Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The microbial degradation of the lignin carbohydrate complex of plant material is only partially understood. Lignin carbohydrate complex was extracted from wheat straw and subsequently analysed. An adjustment to the standard protocol was required to increase the amount of lignin carbohydrate complex extracted from wheat straw. Characterization of the lignin carbohydrate complex after trifluoacetic acid hydrolysis was done by capillary electrophoresis. HPLC proved ineffective, as baseline separation could not be achieved. Characterization of the lignin carbohydrate complex revealed that a large portion (68 %) consisted of carbohydrate and lignin (20 %). Capillary electrophoresis of the trifluoroacetic acid hydrolysates of the lignin carbohydrate complex revealed that the carbohydrates consisted of mannose, xylose, arabinose, galactose, glucose and rhamnose. The major monosaccharide present in the lignin carbohydrate complex was mannose which made up 34 % of the total carbohydrate composition. Ferulic and p-coumaric acid were present in the lignin carbohydrate complex, but in concentrations less than 1 % of the lignin carbohydrate complex. The lignin carbohydrate complex of wheat straw probably had a heterogenous structure consisting of a variety of molecules, as molecular weight determination could not be accurately determined. An estimated molecular weight of 5.9 kOa was determined. Ten fungal strains (Aspergillus niger, Aureobasidium pul/u/ans, Bjerkandera adusta, Corio/us versicolor, Lenzitus betu/ina, Phanerochaete chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 and Trichoderma reeseï; were evaluated for growth on the lignin carbohydrate complex. All strains except B. adusta showed growth after 5 days with A. niger, A. pul/u/ans, C. versicolor, P. chrysosoporium and T. reesei showing the best growth on the lignin carbohydrate complex. The culture fluid revealed a number of proteins secreted by these organisms. The protein determination was confirmed by SOS-PAGE which revealed an array of proteins ranging from 8 kOa to 180 kOA. Prominent bands between 26 kOa and 80 kOa could be observed in the culture fluid of A. niger, A. pul/ulans and T. reesei, but not in C. versicolor. Activity studies on the culture fluid of these four strains revealed activity for xylanase, xylosidase, arabinofuranosidase, ferulic acid esterase and laccase, with vast differences between the activities of the various fungi. The enzymes of these fungal strains were also evaluated for their ability to degrade xylan and sugar cane bagasse using capillary electrophoresis. It appeared that all the organisms produced enzymes to degrade birchwood xylan. However, the electropherograms revealed that the degradation patterns of birchwood xylan differed among these organisms over the same time interval, as xylotetraose, xylotriose, xylobiose and xylose were released in various concentrations. The electropherograms obtained from the enzyme hydrolysates of sugar cane bagasse, pointed to the substrate being inaccessible. Electropherograms of the culture fluid of A. niger, A. pul/ulans, C. versicolor and T. reesei, when incubated on the lignin carbohydrate complex indicated similar peaks to those obtained and identified in the trifluoroacetic acid hydrolysates. However, the electropherograms of the culture fluid of these organisms revealed additional smaller peaks which could not be identified. The electropherograms of the culture fluid of the various organisms also indicated a complete release of some sugars, using the trifluoacetic acid hydrolysate of the lignin carbohydrate complex as a control for the amount of sugars present. HPLC analyses revealed that after 72 h, no apparent degradation of the lignin carbohydrate complex took place as peak height and areas appeared to be similar. These peaks could however not be identified due to a lack of standards as well as baseline separation which could not be achieved. / AFRIKAANSE OPSOMMING: Tans word die mikrobiese afbraak van die lignienkoolhidraatkompleks van plant materiaal slegs gedeeltelik verstaan. Lignienkoolhidraatkompleks was vanaf koringstrooi geïsoleer en gevolglik geanaliseer. Daar moes van die standaard prosedure vir die ekstraksie van lignienkoolhidraatkompleks afgewyk word ten einde beter lignienkoolhidraatkompleks opbrengs te lewer. Karakterisering van die lignienkoolhidraatkompleks na trifluoroasynsuurvertering was deur kapillêre elektroforese bepaal. Dit wou voorkom asof kapillêre elektroforese "n beter opsie vir die analise van die verteerde monster van lignienkoolhidraatkompleks is, vergeleke met hoëdruk vloeistof chromatografie. Daar was gevind dat die lignienkoolhidraatkompleks uit 68 % koolhidraat en 20 % lignien bestaan. Kapillêre elektroforese het die teenwoordigheid van die volgende suikers bevestig naamlik, mannose, xilose, arabinose, glukose, galaktose en ramnose. Mannose was die dominerende suiker in die lignienkoolhidraatkompleks wat 34 % van die totale koolhidraat opbrengs uitgemaak het. Ferulien- en p-kumaarsuur kon ook identifiseer word, maar die twee sure het minder as 1 % van die totale inhoud van die lignienkoolhidraatkompleks uitgemaak. Vanuit resultate bekom wil dit voorkom dat die lignienkoolhidraatkompleks "n heterogene molekuul is omdat die molekulêre gewig daarvan nie akkuraat bepaal kon word nie. 'n Geskatte molekulêre grootte van ongeveer 5.9 kDa was bepaal met verwysing na die hoogste piek wat in die chromatogram waargeneem was. Tien fungus kulture was in die studie gebruik om hul vermoë te toets om op die lignienkoolhidraatkompleks te groei, naamlik Aspergillus niger, Aureobasidium pullulans, Bjerkandera adusfa, Goriolus versicolor, Lenziius betuline. Phanerochaefe chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 en Trichoderma reesei. B. eauste het nie groei na 5 dae getoon nie, en dit wou voorkom asof A. niger, A. pul/ulans, G. versicolor, P. chrysosoporium en T. reesei die beste kon groei op die substraat na 5 dae. Die kultuurvloeistof van die vier kulture het getoon dat proteïene deur hierdie organisms uitgeskei was. Hierdie proteinbepaling was ook bevestig deur SOS-PAGE, wat bande tussen 8 kDa en 180 kDa gelewer het. Prominente bande tussen 26 kDa en 80 kDa kon waargeneem word in die kultuurvloeistof van A. niger, A. pul/ulans, en T. reesei, maar nie in die kultuurvloeistof van C. versicolor nie. Aktiwiteitstudies op die kultuur vloeistowwe het getoon dat daar aktiwiteit was vir die volgende ensieme, naamlik xilanase, xilosidase, arabinofuranosidase en feruliensuur esterase. Hierdie aktiwiteit het aansienlik verskil tussen die verskillende organismes. Die ensieme van die vier organismes was ook gebruik om hul vermoë te toets om xilaan en suikerriet af te breek. Daar was gevind dat xilaanafbraak verskillend was vir die organisms oor dieselfde tydperk. Xilotetraose, xilotriose, xilobiose en xilose was in verskillende konsentrasies gevind vir die verskillende organismes. Die elektroferogramme van die kultuurvloeistof op suikerriet van die verskillende organismes het getoon dat die substraat nie toeganklik vir die ensieme was nie. Die elektroferogramme van die kultuurvloeistof op lignienkoolhidraatkompleks van die verskillende organismes het dieselfde pieke getoon soos geïdentifiseer in die elektroferogramme van die trifluoroasynsuur vertering. Die elektroferogramme met die ensiem vertering het egter addisionele pieke getoon wat nie sigbaar op die elektroferogramme van die trifluoroasynsuur vertering was nie. Hierdie pieke het min of meer dieselfde tyd ge-elueer as die monosakkariede. Kapillêre elektroforese het ook getoon dat die ensiematiese afbraak van die lignienkoolhidraatkompleks gelei het tot algehele vrystelling van sommige suikers, wanneer die trifluoroasynsuur vertering as maatstaaf dien vir die hoeveelheid suikers teenwoordig in die lignienkoolhidraatkompleks. Hoëdruk vloeistof chromatografie het getoon dat geen sigbare afbraak na 72 h van inkubasie met die ensieme op die lignienkoolhidraatkompleks plaasgevind het nie aangesien die piek hoogtes konstant gebly het. Hierdie pieke kon egter nie geïdentifiseer word nie as gevolg van lae resolusie van die pieke asook standaarde wat nie beskikbaar was nie.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Biocompatible composites, generated from renewable biomass feedstock, are regarded as promising materials that could replace synthetic polymers and reduce global dependence on fossil fuel sources. Wood cellulose, the most abundant biopolymer on earth, holds great potential as a renewable biomass feedstock. To unlock the entire scope of potential benefits of this feedstock, the wood components - namely cellulose, hemicellulose and lignin - need to be separated and processed individually. Current methods to separate wood components, such as Kraft pulping for example, suffer considerable drawbacks and cannot be considered environmentally benign. This thesis aims to increase our understanding of the interaction between ionic liquids (ILs) and biomass, in order to develop superior biomass processing techniques necessary to ensure a rapid transformation of our society towards full sustainability. The first part of this work deals with the particular interaction of ILs with cellulose, and aims to investigate the structural requirements of ILs in order to qualify as a cellulose solvent. The cellulose-dissolving behaviour of selected alkanolammonium ILs was studied, and, combined with the results of an extensive literature review, a novel concept for the interaction of cellulose-dissolving ILs with cellulose was developed. It was postulated that efficient cellulose solvents need to position themselves in a distinct manner - with respect to the cellulose chain - in order to offer H-bond interaction sites with enhanced stability. As a result, alternative ions for cellulose-dissolving ILs were proposed, including oxazolium, 1,3-oxaphospholium, dimethylcarbamate, phosphate, nitrate, and nitrite. The second part of the work investigated the use of food-additive based ILs for the separation of wood lignin, and studied the influence of selected process parameters, such as extraction time, extraction temperature, IL moisture content, wood particle size, wood species, IL cation species, solvent composition, and IL recyclability on the lignin extraction efficiency. The lignin extract and the wood residues were characterised via infrared spectroscopy, elemental analysis, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction, and gel permeation chromatography. An extraction efficiency of e = 0.43 of wood lignin was achieved in one gentle extraction step ( T = 373 K, t = 2 h), and it was found that the presence of a co-solvent increased the extraction efficiency to e = 0.60. Gentle conditions during IL treatment did not decrease the crystallinity of the wood sample, and the extracted lignin had both a larger molecular mass and a more uniform molecular mass distribution, compared to commercially available Kraft lignin. The outcomes of both studies were critically evaluated, addressing existing drawbacks and restrictions that need to be considered, and possibilities for future work were suggested.

Submitted in fulfillment of the academic requirements for the degree of Master of Applied Sciences (Chemistry), Durban University of Technology, Durban, South Africa, 2016. / Globally there is a drive for the use of renewable materials for the production of biofuels or high-end value chemicals. The current production of chemicals from crude oil refining is unsustainable and leads to global warming effects. Biomass is the most attractive renewable energy source for biofuel or fine chemical production. Sugarcane bagasse is a by-product of the sugar milling industry and is abundantly available. In this study lignin was sequentially extracted using ionic liquids. The ionic liquids (ILs) 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) and triethylammonium hydrogen sulfate ([HNEt3][HSO4]) were used to fractionate the sugarcane bagasse. The pre-treatment of sugarcane bagasse was carried out at different temperatures ranging from 90 - 150 0C and reaction times ranging from 1 - 24 h in a convection oven at a 10 % biomass loading. Both ILs were able to dissolve the raw bagasse samples at 120 0C with [Emim][OAc] giving a lignin maxima of 28.8 % and a low pulp yield of 57 % after 12 h; [HNEt3][HSO4] gave a lignin recovery of 17.2 % and low pulp yield of 58.5 % after 6 h. Regenerated lignin was obtained by adding ethanol/ water to the mixture followed by vacuum filtration. The regenerated pulp materials were characterized by Scanning Electron Microscope (SEM) to study the morphology; Fourier Transform Infrared Spectroscopy (FTIR) to study the characteristic bands and thermal analysis to study the thermal stability. / M

Orientador: Mario de Oliveira Neto / Resumo: A busca por fontes de energias renováveis vêm ganhando cada vez mais importância, pois, com a eminente escassez dos combustíveis fósseis, que são as principais fontes energéticas utilizadas mundialmente e ao mesmo tempo com os problemas ambientais causados por esses combustíveis, o etanol de segunda geração (2G) surge como uma alternativa de energia renovável já que é obtido a partir de biomassa lignocelulósica. Entretanto, as tecnologias para a conversão desta biomassa em açúcares fermentáveis ainda possuem fatores limitantes. Um dos grandes problemas do processo de obtenção do etanol 2G está relacionado ao custo das enzimas celulolíticas. Sendo assim, desenvolver estratégias para a recuperação dessas enzimas é necessário para assegurar a viabilidade econômica do processo de conversão de biomassa. Diversas técnicas estão sendo estudadas e colocadas em prática para promover a recuperação, reciclagem e imobilização enzimática. Entretanto, nenhuma das metodologias utilizadas demonstram, de maneira geral, alta eficiência, custo baixo, rapidez e manutenção da atividade enzimática. A partir da lignina, subproduto da indústria de papel e celulose, pode-se sintetizar o lignocresol. Este, apresenta grandes expectativas de interação enzimática uma vez que apresenta características físico-químicas favoráveis à adsorção. Frente a isto, propusemos a produção deste composto para promover a interação com enzimas para promover recuperação enzimática. A síntese de lignocresol, oriundo da lig... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The search for renewable energy sources is gaining increasing importance because, with the imminent shortage of fossil fuels, which are the main energy sources used worldwide and at the same time with the environmental problems caused by these fuels, second-generation ethanol (2G) arises as a renewable energy alternative since it is obtained from lignocellulosic biomass. However, the technologies for the conversion of this biomass to fermentable sugars still have limiting factors. One of the major problems in the process of obtaining 2G ethanol is related to the cost of cellulolytic enzymes. Thus, developing strategies for the recovery of these enzymes is necessary to ensure the economic viability of the biomass conversion process. Several techniques are being studied and put into practice to promote recovery, recycling, and enzymatic immobilization. However, none of the methodologies used shows, in general, high efficiency, low cost, fastness and maintenance of the enzymatic activity. From lignin, a byproduct of the pulp and paper industry, lignocresol can be synthesized. This one presents high expectations of enzymatic interaction since it presents physicochemical characteristics favorable to the adsorption. In view of this, we proposed the production of this compound to promote interaction with enzymes to promote enzymatic recovery. The lignocresol synthesis from Pinus spp. Kraft lignin was made by adapting to the phase separation system methodology. In this process, more ... (Complete abstract click electronic access below) / Doutor

In der vorliegenden Arbeit wurden die für den radialen Stofftransport durch die Wurzel Höherer Pflanzen wichtigen apoplastischen Barrieren der Wurzeln von sieben Pflanzenarten (Vicia faba L.; Typha glauca Godr.; Ricinus communis L.; Quercus petraea (Matt.) Liebl.; Fagus sylvatica L.; Picea abies (L.) Karst.; Zea mays L.) mikroskopisch charakterisiert und chemisch analysiert. Nach enyzmatischer Isolation der Gewebe wurde die Biopolymerzusammensetzung von Suberin und Lignin der isolierten Zellwände nach Depolymerisierung durch Umesterungsreaktion (Abbau von Suberin) oder Thioacidolyse (Abbau von Lignin) mittels Gaschromatographie und Massenspektroskopie aufgeklärt. Außerdem wurde Sprossknollenperiderm der Kartoffel (Solanum tuberosum L.) verschiedener post harvest Luftfeuchtebedingungen, sowie neugeformtes Wundperiderm chemisch-analytisch und auf die Permeabilität für Wasser hin untersucht. Zusätzlich zu den mikroskopischen und chemischen Analysen wurden die hydraulischen Leitfähigkeiten von Maiswurzeln verschiedener Kulturbedingungen und die Aufnahme von Rubidium-Ionen über die Maiswurzeln untersucht. Dabei wurde die Auswirkung von Salzstress (100mM NaCl), und eine Applikation des Phytohormons Abscisinsäure (10µM ABA) bei der Aufzucht der Pflanzen auf apoplastische Barrieren untersucht. Auch die Rubidiumaufnahme von bei Nitratmangel (0.00 M NO3-) aufgewachsenen Rizinuspflanzen wurde ermittelt und mit der chemischen Zusammensetzung der apoplastischen Barrieren korreliert. Die Ergebnisse dieser Arbeit zeigen, dass: -monocotyle Pflanzen wesentlich höhere Aromatenanteile im Suberin apoplastischer Barrieren besitzen als dicotyle Pflanzen; -bei der Bewertung des Suberingehaltes apoplastischer Barrieren histochemische Methoden unzureichend sind; -die Flächenbelegung mit Suberin auch innerhalb gleicher Entwicklungsstadien bei verschiedenen Pflanzen stark unterschiedlich sein kann; -der Verknüpfungsgrad der Monomeren im Suberin stark unterschiedlich sein kann; -Suberin keine 100%ige Barriere für Wasser und Ionen darstellt; -Suberin auch eine Barriere gegen unkontrollierte Gasdiffusion darstellen kann; -der Stofftransport (z.B. Rb-Ionen) durch zusätzliche Suberinmengen verlangsamt werden kann, geringere Suberinmengen den Stofffluss aber nicht signifikant erhöhen wie bei Nitratmangelpflanzen gezeigt wurde; -eine direkte Ableitung der Funktion für den Wasser und Stofftransport aus dem Suberingehalt nicht ohne eine Extraktanalyse der Gewebe möglich ist, und in jedem Fall die Notwendigkeit besteht eine Flächenbelegung mit Suberin oder Wachsen zu ermitteln; -die Variabilität von Pflanzen verschiedenen Genotyps und die Entwicklung vieler verschiedener Anpassungsstrategien zum Schutz vor Stress eine Abschätzung funktioneller Aspekte aus monokausaler Sichtweise (z.B.: Suberingehalt) unmöglich macht. Um der Vielfältigkeit pflanzlicher Strategien gerecht zu werden, ist daher die Integration vieler unterschiedlicher Untersuchungsmethoden in interdisziplinärer Arbeitsweise notwendig. / The aim of this thesis was to examine the chemical composition of suberised apoplastic barriers in roots of higher plants and to relate the obtained results to the function of these barrier tissues for the diffusive transport of ions and water. For the analysis of the chemical composition (mainly suberin and also lignin), the roots were digested enzymatically, and the remaining material was separated in two fractions: one fraction consisting of rhizodermal and hypodermal cell walls (RHCW) and the second one of endodermal cell walls (ECW). Xylem vessels were not analised. Suberin content of the isolated cell walls was determined after transesterification and GC/FID and GC/MS analysis of the monomers. Prior to the chemical analysis, the anatomical structures of the roots were thoroughly examined with light microscopy using histochemical dyes and UV-light as well as scanning electron microscopy. Analysis were performed on the roots of several species: Vicia faba L., Typha glauca Godr., Quercus petraea (Matt.) Liebl., Fagus sylvatica L., Picea abies (L.) Karst., Rhicinus communis L. and Zea mays L. To reveal functional aspects for the transport of water through suberised barriers, potato tuber (Solanum tuberosum L.) skin was used as a model system because it is easy to obtain and consists to more than 20% of suberin. Additionally it is impregnated with aliphatic wax like materials. Experiments with freshly harvested potatoes showed, that suberin and wax content increased in the first days after cutting the tubers from the stele, but no additional cell layers were build. As the content of aliphatic components increased in the peridermal cell walls, the water permeability decreased. Removal of the waxes increased the water permeability of potato periderm to more than a 100 fold from 5.4∙10-11 m s-1 to 8.0∙10-9 m s-1 for the extracted ones. Surprisingly, there was no significant difference in the water permeability of periderms with different suberin content. 28 day old periderms had approx. twice as much as the suberin from freshly harvested ones, but the difference in permeability was smaller than 0.4 units. These results are showing, that suberin is not a water tight barrier as it is discussed in common text books, but to a certain extend it controls the water flux. Suberin amounts and permeabilities where used to estimate hypothetical transport data for root tissues, but the calculated values were only roughly in the range of measured root hydraulic permeabilities obtained from pressure probe experiments. Pressure probe experiments with roots of 8 day old maize seedlings showed, that a higher suberin content in the exodermis significantly reduces the radial water flow through the roots. Even the ion flux in maize roots is reduced, when barrier tissues contained more suberin. These results were obtained in experiments with rubidium-ions as a tracer for potassium-ions in maize seedlings. Apoplastic root barrier tissues, especially the endodermis were strongly enhanced, when the plants were grown under 100 mM NaCl or with 10 µM of the stress-hormone abscisic acid in the hydroponic growth-solution. On the other hand, it was shown that plants of the same species but with different genotypes responded each in a different way to stress situations. For example, whilest maize genotype Pioneer 3906 was able to cope with high salt concentrations, without altering apoplastic barriers, the genotype Across 8023 showed strong enhancement with high suberin amounts of the endodermal cell walls at the same salt concentrations. Lack of nutrients (e.g.: nitrate) in castor bean hydroponic culture leads to low suberin content in the endodermis but the uptake ability for rubidium-ions was not affected at all. Analysis of broad bean root nodules showed the relevance of high suberin amounts as a barrier for water and solutes as well as for gases. Suberin content in nodules were much higher than in the root apoplastic barriers, for that it would concluded, that the uncontrolled diffusion of oxygen into the infected zone of the nodule was inhibited by large amounts of suberin in the nodule endodermis. Another hint to this idea was the analysis of Typha glauca roots and rhizomes, which had more suberin in the exodermis than in the endodermis (in maize it was vice versa), because the submerged tissues are full of air-filled intercellular spaces and the function of higher suberin amounts in the exodermis may be to prevent oxygen loss. The results of this thesis are showing that suberin is a variable material with several functions, not only as a transport barrier for water, ions and gases which controls to a certain extend the fluxes in and out of the apoplast, but also as a barrier against pathogens with high contents of aromatic materials.

The aim of this study was to optimize methane production by investigating hydrothermal pretreatment of sugarcane bagasse impregnated with acid (H2SO4) and alkaline H2O2 using substrate (g kg-1) -inoculum (g kg-1) ratio of 1:2. Batch reactors were realized under mesophilic conditions (37 °C). A central composite design (CCD) involving three factors; temperature (°C), time (min), and chemical compound concentration (H2O2 (%v/v) and H2SO4 (%w/v)) was utilized to optimize hydrothermal pretreatment. Thirty-two hydrothermal pretreatments were conducted according to CCD. H2O2 assisted hydrothermal pretreatment resulted in higher solid recovery (93.13%), higher percent glucan increase (139.52%), and lower lignin recovery (76.48%) in pretreated solid fraction in comparison to H2SO4 impregnated hydrothermal pretreatment. In the latter case, lower solid yield (12.27%), glucan recovery (187.01%) and higher lignin recovery (358.85%) was recorded. Higher COD solubilization (25.20 g L-1), lower total phenolic (content 658.13 ppm), higher sulfate (7240 mg L-1), furfural (925.77-2216.47 mg L-1) and 5-hydroxymehtylfurfural (70.95-970.08 mg L-1) were observed in liquid hydrolysate of H2SO4 assisted hydrothermal pretreatment. While lower COD solubilization (17.75 mg L-1), higher total phenolic content (3005.63 ppm), lower concentration of furfural (0-56.91 mg L-1) and 5-hydroxymethylfurfural (2.56-56.60 mg L-1) was recorded with H2O2 assisted hydrothermal pretreatment. Concerning methane production for H2O2 assisted conditions, 5.59 Nmmol g-1 TVS (2% H2O2) to 13.49 Nmmol g-1 TVS (6% H2O2) was recorded. However, pretreatment with 7.36% H2O2 resulted in 14.43 Nmmol g-1 TVS, which was 118.16% higher comparing to untreated sugarcane bagasse (6.60 Nmmol g-1 TVS). Methanogenic inhibition was recorded for most of the H2SO4.pretreated reactor (1 – 3 %w/v H2SO4). Minimum CH4 production observed was 0.58 Nmmol g-1 TVS in pretreatment O-HSO (2% w/v H2SO4). Acetic acid was the predominant volatile fatty acid observed in digestion process of H2O2 treated batch reactor however was not recorded in H2SO4 treated batch reactors. Microbial community analysis indicated the prevalence of unclassified AUTHM297, Clostridium, and Treponema related genera in H2O2 treated reactors. Genera related aromatic compound degradation were identified and abundant in H2SO4 treated reactors. Methanolinea, Methanobacterium, and Methanosaeta were abundant methanogens in both pretreatments. Hydrogen peroxide assisted hydrothermal pretreatment was verified as a better choice for methane production comparing to sulfuric acid assisted hydrothermal pretreatment primarily on account of higher lignin solubilization, higher glucan recovery, and lower furanic compounds production. / O objetivo deste estudo foi otimizar a produção de metano investigando as condições do pré-tratamento hidrotérmico assistido do bagaço da cana de açúcar sob impregnação de ácido (H2SO4) e álcali (H2O2) utilizando-se a razão substrato (g kg-1) inóculo (g kg-1) de 1:2. Os reatores em batelada foram mantidos em condições mesofílicas (37 ºC). Para otimizar as condições de pré-tratamento hidrotérmico, o design de composto central rotacional (DCCR) foi realizado utilizando três fatores: temperatura (ºC), tempo (min) e concentração do composto químico (H2O2 (%v/v) e H2SO4 (% p/v)). Trinta e dois pré-tratamentos hidrotérmicos foram realizados de acordo com a concepção do DCCR. O pré-tratamento hidrotérmico assistido do bagaço, com H2O2 resultou em maior recuperação de sólidos (93,13%), elevado percentual de glicana (139,52%) e menor recuperação de lignina (76,48%) da fração sólida pré-tratada, se comparada aquele com H2SO4. Nesse último caso, observou-se menor rendimento de sólidos (12,27%) e glucanas (187,01%) e maior recuperação de lignina (358,85%). No líquido hidrolisado do pré-tratamento hidrotérmico assistido do bagaço com H2SO4 foi observada elevada solubilização de DQO (25,20 g L-1), menor teor de fenóis totais (658,13 mg L-1), elevado sulfato (7240 mg L-1), furfural (925,77 - 2216,47 mg L-1 e 5-hidroximetilfurfural (70,95 - 970,08 mg L-1). Enquanto, foi registrado menor solubilização de DQO (17,27 g L-1), maior teor de fenóis totais (3005,63 ppm), e menor concentração de furfural (0 - 56.91 mg L-1), 5-hidroximetilfurfural (2,56 - 50,60 mg L--1 com H2O2. Em relação ao a produção de metano nas condições com H2O2, observou-se 5.59 Nmmol g-1 STV (2%v/v H2O2) a 13.49 Nmmol g-1 STV (6%v/v H2O2). No tratamento com 7.36% de H2O2 observou-se 14,43 Nmmol g-1 STV que foi 118.16% maior se comparado com o bagaço não-tratado (6,60 Nmmol g-1 STV). Inibição metanogênica foi observada na maioria dos reatores pré-tratados com H2SO4 (1 – 3% p/v), e a produção mínima observada foi de 0.58 Nmmol g-1 TVS no pré-tratamento com 2% p/v de H2SO4. Ácido acético foi o principal ácido orgânico volátil observado somente no reatores por tratamento de H2O2. Por meio da A análise da comunidade microbiana, para o domínio Bacteria, foi observado prevalência dos gêneros AUTHM297, Clostridium e Treponema nos reatores cujo substrato foi pré-tratado com H2O2. Gêneros relacionados à degradação de compostos aromáticos foram identificados e estiveram em maior abundância nos reatores cujo substrato foi pré-tratado com H2SO4. Methanolinea, Methanobacterium, e Methanosaeta foram os microrganismos do domínio Archaea mais abundantes e identificados em ambos os pré-tratamentos. O pré-tratamento hidrotérmico assistido com H2O2 foi a melhor opção em relação ao H2SO4, devido a maior solubilização de lignina, maior recuperação de glucano e baixa produção de compostos furânicos.

Neste trabalho, 2 diferentes procedimentosforam utilizados para a extração de lignina a partir do licor negro, caracterizando-se quimicamente as amostras de lignina obtidas em relação a umalignina comercial, visando a aplicação no desenvolvimento de sensores voltamétricos. A análise elementar, espectroscopia na região do infravermelho, análise térmica e caracterização eletroquímica do material mostraram que, tanto a origem da lignina quanto a metodologia de obtenção da mesma, a partir do licor negro, podem fornecer materiais com propriedades químicas distintas, embora possuam comportamento eletroquímico similar. Observou-se, também, que a lignina só pode ser armazenada na forma sólida,devido à oxidação pelo oxigênio dissolvidodurante o tempo em que a solução mãe é armazenada.No entanto, a oxidação dalignina é necessária quando se tem por objetivo o desenvolvimento de sensores voltamétricos, devido a predominância de carbonos sp2na estrutura químicaoxidada, condição em que se obtém maior condutividade. Constatou-se também a necessidade de utilizar um transdutor metálico para o desenvolvimento de eletrodos qumicamente modificados com este material, visto que os eletrodos de carbono modificados com lignina oxidada ou não oxidada não apresentaram atividade eletroquímica. Devido à pequena porcentagem de enxofre existente na estrutura química, a lignina oxidada tende a se organizar pelos grupamentos SH quando na presença de ouro, expondo os grupamentos quinônicos eletroativos. A lignina oxidada ainda foi utilizada no preparo de eletrodo de pasta de carbono com nanopartículas de ouro, na qual a lignina oxidada impregnada no grafite atua como redutor \"in-situ\" do ouro, permitindo o preparo de um sensor voltamétrico versátil, capaz de realizar a determinação de ácido ascórbico, dopamina, nitrito e iodato. No que tange ao comportamento eletroquímico de fármacos e estudos de interação fármaco-DNA, eletrodos de carbono foram modificados com DNAdupla fita com a finalidade de monitorar a interação DNA-Gemcitabina.O fármaco não apresentou atividade eletroquímica tanto na região positiva quanto na região negativa de potencial. A interação do mesmo com o DNA promove a condensação/agregação das duplas fitas do DNA em uma primeiraetapa, seguida da clivagem do nucleosídeo guanosina, formando guanina livre. O comportamento eletroquímico de leflunomida e sulfasalazina, dois fármacos aplicados ao tratamento da artrite reumatóide, foi estudado e mecanismos de oxidação foram propostos para cada fármaco. / The chemical properties of samples of lignin, which were precipitated from black liquor using two different methodologies (precipitation with CO2 and H2SO4), were studied and the results compared to those obtained from a commercial lignin sample in order to prepare voltammetric sensors. The elementary analysis, infrared spectroscopy, thermal analysis and electrochemical characterization of the material demonstrated that both, the source of lignin and the precipitation method from the black liquor, can provide lignin samples with different chemical properties, although the electrochemical behavior of all samples has been the same. Lignin could only be stored in solid form as lignin in the black liquor is slowly and quantitatively oxidized by dissolved oxygen, preventing the extraction procedures. However, the lignin as extracted from black liquor cannot be used to modify solid electrodes due its high resistivity. The previous oxidation of the all material was necessary when the aim was its application on the sensors development. The electrical conductivity in the oxidized lignin was achieved, probably due to the predominance of sp2 hybridized carbon atoms, which improved orbital overlapping in the material. In addition, it was necessary to use a metallic transducer to produce electrodes modified with films of lignin with good electrochemical activity. The films drying time was also important parameter, which suggested a specific organization of lignin macromolecules over the electrode surface. Due to the small percentage of sulfur in the material, the oxidized lignin tended to be organized by the SH groups in the presence of metallic substrates, exposing its electroactive quinone groups. The oxidized lignin was further used to prepare carbon paste electrodes modified with gold nanoparticles, in which the impregnated oxidized lignin on graphite acted as an \"in situ\" reducing agent towards HAuCl4.The resulting composite allowed the preparation of a versatile voltammetric sensor, capable of detecting ascorbic acid, dopamine, nitrite and iodate. Regarding the electrochemical behavior and drug interaction studies DNA-molecule, carbon electrodes were modified with double strand DNA with the purpose of monitoring Gemcitabine-DNA interaction. The drug showed no electrochemical activity both, in the positive and the negative potential. The Gemcitabine-DNA interaction promoted condensation / aggregation of the double strand DNA in a first step, followed by cleavage of the nucleoside guanosine in the form of free guanine. In addition, the electrochemical behavior of sulfasalazine and leflunomide, two pharmacological compounds applied to the treatment of rheumathoid arthritis, were studied and their oxidation mechanisms were proposed.

A parede celular vegetal é composta primariamente de celulose, hemicelulose e lignina. A lignina é o segundo biopolímero mais abundante na biosfera, atrás apenas de celulose, e é formado principalmente a partir da ligação entre três monômeros chamados monolignóis. A formação desses monolignóis é catalisada por pelo menos 10 enzimas membros das famílias gênicas AMP_binding (gene 4CL), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL), que compõem a via de biossíntese dos monolignóis. Até o momento, cerca de 25 sequencias da via de biossíntese de lignina já foram identificados em cana-de-açúcar (Saccharum spp). Ainda, o sequenciamento do genoma desta espécie se mostra uma difícil tarefa devido ao tamanho do genoma e ploidia nuclear. Em virtude da disponibilidade de transcriptomas oriundos de diferentes órgãos de cana-de-açúcar e a importância na identificação correta das sequencias ortólogas, o presente trabalho visa à identificação, anotação e análise filogenética dos genes das famílias gênicas envolvidas na biossíntese de lignina em cana-de-açúcar. Para isso, genes destas famílias gênicas da planta modelo de eudicotiledôneas Arabidopsis thaliana e gramíneas tais como Oryza sativa (arroz), Brachypodium distachyon, Zea mays (milho) e Sorghum bicolor (sorgo) foram identificados, anotados e filogeneticamente categorizados. Em seguida, as sequências de cana-de-açúcar foram identificadas em cinco transcriptomas distintos, anotados e avaliados quanto à identidade e cobertura em relação às proteínas de sorgo. As análises filogenômicas entre cana-de-açúcar e as demais espécies estudadas revelaram 23 sequências candidatas envolvidos na biossíntese de lignina em cana-de-açúcar. / Plant cell wall is composed of cellulose, hemicellulose, and lignin. Lignin is the second most abundant biopolymer on biosphere, after only cellulose, and is formed primarily from three monomers, called monolignols. Monolignol biosyntesis is catalysed by at least 10 enzymes members of AMP_binding (4CL enzyme), p450 (C3H, C4H e F5H), ADH_N (CAD), Epimerase (CCR), Methyltransf_3 (CCoAOMT), Methyltransf_2 (COMT), Transferase (HCT) e Lyase_aromatic (PAL) gene families involved in the biosynthetic pathway of monolignols. So far, there is 25 sugarcane (Saccharum spp.) genes identified involved in the lignin biosynthesis. Sequencing sugarcane\'s genome is still a difficult task due to large genome and ploidy level. Due to the availability of transcriptome data from distinct organs of sugarcane and the importance in the accuracy on the identification of the orthologous sequences, this work aim the identification, annotation and phylogenetic analysis of proteins members of distinct gene family involved in monolignol biosynthesis in sugarcane. To conduct it, we identified and annotated all genes from the model plant eudicots Arabidopsis thaliana and grasses such as Oryza sativa (rice), Brachypodium distachyon, Zea mays (mayze) and Sorghum bicolor (sorghum) and analysed the phylogenic relationship among them. The sugarcane sequences were retrieved and annotated from five distinct transcriptome data and analysed by the identity and coverage against the best sorghum orthologous genes. The phylogenomics analysis revealed a total of 23 sugarcane sequences putatively involved in the biosynthesis of lignin.

A biomassa lignicelulósica tem sua estrutura composta por celulose, hemicelulose e lignina. Dentre essas, a lignina tem se mostrado interessante por ser uma fonte precursora sustentável de fragmentos aromáticos antes obtidos apenas de combustíveis fósseis. Sua estrutura é composta por resíduos de fenilpropanóides p-hidroxibenzeno (H), guaiacil (G) e siringil (S) unidas por ligações C–C e C–O–C em que a ligação β–O–4 é a predominante (mais de 50%). Devido à sua complexidade estrutural e conformacional, a clivagem de suas ligações é pouco seletiva e a caracterização dos fragmentos resultantes é complexa. Uma estratégia comumente empregada para evitar esses desafios é o uso de modelos mais simples. Entretanto, poucas metodologias são reportadas na literatura para a sua síntese e a maioria delas envolve o emprego de halocetonas. O presente trabalho desenvolveu duas novas metodologias promissoras para síntese desses oligômeros, contendo ligação β–O–4 por meio da química de diazo: (a) reação de inserção O–H entre fenol e α–aril diazocetonas, e (b) compostos α–diazo β-cetoéster. Ademais, a utilização de monômeros contendo a função fenol e diazocetona no mesmo anel permitiria a síntese de cadeias de diversos tamanhos em uma única etapa. Como ponto de partida para o estudo, limitou-se à síntese de dímeros, visando entender a reação de inserção O–H. Os produtos desejados foram obtidos em rendimentos de 27–51% após catálise com Cu(hfac)2. Por fim, os modelos de lignina propriamente ditos foram sintetizados após simples adição aldólica e redução em rendimentos globais de 51–78%. Os estudos envolvendo a inserção de fenol em α–diazo β-cetoéster mostraram resultados promissores, corroborando para uma nova estratégia sintética para a obtenção de modelos de lignina. Novos estudos em nosso laboratório estão sendo desenvolvidos para se obter resultados mais conclusivos. / Lignocellulosic biomass is composed of cellulose, hemicellulose and lignin. Among these, the macromolecule lignin has widely received attention as an interesting source of aromatic building blocks to replace those currently obtained from fossil–based fuels. Lignin is composed by units of phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) linked by C–C and C–O bonds which β–O–4 linkages are dominant by more than 50%. Unfortunately, due to the complex structure and 3D arrangement of lignin, it is difficult to control selectivity during its degradation as well as characterize the resulting fragment. A common strategy to avoid these challenges is the use of model substrates; however, few methods exist for the synthesis of these models oligomers. The present work developed two promising routes towards the synthesis of these oligomers containing β–O–4 linkages through use of diazo chemistry: (a) O–H insertion with a phenol and α–aryldiazoketones, and (b) α–diazo β–ketoether compounds. Additionally, monomers containing a phenol and diazoketone unit on the same aromatic ring could allow for the synthesis of a variety of side chains in a single step. We began our studies on the synthesis of dimers via O–H insertion and obtained 27–51% yield of the desired products using catalytic Cu(hfac)2. The mimetic lignin models were synthesized after simple Aldol addition and reduction in 51–78% overall yield. Our work involving this O–H insertion into α–diazo β–ketoether is a promising strategy to rapidly synthesize lignin model oligomers and is an active area of research within our lab.