in the flash drier lowered the level of radicals, but the number of radicals was still significantly higher compared to the untreated control. The level of radicals shows that even though no free liquid water was present during the process, the enzyme was active and catalyzed a thorough oxidation of the fibers. This is an important observation as it shows that the enzyme can be applied under industrial type conditions, with a moisture content of 50–60%. In a previous work [15], laccase was added to fibers at the end of the blow line in order to use the existing processing equipment for MDF-boards, and to facilitate adaptation to an industrial process. The properties of the fiber boards were not improved and no excess radicals could be detected, probably because the high temperature, pressure and shear forces in the blow line inactivated the enzyme.
An important question in laccase catalyzed bonding is whether covalent bonding of the lignin component is actually taking place. The difficulty in analyzing the lignin properties in hot-pressed fiberboards is among others, that a number of process parameters such as temperature, time, pressure, and moisture content will influence the lignin flow and degree of cross-linking. As the series of boards were obtained from a continuous process using consistent processing parameters, it was possible to do a valid comparison of the lignin molecular mass in untreated and laccase treated boards. Fig. 3 shows GPC chromatograms of lignin isolated from fibers and MDF boards treated with and without laccase, respectively. The corresponding values for Mw and Mn are shown in Table 4. It was assumed that during disintegration of the boards for preparing samples, rupture of inter-fiber bonds dominated over cleavage of fibers so the exposed surface resembled the fiber surface. This is supported by the similar retention time of the peak maximum found for lignin isolated from pulp and boards.