Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-08-30T17:21:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-08-30T18:48:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) / Made available in DSpace on 2017-08-30T18:48:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / As pentoses oriundas da hidr?lise da fra??o hemicelul?sica de biomassas vegetais podem ser fermentadas a etanol. Entretanto, s?o poucas as esp?cies conhecidas de micro-organismos capazes de converter pentoses a etanol e, mesmo essas, mostram-se ineficientes e suscept?veis ? presen?a de inibidores produzidos durante o pr?-tratamento das biomassas lignocelul?sicas. Neste contexto, o presente trabalho teve por objetivo identificar e avaliar seis linhagens de leveduras isoladas a partir de biomassas vegetais quanto a capacidade de realizar a fermenta??o alco?lica da D-xilose utilizando diferentes formula??es de meios sint?ticos. As linhagens foram submetidas a identifica??o morfol?gica e molecular. Os processos fermentativos foram conduzidos em batelada simples em frascos c?nicos sob agita??o a 150 rpm e temperatura de 30?C por at? 145 horas. Os bioprocessos foram monitorados a intervalos regulares de tempo quanto ? concentra??o de a??cares redutores, glicose, etanol e biomassa celular. Ao final do processo, foram determinados os valores das vari?veis de resposta rendimento (YP/S), produtividade (QP) e taxa de consumo de a??cares (qS). Todas as linhagens avaliadas foram capazes de produzir ?lcool em meio sint?tico contendo somente xilose como carboidrato. Os maiores valores de produ??o e rendimento alc?olico foram obtidos em meios que continham al?m da xilose, a glicose. O maior valor de YP/S observado foi de 0,38, em condi??es experimentais com produ??o de 3,33 g L-1 de etanol. A composi??o de microelementos e fontes de nitrog?nio afetou a fermenta??o alc?olica. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The pentoses from the hydrolysis of the hemicellulosic fraction of plant biomass can be fermented to ethanol. However, few known species of microorganisms are capable of converting pentoses to ethanol, and even those, are inefficient and susceptible to the presence of inhibitors produced during pretreatment of lignocellulosic biomasses. In this context, main objective of this work is to identify and evaluate six yeast strains isolated from plant biomasses in order to perform the alcoholic fermentation of D-xylose using different formulations of synthetic media. The strains were submitted to morphological and molecular identification at the species level. The fermentative processes were conducted in single batch in conical flasks under agitation at 150 rpm and at 30 ?C for up to 145 hours. The bioprocesses were monitored at regular intervals for the concentration of reducing sugars, glucose, ethanol and cell biomass. At the end of the process, the values of the response variables YP/S, QP e qS were determined. All the evaluated strains were able to produce alcohol in synthetic medium containing only xylose as a carbohydrate. The highest values of production and alcoholic yield were obtained in media that contained beyond xylose, glucose. The highest value of YP/S observed was 0.38, under experimental conditions with production of 3.33 g L-1 of ethanol. The composition of microelements and nitrogen sources affected the alcoholic fermentation.

Orientador: Francisco Maugeri Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T06:57:12Z (GMT). No. of bitstreams: 1 Zautsen_RemigiusReinerusMaria_D.pdf: 4385252 bytes, checksum: 3906f1725b3c49ed542429ca357c57ea (MD5) Previous issue date: 2011 / Resumo: Na fermentação de produtos como etanol, utilizando biomassa lignocelulósica como matéria-prima, existem dois fatores principais que limitam a produtividade e eficiência do processo: inibição pelo produto e inibição por substâncias no caldo hidrolítico provenientes da hidrólise. Neste trabalho, é proposta a remoção simultânea de ambos os fatores para eliminar seus efeitos negativos na fermentação alcoólica. Produtos de fermentação prejudicam muitas vezes a integridade da membrana celular do micro-organismo utilizado como fermento. Portanto, a toxidez do produto não permite que a fermentação ocorra de forma ilimitada, uma vez que o produto está presente no meio em certa concentração. O crescimento do micro-organismo, a produtividade e o rendimento são prejudicados pela presença do mesmo. Compostos como furfural, hidroximetil furfural, compostos fenólicos e ácidos, que são produzidos durante o pré-tratamento ou hidrólise da biomassa lignocelulósica, introduzem outros efeitos inibidores, como a extensão da fase lag da levedura, prejudicam o crescimento e a produção. Esta tese propõe empregar um solvente orgânico na dorna do biorreator, com o fim de extrair o produto inibidor e todos os componentes inibidores existentes no substrato, de tal forma que o processo de fermentação não seja prejudicado. Com esse objetivo, primeiramente foi definida a relação entre o tamanho molecular de agentes extrativos, bio-compatibilidade e propriedades extrativas dos mesmos. Em seguida, um solvente foi escolhido, sendo o biodiesel à base de óleo de mamona, através de características como biocompatibilidade, coeficientes de partição, seletividade, alta disponibilidade e reutilização. Foram feitas fermentações em regime batelada em fermentadores de bancada, utilizando o biodiesel como agente extrativo, demonstrando os efeitos positivos no desempenho da fermentação de um licor hidrolítico. Adicionalmente, o comportamento de uma cepa de levedura industrial foi estudado na presença de inibidores e foi construído um modelo matemático que descreve as taxas de conversão dos principais inibidores e as condições em que a levedura, ao invés de manter uma fase lag, inicia a produção de biomassa e etanol. Finalmente, foi elaborado, como exemplo da utilização da tecnologia proposta, um modelo do sistema contínuo de fermentação alcoólica com a extração líquido-líquido, incluindo a recuperação do produto e resfriamento do meio de fermentação pelo próprio solvente orgânico. Por meio desta modelagem e uma série de simulações, foram determinadas as faixas ideais das principais variáveis na produção de etanol pelo sistema bifásico, sendo elas a fração de licor hidrolítico no mosto, concentração de substrato, temperatura de fermentação, e taxa de diluição do solvente. Assim, o trabalho demonstra as vantagens, efeitos positivos e os limites da utilização de extração líquido-líquido na fermentação de substrato da segunda geração. Entre as vantagens se destacam: maior tolerância de caldo hidrolítico no mosto, elevada produtividade, maior rendimento e maior custo-benefício do substrato / Abstract: There are two main factors that limit fermentation productivity and eficiency during the production of chemicals like ethanol when using lignocelulosic biomass as raw material: product inhibition and inhibition by substances in hydrolitic liquor generated during hydrolyzis. In this work, the simultaneous removal of both factors is proposed to eliminate their negative effects on ethanol fermentation. Fermentation products often damage the cellular wall of the micro-organism that is used as ferment. As a result, the toxicicity of the product does not permit that the fermentation continues unhindered once the product concentration has reached a certain level; growth of the micro-organism, productivity and yield are effected. Substances like furfural, hydroximetil furfural, phenolic compounds and organic acids, that are produced or released during pre-treatment or hydrolyzis of ligno-celulosic biomass, introduce other inhibiting effects, like the extension of the lag phase of the ferment or decreasing growth and production. This thesis proposes the use of an organic solvent as a second liquid phase in the bioreactor, to extract both the inhibiting product and all inhibiting compounds present in the substrate, such that the fermentation process remains unhindered. With this objective, first the relation between the molecular size of an extractive agent and its biocompatibility and extractive properties was determined. Next, a solvent was chosen, being biodiesel based on castor oil, by prioritizing characteristics as biocompatibility, partition coeficients, selectivity, availability and possibilities for recycling and reuse. Batch fermentations were executed in bench-scale, using biodiesel as extractive agent, demonstrating the improvements of fermentation of hydrolytic liquor. Aditionaly, the performance of an industrial yeast strain was studied in the presence of inhibitors and a mathematical model was constructed that descibes the conversion rates of the main inhibitors and conditions at which the yeast, instead of maintaining a lag phase, starts production of biomass and ethanol. Finally, as a practical example of the proposed technology, simulations were performed for an integrated process including continuous ethanol fermentation with liquid-liquid extraction, product recovery and cooling of the fermentation broth by the extractive agent itself. The simulation results reveiled the optimal ranges for the most important variables of the two-phase ethanol production process, i.e. fraction of hydrolitic liquor in the must, substrate concentration, fermentation temperature and dilution rate of the solvent. In all, the work shows the advantages, positive effects of and limits to the use of liquid-liquid extraction in fermentation of second-generation substrate. Advantages are, among others, higher tolerance of hydrolyzate in the must, higher yield, higher productivity and higher return on investment of raw-material / Doutorado / Engenharia de Alimentos / Doutor em Engenharia de Alimentos

Orientadores: Aline Carvalho da Costa, José Geraldo da Cruz Pradella / Dissertação (mestrado) - Universidade Estadual de Campoinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-21T13:46:58Z (GMT). No. of bitstreams: 1 Pereira_BeatrizMerchelPiovesan_M.pdf: 1535419 bytes, checksum: fa059ca3da52ab10581615a140f2bc82 (MD5) Previous issue date: 2012 / Resumo: A produção de enzimas lignocelulolíticas por Trichoderma reesei RUT-C30 foi otimizada em frascos agitados e bioreatores de 0,5 e 3L visando maximizar os títulos enzimáticos e produtividade volumétrica. Para isso, foram testadas como fontes de carbono (1% m/v) bagaço de cana-de-açúcar pré-tratado por processo hidrotérmico (BH) ou por explosão a vapor, com (BED) e sem (BEX) deslignificação, em meio contendo proteose peptona, tween 80 e solução salina. Celulose comercial Celufloc200 (CE) foi testada para comparação. Maior produção de enzimas celulolíticas foi obtida com a utilização de BED (1,38 ± 0,11 FPU/mL) quando em comparação com CE (0,78 ± 0,14 FPU/mL) em frascos agitados, sendo esse material utilizado como fonte de carbono nos demais ensaios. A produção de hemicelulases (xilanases) foi similar para os dois meios (em U/mL): 18,03 ± 1,56 para BED e 20,04 ± 1,50 para CE. A variação da concentração da solução salina, da fonte de carbono e dos nutrientes permitiu aumento da produção de enzimas celulolíticas para 1,89 ± 0,12 (meio com o dobro de solução salina) e 2,73 ± 0,09 (meio com 2% m/v de BED e nutrientes proporcionais) em frascos agitados. A suplementação da fonte de carbono com farelo de soja, sacarose, licor de pré-tratamento, lactose e glicerol foi estudada e farelo de soja foi selecionado como suplemento do meio. A elaboração de um meio de mistura contendo o dobro de solução salina, farelo de soja e nutrientes proporcionais à concentração da fonte de carbono (meio MIX) permitiu o aumento da produção de enzimas para, em FPU/mL: 3,33 ± 0,10 (MIX15: contém 1,5% m/v de BED), 3,78 ± 0,33 (MIX20) e 3,67 ± 0,34 (MIX30) em frascos agitados. As atividades de xilanases foram superiores a 130 U/Ml utilizando os meios de mistura. Em bioreator de 3L a produção de enzimas celulolíticas utilizando o meio MIX15 atingiu 2,29 ± 0,20 FPU/mL. Para o meio padrão (BED 1% m/v) o pico de atividade obtido foi de 1,14 ± 0,32 FPU/mL. O aumento da concentração da fonte de carbono em bioreator para 3% (m/v) a partir do meio MIX15 resultou no aumento da atividade celulolítica para 4,20 ± 0,34 FPU/mL. Os picos de atividade de xilanases atingiram valores superiores a 180 U/mL em bioreator. O desempenho do coquetel enzimático produzido no meio MIX15 foi avaliado na hidrólise de BED e BH, e comparado ao coquetel produzido no meio padrão e a um coquetel comercialmente disponível (Sigma). Os valores de conversão de celulose em glicose foram superiores para o coquetel MIX15 em relação aos demais coquetéis ao se utilizar 3 ou 5% de sólidos, com ou sem adição de beta-glucosidase comercial (Novozym 188) / Abstract: The production of lignocellulolytic enzymes by Trichoderma reesei RUT-C30 was optimized in shake flasks and 0.5 and 3L bioreactors to maximize the enzymatic titles and volumetric productivity. The carbon sources considered were sugar cane bagasse (1% w/v) pretreated by the hydrothermal process (BH) or steam explosion, with (BED) and without (BEX) delignification. The medium contained proteose peptone, Tween 80 and saline solution. Commercial cellulose Celufloc200 (CE) was used for comparison. Increased production of cellulolytic enzymes in flasks was obtained with BED as carbon source (1.38 ± 0.11 FPU / ml) when compared to CE (0.78 ± 0.14 FPU / ml), and this material was selected as carbon source for further studies. The production of hemicellulases (xylanases) was similar for the two carbon sources (U / mL): 18.03 ± 1.56 with BED and 20.04 ± 1.50 with CE. Variation of the concentration of the salt solution, carbon source and nutrients led to an increased production of cellulolytic enzymes: 1.89 ± 0.12 (medium with doubled saline solution concentration) and 2.73 ± 0.09 (medium with 2% w/v BED and nutrients proportional to the carbon source) in shake flasks. Supplementation of the carbon source with soybean meal, sucrose, pretreatment liquor, lactose and glycerol was studied and soybean meal has been selected as supplement. The preparation of a mixture medium containing doubled saline solution, soybean meal and nutrients proportional to the concentration of the carbon source allowed increasing the production of enzymes for (in FPU / ml): 3.33 ± 0.10 (MIX15 - containing 1.5% w/v BED) , 3.78 ± 0.33 (MIX20) and 3.67 ± 0.34 (MIX30) in shake flasks. Xylanase activities were higher than 130 U/mL. In a 3L bioreactor, production of cellulolytic enzymes using MIX15 medium reached 2.29 ± 0.20 FPU / mL. For the standard medium (BED 1% w/v) the peak activity was 1.14 ± 0.32 FPU / mL. Increasing the concentration of the carbon source in the bioreactor to 3% w/v starting from MIX15 resulted in a cellulolytic activity of 4.20 ± 0.34 FPU / mL. Xylanase activity reached values higher than 180 U/mL in the bioreactor. The performance of the enzyme cocktail produced in MIX15 medium was evaluated for the hydrolysis of BED and BH, and compared to the cocktail produced in the standard medium and to a cocktail commercially available (Sigma). The values of conversion of cellulose to glucose were higher for the cocktail MIX15 compared to the other cocktails when using 3 or 5% solids, with or without adding commercial beta-glucosidase (Novozym 188) / Mestrado / Desenvolvimento de Processos Químicos / Mestra em Engenharia Química

>Magister Scientiae - MSc / The negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate. / National Research Foundation

The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.

Bagasse is the fibrous derivative of sugar cane, that is grown on a commercial scale in many tropical and sub-tropical countries, where ideal climatic conditions are experienced. The seasonality of sugar cane presents storage problems for bagasse, since this lignocellulosic material is susceptible to degradation by a diverse range of microorganisms, mainly fungi. The decay that is brought about contributes largely to the losses of fibre in a bagasse pile. The surrounding microclimate, and conditions within the pile, needs to be carefully monitored in order to understand the factors that support the fungal populations and biochemical activity. The microclimate at the surface and inside the bagasse pile at a paper mill in Stanger (South Africa) was carefully monitored over a one-year storage period. Significant changes were noted in temperature, pH and moisture content, between the surface and the inside of the pile, as the pile aged. The data were compared to established parameters for bagasse preservation, and it was found that the temperature was lower than expected, thus promoting fungal growth. The pH was much higher (promoting bacteria and actinomycetes) and the moisture content was too low to produce anaerobic conditions. The environmental conditions in the bagasse pile at Stanger, therefore, promoted the proliferation of microbes, and consequently decay. Fungi that were present in the pile, were enumerated in order to investigate the diversity and fungal succession. There was a wider variety of species and higher numbers of fungi at the surface than inside the bagasse pile and the Shannon and Berger-Parker diversity confirmed these observations. Sorensons measure also showed that the types of fungal communities at the surface and inside the pile only started becoming similar toward the latter part of storage. When compared to models for abundance of species, conditions on the surface of the pile allowed maximum niche occupation at the beginning of storage, followed by the establishment of a mature community. The inside of the pile displayed minimal niche pre-emption followed by a state where most fungal species shared the domain. This study indicated that, as the storage time increased, the microbial communities became better established. Bagasse is rich in holocellulose, the basic raw material used for paper-making. Since there were many species of holocellulolytic fungi found growing on the surface and the inside of the bagasse pile, the activity of cellulases and xylanases were determined. These enzymes were found to be active at the surface and inside the pile. However, higher activities of both enzymes were noted inside the bagasse pile than on the surface. The higher levels of activity inside the pile, despite lower fungal numbers, suggested that fungal counts were not a clear indication of biomass or biochemical activity. It appeared that the environment on the inside of the bagasse pile promoted the establishment of specific fungal populations that bring about a high degree of degradation to fibre inside the bagasse pile. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Energy utilization is a determining factor for the standards of living around the world, and the current primary source of energy is fossil fuels. A potential source of liquid fuels that could ease the strain caused by diminishing petroleum resources is bioethanol. Effective exploitation of biomass materials requires a pretreatment to disrupt the lignin and cellulose matrix. The pretreatment utilized for this research was oxygen delignification, which is a standard process stage in the production of bleached chemical pulp. The model substrate utilized as a feedstock for bioethanol was recovered fibre. An analysis of the substrates digestibility resulted in a hexose yield of approximately 23%, which justified the need for an effective pretreatment. An experimental design was performed to optimize the delignification conditions by performing experiments over a range of temperature, caustic loadings, and reaction times. Equations were developed that outline the dependence of various response parameters on the experimental variables. An empirical model that can predict sugar concentrations from enzymatic hydrolysis based on the Kappa number, enzyme loading, and initial fibre concentration was also developed. A study of hydrolysis feeding regimes for untreated recovered fibre (87 Kappa), pretreated recovered fibre (17 Kappa), and bleached pulp (6 Kappa) showed that the batch feeding regime offers reduced complexity and high sugar yields for lower Kappa substrates. In order to evaluate the possibility of lignin recovery, the pH of delignification liquor was reduced by the addition of CO₂ and H₂SO₄, resulting in up to 25% lignin yield. An experiment that looked at effect of post-delignification fibre washing on downstream hydrolysis found that a washing efficiency of approximately 90% is required in order to achieve a hexose sugar yield of 85%. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

Magister Scientiae - MSc / To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. α-L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones. / South Africa

Orientador: José Maria Ferreira Jardim da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Economia / Made available in DSpace on 2018-08-27T10:45:02Z (GMT). No. of bitstreams: 1 Murakami_ThaysGoncalvesdeLima_D.pdf: 10562342 bytes, checksum: 117ec4b1509fe2b8e1fb5ba7b3f11b9b (MD5) Previous issue date: 2015 / Resumo: A pesquisa tem por objetivo investigar as trajetórias tecnológicas que estão se formando na etapa de hidrólise enzimática para a produção de bioetanol de 2ª geração (também denominado bioetanol lignocelulósico). A escolha do bioetanol lignocelulósico como tema geral dessa tese mostra-se pertinente, haja vista que sua produção ainda se encontra em estágio inicial de desenvolvimento, criando oportunidade principalmente para países em desenvolvimento, que podem adequar suas políticas energéticas às especificidades locais (a partir da escolha de matérias-primas abundantes internamente a serem usadas como biomassa) e minimizar sua dependência de fontes fósseis. Particularmente o Brasil pode se beneficiar da produção de bioetanol lignocelulósico partindo do conhecimento e da infraestrutura já aplicados à produção de bioetanol da cana. A motivação para a seleção da etapa de hidrólise como tema específico está no fato de que esse processo ainda carrega muitas incertezas, não havendo tecnologia(s) líder(es) ou trajetória(s) dominante(s). Ao contrário, há um conjunto de rotas tecnológicas possíveis que precisam mostrar sua viabilidade técnica e econômica para se tornarem ideais. O estudo da rota enzimática, em detrimento da rota ácida, deve-se ao fato de que ela tem sido considerada pela literatura a mais promissora. Todavia, mesmo na hidrólise enzimática há enormes indefinições decorrentes da ampla diversidade de micro-organismos com potencial para degradação de material lignocelulósico que, no entanto, metabolizam as enzimas em diferentes proporções e condições ambientais. As incertezas decorrem também da ampla gama de enzimas degradadoras de matéria lignocelulósica e da ação sinérgica entre elas. Dado que cada matéria usada como biomassa possui uma composição lignocelulósica particular, a eleição do grupo de enzimas a serem usadas no processo de degradação se torna mais complexa. Com vistas a atender aos propósitos desta pesquisa ¿ de investigar as trajetórias tecnológicas em hidrólise enzimática para produção de bioetanol ¿ foi usada a base da Derwent Innovations Index durante o período de 1970 a 2014 para a seleção de patentes relacionadas ao tema. A partir da reunião das patentes de interesse, foram extraídas, sistematizadas e analisadas três categorias de informação dos documentos patentários, a saber, o(s) tipo(s) de pesquisa empreendido(s) (ou conteúdo da patente), o(s) micro-organismo(s) envolvido(s) na pesquisa e a(s) enzima(s) de interesse comercial. Com essas três categorias de informação, aplicou-se o modelo NK para detectar a presença de padrões de especialização entre `conteúdo-micro-organismo-enzima¿. Isso porque, espera-se que com o amadurecimento da rota de hidrólise enzimática, haja o afunilamento dos micro-organismos estudados, a paulatina eleição dos mais aptos à produção de bioetanol lignocelulósico e o direcionamento a determinadas enzimas, reduzindo o grau de incerteza que envolve o processo. O que se pretende com a investigação dessas três dimensões, portanto, é compreender em que direção os agentes públicos e privados estão focalizando seus esforços em termos de métodos/técnicas de pesquisa, em quais micro-organismos esses agentes têm apostado suas expectativas e em quais enzimas tem havido crescente interesse comercial. Essas dimensões, em seu conjunto, delineiam as trajetórias tecnológicas em hidrólise enzimática e dão pistas do atual estágio dessa rota de uma perspectiva setorial / Abstract: This research aims to investigate the technological trajectories in enzymatic hydrolysis process for the 2nd generation bioethanol production (also known as lignocellulosic bioethanol). The choice of the lignocellulosic bioethanol as the general topic of this work proves to be relevant, given that its production is still in early stage of development, creating opportunity especially for developing countries inasmuch as they can adjust their energetic policies to local conditions (choosing internally abundant raw materials to be used as biomass) and minimize their dependence on fossil fuels. Brazil particularly can benefit from lignocellulosic bioethanol production taking advantages from the knowledge and infrastructure already applied to the production of sugarcane bioethanol. The motivation for the study of the hydrolysis process is due to the uncertainties around it, to the extent that there is no dominant technology or trajectory. On the contrary, there are a number of possible technological routes that need to prove their technical and economic feasibility to become ideals, being the enzymatic route considered by the literature the most promising of them. Nevertheless, even in the enzymatic hydrolysis there are huge uncertainties arising from the wide range of micro-organisms with potential for the degradation of lignocellulosic matter. These microorganisms metabolize enzymes at different rates and at different environmental conditions. Uncertainties also arise from the wide range of enzymes needed to degrade the lignocellulosic matter and to the synergic action between them. Since each raw material used as biomass has a specific lignocellulosic composition, the choice of the group of enzymes to be used in the degradation process becomes more complex. In order to fulfill the purposes of this research ¿ to investigate the technological trajectories in enzymatic hydrolysis for bioethanol production ¿ we selected patents available in the Derwent Innovations Index database during the period 1970-2014. From the patent documents of interest we extracted, systematized and analyzed three categories of information, namely the type of the research (or patent content), the microorganism involved in the research and the enzyme of commercial interest. With these three categories of information, we applied the NK-model to examine the presence of specialization patterns among `type-microorganism-enzyme¿. It is expected that with the maturation of enzymatic hydrolysis route, there will be a reduction in the number of microorganisms studied, a gradual choice of the most suitable microorganisms and a focus on certain enzymes, reducing the degree of uncertainty involved in the process. The investigation of these three dimensions, therefore, is to understand in what direction the public and private actors are focusing their efforts in terms of methods/research techniques, microorganisms and enzymes. These dimensions, as a whole, outline the technological trajectories in enzymatic hydrolysis and track the current stage of this route in a sectorial perspective / Doutorado / Teoria Economica / Doutora em Ciências Econômicas

>Magister Scientiae - MSc / The complete degradation of recalcitrant lignocellulose biomass into value-added products requires the efficient and synergistic action of lignocellulose degrading enzymes. This has resulted in a need for the discovery of new hydrolytic enzymes which are more effective than commonly used ones. β-xylosidases and β-glucosidases are key glycoside hydrolases (GHs) that catalyse the final hydrolytic steps of xylan and cellulose degradation, essential for the complete degradation of lignocellulose. Functional-based metagenomics has been employed successfully for the identification and discovery of novel GH genes from a metagenome library. Therefore, this approach was used in this study to increase the chances of discovering novel glycoside hydrolase genes from a horse manure metagenomic DNA library constructed in a previous study. Three fosmid clones P55E4, P81G1, and P89A4 exhibiting β-xylosidase activity were found to encode putative glycosyl hydrolases designated XylP55, XylP81, and BglP89. Amino acid sequence analysis revealed that XylP55, XylP81, and BglP89 are members of the GH43, GH39, and GH3 glycoside hydrolase families, respectively. Phylogenetic analysis of XylP81 and BglP89 indicated that these showed relatively low sequence similarities to other homologues in the respective GH families. The enzymes were expressed and purified, and only XylP81 and BglP89 were biochemically characterized. XylP81 (~58 kDa) and BglP89 (~84 kDa) both showed optimum activity at pH 6 and 50℃ and retained 100% residual activity at 55℃ after 1-hour indicating that they are moderately thermostable. XylP81 had high specific activity against 4-nitrophenyl-β-D-xylopyranoside (pNPX; 122 U/mg) with a KM value of 5.3 mM, kcat/KM of 20.3 s-1mM-1, and it showed enzyme activity against α-L-arabinofuranosidase, β-galactosidase, and β-glucosidase activity. BglP89 had a high specific activity for 4-nitrophenyl-β-D-glucopyranoside (pNPG; 133.5 U/mg) with a KM value of 8.4 mM, kcat/KM of 22 s-1mM-1 and also showed α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and low β-xylosidase activity. BglP89 also showed low hydrolytic activity on cellobiose, β-glucan, and lichenan indicating that it is a broad specificity β-glucosidase. XylP81 retained ~40% activity in the presence of 3 M xylose whilst BglP89 showed considerable glucose tolerance at 150 mM glucose and retained ~46% residual activity. This study reveals two metagenomic derived enzymes (β-xylosidase and β-glucosidase) showing characteristics that could make them potential candidates for lignocellulose biomass degradation in biotechnological and industrial applications.