Investigation of Biological Process for the Conversion of Bark Biomass to Bio-based Polyphenols

Abstract

Due to increasing waste production and disposal problems arising from synthetic polymer production, there is a critical need to substitute these materials with biodegradable and renewable resources. The concept of green polymers has become more appealing due to the presence of large volumes of processing residuals from the timber and pulp industries. This, in turn, supports the idea of developing new polymers based on bark extractives. In this thesis, three comparative treatments i.e., enzymatic, alkaline, and UV/H2O2, have been conducted for the extraction of beetle infested lodgepole pine (BILP) and mixed aspen barks polyphenolic extractives. Use of laccases as biocatalysts to affect and enhance the catalytic properties of enzymes has been shown to be a promising solution for bark depolymerization. Furthermore, laccases are suitable for biotechnological applications that transform bark biomass into high valued bark biochemicals. The industrial and biotechnological application of ligninases is constantly increasing due to their multiple uses and applications in a diversity of processes. Bark depolymerization was conducted in submerged fermentation (SF) and we identified polyphenols/polyaromatic compounds after four weeks when the production media (PM) was induced with 50mg/100ml of each type of bark during the lag-phase. During SF where honey was used as a natural mediator substitute (NMS) in the PM, laccase activities were about 1.5 times higher than those found in comparable cultures without honey in the PM. These samples were analyzed by GC-MS. The laccase enzyme was purified using UNO sphere Q-1 anion exchange chromatography and the molecular weight was determined to be ~50kDa on 10% SDS-PAGE and laccase kinetic parameters including maximal velocity (Vmax), Michaelis constant (Km), and turnover number (Kcat) were calculated from a Lineweaver Burk plot. All calculated kinetic parameters of the laccase activity are substrate (ABTS) specific. Py-GC-MS analysis of bark showed differing effects of fungal activity on bark composition. Polyphenolics were separated in reverse-phase mode using HPLC with two selected wavelengths of 290 and 340 nm to improve separation. The replacement of conventional natural mediators (NM) by monofloral honey in production media, and investigation of the effect of fungi-derived laccases on bark polyphenols are studied for the first time by this thesis work.Ph.D