O Brasil possui uma posição privilegiada quando se refere à produção de etanol. Por questões históricas e geográficas o país é responsável por mais de 30 % da produção mundial de etanol, com uma produção nacional de mais de 28 bilhões de litros em 2014. Para maximizar o rendimento desse processo, está em desenvolvimento a tecnologia associada ao etanol de segunda geração ou etanol lignocelulósico. Os principais desafios desta tecnologia são: melhorar a eficiência de conversão do substrato em produto e a produção em grande escala utilizando substratos de baixo custo. Com o objetivo de melhorar a eficiência do processo de conversão foram estudadas proteínas auxiliares (expansinas) que, em conjunto com celulases, melhoram a despolimerização de biomassa lignocelulósica em açúcares fermentescíveis. Além disso, realizou-se também a caracterização de enzimas ativas de carboidratos (CAZymes) de origem termofílica do organismo Thermogemmatispora sp. T81, devido a capacidade que estas proteínas apresentam de manter a atividade e conformação estrutural em altas temperaturas por um prolongado período de tempo. A partir de análises utilizando bioinformática, os genes que codificam para expansinas de Xanthomonas campestris, Bacillus licheniformis e Trichoderma reesei foram clonados e expressos em E. coli, e seus produtos gênicos (as expansinas) tiveram seus índices de sinergismo (devido atuação conjunta com coquetéis comerciais) e atividade catalítica determinados. Adicionalmente, dispondo de alinhamentos estruturais, foi proposto um mecanismo hidrolítico para elas. Em relação à bactéria Thermogemmatispora sp. T81, foram realizadas análises genômicas e proteômicas, a fim de selecionar enzimas superexpressas em meio celulósico. Seus genes foram clonados heterologamente em E. coli e o produto de expressão caracterizado bioquimicamente (cromatografia, ensaios de atividade e perfil de hidrólise) e estruturalmente (SAXS e dicroísmo circular). Os índices de sinergismo determinados foram de 2,47; 1,96 e 2,44 para as expansinas de Xanthomonas campestris, Bacillus licheniformis e Trichoderma reesei, respectivamente. A partir dos alinhamentos estruturais foi proposto a díade Asp/Glu como sitio catalítico em expansinas. As análises de proteômica possibilitaram a seleção de quatro alvos de clonagem, por apresentarem alto índice de expressão quando a bactéria foi cultivada em meio celulósico. Estas proteínas foram caracterizadas quanto a atividade e apresentaram um perfil comum: temperatura ótima de ação (de 70 a 75 °C), pH ótimo de 5, e hidrolisam preferencialmente substratos hemicelulósicos (xilano). A porcentagem de estruturais secundárias das proteínas em estudo foram confirmadas com predições teóricas ao se utilizar a técnica de dicroísmo circular. Desta maneira, os objetivos iniciais propostos neste projeto foram concluídos com a determinação do grau de sinergismo das proteínas expansinas em estudo e a proposição de um mecanismo de hidrólise para as mesmas, considerando que tais proteínas por mais de 20 anos tiveram sua atividade definida exclusivamente como acessória. Além disso, este estudo contribui com a identificação e seleção de genes para CAZymes termofilícas com aplicação biotecnológica devido às propriedades termoestáveis apresentadas. / Brazil holds a privileged position regarding the production of ethanol. Due to geographical and historical reasons the country produces more than 30% of the world’s ethanol, with a national yield of more than 28 billion liters in 2014 alone. To further increase gain in production, technology related to second generation (or lignocellulosic) ethanol is currently under development. The main challenges of this technology are: to improve the substrate-product conversion and large-scale production using low-cost substrates. In order to improve the efficiency of the former, auxiliary proteins (expansins), which enhance lignocellulosic biomass depolymerization to fermentable sugars when associated to celullases, were studied. Besides, due to structural and catalytic resilience when subjected to high temperature, the characterization of carbohydrate-active enzymes (CAZymes) of thermophilic origin from Thermogemmatispora sp. T81 organism was performed. Through the application of bioinformatics, genes coding for Xanthomonas campestris, Bacillus licheniformis e Trichoderma reesei expansins were cloned and expressed in E. coli, being the products assessed regarding their synergism (due to joint action with commercially available enzyme cocktails) and catalytic activity. Additionally, a hydrolytic mechanism was proposed based on structural alignments. Concerning the Thermogemmatispora sp. T81 bacteria, genomic and proteomic analysis were performed in order to select overexpressed enzymes in cellulosic medium. The heterologous cloning of the respective genes was then performed in E. coli, being the products characterized biochemically (utilizing chromatography, activity assay and hydrolysis profile) and structurally (through SAXS and circular dichroism). Determined synergistic indices were 2.47; 1.96 and 2.44 for Xanthomonas campestris, Bacillus licheniformis e Trichoderma reesei expansins, respectively. From structural alignments, the dyad Asp/Glu was proposed as the catalytic site in expansins. Proteomic analysis allowed the selection of four proteins for cloning, due to high expression levels when the bacteria were cultivated in a cellulosic medium. These proteins were characterized based on their activity and showed similar trends: optimal functional temperature (70-75 °C), optimal pH of 5, and preferential hydrolysis of hemicellulosic substrates (xylan). Theoretical predictions of secondary structure percentages of studied proteins were confirmed through circular dichroism technique. Therefore, the initially proposed objectives in this project were accomplished with the determination of synergistic level and proposed hydrolytic mechanism for the expansins, considering that the role of these proteins were deemed marginal for over 20 years. In addition, this study contributes with the identification and selection of genes of thermophilic CAZymes with biotechnological applications due to shown thermostability properties.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Cellulases are enzyme complex composed of endoglucanases, exoglucanases and β-glucosidases with several biotechnological applications. However, their production cost is a major obstacle for its industrial application. About 40% of the total cellulase production cost is related to the culture medium used for the microorganism growth. In this context, efficient processes for cellulolytic enzyme production are of technical and economical interest. Thus, the present study aimed to optimize the production of cellulases by Melanoporia sp. using coconut shell powder as substrate in submerged fermentation. The influence of pH and temperature on the enzyme activity was evaluated by univariate experimental design. Then, the composition of the culture medium was sequentially optimized through Plaket -Burman followed by Central Composite experimental designs. The fermentation under optimized conditions was subsequently conducted in bioreactor to evaluate the influen ce of pH control and aeration on enzyme production. The stability of the enzyme was evaluated for 6 and 8 months at 4 ÂC and - 20 ÂC, respectively. The ability of the enzyme to hydrolyze coconut shell powder was evaluated at 65 ÂC and 80 ÂC using the crude enzyme extract produced by Melanoporia sp. The enzyme activity was determined by the quantification of reducing sugars using DNS method at pH 5.5 and 80 ÂC (optimum conditions). The composition of the culture medium which provided the highest enzyme yield was: 5 g/L of coconut shell; 15 g/L lactose; 3% tween 80; 1 g/L of KH2PO4 and 0.05 g/L FeSO4; pH 6.5 at 30ÂC for 72 hours. For batch enzyme production, the cult ure medium using non-delignified substrate, with pH controlled at 6.5, without aeration resulted in an increase of 90% in enzyme activity compared to the fermentation in a rotatory shaker. Under these conditions, the maximal enzyme production was obtained after 24 hours of fermentation. The crude enzyme extract produced by Melanoporia sp. was able to hydrolyze cellulose (coconut shell powder) efficiently, presenting industrial potential for the degradation of lignocellulosic residues. Unlike most of the cellulases produced by Trichoderma species, the strain reported as one of the best producers, the microorganism was capable of producing cellulases efficiently without the need of substrate pretreatment. Another feature of this enzyme complex is its high stability in the crude broth at-20ÂC e 4 ÂC / Celulases sÃo um complexo enzimÃtico constituÃdo por endoglucanases, exoglucanases e β-glicosidases com diversas aplicaÃÃes biotecnolÃgicas. No entanto, o elevado custo de produÃÃo dessas enzimas à o principal obstÃculo para sua aplicaÃÃo industrial. Estima-se que cerca de 40% do custo total de produÃÃo de celulases esteja relacionado ao meio de cultura utilizado para o crescimento do micro-organismo. Nesse contexto, à de fundamental importÃncia o desenvolvimento de processos para a produÃÃo de enzimas do complexo celulolÃtico que se mostrem tÃcnico e economicamente viÃveis. Diante do exposto, o presente estudo teve como objetivo avaliar a produÃÃo de celulases produzidas por Melanoporia sp. utilizando o pà da casca de coco como substrato em fermentaÃÃo submersa. A influÃncia dos parÃmetros pH e temperatura na determinaÃÃo da atividade da enzima foi avaliada atravÃs de planejamento experimental univariado. Em seguida, a composiÃÃo do meio de cultura foi otimizada atravÃs dos planejamentos experimentais Plaket-Burman e Composto Central. A fermentaÃÃo em condiÃÃes otimizadas foi posteriormente conduzida em fermentador para avaliar a influÃncia do controle de pH e oxigÃnio na produÃÃo da enzima. A estabilidade da enzima foi avaliada por 6 e 8 meses nas temperaturas de 4 ÂC e -20 ÂC, respectivamente. A capacidade das enzimas em hidrolisar o pà da casca do coco foi avaliada nas temperaturas de 65 ÂC e 80 ÂC utilizando o extrato enzimÃtico bruto produzido por Melanoporia sp. A atividade da enzima foi determinada atravÃs da quantificaÃÃo de aÃÃcares redutores pelo mÃtodo de DNS. O pH e a temperatura de determinaÃÃo da atividade enzimÃtica foram pH 5,5 e 80 ÂC, respectivamente. A composiÃÃo do meio de cultura que proporcionou o maior rendimento de produÃÃo da enzima foi: 5 g/L de casca de coco; 15 g/L de lactose; 3% de tween 80; 1 g/L de KH2PO4 e 0,05 g/L de FeSO4; pH 6,5 a 30 ÂC em 72 horas. Para a produÃÃo da enzima em fermentador, o meio de cultura utilizando substrato nÃo deslignificado, com controle do pH em 6,5, sem aeraÃÃo proporcionou um aumento de 90% na atividade da enzima, comparado à fermentaÃÃo em shaker. Nessas condiÃÃes, a mÃxima produÃÃo da enzima foi obtida apÃs 24 horas de fermentaÃÃo. O extrato enzimÃtico bruto produzido por Melanoporia sp. exibiu capacidade de hidrolisar celulose presente na casca de coco com eficiÃncia, apresentando potencial industrial para a degradaÃÃo de resÃduos lignocelulÃsicos. Diferentemente da maior parte das celulases produzidas por espÃcies de Trichoderma, micro-organismo reportado como bom produtor de enzimas celulolÃticas, o micro-organismo utilizado neste trabalho à capaz de produzir celulases de forma eficiente, sem necessidade de prÃ-tratamento do substrato. Outra caracterÃstica diferencial desta enzima à sua elevada estabilidade nas temperaturas de -20 ÂC e 4 ÂC no caldo bruto.

Decolorization of Azo dyes in synthetic wastewater composition which is similar to real textile wastewater was carried out by microbial process. Experiments were performed in two continuous systems. Experiments were performed under anaerobic conditions in order to break the nitrogen bond of the azo group (-N=N-). A synthetic dye solution which contained 200 mg/L Reactive Black 5, 200 mg/L Procion Red MX-5B and 1 g/L yeast extract was prepared. In this study, living microorganisms were used to degrade the dyes in wastewater. Rice husks which contain bacteria and fungi were used in the reactors of continuous systems. The parameters tested on continuous system were wastewater composition, the number of reactors, the amount of yeast extract in wastewater composition, the wastewater flowrate, washing the system with wood chips solution, addition of yeast extract solution.  Results have shown that increasing the number of reactors, the retention time, the amount of yeast extract and washing the system with wood chips solution had positive effects for degradation of the dyes from wastewater. When the flowrate was increased the retention time has decreased so degradation of dyes has decreased but although the flowrate increased twice, % degradation hasn’t decreased as the same ratio. Therefore this result showed that this process can be worked for faster flowrates. Microbial process is a promising technology which might be used to treat wastewater containing azo dyes with good performance.

Thesis (M. Sc. (Microbiology)) --University of Limpopo, 2017 / The recent oil crisis and environmental concerns over fossil fuels has led to the development of biofuels from lignocellulosic materials. Two main sugars from lignocellulose that can be used for bioethanol production are glucose and xylose. Xylose is problematic, because there are few yeasts that can utilise and ferment it. Xylose fermentation is not as efficient compared to glucose fermentation. Some of the factors that affect xylose fermentation include rate of xylose consumption, aeration, temperature and inhibitors. To improve ethanol production and fermentations and to make the process economically viable at industrial scale, there is a need to find a robust microorganism that can ferment efficiently in harsh industrial conditions. Therefore, the aim of this study was to investigate by means of evolutionary engineering (adaptation), the adaptability of seven locally isolated yeasts in terms of growth on high xylose concentration, in the presence of acetic acid as well as at elevated temperatures. Seven yeast strains (Candida guilliermondii MBI2, Candida sp. Kp6.2ey, Candida tropicalis Kp21ey, Candida tropicalis Kp42ey, Candida tropicalis Kp43ey, Ogatea methanolica Kp2ey and Pichia kudriavzevii Kp34ey) were adapted to ferment 60 g/L xylose as sole carbon source in the presence of 3 g/L acetic acid at 37°C. P. kudriavzevii Kp34ey was the only yeast to adapt to these conditions. The adapted P. kudriavzevii Kp34ey was compared with the parental strain (unadapted) and a reference strain, Scheffersomyces stipitis NRRLY-7124, using different volumetric oxygen transfer coefficient (KLa) rates. P. kudriavzevii Kp34ey (adapted and parental strain) and S. stipitis NRRLY-7124 produced the highest ethanol concentrations at a KLa value of 3.3. Overall, for all KLa values tested, the adapted strain performed better than the parental strain and S. stipitis NRRLY-7124. The adapted P. kudriavzevii Kp34ey yielded 4.03 g/L ethanol on 60 g/L xylose with 3 g/l acetic acid at 37°C at a KLa value of 3.3 and was the only yeast tested to grow under these conditions.

Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.

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Lignocellulose is the most abundant biopolymer on earth and offers excellent potential for sustainable manufacturing. Because lignocellulose is structurally complex and resistant to decomposition, innovative degradation strategies are necessary to unlock its value. In this dissertation, a green manufacturing process through enzyme-triggered self-cultured bacteria retting for lignocellulosic fiber was developed and investigated. The mechanism of the lignocellulosic fiber retting at a controlled degradation strategy was studied. This enzymatic degradation strategy utilizes a small amount of enzyme to trigger a large aggregation of specific bacteria to obtain clean fibers. Industrial hemp (Cannabis sativa L.) fiber was successfully retted with this strategy. The degradation of pectin was proved through an environmental scanning electron microscope and reducing sugar analysis. The bacterial successions were identified by 16S rRNA gene metagenomic sequencing. The results showed that Bacillaceae dominated the hemp retting conditions containing 1% pectinase, suggesting that pectinase can manipulate bacterial community succession by changing the nutrients available to bacteria through the degradation of pectin. This degradation strategy has 20-25% less environmental impact than the thermochemical degradation strategy, resulting in better fiber consistency and much shorter processing time (3-5 days) than the traditional water degradation strategy. The study on the degradation of lignin-rich lignocellulose also contributes to the understanding of the natural formaldehyde release mechanisms from wood.