Scaling biological pretreatment from the bench scale to the production scale may be more economical if unsterilized feedstock are used, however these allow for microbial competition from contaminates. An accurate and rapid method for identifying the desired biological pretreatment organism is necessary to confirm the presence of the desired organism when contaminates are morphologically similar to the target organism. Traditional methods, such as visual identification, sequencing, and selective plating can be time consuming and are sometimes still inconclusive. Based on methods described in the literature, plasmid DNA containing the marker genes gus (�-glucuronidase), LacZ, and gfp (green fluorescence protein) incorporated into the lignin-degrading basidiomycete Phanerochaete chrysosporium would result in a rapid genetic test for the desired organism. The presence of these genes can be confirmed either through an X-Gluc (cyclohexylammonia salt), X-Gal histochemical assay or observing the gfp’s fluorescence by a specially equipped confocal microscope. Each reporter systems will allow for rapid, reliable identification of the target species. This study will report on the success of the transformation methods in creating a transformed fungus to be used in the context of a large-scale fermentation operation. Additionally, a novel in-harvest lignocellulose feedstock biological pretreatment inoculation trial was performed comparing lignolytic performance between fungal inoculum application techniques. Optimization of carbohydrate availability for enhanced saccharification was determined by analyzing glucose release by treated and non-treated unsterilized switchgrass. This study also focused on identifying parameters to enhance saccharification efficacy at the farm-scale.

The following full-factorial study compared fungal activity on lignocellulosic biomass that was inoculated with three different amounts of fungus, and grown using three different airflow rates. These treatments were compared to a control which consisted of biomass that was not inoculated but was exposed to the same growth conditions in the environmental chamber. The objectives of the following experiment were to determine the inoculum density and airflow rate required to optimize Phanerochaete chrysosporium lignin degradation. Additionally, this study quantifies the saccharification yield from the pretreated switchgrass. The impact of substrate bulk density and substrate particle size on fungal growth were compared to determine if the particle size or the substrate bulk density has the predominant influence on the growth of the fungus, and subsequent pretreatment effectiveness quantified as an increase in glucose yields and lignin degradation. The particle size tests were controlled for bulk density; all three particle sizes were tested at a bulk density of 80 kg/m3. To test the density, three different bale densities were prepared controlling for particle size. The density tests were performed on small-scale bales made of 4 inch cut pieces of switchgrass compressed to the correct density. Therefore; density tests had the same particle size throughout all treatments, and particle size tests had the same density through all treatments. Carbohydrate accessibility post-pretreatment was examined through enzymatic saccharification and determination of glucose yields in the treatments and controls.

Made available in DSpace on 2014-06-12T18:07:11Z (GMT). No. of bitstreams: 2 arquivo7922_1.pdf: 2006759 bytes, checksum: e658b40f8129b04ae2bab24568ef6746 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / As blendas poliméricas compostas por polímeros sintéticos e amido vêm ganhando importância na área de materiais por apresentarem boas propriedades e maior biodegradabilidade quando descartadas no meio ambiente. O presente trabalho teve por objetivo avaliar a capacidade biodegradadora de uma cultura mista dos fungos Talaromyces wortmannii e Phanerochaete chrysosporium numa blenda biodegradável de polietileno de baixa densidade (PEBD)/Amido, com aplicação na indústria de embalagens. A princípio foi feita uma seleção do tipo de amido pela avaliação do crescimento dos microrganismos. Em seguida, a blenda foi obtida pela mistura de PEBD/Amido 80/20% m/m num reômetro HAAKE a temperatura de 140oC por 10 min. O material foi exposto à cultura mista por um período de 165 dias. Os níveis de biodegradação dos filmes foram caracterizados antes e após a inoculação dos fungos através de análises de variação de massa, calorimetria diferencial exploratória-DSC, microscopia eletrônica de varredura-SEM e determinação da resistência à tração na ruptura e alongamento. Ainda foi realizado um monitoramento da produção de CO2 no processo de biodegradação por 90 dias, e um estudo cinético do crescimento dos fungos em meio Sabouraud modificado com amido. Os resultados mostraram que o amido do tipo anfótero (Foxhead®5901) apresentou um bom desempenho como componente para esse tipo de blenda. O consumo crescente e gradativo da blenda como fonte de carbono alternativa ficou evidente no acompanhamento da produção de CO2. Houve um acréscimo na massa dos filmes após exposição aos fungos pela sua impregnação no material. As análises de DSC mostraram que a processabilidade do PEBD não foi alterada com a adição de amido. O material apresentou uma significativa perda em propriedades mecânicas após contato com os fungos que, segundo evidenciado na SEM, consumiram o amido presente na matriz polimérica, gerando vazios e permitindo uma reorganização de sua estrutura. O fungo Phanerochaete chrysosporium apresentou como parâmetros cinéticos μmáx (h-1) = 0,10 e KS (g/L) = 8,50, enquanto que o fungo Talaromyces wortmannii μmáx (h-1) = 0,04 e KS (g/L) = 0,18, mostrando o quanto pode variar o comportamento de duas espécies de fungos distintas crescendo sobre as mesmas condições

The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.

Effluents with high concentration of organic matter and xenobiotic compounds, especially in the case of the agrochemicals, cause environmental concern. In the present work, the adaptative ability of two filamentous fungi, which cause white rot (Geotrichum candidum Link & Pers. and Phanerochaete chrysosporium Burds.), to tolerate high concentration of xenobiotic substances was analyzed. In solid medium, P. chrysosporium and G. candidum presented excellent development under aerated and not aerated conditions (30ºC and dark), in potato-dextrose-agar and malt extract-agar, respectively. In liquid medium, the same was observed in broth-potato-dextrose and malt extract. The fungi also developed well in liquid media deficient in proteins and carbohydrates, but with high concentrations (10 and 25 mg/L) of the agrochemicals Boral, Decanote, Gliphosate, Paraquat and Plenum - all used in fields of sugar-cane, as the ones of the "S.A Usina Coruripe Açúcar e Álcool" (Coruripe - AL). These chemicals are classified as persistent, belong to the toxicological classes II and III, and have different solubilities. The fungi presented tanninolitic activity when inoculated in samples of the crude effluent with supplement of nutrients, under aeration or not. The activity of the laccase of G. candidum in the same medium was higher in the 3rd day of the growth without aeration, occurring a reduction of that in the 5th day. On the other hand, the fungus P. chrysosporium presented a higher activity of laccase in the 7th day of the growth, also without aeration. Both fungi did not interfere in the color, turbidity, concentration of nitrates and nitrites of this medium. G.candidum revealed equal efficiency in the reduction of the concentration of phosphates of the crude effluent, under aeration (78.18%) and not aeration (79.33%) conditions. However, it did not reduce the COD. P. chrysosporium was efficient in the reduction of this COD, and the difference between the aerated growth (26.77%) and the not aerated one (29.55%) was very small. It did not reduce the total tanning bark concentration, but it diminished the concentration of phosphates under aerated (55.55%) and under not aerated (61.65%) condition. The addition of nutrients in the medium of diluted effluent (2 X, 4 X and 8 X) showed that there is a direct effect of the concentration of the COD on its percentage of removal by the inoculated fungi. There is also a direct effect on the remotion of total phenolic compounds and reduced glycids. The conversion of COD in the diluted effluent (8X) was higher in the growth of P. chrysosporium (65.66%) than in the one of G.candidum (57%). The same it can be said regarding to the best rates of phenol degradation (59.66% and 50.33%, respectively). The pH measured in the media supplemented with nutrients was always higher (7,56-7,8) than the initial one (5,5). In the higher dilution of the effluent (8X) plus the supplement of nutrients, the maximum removal of COD and total phenols after the treatment occurred in 7th day of incubation of both fungi. In all the cases of diluted samples plus supplement of nutrients, the discoloration drastically increased after the inoculation of the fungi. Therefore, the use of these microorganisms in the bioremediation of the effluent of sugar-alcohol industry is recommended only after a correction of the ratio C: N: P to about 100:1,6:1. / Efluentes com alta concentração de matéria orgânica e compostos xenobióticos, especialmente no caso dos agroquímicos, causam preocupação ambiental. No presente trabalho, dois fungos filamentosos causadores de podridão branca (Geotrichum candidum Link e Pers. e Phanerochaete chrysosporium Burds.) mostraram-se capazes de se adaptar a meios com elevados teores de matéria orgânica xenobiótica ou não. Em meio sólido, verificou-se que P. chrysosporium e G. candidum, apresentaram ótimo desenvolvimento em condições aeradas e não aeradas, a 30ºC e escuro, em meios batata-dextrose-ágar e extrato de malte-ágar, respectivamente. Em meio líquido, o mesmo foi observado em caldo-batatadextrose e meio extrato de malte. Os fungos também desenvolveram-se bem em cultivos líquidos deficientes em proteínas e carboidratos, e contendo altas concentrações (10 e 25 mg/L) dos agroquímicos Boral, Decanote, Glifosato, Paraquat e Plenum, todos utilizados nas lavouras de cana de açúcar da S.A Usina Coruripe Açúcar e Álcool (Coruripe AL), classificados como persistentes, de classes toxicológicas II e III, e com diferentes solubilidades. Ambos os fungos apresentaram atividade taninolítica quando inoculados em amostras de efluente de agroindústria sucro-alcooleira não diluídas e não acrescida de nutrientes, sob aeração ou não. A atividade da lacase do fungo G. candidum, nesse mesmo meio, foi maior no 3o dia de cultivo sem aeração, ocorrendo uma diminuição no 5o dia. Já o fungo P. chrysosporium, apresentou maior atividade da lacase no 7o dia de cultivo sem aeração. Em relação à cor, turbidez, concentração de nitratos e nitritos, não houve variação desses parâmetros nas amostras inoculadas por ambos os fungos. G. candidum mostrou-se igualmente eficiente na redução da concentração de fosfatos, em cultivos aerados (78,18%) e não aerados (79,33%) do efluente bruto. Todavia, não reduziu a DQO do mesmo. P. chrysosporium mostrou-se eficiente na redução da DQO, sendo pequena a diferença entre os cultivos aerados (26,77%) com relação aos não aerados (29,55%), e não reduziu o teor de taninos totais, mas diminuiu a concentração de fosfatos sob aeração (55,55%) e não aerado (61,65%). A adição de nutrientes no cultivo dos fungos em efluente agroindustrial diluído (2X, 4X e 8X), mostrou que houve um efeito direto da concentração da DQO sobre sua porcentagem de remoção e sobre a redução de fenóis totais e glicídeos redutores. A conversão da DQO em efluente diluído (8X) foi maior no cultivo de P. chrysosporium (65,66%) do que no de G. candidum (57%). O mesmo pode ser dito quanto às melhores taxas de degradação de fenóis (59,66% e 50,33%, respectivamente). Quanto ao pH mensurado nos cultivos suplementados com nutrientes, foi sempre maior (7,56-7,8) que o inicial (5,5). Já nos cultivos utilizando a maior diluição (8X) e o suplemento de nutrientes, a máxima remoção de DQO e de fenóis totais após o tratamento ocorreu no 7º dia do cultivo para ambos os fungos. Em todos os casos de amostras diluídas e acrescidas de nutrientes, a descoloração aumentou drasticamente após o cultivo dos fungos estudados. Portanto, recomenda-se a utilização desses microrganismos na biorremediação de efluentes sucro-alcooleiros desde que haja uma correção da proporção C:N:P de cerca de 100:1,6:1.

Durant ces dernières décennies, les systèmes enzymatiques de dégradation du bois sécrétés par les champignons ont fait l’objet de nombreuses études aboutissant à la caractérisation fonctionnelle et biochimique des enzymes extracellulaires majeures agissant directement sur le polymère. Cependant, les systèmes annexes associés au processus de dégradation n’ont à l’heure actuelle été que peu étudiés. En particulier, les systèmes de détoxication et de réponses des champignons au stress généré par le processus de dégradation ainsi que les mécanismes lui permettant de croître dans cet environnement hostile sont encore peu connus. Ce stress est majoritairement dû à la présence de radicaux et d’extractibles. Les extractibles sont des molécules issues du métabolisme secondaire de l’arbre qui sont synthétisés pour augmenter la durabilité du bois face aux attaques biotiques et abiotiques. Une analyse transcriptomique réalisée au laboratoire a mis en évidence la surexpression de gènes codant des petites protéines sécrétées (SSP) chez Phanerochaete chrysosporium lors d’une culture en présence d’extractibles de chêne. La fonction de ce type de protéines chez les champignons lignolytiques est inconnue. Mon projet de thèse a porté sur la caractérisation d’une de ces SSP (SSP1). Les résultats obtenus ont révélé des propriétés biochimiques atypiques pour cette protéine qui est capable de former une structure fibrillaire, notamment grâce à la présence d’un domaine C-terminal riche en alanine et glycine. De plus, nous avons pu montrer que cette protéine présentait une activité β-glucuronidase in vitro, qui est dépendante de son état d’oligomérisation. Une approche physiologique a également été abordée grâce à l’obtention de mutants knock-out de SSP de Podospora anserina. La caractérisation de ces mutants a montré un défaut de croissance en condition de stress oxydant et en présence de molécules perturbant l’intégrité de la paroi cellulaire. Enfin, une analyse in silico des orthologues de SSP1 a montré la présence de ce gène dans les génomes d’organismes saprophytes, ectomycorhiziens ou pathogènes suggérant un rôle indirect de cette protéine dans les processus de dégradation du bois, probablement en lien avec la gestion du stress associé / During the last decades, the enzymatic systems involved in wood degradation have been intensively studied in fungi. This has led to functional and biochemical characterization of the main extracellular enzymes that are involved in the process. However, other systems associated to the degradation mechanisms have been poorly studied. In particular, the detoxification and stress response pathways allowing the fungus to grow in and resist the toxic conditions that are associated to the degradative process are still unknown. This stress is mostly due to the presence of radicals and extractives. Extractives are putative toxic compounds produced as secondary metabolites in tree to enhance wood durability against biotic and abiotic attacks. A transcriptomic analysis performed in the laboratory highlighted the up-regulation of genes coding for small secreted proteins (SSP) in Phanerochaete chrysosporium in presence of oak extractives. The functions of these SSP are unknown in lignolytic fungi. My PhD project was focused on the characterization of one of these SSP (namely SSP1) of P. chrysosporium. The biochemical data revealed atypical features for SSP1. Indeed, it is able to form fibrilar structure, thanks to an alanine-rich and glycine-rich C-terminal domain. Moreover, we have shown that this protein exhibits β-glucuronidase activity in vitro which is dependent on its oligomerization state. Physiological data were obtained thanks to the obtention of SSP knock-out mutants in Podospora anserina. These mutants have growth defect in oxidizing stress condition and in presence of cell wall-disruptive compounds. Finally, the in silico analysis of SSP1 orthologues revealed the presence of this gene in genomes of saprophytic, ectomycorrhizal or pathogenic fungi, suggesting an indirect role of this protein in wood degradation processes, probably linked to the associated stress

Phanerochaete chrysosporium est un champignon ligninolytique largement étudié pour ses capacités à dégrader la lignine et certains xénobiotiques grâce à un important système d'enzymes extracellulaires. Son génome est entièrement séquencé et constitue un inventaire de séquences protéiques prédites qui a permis la description de nombreuses superfamilles de protéines. Parmi elles, les Glutathion S-transférases sont essentiellement impliquées dans le métabolisme secondaire du champignon. Cependant, malgré les nombreux travaux montrant l'implication de ces enzymes dans la réponse aux stress, le développement cellulaire et plus globalement dans certaines fonctions métaboliques, leurs réelles fonctions restent inconnues à cause de leur grande diversité et le manque de données concernant leurs spécificités catalytiques. P. chrysosporium possède 27 isoformes de GSTs qui se regroupent en 7 classes. Parmi elles, 3 sont étendues chez les champignons saprophytes : les classes Omega, Ure2p et ethérase. Deux membres de la classe Omega ont été caractérisés au niveau biochimique et montrent desspécificités de substrat. En effet, PcGTO1 fait partie d'une nouvelle classe appelée S-glutathionyl-phydroquinone reductase, alors que PcGTO3 est plutôt active avec le phenylacetophenone. La structure tridimensionnelle de PcGTO1 suggère que l'enzyme appartient également à une nouvelle classe structurale que nous avons appelée xi. La deuxième classe majoritaire que nous avons étudiée est la classe des Ure2p qui est composée de 9 isoformes et se regroupent en 2 sous-classes. Trois isoformes ont été étudiées au niveau transcriptionnel, biochimique et physiologique. PcUre2p4 et PcUre2p6 appartenant à la première sous-classe sont spécifiquement exprimés dans des cultures fongiques en présence d'hydrocarbures aromatiques polycycliques et l'activité des protéines recombinantes correspondantes est classique des GSTs à savoir le transfert de glutathion sur un substrat hydrophobe. A l'inverse, PcUre2p1 qui appartient à la deuxième sous-classe est exprimé de manière constitutive au niveau transcriptionnel et la protéine présente une activité thiol transférase comparable aux protéines de la classe Omega. Les analyses physiologiques menées grâce à la complémentation de souche déficience de Saccharomyces cerevisiae ont montré que PcUre2p1, PcUre2p4 et PcUre2p6 n'avaient pas la même fonction que l'isoforme de la levure puisqu'aucune complémentation n'a été détectée en ce qui concerne la résistance au stress ou la régulation du métabolisme azoté. Ces résultats suggèrent que leschampignons, en particulier ceux qui présentent des propriétés saprophytes ont développé des spécificités de fonction de leur GSTs probablement en réponse à des contraintes environnementales. / Phanerochaete chrysosporium is a ligninolytic fungus widely studied because of its capacities to degrade wood and xenobiotics through an extracellular enzymatic system. Its genome has been sequenced and has provided researchers with a complete inventory of the predicted proteins produced by this organism. This has allowed the description of many protein superfamilies. Among them, Glutathione S-transferases (GSTs) constitute a complex and widespread superfamily classified as enzymes of secondary metabolism. However, despite the numerous associations of GSTs with stress responses, cell development and metabolism in various organisms, the functions of these enzymes remain usually evasive mainly due to their high diversity and also to the lack of knowledge about their catalytic specificities. In P. chrysosporium 27 GST isoforms have been highlighted and clustered into seven classes. Among them three are extended in saprophytic fungi: the Omega, the Ure2p and the etherase classes. Two members of the Omega class have been characterized at the biochemical level showing difference in substrate specificities. Indeed, PcGTO1 is member of a new class of Sglutathionyl- p-hydroquinone reductase, while PcGTO3 is rather active with phenylacetophenone. The three-dimensional structure of PcGTO1 confirms the hypothesis not only of a new biological class, but also of a new structural class that we propose to name GST xi. The second extended class we have studied is the Ure2p one. It is composed of nine isoforms in P. chrysosporium and clusters into two subclasses. Three Ure2p class members have been studied in more details at transcriptional, biochemical and physiological levels. PcUre2p4 and PcUre2p6 of the first subclass are specifically expressed in cultures treated with polycyclic aromatic hydrocarbons and the recombinant proteins are active as typical glutathione transferases. By contrast, PcUre2p1, which belongs to the second subclass is constitutively expressed whatever the condition tested and is active with small molecules as substrate, such as proteins from the Omega class. Physiological studies have revealed that these proteins do not have the same function than the Saccharomyce cerevisiae isoform, concerning both the response to oxidative stress and its involvement in the nitrogen catabolite repression. These results suggest that fungi, especially those with saprophytic capabilities, have developed specificities of GST function as an adaptation to environmental constraints

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.

Phanerochaete chrysosporium est un champignon modèle étudié en particulier pour ses capacités ligninolytiques et son aptitude à dégrader un grand nombre de polymères aromatiques toxiques dérivés notamment des hydrocarbures polycycliques. Durant ces processus de dégradation, une multitude de composés hautement réactifs et toxiques pour les cellules vont dans un premier temps être générés puis dégradés ou excrétés hors de la cellule. Le séquençage complet du génome de P. chrysosporium a permis d'identifier plusieurs superfamilles d'enzymes impliquées dans les mécanismes de tolérance à ces composés toxiques. Parmi elles, les glutathion transférases sont présentes au sein de tous les règnes du vivant et constituent une superfamille multigénique jouant un rôle dans la protection cellulaire, le métabolisme secondaire et la détoxication. Cependant, en dépit de nombreuses études réalisées en particulier chez les vertébrés, le rôle joué par ces enzymes dans la détoxication des dérivés aromatiques toxiques est encore inconnu chez les basidiomycètes. L'analyse comparative des séquences de GST présentes au sein des différents règnes du vivant révèle que les GST fongiques ont évolué différemment de leurs orthologues, notamment via l'extension de sous-classes très peu représentées chez les vertébrés. Parmi elles, les GST à cystéine catalytique représentent 30 % de cette superfamille d'enzymes chez P. chrysosporium. Trois isoformes fongiques ainsi qu'une protéine orthologue exprimée chez une bactérie lignivore ont été caractérisées in vitro tant au niveau biochimique que structural. Ces enzymes sont impliquées dans la déglutathionylation d'une grande variété de molécules électrophiles potentiellement toxiques et issues notamment de la dégradation de polymères aromatiques halogénés. La recherche de substrats a permis d'identifier plusieurs classes fonctionnelles, néanmoins l'activité des quatre isoformes s'effectue via l'attaque directe du conjugué glutathionylé par la cystéine catalytique, qui est dans un deuxième temps régénérée par un réducteur. L'analyse comparée prouve également l'existence d'une nouvelle classe structurale et fonctionnelle appelée glutathionyl hydroquinone réductase absente chez les vertébrés. Ces protéines présentent un mode de dimérisation original ainsi que la capacité tout à fait particulière de déglutathionyler les quinones. Ces résultats suggèrent que les champignons ont développé des mécanismes de résistance en réponse à des contraintes environnementales, notamment via l'évolution de familles multigéniques telles que les GST à cystéine catalytique qui sont impliquées dans le métabolisme et la tolérance vis-à-vis d'une grande variété de composés d'origine exogène ou endogène / Phanerochaete chrysosporium is a model fungus well studied for its lignolytic properties towards wood compounds and various toxic aromatic derivatives such as polycyclic aromatic hydrocarbons. These degradation processes lead first to the formation of highly reactive and toxic compounds, which are then catabolized or excreted outside the cell. Genomic data allowed the identification of genes coding for superfamilies of enzymes putatively involved in these tolerance mechanisms. Among them, glutathione transferases are present in all kingdoms and constitute a multigenic superfamily of enzymes involved in cell protection and detoxification. However, although numerous studies have been performed on vertebrate enzymes, the role of these enzymes in the detoxication of toxic aromatic compounds is still unknown in basidiomycetes. The comparative analysis of GST sequences from various kingdoms of life reveals that fungal GSTs have evolved differently from their orthologs, in particular through the expansion of sub-classes poorly represented in vertebrates. Among them, GSTs with a catalytic cysteine represent 30% of this superfamily of enzymes in P. chrysosporium. Three Cys containing fungal isoforms have been characterized at the biochemical and structural levels, including an orthologue from lignolytic bacteria. All these enzymes are involved in deglutathionylation processes using a wide range of aromatic halogenated electrophilic compounds, including potentially toxic derivatives arising from the degradation of halogenated aromatic polymers. This GSTs family can be organized in various functional groups based on their substrate specificities, but still the catalytic process remains the same with the direct attack of the glutathionylated compound by the catalytic cysteine which is then reduced and regenerated. The comparative analysis of three isoforms revealed a new structural and functional class called glutathionyl hydroquinone reductase absent in vertebrates. These proteins exhibit a new mode of dimerization as well as the ability to deglutathionylate quinones. These results suggest that fungi have developed resistance mechanisms in response to environmental stresses, notably through the evolution of multigenic families such as catalytic cysteine bearing GSTs which are likely involved in the metabolism and tolerance towards a wide range of exogenous or endogenous compounds

In this study, the aim was enzymatic xylooligosaccharide production from cotton and sunflower stalks, two of main agricultural residues in Turkey. In first two parts of the study, alkali extracted xylan from both of the stalks was hydrolyzed by commercial xylanases Veron and Shearzyme. The effect of temperature, pH, enzyme and substrate concentrations were investigated to determine optimum enzymatic hydrolysis conditions of xylan. Sunflower and cotton stalk xylans were hydrolyzed by Shearzyme more efficiently than Veron under the conditions studied. Shearzyme produced different product profiles containing xylobiose (X2), xylotriose (X3), xylotetrose (X4) and xylopentose (X5) from cotton and sunflower stalk xylan. On the other hand, Veron hydrolyzed both xylan types to produce X2, X3, X5, X6 and larger xylooligosaccharides without any change in product profiles. In the third part of the study, home produced xylanase from Bacillus pumilus SB-M13, was also investigated for the production of xylooligosaccharides from both cotton and sunflower stalk xylan. The main products obtained by hydrolysis of both substrates by pure B. pumilus xylanase were X5 and X6, while crude B. pumilus xylanase generated X4 and X5 as the main products. Xylooligosaccharide production from pretreated cotton stalk without alkali extraction of xylan was the final part of the study. Three different pretreatment methods including biomass pretreatment by Phanerochaete chrysosporium fermentation, cellulase pretreatment and hydrothermal pretreatment were investigated to break down complex lignocellulosic structure of cotton stalk to improve the subsequent enzymatic hydrolysis of xylan in pretreated cotton stalk for xylooligosaccharide production. However, xylooligosaccharide was not effectively produced from pretreated cotton stalk. Shearzyme inhibiton was observed after all the pretreatment methods during further hydrolysis of pretreated cotton stalk probably due to production of inhibitory compounds of the enzyme.