Surface oxidation precedes depolymerization of lignocellulosic biopolymers during fungal decay. We have observed that the metachromic fluorescent dye acridine orange (AO) indicates the oxidation state of wood surfaces. Fresh wood adsorbs AO in a manner that results in green emission. In the first three panels of the figure, thin sections exposed to a white-rot fungus, Phanerochaete chrysosporium exhibit fluorescence that begins as green and progresses to red by seven days. Acid-chlorite treatment and UV exposure also result in red emission (panels 4,5). AO has found wide application as a microscopy stain, due to its metachromic behavior. The association of poly nucleic acids with AO appears to have two mechanisms. With double-stranded DNA and RNA, AO is electronically isolated and emits green. In contrast, charged interactions dominate interactions with single strands, which favors stacking of AO molecules. Stacking results in a combined electronic state between two AO molecules giving a red fluorescent emission and fluorescent energy transfer (FRET) quenching of any isolated AO molecules within the Förster distance. We wish to understand the chemical modifications that are responsible for the shift in AO-wood interaction. Apparently, unmodified wood adsorbs AO as isolated molecules. Microcrystalline cellulose, Sigmacell, and milled wood enzyme lignin fluoresce green. Carboxy methylated agarose beads, however, fluoresce red. These observations lead to the hypothesis that carboxylic acid sites are important for associations leading to red fluorescence, which may be produced during oxidation. This is consistent with isothermal titration calorimetry data, which indicate strong binding sites and elimination of red fluorescence by competitive adsorption by calcium. Fluorescence lifetime measurements also indicate FRET between the green and red fluorescent species on wood. Supported by DE-SC0006929 from the U.S. DOE, BER

Exquisite sensitivity of Acridine Orange to lignocellulosic oxidation and mechanistic investigation

249th National Meeting (2015)

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