A produção de biodiesel etílico é uma questão estratégica para a política energética nacional, uma vez que o Brasil é um dos maiores produtores de oleaginosas do mundo e o segundo maior produtor de etanol, álcool de origem renovável. Neste sentido, o presente trabalho buscou promover a transesterificação de óleos vegetais por etanol assistido por líquidos Iônicos sob diferentes fontes de aquecimento. O glicerol foi transformado em compostos de valor agregado e de interesse industrial. O mesmo foi modificado in situ após a reação de transesterificação gerando biodiesel aditivado pelos derivados da glicerina, conforme esquema 1. (Ver arquivo) / The production of ethyl biodiesel is a strategic issue for the national energy policy, because Brazil is one of the largest producers of oilseeds and the second largest producer of ethanol, a renewable alcohol. In this way, this work aimed to promote the transesterification of vegetable oils with ethanol assisted by ionic liquids under different heat sources. Glycerol was transformed into valuable compounds of industrial interest. It was also modified in situ after the transesterification reaction generating additive ethyl biodiesel, as shown below. (See file)

Doctor of Philosophy / Department of Chemical Engineering / Bin Liu / In the course of mitigating our dependence on fossil energy, it has become an urgent issue to develop unconventional and innovative technologies based on renewable energy utilization for fuels and chemicals production. Due to the lack of fundamental understanding of catalytic behaviors of the novel chemical compounds involved, the task to design and engineer effective catalytic systems is extremely challenging and time-consuming. One central challenge is that an intricate balance among catalytic reactivity, selectivity, durability, and affordability must be achieved pertinent to any successful design. In this dissertation, density functional theory (DFT), coupled with modeling techniques derived from DFT, is employed to gain insights into molecular interactions between elusive intermediates and targeted functional catalytic materials for novel electrochemical and heterogeneous catalytic processes. Two case studies, i.e., electroreduction of furfural and step-catalysis for cyclic ammonia production, will be discussed to demonstrate the capability and utility of DFT-based theoretical modeling toolkits and strategies. Transition metal cathodes such as silver, lead, and nickel were evaluated for furfuryl alcohol and 2-methylfuran production through detailed DFT modeling. Investigation of the molecular mechanisms revealed that two intermediates, mh6 and mh7 from mono-hydrogenation of furfural, are the key intermediates that will determine the product formation activities and selectivities. Nickel breaks the trends from other metals as DFT calculations suggested the 2-methylfuran formation pathway is most likely different from other cathodes. In this work, the Brønsted–Evans–Polanyi relationship, derived from DFT energy barrier calculations, has been found to be particularly reliable and computationally efficient for C-O bond activation trend predictions. To obtain the solvation effect on the adsorptions of biomass-derived compounds (e.g., furfural and glycerol), influence of explicit solvent was probed using periodic DFT calculations. The adsorptions of glycerol and its dehydrogenation intermediates at the water-platinum surface were understood via various water–adsorbate, water–water, and water–metal interactions. Interestingly, the bond-order-based scaling relationship established in solvent-free environment is found to remain valid based on our explicit solvent models. In the second case study, step-catalysis that relies on manganese’s ability to dissociate molecular nitrogen and as a nitrogen carrier emerges as an alternative route for ammonia production to the conventional Haber-Bosch process. In this collaborative project, DFT was used as the primary tool to produce the mechanistic understanding of NH3 formation via hydrogen reduction on various manganese nitride systems (e.g., Mn4N and Mn2N). Both nickel and iron dopants have the potential to facilitate NH3 formation. A broader consideration of a wide range of nitride configurations revealed a rather complex pattern. Materials screening strategies, supported by linear scaling relationships, suggested the linear correlations between NHx (x=0, 1, 2) species must be broken in the development of optimal step catalysis materials. These fundamental findings are expected to significantly guide and accelerate the experimental material design. Overall, molecular modeling based on DFT has clearly demonstrated its remarkable value beyond just a validation tool. More importantly, its unique predictive power should be prized as an avenue for scientific advance through the fundamental knowledge in novel catalysts design.

O glicerol é o principal produto obtido da síntese do biodiesel a partir de óleos vegetais e gordura animal, sendo gerado em 10 % da produção do biodiesel. Glicerol é um subproduto de muitos processos industriais tornando-se um substrato potencialmente atrativo para a produção de produtos de valor agregado através de ação bacteriana. Devido a sua alta produção, e sendo, consequentemente, um problema futuro caso este não seja consumido, uma alternativa para utilização do glicerol é a sua conversão em produtos de alta utilidade, tais como o hidrogênio, etanol, 1,3-propanodiol etc. A transformação do glicerol por processos biotecnológicos pode ser socialmente atrativa visto que existe grande interesse em buscas de alternativas para a utilização deste produto evitando seu acúmulo no meio ambiente. O presente trabalho teve como objetivo selecionar bactérias capazes de degradar glicerol residual da síntese química de biodiesel, e ter como subprodutos a formação de H2 e etanol, produtos de alto interesse social. Vários microrganismos foram testados quanto à ação de degradação do glicerol. Inicialmente, realizaram-se testes com consórcio microbiano ambiental (lodo), com isolamento e identificação das bactérias nele presentes. A partir desta etapa foi selecionada, através de fermentações anaeróbias, a cepa (bactéria) de maior potencial na degradação do glicerol e conversão em bioprodutos tais como, hidrogênio e etanol, sendo a bactéria Klebsiella pneumoniae BLb01 a que apresentou 100 % de degradação do glicerol residual com a melhor capacidade de produção de H2 e etanol. Após a seleção da cepa, realizaram-se testes de otimização das condições de cultivo para a produção de hidrogênio, utilizando planejamento experimental do tipo Plackett- Burman, Fatorial Fracionário e Delineamento Composto Central Rotacional. Os resultados obtidos mostraram que a maior produção de hidrogênio (45 % mol) foi obtida com o meio de cultivo constituído da seguinte composição: (30 g.L-1glicerol, pH 9, T de 39 °C, 3 g.L-1de extrato de levedura, 3 g.L-1 de K2HPO4 g.L-1 e 1,0 g.L-1 KH2PO4). Paralelamente foram realizados estudos utilizando este mesmo consórcio microbiano ambiental com diferentes pré-tratamentos (dessecação, térmico, básico, ácido e congelamento). O tratamento de dessecação apresentou o melhor desempenho na degradação do glicerol (66 %) e produção de H2 (34 %mol). / Glycerol is the main product resulting from the biodiesel synthesis from vegetable oils and animal fat, being equal to 10% of the biodiesel production. Glycerol is a byproduct of many industrial processes becoming a potentially attractive substrate for obtaining higher aggregate value products through bacterial action. Due to its high production and being, as a consequence, a future problem if not consumed, an alternative use for glycerol is its conversion in more useful products as hydrogen, ethanol, 1-3-propanediol etc. The transformation of glycerol by biotechnological processes can be socially attractive since there is a great interest in seeking alternatives for using this product avoiding its accumulation in the environment. The present work aims to select bacteria able to degrade residual glycerol from biodiesel synthesis forming hydrogen and ethanol, products of high social interest, as byproducts. Several microorganisms were tested as far as glycerol degrading is concerned. Initially, tests with environmental microbial consortium (sludge) were carried out isolating and identifying the bacteria present in it. Afterwards, through anaerobic fermentations, the strain with the greatest glycerol degradation potential and conversion in byproducts as hydrogen and ethanol was selected. The bacterium Klebsiella pneumoniae BLb01 presented 100% glycerol degradation with the best capacity of hydrogen and ethanol production. After the strain selection, tests of growing conditions optimization for hydrogen production were carried out using experimental planning type Placket-Burman, Fractional Factorial and Central Rotational Compound Delineation. The results showed that the highest hydrogen production (45 mol%) was achieved with the growing medium consisting of: glycerol 30 g.L–1, pH = 9, T = 39°C, yeast extract 3 g.L–1, K2HPO4 3 g.L–1. In parallel studies using the same environmental microbial consortium with different pre treatments (drying, thermal, basic, acid and freezing) were carried out. The drying treatment presented the best result for glycerol degrading (66% and hydrogen production (34 mol%).

Os polissacarídeos possuem diversas aplicações industriais devido a sua ampla variedade de propriedades físico-químicas. Além desses empregos tradicionais, pesquisas mais recentes estão impulsionando o uso de polissacarídeos para novas aplicações, principalmente na área de terapia farmacêutica. Com o objetivo de ampliar a produção industrial de polissacarídeos microbianos, pesquisas têm se concentrado nos exopolissacarídeos (EPS), que apresentam produtividade elevada e processos de extração e purificação mais simples quando comparados aos polímeros tradicionais. A lasiodiplodana, uma ?-(1->6)-D-glucana, é um EPS produzido por Lasiodiplodia theobromae, um fungo filamentoso característico de regiões tropicais e patógeno de mais de 500 espécies vegetais. Na produção de EPS observa-se que, como resultado do crescimento e da produção do biopolímero, o meio de cultivo torna-se mais viscoso, tornando difícil a manutenção da homogeneidade no biorreator e, consequentemente, prejudicando a transferência de massa e oxigênio no meio. Nos cultivos em que ocorrem mudanças na reologia do meio e nos quais são utilizados fungos filamentosos, sensíveis ao cisalhamento, o uso de biorreatores convencionais, como o Stirred Tank Reactor (STR) e o Airlift, não é adequado. Nesse contexto, no presente estudo tem-se como objetivo estudar a cinética de crescimento, consumo de substrato e produção de EPS pelo fungo filamentoso Lasiodiplodia theobromae, a partir de glicose e de glicerol, no Biorreator Agitado e Aerado de Baixo Cisalhamento (BAABC) e compará-lo com o STR. No estudo inicial, realizado em frascos agitados, a maior produção de EPS (6,49 ± 0,03 g/L) foi alcançada pelo ensaio G, que continha a maior concentração glicose testada (50 g/L) e a menor concentração de extrato de levedura (3 g/L). Quando se utilizou glicerol como fonte de carbono, a maior produção de EPS (3,39 ± 0,06 g/L) foi observada no Ensaio O, que continha 30 g/L de glicerol e a maior concentração de fonte de nitrogênio testada (12 g/L de extrato de levedura). Nos ensaios em biorreatores, quando utilizou-se glicose como fonte de carbono observou-se que o BAABC com controle de temperatura proporcionou uma produção de 3,17 ± 0,16 g/L de EPS, concentração inferior à obtida em frascos, porém bastante superior à obtida no biorreator STR (0,70 ± 0,12 g/L). Para os meios contendo glicerol, o biorreator STR proporcionou uma produção de EPS de 3,02 ± 0,19 g/L, enquanto no BAABC a concentração de EPS obtida foi muito menor (1,45 ± 0,25 g/L). Analisando-se, porém, a concentração máxima de biomassa obtida (28,86 ± 1,46 g/L) bem como os parâmetros cinéticos relacionados (YX/S e QX), nota-se que a produção de biomassa foi muito superior e mais eficiente no BAABC. Apesar das diferenças morfológicas visualmente observadas e confirmadas por meio de microscopia óptica nos EPS obtidos a partir de glicose e de glicerol, as análises de Difração de raios-X e Espectroscopia de absorção na região do infravermelho com Transformada de Fourier permitiram evidenciar a similaridade estrutural entre ambos os EPS. / Polysaccharides have several industrial applications because of their wide variety of physicochemical properties. In addition to these traditional applications, recent research is driving the use of polysaccharides towards new applications, especially in the field of pharmaceutical therapy. With the aim of increasing the industrial production of microbial polysaccharides, research has focused on exopolysaccharides (EPS), which have high productivity and simpler extraction and purification processes when compared to traditional polymers. Lasiodiplodana, a ?-(1->6)-D-glucan, is an EPS produced by Lasiodiplodia theobromae, a filamentous fungus characteristic of tropical areas and pathogenic of more than 500 plant species. In EPS production, as a result of the growth and biopolymer production, the culture medium becomes more viscous, making it difficult to maintain homogeneity inside the bioreactor and, consequently, harming the transfer of mass and oxygen in the medium. In cultures where changes in media rheology occur and in which shear-sensitive filamentous fungi are used, the use of conventional bioreactors, such as Stirred Tank Reactor (STR) and Airlift, is not appropriate. In this context, the objective of the present study was to study the growth kinetics, substrate consumption and EPS production by the Lasiodiplodia theobromae filamentous fungus, from glucose and glycerol, in the Low-Shear Aerated-Agitated Bioreactor (LSAAB) and compare it with the STR. In the initial study, conducted in shaken flasks, the highest EPS production (6.49 ± 0.03 g/L) was achieved by the G test, which contained the highest glucose concentration tested (50 g/L) and the lowest concentration of yeast extract (3 g/L). When glycerol was used as the carbon source, the highest EPS yield (3.39 ± 0.06 g/L) was observed in Test O, which contained 30 g/L glycerol and the highest concentration of nitrogen source tested (12 g/L of yeast extract). In the bioreactor trials, when glucose was used as the carbon source, it was observed that the LSAAB with temperature control provided a production of 3.17 ± 0.16 g/L of EPS, a concentration lower than that obtained in flasks, but rather higher than that obtained in the STR bioreactor (0.70 ± 0.12 g/L). For the glycerol-containing media, the STR bioreactor produced 3.02 ± 0.19 g/L of EPS, while in LSAAB the EPS concentration obtained was much lower (1.45 ± 0.25 g/L). However, analyzing the maximum concentration of biomass obtained (28.86 ± 1.46 g/L) as well as the related kinetic parameters (YX/S and QX), biomass production was much higher and more efficient in LSAAB. In spite of the visually observed morphological differences confirmed by optical microscopy in EPS obtained from glucose and glycerol, X-ray diffraction and absorption spectroscopy analyzes in the infrared region with Fourier transform showed the structural similarity between both EPS.

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.

High-protein nutrition (HPN) bars (≥ 30% protein) have limited shelf life and become excessively hard during storage. Various mechanisms have been proposed to explain the hardening. The objectives of this research were to investigate the chemistry of HPN bar hardening and propose solutions for slowing it and improving bar texture. In phase 1, HPN bars were made containing 34% whey protein isolate (WPI) or milk protein concentrate (MPC) powder, along with either sorbitol syrup or glycerol, and vegetable shortening or cocoa butter. Substituting MPC for WPI made the bars brittle and crumbly. Using glycerol initially made bars softer but accelerated hardening. Cocoa butter increased bar hardness because of its higher solid to liquid content. Most water (~99%) in HPN bars made using sorbitol syrup is present as bound water, with ~0.9% as intermediate water and ~0.1% as bulk water. During storage bound water increased ~0.02 g/100 g of solids while intermediate water decreased, suggesting changes in state of water taking place at protein surfaces. During storage, there were changes in protein conformation indicated by an increase (~4°C) in heat denaturation temperature of β-lactoglobulin and α-lactalbumin and a 15 to 40% decrease in denaturation enthalpy. In phase 2, various bar formulations were tested involving different proportions of proteins, lactose, glycerol, and sorbitol syrup, as well as type of lipid component, and disulfide bonds inhibition. Decreases in bar hardening occurred when MPC and WPI and sorbitol syrup and glycerol were used in combination. In phase 3, HPN bars made with 38% protein powder as a 50:50 combinations of WPI and MPC and with 20% of sorbitol syrup substituted with glycerol, had good texture and minimal hardening during storage. Bar hardening was not caused by phase separation of protein and sorbitol, Maillard browning, or formation of inter-molecular disulfide bonds. Minimizing bar hardening requires prevention of entropy-induced protein aggregation by masking hydrophobic regions on protein surfaces and preventing formation of extended protein networks. It is proposed that preferential exclusion of cosolvents causes glycerol to be oriented at protein surfaces such that its carbon backbone masks hydrophobic regions thus avoiding a decrease in entropy of water molecules. (229 pages)

Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.

Due to an increased environmental awareness and thus, concerns over the use of fossil-based monomer for polymer production, there is an ongoing effort to find alternatives to non-renewable traditional monomers. This has ushered in the rapid growth in the development of bio-based materials such as green monomers and biodegradable polymers from vegetable and animal resources. Glycerol, as a renewable bio-based monomer, is an interesting candidate for sustainable polymer production. Glycerol is a renewable material that is a by-product of the transesterification of vegetable oils to biodiesel. Utilization of the excess glycerol derived from the growing biodiesel industry is important to oleochemical industries. The main objective of this thesis was to produce high molecular weight polyglycerol from glycerol and synthesize stimuli-responsive polyglycerol hydrogels. The work began with an investigation of the step-growth polymerization of glycerol to relatively high molecular weight polyglycerol using several catalysts. The catalytic reaction mechanisms were compared and the polymer products were fully analyzed. High molecular weight partially branched polyglycerol with multimodal molecular weight distributions was obtained. The polymerization of glycerol proceeded fastest with sulphuric acid as catalyst as indicated by the highest observed conversion of monomer along with the highest molecular weights. Theoretical models were used to predict the gel point and to calculate monomer functionality. High molecular weight polyglycerol was used to synthesize novel stimuli-responsive hydrogels. Real-time monitoring of step-growth polymerization of glycerol was investigated using in-line and off-line Attenuated Total Reflectance/Fourier Transform infrared (ATR-FTIR) technique.

<p>Glycerol is one of the by-products of transesterification of fatty acids for the production of bio-diesel. Value-added products such as hydrogen, wood stabilizers and liquid chemicals from catalytic treatment of glycerol can improve the economics of the bio-diesel production process. Catalytic conversion of glycerol can be used for production of value-added liquid chemicals. In this work, a systematic study has been conducted to evaluate the effects of operating conditions on glycerol conversion to liquid chemical products in the presence of acid catalysts. </p><p>Central composite design for response surface method was used to design the experimental plan. Experiments were performed in a fixed-bed reactor using HZSM-5, HY, silica-alumina and ã-alumina catalysts. The temperature, carrier gas flow rate and weight hourly space velocity (WHSV) were maintained in the range of 350-500 oC, 20-50 mL/min and 5.40-21.60 h -1, respectively. </p><p>The main liquid chemicals detected in liquid product were acetaldehyde, acrolein, formaldehyde and hydroxyacetone. Under all experimental conditions complete glycerol conversion was obtained over silica-alumina and ã-alumina. A maximum liquid product yield of approximately 83 g/100g feed was obtained with these two catalysts when the operating conditions were maintained at 380 oC, 26 mL/min and 8.68 h-1. Maximum glycerol conversions of 100 wt% and 78.8 wt% were obtained in the presence of HY and HZSM-5 at temperature, carrier gas flow rate and WHSV of 470 oC, 26 mL/min and 8.68 h-1. HY and HZSM-5 produced maximum liquid product of 80.9 and 59.0 g/100 g feed at temperature of 425 and 470 oC, respectively.</p><p>Silica-alumina produced the maximum acetaldehyde (~24.5 g/100 g feed) whereas ã-alumina produced the maximum acrolein (~25 g/100 g feed). Also, silica-alumina produced highest formaldehyde yield of 9g/100 g feed whereas HY produced highest acetol yield of 14.7 g/100 g feed. The effect of pore size of these catalysts was studied on optimum glycerol conversion and liquid product yield. Optimum conversion increased from 80 to 100 wt% and optimum liquid product increased from 59 to 83.3 g/100 g feed when the pore size of catalyst was increased from 0.54 in case of HZSM-5 to 0.74 nm in case of HY, after which the effect of pore size was minimal.

Due to an increased environmental awareness and thus, concerns over the use of fossil-based monomer for polymer production, there is an ongoing effort to find alternatives to non-renewable traditional monomers. This has ushered in the rapid growth in the development of bio-based materials such as green monomers and biodegradable polymers from vegetable and animal resources. Glycerol, as a renewable bio-based monomer, is an interesting candidate for sustainable polymer production. Glycerol is a renewable material that is a by-product of the transesterification of vegetable oils to biodiesel. Utilization of the excess glycerol derived from the growing biodiesel industry is important to oleochemical industries. The main objective of this thesis was to produce high molecular weight polyglycerol from glycerol and synthesize stimuli-responsive polyglycerol hydrogels. The work began with an investigation of the step-growth polymerization of glycerol to relatively high molecular weight polyglycerol using several catalysts. The catalytic reaction mechanisms were compared and the polymer products were fully analyzed. High molecular weight partially branched polyglycerol with multimodal molecular weight distributions was obtained. The polymerization of glycerol proceeded fastest with sulphuric acid as catalyst as indicated by the highest observed conversion of monomer along with the highest molecular weights. Theoretical models were used to predict the gel point and to calculate monomer functionality. High molecular weight polyglycerol was used to synthesize novel stimuli-responsive hydrogels. Real-time monitoring of step-growth polymerization of glycerol was investigated using in-line and off-line Attenuated Total Reflectance/Fourier Transform infrared (ATR-FTIR) technique.