Made available in DSpace on 2014-12-17T15:01:21Z (GMT). No. of bitstreams: 1 RodrigoCG_DISSERT_2010.pdf: 836774 bytes, checksum: 86ff894110e71c4fda4b7950daeb0674 (MD5) Previous issue date: 2010-02-26 / Nowadays generation ethanol second, that t is obtained from fermentation of sugars of hydrolyses of cellulose, is gaining attention worldwide as a viable alternative to petroleum mainly for being a renewable resource. The increase of first generation ethanol production i.e. that obtained from sugar-cane molasses could lead to a reduction of lands sustainable for crops and food production. However, second generation ethanol needs technologic pathway for reduce the bottlenecks as production of enzymes to hydrolysis the cellulose to glucose i.e. the cellulases as well as the development of efficient biomass pretreatment and of low-cost. In this work Trichoderma reesei ATCC 2768 was cultivated under submerged fermentation to produce cellulases using as substrates waste of lignocellulosic material such as cashew apple bagasse as well as coconut bagasse with and without pretreatment. For pretreatment the bagasses were treated with 1 M NaOH and by explosion at high pressure. Enzyme production was carried out in shaker (temperature of 27?C, 150 rpm and initial medium pH of 4.8). Results showed that T.reesei ATCC 2768 showed the higher cellulase production when the cashew apple bagasse was treated with 1M NaOH (2.160 UI/mL of CMCase and 0.215 UI/mL of FPase), in which the conversion of cellulose, in terms of total reducing sugars, was of 98.38%, when compared to pretreatment by explosion at high pressure (0.853 UI/mL of CMCase and 0.172 UI/mL of Fpase) showing a conversion of 47.39% of total reducing sugars. Cellulase production is lower for the medium containing coconut bagasse treated with 1M NaOH (0.480 UI/mL of CMcase and 0.073 UI/mL of FPase), giving a conversion of 49.5% in terms of total reducing sugars. Cashew apple bagasse without pretreatment showed cellulase activities lower (0.535 UI/mL of CMCase and 0,152 UI/mL of FPase) then pretreated bagasse while the coconut bagasse without pretreatment did not show any enzymatic activity. Maximum cell concentration was obtained using cashew nut bagasse as well as coconut shell bagasse treated with 1M NaOH, with 2.92 g/L and 1.97 g/L, respectively. These were higher than for the experiments in which the substrates were treated by explosion at high pressure, 1.93 g/L and 1.17 g/L. Cashew apple is a potential inducer for cellulolytic enzymes synthysis showing better results than coconut bagasse. Pretreatment improves the process for the cellulolytic enzyme production / Recentemente o etanol de segunda gera??o, obtido da fermenta??o dos hidrolisados de celulose, vem despertando a aten??o mundial por ser uma alternativa vi?vel e ambientalmente correta ao petr?leo, uma vez que aumentar a produ??o de etanol a partir do mela?o da cana-de-a??car, o etanol de primeira gera??o, implicaria na utiliza??o de terras destinadas a produ??o de alimentos. Entretanto, o etanol de celulose exige rotas tecnol?gicas que ainda o tornam pouco competitivo, como a produ??o de enzimas que quebrem a celulose em glicose, as celulases, e a ado??o de um processo de pr?-tratamento eficiente e de baixo custo capaz de diminuir o grau de intera??o que existe nas fibras vegetais. No presente trabalho, Trichoderma reesei ATCC 2768 foi cultivada em meio submerso para produ??o de celulases utilizando como substratos res?duos lignocelul?sicos, baga?o do ped?nculo de caju e baga?o de coco, sem tratamento e tratados com NaOH 1M e por explos?o a alta press?o. Os experimentos de produ??o das enzimas foram realizados em meio submerso e conduzidos em incubador rotat?rio (temperatura de 27?C, velocidade de agita??o de 150 rpm e pH inicial do meio de 4,8). Os resultados mostraram que T. reesei ATCC 2768 apresentou maior produ??o de celulases em meio contendo baga?o do ped?nculo de caju tratado com NaOH 1M (2,160 UI/mL de CMCase e 0,215 UI/mL de FPase), onde o consumo de celulose em forma de a??cares redutores totais foi de 98,38%, quando comparado com o mesmo tratado por explos?o a alta press?o (0,853 UI/mL de CMCase e 0,172 UI/mL de Fpase), apresentando um consumo de 47,39% de ART s. Os resultados mostraram ainda que a produ??o de celulase ? menor em meio contendo baga?o de coco tratado com NaOH 1M (0,480 UI/mL de CMcase e 0,073 UI/mL de FPase), chegando-se a 49,5% de ART s consumidos. O baga?o do ped?nculo de caju sem tratamento apresentou uma atividade bem inferior (0,535 UI/mL de CMCase e 0,152 UI/mL de FPase) quando comparado aos baga?os com tratamento e o baga?o de coco n?o-tratado n?o apresentou atividade enzim?tica. A concentra??o m?xima de c?lulas foi maior quando se utilizou como substrato os baga?os do ped?nculo de caju e casca de coco tratados alcalinamente, 2,92 g/L e 1,97 g/L, respectivamente, do que aqueles tratados por explos?o a alta press?o, 1,93 g/L e 1,17 g/L. Conclui-se que o ped?nculo de caju ? um potencial indutor ? s?ntese de enzimas celulol?ticas, apresentando-se melhor que o baga?o de coco bem como o tratamento melhora bastante o processo de s?ntese das enzimas.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-06-05T14:54:01Z No. of bitstreams: 1 2010 - Samuel_Ribeiro_Passos.pdf: 16768295 bytes, checksum: 38c2614ad26085e9a101652551af84b5 (MD5) / Made available in DSpace on 2017-06-05T14:54:01Z (GMT). No. of bitstreams: 1 2010 - Samuel_Ribeiro_Passos.pdf: 16768295 bytes, checksum: 38c2614ad26085e9a101652551af84b5 (MD5) Previous issue date: 2010-02-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES, Brasil. / The increasing demand for biological processes alternative, environmentally friendly and efficient in converting lignocellulosic material, expanding their application potential for agribusiness, motivates researches worldwide. Thus, organisms isolated in nature, in specific ecosystems, become increasingly important because of their physiological and metabolic diversity, which gives them a great potential in the development of biotechnological processes of interest to society. The aim of this study was to assess the microbial community associated with the intestinal tract of millipede Trigoniulus corallinus and bioprospecting for microorganisms with cellulolytic capacity. The millipedes were collected and incubated with litter in diets of grass (Paspalum notatum) and ?sabia? (Mimosa caesalpinifolia). Sampling occurred at 15, 30, 45, and 75 days of incubation. The intestinal tract of five individuals was removed, sectioned the posterior third, processed and stored in ultrasound. DNA from microbes associated with the intestinal tract, litter and coprolite was extracted, and DGGE analysis using 16S rDNA, DGGE group actinomycetes, and it was evaluated the presence of nifH genes. The 16s gene analysis by DGGE revealed a microbial diversity conditioned by the diet offered to 45 days. After this period, this effect was no longer visible. The community associated with coprolites and the type of litter was distributed in separate clusters of samples from the intestinal tract. This effect was not observed in the community assessment of actinomycetes, where the big difference for division of groups was the diet. The animals fed on grass litter showed a diverse community, and they were not influenced by time or compartmentalization. The samples associated with litter and coprolites were 80% similar to samples from the intestinal tract. In millipedes fed with material form Mimosa caesalpinifolia, the result was different, the samples of litter and coprolites where 50% similar to the intestinal tract. All samples had nifH genes detected by polymerase chain reaction. Samples collected at 45 days were also inoculated in mineral minimum medium of Busnell-Hass added carboxymethyl-cellulose (CMC) as sole carbon source. Colonies were evaluated for their ability to breakdown cellulose enzyme and 15 had an index greater than 1. The isolate that showed the highest rate (3.65) was subjected to further analysis. The microscope observation suggested that this was not an isolated but a complex of microorganisms acting on the degradation of cellulose. There is evidence of BNF in the intestinal tract of the millipede and microorganisms proliferated in CMC through the proper amplification of nifH genes and proliferation in medium within nitrogen. The community of prokaryotes was influenced by the diet offered to the community up to 45 days, and the actinomycetes community was conditioned by the diet. It was possible to isolate microorganisms and complexes of microorganisms with cellulolytic capacity, with great potential in the search for environmentally friendly technologies in generating agrobioenergy. / A crescente demanda por processos biol?gicos alternativos, ambientalmente favor?veis e eficientes na transforma??o de material ligninocelul?sico, ampliando seu potencial de aplica??o agroindustrial, estimula pesquisas em todo o mundo. Assim, microrganismos isolados na natureza, em ecossistemas espec?ficos, tornam-se cada vez mais importantes pela sua diversidade metab?lica e fisiol?gica, que lhes confere grande potencialidade no desenvolvimento de processos biotecnol?gicos de interesse ? sociedade. O objetivo deste trabalho foi avaliar a comunidade microbiana associada ao trato intestinal do dipl?pode Trigoniulus corallinus e a bioprospec??o de microrganismos com capacidade celulol?tica. Os dipl?podes foram coletados e incubados em dietas com serrapilheira de grama batatais (Paspalum notatum) e sabi? (Mimosa caesalpinifolia). As amostragens aconteceram aos 15, 30, 45 e 75 dias de incuba??o. O trato intestinal de cinco indiv?duos foi removido e seccionado o ter?o posterior tratado em ultrasom e estocado. Procedeu-se a extra??o de DNA da microbiota associada ao trato intestinal, serrapilheira e copr?lito, com an?lise por DGGE utilizando o gene 16S rDNA, DGGE para grupo actinomicetos e avalia??o da presen?a de genes nifH. A an?lise do gene 16s por DGGE revelou diversidade microbiana condicionada pela dieta oferecida at? os 45 dias. Ap?s este per?odo o efeito n?o foi mais vis?vel. A comunidade associada aos copr?litos e ao tipo de serrapilheira distribui-se em grupamentos separados das amostras oriundas do trato intestinal. O mesmo n?o foi observado na avalia??o da comunidade de actinomicetos, onde o grande diferencial para divis?o de grupos foi a dieta. Os animais alimentados com serrapilheira de grama mostraram uma comunidade diversa e n?o influenciada pelo tempo ou compartimentaliza??o. As amostras associadas ? serrapilheira e aos copr?litos foram 80% similares ?s do trato intestinal. Nos dipl?podes alimentados com sabi?, o resultado foi diferente, sendo as amostras de serrapilheira e copr?litos 50% similares ?s do trato intestinal. Todas as amostragens tiveram genes nifH detectados via PCR. Amostras coletadas aos 45 dias foram tamb?m inoculadas em meio mineral m?nimo de Busnell-Hass adicionado de carboxi-metil-celulose (CMC) como ?nica fonte de carbono. Os microrganismos isolados foram avaliados quanto ? capacidade de degrada??o de celulose e 15 apresentaram ?ndice enzim?tico maior que 1. O isolado com o maior ?ndice (3,65) foi alvo de outras an?lises. A visualiza??o em microsc?pio sugeriu que n?o se tratava de um isolado e sim de um complexo de microrganismos atuando na degrada??o da celulose. H? evidencias de FBN no trato intestinal do dipl?pode e microrganismos proliferados em meio CMC pela boa amplifica??o de genes nifH e prolifera??o em meio com aus?ncia de nitrog?nio. A comunidade de procariotos foi influenciada pela dieta oferecida at? os 45 dias e a comunidade de actinomicetos foi condicionada em fun??o da dieta. Foram isolados microrganismos e complexos de microrganismos com capacidade celulol?tica, com grande potencial para a busca de tecnologias ambientalmente sustent?veis na gera??o de agrobioenergia.