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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23521号 / 農博第2468号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5352(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Pentachlorophenol (PCP) is a biocide used by the NZ forestry industry until 1988. Its use was discontinued due to its toxicity to humans and animals. White-rot fungi have been shown to degrade PCP in laboratory and field trials. New Zealand native white-rot fungi were screened to identify organisms suitable for the clean up of PCP contaminated sites. Four criteria were used for the screening: fungal growth at different temperatures, PCP and creosote resistance and PCP degradation in standard liquid medium. Twenty isolates were identified as potentially useful from over 200 that were screened. One unique isolate, Trametes sp. HR577, was chosen for intensive study because it produced the well known laccases previously described from other PCP-degrading white-rot fungi. The white-rot isolate HR577 was assigned to the genus Trametes based on morphological characteristics and gene sequencing studies. The latter showed that the partial laccase gene sequences from Trametes sp. HR 577 had high sequence homology to laccases from other Trametes species, especially T. versicolor and T. villosa. Two laccase isozymes, designated L1c and L2, were purified from Trametes sp. HR577. These isozymes had similar biological properties to other Trametes species laccase isozymes. Both isozymes had a relatively high temperature optima, however, they were not very stable at elevated temperature. The dependence of laccase on dissolved oxygen for catalysis was demonstrated for isozyme L2. Laccase activity was severely inhibited in the absence of dissolved oxygen. This could be restored by reoxygenation into the assay system. Whole cultures of Trametes sp. HR577 grown in liquid culture removed up to 76% of PCP after 72 hours. PCP removal was mostly due to degradation rather than adsorption of PCP to fungal mycelium. Addition of purified and crude laccase isozymes (100 U mL-1) did not enhance PCP degradation. 6-15% of PCP was removed from solutions containing solely purified isozyme L1c or L2 in acetate buffer over 72 hours. Addition of ethanol or the laccase mediator compound 2,2' azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) resulted in increased PCP disappearance from purified laccase cultures. These studies show that the white-rot Trametes sp. HR577 has potential to be used for the clean up of PCP contaminated sites in NZ.

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatalyseur facile à utiliser ayant une faible activité massique et des propriétés cinétiques moindres que celle de l’enzyme libre. La formation de CLEAs de laccase a permis d’obtenir une activité massique élevée et des propriétés cinétiques supérieures à celle de l’enzyme soluble. Ces biocatalyseurs solides ont étés utilisés pour éliminer en continu le NP, BPA et TCS dans différents types de bioréacteur. Le biocatalyseur sur silice a été utilisé pour éliminer ces substances dans un réacteur garni, tandis que les CLEAs ont été utilisés dans un réacteur à lit fluidisé et un réacteur à perfusion développé au cours de ce projet. Ces différentes configurations de bioréacteur ont permis d’éliminer efficacement ces différents perturbateurs endocriniens. Globalement, les différents résultats obtenus, à l’échelle de laboratoire, au cours de ce projet de recherche démontrent que la laccase et particulièrement les biocatalyseurs formés via les différentes stratégies d’immobilisation testées représentent des approches extrêmement prometteuses pour le développement de biotechnologies environnementales vouées à l’élimination des perturbateurs endocriniens phénoliques.

Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium, the lignocellulolytic activity of P. carnosa has not been investigated. An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium, it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels.

Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium, the lignocellulolytic activity of P. carnosa has not been investigated. An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium, it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels.

Resumo: O eucalipto é a arbórea mais plantada no Brasil, devido ao seu amplo uso e após seu corte, os tocos e raízes que permanecem no local, dificultam o manejo da cultura. Com base nessa necessidade, o objetivo do presente estudo foi avaliar as características fisiológicas e funcionais de fungos basidiomicotas, isolados de campos de reflorestamento de Eucalyptus spp., com vistas à possibilidade do uso destes na destoca natural. Os fungos Pycnoporus sanguineus, Lentinus bertieri, Xylaria sp. e Lentinula edodes e um isolado não identificado foram estudados. No primeiro experimento, avaliou-se a patogenicidade dos mesmos em plantas de eucalipto urograndis (10 meses de idade), através da inoculação de disco de meio de cultura colonizado e não colonizado (Testemunha). As avaliações foram feitas aos 30 e 60 dias após a inoculação, com base no tamanho das lesões internas e externas. No segundo experimento, avaliou-se o crescimento micelial dos fungos P. sanguineus, L. bertieri, Xylaria sp., L. edodes e S. ostrea, em substrato à base de serragem enriquecida com farelos, e em três composições de meios de cultura, Batata-Dextrose-Ágar (BDA); Malte-Ágar (MA) e Serragem-Dextrose-Ágar (SDA), e incubados a 23, 27 e 31ºC. No terceiro experimento, avaliou-se a produção das enzimas ligninocelulolíticas lacase e celulase, e outras ligadas à degradação de tecidos vegetais, as proteases, pectinases e lipase, em meios de cultura específicos. No quarto experimento, estudou-se, pelo método "soil block", a perda de massa (%) de corpos-de-prova de eucalipto urograndis, inoculados com os fungos P. sanguineus, L. bertieri, Xylaria sp. e L. edodes, mantidos sob dois regimes de umidade (50 e 100%), e avaliados aos 30, 60, 90 e 120 após inoculação. Estudaram-se também as características químicas da madeira, após 120 dias da inoculação / Abstract: The Eucalyptus spp. tree is the most planted in Brazil, due to its widespread use and after its use, the stumps and roots that remains in the area, hinder crop management. Based on this need, the purpose of this work was evaluate the physiological and functional characteristics of basidiomycetes fungi, obtained in reforestation fields of Eucalyptus spp., with the possibility of using the fungi in natural stump removal. Pycnoporus sanguineus, Lentinus bertieri, Xylaria sp., Stereum ostrea and a unidentified isolate, were studied. In the first, was evaluate the fungi pathogenicity in eucalipt urograndis plants (10 months old), by means of disk inoculation into culture medium colonized and non-colonized (control) by fungi. Evaluations were performed at 30 and 60 days after inoculation based on the external and internal size of lesions. In experiment 2, the mycelial growth of P. sanguineus, L. bertieri, Xylaria sp., L. edodes and S. ostrea was evaluated in bran and sawdust-based substrate, as well as in three culture media: Potato-Dextrose-Agar (PDA), Malt-Agar (MA) and Sawdust-Dextrose-Agar (SDA), and at three incubation temperatures, at 23, 27 and 31ºC. In experiment 3, the production of lignolytic enzymes lacase and cellulase, and other enzymes related to degradation of plant tissues, like protease, pectinases and lipase was assessed in specific culture media. In experiment 4, mass loss (%) and chemical features of eucalipt urograndis wood degraded by the soil block method were evaluated by the P. sanguineus, L. bertieri, Xylaria sp. and L. edodes fungi. Thus, test specimens were inoculated with the fungi and kept under two moisture regimes, 50 and 100%, and incubated at 25 ±2ºC. Mass loss was assessed at 30, 60, 90 and 120 days after inoculation. The chemical features of eucalipt urograndis test specimens were only analyzed at 120 days / Orientador: Marli Teixeira de Almeida Minhoni / Coorientador: Edson Luiz Furtado / Banca: Celso Garcia Auer / Banca: Elias Taylor Durgante Severo / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22854号 / 農博第2437号 / 新制||農||1082(附属図書館) / 学位論文||R2||N5314(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

To gain a better understanding of the mechanisms by which the white-rot decay fungus Phanerochaete chrysosporium regulates selective delignification versus simultaneous decay, differential gene expression of its two key enzymes were measured over the course of aspen and birch wood decay tests. The type of decay was determined by differential staining and scanning electron microscopy. Real-time qPCR assessed gene expression of four lignin peroxidase genes and two manganese peroxidase genes at each stage of decay. Differential expression was significant in the mnp2 gene between aspen and birch decay tests during incipient decay; abundant expression of mnp2 in aspen corresponded to early initiation of selective delignification, whereas birch underwent initial simultaneous decay in the absence of abundant mnp2 expression. The lipC gene was the most abundantly expressed lip gene at all time points in both wood species and likely plays an important role in regulating wood decay.

Hydrogen is re-emerging as a serious alternative to fossil fuels. It is a clean gas with high energy density and its combustion only generates water vapour. Nevertheless, the hydrogen industry has a significant carbon footprint since this gas is mostly derived from fossil fuels reforming processes. This project focusses on the development of sustainable alternatives to conventional hydrogen production, in which approaches based on dark fermentation (DF) using an inexpensive residue from the brewery industry as primary feedstock are presented. Firstly, a fungal pre-treatment (FT) was proposed to degrade a high-strength brewery waste slurry (BWS) to obtain an effluent with a lower concentration of chemical oxygen demand (COD) but rich in readily fermentable sugars for the ensuing DF, thus improving hydrogen yields (HY). Secondly, microbial electrolysis and fuel cells (MECs and MFCs) were proposed to assist DF, generating electricity in MFCs while improving HY by MECs. Coupling both microbial electrochemical technologies sequentially after DF did not show any advantage. However, promising results were obtained for electricity and hydrogen production when taking a single-staged approach. Treating BWS directly by MFCs produced 2.0 watts/g COD consumed, while the DF process assisted simultaneously by MECs (DF/MEC) produced 1.6 times more hydrogen than DF alone. An average HY of 2.32 ± 0.06 mol H₂/mol glucose was attained between both DF/MEC and DF after FT, hence approaching the theoretical value of 2.4 mol H₂/mol glucose, representing roughly a 50% improvement compared to DF alone. With an overall COD reduction above 76%, the DF after FT exhibited the highest energy conversion rate per substrate consumed (6.3 kJ/g COD). As valuable by-products obtained, up to 31 g/L of fungal biomass, which is appreciated in many state-of-the-art biomaterials applications, was produced by using BWS. While in the DF/MEC process, 18 g/L of butyric acid were generated, which is three times more than with DF alone. Butyric acid being the precursor to butanol and building block of biodegradable thermoplastics, this result is not without significance. The proposed approaches not only valorize BWS but also validate their economic and environmental attractiveness as promising alternative hydrogen production methods.