The most abundant organic compound produced by plants is cellulose, however it has long been accepted that animals do not secrete the hydrolytic enzymes required for its degradation, but rely instead on cellulases produced by symbiotic microbes. The recent discovery of an endogenous cDNA transcript encoding a putative GHF9 endoglucanase in the parastacid crayfish Cherax quadricarinatus (Byrne et al., 1999) suggests that similar cellulase genes may have been inherited by a range of crustacean taxa. In this study, the evolutionary history of the C. quadricarinatus endoglucanase gene and the presence of additional GHF9 genes in other decapod species were investigated. The activity of endoglucanase and endoxylanase enzymes within several cultured decapod species were also compared. The evolutionary history of the C. quadricarinatus endoglucanase gene was assessed by comparing intron/exon structure with that of other invertebrate and plant GHF9 genes. The coding region of the gene was found to be interrupted by eleven introns ranging in size from 102-902 bp, the position of which was largely conserved in both termite and abalone GHF9 genes. These structural similarities suggest GHF9 genes in crustaceans and other invertebrate taxa share a common ancestry. In addition, two introns were observed to share similar positions in plant GHF9 genes, which indicates this enzyme class may have been present in ancient eukaryote organisms. The presence of GHF9 genes in C. quadricarinatus and various other decapod species was then explored via degenerate primer PCR. Two distinct GHF9 gene fragments were determined for C. quadricarinatus and several other Cherax and Euastacus parastacid freshwater crayfish species, and a single GHF9 gene fragment was also determined for the palaemonid freshwater prawn Macrobrachium lar. Phylogenetic analyses of these fragments confirmed the presence of two endoglucanase genes within the Parastacidae, termed EG-1 and EG-2. The duplication event that produced these two genes appears to have occurred prior to the evolution of freshwater crayfish. In addition, EG-2 genes appear to have duplicated more recently within the Cherax lineage. The presence of multiple GHF9 endoglucanase enzymes within the digestive tract of some decapod species may enable more efficient processing of cellulose substrates present in dietary plant material. Endoglucanase and endoxylanase enzyme activities were compared in several parastacid crayfish and penaeid prawn species using dye-linked substrates. Endoglucanase activity levels were higher in crayfish compared with prawn species, which corresponds with the known dietary preferences of these taxa. Endoglucanase temperature and pH profiles were found to be very similar for all species examined, with optimum activity occurring at 60°C and pH 5.0. These results suggest endoglucanase activity in penaeid prawns may also be derived from endogenous sources. Additional in vitro studies further demonstrated crayfish and prawn species liberate comparable amounts of glucose from carboxymethyl-cellulose, which indicates both taxa may utilise cellulose substrates as a source of energy. Endoxylanase temperature and pH profiles were also similar for all crayfish species examined, with optimal activity occurring at 50°C and pH 5.0. These results suggest xylanase activity in crayfish may originate from endogenous enzymes, although it is unclear whether this activity is derived from GHF9 enzymes or a different xylanase enzyme class. In contrast, no endoxylanase activity was detected in the three prawn species examined. Together, these findings suggest a wide range of decapod crustacean species may possess endogenous GHF9 endoglucanase genes and enzymes. Endoglucanases may be secreted by various decapod species in order to digest soluble or amorphous cellulose substrates present in consumed plant material. Further biochemical studies may confirm the presence and functional attributes of additional endoglucanase genes and enzymes in decapods, which may ultimately assist in the design of optimal plant based crustacean aquaculture feeds.

O mundo se depara atualmente com a perspectiva de um significativo aumento na demanda por etanol combustível. O bagaço de cana está entre os maiores subprodutos agroindustriais no Brasil, sendo uma das alternativas na utilização para a produção do etanol de segunda geração. A degradação do bagaço de cana requer a ação de muitas enzimas diferentes que são reguladas transcripcionalmente. Considerando-se que o custo de celulases e hemicelulases contribuem substancialmente no preço do bioetanol, novos estudos visando o entendimento da eficiência e produtividade de celulases são de grande importância. Para entender como melhorar coquetéis de enzimas que podem hidrolizar o bagaço de cana-de-açúcar pré-tratado, uitlizou-se um experimento de genômica para investigar-se quais genes e vias são transcripcionalmente moduladas durante o crescimento de A. niger em bagaço de cana-de-açúcar explodido. Neste trabalho foram identificados genes que codificam celulases e hemicelulases com aumento da expresão durante o crescimento em bagaço de cana-de-açúcar explodido. Foi também realizada a determinação do acúmulo de mRNA de diversos genes que codificam transportadores para verificar se estes eram induzidos por xilose e por depedência de glicose. Foram identificados 18 genes que corresponde a 58% de celulases preditas em A. niger e 21 genes que correponde a 58% de hemicelulases preditas em A. niger os quias foram altamente expressos durante o crescimento em bagaço de cana-de-açúcar explodido. Foi investigado também o papel central realizado pelas proteínas quinases e fosfatases não essenciais (NPKs e NPPs, respectivamente) quando em presença de celulose como fonte de carbono, no sensoriamento do estado energético e na subsequente via de sinalização no fungo filamentoso modelo Aspergillus nidulans. O estudo com A. nidulans identificou 11 quinases e 7 fosfatases não essências, NPKs e NPPs, respectivamente, envolvidas na produção de celulases e em alguns casos, na produção também de hemicelulases. O envolvimento destas NPKs identificadas na resposta induzida por avicel e na desrepressão foram acessados pela análise do transcriptoma da cepa selvagem e por microscopia de fluorescência através da cepa de fusão CreA::GFP expressa no selvagem e no background dos mutantes de NPKs. A ausência das quinases snfA e schA reduziu dramaticamente a resposta transcricional induzida por celulose incluindo a expressão de enzimas hidrolíticas e transportadores, enquanto que a ausência de snfA resultou em uma quase completa modulação gênica induzida por celulose. O mecanismo pelo qual essas duas quinases controlam a transcrição gênica foi identificado, onde os dois mutantes de quinases foram capazes de desbloquear o CreA mediante a repressão catabólica do carbono (CCR), sob condições de desrepressão, como em baixa presença de carbono ou crescimento em celulose. Desta forma, este trabalho abriu novas possibilidades para o entendimento da sacarificação do bagaço de cana-de-açúcar por hidrolases de A. niger e para a construção de coquetéis de enzimas mais eficientes para a obtenção do etanol de segunda geração. Também possibilitou a identificação de muitas quinases e fosfatases envolvidas no sensoriamento do carbono e do estado energético, as quais demonstraram papéis sobrespostos e distintos de snfA e schA na regulação da desrepressão de CreA e na produção de enzimas hidrolíticas em A. nidulans. / The world today is faced with the prospect of a significant increase in demand for fuel ethanol. Sugarcane bagasse is among the largest agro-industrial by-products in Brazil, one of the alternatives in use for the production of second generation ethanol. Degradation of sugarcane bagasse requires the action of many different enzymes which are transcriptionally regulated. Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse. We also sought to determine whether the mRNA accumulation of several steam-exploded sugarcane bagasseinduced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 genes that corresponds to 58% of A. niger predicted cellulases and 21 genes that correspond to 58% of A. niger predicted hemicellulases, that were highly expressed during growth on sugarcane bagasse. The central role performed by nonessential protein kinases (NPK) and phosphatases (NPP) when grown on cellulose as a sole carbon source, in the sensing energetic status and the subsequent signalling pathways was assessed in the model filamentous fungus Aspergillus nidulans. This study identified multiple kinases and phosphatases (NPKs and NPPs, respectively) involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans. The involvement of the identified NPKs in avicel-induced responses and CreA derepression was assessed by genome-wide transcriptomics and fluorescent microscopy of a CreA::GFP fusion proteinexpressed in the wild-type and NPK-deficient mutant backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses including the expression of hydrolytic enzymes and transporters, while the absence snfA resulted in a near complete loss of wild-typecellulose-induced gene modulation. The mechanism by which these two NPKs controlled gene transcription was identified, as neither of NPK-deficient mutants were able to unlock CreA-mediated carbon catabolite repression, under derepressing conditions, such as carbon starvation or growth on cellulose. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. This work also enable the identification of multiple kinases and phosphatases involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans.

Made available in DSpace on 2014-12-17T15:01:34Z (GMT). No. of bitstreams: 1 PedroFSF_DISSERT.pdf: 2707475 bytes, checksum: 2a6ef9226a3d6d607615f74305bb9e07 (MD5) Previous issue date: 2014-05-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The need for new sources of energy and the concern about the environment have pushed the search for renewable energy sources such as ethanol. The use of lignocellulosic biomass as substrate appears as an important alternative because of the abundance of this raw material and for it does not compete with food production. However, the process still meets difficulties of implementation, including the cost for production of enzymes that degrade cellulose to fermentable sugars. The aim of this study was to evaluate the behavior of the species of cactus pear Opuntia ficus indica and Nopalea cochenillifera, commonly found in northeastern Brazil, as raw materials for the production of: 1) cellulosic ethanol by simultaneous saccharification and fermentation (SSF) process, using two different strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11), and 2) cellulolytic enzymes by semi-solid state fermentation (SSSF) using the filamentous fungus Penicillium chrysogenum. Before alcoholic fermentation process, the material was conditioned and pretreated by three different strategies: alkaline hydrogen peroxide, alkaline using NaOH and acid using H2SO4 followed by alkaline delignification with NaOH. Analysis of composition, crystallinity and enzymatic digestibility were carried out with the material before and after pretreatment. In addition, scanning electron microscopy images were used to compare qualitatively the material and observe the effects of pretreatments. An experimental design 2? with triplicate at the central point was used to evaluate the influence of temperature (30, 40 and 45 ?C) and the initial charge of substrate (3, 4 and 5% cellulose) in the SSF process using the material obtained through the best condition and testing both strains of S. cerevisiae, one of them flocculent (LNF CA-11). For cellulase production, the filamentous fungus P. chrysogenum was tested with N. cochenillifera in the raw condition (without pretreatment) and pretrated hydrothermically, varying the pH of the fermentative medium (3, 5 and 7). The characterization of cactus pear resulted in 31.55% cellulose, 17.12% hemicellulose and 10.25% lignin for N. cochenillifera and 34.86% cellulose, 19.97% hemicellulose and 15.72% lignin for O. ficus indica. It has also been determined, to N. cochenillifera and O. ficus indica, the content of pectin (5.44% and 5.55% of calcium pectate, respectively), extractives (26.90% and 9.69%, respectively) and ashes (5.40% and 5.95%). Pretreatment using alkaline hydrogen peroxide resulted in the best cellulose recovery results (86.16% for N. cochenillifera and 93.59% for O. ficus indica) and delignification (48.79% and 23.84% for N. cochenillifera and O. ficus indica, respectively). This pretreatment was also the only one which did not increase the crystallinity index of the samples, in the case of O. ficus indica. However, when analyzing the enzymatic digestibility of cellulose, alkali pretreatment was the one which showed the best yields and therefore it was chosen for the tests in SSF. The experiments showed higher yield of conversion of cellulose to ethanol by PE-2 strain using the pretreated N. cochenillifera (93.81%) at 40 ?C using 4% initial charge of cellulose. N. cochenillifera gave better yields than O. ficus indica and PE-2 strain showed better performance than CA-11. N. cochenillifera proved to be a substrate that can be used in the SSSF for enzymes production, reaching values of 1.00 U/g of CMCase and 0.85 FPU/g. The pretreatment was not effective to increase the enzymatic activity values / A necessidade de novas fontes de energia e a preocupa??o com o meio-ambiente t?m impulsionado a pesquisa por fontes renov?veis de energia, como o etanol. O uso de biomassa lignocelul?sica como substrato aparece como uma importante alternativa devido ? abund?ncia desta mat?ria-prima e por n?o concorrer com a produ??o de alimentos. Entretanto, o processo ainda encontra dificuldades de implementa??o, entre elas o custo para produ??o das enzimas que degradam a celulose em a??cares fermentesc?veis. O objetivo deste trabalho foi avaliar o comportamento das esp?cies de palma forrageira Opuntia ficus indica (gigante) e Nopalea cochenillifera (mi?da), comumente encontradas na regi?o Nordeste do Brasil, como mat?rias-primas para produ??o de: 1) etanol celul?sico pelo processo de sacarifica??o e fermenta??o simult?neas (SFS) usando duas cepas diferentes de Saccharomyces cerevisiae (PE-2 e LNF CA-11) e 2) enzimas celulol?ticas atrav?s da fermenta??o em estado semiss?lido (FES) usando o fungo filamentoso Penicillium chrysogenum. Antes do processo de fermenta??o alco?lica, o material foi condicionado e pr?-tratado por tr?s diferentes estrat?gias: per?xido de hidrog?nio alcalino, alcalino usando NaOH e ?cido usando H2SO4 seguido de deslignifica??o alcalina com NaOH. An?lises de composi??o, cristalinidade e digestibilidade enzim?tica foram feitas com o material antes e depois do pr?-tratamento. Adicionalmente, imagens de microscopia eletr?nica de varredura foram usadas para comparar qualitativamente o material e observar os efeitos dos pr?-tratamentos. Um planejamento fatorial 2? com triplicata no ponto central foi utilizado para avaliar a influ?ncia da temperatura (30, 40 e 45 ?C) e da carga inicial de substrato (3, 4 e 5% de celulose) no processo SFS, usando o material obtido nas melhores condi??es de pr?-tratamento e testando duas cepas de S. cerevisiae, sendo uma delas floculante (LNF CA-11). Para a produ??o de celulase, o fungo filamentoso P. chrysogenum foi testado com a esp?cie de palma N. cochenillifera no estado in-natura (sem pr?-tratamento) e submetida a um pr?-tratamento hidrot?rmico, variando-se o pH do meio fermentativo (3, 5 e 7). A caracteriza??o das palmas forrageiras resultou em 31,55% de celulose, 17,12% de hemicelulose e 10,25% de lignina para a esp?cie N. cochenillifera e 34,86% de celulose, 19,97% de hemicelulose e 15,72% de lignina para a esp?cie O. ficus indica. Analisou-se ainda, para as palmas mi?da e gigante, o teor de pectina (5,44% e 5,55% de pectato de c?lcio, respectivamente), extrativos (26,90% e 9,69%, respectivamente) e cinzas (5,40% e 5,95%). O pr?-tratamento usando per?xido de hidrog?nio alcalino apresentou os melhores resultados de recupera??o de celulose (86,16% para a palma mi?da e 93,59% para a palma gigante) e de deslignifica??o (48,79% e 23,84% para as palmas mi?da e gigante, respectivamente). Este pr?-tratamento foi tamb?m o ?nico a n?o elevar o ?ndice de cristalinidade das amostras, no caso da palma gigante. Entretanto, quando analisada a digestibilidade enzim?tica da celulose, o pr?-tratamento alcalino foi o que proporcionou os melhores rendimentos e, portanto, este foi o escolhido para os testes de SFS. Os experimentos demonstraram maior rendimento da convers?o de celulose em etanol pela cepa PE-2 usando a palma mi?da pr?-tratada (93,81%) a 40 ?C e usando 4% de carga inicial de celulose. A palma mi?da demonstrou melhores rendimentos que a gigante e a cepa PE-2 resultou melhor desempenho que a CA-11. A palma mi?da se mostrou um substrato poss?vel de ser usado na FES para produ??o de enzimas, alcan?ando valores de 1,00 U/g de CMCase e 0,85 FPU/g. O pr?-tratamento n?o se mostrou eficaz para aumentar os valores de atividade enzim?tica

Made available in DSpace on 2017-07-10T18:08:12Z (GMT). No. of bitstreams: 1 Caroline Mariana de Aguiar.pdf: 5014872 bytes, checksum: 64beb98ea03bbb601831a3ce234b8c31 (MD5) Previous issue date: 2010-02-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lignocellulosic materials are the most abundant residues in the world and there is a worldwide concern to use them as raw material for bioethanol production. This is possible because these materials are rich in cellulose. Cellulose is a biopolymer composed of glucose molecules linked by ß-1-4 glycosidic bonds. Glucose can be converted into ethanol by fermentation and can be obtained from cellulose by enzymatic hydrolysis using cellulases. The cellulases can be produced by several microorganisms under appropriate environmental conditions. Amongst these microorganisms is the fungus Aspergillus niger. In this work, cellulases were obtained by fermentation cultivating A. niger in broth containing pretreated lignocellulosic materials such as sugarcane bagasse, corn straw or wheat straw as the only carbon source. The fermentation kinetic was observed when the pretreated sugarcane bagasse was used as the carbon source. Several variables that affect the enzymatic hydrolysis were analyzed using the three pretreated lignocellulosic materials as hydrolysis substrate. The variables analyzed were: pH, temperature, time of the hydrolysis, mass fraction of the substrate and dilution of the enzymatic broth. The pretreatment of the lignocellulosic materials is paramount for exposing the cellulose chain. Pretreatment consisted of using 4%w/w NaOH solution or 1%w/w H2O2 and their efficiency for removing the lignin from the residues were evaluated. The enzymatic activity also was evaluated by submeting the lignocellulosic materials to successive enzymatic hydrolysis. The enzyme deactivation was evaluated by cooling or freezing the enzymatic broth. It was concluded that Aspergillus niger produces cellulases when grown on medium with pretreated lignocellulosic materials as carbon source. Considering the fermentation kinetic, the ideal time to collect the enzymatic broth with maximum productivity was about 7 days. The cellulase complex does not suffer considerable deactivation when stored at -18°C (freezer) for 43 days, however, the broth activity drops by 43% after 48 hours when stored at 4°C (fridge). The corn straw showed better results as carbon source in fermentation and as substrate hydrolysis, compared with the other materials, with enzymatic activity of 0.895 U/ml. The ideal pH to conduct the enzymatic hydrolysis was 4.8 at 50°C for 50 minutes. The mass fraction of the substrate and enzyme concentration affects the enzymatic activity by a linear dependence. The pretreated materials provided higher enzymatic activity results than the untreated materials. The highest activity enzymatic results were obtained with H2O2 treated substrates, with enzymatic activity of 0.655 U/ml for the sugarcane bagasse, 0.892 U/ml for the corn straw and 0.801 U/ml for the wheat straw. Also, the results show that the H2O2 pretreated materials can be submitted up to, at least, four successive hydrolysis with the second one yielding the highest enzymatic activity for all pretreated residues. / Os resíduos lignocelulósicos são os mais abundantes no mundo e atualmente há uma preocupação mundial em aproveitá-los como matéria-prima na produção de bioetanol. Isto é possível visto que tais resíduos são ricos em celulose. A celulose é um biopolímero composto por moléculas de glicose unidas por ligações glicosídicas ß-1-4. A glicose pode ser transformada em etanol por via fermentativa e pode ser obtida da celulose via hidrólise enzimática utilizando as enzimas celulases. As celulases podem ser produzidas por diversos micro-organismos sob condições adequadas. Dentre esses micro-organismos, destaca-se o fungo Aspergillus niger. Neste trabalho, celulases foram obtidas cultivando-se A. niger em meio de cultura com os resíduos lignocelulósicos bagaço de cana-de-açúcar, palha de milho e palha de trigo pré-tratados com NaOH 4% como única fonte de carbono. Observou-se a cinética da fermentação com bagaço de cana-de-açúcar pré-tratado com NaOH 4% como fonte de carbono. Foram analisadas diversas variáveis que afetam a hidrólise enzimática utilizando os três resíduos lignocelulósicos pré-tratados com NaOH 4% como substrato. As variáveis analisadas foram: pH, temperatura, tempo de hidrólise enzimática, fração mássica de substrato e diluição do caldo enzimático. Avaliou-se a eficiência dos pré-tratamentos dos resíduos com NaOH 4% e com H2O2 1%. Avaliou-se o comportamento da atividade enzimática submetendo os resíduos lignocelulósicos a hidrólises enzimáticas sucessivas. A desativação enzimática foi avaliada nas condições de resfriamento e congelamento do caldo enzimático. Nas condições estudadas, foi concluído que o Aspergillus niger produz celulases quando cultivado em meio com resíduos lignocelulósicos pré-tratados como fonte de carbono. O tempo ideal para coleta do caldo enzimático, com produtividade máxima, foi de aproximadamente 7 dias. O complexo celulásico não sofre desativação se armazenado a temperatura de -18°C (freezer) por 43 dias, mas perde sua atividade em 43% após 48 h se armazenado a 4°C (geladeira). A palha de milho apresentou melhores resultados como fonte de carbono na fermentação e como substrato na hidrólise, comparada com os outros resíduos, com atividade enzimática de 0,895 U/mL. O pH ideal para se conduzir a hidrólise foi 4,8 na temperatura de 50ºC por 50 minutos. A fração de substrato e a concentração das enzimas afetam linearmente a atividade enzimática. Os resíduos pré-tratados proporcionaram melhores resultados de atividade enzimática do que os resíduos não tratados. Os melhores resultados de atividade foram obtidos com os resíduos tratados com solução de H2O2 1%, com atividade de 0,655 U/mL para o bagaço de cana, 0,892 U/mL para a palha de milho e 0,801 U/mL para a palha de trigo. Além disso, os resíduos tratados com H2O2 podem sofrer quatro processos de hidrólise sucessivos, com o segundo processo rendendo a maior atividade enzimática para todos os resíduos pré-tratados.

New technologies, such as an efficient vapor-compression evaporator, a stationary lime kiln (SLK), and the MixAlco process, compelled us to re-evaluate methods for producing sugar from cane. These technologies allow more water and lime to be used, and they add more value to bagasse. Extracting and preserving the sugars, and lime pretreating the bagasse to enhance biodigestibility, all at the same time in a pile, was demonstrated to be unfeasible; therefore, sugar extraction must occur before lime treating the bagasse. Sugar extraction should occur countercurrently by lixiviation, where liquid moves in stages opposite to the soaked bagasse (megasse), which is conveyed by screw-press conveyors that gently squeeze the fiber in each stage, improving extraction. The performance of a pilot-scale screw-press conveyor was tested for dewatering capabilities and power consumption. The unoptimized equipment decreased megasse moisture from 96 to 89%. Simulation of the process suggested that eight stages are necessary to achieve 98% recovery from typical sugarcane. The cumulative power for the screw-press conveyor system was 17.0±2.1 hp•h/ton dry fiber. Thin raw juice preserved with lime for several months showed no sucrose degradation and no quality deterioration, except for reducing sugar destruction. The lime loading needed for 1-year preservation is 0.20 g Ca(OH)2/g sucrose. Shorter times require less lime. After preservation, the juice was carbonated and filtered, and the resulting sludge pelletized. Due to their high organic content, the pellets were too weak for calcination temperatures used in the SLK. The organics must be decreased prior to pelletization and sodium must be supplemented as a binding agent. Long-term lime pretreatment of bagasse showed two delignification phases: bulk (rapid) and residual (slow). These were modeled by two simultaneous first-order reactions. Treatments with air purging and higher temperatures (50 – 57oC) delignified more effectively, especially during the residual phase, thus yielding higher cellulase-enzyme digestibilities after 2 – 8 weeks of treatment. At temperatures > 60oC, pure oxygen purging is preferred. Fresh bagasse was of better quality than old bagasse. Treatment with NaOH yielded a larger bulk delignification phase than Ca(OH)2. Long-term lime pulping of bagasse was unsuitable for copy-quality paper, but it was appropriate for strawboard and other filler applications.