Made available in DSpace on 2016-06-02T19:32:09Z (GMT). No. of bitstreams: 1 5754.pdf: 3428035 bytes, checksum: 957ede5a2163383ade9568169703049f (MD5) Previous issue date: 2014-02-11 / Financiadora de Estudos e Projetos / The cave environments are oligotrophic and have specific characteristics that determine the native microbiota. Few studies have investigated the composition and function of soil microbiota of subterranean environments. This study aimed to expand the knowledge on the cave microbiota, discuss the functional role of the microbiota in these environments, isolate and select fungal strains with biotechnological potential for the degradation of cellulose. Soil epigean environment and two poins inside the cave Catão, São Desiderio BA, were sampled. The amount of total organic carbon, carbon and nitrogen in the microbial biomass and the microbial density was evaluated in the soil. All strains were evaluated for the activities of endoglucanase, β-glucosidase and total cellulase by submerged fermentation. Of these three strains of Aspergillus strains, one strain standard, and a Penicillium the process of solid state fermentation using wheat bran as substrate were selected. We observed a significant difference between epigeal and subterranean environment in relation to physical, chemical and biological soil parameters evaluated. We obtained 20 isolates belonging of the genus Aspergillus (SDC1.1, SDC 1.2, SDC 1.4, SDC 1.6, SDC 2.4, SDC 2.6, SDC 2.8, SDC 2.10, SDC 2.11, SDC 2.12), Penicillium (SDC 1.3, SDC 1.7, SDC 2.2, SDC 2.7, SDC2.9, SDC 2.13), Trichoderma (SDC 2.3), Scopulariopses (SDC 2.1) e Purpureocillium (SDC 2.5). Of all isolates showed 90% cellulolytic activity. During the submerged fermentation endoglucanase activity showed maximum values of 6.39 IU/mL in a strain of Aspergillus sp6 and maximum total cellulase activity was 1.7 IU/ml for Aspergillus sp8 strain. The strain Aspergillus sp6 showed maximum β- glucosidase activity of 13.67 IU/mL. Compared to solid-state fermentation, the Aspergillus sp6 and Aspergillus sp8 strains stood out compared to mutant strain Aspergillus niger 3T5B8, with values of endoglucanase and total cellulase activity higher whole with a lower protein content in their enzymatic extracts. From these resulados, we conclude that the cave environment presents singularities compared to epigean environment which probably allows you to select naturally microorganisms that use alternative sources for energy and nutrients. / Os ambientes cavernícolas são oligotróficos e possuem características específicas que determinam a microbiota local. Poucos estudos têm investigado a composição e funcionalidade da microbiota edáfica de ambientes subterrâneos. Este trabalho teve como objetivo ampliar o conhecimento sobre a microbiota cavernícola, discutir o papel funcional da microbiota nesses ambientes, isolar e selecionar linhagens fúngicas com potencial biotecnológico para a degradação de celulose. Foram coletadas amostras de solo do meio epígeo e de dois pontos no interior da caverna do Catão, São Desidério-BA. Nas amostras foram avaliadas as quantidades de carbono orgânico total, a densidade microbiana, carbono e nitrogênio da biomassa microbiana, bem como isoladas linhagens de fungos filamentosos. Todas as linhagens foram avaliadas quanto as atividades de endoglucanase, celulase total e β-glucosidases por meio de fermentação submersa. Dessas linhagens foram selecioandas três linhagens de Aspergillus, sendo uma linhagem padrão, e uma linhagem de Penicillium para o processo de fermentação em estado sólido, utilizando farelo de trigo como substrato. Observamos uma diferença significativa entre o meio epígeo e subterrâneo em relação aos parâmetros físicos, químicos e biológicos do solo avaliados. Obtivemos 20 isolados pertencentes aos gêneros Aspergillus (SDC1.1, SDC 1.2, SDC 1.4, SDC 1.6, SDC 2.4, SDC 2.6, SDC 2.8, SDC 2.10, SDC 2.11, SDC 2.12), Penicillium (SDC 1.3, SDC 1.7, SDC 2.2, SDC 2.7, SDC2.9, SDC 2.13), Trichoderma (SDC 2.3), Scopulariopses (SDC 2.1) e Purpureocillium (SDC 2.5). De todos os isolados 90% apresentaram atividade celulolítica. Durante a fermentação submersa a atividade endoglucanase apresentou valores máximos de 6,39 IU/mL em uma linhagem de Aspergillus sp6 e a atividade máxima de celulase total foi de 1,7 IU/mL para uma linhagem Aspergillus sp8. A linhagem Aspergillus sp6 apresentou atividade β-glucosidase máxima de 13,67 IU/mL. Em relação a fermentação em estado sólido, as linhagens Aspergillus sp6 e Aspergillus sp8 se destacaram em relação a linhagem padrão Aspergillus niger 3T5B8, com valores de atividade endoglucanase e celulase total superiores e com um menor conteúdo proteico em seus extratos enzimáticos. A partir desses resultados, concluímos que o ambiente cavernícola apresenta singularidades em relação ao meio epígeo e que provavelmente permite selecionar naturalmente microrganismos que utilizam fontes alternativas para obtenção de energia e nutrientes.

A total of 30 fungal fruiting bodies were collected from decaying plant materials (barks and litter) from the wild based on morphological variations. Nine of these fungi purified to monoaxenic cultures were included in the present study and also a type strain Ganoderma lucidum ATCC- 32471. These fungi were screened for lignocellulolytic activities, five of these organisms produced ligninolytic enzymes when exposed to two different concentrations of guaiacol (0.02% and 0.2%) on two different media (MEA and PDA). All ten fungal isolates screened for cellulolytic activity were positive for the production of the cellulase enzyme. The fungal isolates were characterised using morphological and molecular methods. Molecular characterization using ITS1 and ITS4 primers was able to identify these fungal isolates to degrees of accuracy ranging from 98% to 100%. The phylogenetic and lineage analysis showed that the species varied amongst phylum Basidiomycota, Ascomycota and early diverging fungal lineages Mucormycotina. Both monocultures and dual cultures of these 10 fungal species were cultivated for the purpose of spectrophotometrically quantifying and evaluating enzyme production on agricultural waste residues; corn cob, sugar cane bagasse and wheat straw. A pattern of antagonistic invasion interaction was identified to demonstrate increased enzyme production on dual cultures. Four of these fungal species, Trichoderma sp. KN10, Rhizopus microsporus KN2, Fomitopsis sp. KN1 and Coriolopsis sp. KN6 demonstrated tendencies of invasion and replacement in co-cultures. The fungi and their dual cultures showed varying levels of enzyme production. Analysis of mean showed dual culture interactions involving KN10 with values for MnP production approximately at 1.46U/ml compared to monoculture of 0.06U/ml. Further, dual laccase values approximately at 0.09U/ml compared to monocultures of 0.05U/ml. Overall the highest enzyme activity was observed using wheat straw. This study demonstrated and proved that agricultural waste residues can be used for lignocellulytic enzyme production and that antagonistic invasion by some fungi (in particular Trichoderma sp. KN10) in co-cultures can increase production of one or more of the three enzyme laccase, lignin peroxidase and manganese peroxidase. / Environmental Sciences / Ph. D. (Environmental Science)

Made available in DSpace on 2016-06-02T19:31:52Z (GMT). No. of bitstreams: 1 3245.pdf: 5126286 bytes, checksum: f1bcfa952e188a3e24d1b0431f77a619 (MD5) Previous issue date: 2010-08-12 / Universidade Federal de Sao Carlos / Enzymatic activity during decomposition is extremely important to hydrolyze molecules that are assimilated by microorganisms. During aquatic macrophytes decomposition, enzymes act mainly in the breakdown of lignocellulolytic matrix fibers (i.e. cellulose, hemicellulose and lignin) that encompass the refractory fraction from organic matter. Considering the importance of enzymatic activities role in decomposition processes, this study aimed to describe the temporal changes of xylanase and cellulase activities during anaerobic decomposition of the aquatic macrophytes community located at Óleo Lagoon, placed in Ecological Station of Jataí, Luiz Antonio, São Paulo, Brazil. After a survey of existing aquatic macrophytes, 10 species were indentified in the lagoon. They were collected and bioassays were accomplished in the laboratory. Decomposition chambers from each species (n = 10) were set up with dried macrophyte fragments and filtered Óleo Lagoon water. The chambers were incubated at 22.5oC, in the dark and under anaerobic conditions. Enzymatic activities and remaining organic matter were measured periodically during 90 days. Besides this analysis, pH, humification index and carbon types were evaluated. Initial cellulose and hemicellulose was measured for each species. For all the plants xylanase s activitiy was greater than cellulase activity. The results showed no correlation between organic matter decay and enzyme production. This could have occurred because of lignocellulolytic matrix fibers structural differences and also because of physical, chemical and biological characteristics. The incubations showed a low pH, what agrees with Óleo lagoon characteristics, supporting that aquatic macrophytes decomposition interfers in its physical and chemical characteristics. Egeria najas presented a dissolved organic carbon (DOC) accumulation pattern during its decomposition which differed from the other species. This pattern tends to accumulate humic substances and consequently alters some water characteristics. Besides it, E. najas presented the largest distribution over Óleo lagoon area. Emergent species presented the greater amount of particulate organic matter (POM) accumulated in sediment, configuring the main available substrate to anaerobic microorganisms. / As atividades enzimáticas durante a decomposição em ambientes aquáticos são extremamente importantes para hidrolizar as moléculas a serem assimiladas pelos microrganismos. Durante a decomposição das macrófitas aquáticas, as enzimas atuam principalmente na hidrólise das fibras presentes na matriz lignocelulósica (como celulose, hemicelulose e lignina), presentes na fração refratária da matéria orgânica. Considerando a importância do papel das atividades enzimáticas no processo de decomposição, esse estudo teve como objetivo quantificar e descrever as atividades das enzimas xilanase e celulase durante o processo de decomposição anaeróbia de uma comunidade de macrófitas aquáticas, bem como relacionar essas atividades às perdas de massa, baseando-se na hipótese de que as plantas com menores teores de tecidos de sustentação (celulose e hemicelulose), geralmente as de hábito submerso, apresentariam menor atividade destas enzimas em comparação às emergentes e flutuantes. A comunidade de macrófitas selecionada foi proveniente da lagoa do Óleo, situada na Estação Ecológica de Jataí, Luíz Antônio, São Paulo, Brasil. Após um levantamento das espécies de macrófitas aquáticas, foram identificadas 10 espécies na lagoa. Exemplares de cada espécie foram coletados e lavados ao laboratório, onde foram realizados alguns bioensaios e análises. Em laboratório foram preparadas câmaras de decomposição para cada espécie (n=10) contendo fragmentos secos e amostras de água filtrada da lagoa do Óleo. As câmaras foram incubadas a 22,5º C, no escuro e sob condições anaeróbias. As atividades enzimáticas e os teores de matéria orgânica remanescente foram avaliados periodicamente durante 90 dias. Além disto, durante este período, também foram determinados: o pH, o índice de humificação e as rotas de mineralização do carbono. Os teores iniciais de celulose e hemicelulose também foram quantificados. Os resultados mostraram maior produção de xilanase que celulase em todos os casos; além de uma maior produção de enzimas no início do processo. Não foram encontradas correlações entre as maiores perdas de massa e as maiores produções enzimáticas, o que pode ter ocorrido devido às características estruturais destas fibras na matriz lignocelulósica e também por fatores físicos, químicos e biológicos (comunidade microbiana presente). O pH das incubações apresentou-se baixo, o que condiz com a característica ácida da lagoa do Óleo e permitem inferir que a decomposição das macrófitas aquáticas interfere nas suas características físicas e químicas. O padrão de acúmulo de COD observado na decomposição de Egeria najas permitiu inferir sua grande influência nas características físicas e químicas da lagoa do Óleo, devido ao acúmulo de substâncias húmicas e por apresentar maior ocupação na área da lagoa. As espécies emergentes são principalmente responsáveis pelo acúmulo de MOP no sedimento, servindo como importante fonte de substrato para a microbiota anaeróbia.

Made available in DSpace on 2016-06-02T19:56:28Z (GMT). No. of bitstreams: 1 DissREM.pdf: 1870524 bytes, checksum: 11256ff68568004b18adba99fa4ba12b (MD5) Previous issue date: 2007-01-29 / Cellulases are enzymes that catalyze the hydrolysis of cellulose producing glucose. Immobilization and stabilization of these enzymes could improve enzymatic process making it more attractive by reducing costs during their use. In this work, Celluclast (Novozymes A/S, Denmark) and CG 200 (Genencor) were immobilized into chitosan and into a chitosan/alginate hybrid support, after activation of the support with glutaraldehyde and glycidol under different conditions of immobilization such as temperature and pH. Immobilization process was evaluated by calculating the immobilization yield, coupling yield and thermal stability at 65°C. Temperature and pH optimum of enzymatic activity were analyzed as well. The best derivatives were obtained with chitosan alginate activated with glycidol, after immobilization at 27°C, pH 7.0, for 16 hours. The biocatalyst presented 56.3% of immobilization yield, 40.6% of coupling yield and it 11.2 fold more stable than free enzyme at 65°C. The temperature and pH for maximum activity of the derivatives and soluble enzyme was 60°C and 50°C, pH 2.5-3.0 and 4.2, respectively. Finally, the performance of the best biocatalyst developed in this work in the hydrolysis of sugarcane bagasse was compared to one observed using soluble enzyme. Although a higher conversion has been obtained with soluble enzyme in the beginning of the reaction, the immobilized enzyme allowed to reach a higher final conversion, 70%, than the one obtained with free Celluclast, 38.9%. / Celulases catalisam hidrólise de celulose à glicose e a imobilização e estabilização da mesma podem viabilizar o processo enzimático, pois reduz custos em sua utilização. Neste trabalho, Celluclast 1.5L (Novozymes A/S, Denmark) e CG 200 (Genencor) foram imobilizadas em géis de quitosana e quitosana-alginato ativados com glutaraldeído e glicidol para posterior hidrólise do bagaço de cana-de-açúcar. O processo de imobilização foi acompanhado medindo-se proteína e atividade hidrolítica da enzima em papel de filtro. Avaliaram-se rendimento de imobilização, atividade recuperada, estabilidade térmica, temperatura e pH ótimo de atividade enzimática. Os melhores derivados foram obtidos com quitosana-alginato, ativados com glicidol com temperatura de imobilização de 27°C e pH 7,0 sendo 11,2 vezes mais estáveis que a enzima livre, com rendimento em torno de 40,6% e atividade recuperada em torno de 56,3%. A temperatura ótima para os derivados foi cerca de 10 graus maior que a da enzima livre (50°C), enquanto que o pH deslocou-se de 4,2 (enzima livre) para a região entre 2,5 e 3, para os derivados. Com o melhor derivado, realizou-se a hidrólise do bagaço de cana-de-açúcar, que apresentou conversão de 1,8 vezes (70,0%) superior à enzima livre (38,9%).

A hidrólise enzimática de celulose representa, em relação à hidrólise ácida, uma alternativa limpa para produção de etanol. No entanto, existem duas dificuldades: o alto custo das enzimas e recalcitrância das regiões cristalinas da celulose. Para o primeiro problema, propomos a imobilização de celulase, um complexo enzimático que sinergicamente promove a degradação da celulose em glicose e celobiose, sobre wafers de silício. Apesar da atividade enzimática de celulase adsorvida ser em geral menor que a de celulase livre, a imobilização de celulases provou ser vantajosa, pois permite até seis reusos, mantendo um nível de atividade apenas 20% inferior ao original. Quanto à questão da recalcitrância das regiões cristalinas da celulose, utilizamos diferentes pré-tratamentos de celulose, a fim de reduzir a sua cristalinidade e o seu grau de polimerização, além de também modificar a estrutura supramolecular da celulose e a quantidade de poros que esta apresenta, avaliando todos estes parâmetros quantitativamente frente à atividade enzimática livre e imobilizada. A sacarificação enzimática de celulase livre e imobilizada foi determinada na hidrólise de celulose microcristalina (Avicel), e dois tipos de celulose nativa, algodão e eucalipto. Avicel foi pré-tratada a partir da (i) dissolução e degradação em ácido fosfórico, (ii) dissolução em acetato de 1-etil-3-metil-imidazólio (EMIMAc), e (iii) da hidrólise por endoglucanases adsorvidas, uma enzima do complexo enzimático celulase. Celulose de eucalipto e algodão foram mercerizadas a fim de se retirar contaminantes. A hidrólise com celulase livre levou a taxas de conversão de celulose à glicose que não apresentaram correlação com o índice de cristalinidade, nem com o grau de polimerização, mas apresentaram correlação direta com a capacidade de absorção de água, também chamada de constante de capilaridade. As taxas de conversão obtidas na presença de celulase adsorvida apresentaram correlação inversa com a constante de capilaridade, evidenciando que o mecanismo de hidrólise neste caso é fortemente dependente da camada de hidratação da celulose. / Enzymatic hydrolysis of cellulose represents, in relation to acid hydrolysis, a clean alternative for production of ethanol. However, there are two difficulties: the high cost of enzymes and the recalcitrance of the crystalline regions of cellulose. For the first problem, we propose the immobilization of cellulase, an enzymatic complex which synergistically promotes the degradation of cellulose to glucose and cellobiose, onto Si wafers. Although the enzymatic activity of immobilized cellulase is generally lower than that of free cellulase, immobilization has proven to be advantageous since it allows up to six reuses maintaining the activity level at 80% of the original one. Concerning the recalcitrance of the crystalline regions of cellulose, we used different cellulose pretreatments in order to reduce its crystallinity and degree of polymerization, and to modify the cellulose supramolecular structure along with the amount of pores. All these parameters were quantitatively correlated with the activity of free and immobilized cellulase. The enzymatic activity of free and immobilized enzyme was determined by the hydrolysis of microcrystalline cellulose (Avicel), and two types of native cellulose, cotton and eucalyptus. Avicel was pretreated in three different ways: (i) dissolution and degradation in phosphoric acid, (ii) dissolution in 1-ethyl-3-methyl-imidazolium acetate (EMIMAc), and (iii) hydrolysis by immobilized endoglucanase, an enzyme that is part of the cellulase enzyme complex. Eucalyptus and cotton pulp were mercerized in order to remove contaminants. Hydrolysis with free cellulase yielded cellulose to glucose conversions that were neither correlated with the crystallinity index nor with the degree of polymerization, but were directly correlated with the cellulose ability to absorb water (capillary constant). The conversion rates obtained in the presence of immobilized cellulase correlated inversely with the capillary constant values, indicating that hydrolysis mechanism in this case is strongly dependent on the hydration layer of cellulose

The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.

Orientador: Luis Henrique Souza Guimarães / Banca: Daniela Alonso Bocchini / Banca: Valéria de Carvalho Santos Ebinuma / Resumo: A prospecção de enzimas fúngicas tem sido amplamente estudada nos ultimos anos, principalmente utilizando a Fermentação em Estado Sólido (FES). A procura por enzimas microbianas, principalmente de fungos filamentosos, capazes de degradar material lignocelulósico, tem despertado grande interesse para processos biotecnológicos como, por exemplo, na produção de bioetanol, a partir do bagaço de cana-de-açúcar. Fungos do gênero Aspergillus são reconhecidos como ótimos produtores de enzimas do complexo celulolítico e hemicelulolítico, e a busca por novas linhagens com potencial de produção destas torna-se um grande desafio. Aspergillus labruscus ITAL 22.223 é um fungo filamentoso recentemente isolado no sul do Brasil e, por este motivo, não há estudos na literatura sobre seu potencial de produzir enzimas celulolíticas e hemicelulolíticas. Diante do exposto, este estudo visou a produção e quantificação de enzimas do complexo celulolítico (celulase total, endoglucanase e β-glicosidase) e hemicelulolítico (xilanase), a partir de fermentação em estado sólido utilizando resíduos/produtos agroindustriais como substratos. Neste contexto, a maior atividade enzimática de xilanase foi obtida na presença de farelo de trigo (74,83 U/g de substrato) e de β-glicosidase em farelo de aveia (6,35 U/g de substrato) como substratos/fontes de carbono. Sendo a produção de xilanase em FES a que mais se destacou, algumas características da enzima contida no extrato bruto foram determinadas. Apresentou te... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prospecting of fungal enzymes has been largely studied in recent years, especially by means of Solid-State Fermentation (SSF). The search for microorganisms' enzymes able to degrade lignocellulosic material, mainly those of filamentous fungi, has attracted interest for application in biotechnological processes, such as in production of cellulosic ethanol from sugarcane bagasse. Aspergillus species are well known for their capability to produce cellulosic and hemicellulosic enzymes complex and the search for strains with this potential is an important challenge. Aspergillus labruscus ITAL 22.223 is a filamentous fungus recently isolated in south of Brazil with unknown potential to produce cellulolytic or hemicellulolytic enzymes. This study aimed the production and quantitation of enzymes of the complex cellulolytic (total cellulose, endoglucanase and and β-glucosidase) and hemicellulolytic (xylanse) obtained under solid-state fermentation using agroindustrial waste and product as substrates. According to this, the greatest enzymatic productions of xylanase (74.83 U/g of substrate) and β-glucosidase (6.35 U/g of substrate) were obtained using wheat bran and oat bran as substrates during SSF fermentation, respectively. Taking in account the best production of xylanase in SSF, some biochemical characteristics were determined for the enzyme contained in the crude extract. Optimal of temperature for enzyme activity was 55ºC and optimal pH was 5.5. Regarding its thermal stability, ... (Complete abstract click electronic access below) / Mestre

Production of cellulase and polygalacturonase by Aspergillus niger and Aureobasidium pullulans was studied in submerged (SmF) and solid state fermentation (SSF) systems. Substrates used in fermentation systems were mandarin peels and grape pomace. With Aspergillus niger used on grape pomace as a sole carbon source, cellulase production was detected after 72 hours in SSF and after 24 hours in SmF systems. The activity of cellulase per gram of substrate was higher in a submerged than in a solid state fermentation system. The longer time for higher polygalacturonase production was necessary in submerged fermentation systems and polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 72 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. With Aureobasidium pullulans used on grape pomace as a sole carbon source, cellulase production was detected after 48 hours in SmF and SSF fermentation systems. The activity of cellulase per gram of substrate was higher in solid state system than in a submerged fermentation system. Longer time for higher polygalacturonase production was necessary in both fermentation systems. Polygalacturonase activity was higher in SmF. The SSF fermentation with mandarin peels as a sole carbon source was similar, the highest detected activity of cellulase was determined after 48 hours. Different production of polygalacturonase was observed on mandarin peels in SmF systems. A comparison of enzyme productivities on grape pomace and mandarin peels showed that polygalacturonase activity per gram of substrate is highest in SmF system with mandarin peels as a sole carbon source. For both systems and both substrates manganese-dependent peroxidase was detected for the first time. Differences in the enzyme synthesis by Aspergillus niger and Aureobasidium pullulans depend on both the substrates used as well as on the fermentation system.

In diploma thesis the process of enzymatic hydrolysis of waste paper as a source for the production of liquid biofuels is discused. It follows directly the homonymous diploma thesis from Ing. Brummer, and it is based on the findings, which were solved and decided in previous work. In the theoretical part there is a summarization of basic information on the enzymatic hydrolysis of waste paper and the associated influences of various factors of the rate and degree of hydrolysis. Higher attention is paid to a waste cardboard and its pretreatment methods due to the maximalization of the yield of hydrolysis. The next part summarizes options of the fermentative production of biofuels, focusing on the method of simultaneous saccharification and fermentation, where the further appropriate organism for ethanol fermentation is discussed. The last part is about the technological process from the raw material input to the separation of ethanol. In the experimental section the pre-treatment of waste paper in order to maximize the efficiency of hydrolysis was examined. The best results were achieved using a vibratory mill. In addition, various parameters for simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the yeast Saccharomyces cerevisiae. The conversion rate of waste paper cellulose to reducing sugars was observed by spectrophotometric method by Somogyi - Nelson and the amount of produced ethanol was quantified using HPLC / RI. As a part of this thesis some conditions (amount of enzyme, substrate, nutrients, yeasts, temperature, pH, type of buffer) were optimized to maximize the effectiveness of the overall process. All experiments were carried out on corrugated cardboard, which was chosen as the most promising material for hydrolysis that was among the waste paper pulp in diploma thesis by Ing. Brummer.

In the diploma thesis are discussed the process of enzymatic hydrolysis of waste paper as a source for the production of bioethanol by bacteria Zymomonas mobilis. In the theoretical part summarize basic information about particular methods of hydrolysis, about paper used as a raw material for enzymatic hydrolysis, about possibilities of the fermentative production of bioethanol focusing on the method of simultaneous saccharification and fermentation comparison with enzymatic hydrolysis and fermentation. Suitable microorganisms for ethanolic fermentation and simultaneous saccharification and fermentation and their advantages and disadvantages, are further discussed in this part as well. The theoretical part ends with the suggestion of the technological process for production of bioetanol. It covers all necessary steps from the input of raw material to the separation of produced ethanol. In the experimental part various parameters of hydrolysis, fermentation and simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the Zymomonas mobilis CCM2770 and Zymomonas mobilis LMG457 bacterium. The conversion rate of paper cellulose to gluckose and production of ethanol were observed by HPLC/RI method. Type of buffer, quantity of cells, enzyme and substrate were optimized in order to maximize the efficiency of the process. All experiments were performed on paper containing high amount of cellulose and for comparison on standard medium which contains gluckose. The highest yields was achieved with the use of Novozymes® Cellulosic ethanol enzyme Kit. The strain Zymomonas mobilis LMG457 has demonstrated as a better producer.