Woody biomass is an important material for the growing bioeconomy, and has gained significant attention as a feedstock for second-generation biorefineries. Wood has traditionally been used as a building material for millennia, but due to its biogenic nature is susceptible to degradation by wood decaying fungi. The biochemistry used by these fungi to degrade wood is of interest, both from a wood protection perspective, and as potential bioprocessing tools. In Nature, wood-degrading basidiomycetes, which can be grouped as white- or brown-rot fungi, are the only organism known to fully degrade the polysaccharides of lignified woody biomass. Brown-rot fungi are unique, in that they successfully remove holocellulose without the mineralization of lignin, unlike white-rot fungi, which degrade both holocellulose and lignin. The objective of this thesis is the study of fundamental brown-rot fungal decay mechanisms for applied utilization.
This thesis describes studies on brown-rot decay from three perspectives; 1) the oxidative non-enzymatic early decay mechanisms as potential pretreatment of wood, 2) the expression of brown-rot decay associated genes on modified wood and 3) the interplay of cellulose-oxidizing lytic polysaccharide monooxygenases with hydrogen peroxide and reductants.
In Paper I the early decay mechanisms of brown-rot fungi was studied as a potential pretreatment for Norway spruce wood. We show that Norway spruce pretreated with two species of brown-rot fungi yielded more than 250% increases in glucose release when subsequently treated with a commercial enzyme cocktail. A series of experiments were performed that aimed at mimicking the brown-rot pretreatment, using a modified version of the Fenton reaction. After pretreatment, where the aim was to generate reactive oxygen species within the wood cell wall matrix, a small increase in digestibility was observed, Further experiments were performed to assess the possibility of performing pretreatment and saccharification in a single system to avoid loss of solubilized sugars, but the results indicated the need for a complete separation of oxidative pretreatment and saccharification. We conclude that a biomimicking approach to pretreatment of softwoods using brown-rot fungal mechanisms is possible, but that there are additional factors of the system that need to be known and optimized before serious advances can be made to compete with already existing pretreatment methods.
In Paper II, the aim was to determine the effect of acetylation of Pinus radiata wood (a type of wood modification), on the expression of genes involved in wood decay by brown-rot fungus Rhodonia placenta. The initiation of decay was delayed as a result the degree of acetylation, and gene expression analysis using qRT-PCR captured incipient to advanced decay stages. Once decay was established, the rate of degradation in acetylated samples was similar to that of unmodified wood. This suggests a delay in decay, rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of hydrolytic enzymes was delayed in acetylated samples compared to untreated samples. Enzymes involved in hemicellulose and pectin degradation have previously not been the focus of studies on degradation of acetylated wood. Interestingly, we observed that a CE16 carbohydrate esterase assumed to be involved in deacetylation of carbohydrates was expressed significantly higher in untreated samples compared to highly acetylated samples. We hypothesize that this enzyme might be regulated through a negative feedback system, where acetic acid suppresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously reported, resulting in delayed initiation of decay.
In Paper III we purified and characterized a recombinant family AA9 lytic polysaccharide monooxygenase from Gloeophyllum trabeum, GtLPMO9B, which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid and 2,3-dihydroxybenzoic acid (2,3-DHBA). When using standard aerobic conditions typically used in LPMO experiments, the former two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, sub-stoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2. At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2 and this ability overlaps entirely with endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a co-substrate of LPMOs, and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant
