Orientadores: Maria Regina Wolf Maciel, Reinaldo Gaspar Bastos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T08:48:28Z (GMT). No. of bitstreams: 1 Klein_BrunoColling_M.pdf: 2562214 bytes, checksum: f891de86d253786cf5d2101fec1f3eba (MD5) Previous issue date: 2013 / Resumo: A busca por uma maior sustentabilidade tem levado a uma mudança em direção à utilização de fontes renováveis para geração de energia em detrimento do uso de combustíveis fósseis, visando a uma modificação na matriz energética global. A utilização da biomassa de microalgas para produção de biocombustíveis vem sendo vista como uma alternativa promissora, uma vez que o seu cultivo proporciona produtividades em carboidratos e lipídios superiores às matérias-primas vegetais convencionalmente utilizadas na obtenção de etanol e biodiesel. Neste contexto, o objetivo da presente dissertação de mestrado foi avaliar a produção de biomassa da microalga clorofícea Chlorella vulgaris em fotobiorreator de placa plana em diferentes condições de fluxo luminoso, concentração de CO2 na alimentação gasosa e concentração de NaNO3 no meio de cultivo, visando o acúmulo de carboidratos para obtenção de bioetanol de terceira geração. As influências das variáveis nutricionais e de processo sobre a eficiência fotossintética das microalgas também foram estimadas para determinação do estado fisiológico das culturas. A produtividade média de biomassa e a concentração máxima final das microalgas foram significativamente afetadas pela incidência de radiação luminosa e pela suplementação de CO2 gasoso, obtendo-se maiores produtividades de carboidratos em cultivos com alto fluxo luminoso e concentrações de CO2 intermediárias (7,5%). Também foi observado o efeito positivo do aumento do fotoperíodo sobre o crescimento das microalgas. Através de hidrólise ácida foi possível atingir concentrações de até 2 g L-1 de açúcares fermentescíveis no hidrolisado a partir de biomassa de microalgas cultivadas em meio com baixo teor de nitrogênio. A fermentação etanólica foi então conduzida com a levedura Dekkerabruxellensis capaz de converter diferentes hexoses e pentoses em bioetanol, dada a presença de ambos os tipos de açúcares no hidrolisado / Abstract: The search for industrial processes with higher sustainability has led to a change towards the utilization of renewable sources for energy generation in substitution of fossil fuels, aiming the modification of the global energy matrix. The utilization of microalgal biomass for the production of biofuels is viewed as a promising alternative, since its cultivation yields carbohydrate and lipid productivities superior to those of conventional sources used in the obtention of bioethanol and biodiesel. In this context, the goal of this master thesis was to evaluate the biomass production of the chlorophycean microalga Chlorella vulgaris in a flat plate photobioreactor under different conditions of light flux, CO2 concentration in the gas feed and NaNO3 concentration in the culture medium, aiming carbohydrate accumulation for the production of third generation bioethanol. The influences of both process and nutritional variables on the photosynthetic efficiency of microalgae were estimated for the determination of the physiological condition of the cultures. The mean biomass productivity and the maximum final microalgae concentration were significantly affected by the incidence of light radiation and by the supplementation of gaseous CO2, the highest carbohydrate productivities being obtained in cultivations with high light flux and intermediate CO2 concentrations (7,5%). It was also observed the positive effect of increasing the photoperiod over microalgae growth. Through acid hydrolysis, it was possible to attain fermentable sugar concentration of up to 2 g L-1 from biomass of microalgae cultivated in low-nitrogen medium. The ethanolic fermentation was then carried out with the Dekkerabruxellensis yeast, capable of converting different hexoses and pentoses into ethanol, due to the presence of both sugar types in the hydrolysate / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T07:03:56Z (GMT). No. of bitstreams: 1 Gomes_ThiagoCostaFerreira_D.pdf: 13438556 bytes, checksum: add103a09a2f775dbe590afd1507f3e4 (MD5) Previous issue date: 2013 / Resumo: Nesta Tese foram investigados sistemas biológicos relacionados à hidrólise da biomassa lignocelulósica. O trabalho de tese está subdividido em três partes. Na primeira parte foram investigadas, mediante simulações por dinâmica molecular, as características estruturais e dinâmicas que conferem termoestabilidade a laminarinases, enzimas da família das glicosideo hidrolases de grande interesse no desenvolvimento de tecnologias viáveis para a produção industrial de etanol celulósico. Na segunda parte do trabalho, desenvolvemos e tornamos publicamente disponível um programa de computador, denominado cellulose-builder, que visa automatizar a construção de arquivos em formato PDB que representem cristalitos de celulose. Os arquivos em formato PDB gerados pelos nosso programa podem ser utilizados como configuração inicial em simulações por dinâmica molecular de sistemas que contenham fases cristalinas da celulose. O programa permite gerar cristalitos de forma e tamanho arbitrários, permitindo expor qualquer face cristalográfica de quatro alomorfos da celulose cristalina. Visando contribuir para o melhor entendimento em nível molecular da estrutura da parede celular vegetal e elucidar os modos de interação entre a celulose e a hemicelulose (dois componentes importantes da parede celular vegetal), na terceira parte deste trabalho empregamos o programa cellulose-builder para construir cristais de celulose com xilanos (hemiceluloses) adsorvidos em diversas faces cristalográficas e utilizamos simulações por dinâmica molecular para estudar em nível atômico a interação entre xilanos e diversas faces cristalográficas do alomorfo I-beta da celulose. Também investigamos o efeito de substituintes comumente presentes em hemiceluloses (acetil e glucuronil) nos parâmetros geométricos e energéticos da interação entre celulose e hemicelulose / Abstract: This Thesis comprises the study of biological systems related to the hydrolysis of lignocellulosic biomass. The work is divided in three parts. In the first part we investigated, by means of molecular dynamics simulations, the structural and dynamical features that confer thermostability to laminarinases, enzymes belonging to the glycoside hydrolase family, which are of great interest in the development of viable tecnologies aiming the industrial production of cellulosic ethanol. In the second part of the Thesis work we developed and made publicly available a computer program, named cellulose-builder, whose purpose is to automatize the construction of PDB-format files representing cellulose crystallites. The PDB-format files generated by our program can be used as starting configurations in molecular dynamics simulations of systems containing crystalline phases of cellulose. The program enables one to generate crystallites of arbitrary size and shape, making it possible to expose any crystallographic face of four cellulose allomorphs. Aiming to contribute to a better understanding of the plant cell wall structure at the molecular level and to elucidate the modes of interaction between cellulose and hemicellulose (two major components of the plant cell wall), in the third part of this work we employed the computer program cellulose-builder to set up cellulose crystals with xylans (hemicelluloses) adsorbed onto several crystallographic faces and applied molecular dynamics simulations to study, at the atomic level, the interactions between xylans and several crystallographic faces of cellulose's allomorph I-beta. We also investigated the effect of substituents commonly occurring in hemicelluloses (namely acetyl and glucuronyl) in geometric and energetic parameters resulting from the interaction between cellulose and hemicellulose / Doutorado / Físico-Química / Doutor em Quimica

Orientadores: Anete Pereira de Souza, Sindélia Freitas Azzoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T11:06:28Z (GMT). No. of bitstreams: 1 Crucello_Aline_D.pdf: 3829574 bytes, checksum: b0eb3f1e3e0030f42a0081e3bf8b5ef8 (MD5) Previous issue date: 2014 / Resumo: O Brasil é hoje referência mundial na produção de etanol produzido a partir da cana-de-açúcar, cujo consumo tem aumentado significativamente nos últimos anos. Entretanto, a produção atual de etanol a partir do suco da cana-de-açúcar é insuficiente para atender a demanda do mercado nacional e internacional. Nesse contexto, a produção de etanol celulósico (de segunda geração) emergiu como uma alternativa promissora ao bioetanol de primeira geração. O fungo filamentoso Trichoderma harzianum IOC-3844 é uma linhagem brasileira que se destaca pela alta capacidade de produção de enzimas do complexo das celulases e hemicelulases, característica de grande interesse em biocatálise para conversão de biomassa em monômeros de açúcar fermentáveis. Apesar de seu comprovado potencial, há poucos dados de literatura disponíveis a respeito de sua capacidade celulolítica. Desta forma, este projeto teve como objetivo principal contribuir para o conhecimento básico sobre regiões do genoma de T. harzianum IOC-3844 envolvidas na via de hidrólise de compostos celulósicos, através da construção de uma biblioteca genômica de BAC (bacterial artificial chromosome). A biblioteca de BACs conta com 5760 clones, com insertos de DNA de tamanho médio de 90 kb, o que dá uma cobertura de aproximadamente 12 vezes o genoma de T. harzianum. Através da seleção de clones contendo genes de interesse, foram identificadas regiões com altas concentrações de genes relacionados à hidrólise de biomassa. Além disso, a combinação de dados genômicos, obtidos através da biblioteca de BACs, juntamente com dados de transcriptoma possibilitou a identificação de novos potenciais genes regulatórios. Os resultados trazem grande contribuição para a pesquisa associada a T. harzianum e à genômica de fungos relacionada à produção de etanol de segunda geração / Abstract: Brazil is a world reference in sugarcane ethanol production, whose consumption has increased significantly in recent years. However, the current production of ethanol from sugarcane juice is insufficient to meet the demand of national and international market. In this context, the production of cellulosic ethanol (second generation) has emerged as a promising alternative to first-generation bioethanol. The filamentous fungus Trichoderma harzianum IOC-3844 is a Brazilian strain known for its high ability to produce enzymes of cellulosic and hemicellulosic complex, characteristic of great interest in biocatalysis for conversion of biomass into fermentable sugar monomers. Despite its potential, there are few published data available regarding its cellulolytic ability. Thus, this project aimed to contribute to the basic knowledge about regions of the genome of T. harzianum IOC-3844 involved in the hydrolysis of cellulosic compounds pathway, through the construction of a genomic BAC (bacterial artificial chromosome) library. The BAC library comprises 5,760 clones with an average DNA insert size of 90 kb, which represents about 12-fold coverage of the T. harzianum genome. Through the selection of clones containing genes of interest, regions containing high concentrations of genes related to biomass hydrolysis were identified. Furthermore, the combination of genomic data obtained from BAC library together with transcriptome data allowed the identification of novel potential regulatory genes. The results bring great contribution to studies related to T. harzianum and to fungal genomics regarding second generation bioethanol production / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-20T14:35:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Ribas tese vf.pdf: 2036236 bytes, checksum: 4d1f446720dbedcab34a1394932ee78f (MD5) / Made available in DSpace on 2016-04-20T14:35:10Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Ribas tese vf.pdf: 2036236 bytes, checksum: 4d1f446720dbedcab34a1394932ee78f (MD5) Previous issue date: 2015-03-06 / FACEPE / O termo biocombustível refere-se aos biocombustíveis sólidos, líquidos ou gasosos que são produzidos predominantemente por matérias-primas. Atualmente, dois tipos de combustíveis são utilizados para o transporte a nível global, o bioetanol e o biodiesel. Destacando-se o bioetanol, sendo este o mais consumido mundialmente. A produção desse biocombustível a partir da cana-de-açúcar no Brasil e milho nos Estados Unidos apresenta um mercado maduro com os dois países liderando a produção mundial. Contudo as destilarias de todo mundo que produzem etanol a partir dessas matérias primas ou outros cereais tem presenciado diminuição no rendimento industrial, e uma das causas é a presença de contaminantes bacterianos. Os episódios de contaminação são normalmente causados por bactérias láticas (LAB), devido à sua capacidade de adaptação às condições adversas impostas pelo processo industrial. Além disso, o aumento da população de espécies de leveduras contaminantes, podem também contribuir para a redução da produção de etanol. O presente trabalho avaliou a possível relação ecológica entre a levedura comercialmente utilizada para o processo fermentativo das destilarias de etanol a S. cerevisiae e dois microrganismos freqüentemente encontrados no processo: a levedura Dekkera bruxellensis e a bactéria Lactobacillus vini. Os resultados mostraram que o elevado teor de etanol no meio produzido por S. cerevisiae, pode controlar a população de bactérias, enquanto que a diminuição deste metabolito causada pelo aumento de contagem de células de D. bruxellensis poderia favorecer o crescimento bacteriano. Além disso, o elevado número de células bacterianas promoveu o aumento na produção de ácido orgânico que reduz o crescimento de levedura. No entanto, este fenômeno pareceu dependente do pH do meio. Por fim, foi realizado um estudo acerca do metabolismo de carboidratos de L. vini em meio sintético similar ao Manga Rosa Shape, com o objetivo de analisar a velocidade específica de L.vini e a sua biomassa final, tendo sido demonstrado uma alta biomassa quando celobiose, glicose, frutose e sacarose foram utilizados em relação a lactose e maltose, quando citrato de amônio estava presente no meio. Contudo a velocidade específica de crescimento de L.vini e sua biomassa variaram para os diferentes carboidratos com a mudança de citrato de amônio para sulfato de amônio, nitrato de sódio ou glutamato. A velocidade específica de crescimento e a biomassa de L.vini também foram analisadas em caldo e melaço da cana de açúcar com Brix 12, adicionados ou não de suplementos nutricionais como peptona, extrato de levedura, extrato de carne, citrato de amônia, Tween 80 e vitaminas, assim como com lisado celular de S.cerevisiae, interessantemente L.vini cresceu em caldo porém não em melaço quando adicionados do lisado de S.cerevisiae. Dessa forma, estamos apresentando um dos principais aspectos ecológicos da relação entre leveduras e bactérias que tem um enorme significado para o processo de produção de etanol. / The biofuel term refers to solid, liquid, or gaseous biofuels that are made predominantly of raw materials. Currently, two fuels are used to transport the global level, bioethanol and biodiesel. Highlighting bioethanol, which is the most consumed worldwide. The production of this biofuel from sugar cane in Brazil and corn in the United States has a mature market with more advanced technology. However distilleries worldwide that produce ethanol from these raw materials or other cereals has witnessed decline in industrial output, and one reason is the presence of bacterial contaminants. The episodes of contamination are normally caused by Lactic Acid Bacteria (LAB) due to their adaptability to the rough conditions imposed by the industrial process. Furthermore, the increase in the population of contaminating yeast species may also contribute to the reduction of ethanol production. This study evaluated the possible ecological relationship between the yeast commercially used for the fermentation of ethanol distilleries S. cerevisiae and two microorganisms frequently found in the process: the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini. The results showed that high ethanol content in the medium produced by S. cerevisiae can control bacterial population, while the decrease of this metabolite caused by the raising of D. bruxellensis cell count could stimulate bacterial growth. Furthermore, the high number of bacterial cell promoted the increase in organic acid production that reduces yeast growth. However, this phenomenon seemed dependent on the medium pH. Finally, a study was conducted on the L.vini carbohydrate metabolism in synthetic medium, in order to analyze the specific growth rate and biomass of L.vini, it has been demonstrated high biomass while cellobiose, glucose, fructose and sucrose were used in connection with the lactose and maltose, while ammonium citrate was present in the medium. However, the specific growth rate and biomass of L.vini varied for different carbohydrates with a change in ammonium citrate to ammonium sulfate, sodium nitrate or glutamate. The specific growth rate and biomass of L.vini were also analyzed in broth and molasses from sugar cane with Brix 12, with or without nutritional supplements as peptone, yeast extract, meat extract, ammonium citrate, Tween 80 and vitamins, as well as with cell lysate of S. cerevisiae, interestingly L.vini grown in broth but not in molasses when added lysate of S. cerevisiae. Thus, we are presenting one of the major ecological aspects of the relationship between yeasts and bacteria that has an enormous significance to ethanol production process.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-22T16:39:29Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Ribas tese vf.pdf: 2036236 bytes, checksum: 4d1f446720dbedcab34a1394932ee78f (MD5) / Made available in DSpace on 2016-04-22T16:39:29Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Ribas tese vf.pdf: 2036236 bytes, checksum: 4d1f446720dbedcab34a1394932ee78f (MD5) Previous issue date: 2015-03-06 / FACEPE / O termo biocombustível refere-se aos biocombustíveis sólidos, líquidos ou gasosos que são produzidos predominantemente por matérias-primas. Atualmente, dois tipos de combustíveis são utilizados para o transporte a nível global, o bioetanol e o biodiesel. Destacando-se o bioetanol, sendo este o mais consumido mundialmente. A produção desse biocombustível a partir da cana-de-açúcar no Brasil e milho nos Estados Unidos apresenta um mercado maduro com os dois países liderando a produção mundial. Contudo as destilarias de todo mundo que produzem etanol a partir dessas matérias primas ou outros cereais tem presenciado diminuição no rendimento industrial, e uma das causas é a presença de contaminantes bacterianos. Os episódios de contaminação são normalmente causados por bactérias láticas (LAB), devido à sua capacidade de adaptação às condições adversas impostas pelo processo industrial. Além disso, o aumento da população de espécies de leveduras contaminantes, podem também contribuir para a redução da produção de etanol. O presente trabalho avaliou a possível relação ecológica entre a levedura comercialmente utilizada para o processo fermentativo das destilarias de etanol a S. cerevisiae e dois microrganismos freqüentemente encontrados no processo: a levedura Dekkera bruxellensis e a bactéria Lactobacillus vini. Os resultados mostraram que o elevado teor de etanol no meio produzido por S. cerevisiae, pode controlar a população de bactérias, enquanto que a diminuição deste metabolito causada pelo aumento de contagem de células de D. bruxellensis poderia favorecer o crescimento bacteriano. Além disso, o elevado número de células bacterianas promoveu o aumento na produção de ácido orgânico que reduz o crescimento de levedura. No entanto, este fenômeno pareceu dependente do pH do meio. Por fim, foi realizado um estudo acerca do metabolismo de carboidratos de L. vini em meio sintético similar ao Manga Rosa Shape, com o objetivo de analisar a velocidade específica de L.vini e a sua biomassa final, tendo sido demonstrado uma alta biomassa quando celobiose, glicose, frutose e sacarose foram utilizados em relação a lactose e maltose, quando citrato de amônio estava presente no meio. Contudo a velocidade específica de crescimento de L.vini e sua biomassa variaram para os diferentes carboidratos com a mudança de citrato de amônio para sulfato de amônio, nitrato de sódio ou glutamato. A velocidade específica de crescimento e a biomassa de L.vini também foram analisadas em caldo e melaço da cana de açúcar com Brix 12, adicionados ou não de suplementos nutricionais como peptona, extrato de levedura, extrato de carne, citrato de amônia, Tween 80 e vitaminas, assim como com lisado celular de S.cerevisiae, interessantemente L.vini cresceu em caldo porém não em melaço quando adicionados do lisado de S.cerevisiae. Dessa forma, estamos apresentando um dos principais aspectos ecológicos da relação entre leveduras e bactérias que tem um enorme significado para o processo de produção de etanol. / The biofuel term refers to solid, liquid, or gaseous biofuels that are made predominantly of raw materials. Currently, two fuels are used to transport the global level, bioethanol and biodiesel. Highlighting bioethanol, which is the most consumed worldwide. The production of this biofuel from sugar cane in Brazil and corn in the United States has a mature market with more advanced technology. However distilleries worldwide that produce ethanol from these raw materials or other cereals has witnessed decline in industrial output, and one reason is the presence of bacterial contaminants. The episodes of contamination are normally caused by Lactic Acid Bacteria (LAB) due to their adaptability to the rough conditions imposed by the industrial process. Furthermore, the increase in the population of contaminating yeast species may also contribute to the reduction of ethanol production. This study evaluated the possible ecological relationship between the yeast commercially used for the fermentation of ethanol distilleries S. cerevisiae and two microorganisms frequently found in the process: the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini. The results showed that high ethanol content in the medium produced by S. cerevisiae can control bacterial population, while the decrease of this metabolite caused by the raising of D. bruxellensis cell count could stimulate bacterial growth. Furthermore, the high number of bacterial cell promoted the increase in organic acid production that reduces yeast growth. However, this phenomenon seemed dependent on the medium pH. Finally, a study was conducted on the L.vini carbohydrate metabolism in synthetic medium, in order to analyze the specific growth rate and biomass of L.vini, it has been demonstrated high biomass while cellobiose, glucose, fructose and sucrose were used in connection with the lactose and maltose, while ammonium citrate was present in the medium. However, the specific growth rate and biomass of L.vini varied for different carbohydrates with a change in ammonium citrate to ammonium sulfate, sodium nitrate or glutamate. The specific growth rate and biomass of L.vini were also analyzed in broth and molasses from sugar cane with Brix 12, with or without nutritional supplements as peptone, yeast extract, meat extract, ammonium citrate, Tween 80 and vitamins, as well as with cell lysate of S. cerevisiae, interestingly L.vini grown in broth but not in molasses when added lysate of S. cerevisiae. Thus, we are presenting one of the major ecological aspects of the relationship between yeasts and bacteria that has an enormous significance to ethanol production process.

Resíduos celulósicos por erem ricos em celulose e hemicelulose estão sendo apontados como promissoras fontes para a produção de etanol. Com isso, este trabalho tem como objetivo hidrolisar e fermentar resíduos celulósicos visando a produção de etanol. Para estudo da hidrólise do bagaço de cana-de-açúcar, foram isolados 22 fungos da cidade de Araras-SP e estes testados para produção de endoglucanases (EG). Foram testadas temperatura e pH ótimos, diferentes tampões, estabilidade do coquetel e influência de sais, EDTA e ?-mercaptoetanol para lacase, xilanase, endo-?-1,4-glucanase, celobiohidrolase I, ?-glucosidase e glucoamilase. As condições padronizadas para aplicação do coquetel enzimático foi 55ºC, tampão citrato de sódio 50 mM e pH 5,0. Para elaboração do coquetel enzimático foi realizado um Plackett-Burman seguido de um delineamento composto central rotacional, em que as condições padronizadas para aplicação foram (U/g de bagaço de cana-deaçúcar): 0,122 de lacase; 7 de xilanase; 5 de endoglucanase; 14 de celobiohidrolase e 9 de ?-glucosidase. Foram liberados 4,405 µmol de açúcares redutores por mL, após 48 horas de hidrólise do bagaço a 55ºC, em tampão citrato de sódio 50 mM (7 mL), pH 5,0, agitação de 110 rpm. O hidrolisado obtido foi fermentado pelas leveduras Saccharomyces cerevisiae PE-2 e Meyerozyma guilliermondii 311 (CCT7783), separadamente e em cultivo misto, averiguando a necessidade de suplementação do meio, para a produção de etanol. Os resultados mostraram a necessidade de suplementação para aumento da viabilidade das leveduras. Observou-se que a suplementação manteve a viabilidade acima de 90%. A fermentação com Meyerozyma guilliermondii 311 (CCT7783), sem a necessidade de suplementação, possibilitou a produção de 12,66% de etanol; com a Saccharomyces cerevisiae a produção de etanol foi de 7,03%, quando o meio foi suplementado com 3 g/L de extrato de levedura. A fermentação com cultura mista produziu 0,53% e 1,18% de etanol em 24 e 48 horas de cultivo, respectivamente. Para aplicação enzimática na hidrólise do lodo branco foram utilizadas amilase de Aspergillus carbonarius e celulase comercial. Os produtos de hidrólise enzimática do lodo branco detectados HPLC mostraram a formação de glicose, xilose e traços de maltose. A viabilidade celular da levedura Saccharomyces cerevisiae durante a fermentação se manteve bastante elevada, acima de 95 %. A fermentação do lodo branco, quando o meio foi suplementado com 3g/L de extrato de levedura, foi considerada satisfatória, com 2,37 g/L de etanol. Foram realizadas análise de superfície por ion-tof e microscopia eletrônica de varredura nos resíduos celulósicos. Desta maneira, com este trabalho foi possível estudar a importância da aplicação de enzimas fúngicas para degradação de biomassa lignocelulósica para produção de etanol de segunda geração. / As cellulosic wastes are rich in cellulose and hemicellulose they have been pointed as promising sources for ethanol production. Thereby, this work aimed to hydrolyze and ferment cellulosic residues to produce ethanol. In order to study the hydrolyzes of sugar-cane bagasse and ferment the sugars obtained from this process, twenty-two fungi were isolated in the city of Araras-SP and tested for endoglucanase (EG) production. Aiming to optmize an enzyme cocktail, variables as temperature, optimum pH, different buffers, stability, salt influence, EDTA and ?-mercaptoethanol were tested for laccase, xylanase, endo- ?-1,4-glucanase, cellobiohydrolase I, ?-glucosidase and glucoamylase. The standard conditions for applying the enzyme cocktail were 55°C, sodium citrate buffer 50 mM pH 5.0. For the preparation of the enzyme cocktail a Plackett-Burman was made followed by a central composite design, in which the standard conditions for application were (U/g of sugarcane bagasse): 0.122 of laccase; 7 of xylanase; 5 of endoglucanase; 14 of cellobiohydrolase and 9 of ?-glucosidase per gram of sugarcane bagasse. After 48 hours of hydrolyzes in the following conditions, 55ºC, sodium citrate buffer 50 mM (7 mL), pH 7.0, 110 rpm, 4.405 µmol of reducing sugar per mL were released. The hydrolyzate obtained was separately fermented by the yeasts Saccharomyces cerevisiae PE-2, Meyerozyma guilliermondii 311 (CCT7783) and with both yeasts in a mixed cultivation, in order to investigate the need of medium supplementation for ethanol production. The results showed the need of supplementation to increase the viability of the yeasts. It was observed that this procedure kept the viability over 90%. The fermentation with Meyerozyma guilliermondii 311 (CCT7783), without supplementation, allowed the production of 12.66% of ethanol and with Saccharomyces cerevisiae the production was 7.03% with a supplementation of 3 g/L of yeast extract. The fermentation with mixed cultivation produced 0.53% and 1.18% in 24 and 48 hours of cultivation, respectively. Another objective of this work was the hydrolysis of paper sludge which was obtained by using amylases from Aspergillus carbonarius and commercial cellulases. The products of enzymatic hydrolysis of the white sludge detected in HPLC showed the formation of glucose, xylose and maltose traits. Cell viability counting of the yeast, analyzes of pH, reducing sugar and alcohol content were carried out. Cellular viability of Saccharomyces cerevisiae along the fermentation was kept over 95%. Paper sludge fermentation using Saccharomyces cerevisiae, in medium supplemented with yeast extract 3 g/L, was considered satisfactory, showing 2.37 g/L of ethanol. Surface analysis was performed by Ion-tof and scanning electron microscopy in the cellulosic waste. Thus, this work made possible to study the importance of fungal enzymes application in lignocellulosic biomass degradation for the production of second generation ethanol.

Dans le but d’identifier des critères de sélection du blé (T. aestivum L.) destiné à la production de bioéthanol, les objectifs de cette thèse étaient (1) de mettre en évidence les caractéristiques physico-chimiques du grain associées aux rendements en glucose et éthanol, et (2), d’étudier par approche protéomique l’effet de variants génétiques affectant la quantité d’amylose sur le métabolisme des sucres et de l’amidon. L’analyse de trente variétés implantées dans un essai multilocal pluriannuel a mis en évidence l’importance du taux de protéines, de la dureté et de la distribution des granules d’amidon sur les rendements en glucose et éthanol. Dans une moindre mesure, la composition allélique des protéines de réserves et la viscosité des arabinoxylanes ont également un effet lors de l’étape de transformation de l’amidon en sucres fermentescibles. Huit lignées isogéniques waxy de trois variétés françaises ont été implantées dans un essai multilocal. Les lignées dépourvues d’amylose ont produit moins de glucose et d’éthanol que les variétés normales. Les analyses protéomiques des protéines de l’albumen (albumines, globulines et amphiphiles) ainsi que des protéines associées aux granules d’amidon des grains matures des lignées isogéniques de la variété Trémie ont mis en évidence : (1) une relation entre le volume spécifique des GBSS et la quantité d’amylose et (2) une modification de l’expression d’enzymes impliquées dans le métabolisme des sucres et de l’amidon (Susy, AGPase, fructose biphosphate aldolase) mais aussi de protéines de stress et de défense (serpines et HSP). Ces observations suggèrent un développement du grain incomplet pour la lignée dépourvue d’amylose. / To identify selection criteria for wheat (T. aestivum L.) used for bioethanol production, our objectives were (1) to identify the physicochemical grain characteristics associated with glucose and ethanol yields and (2) using a proteomic approach, to study the impact on sugar and starch metabolism of genetic variants that influence the quantity of amylose. The analysis of 30 wheat varieties grown in a 3-year multi-local field trial underlined the importance of protein content, grain hardness and starch granule size distribution for glucose and ethanol yields. A minor effect of the allelic composition of the storage proteins and of viscosity on the transformation of starch in fermentable sugars was also revealed. Eight waxy isogenic lines of three French varieties were grown in a multi-local field trial. The amylose-free lines produced less glucose and ethanol than normal varieties. Proteomic analyses of endosperm proteins (albumins, globulins and amphiphilic proteins) and of proteins associated with starch granules in mature grains of isogenic lines of the Tremie variety revealed : (1) a relation between the specific volume of the GBSS and amylose quantity and (2) a modification of the expression of enzymes involved in starch and sugar metabolism (Susy, AGPase, Fructose biphosphate aldolase) and also in stress and defence proteins (serpins and HSP). These observations suggest incomplete grain development in the line without amylose.

Given the increasing demand for energy, climate change, and environmental concern of fossil fuels, it is becoming increasingly significant to find alternative renewable energy sources. Bioethanol as one sort of cellulosic biofuel produced from lignocellulosic biomass feedstocks has shown great potential as a renewable resource. Delivering a competitive, sustainable biofuel product requires comprehensive supply chain planning and design. Developing economically and environmentally optimal supply chain models is necessary in this context. Also, designing biomass bioethanol supply chain (BBSC) models addressing social issues requires using second-generation biomass which is not a source of food for humans. Currently, corn as a first-generation feedstock is the primary source of bioethanol in the United States which has given growth to new social issues such as the food versus fuel debate. Considering incentives for first-generation bioethanol producers to switch to second-generation biomass and associated production technologies will help to address such social issues. The scope of this study focuses on analyzing economic and environmental market incentives for second-generation bioethanol producers while considering different carbon policies as penalties and restrictions for emissions coming from BBSC activities. First, we develop an integrated life cycle emission and energy optimization model for analyzing an entire second-generation bioethanol supply chain using switchgrass as the source of biomass while finding the most appropriate potential locations for building new cellulosic biorefineries in North Dakota. Second, we propose a supply chain model by comparing a first-generation (corn) and a second-generation (corn stover) bioethanol supply chain to analyze how policymakers can incentivize first-generation bioethanol producers to switch their technology and biomass supply from first-generation to second-generation biomass. Third, we develop the model further by investigating the impact of four different carbon policies including the carbon tax, carbon cap, carbon cap-and-trade, and carbon offset on the supply chain strategic and operational decisions. This research will help to design robust BBSCs focused on sustainability in order to optimally utilize second-generation biomass resources in the future. The findings can be utilized by renewable energy policy decision makers, bioethanol producers, and investors to operate in a competitive market while protecting the environment.

The aim of this diploma thesis was to study the possibility of using waste bread to bioproduction of selected metabolites. As bakery waste was used waste bread that came from coffee-house “Zastávka”. Bread was pre-treated by grinding into small particles and then it was made to form 15% w/v suspension, which was subjected to enzymatic hydrolysis. For the hydrolysis has been used the -amylase for liquefaction of the substrate and that was followed by a glucoamylase which sacharificated the substrate. There have been several methods of hydrolysis from which was chosen the optimal method for pre-treatment of the substrate prior to fermentation. The effectivity and a process of hydrolysis were determined spectrophotometrically by Somogyi-Nelson method. Final yields of glucose from hydrolysis were determined by HPLC method. Enzymatic hydrolysis was followed by fermentation, which was carried out in two ways, namely by adjusting the pH of the hydrolyzate to pH 5, and without pH adjustment. During the fermentation was carried out sampling in which was determined the content of glucose, glycerol and ethanol by HPLC method. The yeasts Saccharomyces cerevisiae were used for the fermentation which was performed at 30 °C. High yield of glucose was achieved by hydrolysis in two steps (70,28 gl-1), but for the fermentation was used mixed hydrolysis (second method of mixed hydrolysis) with yield 67,94 gl-1. High ethanol yield was achieved during fermentation without treatment pH, namely 31,5 gl-1.

Yeasts are like other organisms constantly exposed to environmental influences. Their survival depends on the skills to adapt to environmental changes, including the ability to use various alternative sources of nutrients. In presented PhD thesis carotenogenic yeast belonging to the genera Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates, and also subjected to several types of exogenous stress effects and mutations in order to increase the production of microbial biomass enriched with specific metabolites. As alternative nutrient sources derived from waste substrates from agricultural and farm production apple peel, pulp, corn germ and more were tested. Yeasts were also exposed to osmotic, oxidative and combined stress (benefits of various concentrations of NaCl and H2O2 to the culture media), followed by metal ions of selenium and chromium in concentrations of 0.01 mM, 0.1 mM and 1 mM. The effect of mutagen methanesulfonic acid ethyl ester was tested too. In all experiments the adaptivity of cells, morphological changes, color pigments produced by the media while some important fungal metabolites production and changes in chromosomal DNA fragmentation were analyzed. In order to evaluate potential changes in the yeast genome after treatment with mutagen and stress factors methods for isolation of intact chromosomal DNA and DNA analysis by pulsed field gel electrophoresis was optimized. The amount of produced metabolites was mainly analyzed by RP-HPLC with UV/VIS and MS detection. The work has been shown that most strains are able to use waste substrates and produced selected target metabolites. Biomass, for example, in R. aurantiaca on apple fiber was about 7 g/l and in C. capitatum cultivated on modified whey reached to 9 g/l. Amount of produced carotenoids by R. aurantiaca cultivated on wheat germ and maize after enzymatic hydrolysis by F. solani was 1.01 mg/g and S. roseus on pasta 4.3 mg/g. The values of ergosterol synthesis in R. aurantiaca are on the apple shells around 4.8 mg/g, in S. roseus on pasta with the enzymatic hydrolysis of P. chrysosporium 8.9 mg/g. The best substrate for biomass production and induction of carotenoids are waste substartes containing a mixture of simple and complex carbohydrates enriched with the addition of nitrogen compounds. Potential cytotoxic effect of stress factors of low concentrations was demonstrated. Red yeast genome was able to distribute by optimized PFGE, the karyotype of tested yeasts contain 11 or more chromosomes with visible differences between yeast species and genera. During exchange internship the ability of recombinant yeast S. cerevisiae to convert xylose to xylitol, which would be achieved by increasing the production of bioethanol as alternative fuel sources was studied. It turned out that both ligninocellulose materials to bioethanol production, as well as various waste substrates for microbial synthesis of carotenoids would reduce costs for industrial production of yeast metabolites, as well as to reduce the negative burden on the environment.