Anne Steenkjær Hastrup:
Aspects of Cellulose Degradation by Brown Rot Fungi

Date: 15-06-2011    Supervisor: Bo Jensen

The body of my PhD work is focused on aspects of brown rot degradation and the effect on the wood structures in particular during depolymerization of cellulose. The mechanisms involved in brown rot decay can be devided into two mechanisms. An initial non-enzymatic process that opens op the structure and a following enzymatic hydrolyses of the wood that liberates digestible sugars. The two pathways are highly linked and essential for the efficient depolymerization observed during wood decay. The manuscripts presented in this thesis approach multiple facets involved in the process of biomass degradation by brown rot fungi and the effects on the wood components.

The aspects related to the non-enzymatic processes was analyzed in terms of the direct influence on cotton cellulose of the individual components involved: Iron, hydrogen peroxide, oxalic acid, and iron chelator (Manuscript 1). The study revealed high depolymerization of cellulose by the components directly involved in the Fenton reaction. The combination of iron and hydrogen peroxide, but also the individual components were able to reduce the degree of polymerization from 10,000 to less than 500 in 24 hour. The oxalic acid concentration reported from wood degraded by brown rot fungi (10 mM) also showed significant depolymerization effect. The results confirmed the capability of these low molecular weight compounds in cellulose depolymerizing and the possible involvement during incipient brown rot decay.

A related study looked further into the presence of iron-chelating and iron-reducing compounds from four brown-rot fungi: Meruliporia incrassata, Gloeophyllum trabeum, Coniophora puteana and Serpula lacrymans (Manuscript 2). This was done quantitatively and qualitatively from aliquots of liquid growth media, decayed wood extractions, and actively growing plate cultures. Iron-chelating compounds were detected from all four species with the highest reactivity in G. trabeum and S. lacrymans and lowest in M. incrassata. Iron-reducing activity was detected in liquid medium and wood extractions of all four fungi, although no activity was detected from S. lacrymans in the latter. The presence of both phenolate and hydroxamate derivate chelators was indicated in all four species with cultures of G. trabeum showed the highest concentration of extracellular chelators.

The presence and accumulation of metals for the Fenton reaction and possible oxalic acid including other aspects of brown rot decay was elaluated in a study measuring the concentration of iron, manganese, calcium, and copper in wood after colonization with the three brown rot fungi Serpula lacrymans, Meruliporia incrassata, and Coniophora puteana (Manuscript 3). The growth media were supplemented with materials present in the built environment. Serpula lacrymans caused a significantly higher uptake of iron and only little copper accumulation compared to M. incrassate, which showed the opposite tendency despite similar weight losses. The ability to extract calcium from different substrates varies between the species and is not correlated with the actual content of the particular metal in the substrate. None of the metals tested revealed what caused the increased wood decay in blocks inoculated with C. puteana and incubated in soil blocks jars in the presence of mortar, E-glass and in particular gypsum board.

The possible regulation of oxalic acid in brown rot fungi was analyzed through an evaluation of accumulation of oxalic acid related to wood decay, pH regulation, synthesis of formic acid, carbon oxalate crystals, and oxalic acid decarboxylating activity from wood extracts (Manuscript 4). Analysis indicated the presence of the oxalic acid degrading enzyme oxalate decarboxylase (EC in particular from extractions of G. trabeum. A preliminary study detected the presence of a protein with oxalic acid degrading ablitity from liquid cultures of G. trabeum. The study further sequenced a gene in G. trabeum, when blasted was found to be higly similar to sequenses known to code for ODC. This strongly indicates that G. trabeum express the enzyme known to regulate oxalic acid accumulation during growth and wood decay. Further studies on the isolated protein and detection of mRNA coding for ODC is being conducted at the moment.

The degradation of wood by fungi has been studied intensely for many years due to its importance in preservation of in-service wood and nutrient cycling in forests and other ecosystems. Lately wood and cellulose inparticular are being considered as a major potential feedstock for renewable fuels and chemicals biomass. Understanding the nature of cellulose nanostructures in the fibers of higher plants and how these structures are modified and degraded is one of the key challenges. Therefore the objective of a large part of my research was to explore the organization of cellulose on the supramolecular level before and during the breakdown to fermentable sugars through the action of brown, white and soft rot fungi and to compare the changes between fungal species and fungal groups (Manuscript 5, 6, 7, 8).

At the time of our 2007-paper there were no publications using x-ray diffraction data to describing the effects of soft rot fungi on cellulose crystallinity and crystallite size. The purpose of that study was gain understanding of the changes occurring to cellulose crystallinity during degradation by two types of decay fungi but also to develop the techniques of x-ray diffraction for investigating the structural changes of wood during decay (Manuscript 5). Results showed large disruption in the cellulose crystals in wood decayed by the brown rot fungus Meruliporia incrassata, whereas the soft rot fungus Chaetomium elatum only caused minor changes.

This lead to an elaborated study on the changes in wood crystallinity caused by three brown rot species Gloeophyllum trabeum, Coniophora puteana, and Serpula lacrymans, and a comparison of how the result relate to the changes in weight loss and wood sugar composition (Manuscript 6). All fungi were seen to increase the relative crystallinity in wood at incipient decay followed by a decrease. The structural proporties of the cellulose was found to change during the decay as an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes was observed after roughly 20% weight loss. This led us to speculate if this was due to a disruption of the outer semicrystalline layers of the cellulose microfibril.

Mechanical and biological pretreatments are used for bioremediation of wood components. A study was conducted to examine structural modifications of crystalline cellulose in wood when exposed to hot-water extraction or either brown or white rot decay using a combination of X-ray diffraction (XRD) and 13C solid-state nuclear magnetic resonance (NMR) spectroscopy (Manuscript 7). Both hot water treatments and the two brown rot fungi caused a significant decrease in the 200 crystal plane spacing (d-spacing), which was not seen for the white-rotted samples. The effect was found to be additive and suggested that the two treatment methods facilitate the decrease in d-spacing in different ways. The NMR results supported the conclusion of differing structural effects, suggesting that the hot-water extraction procedure was causing cocrystallization of existing crystalline domains, in which adjacent crystalline domains are joined together following e.g. removal of non-crystalline material separating them, while the brown rot decay was depolymerizing cellulose chains of the crystals, possibly allowing the remaining crystalline material the freedom to relax into a more energetically favorable, tightly packed state. The differences occurring to the structural propeties of wood when colonized either by brown and white rot fungi were evident (Manuscript 7) and needed further analysis to elucidate the way they alter cellulose on the molecular level. Analysis by X-ray diffraction and 13C CP/MAS NMR spectroscopy were used to compare the effects of the mechanisms employed by these two groups of fungi (Manuscript 8). The crystallinity of the wood undergoing decay by the two brown rot fungi appeared to be dependent on the stage of decay as measured by wood weight loss whereas wood degraded by white rot fungi, crystallinity was less altered but appeared to be dependent on the type of white rot, i.e. selective white rot or simultaneous white rot. However, a greater reduction in cellulose crystallinity was seen for the two brown rot fungi compared to the two white rot fungi, Samples decayed by G.  trabeum show changes in lignin content similar to the two white rot fungi. This was not observed in wood degraded by M. incrassata and revealed difference in the method of attack between the two brown rot fungi tested. As a follow up on the conclusions from Manuscript 7 on the changes in the spacing between the cellulose crystal planes the decrease in wood degraded by brown rot was confirmed, whereas changes observed in wood degraded by the white rot fungi varied according to the type. The results are suggested to be correlated to differences in degradation methods: initial non-enzymatic degradation in brown rot versus enzymatic degradation in white rot.

The results on biomimetic and biological degradation by different fungi presented in this thesis are of great interest to those seeking to optimize lignocellulose biorefineries for the sustainable production of chemicals, materials, or fuels from renewable biomass as well as those concerned with wood degradation and wood protection. The results show parameters that have implications for a further division of the species within the group of brown rot fungi, as they reveal new information on the differences present in the degradative approach and the mechanisms behind.