Decay mechanisms of brown-rot fungi: Dissertation

Brown-rot fungi, e.g. the dryrot fungus (Serpula lacrymans), are the most harmful microorganisms in timber in service in Finland and in temperate regions. Brown-rot fungi cause wood decay primarily by attacking the carbohydrates of the cell walls, leaving lignin essentially undigested. At the initial stage of decay, brown-rot fungi seem to operate by a mechanism which causes extensive changes in the wood cell wall structure, leading to a rapid decline in strength properties. It has been suggested that brown-rot fungi produce a low molecular degradation agent which is capable of penetrating into the cell wall structures. Research on the brown-rot decay mechanism has focused on identifying the low molecular weight compounds enhancing cellulose depolymerization in the initial stages of brown-rot decay. The production of extracellular hydrogen peroxide by brown-rot fungi was qualitatively and quantitatively detected by using chromogen ABTS (2,2-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) and horseradish peroxidase. Two brown-rot fungi, Poria placenta and Serpula lacrymans were found to produce hydrogen peroxide on solid spruce sawdust medium. The production of hydrogen peroxide by P. placenta was observed in liquid culture media containing either amorphous or crystalline cellulose as a carbon source. The production of hydrogen peroxide and oxalic acid occured to be simultaneous on crystalline and amorphous cellulose, and the highest amount of hydrogen peroxide was detected on amorphous cellulose. The production of hydrogen peroxide by P. placenta depended on the formation of acid pH of the culture medium. The accumulation of hydrogen peroxide was preceded by a drop of pH of the culture medium, which was due to the production of oxalic acid. As a small diffusible molecule, hydrogen peroxide can act as a degradation agent providing reactive hydroxyl or other oxygen radicals through the Fenton type of reaction which leads to the degradation of wood cellulose. The enzymatic hydrolysis of wood polysaccharides by Gloeophyllum trabeum was detected by following the production of cellulases, hemicellulases and extracellular protein on spruce sawdust or microcrystalline cellulose media. The production of endo-beeta-1,4-glucanase and endo-beeta-1,4-xylanase was most pronounced on both media. Brown-rot fungi differ from other cellulolytic fungi by lacking enzyme activites needed for the enzymatic degradation of crystalline cellulose. The endoglucanase activities produced by P. placenta were most pronouced on glucose medium, thus indicating that the brown-rot cellulases are constitutive and not repressed by glucose. The degradation of hemicellulose is believed to be an important initial reaction taking place in brown-rot decay. The endo-beeta-1,4-xylanase produced by G. trabeum was purified and characterized and appeared to be a protein with a molecular mass of 39 - 42 kDa. The endo-beeta-1,4 -xylanase of G. trabeum has its pH optimum at pH 4 and it is found to have a very high temperature optimum (80 oC). A biomimetic approach was used to clarify the role and importance of the Fenton-type reaction in carbohydrate degradation by brown-rot fungi. Spruce sawdust and microcrystalline cellulose were modified by H2O2/Fe(II) treatment. The degree of hydrolysis of the pretreated spruce sawdust was clearly increased with complete cellulase (Econase), purified endoglucanase from Trichoderma reesei and endoglucanase of P. placenta. The oxidative pretreatment of microcrystalline cellulose decreased the hydrolyzability of pure cellulose with complete cellulase, but the hydrolyzability with both purified endoglucanase of T. reesei and endoglucanase from P. placenta was increased. Thus, after oxidative treatment with Fenton s reagent the hydrolysis of both pure cellulose and wood was substantially increased.