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Saccharification of lignocelluloseWarsame, Mohamed January 2012 (has links)
Den ökande efterfrågan på energi och den förväntade nedgången i råoljeproduktion har lett till ett enormt sökande efter nya energikällor.Cellväggen i växter består till stor del av lignocellulosa som i sin tur innehåller cellulosa och hemicellulosa. Dessa polysackarider är av stor betydelse för sökandet efter förnyelsebar energi.Cellväggen måste förbehandlas innan den kan brytas ner till enkla sockerarter. Efter nedbrytning kan monosackariderna användas till produktion av etanol eller biogas genom väl etablerade fermenteringstekniker. Syftet med denna studie var att jämföra och utvärdera några metoder som används vid degradering av lignocellulosa. Tre behandlingar har jämfört för att se vilken som ger mest avkastning i form av monosackarider. Vetehalm användes som substrat och hydrolyseras med hjälp av tre kommersiella enzymblandningar. Proverna förbehandlades före den enzymatiska reaktionen med antingen mikrovågor eller ångexplosion.Resultaten visade att en behandling med mikrovågsbestrålning eller ångexplosion kombinerad med enzymhydrolys gav högst avkastning. De slutsatser som kan dras är att en mekanisk förbehandling ökar utbytet drastiskt men är otillräcklig i sig. Ytterligare enzymatisk behandling är nödvändig att erhålla större mängder enkla sockerarter från lignocellulosa. / The increasing energy demand and the anticipated decline in crude oil production has led to an immense search for new energy sources. Plant cell walls contain lignocellulose that conserve great amounts of energy. These polysaccharides are of high importance for the search of renewable energy sources. Pretreatment of the cell wall is necessary in order to hydrolyse it to its component sugars. Once degraded to monomeric sugars it can be fermented to either ethanol or biogas through established fermentation technologies.The aim of this thesis was to compare and evaluate some of the methods used for sacchrification of lignocellulose. Three treatments where compared to determine which is highest yielding. These are enzymatic hydrolysis, microwave irradiation and steam explosion.Wheat straw was used as substrate and hydrolysed by three commercial enzyme mixtures. Samples were pretreated before the enzymatic reaction with either microwave or steam explosion. Results showed that a treatment of either microwave irradiation or steam explosion combined with enzyme hydrolysis gives the highest yield in monomeric sugars. The conclusions that can be drawn are that mechanical pretreatment increases yield drastically but is insufficient in its self. Further enzymatic treatment of wheat straw is necessary to obtain high amounts of simple sugars.
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Characterization and Saccharification of Ionic Liquid Pretreated Lignocellulosic BiomassSamayam, Indira Priya January 2011 (has links)
No description available.
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Lignocellulose deconstruction using glyceline and a chelator-mediated Fenton systemOrejuela, Lourdes Magdalena 15 December 2017 (has links)
Non-edible plant biomass (lignocellulose) is a valuable precursor for liquid biofuels, through the processes of pretreatment and saccharification followed by fermentation into products such as ethanol or butanol. However, it is difficult to gain access to the fermentable sugars in lignocellulose, and this problem is principally associated with limited enzyme accessibility. Hence, biomass pretreatments that destroy native cell wall structure and allows enzyme access are required for effective biomass conversion techniques. This research studied two novel pretreatment methods on two wood species: 1) a deep eutectic solvent (DES) that, under heat, swells lignocellulose and partially solubilizes cell wall materials by causing breakage of lignin-carbohydrate linkages and depolymerization of the biomass components, and 2) a chelator-mediated Fenton reaction (CMF) that chemically modifies the nanostructure of the cell wall through a non-enzymatic cell wall deconstruction. After pretreatment, utilizing analytical techniques such as nuclear magnetic spectroscopy, wide angle x-ray scattering, and gel permeation chromatography, samples were analyzed for chemical and structural changes in the solubilized and residual materials.
After single stage DES (choline-chloride-glycerol) and two stage, CMF followed by DES pretreatments, lignin/carbohydrate fractions were recovered, leaving a cellulose-rich fraction with reduced lignin and hemicellulose content as determined by compositional analysis. Lignin and heteropolysaccharide removal by DES was quantified and the aromatic-rich solubilized biopolymer fragments were analyzed as water insoluble high molecular weight fractions and water-ethanol soluble low molecular weight compounds. After pretreatment for the hardwood sample, enzyme digestibility reached a saccharification yield of 78% (a 13-fold increase) for the two stage (DES/CMF) pretreated biomass even with the presence of some lignin and xylan remained on the pretreated fiber; only a 9-fold increase was observed after the other sequence of CMF followed by DES treatment. Single stage CMF treatment or single stage DES pretreatment improved 5-fold glucose yield compared to the untreated sample for the hardwood sample. The enhancement of enzymatic saccharification for softwood was less than that of hardwoods with only 4-fold increase for the sequence CMF followed by DES treatment. The other sequence of treatments reached up to 2.5-fold improvement. A similar result was determined for the single stage CMF treatment while the single stage DES treatment reached only 1.4-fold increase compared to the untreated softwood. Hence, all these pretreatments presented different degrees of biopolymer removal from the cell wall and subsequent digestibility levels; synergistic effects were observed for hardwood particularly in the sequence DES followed by CMF treatment while softwoods remained relatively recalcitrant. Overall, these studies revealed insight into two novel methods to enhance lignocellulosic digestibility of biomass adding to the methodology to deconstruct cell walls for fermentable sugars. / Ph. D. / Wood is a valuable material that can be used to produce liquid biofuels. Wood main components are biopolymers cellulose, hemicellulose and lignin that form a complex structure. Nature has locked up cellulose in a protective assembly that needs to be destroyed to gain access to cellulose, convert it to glucose and then ferment it to bioalcohol. This process is principally associated with limited enzyme accessibility. Therefore, biomass pretreatments that deconstruct native cell wall structure and allow enzyme access are required for effective biomass conversion techniques. This research studied two novel pretreatment methods on two wood species: 1) a deep eutectic solvent called glyceline that, under heat, swells wood and partially solubilizes cell wall materials by causing breakage of bonds and converting it into smaller molecules (monomers and oligomers), and 2) a chelator-mediated Fenton system (CMF) that chemically modifies the structure of the cell wall. Pretreatments were tested individually and in sequence in sweetgum and southern yellow pine. After pretreatments, utilizing analytical techniques, fractions were investigated for chemical and structural changes in the solubilized and residual materials. Treated wood samples were exposed to enzymatic conversion. A maximum 78% of glucose yield was obtained for the glyceline followed by CMF pretreated wood. For yellow pine only a 24% of glucose yield was obtained for the CMF followed by glyceline treatment. All these pretreatments presented different degrees of biopolymer removal from the cell wall and subsequent enzyme conversion levels. Overall, these studies revealed insight into two novel methods to enhance wood conversion adding to the methodology to deconstruct cell walls for fermentable sugars.
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Etude de la voie de biosynthese des monolignols chez brachypodium distachyon / Identification of genes involved in the biosynthesis of monolignols in Brachypodium distachyonBouvier d'yvoire, Madeleine 19 December 2011 (has links)
La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l’étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l’obstacle majeur à l’exploitation industrielle de la biomasse lignocellulosique. L’acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d’identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l’implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l’isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d’acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l’isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d’acide p-coumarique, une baisse du rapport S/G ainsi que l’accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d’un quart par rapport au sauvage. / Brachypodium distachyon was recently adopted as an experimental model for grass species. As such, it is used to study grass cell wall, in particular in the context of their use as renewable feedstock for the production of second generation bioethanol. Lignins are polymers of three main units (H, G and S) originating from the polymerization of monolignols, and are linked to hydroxycinnamic acids in grasses. They constitute the main bottleneck to industrial processes targeting lignocellulosic biomass and improving the understanding of the mechanisms directing their structure and deposition could lead to the identification of the factors modulating associated production yields. Four gene families were studied and the involvement of three genes in the monolignols biosynthetic pathway was shown: BdF5H2 displays a ferulate-5-hydroxylase activity enabling the synthesis of the S lignin units, BdCOMT3 is the main caffeic acid O-methyltransferase and its partial loss of function in two independent mutant lines leads to the reduction of lignin content, the modification of the S/G units ratio and a decrease in p-coumaric acid accumulation. BdCAD1 is the main cinnamyl alcohol dehydrogenase isoform: its loss of function in two independent mutant lines results in a decrease in lignin content and of the S/G ratio and the accumulation of sinapaldehyde. Moreover, these two lines display significatively increased saccharification yields.
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Three-dimensional modelling of simultaneous saccharification and fermentation of cellulose to ethanolVan Zyl, Josebus Maree 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Second-generation bioethanol is an alternative transportation fuel currently being
investigated whereby cellulose, specifically lignocellulosic (woody) portions, of
any plant mass can be converted to ethanol. To date, the technology had only been
successfully implemented with demonstration scale facilities. Despite intensive
research efforts at laboratory scale, no-one is certain what the secondary effects of
scale-up to large systems are. The objective of this project was to develop threedimensional
numerical models of a laboratory scale fermenter which could predict
the effects of particulate mixing and reaction kinetics for future scale-up
investigations.
A numerical model of the reaction kinetics for simultaneous saccharification and
fermentation of Avicel (microcrystalline cellulose) particles to ethanol is
presented. The novelty of this model is the separation of the two primary
cellulase enzyme-kinetics, which generated the capability to predict the
heterogeneous behaviour of the enzyme-substrate interactions. This model
improves the understanding of these systems while maintaining sufficient
simplicity for implementation alongside a commercial computational fluid
dynamics environment.
Effects of the various fermentation medium constituents and the influence of each
on the dynamic viscosity of the medium were also investigated. Results indicated
that particle volume fraction had the dominant effect on the apparent dynamic
viscosity resulting in further research of the particle properties. Due to the
irregular shapes of Avicel particles, tests were conducted to determine drag and
settling behaviour, which led to the development and modification of models to
account for these phenomena. This investigation is unique as it allows a more
accurate calculation of particle transportation through a three-dimensional
environment including the effects of natural packing density. At lower particle
volume fraction the concentration of ethanol and glycerol had the greatest effect
on the apparent dynamic viscosity and was calculated from models obtained from
literature.
Validation of the physics and the incorporation thereof in the simulations resulted
in the modification of various generic models which either improved numerical
stability or accuracy, or both. Contributions included a modified form of the
pressure force model, which proved significantly more stable and accurate than
previous models proposed in literature. The models developed for capturing the
effects of particles on the apparent dynamic viscosity proved effective for this
specific substrate.
Results from cross-coupling the reaction models with computational fluid
dynamic simulations provide a novel approach to capturing the secondary effect
of substrate conversion and particle distribution on the performance of the
fermentation vessels. This is the first time where that biological reactions were successfully combined with particle dynamics and fluid flow fields to investigate
the secondary effects which occur in fermenters.
This work served as a foundation for future research and development within the
bioethanol field with significant potential for expansion into other biochemical
disciplines. / AFRIKAANSE OPSOMMING: Tweede-generasie bioetanol is ’n alternatiewe vervoerbrandstof wat tans
ondersoek word waar sellulose, spesifiek lignosellulosiese (houtagtige) gedeeltes,
van enige plantmassa na etanol omgesit kan word. Tot op hede was die
tegnologie slegs suksesvol geïmplimenteer in demonstrasieskaal fasiliteite. Ten
spyte van intensiewe navorsingpogings op laboratoriumskaal, is niemand seker
wat die sekondêre effekte van die opskaal tot groot stelsels sal wees nie. Die
doelwit van die projek was om drie-dimensionele modelle te ontwikkel van ’n
laboratoriumskaal fermentor wat die effekte van partikulêre vermenging en
reaksiekinetika kan voorspel vir toekomstige opskaal navorsing.
’n Numeriese model van die reaksiekinetika vir gelyktydige versuikering en
fermentasie van Avicel (mikrokristallyne sellulose) partikels tot etanol word
aangebied. Die oorspronklikheid van die model is geleë in die skeiding van die
twee primêre sellulase ensiemkinetika, wat lei tot die vermoë om die heterogene
gedrag van die ensiem-substraat interaksies te voorspel. Hierdie model verbeter
die kennis van die stelsels, terwyl voldoende eenvoud behoue bly vir
implementering parallel aan kommersiële berekeningsvloeidinamika sagteware.
Effekte van die verskillende bestanddele van die fermentasiemedium en die
invloed van elk op die dinamiese viskositeit van die medium is ook ondersoek.
Resultate dui aan dat partikel volume fraksie die dominante invloed op die
skynbare dinamiese viskositeit het, wat gelei het tot verdere ondersoek van die
partikel eienskappe. As gevolg van die onreëlmatige vorms van Avicel partikels,
is toetse gedoen om die sleur-en uitsakkingsgedrag te bepaal, wat gelei het tot die
ontwikkeling en aanpassing van modelle om hierdie verskynsels in ag te neem.
Hierdie ondersoek is uniek, want dit laat meer akkurate berekening van
partikelvervoer deur ’n drie-dimensionele omgewing toe, insluitend die effekte
van natuurlike verpakkingsdigtheid. By laer partikel volume fraksie het die
konsentrasie van etanol en gliserol die grootste effek op die skynbare dinamiese
viskositeit gehad en was bereken vanaf modelle in die literatuur.
Bevestiging van die fisika en die insluiting daarvan in die simulasies het gelei tot
die aanpasing van verskillende generiese modelle wat óf numeriese stabiliteit óf
akkuraatheid óf beide verbeter. Bydraes gemaak sluit ’n aangepaste vorm van die
drukkragmodel in, wat heelwat meer stabiel en akkuraat was as die vorige modelle
voorgestel in die literatuur. Die modelle wat ontwikkel is om die effek van
partikels op die skynbare viskositeit vas te vang, was effektief bewys vir hierdie
spesifieke substraat.
Resultate van die kruiskoppeling van inligting vanaf die reaksiemodelle met
berekeningsvloeidinamika simulasies lewer ’n nuwe benadering tot die bepaling
van die sekondêre effek van substraatomskakeling en partikeldistribusie op die
uitvoering van die fermentasie toestel. Hierdie is die eerste poging om biologiese reaksies met partikel dinamika en vloeivelde te kombineer om die sekondêre
effekte wat in fermenter plaasvind, te ondersoek.
Hierdie werk dien as ’n grondslag vir toekomstige navorsing en ontwikkeling
binne die bioetanolveld, met beduidende potensiaal vir uitbreiding na ander
biochemiese dissiplines.
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Purificação, caracterização bioquímica e potencial de aplicação biotecnológica de uma xilanase halotolerante e termoestável de Colletotrichum graminicola / Purification, biochemical characterization and potential biotechnological applications of a salt- tolerant and thermostable xylanase Colletotrichum graminicolaCarli, Sibeli de 04 August 2016 (has links)
A viabilidade econômica da produção de etanol de segunda geração (2G) depende do desenvolvimento de tecnologias eficientes e baratas para a hidrólise da biomassa lignocelulósica. Em particular, o grande consumo de água nas plantas de processamento de biomassa pode inviabilizar o processo. As xilanases são enzimas chave na hidrólise enzimática da xilana para a produção de etanol 2G e atualmente é grande o interesse na identificação de xilanases tolerantes a altas concentrações salinas, bem como aos subprodutos de etapas de pré-tratamento da biomassa, o que permitiria reduzir o volume de água empregado em etapas de lavagem e/ou a substituição da água doce pela água do mar. Neste contexto, os objetivos deste trabalho foram a purificação e caracterização bioquímica e cinética de uma endo-xilanase halotolerante e termoestável produzida por uma linhagem de C. graminicola isolada da floresta Amazônica (Brasil). A enzima pura (Excg1) apresentou massa molecular aparente de 20,0 ± 2,4 kDa em SDS-PAGE e 17,3 ± 1,9 kDa por filtração em gel, sugerindo que a enzima nativa é monomérica. O teor de carboidratos totais de Excg1 foi de 97,0 ± 3,7 % (m/m) e o seu ponto isoelétrico correspondeu a 9,200 ± 0,018. A enzima manteve cerca de 85% da atividade controle na presença de NaCl 0,5 mol/L. Em ausência e presença de NaCl em concentração 0,5 mol/L a temperatura e o pH ótimos de atividade de Excg1 foram 65 ºC e 5,5, respectivamente, enquanto na presença de NaCl 2,5 mol/L o pH ótimo foi alterado para 6,0. Excg1 mostrou-se bastante termoestável a 50ºC, com tempo de meia-vida de 48 h na ausência do substrato. Já na presença de NaCl 2,5 mol/L a termoestabilidade foi substancialmente maior, com atividade residual de 75% após o mesmo intervalo de tempo. Excg1 apresentou excelente estabilidade na faixa de pH de 3,0-10,0 na ausência de sal, mantendo-se também completamente estável entre pH 4,0 e 10,0 na presença de NaCl em concentração 0,5 e 2,5 mol/L. Os parâmetros cinéticos determinados para a hidrólise de xilana Beechwood por Excg1 na ausência de NaCl foram Vmax= 481,3 ± 34,0 U/mg e KM= 3,7 ± 0,3 mg/mL, resultando em eficiência catalítica (kcat/KM) de 36,9 mL/s.mg. Parâmetros muito similares foram determinados na presença de NaCl 0,5 mol/L, porém em presença do sal em concentração 2,5 mol/L ocorreu diminuição da afinidade aparente pelo substrato e redução da velocidade máxima, resultando em eficiência catalítica 2,3 vezes menor. Já a hidrólise de xilana Birchwood em ausência de sal ocorreu com constante de afinidade aparente similar e eficiência catalítica cerca de 18% maior, se comparada à hidrolise de xilana Beechwood na mesma condição. Excg1 foi tolerante a K+, Na+, Pb2+, Ni2+, Zn2+, Mn2+, Mg2+, Co2+, Ca2+ e Sr2+ em concentração 10 mmol/L, além de Cu2+, Al3+, Cr3+ e Fe3+ em concentração 1 mmol/L. Além disso, Excg1 foi tolerante a diferentes solventes orgânicos e a acetato. A análise dos produtos de hidrólise de xilana Beechwood por Excg1 revelou que os produtos principais formados foram xilobiose e xilotriose com uma ramificação de ácido 4-O-metilglucurônico. A presença de NaCl 0,5 mol/L não afetou este padrão de hidrólise e a enzima mostrou também boa tolerância aos produtos de hidrólise. Em conjunto, as propriedades de Excg1 sugeriram bom potencial de aplicação na sacarificação da biomassa lignocelulósica, particularmente em condições de salinidade elevada e/ou em presença de resíduos de etapas de pré-tratamento, o que é potencialmente interessante para viabilizar economicamente processos de produção de etanol 2G. / The economic viability of the production of second-generation (2G) ethanol depends on the development of efficient and inexpensive technologies for the hydrolysis of lignocellulosic biomass. In particular, the large consumption of water in the biomass processing plants can make the process unfeasible. The xylanases are key enzymes in the enzymatic hydrolysis of xylan aiming the production of 2G ethanol and there is currently great interest in identifying xylanases tolerant to high salt concentrations and to the byproducts from biomass pretreatment steps, allowing the reduction of the volume of water used in washing steps and/or the replacement of fresh water by sea water. In this context, the objectives of this work were the purification and the biochemical and kinetic characterization of a salt-tolerant and thermostable endoxylanase produced by a strain of C. graminicola isolated from the Amazon forest (Brazil). The pure enzyme (Excg1) showed apparent molecular mass of 20.0 ± 2.4 kDa by SDS-PAGE and 17.3 ± 1.9 kDa by gel filtration, suggesting that the native enzyme is monomeric. The total carbohydrate content of Excg1 was 97.0 ± 3.7% (m/m) and its isoelectric point corresponded to 9.200 ± 0.018. The enzyme retained approximately 85% of control activity in the presence of 0.5 mol.L-1 NaCl. In the absence and presence of NaCl at 0.5 mol.L-1 concentration, the optimum reaction temperature and pH of Excg1 were 65 ° C and 5.5, respectively, while in the presence of 2.5 mol.L-1 NaCl the optimum pH was altered to 6.0. Excg1 was highly thermostable at 50 °C, with a half-life of 48 h in the absence of substrate. In the presence of 2.5 mol.L-1 NaCl the thermal stability was greatly increased, and a residual activity of 75% was determined after 48 h at 50 ºC. Excg1 showed excellent stability in the pH range from 3.0 to 10.0 in the absence of salt, and was likewise completely stable at pH 4.0-10.0 in the presence of NaCl at the concentrations 0.5 mol.L-1 and 2.5 mol.L-1. The kinetic parameters for the hydrolysis of Beechwood xylan by Excg1 in the absence of salt were Vmax = 481.3 ± 34.0 U.mg-1 and KM = 3.7 ± 0.3 mg.mL-1, with a catalytic efficiency (kcat/KM) of 36.9 mL.s-1.mg-1. Similar parameters were determined in the presence of 0.5 mol.L-1 NaCl, while in the presence of a higher salt concentration (2.5 mol.L-1) decreases in the apparent affinity for the substrate and in the maximum velocity were observed, resulting in a catalytic efficiency 2.3 fold lower. In comparison with Beechwood xylan, the hydrolysis of Birchwood xylan in the absence of salt occurred with similar apparent affinity and catalytic efficiency about 18% greater. Excg1 was tolerant to 10 mmol.L-1 K+, Na+, Pb2+, Ni2+, Zn2+, Mn2+, Mg2+, Co2+, Ca2+ or Sr2+, and also to Cu2+, Al3+, Cr3+ e Fe3+ at 1 mmol.L-1 concentration. Furthermore, the enzyme was tolerant to various organic solvents and acetate. The analysis of Beechwood xylan hydrolysis products by Excg1 revealed that the main products were xylobiose and xylotriose with a 4-O-methylglucuronic acid branch. The presence of 0.5 mol.L-1 NaCl has not affected the hydrolysis pattern and the enzyme showed good tolerance to the hydrolysis products. Altogether, the properties of Excg1 suggested good potential for the saccharification of lignocellulosic biomass, particularly under high salinity conditions and/or in the presence of residues of pre-treatment steps, which is potentially interesting for the economically viable production of 2G ethanol.
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Caracterização ultraestrutural e hidrólise enzimática de cana-de-açúcar e bagaço pré-tratado quimio-mecanicamente / Ultrasctructural characterization and enzymatic hydrolysis of chemomechanical pretreated sugarcane and sugarcane bagasse.Fernanda Machado Mendes Carvalho 21 August 2014 (has links)
O presente trabalho tem como objetivo estudar as modificações ocorridas na cana-de-açúcar, com diferentes composições químicas e estruturais, pelo pré-tratamento sulfito alcalino. A remoção de lignina e hemicelulose, bem como a introdução de grupos sulfônicos em cana-de-açúcar que ocorrem durante o pré-tratamento sulfito alcalino tornam mais fácil a hidrólise da celulose. A compreensão das modificações químicas e físicas em materiais lignocelulósicos que ocorrem durante este pré-tratamento é fundamental para a geração de processos mais eficazes. Neste trabalho, bagaço e entrenós de cana-de-açúcar, selecionados de plantas híbridas com composição química variada, foram pré-tratados em condições brandas com 10% de sulfito e 5% de hidróxido de sódio por diferentes tempos. No início do pré-tratamento, a deslignificação aumentou rapidamente, o mesmo não aconteceu com a hemicelulose. Nos primeiros 30 min de pré-tratamento do bagaço de cana-de-açúcar houve remoção de 50% da lignina inicial e 30% da hemicelulose, o que ocasionou uma melhora significativa na conversão de celulose, atingindo 64%. Mesmo sem remoção adicional de lignina e hemicelulose, o processo continuou a introduzir os grupos ácidos, o que contribuiu para o inchamento da fibra. A largura da fibra do bagaço não tratado aumentou de 10,4 ?m para 30 ?m no material pré-tratado com 120 min. Estas modificações na fibra foram responsáveis pelo aumento na eficiência da hidrólise enzimática da celulose, a qual atingiu 92%. Híbridos experimentais com teores reduzidos de lignina apresentaram taxas iniciais de hidrólise mais elevadas e um menor tempo de pré-tratamento para alcançar a conversão total de celulose do que a cana de referência. Diferentes regiões (medula, interface, córtex e fração externa) dos entrenós das canas foram hidrolisadas por celulases. O pré-tratamento da interface, córtex e fração externa com sulfito-alcalino produziu substratos menos recalcitrantes com o aumento do tempo de reação e resultou na melhora da hidrólise enzimática. Foram utilizadas várias técnicas para avaliar as mudanças que ocorreram durante o pré-tratamento, as quais foram capazes de estudar a morfologia da superfície e as características químicas das amostras. O tratamento químico ocasionou uma intensa deslignificação e alterações morfológicas nas superfícies das fibras da cana-de-açúcar. A redução na absorção a 285 nm e 315 nm das paredes celulares das fibras, parênquima e dos vasos aumentou substancialmente os valores de conversão enzimática da celulose e da hemicelulose. Microscopia eletrônica de varredura por emissão de campo (FE-SEM) revelou que as fibras da região do córtex e, especialmente, da interface mostrou paredes celulares colapsadas após a parcial deslignificação. Após o tratamento sulfito alcalino, os dados de espectroscopia fotoelétrica de raio-X (XPS) e espectrometria de massa de íons secundários por tempo de vôo (TOF-SIMS) apresentaram um aumento das intensidades dos sinais nas superfícies das fibras, os quais foram atribuídos à presença de carboidratos em algumas amostras. Em conformidade, os sinais de lignina diminuíram nas superfícies das fibras das mesmas amostras. / The present work aims to study the changes occurring in sugar cane, with different in structure and chemical compositions, by sulfite-alkaline pre-treatment. Removing lignin and hemicellulose as well as introducing sulfonic groups in sugar cane pretreated with alkaline sulfite made cellulose hydrolysis easier. Understanding the chemical and physical alterations occurring during this pretreatment of lignocellulosic materials is fundamental for the generation of effective pretreatment methods. In the present work, sugarcane bagasse and also sugar cane internodes, selected from experimental hybrid plants, were pretreated with the alkaline-sulfite process under mild conditions with varied cooking times. The first 30 min of pretreatment of sugar cane bagasse, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly and hemicellulose removal was not enhanced. However, the process continued to introduce acid groups into the residual lignin that enhanced the fiber swelling up to 120 min of cooking. The fiber widths increased from 10,4 ?m in the untreated bagasse to 30 ?m in the 120 min-pretreated material. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92%. Experimental hybrids with less original lignin presented higher initial hydrolysis rates than reference sugar cane and required lower time of pretreatment to achieve the total cellulose conversion. Different regions (pith, interface, rind and outermost fraction) of the internodes of types of sugarcanes were hydrolyzed by cellulases. The pretreatment of the interface, rind and outermost fraction with alkaline sulfite produced less recalcitrant substrates with increasing reaction time and resulted in improvement enzymatic hydrolysis. Several techniques enabling the study of surface morphological and chemical characteristics were used to evaluate the changes occurring during the pretreatment step. The chemical treatment caused intense delignification and morphological changes on the sugar cane fiber surfaces. The reduction in the absorption at 285 nm and 315 nm of the cell walls of the fibers, parenchyma and vessel, substantially increased the values of enzymatic conversion of cellulose and hemicellulose. Field emission scanning electron microscopy (FE-SEM) indicated that the fibers from rind regions and especially from the interface showed collapsed cell walls after partial delignification. After the alkaline sulfite treatment, X-ray photoelectrom spectroscopy (XPS) and time-of-flight-secondary ion mass spectrometry (ToF-SIMS) data showed increased signal intensities on the fibers surfaces assigned to carbohydrates of some samples. In accordance, the lignin signals diminished on the fiber surfaces of the same samples.
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Produção e caracterização bioquímica de enzimas lignocelulolíticas fúngicas e sua aplicação na sacarificação de biomassa lignocelulósica / Production and biochemical characterization of fungal enzymes lignocellulolytic and its application in saccharification of biomass lignocellulosicZimbardi, Ana Lucia Ribeiro Latorre 05 August 2014 (has links)
Atualmente há grande interesse no desenvolvimento de processos enzimáticos eficientes para a hidrólise da biomassa lignocelulósica. O objetivo deste trabalho foi a otimização da produção por fermentação em estado sólido e a caracterização bioquímica, no extrato bruto, das -glucosidases, -xilosidases e xilanases produzidas por Colletotrichum graminicola e das lacases produzidas por Pycnoporus sanguineus. Também foi avaliado o potencial de aplicação dos extratos obtidos em coquetéis enzimáticos para a sacarificação de resíduos agroindustriais. A otimização das condições de cultivo, empregando a Metodologia de Superfície de Resposta, levou à produção de 159,3 ± 12,7 U g-1, 125,88 ± 6,4 U g-1, 378,1 ± 23,3 U g-1 e 138,6 ± 6,4 U g-1 de -glucosidases, -xilosidases, xilanases e lacases, respectivamente. Os meios de cultivo empregados foram constituídos por farelo de trigo suplementado com resíduos agroindustriais. Todas as enzimas produzidas apresentaram pH e temperatura ótimos de reação de 4,5-5,0 e 65ºC, respectivamente, bem como boa estabilidade térmica e ao pH. O coquetel composto pelos extratos brutos obtidos em condições otimizadas para a produção de xilanases (ECg) e lacases (EPs), em mistura com um extrato bruto de Trichoderma reesei rico em celulases (ETr) foi muito eficiente na sacarificação de palha de cana e papelão, sem pré-tratamento, atingindo rendimentos de 41,4 e 71,1% em glicose, respectivamente. Além disso, este coquetel foi mais eficiente na sacarificação de bagaço de cana explodido e in natura bem como de palha de cana in natura, quando comparado a um coquetel contendo celulases comerciais (Celluclast®) em mistura com ECg e EPs. Visando estudos futuros da ação individual de cada enzima sobre a biomassa, foi purificada uma -glucosidase majoritária de C. graminicola. A enzima mostrou temperatura e pH ótimos de reação de 5,0 e 65ºC, respectivamente, boa estabilidade térmica e ao pH, além da estimulação por xilose, propriedade muito interessante para emprego em coquetéis mistos de celulases e xilanases. Os resultados encontrados sugerem que as enzimas produzidas por C. graminicola e P. sanguineus, assim como os coquetéis enzimáticos avaliados, apresentam características muito interessantes para aplicações biotecnológicas, particularmente em processos de sacarificação da biomassa para obtenção de etanol celulósico. / There is currently a great interest in developing efficient processes for the enzymatic hydrolysis of lignocellulosic biomass. The objective of this study was the optimization of the culture conditions for the production of -glucosidases, xylanases and -xylosidases by Colletotrichum graminicola and laccases by Pycnoporus sanguineus under solid state fermentation, followed by the biochemical characterization of the enzymes in the crude extracts. The potential of application of the extracts to compose enzyme cocktails for the saccharification of agroindustrial residues was also investigated. Optimization of the culture conditions using the Response Surface Methodology led to the production of 159.3 ± 12.7 U g - 1, 125.88 ± 6.4 U g- 1, 378.1 ± 23.3 U g - 1 and 138.6 ± 6.4 U g - 1 of -glucosidases, -xylosidases, xylanases and laccases, respectively. The culture media employed consisted mainly of wheat bran, supplemented with agroindustrial residues. All enzymes produced showed optimum pH and temperature of 4.5-5.0 and 65° C, respectively, as well as good thermal and pH stability. A cocktail composed of the crude extracts obtained under optimized conditions for the production of xylanases (ECg) and laccases (EPs), mixed with a Trichoderma reesei crude extract (ETr), rich in cellulases, was highly efficient for the saccharification of sugarcane trash and cardboard, without pretreatment, reaching yields of 41.4% and 71.1% in glucose, respectively. Moreover, this cocktail was more efficient than a cocktail composed of commercial cellulases (Celluclast ®) in combination with ECg and EPs for the saccharification of raw and steam exploded sugarcane bagasse, as well as raw sugarcane trash. Aiming future studies on the individual action of each enzyme on biomass, a majoritary -glucosidase from C. graminicola was purified. The enzyme showed optima of temperature and pH of 5.0 and 65° C, respectively, good thermal and pH stability, as well as stimulation by xylose, a very interesting property for its application in mixed cellulase-xylanase cocktails. The results suggested that the enzymes produced by C. graminicola and P. sanguineus, as well as the cocktails employed in this study, have good potential for biotechnological applications, particularly in biomass saccharification processes for cellulosic ethanol production.
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Caracterização ultraestrutural e hidrólise enzimática de cana-de-açúcar e bagaço pré-tratado quimio-mecanicamente / Ultrasctructural characterization and enzymatic hydrolysis of chemomechanical pretreated sugarcane and sugarcane bagasse.Carvalho, Fernanda Machado Mendes 21 August 2014 (has links)
O presente trabalho tem como objetivo estudar as modificações ocorridas na cana-de-açúcar, com diferentes composições químicas e estruturais, pelo pré-tratamento sulfito alcalino. A remoção de lignina e hemicelulose, bem como a introdução de grupos sulfônicos em cana-de-açúcar que ocorrem durante o pré-tratamento sulfito alcalino tornam mais fácil a hidrólise da celulose. A compreensão das modificações químicas e físicas em materiais lignocelulósicos que ocorrem durante este pré-tratamento é fundamental para a geração de processos mais eficazes. Neste trabalho, bagaço e entrenós de cana-de-açúcar, selecionados de plantas híbridas com composição química variada, foram pré-tratados em condições brandas com 10% de sulfito e 5% de hidróxido de sódio por diferentes tempos. No início do pré-tratamento, a deslignificação aumentou rapidamente, o mesmo não aconteceu com a hemicelulose. Nos primeiros 30 min de pré-tratamento do bagaço de cana-de-açúcar houve remoção de 50% da lignina inicial e 30% da hemicelulose, o que ocasionou uma melhora significativa na conversão de celulose, atingindo 64%. Mesmo sem remoção adicional de lignina e hemicelulose, o processo continuou a introduzir os grupos ácidos, o que contribuiu para o inchamento da fibra. A largura da fibra do bagaço não tratado aumentou de 10,4 ?m para 30 ?m no material pré-tratado com 120 min. Estas modificações na fibra foram responsáveis pelo aumento na eficiência da hidrólise enzimática da celulose, a qual atingiu 92%. Híbridos experimentais com teores reduzidos de lignina apresentaram taxas iniciais de hidrólise mais elevadas e um menor tempo de pré-tratamento para alcançar a conversão total de celulose do que a cana de referência. Diferentes regiões (medula, interface, córtex e fração externa) dos entrenós das canas foram hidrolisadas por celulases. O pré-tratamento da interface, córtex e fração externa com sulfito-alcalino produziu substratos menos recalcitrantes com o aumento do tempo de reação e resultou na melhora da hidrólise enzimática. Foram utilizadas várias técnicas para avaliar as mudanças que ocorreram durante o pré-tratamento, as quais foram capazes de estudar a morfologia da superfície e as características químicas das amostras. O tratamento químico ocasionou uma intensa deslignificação e alterações morfológicas nas superfícies das fibras da cana-de-açúcar. A redução na absorção a 285 nm e 315 nm das paredes celulares das fibras, parênquima e dos vasos aumentou substancialmente os valores de conversão enzimática da celulose e da hemicelulose. Microscopia eletrônica de varredura por emissão de campo (FE-SEM) revelou que as fibras da região do córtex e, especialmente, da interface mostrou paredes celulares colapsadas após a parcial deslignificação. Após o tratamento sulfito alcalino, os dados de espectroscopia fotoelétrica de raio-X (XPS) e espectrometria de massa de íons secundários por tempo de vôo (TOF-SIMS) apresentaram um aumento das intensidades dos sinais nas superfícies das fibras, os quais foram atribuídos à presença de carboidratos em algumas amostras. Em conformidade, os sinais de lignina diminuíram nas superfícies das fibras das mesmas amostras. / The present work aims to study the changes occurring in sugar cane, with different in structure and chemical compositions, by sulfite-alkaline pre-treatment. Removing lignin and hemicellulose as well as introducing sulfonic groups in sugar cane pretreated with alkaline sulfite made cellulose hydrolysis easier. Understanding the chemical and physical alterations occurring during this pretreatment of lignocellulosic materials is fundamental for the generation of effective pretreatment methods. In the present work, sugarcane bagasse and also sugar cane internodes, selected from experimental hybrid plants, were pretreated with the alkaline-sulfite process under mild conditions with varied cooking times. The first 30 min of pretreatment of sugar cane bagasse, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly and hemicellulose removal was not enhanced. However, the process continued to introduce acid groups into the residual lignin that enhanced the fiber swelling up to 120 min of cooking. The fiber widths increased from 10,4 ?m in the untreated bagasse to 30 ?m in the 120 min-pretreated material. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92%. Experimental hybrids with less original lignin presented higher initial hydrolysis rates than reference sugar cane and required lower time of pretreatment to achieve the total cellulose conversion. Different regions (pith, interface, rind and outermost fraction) of the internodes of types of sugarcanes were hydrolyzed by cellulases. The pretreatment of the interface, rind and outermost fraction with alkaline sulfite produced less recalcitrant substrates with increasing reaction time and resulted in improvement enzymatic hydrolysis. Several techniques enabling the study of surface morphological and chemical characteristics were used to evaluate the changes occurring during the pretreatment step. The chemical treatment caused intense delignification and morphological changes on the sugar cane fiber surfaces. The reduction in the absorption at 285 nm and 315 nm of the cell walls of the fibers, parenchyma and vessel, substantially increased the values of enzymatic conversion of cellulose and hemicellulose. Field emission scanning electron microscopy (FE-SEM) indicated that the fibers from rind regions and especially from the interface showed collapsed cell walls after partial delignification. After the alkaline sulfite treatment, X-ray photoelectrom spectroscopy (XPS) and time-of-flight-secondary ion mass spectrometry (ToF-SIMS) data showed increased signal intensities on the fibers surfaces assigned to carbohydrates of some samples. In accordance, the lignin signals diminished on the fiber surfaces of the same samples.
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Caracterização química e enzimática do processo de adoçamento da manga \'Keitt\' / Chemical and enzymatic characterization of the mango Keitt sweetening processSilva, Ana Paula Fioravante Bernardes 16 June 2004 (has links)
Dentre as características que definem um fruto maduro, o adoçamento é um dos mais importantes. Porém, no que concerne à manga, os dados existentes são escassos e pouco esclarecedores. Neste trabalho foi estudada a síntese e a degradação do amido da manga \'Keitt\', nos aspectos químicos (teores de amido, de amilose e de açúcares solúveis), nos aspectos bioquímicos (atividade de enzimas relacionadas à degradação do amido, perfil de enzimas ligadas ao grânulo de amido) e aspecto morfológico do grânulo de amido (preliminar), e o amadurecimento do fruto. A manga \'Keitt\' teve um padrão atípico de fruto climatérico, com a produção de pequenas quantidades de etileno e C02, culminando em picos após o processo de amadurecimento ter sido iniciado. Todo o amido acumulado (cerca de 8 %) durante o desenvolvimento até os 3 dias após a colheita (dpc), foi totalmente degradado a partir dos 5 dpc chegando ao final do amadurecimento com apenas traços do seu conteúdo inicial. Ao mesmo tempo acumulou cerca de 10 % de açúcares solúveis, com predominância da sacarose. As enzimas que potencialmente podem degradar o amido, tiveram perfil de atividade compatível com a sua atuação. Houve um aumento bastante significativo de atividade da α-amilase durante a formação do fruto e da β--amilase durante o amadurecimento do fruto. As fosforilases e isoamilases, embora tivessem atividade suficiente para atuar durante a degradação do amido, demonstraram pelo perfil de atividade terem bastante importância durante a síntese do amido. Os grânulos de amido, como observados por microscopia eletrônica de varredura, tem grânulos redondos, lisos e pequenos (até 20 µm), que diminuem de tamanho durante o amadurecimento da manga. As proteínas ligadas ao grânulo, como visto por eletroforese em condições dissociantes, aumentaram em número e quantidade depois da colheita da manga, mostrando que várias proteínas de alto e baixo peso molecular, aderiram ao grânulo antes do início da degradação. / Sweetening is one of the most important characteristics concerning ripe fruit. However, physiological and biochemical changes associated to mango fruit ripening process, including mango sweetening, is still poor understood. In this work the synthesis and breakdown of the starch were studied focusing starch, amylose, and soluble sugars content. Also the activities of some enzymes that participate in starch metabolism were evaluated during development and ripening of mango \'Keitt\'. Thought Scanning Electron Microscopy, granule starch shape and superficial changes related to the degree of mango ripening, were investigated. Results shown that mango fruit (cv. Keitt) has not a typical profile of ethylene and respiration during the ripening process, with very low leveis of both parameters. There was a massive conversion of the starch accumulated (~8 % )during fruit development to soluble sugar (~10 %), with a predominance of sucrose. The activity of α-amylase increased at least 20 times during fruit development while β:-amylase showed detectable activity after fruit harvesting. Starchphosphorylases and isoamylases activities can be linked with both starch synthesis and degradation. Granule starch isolated from mango, showed spherical shape and small size (~20 µm) when the mango achieve full starch content (~3 days after harvest), with about 25 % of amylose. During fruit ripening, granule size suffered superficial corrosion and decreased in size (~6 µm). SDS-PAGE gels showed that after harvesting increased the number of starch bounded proteins with low and high molecular weight, inclusive inside the granule. These proteins can be associated with starch degradation process.
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