The LignoBoost process is a valuable supplement to the Kraft process. It can increase the pulp production rate of a Kraft mill and it enables lignin separation from black liquor with a high degree of purity. However, residual xylan in black liquor has been observed to increase filtration resistance of lignin during the LignoBoost process. In order to uncover underlying mechanisms, this thesis investigates the potential influence of xylan during lignin precipitation and filtration, which are the two main steps of the LignoBoost process. For this purpose experiments based on a model system were designed. Model liquors consisted of lignin and xylan as the only organic compounds and contained lower salt concentrations (4.2-5.9 wt%) compared to black liquor. Furthermore, reference liquors were prepared without xylan addition. Precipitation mechanisms were studied in the onset precipitation region (i.e. alkaline regime) by in-situ focused beam reflectance measurements (FBRM) during step-by-step acidic precipitation of the model liquor. It was found that the onset precipitation pH does not change with the presence of xylan as all liquors started precipitation around pH 9.15. The filtration process was investigated on model liquors that had been precipitated by fast acidification to acidic regimes (pH 6.5-2.87). The use of FBRM during acid precipitation of model liquors suggested that temperature had a significant influence on the chord length distribution (CLD) of the particles. In all filtration experiments, a decrease in CLD was observed when the temperature was changed from 80 °C to 25 °C. Moreover, this thermal instability of particles seemed to be higher when added xylan was present in the liquor. The investigation of the resulting filer cakes with HPLC showed that xylan was evenly distributed through the cake. Further findings on the influence of xylan were impeded due to variations in ionic strength in the model liquors. It was found that the effect of ionic strength on filtration properties and particle sizes overshadows the effect of xylan. Higher ionic strength was observed to yield a lower filtration resistance, a higher solidosty, larger particles and lower solid surface area, as investigated by filtration measurements, laser diffraction and BET analysis. Finally, xylan was fluorescently tagged (i.e. dyed) with Remazol Brilliant Blue R to investigate xylan position in the ligninxylan filer cake, using a confocal fluorescence microscope. However, due to the autofluorescence of lignin as well as low emission intensity of the synthesized dyed xylan, xylan could not been tracked within the lignin particle. Nevertheless, valuable insight was gained into the preparation of dyed xylan and the bond stability.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The extraction of hemicelluloses prior to pulping that would have been dissolved in black liquor during pulping process, is an attractive alternative for pulp and paper mills as they, in addition to their core products, can increase their revenue by producing biofuels, biopolymers, paper additives and other chemicals. However, the amount of hemicelluloses extracted will be limited by the requirement to maintain pulp yield and pulp quality in comparison to existing pulping processes. In the present study, mild alkaline (NaOH) and dilute sulphuric acid conditions were used to extract hemicelluloses from Eucalyptus grandis, giant bamboo (Bambusa balcooa) and sugarcane (Saccharum officinarum) bagasse (SCB) prior to kraft or sodaAQ pulping processes. The effects of catalyst concentration, temperature and reaction time on hemicelluloses pre-extraction were studied, using a statistical experimental design to investigate conditions under which hemicelluloses could be extracted prior to alkaline pulping with minimal interference on cellulose (glucan) content. Subsequently, selected pre-extracted materials were subjected to kraft or sodaAQ pulping to evaluate the effect of the hemicelluloses pre-extraction on cooking chemicals, pulp yield and properties. This study also included evaluation of hot water hemicelluloses pre-extraction of SCB as it was part of a dilute sulphuric acid experimental design. The pulp yield, cooking chemicals and handsheet strength properties were compared with those obtained from kraft or sodaAQ pulping of non extracted raw materials. The results showed that alkaline pre-extraction options investigated preserves the pulp yield with minimal effect on handsheet strength properties depending on the choice of the subsequent pulping method while a fraction of xylan was extracted in polymeric form. In addition, less active alkali was required to delignify the xylan extracted materials. The integration of hemicelluloses pre-extraction by alkaline methods into a kraft pulping process was preferred for giant bamboo and E. grandis since it maintained pulp yields at desired industrial levels of 50%, and pulps within a bleachable kappa number range. Another advantage observed was the reduction in total cooking active alkali required to delignify alkaline extracted giant bamboo or E. grandis by 8or 3 percentage points respectively. However, the compromise to maintain the pulp yield was obtained when only 13.6% or 12.4% polymeric xylan was solubilised from giant bamboo or E. grandis respectively. Slight improvement in burst index of the handsheet was observed for extracted giant bamboo. On the other hand, pulp viscosity was increased by 13% due to the removal of low molecular weight hemicelluloses, while the breaking strength of the handsheet was also increased by 8.9% for pulps produced from extracted E. grandis. In the case of sugarcane bagasse, hemicelluloses pre-extraction by alkaline methods integrated well with the sodaAQ pulping process. It enabled a xylan recovery of 69.1%, while providing pulp with higher screened pulp yield (45.0%), with an advantageous decrease in kappa number (15.5). The handsheet tear index was superior without reduction in viscosity compared to pulp produced from non extracted SCB. On the contrary, results obtained from optimised dilute sulphuric acid pre-extraction of all the tested feedstocks were found to negatively impact subsequent kraft or sodaAQ pulping processes resulting in lower pulp yields and poorer strengths properties. Nonetheless, the differences were better when sodaAQ pulping was used compared to kraft pulping. SodaAQ protects the carbohydrates against the peeling reaction under alkaline medium. Conversely, pre-extraction of SCB with hot water resulted in low concentration of xylooligomers (5.7%), while the subsequent sodaAQ pulping resulted in no pulp yield reduction. The tear index and optical brightness of the handsheet papers produced from hot water extracted SCB were slightly improved while the breaking length, tensile and burst indexes were similar to those of pulps produced from non extracted SCB fibres. Of equal importance were the observed higher tear and burst indexes of handsheets produced from giant bamboo compared to E. grandis for both extracted and non extracted materials prepared under similar pulping processes. The advantage of bamboo was due to the larger fibre length and different morphological properties to those of hardwoods. However, the pulps produced from giant bamboo showed higher kappa numbers than those pulps produced from E. grandis due to the high condensation behaviour of bamboo lignins under alkaline conditions. Higher kappa numbers explained the higher demand for subsequent bleaching chemicals. In conclusion, the pulp mill biorefinery concept through hemicelluloses pre-extraction with NaOH can be achieved with modified kraft pulping or the sodaAQ pulping processes, but it depends on the type of raw material, extraction method and quality and performance requirements of a particular paper. The low pulping chemicals demand, comparable pulp yields and the improvement in some physico-chemical properties of the pulps from preextracted materials were observed. Furthermore, owing to xylan pre-extraction a larger amount of (extracted) material could be loaded into the digester as when non-extracted materials were used. / AFRIKAANSE OPSOMMING: Ekstraksie van hemiselluloses wat tydens verpulping in die swartloog opgelos word, bied ‘n aantreklike alternatief aan pulp- en papiermeulens om, addisioneel tot hul hoofprodukte, hul inkomste deur die vervaardiging van biobrandstowwe, biopolimere, papierbymiddels en ander chemikalië, daardeur te kan verhoog. Die hoeveelheid hemiselluloses wat ge-ekstraheer kan word, sal egter beperk word deur die vereiste dat pulpopbrengs en –kwaliteit tydens bestaande verpulpingsprosesse gehandhaaf moet word. In hierdie ondersoek is matige alkaliese (NaOH) en verdunde swawelsuurtoestande gebruik om hemiselluloses vóór kraft- of natriumantrakinoonverpulping uit Eucalyptus grandis, reuse bamboes (Bambusa balcooa) en suikerriet (Saccharum officinarum) bagasse, mee te ekstraheer. Die invloed van katalisatorkonsentrasie, temperatuur en reaksietyd is mbv ‘n statistiese, eksperimentele ontwerp ondersoek om die toestande te bepaal waaronder hemiselluloses, met minimale effek op die sellulose (glukaan) –inhoud, vóór alkaliese verpulping ge-ekstraheer kan word. Die pre-ge-ekstraheerde materiale, met hoë glukaan- en voldoende hemisellulosesinhoud, is vervolgens aan kraft- en natriumantrakinoonverpulping onderwerp om die invloed van pre-ekstraksie van hemiselluloses op die verpulpingsreagense, pulpopbrengs en - eienskappe vas te stel. Hierdie studie het ook die evualering van warmwater hemisellulosespre-ekstraksie van suikerrietbagasse, wat deel is van ‘n verdunde swawelsuur eksperimentele uitleg, ingesluit. Pulpopbrengs, die hoeveelheid verpulpingsreagense en handveleienskappe van dieselfde materiale wat nie vooraf ge-ekstraheer is nie, is vergelyk. Die resultate toon dat alkaliese pre-ekstraksie metodes wat ondersoek is die pulpopbrengs met minimale effek op handvel sterkte-eienskappe afhangende van die keuse van daaropvolgende pulpmetode kon handhaaf terwyl ‘n fraksie van xilaan in polimeriese vorm ge-ekstraheer is. Addisioneel, is minder aktiewe alkali benodig om die xilaan ge-ekstraheerde materiale te delignifiseer. Die integrasie van hemisellulosespre-ekstraksie dmv alkaliese metodes tydens ‘n kraft verpulpingsproses is vir reuse bamboes en E. grandis verkies omdat pulpopbrengste op ideale industriële vlakke van 50% gehandhaaf en is en pulp in ‘n bleikbare kappa nommergebied interval kon lewer. ‘n Verdere voordeel wat waargeneem is was die vermindering in die totale gekookte aktiewe alkali benodig vir reuse bamboes of E. grandis met 8 of 3 persentasiepunte onderskeidelik. Die kompromie om die pulpopbrengs te handhaaf is verkry toe slegs 13.6% of 12.4% polimeriese xilaan opgelos is vanuit reuse bamboes of E. grandis onderskeidelik. ‘n Effense verbetering in bars-indeks van die handvelle is waargeneem vir ge-ekstraheerde reuse bamboes. Pulpviskositeit het met 13% gestyg agv die verwydering van die lae molekulêre massa hemiselluloses, terwyl breeksterkte van handvelle ook met 8.9% toegeneem het vir pulp verkry uit pre-gekstraheerde E. grandis. NaOH pre-ekstraksie van 69.1% xilaan (droë massa) uit suikerriet bagasse (SCB) het ‘n hoër natriumantrakinoon, gesifte pulpopbrengs gelewer (45.0%) met ‘n verbeterde afname in kappa-getal (15.5) en uitstekende skeursterkte sonder verlaging in viskositeit, soos vergelyk met nie-ge-ekstraheerde suikkerrietbagasse. Daarteenoor het die resultate verkry met die geoptimeerde verdunde swawelsuur preekstraksie van al die getoetste rumateriale getoon om‘n negatiewe effek te gehad het op die daaropvolgende kraft- of natriumantrakinoonverpulping dws het laer pulpopbrengste en swakker sterkte-eienskappe opgelewer. Die verskille was nietemin kleiner toe natriumantrakinoonverpulping ipv kraftverpulping gebruik is. Antrakinoon beskerm die koolhidrate teen die afskilreaksie in alkaliese medium. Daarteenoor het pre-ekstraksie van suikerrierbagasse met warm water tot 'n lae hoeveelheid (5.7%) xilaanoligomere gelei, terwyl die daaropvolgende natriumantrakinoonverpulping geen verlaging in pulpopbrengs veroorsaak het nie. Skeursterkte en optiese helderheid van handvelle wat uit warm water ge-ekstraheerde suikerrietbagasse vervaardig is, het ietwat verbeter terwyl breek-, trek- en barssterkte dieselfde was as van suikerrietbagasse pulp wat nie ge-ekstraheer is nie. Net so belangrik was die waargenome hoër skeur- en barsindekse van handvelle vervaardig van reuse bamboes in vergelyking met E. grandis van beide geekstraheerde en nie ge-ekstraheerde materiale voorberei onder dieselfde verpulpings toestande. Bamboes se sterker eienskappe was as gevolg van die hoër vesellengte en ander morfologiese eienskappe as diévan loofhout. Pulp wat vervaardig is van reuse bamboes het ‘n hoër kappanommer getoon as pulp van E. grandis as gevolg van die hoë kondensasiegedrag van bamboeslignien onder alkaliese toestande. Hoër kappanommers kon die gepaardgaande hoër aanvraag vir bleikchemikalieë verklaar. Ten slotte, die pulpmeul bio-raffinaderykonsep nl. deur hemisellulosesekstraksie met NaOH gekombineer met óf ‘n gemodifiseerde kraft verpulping óf ‘n gemodifiseerde natriumantrakinoon verpulping, is wel uitvoerbaar. Dit word egter sterk beïnvloed deur die tipe ru-materiaal en die ekstraksie-metode gebruik, asook deur die kwaliteits- en gebruiksvereistes van verskillende tipes papier. ‘n Lae aanvraag vir verpulpingschemikalieë, vergelykbare pulpopbrengste en die verbetering in fisies-chemiese eienskappe van pulp vanaf pre-ge-ekstraheerde materiale is waargeneem. Verder kon, as gevolg van xilaan ekstraksie, meer ge-ekstraheerde materiaal in die verteerder gelaai word as wanneer nie-ge-ekstraheerde materiaal gebruik is.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-17T16:57:36Z No. of bitstreams: 1 PDF - Silvana Cartaxo da Costa.pdf: 2220034 bytes, checksum: fa6fe0b10616cb9ed70f24ac4dc15d62 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:01:05Z (GMT) No. of bitstreams: 1 PDF - Silvana Cartaxo da Costa.pdf: 2220034 bytes, checksum: fa6fe0b10616cb9ed70f24ac4dc15d62 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:01:16Z (GMT) No. of bitstreams: 1 PDF - Silvana Cartaxo da Costa.pdf: 2220034 bytes, checksum: fa6fe0b10616cb9ed70f24ac4dc15d62 (MD5) / Made available in DSpace on 2016-07-22T15:01:16Z (GMT). No. of bitstreams: 1 PDF - Silvana Cartaxo da Costa.pdf: 2220034 bytes, checksum: fa6fe0b10616cb9ed70f24ac4dc15d62 (MD5) Previous issue date: 2014-05-23 / The development of a colon-specific delivery system using polymeric microparticles has received great attention in the pharmaceutical field. An interesting group of polymers with potential properties in this area are the hemicellulose. Xylan is a hemicellulose that has the ability to pass through the digestive tract unchanged and its complex structure requires enzymes that are produced specifically by the human colonic microflora, which makes it an interesting raw material in the production of target drug delivery systems. The microparticulate systems can be developed by various techniques. The interfacial crosslinking polymerization is one of the major techniques to produce polysaccharide based microparticles. However, the use of highly toxic crosslinkers often makes the use of this technique limited. The sodium trimetaphosphate (TSTP), a low toxic crosslinking agent, has no adverse effects reported on human beings. The aim of this study was to develop xylan microparticles using sodium trimetaphosphate. The microparticles were characterized by optical microscopy, SEM, XRD and FT -IR. The influence of different parameters on the diameter of the microparticles was analyzed during their development. Toxicity studies against Artemia saline Leach were made to compare the microparticles produced with terephthaloyl chloride and sodium trimetaphosphate. Xylan microparticles showed to be spherical shape, well individualized and with a smooth surface. All different parameters influenced the in size of the microparticles. The FT-IR spectrum of microparticles was similar to xylan, but with the presence of the peak at 1258 cm -1 , which is typical of phosphate ester bonds, that can be attributed to the bond between TSTP and xylan during the crosslinking process. The xylan microparticles produced in this work showed no toxicity at the concentration studied. It be concluded that TSTP was able to produce xylan microparticles with well defined physicochemical characteristics and low toxicity. / O desenvolvimento de um sistema de liberação cólon-específica utilizando micropartículas poliméricas têm recebido grande atenção no campo farmacêutico. Um grupo interessante de polímeros com potenciais propriedades nessa área são as hemiceluloses. A xilana é uma hemicelulose que tem a capacidade de passar através do trato digestivo inalterada e sua complexa estrutura requer enzimas que são produzidas especificamente pela microflora colônica humana, o que a torna uma interessante matéria-prima na área produção de sistemas de liberação de fármacos. Os sistemas microparticulados podem ser desenvolvidos por várias técnicas. A reticulação polimérica interfacial é uma das principais técnicas para produção de micropartículas à base de polissacarídeos. Porém o uso de reticulantes de alta toxicidade muitas vezes torna o uso desta técnica limitada. O trimetafosfato de sódio (TSTP) é um reticulante de baixa toxicidade, sem efeitos adversos relatados em seres humanos. Esse trabalho teve como objetivo desenvolver micropartículas de xilana utilizando TSTP. As micropartículas foram caracterizadas por microscopia óptica, MEV, DRX e FT-IR. Estudos de toxicidade frente à artemia salina Leach foram feitos para comparar as micropartículas produzidas com cloridrato de tereftaloíla e trimetafosfato de sódio. As micropartículas de xilana apresentaram forma esférica, bem individualizada e com superfície bem definida. Todos os diferentes parâmetros influenciaram no tamanho das micropartículas. O espectro de FT-IR das micropartículas foi semelhante ao da xilana, porém com a presença de um pico em 1258 cm -1 , que é típico de ligações éster-fosfato, que pode ser atribuído a ligação entre TSTP e a xilana durante o processo de reticulação. As micropartículas de xilana produzidas neste trabalho não apresentaram toxicidade na concentração estudada. Podemos concluir que o TSTP foi capaz de produzir micropartículas de xilana com cracterísticas fisico-químicas bem definidas e de baixa toxicidade.

In this thesis the interactions in cellulose systems havebeen studied with emphasis on surface forces and adsorptionphenomenon. The study was limited to surfaces and polymers ofinterest for papermaking. During papermaking both therunability of the paper machine as well as the properties ofthe final paper will depend on the molecular interactionsbetween the components present in the suspension. The objectiveof this work was to gain a better understanding of theseinteraction forces and how they are affected by solutionconditions like pH and electrolyte concentration. The maintechniques employed for this purpose were direct surface forcemeasurements using both the interferometric surface forceapparatus (SFA) and the atomic force microscopy (AFM)colloidal-probe technique. In addition, both electronspectroscopy for chemical analysis (ESCA) and AFM imaging wereused for surface characterisation and to study adsorptionbehaviour. Since a smooth and transparent surface is needed forsurface force studies using SFA, Langmuir-Blodgett films ofcellulose were employed. The interactions between cellulose surfacesand xylansurfaces were studied. Cellulose is the main constituent of thewood fibre. In close association with cellulose in the cellwall are the hemicelluloses, xylan being a commonhemicellulose. Steric forces were found to dominate theinteractions between these substances in aqueous solutions.Still factors like charge density, solution pH and electrolyteconcentration were important. The adhesion between cellulosesurfaces in air depended on the contact area. The effect of acationic polyelectrolyte on the forces between cellulosesurfaces and between cellulose and mineral surfaces wasinvestigated to obtain a better understanding of the effects ofcationic retention and strength additives. In the presence of acationic polyelectrolyte the forces at large distances weredominated by double-layer repulsion. Bridging attraction was insome cases observed, but the presence and magnitude of thisforce was dependent on the properties of the substratesurface. Lignin on the fibre surface affects brightness, swelling andstrength of paper. In addition, dissolved lignin interfereswith added polymers during papermaking. For this reason theadsorption of lignin on cellulose fibre surfaces was explored.The adsorption of lignin was influenced by the concentration oflignin in solution and by the electrolyte composition of thesolution. The retention of lignin was affected by the presenceof a cationic polyelectrolyte and particularly by how thepolyelectrolyte was added. The AFM imaging revealed thestructure of the adsorbed lignin, which varied with the way ofadding the polyelectrolyte, and gave an idea about theadsorption mechanism. The effect of lignin on strengthproperties of paper sheets was also evaluated. Keywords: cellulose, hemicellulose, xylan, lignin, surfaceforces, adsorption, polyelectrolyte, SFA, AFM, ESCA,Langmuir-Blodgett films, chitosan, steric forces,bridging,adhesion

<p>In this thesis the interactions in cellulose systems havebeen studied with emphasis on surface forces and adsorptionphenomenon. The study was limited to surfaces and polymers ofinterest for papermaking. During papermaking both therunability of the paper machine as well as the properties ofthe final paper will depend on the molecular interactionsbetween the components present in the suspension. The objectiveof this work was to gain a better understanding of theseinteraction forces and how they are affected by solutionconditions like pH and electrolyte concentration. The maintechniques employed for this purpose were direct surface forcemeasurements using both the interferometric surface forceapparatus (SFA) and the atomic force microscopy (AFM)colloidal-probe technique. In addition, both electronspectroscopy for chemical analysis (ESCA) and AFM imaging wereused for surface characterisation and to study adsorptionbehaviour. Since a smooth and transparent surface is needed forsurface force studies using SFA, Langmuir-Blodgett films ofcellulose were employed.</p><p>The interactions between cellulose surfacesand xylansurfaces were studied. Cellulose is the main constituent of thewood fibre. In close association with cellulose in the cellwall are the hemicelluloses, xylan being a commonhemicellulose. Steric forces were found to dominate theinteractions between these substances in aqueous solutions.Still factors like charge density, solution pH and electrolyteconcentration were important. The adhesion between cellulosesurfaces in air depended on the contact area. The effect of acationic polyelectrolyte on the forces between cellulosesurfaces and between cellulose and mineral surfaces wasinvestigated to obtain a better understanding of the effects ofcationic retention and strength additives. In the presence of acationic polyelectrolyte the forces at large distances weredominated by double-layer repulsion. Bridging attraction was insome cases observed, but the presence and magnitude of thisforce was dependent on the properties of the substratesurface.</p><p>Lignin on the fibre surface affects brightness, swelling andstrength of paper. In addition, dissolved lignin interfereswith added polymers during papermaking. For this reason theadsorption of lignin on cellulose fibre surfaces was explored.The adsorption of lignin was influenced by the concentration oflignin in solution and by the electrolyte composition of thesolution. The retention of lignin was affected by the presenceof a cationic polyelectrolyte and particularly by how thepolyelectrolyte was added. The AFM imaging revealed thestructure of the adsorbed lignin, which varied with the way ofadding the polyelectrolyte, and gave an idea about theadsorption mechanism. The effect of lignin on strengthproperties of paper sheets was also evaluated.</p><p>Keywords: cellulose, hemicellulose, xylan, lignin, surfaceforces, adsorption, polyelectrolyte, SFA, AFM, ESCA,Langmuir-Blodgett films, chitosan, steric forces,bridging,adhesion</p>

Submitted by Michel Ricardo Gracioli (michel-rg@hotmail.com) on 2018-04-17T15:51:19Z No. of bitstreams: 1 Dissertação Michel.pdf: 1706772 bytes, checksum: 1d66fae75b86a68d89ec8bceca0d2f31 (MD5) / Approved for entry into archive by Adriana Aparecida Puerta null (dripuerta@rc.unesp.br) on 2018-04-18T14:13:36Z (GMT) No. of bitstreams: 1 gracioli_mr_me_rcla.pdf: 1697470 bytes, checksum: 82696e96fe2dfed0fbd786c7230a8994 (MD5) / Made available in DSpace on 2018-04-18T14:13:36Z (GMT). No. of bitstreams: 1 gracioli_mr_me_rcla.pdf: 1697470 bytes, checksum: 82696e96fe2dfed0fbd786c7230a8994 (MD5) Previous issue date: 2018-02-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As enzimas xilanases são de grande interesse biotecnológico em função da sua habilidade na degradação da molécula de xilana, a principal hemicelulose presente na parede celular de plantas. Tais enzimas encontram aplicações nos mais diversos ramos da indústria como na produção de alimentos e bebidas, de cosméticos, de ração animal, de biocombustíveis entre outros. Em razão da sua composição química, os subprodutos lignocelulósicos, produzidos em grande quantidade pela agroindústria, se apresentam como uma potencial fonte sustentável para a produção de valiosos compostos como biocombustíveis, fertilizantes, rações, produtos químicos e enzimas. Assim, tendo em vista esse contexto, foi desenvolvido o presente trabalho, cujo objetivo foi caracterizar e purificar a principal endoxilanase produzida por uma linhagem de Aspergillus hortai cultivado em estado sólido e aplicá-la, nas formas bruta e purificada, na hidrólise de subprodutos agroindustriais. A purificação da enzima foi alcançada após três etapas cromatográficas, respectivamente, uma cromatografia de troca iônica seguida por cromatografias de interação hidrofóbica e exclusão molecular. Ao final das etapas, a enzima purificada apresentou recuperação de 1,1% e fator de purificação de 15,8 vezes. A enzima bruta e purificada se mostrou tolerante ao NaCl na presença de até 4 M do sal. Por outro lado, foi pouco tolerante à presença de íons e alguns compostos como Hg2+, Cu2+, SDS, Triton X-100 e TWEEN 20/80. Em contrapartida, DTT e β-mercaptoetanol a 10 mM estimularam a atividade da enzima bruta em 13 e 19%, respectivamente. Para a enzima bruta, a temperatura e pH ótimos de reação foram, respectivamente, 60 °C e 6,0 tanto na ausência quanto na presença de NaCl 0,5 e 2,5 M. Apresentou boa estabilidade térmica a 40 °C na ausência de NaCl, e foi ativada na presença de 0,5 M e 2,5 M do sal durante as 4 h de incubação. A enzima bruta e purificada foi estável em uma ampla faixa de pHs (3,0-8,0) na ausência e na presença de NaCl e apresentou alta especificidade pelas xilanas birchwood, beechwood e oat spelt, não tendo sido detectadas atividades endoglucanase sobre CMC, exoglucanase em Avicel e β-xilosidase em ρNPX. Após a purificação, os perfis de temperatura e pH ótimos de reação se mantiveram em 60 °C e 6, respectivamente. A enzima pura foi, também, bastante estável a temperatura de 40 °C, mantendo mais de 50% da atividade controle durante as 4 h de incubação, e ao pH, variando de 3,0-8,0, na ausência e na presença de NaCl. Xilobiose e xilo-oligossacarídeos superiores foram os produtos formados na hidrólise da xilana oat spelt pela enzima purificada, sugerindo ação enzimática do tipo endoxilanase. A massa molecular aparente, após a purificação, foi estimada em 25,0 kDa por SDS-PAGE e em 14,8 kDa por exclusão molecular. Os parâmetros cinéticos Km e Vmáx em xilana de beechwood sofreram variações em resposta a presença de NaCl, sendo a maior eficiência catalítica obtida na presença de 2,5 M do sal (kcat/km 40,91). A análise da composição química dos subprodutos - sabugo, palha e folha de milho - revelou, respectivamente, 25,6%, 45,3% e 34,9% de carboidratos, 19,7%, 15,2% e 17,6% de lignina e 9,0%, 17,4% e 24,0% de extrativos para a biomassa in natura e, 33,3%, 25,2% e 38,8% de carboidratos e 6,5%, 8,9% e 12,3% de lignina para a xilana extraída desses subprodutos. A extração das hemiceluloses utilizando tratamento alcalino oxidativo resultou em um rendimento de 64% para o sabugo, 18% para a palha e 50% para a folha. A enzima bruta e purificada foi capaz de hidrolisar as biomassas na forma in natura, bem como as xilanas extraídas desse material produzindo majoritariamente xilopentoses e xilohexoses. Sendo assim, pode-se afirmar que a xilanase bruta e purificada em estudo apresentou propriedades interessantes para a aplicação industrial, especialmente na produção de xilo-oligossacarídeos e em processos conduzidos sob elevada salinidade. / Xylanases raise a biotechnological interest due to its ability to degrade xylan. Such enzymes find use in a variety of industrial processes such as the production of food and beverage, cosmetics, animal feed, second generation ethanol, among many others. As for its chemical composition, lignocellulosic byproducts, widely produced by agroindustry, presents itself as a potential sustainable source for production of valuable composts like soil fertilizers, animal feed, chemical products and enzymes. In this context, the main xylanase produced by an Aspergillus hortae strain, cultivated in solid state (SSF), was characterized, purified and applied in the hydrolysis of hemicellulose from corn by-products. Enzyme purification was achieved after three chromatographyc steps, respectively, an ion exchange chromatography followed by hydrophobic interaction and molecular exclusion chromatographys. After the purification steps, a final yield of 1.1% and purification fold of 15.8 was achieved. Both crude and purified enzyme showed high tolerance to NaCl in the presence of up to 4 M of the salt. The enzyme displayed a very low tolerance to some metal ions and chemical compounds, especially Hg2+, Cu2+, SDS, Triton X-100 and TWEEN 20/80. On the other hand, DTT and β- mercaptoetanol at 10 mM stimulated crude enzyme activity in 13% and 19%, respectively. As for the crude enzyme, optimum temperature and pH were, respectively, 60 °C and 6.0, in the absence and presence of 0.5 and 2.5 M NaCl. It showed good thermal stability at 40 °C in the absence of NaCl and was activated in the presence of 0.5 and 2.5 M of the salt throughout the 4 h incubation time. Crude and purified enzyme was also stable over a wide pH range (3.0 - 8.0) both in the absence and presence of NaCl and presented high specificity for birchwood, beechwood and oat spelt xylans, not showing detectable endoglucanase, exoglucanase and β- xylosidase activities. After purification, optimum temperature and pH profiles remained at 60 °C and 6.0, respectively. Purified enzyme showed good stability at 40 °C, being able to retain more than 50% of the control activity during 4 h of incubation, and to the pH, varying from 3.0 to 8.0, in the absence and in the presence of NaCl. Xylobiose and xilooligosaccharides were the products of oat spelt xylan hydrolysis by the purified enzyme, suggesting an endoxilanase mode of action. The apparent molecular mass, after purification, was estimated to be 25.0 kDa by SDS-PAGE and 14.8 kDa by exclusion chromatography. Kinetic parameters of Km and Vmáx using beechwood xylan were 5.12 mg/mL and 14.25 μmol/min.mL, respectively, in the absence of NaCl, 9.56 mg/mL and 20.80 μmol/min.mL in the presence of 0.5 M and 3.13 mg/mL and 9.22 μmol/min.mL in the presence of 2.5 M of the same salt. Chemical analysis of the biomasses from corn cob, stover and leaves, revealed a composition of, respectively, 25.6%, 45.3%, 34.9% carbohydrates, 19.7%, 15.2%, 17.6% lignin and 9.0%, 17.4%, 24.0% extractives for in natura biomasses and 33.3%, 25.2%, 38.8% carbohydrates and 6.5%, 8.9%, 12.3% lignin for the extracted hemicelluloses. Hemicellulose extraction yields were of 64% for corn cob, 18% for stover and 50% for leaves. Both crude and purified enzyme was capable of hydrolyzing in natura biomasses, as well as the extracted hemicelluloses from these materials, producing mainly xylopentoses and xyloheoses. In summary, the xylanase presented attractive properties for industrial applications, especially in the production of xyloolygossacharides and in those carried out under high NaCl concentration. / CNPq: 130841/2016-1.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed

No description available.

Single stage and two-stage sodium sulfite cooking were carried out on either spruce, pine or pure pine heartwood chips to investigate the influence of several process parameters on the initial phase of such a cook down to about 60 % pulp yield. The cooking experiments were carried out in the laboratory with either a lab-prepared or a mill-prepared cooking acid and the temperature and time were varied. The influences of dissolved organic and inorganic components in the cooking liquor on the final pulp composition and on the extent of side reactions were investigated. Kinetic equations were developed and the activation energies for delignification and carbohydrate dissolution were calculated using the Arrhenius equation. A better understanding of the delignification mechanisms during bisulfite and acid sulfite cooking was obtained by analyzing the lignin carbohydrate complexes (LCC) present in the pulp when different cooking conditions were used. It was found that using a mill-prepared cooking acid beneficial effect with respect to side reactions, extractives removal and higher stability in pH during the cook were observed compared to a lab-prepared cooking acid. However, no significant difference in degrees of delignification or carbohydrate degradation was seen.  The cellulose yield was not affected in the initial phase of the cook however; temperature had an influence on the rates of both delignification and hemicellulose removal. It was also found that the  corresponding activation energies increased in the order:  xylan, glucomannan, lignin and cellulose. The cooking temperature could thus be used to control the cook to a given carbohydrate composition in the final pulp. Lignin condensation reactions were observed during acid sulfite cooking, especially at higher temperatures. The LCC studies indicated the existence of covalent bonds between lignin and hemicellulose components with respect to xylan and glucomannan. LCC in native wood showed the presence of phenyl glycosides, ϒ-esters and α-ethers; whereas the α-ethers  were affected during sulfite pulping. The existence of covalent bonds between lignin and wood polysaccharides might be the rate-limiting factor in sulfite pulping. / The sulfite pulping process is today practised in only a small number of pulp mills around the globe and the number of sulfite mills that use sodium as the base (cation) is less than five. However, due to the increasing interest in the wood based biorefinery concept, the benefits of sulfite pulping and especially the sodium based variety, has recently gained a lot of interest. It was therefore considered to be of high importance to further study the sodium based sulfite process to investigate if its benefits could be better utilized in the future in the production of dissolving pulps. Of specific interest was to investigate how the pulping conditions in the initial part of the cook (≥ 60 % pulp yield) should be performed in the best way. Thus, this thesis is focused on the initial phase of sodium based single stage bisulfite, acid sulfite and two-stage sulfite cooking of either 100 % spruce, 100 % pine or 100 % pine heartwood chips. The cooking experiments were carried out with either a lab prepared or a mill prepared cooking acid and the temperature and cooking time were varied. Activation energies for different wood components were investigated as well as side reactions concerning the formation of thiosulfate. LCC (Lignin carbohydrates complexes) studies were carried out to investigate the influence of different cooking conditions on lignin carbohydrate linkages.