Diplomová práce na téma vybrané ekonomické a environmentální dopady zavádění bioethanolu jako součásti motorových paliv hodnotí některé dopady této legislativou dané povinnosti. Práce se především snaží vysvětlit možné dopady ekonomické, environmentální a sociální (změnu kvality ovduší, zdražování potravin a surovin, energetickou náročnost výroby a samotnou potřebu fosilních paliv při výrobě biosložky). Dále ukazuje přístup jednotlivých složek státního aparátu (MŽP, MPO, MD a MZe) a možnosti finanční podpory výrobcům bioethanolu.

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

This master’s thesis is aimed at process of enzymatic hydrolysis of lignocellulosic material – waste paper as a source of raw material for production of liquid biofuels. In the theoretical part of this work are summarized previously used methods of hydrolysis and lignocellulosic materials used for the process of hydrolysis as a source of fermentable sugars for fermentation technology. The different types of waste paper are evaluated from the composition and usability with consideration to the papermaking process in order to select the appropriate type of waste paper for the enzymatic hydrolysis process. In the next part of this work are suggested technological premises and procedures for the preparation of raw materials and the subsequent enzymatic hydrolysis of these pre–treated materials. In the experimental part were optimized parameters of enzymatic hydrolysis using the Novozymes company enzyme package. Enzymatic degradation of cellulose to reducing sugars was observed using Somogyi – Nelson method. For the verification of hydrolysis conditions were used model materials with high cellulose content – pulp and filter paper. Conditions, which seems to be the best after testing on the model materials, were verified on specific waste paper materials – offset cardboard, recycled paper, matte MYsol paper and for comparison again on model materials – pulp and filter paper. The highest yields was achieved with the use of cardboard, which was further tested using various combinations of pretreatment to material for purpose of increase the yields of hydrolysis.

This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.

The increasing demand for transportion fuels has led most countries around the world to look for alternatives to the predominant fossil fuels in the sector. Furthermore, the needs for security of energy supply, social – economic and sustainable development are among the factors driving the search for alternative fuels with regard to the fact that most oil producing countries are politically unstable. Developing countries are not an exception and are also formulating policies that will enhance development of alternatives to fossil fuels.   This study analyses the opportunities and consequences of the development of the biofuels industry in Zambia from a local, regional and global perspective with regard to policy, technical, socio – economic, environment and sustainability considerations. From analysis of global, regional and local situation, the development of biofuels in Zambia is simulated using Long – range Energy Alternatives Planning system (LEAP) under different policy assumptions with consideration of current and future demands. Furthermore, the socio – economic and environmental consequences are modelled in LEAP using these policies and other assumptions which include demand – cost projections. The environmental loadings are also simulated for the period with the assumption that biofuels are zero net emitters of green house gases. Land requirements are then estimated from these results for the simulated period. A model of illustrative scenarios for implementation of different biofuels policies is developed which can be used to monitor and analyse their consequences by adjusting the major parameters depending on varying circumstances.   The results of this study show that there is considerable potential for feedstock, land, water and mature technology for production of both biodiesel and bioethanol to meet blending demands for the simulated period. There is considerable potential for production of bioethanol from sugar cane and sweet sorghum using the well established fermentation processes. Biodiesel can be produced from jatropha and other oil bearing plants. Most of the current fleet of vehicles in Zambia was manufactured after 1990, thus there is little or no modification required on engines to run on low biofuel blends considered in the simulations. It is clear from the results of the simulation that biofuel demand for blends of 5% in 2015 rising to 10% in 2030 with current yields required for both can be achieved with the available land ad water resources. To achieve the 10% blending target set in the simulated period for bioethanol and biodiesel from sugar cane and jatropha, land required is 4,480Ha and 47,000Ha respectivey for growing this feedstock at current yields for the region. The GHG reduction would be equivalent to the 10% in 2030 if we assume that biofuels are zero net emitters. However, policies have to be formulated and implemented which should include manadatory blending targets and incentives on tax and investments.

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Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Bioethanol production from lignocellulosic biomass is seen as an alternative source of energy. However, large-scale production of bioethanol from lignocellulosic biomass is still not feasible due to the high cost of cellulase and lack of cellulases with a high specific activity that can act on crystalline cellulose. The study aimed at screening for microbial cellulolytic enzymes using a proteomic approach. The objectives were to screen for microbial cellulases with a high specific activity and separate the cellulolytic enzymes using a combination of zymography and two dimensional (2-D) gel electrophoresis followed by tryptic digestion, matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) and bioinformatics analysis. Fungal and bacterial isolates were cultured in M9 minimal and Mandel media for a period of 168 hours at 60°C and 30°C with cellobiose and Avicel as carbon sources. Microbial cells were separated from the supernatants through centrifugation and the crude enzymes from the cultures were used for the determination of cellulase activity, zymography, SDS-PAGE and two-dimensional gel electrophoresis. Five isolates, with lytic action on carbon sources studied were a bacterial strain, (BARK) and fungal strains (VCFF1, VCFF14, VCFF17 and VCFF18). Peak cellulase production by the isolates was found to be 3.8U/ml, 2.09U/ml, 3.38U/ml, 3.18U/ml and 1.95U/ml, respectively. Beta-glucosidase zymography resulted in a dark brown band and clear zones against a dark background for endoglucanase. Affinity precipitation of the VCFF17 isolate’s crude enzyme resulted in seven glycoside hydrolases with a carbohydrate binding module (CBM). The presence of the CBM in the glycoside hydrolases produced by the VCFF17 confer the isolate’s potential to be used in the hydrolysis of plant biomass for bioethanol production. Two-dimensional gel protein maps resulted in the separation and quantitative expression of different proteins by the microbial isolates. MALDI-TOF analysis and database search showed that the expressed proteins in this study closely relate to different glycoside hydrolases produced by other microbial species (Hypocrea jecorina, Emericella nidulans, Trichoderma pseudokoningii and Trichoderma koningii). BARK, VCFF1, VCFF14, VCFF17 and VCFF18 showed great potential as cellulolytic enzyme producers for bioethanol production. The BARK isolate exhibited the highest beta glucosidase activity. The isolates studied may benefit the industry in reducing the costs associated with bioethanol production in consolidated bioprocessing system. / National Research Foundation (NRF)

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