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Hydrophobins in wood biology and biotechnology

The main aims of this research were to study the role of hydrophobins in wood decay processes and to test the ability of these proteins to coat surfaces, particularly wood surfaces. Hydrophobins are small amphiphatic fungal proteins that are known to decrease the surface tension of moist surfaces (water/air interfaces), to help in the formation of aerial hyphae by coating them with a protein film and making them hydrophobic and to support attachment of the fungal hyphae to hydrophobic materials. The SC3 hydrophobin from Schizophyllum commune is the best studied hydrophobin until now. Another protein, SC15, is known to act supportive to the SC3 hydrophobin in the formation of aerial hyphae and their attachment to surfaces and, in the absence of the SC3 hydrophobin, it mediates low levels of hyphal hydrophobicity and attachments. Due to the existence of several SC3 hydrophobin and SC15 mutant strains, the white rot fungus S. commune was selected for this study to study the functions of these proteins in wood colonization and decay. Wood block and saw dust tests with various S. commune strains showed that both fungal monokaryons and dikaryons, including co-isogenic wild type strains, ΔSc3 mutants, ΔSc15 mutants and ΔSc3 ΔSc15 double mutants, were able to grow on beech, birch and pine wood. The obtained results indicate that neither the SC3 hydrophobin nor the SC15 protein are principally necessary for S. commune to enter into the wood, to decay the wood or to affect the strength to the wood. However, there were differences in the overall formation of surface mycelium in correlation of the presence of the SC3 hydrophobin. In most instances, the wood decay by the fungal strains were low (deciduous wood) or there was no decay (pine wood). In wood block and saw dust decay tests, the maximum decay caused by S. commune strains was 4-5% (beech and birch), and 9-16% (beech and birch), respectively. S. commune strains decolorized the pine saw dust but not beech or birch saw dust. Although there was only little or no decay in wood blocks, the strength of the wood was negatively affected by S. commune strains. The maximum strength loss caused by S. commune strains in beech, birch and pine wood was 21, 52 and 35 %, respectively.S. commune is an opportunistic pathogen on weakened trees. An infestation of a Juglans ailantifolia tree by the fungus was observed over a period of 3 years. The fungus caused over the time die off of a number of branches. In at least one branch, S. commune was not the only fungus. Next to S. commune fruiting bodies, carpophores of Trametes hirsuta were observed. Both fungi were isolated from the tree and studied in interactions. Furthermore, SC3 hydrophobin mutants and their co-isogenic wild type strains were included in the study.Interaction studies of S. commune strains with T. hirsuta on agar plates resulted in the deadlock phenomenon in which a barrier is formed between the fungal strains hindering the opponents in further growth. Subsequently with time, the deadlock situation changed by the partial replacement of S. commune by T. hirsuta. As a stress reaction during the combat interaction, a blue pigmentation was observed at the fungal interaction zone. All the S. commune strains (monokaryotic and dikaryotic wild type strains and hydrophobin monokaryon mutants) except the SC3 hydrophobin mutant dikaryon showed the barrier formation and the pigmentation in the interaction zone, indicating that in principle the SC3 hydrophobin is not involved in the stress reactions of S. commune.Further, differences between a S. commune wild type strain and a coisogenic hydrophobin mutant were studied in liquid cultures in relation to the produced protein and polysaccharide. In the liquid cultures, as determined by the FTIR (Fourier Transform Infrared) spectroscopy analysis, there were significant differences between the mycelium of the wild type strain and the mycelium of the hydrophobin mutant with respect to protein and polysaccharide contents. The protein content were higher in the wild type strain than in the mutant, whereas the polysaccharide content was higher over the time in the mutant strain. When growing on beech wood, the mycelium of different S. commune wild type strains and the co-isogenic hydrophobin mutants on the surface of the wood were discriminated by FTIR spectra submitted to a cluster analysis. Inside the wood, however, the FTIR spectra of the mycelia of the different S. commune wild type strains and hydrophobin mutants clustered together. There were no significant differences noted amongst the values from protein peak area values calculated from the FTIR spectra in between the tested strains on the surface of beech wood. On the surface of wood, the polysaccharide peak area value of the dikaryotic Sc3 hydrophobin mutant ∆Sc3 4-39 x ∆Sc3 4-40 was significantly higher than that of the monokaryotic mutants ∆Sc3 4-39 and ∆Sc3 4-40.SC3 hydrophobin of S. commune was purified from fungal mycelium with yields of 0.1% (w/w, 0.5 mg of SC3 hydrophobin from 500 mg of mycelium) and used to coat hydrophobic and hydrophilic materials. Hydrophobic Teflon and form-war film turned hydrophilic and hydrophilic glass turned hydrophobic upon coating with SC3 hydrophobin. Further, the amphipathic nature of films of this protein was tested on solid beech wood. Beech wood turned hydrophilic or hydrophobic, depending on the concentration of SC3 hydrophobin applied. Lower concentrations (50, 25, 15 μg/ml) of SC3 resulted in hydrophobic behavior and higher concentrations (100 to 400 μg/ml) in hydrophilic behavior of beech wood. The surface activity of other class I hydrophobins from different fungal species were also tested on the beech wood. About 0.3 and 0.1% (w/w) purified hydrophobins were obtained from the mycelium of Coprinopsis cinerea and Pleurotus ostreatus cultures, respectively. The hydrophobins of both fungi followed the same pattern of surface activity on wood than the Sc3 hydrophobin. Results from ATR-FTIR (Attenuated Total Reflection - Fourier Transform Infrared) spectra of hydrophobin-treated beech wood showed a higher intensity in the peak associated with amide I band of proteins indicating the binding of hydrophobin protein to the wood components.