Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Lignocellulosic material is composed of three major macromolecule components i.e., cellulose, hemicelluloses and lignin. These components are chemically associated and directly linked to each other through covalent bonding which is scientifically denoted as lignin-carbohydrate complexes (LCCs) and their interaction is fundamentally important as to understand wood formation and reactivity during chemical and biological processing e.g. pulping and enzymatic hydrolysis. The association of lignin with polysaccharides (covalent linkages) has been surrounded by contradictions and controversy in several wood chemistry studies. These linkages exist in lignocellulosic materials from wood to herbaceous plants. In woody plants, they consist of ester and ether linkages through sugar hydroxyl to α-carbonyl of phenyl-propane unit on lignin. However, in herbaceous plants ferulic and p-coumaric acids are esterified to hemicelluloses and lignin respectively. In recent studies, the existence of the bonds has been shown by applying indirect analysis strategies which resulted to low yields and contaminations. The general aim of the current study was to isolate and fractionate LCCs from raw lignocellulosic materials (E. grandis and sugarcane bagasse) and corresponding processed materials (chemical pulps and water-insoluble residues (WIS)) in order to determine the chemical structure of the residual lignin associated with polysaccharides and how they affected industrial processing. The objective of the study is to compile a document that when the development of pulping and bio-ethanol bio-refinery will greatly depends on the detailed wood chemistry on how the components interact with each before and after hemicelluloses pre-extraction prior to pulping and steam explosion pre-treatment prior to enzymatic hydrolysis. The current study was focusing on understanding the effect LCCs isolated from two different industrial processing methods, i.e. pulping and enzymatic hydrolysis (EH). There were two lignocelluloses feedstocks used for pulping, i.e. Eucalyptus grandis and sugarcane bagasse whereas sugarcane bagasse was the only feedstock used for enzymatic hydrolysis. Hemicelluloses pre-extracted (mild alkali or dilute acid and autohydrolysis for sugarcane bagasse) pulps of Kraft or soda AQ from E. grandis and sugarcane bagasse were used to understand the effect of xylan pre-extraction prior to pulping on lignin-carbohydrate complexes has not been reported to the best knowledge of the primary author. Also prior to EH the material was subjected to two different treatment methods, i.e. steam explosion and ionic liquid fractionation in varying conditions. The study illustrated the types of extracted and fractionated LCCs from hemicelluloses pre-extracted pulps and WIS in comparison to the non-extracted pulps and reports from the literature. Lignin-carbohydrate complexes (LCCs) were isolated and fractionated by an inorganic method which yielded reasonable quantification quantities and no contamination and low yields for the hardwood compared to reports of using an enzymatic method. To the best knowledge of the authors, no work has been done on WIS material. The lignocelluloses were subjected to ball milling which was followed by a sequence of inorganic solvents swelling and dissolution into 2 fractions i.e. glucan-lignin and xylan-lignin-glucan. Characterisation of the isolated LCCs was made using a variety of analytical tools such as FTIR-PCA, HPLC, GPC and GC-MS. LCCs were evident when FTIR and HPLC studies were conducted. Residual lignin isolated from the lignocelluloses was assumed to be chemically bonded to carbohydrates and mostly to xylan. Approximately 60% and 30% of the lignin was linked to xylan while for the second and first fractions respectively. It is reported that lignin associated with xylan is more resistant and reduce the delignification process than when linked to glucan that is easily hydrolysable. With the FTIR and GPC analyses of LCC fractions, it was evident that the ester bonds of LCCs were destroyed through pre-extraction and pre-treatment, where this resulted to more cellulose being more accessible to alkaline pulping and enzymatic hydrolysis respectively. The linkages were either partially broken down or completely destroyed leading to significant changes of chemical structures. The polydispersity of the LCCs assisted in determining the structure of lignin, either existing as monolignols on the surfaces of fibres or a as complex two or three-dimensional structure that is linked to carbohydrates as the Mw increased or decreased. In general, these findings may have an important implication for the overall efficiency on bio-refinery. The molecular weights (Mw) of the extracted LCCs were measured by gel permeation chromatography. From the chromatograms, it was observed that the materials that were subjected to pre-processing prior to further processing, the Mw shifted to lower Mws regions. It was found that LCCs isolated from mild alkali pre-extracted pulps had high lignin syringyl to guaiacyl lignin contents than LCCs isolated from dilute acid pre-extracted pulps. High syringyl/guaiacyl ratio (S/G ratio) was an indication of low lignin content as a result of processing which will result to high product yields after downstream processing. The 5 average S/G ratio for the pulps from E. grandis and sugarcane bagasse was ranging between 1.1 to 19.01 and 1.4 to 18.16 respectively, while for the WIS-material generated from ionic liquid fractionated and steam exploded materials ranged from 3.29 to 9.27 and 3.5 to 13.3 respectively. The S/G ratios of the LCCs extracted from E. grandis and sugarcane bagasse pulps ranged from 0.42 to 2.39 and 0.041 to 0.31 was respectively while for the LCCs extracted from water-insoluble-solids (WIS) material generated from steam exploded material was from 4.87 to 10.40. The determination of S/G ratio is recommended for the LCC extraction and characterisation study as an evaluation of residual lignin in processed materials such as pulps and WIS. The obtained saccharifications were low, possibly due to the severity of the steam explosion pre-treatment and ionic liquid fractionation conditions which resulted on high accumulation of acetic acid and increased in cellulose crystallinity respectively. From quantitative analysis of the LCCs perspective it could be concluded that free lignin was present in mild alkali pre-extracted pulps than for the dilute acid pre-extracted pulps. / AFRIKAANSE OPSOMMING: Cellulose materiaal is saamgestel uit drie groot makromolekule komponente naamlik, sellulose, hemisellulose en lignien. Hierdie komponente is chemies verwante en direk met mekaar verbind deur kovalente binding wat wetenskaplik aangedui as lignien-koolhidraat komplekse (LCCs) en hul interaksie is fundamenteel belangrik as hout vorming en reaktiwiteit tydens chemiese en biologiese verwerking bv om te verstaan verpulping en ensiematiese hidrolise. Die vereniging van lignien met polisakkariede (kovalente verbindings) is omring deur teenstrydighede en omstredenheid in verskeie hout chemie studies. Hierdie skakeling bestaan in cellulose materiaal uit hout te kruidagtige plante. In houtagtige plante, hulle bestaan uit ester en eter bindings deur suiker hidroksiel te α-karboniel van feniel-propaan eenheid op lignien. Maar in kruidagtige plante ferulic en p-coumaric sure veresterd te hemisellulose en lignien onderskeidelik. In onlangse studies, het die bestaan van die bande is getoon deur die toepassing van indirekte analise strategieë wat gelei tot lae opbrengste en kontaminasie. Die algemene doel van die huidige studie was om te isoleer en fraksioneer LCCs van rou cellulose materiaal (E. grandis en suikerriet bagasse) en die ooreenstemmende verwerkte materiaal (chemiese pulp en water-oplosbare residue (WIS)) ten einde die chemiese struktuur van die te bepaal oorblywende lignien wat verband hou met polisakkariede en hoe hulle geaffekteerde industriële verwerking. Die doel van die studie is 'n dokument op te stel dat wanneer die ontwikkeling van verpulping en bio-etanol bio-raffinadery sal grootliks afhang van die gedetailleerde hout chemie oor hoe om die komponente met mekaar voor en na hemisellulose pre-onttrekking voor verpulping en stoom ontploffing pre-behandeling voor ensiematiese hidrolise. Die huidige studie was die fokus op die begrip van die effek LCCs geïsoleerd van twee verskillende industriële verwerking, maw verpulping en ensiematiese hidrolise (EH). Daar was twee lignocelluloses voerstowwe gebruik vir verpulping, dws Eucalyptus grandis en suikerriet bagasse terwyl suikerriet bagasse was die enigste grondstof gebruik vir ensiematiese hidrolise. Hemisellulose pre-onttrek (ligte alkali of verdunde suur en autohydrolysis vir suikerriet bagasse) pulp van Kraft of soda AQ van E. grandis en suikerriet bagasse is gebruik om die effek van Xylan pre-onttrekking te voor verstaan verpulping op lignien-koolhidraat komplekse het nie aan die berig is beste kennis van die primêre outeur. Ook voor EH die materiaal is onderworpe aan twee verskillende behandeling metodes, naamlik stoom ontploffing en ioniese vloeistof fraksionering in wisselende toestande. Die studie geïllustreer die tipes onttrek en gefractioneerd LCCs van hemisellulose pre-onttrek pulp en WIS in vergelyking met die nie-onttrek pulp en verslae van die literatuur. Lignien-koolhidraat komplekse (LCCs) is geïsoleer en gefraksioneer deur 'n anorganiese metode wat redelike kwantifisering hoeveelhede en geen besoedeling en lae opbrengste opgelewer vir die hardehout vergelyking met verslae van die gebruik van 'n ensiematiese metode. Na die beste kennis van die skrywers, het geen werk op WIS materiaal gedoen. Die lignocelluloses is onderworpe aan die bal maal wat gevolg is deur 'n reeks van anorganiese oplosmiddels swelling en ontbinding in 2 breuke dws glucan-lignien en Xylan-lignien-glucan. Karakterisering van die geïsoleerde LCCs is gemaak met behulp van 'n verskeidenheid van analitiese gereedskap soos FTIR-PCA, HPLC, GPC en GC-MS. LCCs was duidelik wanneer FTIR en HPLC studies is uitgevoer. Residuele lignien geïsoleerd van die lignocelluloses is aanvaar moet word chemies gebind aan koolhidrate en meestal te xylan. Ongeveer 60% en 30% van die lignien is gekoppel aan xylan terwyl dit vir die tweede en eerste breuke onderskeidelik. Dit is gerapporteer dat lignien wat verband hou met Xylan is meer bestand en die delignification proses as wanneer gekoppel aan glucane wat maklik hidroliseerbare verminder. Met die FTIR en GPC ontledings van LCC breuke, was dit duidelik dat die ester bande van LCCs is deur pre-ontginning en pre-behandeling, waar dit gelei tot meer sellulose om meer toeganklik te alkaliese verpulping en ensiematiese hidrolise onderskeidelik vernietig. Die skakeling is óf gedeeltelik afgebreek of heeltemal vernietig lei tot beduidende veranderinge van chemiese strukture. Die polydispersity van die LCCs bygestaan in die bepaling van die struktuur van lignien, hetsy bestaande as monolignols op die oppervlak van die vesel of 'n as komplekse twee of drie-dimensionele struktuur wat gekoppel is aan koolhidrate as die Mw vermeerder of verminder. In die algemeen, kan hierdie bevindinge het 'n belangrike implikasie vir die algehele doeltreffendheid op bio-raffinadery. Die molekulêre gewigte (Mw) die onttrek LCCs gemeet deur gelpermeasie- chromatografie. Van die chromatograms, was dit opgemerk dat die materiaal wat blootgestel is aan die pre-verwerking voor verdere verwerking, die Mw verskuif MWS streke te verlaag. Daar is gevind dat LCCs geïsoleerd van ligte alkali pre-onttrek pulp het hoë lignien syringyl lignien inhoud as LCCs geïsoleerd van verdunde suur vooraf onttrek pulp te guaiacyl. Hoë syringyl / guaiacyl verhouding (S/G-verhouding) was 'n aanduiding van 'n lae lignien inhoud as 'n resultaat van verwerking wat sal lei tot 'n hoë produk opbrengste ná stroomaf verwerking. Die gemiddelde S/G-verhouding vir die pulp van E. grandis en suikerriet bagasse was wat wissel tussen 1,1-19,01 en 1,4-18,16 onderskeidelik, terwyl dit vir die WIS-materiaal gegenereer uit ioniese vloeistof gefraksioneer en stoom ontplof materiaal het gewissel 3,29-9,27 en 3.5 13,3 onderskeidelik. Die S/G verhoudings van die LCCs onttrek uit E. grandis en suikerriet bagasse pulp gewissel 0,42-2,39 en ,041-,31 was onderskeidelik terwyl dit vir die LCCs onttrek uit water-oplosbare-vastestowwe (WIS) materiaal gegenereer uit stoom ontplof materiaal was van 4,87-10,40. Die bepaling van S/G-verhouding word aanbeveel vir die LCC ontginning en karakterisering studie as 'n evaluering van die oorblywende lignien in verwerkte materiaal soos pulp en WIS. Die verkry saccharifications was laag, moontlik as gevolg van die erns van die stoom ontploffing pre-behandeling en ioniese vloeistof fraksionering voorwaardes wat gelei op 'n hoë opeenhoping van asynsuur en vermeerder in sellulose kristalliniteit.

>Magister Scientiae - MSc / The negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate. / National Research Foundation

Electrically conductive fabrics are one of the major components of smart textile that attracts a lot of attention by the energy, medical, sports and military industry. The principal contributors to the conductivity of the smart textiles are the intrinsic properties of the fiber, functionalization by the addition of conductive particles and the architecture of fibers. In this study, intrinsic properties of non-woven carbon fabric derived from a novel linear lignin, poly-(caffeyl alcohol) (PCFA) discovered in the seeds of the vanilla orchid (Vanilla planifolia) was investigated. In contrast to all known lignins which comprise of polyaromatic networks, the PCFA lignin is a linear polymer. The non-woven fabric was prepared using electrospinning technique, which follows by stabilization and carbonization steps. Results from Raman spectroscopy indicate higher graphitic structure for PCFA carbon as compared to the Kraft lignin, as seen from G/D ratios of 1.92 vs 1.15 which was supported by a high percentage of graphitic (C-C) bond observed from X-ray photoelectron spectroscopy (XPS). Moreover, from the XRD and TEM a larger crystal size (Lc=12.2 nm) for the PCFA fiber was obtained which correlates to the higher modulus and conductivity of the fiber. These plant-sourced carbon fabrics have a valuable impact on zero carbon footprint materials. In order to improve the strength and flexibility of the non-woven carbon fabric, lignin was blended with the synthetic polymer Poly acrylonitrile (PAN) in different concertation, resulting in electrical conductivity up to (7.7 S/cm) on blend composition which is enough for sensing and EMI shielding applications. Next, the design of experiments approach was used to identify the contribution of the carbonization parameters on the conductivity of the fabrics and architecture of the fibers, results show carbonization temperature as the major contributing factor to the conductivity of non-woven fabric. Finally, a manufacturing procedure was develop inspired by the architecture of plant fibers to induce controlled porosity either on the skin or core of fibers which results in stiffness and flexibility in the fibers. Coaxial Electrospinning and Physical foaming (CO2 foaming) techniques were utilized to create the hierarchical fiber architecture. Finite Element model was developed to design for mechanical properties of the bioinspired fiber mesh. Results show the polymers contributes less in a coaxial design as compared to the individual fibers for mechanical properties. This manufacturing method can use for hierarchical functionalization of fibers by adding conductive nanoparticles at different levels of fiber cross-section utilized for sensing applications in sports and medical industry.

Paper and paperboard are the major constituents found in US landfills. Typically paper consists of 79% to 98% of lignocellulose which is considered to be the most abundant source of natural carbon on earth. Lignocellulose decomposition depends on the association of biodegradable cellulose and hemicellulose with lignin. Lignin is a recalcitrant material which hinders cellulose degradation in conventional landfills. Because of this property of lignin cellulose to lignin ratio (C/L) is a common landfill stabilization parameter. Refuse degradation in landfills is a microbiological process and is highly dependent on temperature, moisture, and pH. Bioreactor landfills are designed to enhance biodegradation of refuse by providing favorable conditions for microorganisms. Effect of elevated temperature and moisture on possibility of lignin degradation is studied in this work. Synthetic and newspaper lignin were preheated and then inoculated with anaerobically digested sludge. Newspaper in distilled water exposed to 95°C for 48 hours released 8 times more of solubilized lignin then non preheated newspaper. Moreover lignin monomers were detected as a result of 95°C pretreatment indicating the positive effect of high temperature on the providing lignin in more bioavailable form for microbes. Digested sludge inocula was found to be capable of lignin monomers degradation as well as low but significant mineralization of synthetic lignin with approximately 6% of carbon originated from lignin mineralized into methane and carbon dioxide. An exponentially increasing trend for lignin monomers solubilization as a function of temperature was observed for three types of substrate, synthetic lignin, cardboard, and newspaper with the highest rate of solubilization for newspaper. Results of this study suggest that some lignin degradation can occur at conditions typical for bioreactor landfills. / Master of Science

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There is growing need for technologies to displace traditional petroleum resources. Towards this goal, lignocellulosic biomass is seen as a potential renewable resource for the production of fuels and commodity chemicals. One of the most difficult components of lignocellulose to process is lignin, which is a complex, amorphous aromatic polymer that acts as one of the structural components in plants. Ionic liquids are a class of compounds that are composed completely of anions and cations that, in some cases, can completely dissolve lignocellulosic biomass. The research performed for this dissertation aims to advance the technologies of lignocellulose processing through effective depolymerization of lignin in ionic liquids. Lignin fragments from this depolymerization could be used as a feedstock for further processing into aromatic commodity chemicals or polymers. Additionally, by removing lignin, biomass becomes much more accessible to enzymatic or chemical saccharification as a step towards fermentation into ethanol or other fuels. Both base and acid catalyzed methods were explored, although the base promoted depolymerization of lignin in ionic liquids did not show much promise, as the reaction was never shown to be catalytic. Acidic routes towards lignin depolymerization were more successful. Using the acidic ionic liquid 1-H-3-methylimiazolium chloride, the ether linkages in lignin model compounds could be hydrolyzed with high yields. This technology was also applicable to the whole lignin macromolecule. The mechanisms of this reaction, as well as the effects on lignin were explored with various neutral and acidic ionic liquids, using HPLC, GPC, NMR, FT-IR, and mass spectrometry for analysis of samples. To demonstrate the applications of this technique, pine wood was treated with the acidic ionic liquids to open the structure of the wood to enzymatic saccharification through the removal of lignin and hemicellulose. / text

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