Browsing by Author "Van Heerden, Andrea"

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    Assessment of wood degradation by Pycnoporus sanguineus when co-cultured with selected fungi
    (Stellenbosch : Stellenbosch University, 2007-03) Van Heerden, Andrea; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on plant surfaces. Similarly, on fallen trees an ecological succession of different fungal species is known to occur during wood degradation. Some of these fungi may be pioneer fungi contributing to the initial degradation process, while others may be yeasts associated with the fruiting bodies of macro-fungi which in turn are able to utilize the more recalcitrant polymers in wood. Previously, it was revealed that an increase occurs in the wood degradation rate of certain white-rot fungi when co-cultured with selected yeast species. A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus sanguineus. It was found by some that this fungus is capable of selective delignification while growing on the wood of poplar trees, while other authors found a simultaneous delignification pattern on Eucalyptus grandis trees. In the latter case cellulose and lignin are degraded simultaneously. We were interested in how yeasts occurring on the surface of P. sanguineus fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood degradation by this white-rot fungus. Restriction Fragment Length Polymorphisms (RFLP) analyses were used to obtain an indication of the species composition of the culturable yeast community associated with fruiting bodies of P. sanguineus. The impact of the most dominant of these yeasts species, i.e. Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood degradation by P. sanguineus was then determined by analyzing the major wood components after growth of co-cultures on hot water washed E. grandis wood chips. Co-cultures of P. sanguineus with the other fungi were prepared by inoculating the wood chips, contained in solid state bioreactors and supplemented with molasses and urea, with the an appropriate volume of fungal inoculum, resulting in an initial moisture content of 60%. After two weeks of incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp industry, were then employed to determine the percentage cellulose, Klason Lignin, as well as polar and solvent-borne extractives in the chips. The resulting data were analyzed using box plots, as well as biplots. No degradation of Klason lignin was observed, while the percentage cellulose did decrease during fungal degradation. Taking into account the inherent shortcomings of the Klason Lignin determination, the results supported the findings of others that P. sanguineus shows a simultaneous delignification pattern while growing on E. grandis wood. In addition, it was found that the yeasts played no significant role in the degradation ability of P. sanguineus, while A. flavipes showed an antagonistic effect on P. sanguineus with respect to cellulose degradation. However, it was clear that the analytical methods used in this study were inadequate to accurately determine fungal degradation of wood. In addition, it was obvious that the methods used did not distinguish between fungal biomass and wood components. Nevertheless, the methods provided us with a fingerprint of each culture growing on E. grandis wood, allowing us to compare the chemical composition of the different cultures and the un-inoculated hot water washed wood chips. The question, therefore, arose whether the effect of a particular coculture, on the chemical composition of wood, differs between tree species. Consequently, chemical alterations in different tree species, induced by a P. sanguineus / A. flavipes co-culture, were investigated in the next part of the study. Wood chips originating from four tree species, i.e. Acacia mearnsii, Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with this co-culture. The culture conditions and subsequent analyses of the wood components were the same as in the first part of the study. From the box- and biplots constructed from the resulting data, it was clear that the chemical composition of each tree species were altered in a different manner by the coculture. Lignin content showed an apparent increase in A. mearnsii, while E. dunnii showed a decrease in cellulose content. The results indicate that wood of different tree species are degraded in a different manner and this phenomenon should be taken into account in selecting fungi for biopulping.
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    Interactions of the lignicolous fungus Coniochaeta pulveracea
    (Stellenbosch : Stellenbosch University, 2015-12) Van Heerden, Andrea; Botha, Alfred ; Van Zyl, Willem Heber ; Cruywagen, C. W.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Coniochaeta pulveracea is a microcyclic dimorphic lignicolous fungus that has mostly been isolated from the woody cauloplane. Since the majority of studies conducted on this fungus were taxonomic investigations that focused on macro- and microscopical characterization little is known about their eco-physiology. The latter include morphogenesis as a result of changing environmental conditions, as well as interactions with other fungi, such as yeasts, also occurring on the woody cauloplane. As such, the overall aim of this project was to study the role of Coniochaeta pulveracea’s physiology and morphogenesis in relation to its interactions on the woody cauloplane, including symbioses with yeasts originating from this environment. A representative of the microcyclic fungus C. pulveracea was isolated from a decaying Acacia tree and subsequently identified using morphology, as well as sequence analysis of the D1/D2 (600 – 650 bp) region of the large subunit ribosomal deoxyribonucleic acid gene complex. Plate assays for extracellular hydrolytic enzyme activity indicated that this C. pulveracea isolate, as well as representatives of its closest relatives including Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis, were able to produce cellulase and xylanase. All representatives were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by filamentous growth of Coniochaeta may enhance growth of yeasts previously isolated from wood degrading fungi or decaying wood. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases. Since the above-mentioned interactions were studied whilst the Coniochaeta strains were mostly in their filamentous growth stage, microcycle conidiation of C. pulveracea strains and representatives of the closely related species, C. boothii and C. subcorticalis was also studied under different environmental conditions. The strains were found to exhibit hyphal growth on solid substrates and underwent a dimorphic switch to produce microcycle conidiation upon transfer to a liquid medium which differed in physico-chemical composition compared to the original solid medium. Factors that were found to contribute to this dimorphic switch were temperature, pH and the presence of complex nitrogen sources such as casamino acids and peptone in the medium. However, C. pulveracea showed intraspecific differences with regard to its response to changes in the physico-chemical environment. The interactions of microcyclic Coniochaeta strains with selected yeasts, such as representatives of Meyerozyma guilliermondii and Cryptococcus neoformans, were also studied in complex liquid media and it was found that, depending on medium composition, the microcyclic Coniochaeta exerted different effects on the different yeasts strains. In some co-cultures a positive effect on yeast growth was observed, while in other cases microcyclic Coniochaeta inhibited yeast growth.