A presente dissertação visa compreender a relação das políticas públicas com o desenvolvimento do setor sucroenergético, tendo em vista as exigências da comunidade internacional em relação à produção de combustíveis sustentáveis sob a ótica do mercado globalizado. Partimos da análise das políticas públicas para agricultura, energia e meio ambiente e da política externa brasileira para os biocombustíveis nos governos Fernando Collor de Mello, Itamar Franco, Fernando Henrique Cardoso e Luís Inácio Lula da Silva. Após isso, analisamos as políticas públicas ambientais e de ordenamento territorial através da compreensão dos três zoneamentos existentes para o setor sucroenergético, com um recorte no estado de São Paulo. Finalmente, discutimos os acordos internacionais para o setor de biocombustíveis. / This work aims to understand the relationship between public policy and the development of sugarcane industry, in view of the international communitys regarding sustainable production of fuels from the perspective of the global market. First, we analyze the public policies for agriculture, energy and environment and foreign policy for biofuels in Brazilian government of Fernando Collor de Mello, Itamar Franco, Fernando Henrique Cardoso and Luís Inácio Lula da Silva. After that, we analyze the public policy for environmental and the land use planning, through the understanding of the three existing zoning for the sugarcane industry, with an indentation in the state of Sao Paulo. Finally, we discuss the international agreements for the biofuels sector.

It has been long known that algae have the potential to produce a diverse range of metabolic products including lipid and starch, which could be utilized as a fuel feedstock. Despite the capacity of algae to synthesize and store large amounts of lipids and starch, algae are not currently a commercially viable feedstock for biofuel. The metabolite storage in algae can depend on the availability of nutrients such that nutrient starvation can boost the storage of lipid and carbohydrate. These nutrient-status-induced changes in lipid and starch are underpinned by altered expression of several metabolite-related genes. However, many aspects of fatty acid and carbohydrate biosynthesis are not well understood. Furthermore, the genetic regulators of nutrient starvation-induced carbohydrate and lipid accumulation are unknown in microalgae. Therefore, this PhD focused on screening cultivation conditions, in particular Phosphorus (P) and Nitrogen (N) limited conditions that induce metabolic changes, evaluated a rapid microalgal screening method, which was used to identify putative metabolism regulators, and characterized in detail the role of one P-starvation regulator, called PSR1 (Phosphorus starvation response 1). For establishing suitable culture conditions, the microalga Chlamydomonas reinhardtii was cultured in five different P and N-limited conditions and screened for metabolic changes using Fourier transform infrared spectroscopy (FT-IR) at different phases of growth. The FT-IR spectral changes were visualized by multivariate statistical tools such as principal component analysis (PCA) and principal component-discriminant function analysis (PC-DFA). Clear clustering based on nutrient availability and metabolic changes demonstrates the potential and sensitivity of FT-IR in screening multiple culture conditions. The potential of FT-IR was further tested by screening mutant strains of C. reinhardtii that were defective in response to nutrient starvation. Nine lines with mutation in one or more of the PSR1, SNRK2.1 or SNRK2.2 genes and a wild type were screened by FT-IR for P and N starvation-induced metabolic changes. PCA, PC-DFA and predictive partial least squares discriminant analysis (PLS-DA) of FT-IR spectra, clearly distinguished wild type from mutant strains and clustered mutants with similar genetic backgrounds, demonstrating the potential of FT-IR to detect and differentiate specific genetic traits. The changes in lipid and carbohydrate profile under nutrient stress and in the different strains were validated by biochemical analysis and liquid chromatography-mass spectrometry (LC-MS).This thesis demonstrated that PSR1 is an important regulator of neutral lipid and starch biosynthesis. Transcriptomic analysis on wild type and psr1 mutant under P-starvation was performed to identify transcripts induced by P-starvation that were mis-regulated in psr1. Mainly transcripts encoding starch and triacylglycerol enzymes were affected. To further evaluate the role of PSR1 in regulating lipid and starch metabolism, complementation of psr1 and overexpression by PSR1 was performed. The P-starvation phenotype was clearly rescued in the complementation lines, and overexpression lines showed increased expression of P homeostasis genes and increased Pi accumulation in cells, with an increase in total starch content and number of starch granules. Clear increases in expression of key starch biosynthesis genes such as soluble starch synthase (SSS1, SSS5) and starch phosphorylase (SP1) was observed, which correlated with increased starch content in the overexpression lines. A carbon shift was observed as a decrease in neutral lipid was coupled with the increase in starch content. All together these findings suggest that PSR1 is a key transcriptional regulator of global metabolism, and demonstrated successful transcriptional engineering of microalgae.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-08-31T18:47:24Z No. of bitstreams: 1 LucyRoseMoreira.pdf: 1783051 bytes, checksum: 44ba9b1db5bc1ac27ae2d8e2f01cf6ef (MD5) / Made available in DSpace on 2017-08-31T18:47:24Z (GMT). No. of bitstreams: 1 LucyRoseMoreira.pdf: 1783051 bytes, checksum: 44ba9b1db5bc1ac27ae2d8e2f01cf6ef (MD5) Previous issue date: 2010-08-12 / CAPES / This paper describes an analytical procedure for determination of trace metals copper, cadmium and lead in fuel samples, using Basal Plane Pyrolytic Graphite Modified Electrode (BPPGME) by tetrazolium salts: ([3,3 '- (3-3'-dimethoxy-4, bifenililen 4'-bis [2 - (4-nitrophenyl)-5-phenyl-2H-tetrazolium (NBT) Cl 3 - (4,5 - dimetitiazol-2il) -2,5-diphenyl-2H-Tetrazolium ( MTT) Br]. Samples of fuels (ethanol, biodiesel and gasoline) were previously digested in a microwave oven using nitric acid media and peroxides (biodiesel) and only nitric acid (alcohol and gasoline). The species of tetrazolium employed adsorbed spontaneously and irreversibly at the electrode surface and showed redox peaks characteristic of pH dependent. The metal ions studied reacted at the electrode surface chemically modified to produce marked changes in the corresponding voltammograms. Peak current produced in the voltammograms using Chemically Modified Electrode in the presence of metallic ion was used for quantification of metal in the sample of fuel. In order to determine the species of interest the best results were in pH 5.0 for copper and pH 6.0 for other species. The concentration range for copper was 2,0x10-6 to 1.25x10-5 mol L-1; and for cadmium and lead the range varied from 1,0x10-9 to 1,0x10-8 mol L-1. The limit of detection in samples fuels (ethanol, biodiesel or petrol), by VRA was 2,5x10-6 mol L-1. The accuracy of the proposed technique (VRA) for the determination of copper in ethanol, biodiesel and gasoline was compared with the emission spectroscopy technique Optical inductively coupled plasma (ICP-OES), providing results in agreement among themselves, i.e. the copper concentration in the samples analyzed by both techniques to a level of certainty of 95%, not statistically different. / Este trabalho descreve um procedimento analítico para determinação dos metais traço cobre , cádmio e chumbo em amostras de combustíveis, utilizando eletrodo de grafite pirolítico plano basal modificado pelas espécies sais tetrazólios: ([3,3’-(3-3’- dimetoxi-4,4'-bifenililen-bis[2-(4-nitrofenil)-5-fenil-2H-tetrazolio-(NBT)Cl; 3-(4,5- dimetitiazol-2il)-2,5-difenil-2H Tetrazólio- (MTT)Br]. As amostras de combustíveis (álcool, biodiesel e gasolina) foram previamente digeridas em forno de microondas utilizando-se os meios acido nítrico e peróxidos (biodiesel) e somente acido nítrico (álcool e gasolina). As espécies tetrazólio utilizadas adsorveram espontânea e irreversivelmente na superfície do eletrodo e apresentaram picos de oxi-redução característicos dependentes do pH. Os íons metálicos estudados reagiram na superfície do eletrodo quimicamente modificado produzindo modificações acentuadas nos correspondentes voltamogramas. A corrente de pico produzida no voltamograma do EQM na presença do íon metálicos foi utilizada para quantificação do metal na amostra de combustível. Na determinação das espécies de interesse os melhores resultados foram em pH 5,0 para o cobre e pH 6,0 para as demais espécies. A faixa de concentração estudada para o cobre foi 2,0x10-6 a 1,25x10-5 mol.L-1; e para cádmio e chumbo a faixa variou de 1,0x10-9 a 1,0x10-8 mol.L-1. O limite de detecção nas amostras de combustíveis (álcool, biodiesel gasolina), por VRA foi de 2,5x10-6 mol.L-1 . A exatidão da técnica proposta (VRA) para as determinações de cobre em álcool, biodiesel e gasolina foi comparada com a técnica espectroscopia de emissão óptica com plasma indutivamente acoplado ( ICP-OES), fornecendo resultados concordantes entre si, ou seja o teor de cobre encontrado nas amostras analisadas pelas duas técnicas, para um nível de certeza de 95%, não diferiram significativamente.

Difficulties associated with the study of microbial communities, such as low proportions of cultivable species, have been addressed in recent years with the advent of a range of sequencing technologies and bioinformatic tools. This is enabling previously unexplored communities to be characterised and utilised in a range of biotechnology applications. In this thesis bioinformatic methods were applied to two datasets of biotechnological interest: microbial communities found living with the oil-producing alga Botryococcus braunii and microbial communities in acid mine drainage (AMD). B. braunii is of high interest to the biofuel industry due to its ability to produce high amounts of oils, in the form of hydrocarbons. However, a number of factors, including low growth rates, have prevented its cultivation on an industrial scale. Studies show B. braunii lives in a consortium with numerous bacteria which may influence its growth. This thesis reports both whole genome analysis and 16S rRNA gene sequence analysis to gain a greater understanding of the B. braunii bacterial consortium. Bacteria have been identified, some of which had not previously been documented as living with B. braunii, and evidence is presented for ways in which they may influence growth of the alga, including B-vitamin synthesis and secretion systems. AMD is a worldwide problem, polluting the environment and negatively impacting on human health. This by-product of the mining industry is a problem in the South West of England, where disused metalliferous mines are now a source of AMD. Bioremediation of AMD is an active area of research; sulphur-reducing bacteria and other bacteria which can remove toxic metals from AMD can be utilised for this purpose. Identifying bacteria and archaea that are able to thrive in AMD and which also have these bioremediation properties is therefore of great importance. Metagenomic sequencing has been carried out on the microbial community living in AMD sediment at the Wheal Maid tailings lagoon near Penryn in Cornwall. From these data have been identified a diverse range of bacteria and archaea present at both the sediment surface level and at depth, including microorganisms closely related to taxa reported from metalliferous mines on other continents. Evidence has been found of sulphur-reducing bacteria and of pathways for various other bioremediation-linked processes.

The recent surge in biodiesel production has resulted in a huge surplus of crude glycerol, a by-product of the process to the level of 10% by weight. This is turn has caused the price of glycerol to fall dramatically, and there are now few economically viable channels for using this resource: waste glycerol is usually combusted. Therefore, much interest has arisen in the possibility of making use of glycerol with biotechnology, as this would not only be a more efficient use of resources but also make biodiesel itself more commercially viable. The purple bacterium Rhodopseudomonas palustris is able to metabolize glycerol through photofermentation and thereby produce hydrogen, a commercially useful commodity. R. palustris is of particular interest for this purpose as, in contrast to many other species which have been investigated with a view to fermenting glycerol, it is highly tolerant of crude glycerol. The feedstock requires little purification or dilution to be made suitable for cultivation of R. palustris. Furthermore, the hydrogen gas produced by R. palustris when grown on glycerol is of high purity, and the organism's great metabolic diversity suggests it may be a useful strain for remediation of other waste materials. However, much groundwork is needed to establish R. palustris as a viable chassis organism for biotechnological purposes. This work sets out to establish optimal conditions for cultivating R. palustris in the laboratory, including the design of a suitable batch photobioreactor system. It also determines optimal conditions for electroporation of R. palustris for the purpose of knocking out endogenous genes or introducing heterologous genes. Furthermore, the introduction of heterologous genes is attempted in order to demonstrate the possibility of producing other high-value compounds with R. palustris, and several deletion strains with potential benefits for hydrogen production are created. Finally, several existing deletion strains are investigated to establish their suitability as chassis strains for further genetic manipulation.

Microrganismos simbiontes são essenciais para a exploração de dietas de baixo valor nutricional, o desenvolvimento, crescimento e a reprodução de seus hospedeiros. Insetos que se alimentam de dieta rica em materiais celulósicos, como é o caso de cupins, apresentam protozoários e/ou bactérias associadas ao trato digestivo que auxiliam na quebra do polímero de celulose e na fixação de nitrogênio. A celulose e a hemicelulose são polímeros estruturais formados por unidades de glicose, sendo a hidrólise desses polímeros de grande interesse industrial para a produção de etanol. O modo mais eficiente de hidrolisar a celulose é pelo uso de enzimas, as celulases. Os cupins apresentam grande eficiência na digestão de celulose e hemicelulose, sendo que a compreensão do processo de digestão de celulose por esses insetos pode facilitar o desenvolvimento de tecnologia mais eficiente para a quebra desse polímero. Assim, este trabalho buscou i) isolar, identificar e caracterizar microrganismos associados ao trato digestivo dos cupins Armitermes euamignathus (Isoptera: Termitidae) e Coptotermes gestroi (Isoptera: Rhinotermitidae); ii) verificar o potencial da microbiota na degradação dos principais componentes da lignocelulose (celulose, xilana e pectina); iii) caracterizar o potencial hidrolítico e determinar as condições ótimas de hidrólise (pH e temperatura das diferentes enzimas produzidas). A análise da microbiota cultivável levou à identificação de 14 filotipos para A. euamignathus e de 11 para C. gestroi, distribuídos nos quatro principais filos, Proteobacteria, Firmicutes, Bacteroidetes e Actinobacteria. A caracterização da microbiota não-cultivável levou à identificação de 17 filotipos em operários e três em soldados de A. euamignathus, enquanto que em C. gestroi foi possível identificar seis filotipos em operários e oito em soldados. O filo Firmicutes foi o mais abundante em A. euamignathus, enquanto Proteobacteria predominou em C. gestroi. O isolamento de bactérias em meio seletivo para degradação de celulose, xilana ou pectina levou à seleção de oito filotipos para A. euamignathus e cinco para C. gestroi. Extratos brutos obtidos do cultivo dessas bactérias apresentaram atividade de hidrólise de pectina e xilana, mas não celulose. Ensaios para otimização das reações de degradação indicaram a presença de enzimas que atuam em diferentes faixas de pH ótimo. Assim, a microbiota associada aos cupins estudados foi bastante diversa, apresentando ainda diferenças entre as diferentes castas desses insetos. Essa microbiota também atua em parte do processo de degradação da celulose, demonstrando o potencial que bactérias associadas ao intestino de cupins podem apresentar para a identificação de enzimas digestivas que possam ser utilizadas no processamento da celulose. / Symbionts are essential for insect hosts as they enhance the nutritional value of their host diets and support host development, growth and reproduction. Insects that feed on diets rich in cellulose, such as termites, exhibit protozoa and/or bacteria within their digestive tract that aid in breaking the cellulose and in nitrogen fixation. Cellulose and hemicellulose are polymers formed by units of glucose, and the hydrolysis of these polymers is of great industrial interest for the production of ethanol. Cellulases are the most efficient enzymes to break cellulose. Termites have a huge capacity to digest cellulose and hemicellulose; thefore, understanding the process by which they digest cellulose may allow the development of more suitable technologies devoted to the industrial utilization of cellulose. This work aimed to i) isolate, identify and characterize microorganisms associated with the digestive tract of Armitermes euamignathus (Isoptera: Termitidae) and Coptotermes gestroi (Isoptera: Rhinotermitidae), ii) investigate the potential of symbionts in the degradation of the main components of lignocellulose (cellulose, xylan and pectin); iii) characterize the hydrolytic potential and determine the optimum hydrolysis conditions (pH and temperature) for the different enzymes produced. The analysis of culturable microorganisms led to the identification of 14 phylotypes for A. euamignathus and 11 for C. gestroi, which were distributed in four Phyla, Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. The characterization of the non-culturable microbiota led to the identification of 17 phylotypes in workers and three in soldiers of A. euamignathus, while six phylotypes were identified in workers and eight in soldiers of C. gestroi. Firmicutes was the most abundant in A. euamignathus, while Proteobacteria predominated in C. gestroi. The isolation of bacteria in selective medium to degrade cellulose, xylan or pectin led to the selection of eight phylotypes from A. euamignathus and five from C. gestroi. Crude extracts obtained from the cultivation of these bacteria showed hydrolytic activity towards to xylan and pectin, but not cellulose. Assays for optimization of enzymatic reaction indicated the presence of enzymes that act at different pH ranges great. As a conclusion, symbiont diversity was quite different between the termites species and in between the castes of these species. But the microbiota isolated also acts in the degradation of cellulose, demonstrating the potential for the gut-associated bacteria of termites may present for the identification of digestive enzymes which can be used in the processing of cellulose.

Triacylglycerols (TAGs) are stored in variable amounts (1-90 % by dry weight) in seed and non-seed tissues of various plant species. To gain insights into tissue- and species-specific regulation and biosynthesis of TAG in plants, we used 454 pyro and Illumina sequencing and generated transcriptome for eight species (brassica, castor, nasturtium, euonymus, oil palm, date palm, and avocado). In all tissues analyzed, an increased expression was noted for genes mostly associated with fatty acid biosynthesis in plastid, but much less increase in those for TAG assembly in the endoplasmic reticulum. Furthermore, hexose metabolism in plastid, relative to cytosol, appeared to be crucial in most oil-rich tissues, which is likely associated with directing high pyruvate flux toward plastid fatty acid synthesis. We also identified that WRINKLED1 transcription factor acts as a master switch in regulating oil biosynthesis in most oil-rich seed and non-seed tissues of monocot and dicot plants, suggesting a highly conserved mechanism. Overall, our studies point to distinctive modes of regulation of fatty acid biosynthesis and TAG assembly that are conserved in both seed and non-seed oil-rich plants, which can be extended to other crops for the purpose of generating biofuels.

The production of alternative transportation fuels is imperative to meet future energy demands without contributing to global climate change. Advances in alternative processing techniques that have emerged due to interest in microalgae as a feedstock have led to a variety of potential processing pathways for the production of bio-based fuels. A major hurdle in the algal production process is maintaining a fast and stable algae culture. Monocultures, developed for their high lipid content, suffer from low productivity, are susceptible to crashes and require a constant supply of carbon dioxide to maintain productivity. In an effort to circumvent these obstacles, algal turf scrubber systems (ATS) are now being targeted not only for water purification, but as a means of producing algae feedstocks for fuel conversion. The resulting algae are capable of being harvested at a much higher density, requiring less energy for dewatering purposes. ATS systems do present other drawbacks that downstream technologies need to account for to make this system a viable means for fuel conversion. While polyculture algae species display great growth characteristics, they contain high percentages of nitrogen containing proteins and low lipid content. If not removed this nitrogen pollutes any resulting biocrude making it unacceptable for diesel fuel blends. This study investigates a processing method which reduces the nitrogen content of the resulting fuel by fermenting both carbohydrates and proteins into intermediate compounds. By tuning the E. coli fermentation stain it is hoped that the process will yield higher value co-products than those investigated in this study. The research contained herein incorporates laboratory experimentation with engineering systems modeling to assess the economic feasibility and environmental impacts of generating biofuels from ATS cultivated algae. Results show a minimum fuel selling price of $5.93 per gasoline gallon equivalent and greenhouse gas emissions of -0.0185 kg CO2eq per MJ fuel. Discussion points include process optimization in terms of minimum fuel selling price and global warming potential.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.

<p>Fuel availability and security of supply are two of the most important factors in the well functioning of a company like Gävle Energi. Another important factor is the price of the fuels used. The transportation cost plays also an important role when purchasing fuels from different sources. Currently the fuels used in Gävle Energi are mainly woody biofuels, but waste and peat could also be used in the future.</p><p>The aim of this thesis is to provide an overview of the different available biofuels in the region of Gävle. The fuels considered in the study are:</p><p>- Bark</p><p>- Forest Residues</p><p>- Wood waste</p><p>- Pellets and Briquettes</p><p>- Garbage/waste materials</p><p>- Peat</p><p>The research is focused on the physical properties of the fuels, their price and transportation cost, environmental and legislation issues and the availability in the region of Gävle. A 10-year perspective is defined for an estimated availability of the different fuels in te region.</p>