A demanda por fontes renováveis de energia juntamente com o esgotamento das reservas de combustíveis fósseis e do aquecimento global tem despertado grande interesse. A degradação da biomassa lignocelulósica para a produção de biocombustíveis tem se revelado uma alternativa viável, contudo tem enfrentado desafios em função de sua recalcitrância. Novos preparados enzimáticos envolvendo não só celulases com mecanismo hidrolítico, mas também proteínas com atividade auxiliares têm sido desenvolvidas e comercializadas. Contudo a adição de enzimas oxidativas nestes coquetéis ainda é passível de desenvolvimento e otimização. Nesse âmbito, o objetivo desse trabalho foi analisar três possíveis abordagens para a resolução desse problema. A primeira através do isolamento de fungos filamentosos de compostagem com o intuito de obter novas enzimas com função oxidativa. A segunda, constituiu o estudo de sinergismo de uma enzima acessória da família GH43 com o coquetel enzimático industrial Accellerase e a terceira no estudo da enzima oxidativa monooxigenases lítica de polissacarídeo (Lpmo) assim como sua interação sinérgica com uma endoglucanase de Trichoderma harzianum. A atividade celulolítica dos fungos foi determinada por análise do halo hidrolítico, assim como por reações enzimáticas utilizando substratos específicos. Os potenciais fungos produtores de enzimas oxidativas foram identificados por amplificação e sequenciamento da região ITS3. Dezesseis fungos foram isolados e testados, destes três apresentaram atividade positiva para a enzima lacase. O sequenciamento revelou que todos os fungos pertencem à mesma espécie patogênica Scedosporium prolificans, A enzima BlAbn1 (GH43) foi expressa em células de E. coli (BL21) e purificada para análise do sinergismo com o coquetel enzimático Accellerase em diferentes condições como sinergismo sequencial ou simultâneo, diferentes pH´s, tempo de reação e concentração enzimática. No sinergismo simultâneo e análises em pH 5 e pH 8 a taxa de sinergismo encontrada foi muito baixa apenas 4 %. Contudo no sinergismo sequencial com diferentes tempos e concentrações enzimáticas foi encontrado um aumento de 30 % na liberação de açúcares redutores na reação de 20 horas, mostrando que há ação sinérgica. O estudo da enzima oxidativa Lpmo foi realizado utilizando a enzima purificada após expressão heteróloga em Aspergillus nidulans. Análises com diferentes substratos e doadores de elétrons assim como sinergismo com uma endoglucanases foram realizados. Ácido ascórbico e pirogalol apresentaram-se como doadores de elétrons utilizando celulose amorfa (PASC), xilano e arabinoxilano como substratos. Ação sinérgica entre a endoglucanase e Lpmo foi observada, apresentando um aumento na liberação de açúcares redutores na ordem de 15 %. Em habitats naturais os microrganismos produtores de enzimas hidrolíticas e auxiliares apresentam ação sinérgica como demonstrado para enzimas da família GH43 com coquetéis enzimáticos e de enzimas oxidativas com endoglucanases. Este efeito pode ser explorado e utilizado na melhora da composição dos coquetéis enzimáticos dedicados á produção de biocombustíveis lignocelulósicos. / The demand for renewable energy sources coupled with the depletion of fossil fuel reserves and global warming has aroused great interest. The degradation of lignocellulosic biomass for the production of biofuels has proved to be a viable alternative, however it has faced challenges due to its recalcitrance. New enzymatic preparations involving not only cellulases with a hydrolytic mechanism, but also proteins with an auxiliary activity have been developed and commercialized. However the addition of oxidative enzymes in these cocktails is still amenable to development and optimization. In this context, the objective of this work was to analyze three possible approaches to solve this problem. The first one through the isolation of filamentous fungi from composting in order to obtain new enzymes with oxidative function. The second was the synergism study of an accessory enzyme of the GH43 family with the industrial enzymatic cocktail Accellerase and the third in the study of lytic polysaccharide monooxygenases (Lpmo) oxidative enzyme as well as its synergistic interaction with a Trichoderma harzianum endoglucanase. The cellulolytic activity of the fungi was determined by analysis of the hydrolytic halo, as well as by enzymatic reactions using specific substrates. Potential fungi producing oxidative enzymes were identified by amplification and sequencing of the ITS3 region. Sixteen fungi were isolated and tested, of these three showed positive activity for the lacase enzyme. Sequencing revealed that all fungi belong to the same pathogenic species Scedosporium prolificans. The enzyme BlAbn1 (GH43) was expressed in E. coli cells (BL21) and purified for synergism analysis with the enzymatic cocktail Accellerase under different conditions such as sequential synergism or Different pH, reaction time and enzymatic concentration. In simultaneous synergism and analyzes at pH 5 and pH 8 the synergism rate found was very low, only 4%. However in the sequential synergism with different times and enzymatic concentrations a 30% increase in the release of reducing sugars in the reaction of 20 hours was found, showing that there is synergistic action. The study of the oxidative enzyme Lpmo was performed using the enzyme purified after heterologous expression in Aspergillus nidulans. Analyzes with different substrates and electron donors as well as synergism with an endoglucanase were performed. Ascorbic acid and pyrogallol were presented as electron donors using amorphous cellulose (PASC), xylan and arabino-xylan as substrates. Synergistic action between endoglucanase and Lpmo was observed, with an increase in the release of reducing sugars approximately 15%. In natural habitats, the microorganisms producing hydrolytic and auxiliary enzymes present synergistic action as demonstrated for enzymes of the GH43 family with enzymatic cocktails and oxidative enzymes with endoglucanases. This effect can be exploited and used in improving the composition of the enzymatic cocktails dedicated to the production of lignocellulosic biofuels.

Submitted by WELLINGTON PINE OMORI null (wpomori@hotmail.com) on 2018-03-02T14:43:37Z No. of bitstreams: 1 Tese_Wellington_Pine_Omori.pdf: 3601697 bytes, checksum: 300108bd7832f7e80a842293e639462a (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-05T18:02:24Z (GMT) No. of bitstreams: 1 omori_wp_dr_jabo.pdf: 3601697 bytes, checksum: 300108bd7832f7e80a842293e639462a (MD5) / Made available in DSpace on 2018-03-05T18:02:24Z (GMT). No. of bitstreams: 1 omori_wp_dr_jabo.pdf: 3601697 bytes, checksum: 300108bd7832f7e80a842293e639462a (MD5) Previous issue date: 2018-02-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A torta de filtro apresenta bagaço residual oriundo do processo de extração do caldo de cana-de-açúcar e quando armazenada por longos períodos, se torna um habitat ideal para o desenvolvimento de comunidades microbianas que atuam na desconstrução da lignocelulose. Nossas análises de dados de sequenciamento de DNA metagenômico sugerem que a torta de filtro armazenada por 40 dias possui uma microbiota com características funcionais e ecológicas exclusivas em relação a outros ambientes com elevada disposição de material lignocelulósico. Assim como em ambientes de compostagem, os filos mais abundantes são Actinobacteria, Proteobacteria, Firmicutes e Bacteroidetes. Dentre os principais genes que estes micro-organismos possuem, estão Glicosiltransferases, Carboidrato Esterases e Glicosil Hidrolases, que atuando em conjunto, são passíveis de desconstruírem a lignocelulose e participarem na liberação de açúcares menores, ácidos orgânicos e outros nutrientes. Neste trabalho, identificamos novas enzimas da família AA10 que oxidam a celulose cristalina, demostrando o potencial deste ambiente em possibilitar a adaptação de micro-organismos que expressam enzimas capazes de desestruturar a celulose altamente condensada, possibilitando a liberação de moléculas de glicose. A comunidade microbiana pode acessar nutrientes como Fósforo e Nitrogênio através da despolimerização da biomassa vegetal ou decomposição da microbiota morta. No ciclo biogeoquímico do nitrogênio, a evaporação de amônia é reduzida pela assimilação desta substância pela comunidade microbiana, sendo que a amônia é produzida pela via de amonificação de nitrato e nitrito. Outro ciclo biogeoquímico identificado na torta de filtro foi o do carbono, ocorrendo diminuição da emissão de metano e gás carbônico devido ao uso destas moléculas no metabolismo microbiano. Por apresentar muitas espécies de micro-organismos termofílicos e funções ecológicas similares a compostagens que alcançaram fase termofílica, a torta de filtro armazenada por 40 dias não aparenta conter micro-organismos patogênicos em elevada abundância, o que poderia ser um indício de sua segurança biológica se usado como adubo no solo. No entanto, recomenda-se que novos estudos sejam realizados neste tipo de ambiente agrícola, afim de avaliar como se comportam principalmente os fungos Neosartorya fumigata e Paracoccidioides brasiliensis, os quais são identificados como agentes patogênicos mas que também são encontrados vivendo na natureza como organismos saprofíticos e em interação com alguns mamíferos, sem causar doença. / The filter cake presents residual bagasse from the process of extracting the sugarcane juice and when stored for long periods, it becomes an ideal habitat for the development of microbial communities that act in the deconstruction of lignocellulose. Our analyzes of metagenomic DNA sequencing data suggest that the filter cake stored for 40 days has a microbiota with unique functional and ecological characteristics compared to other environments with high lignocellulosic material. Thus in composting environments, the most abundant phyla are Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Glycosyltransferases, Carbohydrate Estersases and Glycoside Hydrolases, which act together, are capable of deconstructing lignocellulose and participate in the release of smaller sugars, organic acids and other nutrients. In this work, we identify new enzymes of the AA10 family that oxidize crystalline cellulose, demonstrating the potential of this environment to enable the adaptation of microorganisms that express enzymes capable of destabilizing highly condensed cellulose, allowing the release of glucose molecules. The microbial community can access nutrients such as Phosphorus and Nitrogen through the depolymerization of the plant biomass or decomposition of the dead microbiota. In the biogeochemical cycle of nitrogen, the evaporation of ammonia is reduced by the assimilation of this substance by the microbial community, and ammonia is produced by ammonification of nitrate and nitrite. Another biogeochemical cycle identified in the filter cake was that of carbon, with a decrease in the emission of methane and carbon dioxide due to the use of these molecules in microbial metabolism. Because it contains many species of thermophilic microorganisms and ecological functions similar to composting that reached the thermophilic phase, the filter cake stored for 40 days does not appear to contain pathogenic microorganisms in high abundance, which could be an indication of its biological safety if used as soil fertilizer. However, it is recommended that new studies be carried out in this type of agricultural environment, in order to evaluate the behavior of the fungi Neosartorya fumigata and Paracoccidioides brasiliensis, which are identified as pathogens but are also found living in nature as saprophytic organisms and in interaction with some mammals, without causing disease.

A demanda por fontes renováveis de energia juntamente com o esgotamento das reservas de combustíveis fósseis e do aquecimento global tem despertado grande interesse. A degradação da biomassa lignocelulósica para a produção de biocombustíveis tem se revelado uma alternativa viável, contudo tem enfrentado desafios em função de sua recalcitrância. Novos preparados enzimáticos envolvendo não só celulases com mecanismo hidrolítico, mas também proteínas com atividade auxiliares têm sido desenvolvidas e comercializadas. Contudo a adição de enzimas oxidativas nestes coquetéis ainda é passível de desenvolvimento e otimização. Nesse âmbito, o objetivo desse trabalho foi analisar três possíveis abordagens para a resolução desse problema. A primeira através do isolamento de fungos filamentosos de compostagem com o intuito de obter novas enzimas com função oxidativa. A segunda, constituiu o estudo de sinergismo de uma enzima acessória da família GH43 com o coquetel enzimático industrial Accellerase e a terceira no estudo da enzima oxidativa monooxigenases lítica de polissacarídeo (Lpmo) assim como sua interação sinérgica com uma endoglucanase de Trichoderma harzianum. A atividade celulolítica dos fungos foi determinada por análise do halo hidrolítico, assim como por reações enzimáticas utilizando substratos específicos. Os potenciais fungos produtores de enzimas oxidativas foram identificados por amplificação e sequenciamento da região ITS3. Dezesseis fungos foram isolados e testados, destes três apresentaram atividade positiva para a enzima lacase. O sequenciamento revelou que todos os fungos pertencem à mesma espécie patogênica Scedosporium prolificans, A enzima BlAbn1 (GH43) foi expressa em células de E. coli (BL21) e purificada para análise do sinergismo com o coquetel enzimático Accellerase em diferentes condições como sinergismo sequencial ou simultâneo, diferentes pH´s, tempo de reação e concentração enzimática. No sinergismo simultâneo e análises em pH 5 e pH 8 a taxa de sinergismo encontrada foi muito baixa apenas 4 %. Contudo no sinergismo sequencial com diferentes tempos e concentrações enzimáticas foi encontrado um aumento de 30 % na liberação de açúcares redutores na reação de 20 horas, mostrando que há ação sinérgica. O estudo da enzima oxidativa Lpmo foi realizado utilizando a enzima purificada após expressão heteróloga em Aspergillus nidulans. Análises com diferentes substratos e doadores de elétrons assim como sinergismo com uma endoglucanases foram realizados. Ácido ascórbico e pirogalol apresentaram-se como doadores de elétrons utilizando celulose amorfa (PASC), xilano e arabinoxilano como substratos. Ação sinérgica entre a endoglucanase e Lpmo foi observada, apresentando um aumento na liberação de açúcares redutores na ordem de 15 %. Em habitats naturais os microrganismos produtores de enzimas hidrolíticas e auxiliares apresentam ação sinérgica como demonstrado para enzimas da família GH43 com coquetéis enzimáticos e de enzimas oxidativas com endoglucanases. Este efeito pode ser explorado e utilizado na melhora da composição dos coquetéis enzimáticos dedicados á produção de biocombustíveis lignocelulósicos. / The demand for renewable energy sources coupled with the depletion of fossil fuel reserves and global warming has aroused great interest. The degradation of lignocellulosic biomass for the production of biofuels has proved to be a viable alternative, however it has faced challenges due to its recalcitrance. New enzymatic preparations involving not only cellulases with a hydrolytic mechanism, but also proteins with an auxiliary activity have been developed and commercialized. However the addition of oxidative enzymes in these cocktails is still amenable to development and optimization. In this context, the objective of this work was to analyze three possible approaches to solve this problem. The first one through the isolation of filamentous fungi from composting in order to obtain new enzymes with oxidative function. The second was the synergism study of an accessory enzyme of the GH43 family with the industrial enzymatic cocktail Accellerase and the third in the study of lytic polysaccharide monooxygenases (Lpmo) oxidative enzyme as well as its synergistic interaction with a Trichoderma harzianum endoglucanase. The cellulolytic activity of the fungi was determined by analysis of the hydrolytic halo, as well as by enzymatic reactions using specific substrates. Potential fungi producing oxidative enzymes were identified by amplification and sequencing of the ITS3 region. Sixteen fungi were isolated and tested, of these three showed positive activity for the lacase enzyme. Sequencing revealed that all fungi belong to the same pathogenic species Scedosporium prolificans. The enzyme BlAbn1 (GH43) was expressed in E. coli cells (BL21) and purified for synergism analysis with the enzymatic cocktail Accellerase under different conditions such as sequential synergism or Different pH, reaction time and enzymatic concentration. In simultaneous synergism and analyzes at pH 5 and pH 8 the synergism rate found was very low, only 4%. However in the sequential synergism with different times and enzymatic concentrations a 30% increase in the release of reducing sugars in the reaction of 20 hours was found, showing that there is synergistic action. The study of the oxidative enzyme Lpmo was performed using the enzyme purified after heterologous expression in Aspergillus nidulans. Analyzes with different substrates and electron donors as well as synergism with an endoglucanase were performed. Ascorbic acid and pyrogallol were presented as electron donors using amorphous cellulose (PASC), xylan and arabino-xylan as substrates. Synergistic action between endoglucanase and Lpmo was observed, with an increase in the release of reducing sugars approximately 15%. In natural habitats, the microorganisms producing hydrolytic and auxiliary enzymes present synergistic action as demonstrated for enzymes of the GH43 family with enzymatic cocktails and oxidative enzymes with endoglucanases. This effect can be exploited and used in improving the composition of the enzymatic cocktails dedicated to the production of lignocellulosic biofuels.

History teaches us that novel materials, such as chlorofluorocarbon and asbestos, can have dire unintended consequences to human and environmental health. The exponential growth of the field of nanotechnology and the products developed along the way provide the opportunity for a new paradigm of design thinking, in which human and environmental impacts are considered early on in product development. In particular, nanocellulose is touted as a promising green nanomaterial, as it is sourced from an effectively inexhaustible feedstock of wood-based cellulose and is assumed to be harmless to the environment since it is derived from a natural material and assumed to be biodegradable. The various forms of nanocellulose possess an impressive diversity of properties, making it suitable for a wide variety of applications such as drug delivery, reinforcement, food additives, and iridescent make-up. However, as nanomaterials can have different properties relative to their bulk form, it is questionable whether they are truly environmentally friendly, particularly in terms of their biodegradability and potential impacts to receiving environments. Given the projected mass-scale application of nanocellulose and the inevitability of its subsequent release into environment, the purpose of this study was to determine the biodegradability of nanocellulose and the response of environmentally-relevant microbial communities. Specifically, it was hypothesized that cellulose in the nano size range would display distinct biodegradation patterns and rates, relative to larger forms of cellulose. Further, it was hypothesized that modification of nanocellulose, in terms of morphology and surface properties (e.g., charge), would further influence its biodegradability. Wetlands and anaerobic digesters were selected as two environmentally-relevant receiving environments that also play critical roles in global carbon turnover. To examine the biodegradability of nanocellulose, two distinct microbial consortia were enriched from wetland (W) and anaerobic digester (AD) inocula and applied in parallel experiments. The consortia were grown under anaerobic conditions with microcrystalline cellulose as the sole carbon substrate over a period of 246 days before being aliquoted to microcosms for subsequent biodegradation assays. Various forms of nanocellulose were spiked into the microcosms and compared with microcrystalline cellulose as a non nano reference. Microcosms were sacrificed in triplicate with time to monitor cellulose degradation as well as various measures of microbial community response. Microbial communities were characterized in terms of gene markers for total bacteria (16S rRNA genes) and anaerobic cellulose degraders (glycoside hydrolase family 48 genes, i.e., cel48) as well as high throughput amplicon sequencing of 16S rRNA genes (V4 region). A series of three studies examined: 1) the effect of nanocrystalline versus microcrystalline cellulose; 2) the effects of nanocellulose morphology (crystalline rod versus filament) and surface functionalization (cationic and anionic); and 3) metagenomic characterization of cellulose degrading communities using next-generation DNA sequencing. It was found that the nano- size range did not hinder cellulose degradation, in fact, nanocrystalline cellulose degraded slightly faster than microcrystalline cellulose according to 1st order kinetics (1st order decay constants: 0.62±0.08 wk-1 for anionic nanocrystalline cellulose versus 0.39±0.05 wk-1 for microcrystalline cellulose exposed to AD culture; 0.69±0.04 wk-1 for anionic nanocrystalline cellulose versus 0.58±0.05 wk-1 for microcrystalline cellulose exposed to W). Experiments comparing the effects of surface functionalization indicated that anionic nanocellulose degraded faster than cationic cellulose (1st order decay constants for cationic nanocrystalline cellulose: 0.48±0.06 wk-1 and 0.58±0.07 wk-1 on exposure to AD and W cultures respectively). Measurements of 16S rRNA and cel48 genes were consistent with this trend of greater biological growth and cellulose-degrading potential in the anionic nanocellulose condition, suggesting that surface properties can influence biodegradation patterns. Taxonomic characterization of 16S rRNA gene amplicons suggested that taxa known to contain anaerobic cellulose degraders were enriched in both W and AD consortia, which shifted in a distinct manner in response to exposure to the different cellulosic materials. This suggests that distinct groups of microbes may drive the biodegradation of different forms of cellulose. Further, metagenomic investigation provided new insight into taxonomic and functional aspects of anaerobic cellulose degradation, including identification of enzymatic families associated with degradation of the various forms of cellulose. Overall, the findings of this study advance understanding of anaerobic cellulose degradation and indicate that nanocellulose is likely to readily degrade in receiving environments and not pose an environmental concern. / Ph. D.