Thesis (MSc)--University of Stellenbosch, 2007. / 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. / AFRIKAANSE OPSOMMING: Dit is algemeen bekend dat 'n verskeidenheid fungi, insluitend giste, op plantoppervlaktes mag voorkom. Dit is ook bekend dat 'n ekologiese opeenvolging van verskillende fungusspesies tydens hout-afbraak op omgevalle bome voorkom. Van hierdie fungi mag pionierfungi wees wat bydra tot die aanvanklike afbraakproses, terwyl ander giste mag wees wat geassosieer word met die vrugliggame van makro-fungi, wat op hul beurt weer in staat is om die meer weerstandbiedende polimere in hout te benut. Dit is voorheen bekendgemaak dat daar 'n toename plaasvind in die tempo van houtafbraak deur sekere witvrot-fungi wanneer dit in ko-kulture met geselekteerde gisspesies voorkom. 'n Bekende bewoner van verrottende bome is die wit-vrotfungus Pycnoporus sanguineus. Dit is gevind dat hierdie fungus tot selektiewe delignifikasie in staat is terwyl dit op die hout van populierbome groei, terwyl ander outeurs 'n gelyktydige patroon van delignifisering op Eucalyptus grandis bome gevind het. In laasgenoemde geval is sellulose en lignien gelyktydig afgebreek. Ons was geïnteresseerd in die effek van giste op die oppervlak van vrugliggame van P. sanguineus, en die pionierfungus Aspergillus flavipes, op die houtafbraak deur hierdie wit-vrotfungus. Restriction Fragment Length Polymorphisms (RFLP) analises is gevolglik gebruik om 'n aanduiding te kry van die spesiesamestelling van die kweekbare gisgemeenskap wat met die vrugliggame van P. sanguineus geassosieer word. Die impak van die mees dominante van hierdie gisspesies, naamlik Pichia guilliermondii en Rhodotorula glutinis, asook A. flavipes, op houtafbraak deur P. sanguineus is voorts bepaal deur die analise van die belangrikste houtkomponente na die kweek van ko-kulture op E. grandis houtskyfies wat met warm water gewas is. Ko-kulture van P. sanguineus met die ander fungi is voorberei deur die houtskyfies in vaste fase bioreaktore, aangevul met melasse en ureum, te inokuleer met 'n toepaslike volume van die fungus inokulum om 'n aanvanklike voginhoud van 60% te verkry. Na twee weke se inkubasie by 30°C met konstante belugting is die skyfies ge-oes. Standaard protokol (TAPPI Standard Methods), algemeen deur die papier en pulpindustrie gebruik, is ingespan om die persentasie sellulose, Klason Lignien, asook polêre en oplosmiddel-gedraagde ekstrakte in die skyfies te bepaal. Die gevolglike data is geanaliseer deur gebruik te maak van box plots en biplots. Daar is geen afbraak van Klason Lignien bespeur nie, terwyl die persentasie sellulose wel toegeneem het tydens fungus degradasie. Met die inherente tekortkominge van die Klason Lignien bepaling inaggenome, het die resultate die bevindings ondersteun van andere wat getoon het dat P. sanguineus 'n gelyktydige delignifikasiepatroon openbaar terwyl dit op E. grandis hout groei. Daarby is dit gevind dat die giste geen beduidende rol in die afbraakvermoeë van P. sanguineus gespeel het nie, terwyl A. flavipes 'n antagonisiese effek ten opsigte van die sellulose degradering van P. sanguineus getoon het. Dit was egter duidelik dat die analitiese metodes wat in hierdie studie gebruik is, onvoldoende was om die degradering van hout akkuraat te bepaal. Daarby was dit duidelik dat die metodes nie tussen fungus biomassa en houtkomponente kon onderskei nie. Nogtans het die metodes 'n vingerafdruk verskaf van elke kultuur wat op E. grandis hout groei, wat ons toegelaat het om die chemiese samestelling van die verskillende kulture en die ongeïnokuleerde, met warm water gewasde houtskyfies te vergelyk. Die vraag het gevolglik ontstaan of die effek van 'n bepaalde ko-kultuur op die chemiese samestelling van hout van boomspesie tot boomspesie verskil. Gevolglik is die chemiese wisselinge in verskillende boomspesies, geïnduseer deur 'n P. sanguineus / A. flavipes ko-kultuur, in die volgende gedeelte van die studie ondersoek. Houtskyfies van vier boomspesies, naamlik Acacia mearnsii, Eucalyptus dunnii, E. grandis, en Eucalyptus macarthurii, is met hierdie ko-kultuur geïnokuleer. Die kultuurkondisies en daaropvolgende analises van die houtkomponente was dieselfde as in die eerste deel van die studie. Van die box- en biplots wat van die resultate getrek is, is dit duidelik dat die chemiese samestelling van elke boomspesie op 'n verskillende manier deur die ko-kulture verander is. Lignien-inhoud het ’n waarskynlike toename getoon in A. mearnsii, terwyl E. dunnii 'n afname in sellulose-inhoud getoon het. Die resultate toon dat hout van verskillende boomspesies op verskillende maniere afgebreek word en dat hierdie fenomeen in aanmerking geneem moet word wanneer fungi vir bioverpulping geselekteer word.

Thesis (PhD)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their removal of lignin from wood. In this study over 600 Basidiomycete fungi were isolated from indigenous forests as well as from commercial Eucalyptus spp. and Pinus spp. plantations in South Africa. One hundred isolates were identified to genus level. Biochemical tests were done to screen the fungal cultures for characteristics that are favourable for biopulping, e.g. low cellulase activity with concomitant high activity of ligninolytic enzymes. Various Phanerochaete isolates with potentially high ligninolytic activity were identified. Although Phanerochaete chrysosporium Burds. has previously been isolated from the indigenous forest at Knysna in South Africa, this study showed that P. chrysosporium was a natural coloniser of wood chip piles in South Africa, indicating potential for application in industry. A possible new species of Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain PP25) from decayed wood collected in Stellenbosch, South Africa, was described and illustrated. It differs from previously described Phanerochaete species in having smaller basidiospares, and in the formation of few chlamydospores on malt extract agar but more on xylose containing media. The potential of using internal transcribed spacer DNA sequences (ITS) to infer phylogenetic relationships among species of the genus Phanerochaete was investigated. Consensus phylogenetic trees could be presented, but the presence of ambiguous aligning sequences within the ITS made inferring of phylogenetic relationships within the whole genus difficult. Fifty-five South African strains of P. chrysosporium were screened for lignin peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX) enzymes. Manganese peroxidase activity was quantified on agar media. The liP and GLOX activities of 13 selected strains, including control strains and P. pseudomagnoliae (PP25), were also quantified on agar media. Differences in MnP and GLOX activities existed among the strains. Preliminary biochemical characterisation of strain PP25 indicated that the most important difference was the apparent unique regulation of ligninolytic enzymes. Under low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under high nitrogen conditions. Restriction fragment length polymorph isms of the lignin and manganese peroxidase gene DNA fragments showed variability among strains, whereas there was probably only allelic variation for the glox gene DNA fragments. Previous research has indicated xylose oxidation activity within P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose > D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a 1:1 stoichiometric relationship between H202 produced and xylose used. Xylono-1,4-lactone was converted non-enzymatically to xylonate. This suggested that the furanose form of xylose, rather than the pyranose form, is a substrate of GLOX. The production of H202 and the removal of inhibitory compounds by GLOX could enhance ligninolytic activity. . To conclude, unique strains of P. chrysosporium have been isolated from South Africa with potential biotechnological use in paper manufacturing. The relationship of P. pseudomagnoliae nom. provo to other Phanerochaete species was evaluated and light was shed on the possible role of GLOX in lignin degradation. / AFRIKAANSE OPSOMMING: Witvrot swamme is van belang vir die papier en pulp industrie omdat hulle lignin vanaf hout kan verwyder. Meer as 600 Sasidiomiseet fungi, afkomstig vanaf inheemse woude asook kommersiële Eucalyptus spp. en Pinus spp. plantasies, IS geïsoleer. Een honderd isolate is tot op genusvlak geïdentifiseer. Die isolate is biochemies vir eienskappe wat voordelig vir "bioverpulping" kon wees, bv. die gelyktydige produksie van lae sellulosemaar hoë ligninolitiese ensiemaktiwiteit, getoets. Verskeie isolate met potensieel hoë vlakke van ligninolitiese aktiwiteit is verkry. Alhoewel Phanerochaete chrysosporium Surds. vantevore in die Knysna inheemse woud in Suid-Afrika geïsoleer is, het hierdie studie gewys dat P. chrysosporium natuurlik op hope houtblokkies voorgekom, met moontlike toepasing in die industrie. Isolaat PP25, geisoleer vannaf verrottende hout te Stellenbosch, Suid Afrika, is as 'n potensieel nuwe spesie van die genus Phanerochaete beskryf en as Phanerochaete pseudomagnoliae nom. provo benoem. Hierdie isolaat verskil van ander Phanerochaete-spesies daarin dat dit kleiner basidiospore vorm en nie klamydospore op moutekstrakagar produseer nie, maar wel op media wat xilose bevat. Die potensiaal van intern getranskribeerde spasieerder ONS opeenvolging vir die aflei van filogenetiese verhoudings tussen spesies van die genus Phanerochaete is ondersoek. Konsensus filogenetiese bome kon bepaal word, maar die teenwoordigheid van varieerbare areas het die afleiding van filogenetiese verwantskappe vir die hele genus bemoeilik. Vyf-en-vyftig Suid-Afrikaanse isolate van P. chrysosporium is vir die teenwoordigheid van lignienperoksidase- (liP), mangaanperoksidase- (MnP) en glioksaaloksidase (GLOX)-aktiwiteit getoets. Vlakke van MnP-aktiwiteit is op agarplate gekwantifiseer. Vlakke van LiP- en GLOX-ensieme op agarplate is vir 13 geselekteerde isolate, insluitend kontroles en ras PP25, gekwantifiseer. Aktiwiteit van MnP en GLOX het statisties betekenisvol tussen isolate verskil. Lignienperoksidase-aktiwiteit onder lae stikstof toestande het nie statisties betekenisvol van mekaar verskil nie. Onder hoë stikstof toestande het isolaat PP25 wel verhoogde liP-aktiwiteit getoon. Restriksie fragment polimorfismes van die lignien- en mangaanperoxidase-gene het variasie getoon, terwyl waarskynlik slegs alleliese variasie vir die glox geen waargeneem IS. Rekombinante GLOX (GLOX vanaf P. chrysosporium BKM-F-1767) het xilose en glukose (D-xilose > D-glukose) geoksideer met meegaande produksie van H202. Xilose is na xilono-1,4-laktoon geoksideer met 'n 1:1 stoigiometrie tussen H202-produksie en xilose verbruik. Xilono-1,4-laktoon is nieensiematies na xilonaat omgeskakel. Bogenoemde resultaat dui aan dat die furanose vorm van xilose die werklike substraat vir GLOX is. Deur die meegaande produksie van H202 en die verwydering van inhiberende produkte word lignoliese aangehelp. Ten slote, unieke P. chrysosporium rasse met potensiële gebruik in papiervervaardiging is in Suid-Afrika geisoleer. Die genetiese diversiteit van 'n nuwe spesie, P. pseudomagnoliae, is bepaal en nuwe lig is op die potensiële rol van GLOX in lignienafbraak gewerp.