Graduation date: 1996

Under cellulolytic conditions, the white-rot fungus Trametes versicolor produces cellobiose dehydrogenase (CDH), an enzyme with a number of biochemical properties that are potentially relevant to the degradation of lignin and cellulose. To clarify its biochemical properties, CDH was purified from cultures of T. versicolor. Two isoforms of CDH were found: a 97 kDa isoform with both heme and flavin cofactors, and an 81 kDa isoform with a flavin cofactor. Both isoforms of CDH were found to be quite non-specific in their reductive half reactions. The flavin enzyme catalyzed many of the same reactions as the heme/flavin enzyme, but less efficiently. The flavin isoform reduced Fe(III) and Cu(II) only at concentrations well above those found physiologically. Thus the heme/flavin enzyme, but not the flavin enzyme, could be involved in promoting and sustaining the generation of hydroxyl radicals (·OH) by Fenton's chemistry. / To characterize further the structural features of CDH, a genomic clone was isolated and sequenced. CDH was found to consist of 748 amino acids, without its predicted 19 amino acid signal peptide. Consistent with the domain structure of other CDHs, T. versicolor CDH appeared to be divided into an amino terminal heme domain and a carboxy terminal flavin domain, connected by a hydroxyamino acid-rich linker. Within the flavin domain, a putative cellulose-binding domain (CBD) was found by alignment to the hypothesized CBD of P. chrysosporium CDH. The CBD of CDH appeared to be structurally unrelated to other CBDs which have been reported. / A cDNA clone encoding T. versicolor CDH was isolated by RT-PCR. Using this clone, three vectors for the heterologous expression in Aspergillus oryzae of CDH were prepared. These vectors were built by performing in-frame fusions of the cDNA to control sequences from the highly expressed A. oryzae amylase gene. These vectors were transformed into A. oryzae and one strain was isolated which contained the expression construct DNA. / A rapid method for cloning cdh-like genes was developed. Using short stretches of amino acids completely conserved within T. versicolor and P. chrysosporium CDH, PCR primers were designed to amplify a homologous gene from other fungi. The primers were tested using genomic DNA of Pycnoporus cinnabarinus. A 1.8-kb fragment of P. cinnabarinus cdh was thereby amplified and cloned, and its sequence was determined. The three CDHs displayed very high homology at the amino acid level. / Finally, to probe the role of CDH in lignocellulose degradation by T. versicolor, a "knockout" vector was constructed consisting of a phleomycin-resistance cassette inserted into the protein coding sequence of cloned T. versicolor cdh. T. versicolor was transformed with the knockout vector and the transformants were analyzed for their CDH-producing phenotype. Three isolates were found that produced no detectable CDH. Biobleaching and delignification by the CDH(-) strains appeared to be unaffected, suggesting that CDH does not play an important role in these processes.

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.

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published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

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Orientadores: Lucia Helena Innocentini Mei, Nelson Eduardo Duran Caballero / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-13T07:57:38Z (GMT). No. of bitstreams: 1 Camargo_FranciscoAdaode_D.pdf: 5687735 bytes, checksum: 30f309fc3c8612bce1b126f965cd65a9 (MD5) Previous issue date: 2009 / Resumo: A produção de plásticos sintéticos tem aumentado ano após ano, por ser barato, resistente, etc. Mas, os especialistas advertem que o lucro obtido na industrialização e comercialização dos plásticos não pode por em risco o futuro da humanidade. Os fatos demonstram que o planeta já foi prejudicado, principalmente a fauna e a flora marinha. A solução para este problema é o desenvolvimento de materiais biodegradáveis, que garantam um desenvolvimento sustentável, onde o homem e o meio possam conviver sem causar prejuízos entre si. Com este princípio, buscamos neste trabalho o desenvolvimento de blendas biodegradáveis de polímeros naturais, com potencial para substituir alguns dos plásticos convencionais, contribuindo para o binômio produção versus preservação ambiental. A lignina é uma biomacromolécula, assim como o tanino, os quais são disponíveis em grande quantidade no Brasil. O outro componente da blenda, o copolímero de poli(3- hidroxibutirato-co-3-hidroxivalerato) ou PHBV, é uma biomolécula, termoplástico natural, biodegradável e biocompatível. Foi escolhida esta proporção (50:50)% (m/m), esta proporção foi a proporção estudada anteriormente, para a blenda de lignina de bagaço de cana com o PHBV, no aparelho Haake. Os estudos das propriedades mecânicas mostraram boas características de resistência, sendo processada na extrusora dupla rosca e injetada para a confecção dos corpos de prova. Foi estudada a biodegradação das amostras, em meio sólido, por ação dos fungos e bactérias do solo. As caracterizações das blendas (50:50) %, (m/m), ligninas, taninos, PHBV e PHB foram feitas por: Infravermelho com Transformada de Fourier (FT-IR), Calorimetria Diferencial de Varredura (DSC), Análise Dinâmico Mecânica (DMA), Microscopia Eletrônica de Varredura (MEV), Análise Elementar (CHN), Espectroscopia de Fluorescência, Fluorescência de Raios-X e Ressonância Magnética Nuclear de Hidrogênio (RMN- 1H). / Abstract: The production of synthetic plastics has been increasing year after year, for being cheap, resistant, and soon however, the specialists notice that the profit obtained in the industrialization and commercialization of the plastics should not put in risk the Humanity's future. The facts demonstrate that the planet was already harmed, mainly the fauna and the sea flora. The solution for this problem is the development of biodegradable materials to guarantee a sustained development, where the man and the environment have together without causing damages amongst themselves. With this objective, we decided to study in this work the development of biodegradable blends of natural polymer, with potential to substitute some of the conventional plastics, contributing thus the binomial production versus environmental preservation. The lignin is a biomacromolecule, as well as the tannin, theses components are available in great amount in the tropical countries like Brazil. The other component of the blends, the copolymer of poly(3-hydroxybutirate-co-3-hydroxyvalerate) or PHBV, is a natural biomolecule, thermoplastic, biodegradable and biocompatible. It was chosen this proportion (50:50) % (w/w), also this proportion was studied previously in our research group, for the blend of lignin of sugar cane bagasse with PHBV, in the apparatus Haake. The studies of mechanical properties because showed good resistance characteristics. The blends were processed in the twin screw extruders and injected for the making of the test sample. It was studied the biodegradation of the samples, in solid medium, in presence of the fungi and bacteria of the soil. The characterization of blends (50:50%) (w/w), lignins, tannins, PHBV's and PHB's were made by: Fourier Transform Infrared (FT-IR), Differential Scanning Calorimetry (DSC), Dynamic Mechanical Analysis (DMA), Scanning Microscopy Electronic (SEM), Elementary Analysis (CHN), Spectroscopy of Fluorescence, Fluorescence of ray-X and Nuclear Magnetic Resonance of Hydrogen (NMR -1H). / Doutorado / Ciencia e Tecnologia de Materiais / Doutor em Engenharia Química

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The metabolism of white-rot fungi has many proposed biotechnological applications. Their unique capability to depolymerize and catabolize lignin, the most recalcitrant component of lignocellulosic biomass, could be instrumental to the sustainable production of fuels, chemical, and materials from waste biomass feedstocks. The non-specific, oxidative nature of this lignin-degrading metabolism of white-rot fungi renders them capable of degrading a wide range of complex refractory organic compounds beyond lignin, including emerging micropollutants such as pharmaceuticals and pesticides which current wastewater treatment processes were not designed to remove. However, harnessing these metabolic capabilities into engineered bioprocesses has proven to be challenging. Common bioreactor design strategies were developed for traditionally-used unicellular bacteria and yeasts and are not necessarily appropriate for the more complex, filamentous white-rot fungi. Due to a lack of specific engineering strategies and other knowledge gaps, the realization of white-rot fungal bioprocesses has been hampered by low process efficiencies and operational challenges. This dissertation aims to expand the engineering toolbox for harnessing the metabolism of white-rot fungi in bioprocesses. Specifically, it proposes the addition of Fenton chemistry as an avenue to unlock the biotechnological potential of white-rot fungi. The production of hydroxyl radicals through the Fenton reaction is generally understood to be part of the lignin-degrading machinery of white-rot fungi and the addition of Fenton chemistry has been shown to synergistically enhance lignin degradation by white-rot fungi. Overall, the research presented here aims to demonstrate that incorporating Fenton chemistry into white-rot fungal bioprocesses not only synergistically increases lignin degradation efficiency, but also offers a potential solution for the operational challenges that have prevented the implementation of white-rot fungal bioprocesses. This dissertation was guided by five objectives aimed at illustrating the utility of coupling Fenton chemistry and white-rot fungi in engineered bioprocesses. The first objective was to demonstrate, optimize, and uncover the underlying mechanisms driving the synergistic degradation of lignin by white-rot fungi and Fenton chemistry. Through this assessment, it was found that lignin degradation increased synergistically from 58.8% to 80.2% in the presence of Fenton chemistry at the optimum concentration. This work also showed that Fe(II)/Fe(III) cycling and the induction of auxiliary ligninolytic pathways mediate this synergistic interaction. The second objective was to elucidate how Fenton chemistry influences the regulating mechanisms of ligninolytic activity in white-rot fungi, specifically C:N ratio. This showed that C:N ratio significantly influences lignin degradation in the absence of Fenton, but that this effect is blunted in the presence of Fenton. The third objective was to investigate how Fenton chemistry modulates the relationship between the concentration of fungal biomass and the extent of lignin. In the absence of Fenton, fungal biomass concentration was strongly correlated to the extent of lignin degradation. While this was also the case in the presence of Fenton chemistry at very low fungal biomass concentrations, this relationship became uncoupled at sufficiently high fungal biomass concentrations. The fourth objective was to evaluate Fenton chemistry as a selective disinfectant to allow for the persistence or enrichment of white-rot fungi in non-sterile settings. The model competitor E. coli became completely inactivated within hours at the optimal concentration of Fenton reagents, whereas the white-rot fungus P. chrysosporium survived and grew. Lastly, the fifth objective was to demonstrate the long-term performance of a continuously-operated bioreactor which integrated Fenton chemistry and white-rot fungal metabolism. A rotating biological contactor (RBC) combined with a rotating cathode electro-Fenton was constructed and a kinetic model based on batch tests was successfully developed and validated. The reactors were operated for over 100 days and reached stable lignin degradation performance at ~55%. Analysis of the microbial ecology of these reactors showed the persistence of the inoculated P. chrysosporium within the biofilms, as well as the enrichment for other lignin-degrading fungi and bacteria with aromatic catabolism and iron-reduction capabilities. Overall, this research provides insight into the potential and practical implications of integrating Fenton chemistry with white-rot fungi in bioprocesses. The lignin-degrading metabolism of white-rot fungi has long been of interest for biotechnological purposes, but attempts to operationalize them have thus far been unsuccessful at scale. In order to consider scaling white-rot fungi to full-scale operations such as wastewater treatment plants, a better understanding and tighter controls on the growth, ligninolytic activity, and ecological interactions of white-rot fungi are needed. This work proposes Fenton chemistry as a synergetic actor, selective promoter and regulator of white-rot fungal biomass and their production of lignin degrading enzymes.

Using ����C cross polarization magic angle spinning nuclear magnetic resonance techniques on 5 species of dead trees from the northwest (western hemlock, Douglas fir, Sitka spruce, lodgepole pine and ponderosa pine) I tracked the lignin and cellulose content over a 22 to 36 year period in order to determine the effects of decay fungi, if any, that is attacking certain species of tree. I had samples from the wood of the roots, the bark on the roots and, in some cases, the resin core of the roots. The Department of Forest Science at Oregon State University has studied this problem by using wet chemical analysis, and direct visual observation. Mark Harmon and Hua Chen of the Department of Forest Science believe that white rot occurred most frequently in the lodgepole pine and ponderosa pine and brown rot was more frequent in the Douglas-fir and Sitka spruce. Western hemlock seemed to have both brown and white rots active. The Douglas fir bark sample showed definite decomposition consistent with white rot during the first 10 years. The ponderosa pine sap showed decomposition consistent with white rot in the 10 to 22 year period. Sitka Spruce showed some decomposition consistent with white rot in the bark from 7 to 33 years, and the western hemlock showed some decomposition consistent with white rot in the sap in the first 10 years. The decompositions consistent with brown rot were much easier to see in this study. Virtually all the sap and bark samples showed decomposition consistent with brown rot at some point. The Douglas fir was the only species, other than lodgepole pine, not to show any decomposition consistent with brown rot in the bark of the tree, only decomposition consistent with white rot. The Douglas fir did show a decay consistent with brown rot in the sap for the first ten years. Ponderosa pine showed evidence of decay that brown rot would cause for the first 10 years in the sap and the bark. The Sitka spruce species analysis showed brown rot type decay in the bark for the first 7 years and in the sap for the entire time studied of 33 years. The lodgepole pine was the only species to not show any brown rot type decay in the sap or bark for the entire 22 year period studied. The western hemlock was distinct by not showing any definitive brown rot type decay for the first 10 years, but showed massive decay consistent with brown rot in both sap and bark during the following 26 years studied. I used an 8 Tesla magnet and the MAS frequency was at 5 kHz. The recycle time was 1.5 seconds and the contact time was 1 ms. I generally took about 10,000 acquisitions per sample, which added up to about 4 hours total acquisition time per sample. Presence of these rots shows that certain species are more susceptible than others, and also shows that local environmental conditions can contribute to rot susceptibility. / Graduation date: 2003