Thesis (Doctor of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Lignocellulosic biomass (agro-waste) has been recommended as the most promising feedstock for the production of bioalcohols, in the biofuel industry. Furthermore, agro-waste is well-known as the most abundant organic matter in the agricultural and forestry product processing industry. However, the challenge with utilizing agro-waste as a feedstock is its highly recalcitrant structure, which limits hydrolysis to convert the holocelluloses into fermentable sugars. Conventional pre-treatment methods such as dilute acid, alkaline, thermal, hot water and enzymatic, have been used in previous studies. The challenge with these conventional methods is the generation of residual toxicants during the pretreatment process, which inhibits a high bioalcohol yield, by reducing the microbial populations’ (fermenter) ability to be metabolically proficient during fermentation. Numerous studies have been developed to improve the engineered strains, which have shown to have an ability to reduce the inhibition and toxicity of the bioalcohols produced or by-products produced during pre-treatment, while enhancing the bioalcohol production. In the present study (chapter 5), evaluation of common conventional methods for the pretreatment of the mixed agro-waste, i.e. (˃45µm to <100µm) constituted by Citrus sinensis, Malus domestica peels, corn cobs from Zea mays and Quercus robur (oak) yard waste without a pre-rinsing step at a ratio of 1:1 at 25% (w/w) for each waste material, was undertaken, focusing on hot water pre treatment followed by dilute acid (H2SO4) pre-treatment. To further pretreat the mixed agro-waste residue, cellulases were used to further hydrolyse the pre-treated agro-waste in a single pot (batch) multi-reaction process. The TRS concentration of 0.12, 1.43 and 3.22 g/L was achieved with hot water, dilute acid and cellulases hydrolysis as sequential pretreatment steps, respectively, in a single pot multi-reaction system. Furthermore, a commercial strain was used to ascertain low (C1 to C3) and high carbon content (C4+) bioalcohol production under aerobic conditions. Multiple bioproducts were obtained within 48 to 72 h, including bioethanol and 1-Butanol, 3-methyl, which were major products for this study. However, undesirable bio-compounds such as phenolics, were detected post fermentation. Since multiple process units characterised by chemical usage and high energy intensivity have been utilized to overcome delignification and cellulolysis, a sustainable, environmental benign pretreatment process was proposed using N. mirabilis “monkey cup” fluids (extracts) to also reduce fermenter inhibitors from the delignification of mixed agrowaste; a process with minimal thermo physical chemical inputs for which a single pot multi-reaction system strategy was used. Nepenthes mirabilis extracts shown to have ligninolytic, cellulolytic and xylanolytic activities, were used as an enzyme cocktail to pretreat mixed agro-waste, subsequent to the furtherance of TRS production from the agro-waste, by further using cellulase for further hydrolysis. N. mirabilis pod extracts were determined to contained carboxylesterases (529.41±30.50 U/L), β-glucosidases (251.94±11.48 U/L) and xylanases (36.09±18.04 U/L), constituting an enzymatic cocktail with a significant potential for the reduction in total residual phenolic compounds (TRPCs). Furthermore, the results indicated that maximum concentration of TRS obtainable was 310±5.19 mg/L within 168 h, while the TRPCs were reduced from 6.25±0.18 to 4.26 ±0.09 mg/L, which was lower than that observed when conventional methods were used. Overall N. mirabilis extracts were demonstrated to have an ability to support biocatalytic processes for the conversion of agro-waste to produce fermentable TRS in a single unit facilitating multiple reactions with minimised interference with cellulase hydrolysis. Therefore, the digestive enzymes in N. mirabilis pods can be used in an integrated system for a second generation biorefinery.

Submitted by Leticia Schettini (leticia@ufrrj.br) on 2016-09-19T15:16:21Z No. of bitstreams: 1 2010 - Juarez Vicente.pdf: 1012625 bytes, checksum: 30ce03687174fc4cf4226e455adde13a (MD5) / Made available in DSpace on 2016-09-19T15:16:21Z (GMT). No. of bitstreams: 1 2010 - Juarez Vicente.pdf: 1012625 bytes, checksum: 30ce03687174fc4cf4226e455adde13a (MD5) Previous issue date: 2010-05-07 / From the scientific viewpoint, the word ?uncertainty? expresses doubt about an analytic result. The implementation of the concept of uncertainty of measurement is a crucial step that the Brazilian laboratories must take in the process for obtaining the certification of the ability to perform essays in accordance with the Brazilian technical norm ABNT NBR ISO/IEC 17025. Regardless the technical area of application, the determination of the uncertainty of measurement associated with the result of an analysis is extremely important, especially in the food industry. The expanded uncertainty is related with the degree of reliability of an analytical result, and by means of this information it is possible to evaluate if the result of an analysis is consistent, i.e., if the uncertainty associated to the measurement remains inside an acceptable range. When the percentage of uncertainty exceeds a threshold, it is necessary to find the process variables that more intensely contribute to increasing the uncertainty and, in the sequence, to take corrective actions in order to minimize the impact caused for those variables over the uncertainty of measurement. The aim of this study was to detect the uncertainty of measurement associated with the analysis of total reducing sugars (TRS) by the Lane-Eynon method. The equipments used were analytical and semi analytical balances, burettes with 10mL and 25mL, and the food matrix studied was wild honey. To perform the calculations of the standard uncertainty, u, information about the uncertainty of the laboratory materials used in the analysis ? obtained from the certificate of calibration. The combined uncertainty, uc, was obtained by deriving the influence factors and considering, if necessary, the effective degree of freedom, ?eff, as stated in the Welch-Satterthwaite equation. The expanded uncertainty, U, was obtained by multiplying uc by the appropriate coverage factor, k = 2 (at level of confidence of 95,45%). The results showed that the expanded uncertainty obtained for the analysis of TRS was less than 2,0% when the analytical balance was used. The process variable having the greatest impact was the volume (close to 80,0%), followed by the title (close to 20,0%). The analysis of the expanded uncertainty obtained for the analysis of TRS when the semi analytical balance, and 10mL as well as 25mL burettes were used provided reliable results. However, care must be taken in such a combination of equipments, especially when using the 25mL burettes, since the percentage of uncertainty was great than 3,5%, too close to the limit established by the Codex Alimentarius, 4,0%. It was observed that, in this case, the variable having the greatest impact on the uncertainty was the title, with 79,82% (10mL burettes), and 87,10% (25mL burettes). / Do ponto de vista t?cnico-cient?fico, a palavra ?incerteza? expressa a d?vida em rela??o a um resultado anal?tico. A aplica??o do conceito de incerteza de medi??o ? fundamental para os laborat?rios brasileiros que almejam obter o reconhecimento da capacidade de realiza??o de ensaios em conformidade com a norma ABNT NBR ISO/IEC 17025. Independentemente da ?rea de atua??o, ? de extrema import?ncia o estudo e o conhecimento da incerteza de medi??o associada a um resultado de an?lise, em particular quando aplicado ? ind?stria de alimentos. A incerteza expandida informa o grau de confiabilidade de um resultado anal?tico e, atrav?s dela, ? poss?vel avaliar, a um n?vel de abrang?ncia estabelecido, se o resultado de uma an?lise transmite credibilidade, ou seja, se o valor da incerteza est? em uma faixa de concentra??o aceit?vel ou n?o. Quando o percentual de incerteza ultrapassa o limite aceit?vel, ? necess?rio conhecer quais as vari?veis do m?todo que mais contribuem para a incerteza e executar as medidas corretivas a fim de minimizar este impacto e, consequentemente, reduzir a incerteza total. O objetivo geral deste trabalho foi determinar a incerteza expandida associada ? an?lise titulom?trica de a??cares redutores totais (ART) pelo m?todo de Lane-Eynon, tendo como matriz uma amostra de mel, utilizando balan?a anal?tica e semi anal?tica e buretas de 10mL e 25mL. Para o c?lculo da incerteza padr?o (u), utilizaram-se os dados obtidos nos certificados de calibra??o de equipamentos e vidrarias empregados na an?lise. A incerteza combinada ( ) foi obtida derivando os fatores de influ?ncia e considerando, quando necess?rio, o grau de liberdade efetivo (?eff), dado pela Equa??o de Welch-Satterthwaite. Obteve-se a incerteza expandida (U) multiplicando-se por 2 (dois) o valor de para um n?vel de confian?a de 95,45%. Os resultados obtidos mostraram que a incerteza expandida de ART utilizando balan?a anal?tica n?o foi significativa (<2%) e que a vari?vel que mais influenciou foi o volume de amostra gasto (~80%), seguido do T?tulo (~20%). A an?lise da incerteza expandida de ART obtida com balan?a semi anal?tica e buretas de 10mL e 25mL gerou resultados confi?veis. No entanto, deve haver cautela neste caso, especialmente ao se utilizar bureta de 25mL, visto que o valor percentual de incerteza superou 3,5%. Observa-se que a vari?vel que mais contribui para a incerteza foi o T?tulo com 79,82% (bureta de 10mL) e 87,10% (bureta de 25mL).

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Atualmente, tem-se estudado diversos tipos de pré- tratamentos para reduzir a recalcitrância da biomassa lignocelulósica com intuito de aumentar sua digestibilidade química/enzimática, para que esta possa ser utilizada na produção de etanol e/ou outros bioprodutos de valor agregado. Neste estudo avaliou-se o efeito do pré-tratamento de explosão a vapor catalisada por ácido cítrico e hidróxido de sódio, e do pré-tratamento de deslignificação alcalina nas propriedades químicas e estruturais do bagaço de cana-de-açúcar (BCA), bem como sobre o processo de hidrólise enzimática. O pré-tratamento de explosão a vapor foi realizado em reator de 1,4L sob temperatura de 180 °C com tempo de retenção de 5 min. A etapa de deslignificação com NaOH (2% m:m) foi realizada a 120 °C, sob refluxo de 4h. A caracterização química e estrutural da biomassa in natura e pré-tratada foi realizada por FTIR, MEV, DRX. A hidrólise enzimática foi realizada com volume final de 20 mL constituído de 3% de BCA (massa seca), tampão de citrato de sódio 50 mM (pH=5,0) e 10 FPU do complexo enzimático Cellic® CTec 3. Os frascos foram mantidos sob agitação de 150 rpm a 50ºC por 72 h. Retirou-se alíquotas de 1,5 mL após 0, 12, 24, 36, 48 e 72h para determinação de açúcares redutores totais (ART) pelo método do ácido 3,5-dinitrosalicílico (DNS). O bagaço de cana-de-açúcar apresentou 24,22% de lignina, 27,61% de hemiceluloses e 42,77% de celulose, no entanto após o pré-tratamento de explosão a vapor catalisado com ácido cítrico, obteve-se uma biomassa com menor quantidade de hemiceluloses (16,16%) e com formação de fissuras na parede celular da fibra. O bagaço pré-tratado por explosão a vapor com NaOH apresentou completa desestruturação da fibra, remoção de 65% da lignina e preservação da fração hemicelulósica. Depois de submeter à biomassa lignocelulósica sem tratamento e pré-tratada por explosão a vapor ao processo de deslignificação alcalina observou-se a completa desestruturação da matriz lignocelulósica e a solubilização de 85-90% da lignina em todas as amostras. O índice de cristalinidade da biomassa após os prétratamentos teve uma aumento quando comparado ao material in natura, podendo este ser associado à remoção de componentes amorfos, como a lignina e hemiceluloses, e também da fração amorfa da celulose. Frente ao percentual mássico de biomassa utilizada no processo de hidrólise enzimática valor teórico correspondente a 100% de sacarificação equivale, aproximadamente, a 33,0 g.L-1. Após a hidrólise enzimática (72h) da biomassa in natura e pré-tratada por explosão a vapor obteve-se uma maior concentração de ART e um maior percentual de sacarificação para o bagaço de cana obtido a partir da explosão a vapor com NaOH (23,05 g.L-1, 69,15% de sacarificação). Já na hidrólise da biomassa in natura após processo de deslignificação alcalina a concentração de ART aumentou em 18,33 g.L-1, enquanto que para o bagaço pré-tratado por explosão a vapor com água, ácido cítrico e NaOH seguido da deslignificação alcalina o aumento de ART, correspondeu a 19,67 g.L-1, 19,93 g.L-1 e 6,87 g.L-1. Diante da produção de ART após deslignificação notou-se que o percentual de sacarificação para a biomassa sem tratamento elevou-se de 11,88% para 69,15%, enquanto que para o bagaço após explosão a vapor e deslignificação este percentual ficou entre 82,05% - 89,79%. Por fim, cabe ressaltar que no BCA previamente pré-tratado por explosão a vapor com NaOH, a remoção de lignina após o segundo pré-tratamento teve um acréscimo de apenas 20% e a concentração de ART de 6,87 g.L-1. Dessa forma, acredita-se que ao aumentar a concentração da solução de NaOH para realizar a explosão a vapor poderia-se não necessitar da realização da etapa de deslignificação. / Nowadays, there is various types of studied pre-treatments to reduce the lignocellulosic biomass recalcitrance in order to increase its chemical / enzymatic digestibility, so that it can be used in ethanol production and / or other bioproducts value. This study evaluated the effect of steam explosion pretreatment catalyzed by citric acid and sodium hydroxide, and alkaline delignification pretreatment over chemical and structural properties of sugarcane bagasse, as well as the enzymatic hydrolysis process. Steam explosion pretreatment was conducted in a 1.4 L reactor at temperature of 180 °C and 5 min hold time. The delignification step with NaOH (2% m:m) was performed at 120 °C under reflux for 4 hours. Chemical and structural characterization of raw and pretreated biomass was performed by FTIR, SEM and XRD. Enzymatic hydrolysis was performed with final volume of 20 mL consisting 3% SCB on dryweight basis, sodium citrate buffer 50 mM (pH = 5.0) and 10 FPU of Cellic® Ctec 3 enzyme complex. Flasks were kept under agitation of 150 rpm at 50 °C during 72 h. It was analyzed 1.5 mL aliquots after 0, 12, 24, 36, 48 and 72 hours to determinate total reducing sugars (TRS) by acid 3,5-dinitrosalicílic method (DNS). Sugarcane bagasse showed 24.22% lignin, 27.61% hemicelluloses and 42.77% of cellulose, however after steam explosion pretreatment catalyzed by citric acid, it was obtained a biomass with lesser hemicelluloses amount (16.16%) and cracks formation on fiber cell wall were observed. The bagasse pretreated by steam explosion by NaOH showed complete destructuring of fiber and lignin removal was 65%, while hemicellulosic fraction was preserved. After submitting untreated and pretreated lignocellulosic biomass by steam explosion to alkaline delignification process, complete destructuring and solubilization of the lignocellulosic matrix was observed with 85-90% lignin removal in all samples. The crystallinity index of biomass after pretreatments increased when compared to raw material and this could be associated to amorphous components removal, such as lignin and hemicelluloses, and also the amorphous cellulose fraction. The theoretical value corresponding to 100% of saccharification corresponds to approximately 33.0 g·L-1, compared to the mass percentage of biomass used in the enzymatic hydrolysis process. After 72 h enzymatic hydrolysis of raw and pretreated biomass by steam explosion, the highest concentration of total reducing sugars (TRS) and the highest percentage of saccharification were obtained for sugarcane bagasse (23.05 g·L-1, 69,15% saccharification). In in nature biomass hydrolysis, after alkaline delignification process, ART concentration increased 18.33 g·L-1, while pretreated biomass by steam explosion with water, citric acid and NaOH followed by alkaline delignification increase TRS to 19.67 g·L-1, 19.93 g·L-1 and 6.87 g·L-1. After delignification, it was noted that saccharification percentage for untreated biomass increased from 11.88% to 69.15%, while for bagasse after steam explosion and delignification this percentage was between 82.05% - 89.79%. Lastly, it should be noted that in biomass previously pretreated by NaOH steam explosion, lignin solubilization after the second pretreatment had an increase of only 20% and TRS concentration of 6.87 g·L-1. Thus, it is believed that increasing NaOH solution concentration to perform steam explosion could not need to implement delignification step. / Dissertação (Mestrado)