Phenolic residues which accumulate in the environment as a result of agro-industrial practices has resulted in the need to find and use Eco-Friendly techniques, rather than the traditional methods of burning or burying this kind of waste. Bioremediation and bioconversion are attractive alternatives using whole cell or enzyme-based systems. The aims of this project were to isolate and uses thermophilic Actinomycetes, which produce thermo-tolerant oxidoreductase enzymes, which can be used to bioconvert a model industrial phenolic waste commonly genersated in the wine-making industry of South Africa. Current research in bioconversion and bioremediation focuses on mesophilic microbes in that their enzymes can catalyse reactions at higher temperatures without affecting its activity and lower contamination levels. Three novel Actinomycete isolates were isolated (RU-A0l , RU-A03 and RU-A06) from a compost site and characterized using a combination of conventional identification techniques and 16S rDNA methodology to identity the three isolates. All three isolates belong to the Streptomyces clade. In addition, five known Actinomycetes were selected from an internation culture collection and also screened for oxidoreductase activity in comparision to the three novel isolates. Although the five isolates were selected based on their ability to produce oxidoreductase enzymes, unexpectedly, no activity was detected. Screening assays for peroxidase, polyphenol oxidase and laccase on RU-AO 1, RU-A03 and RU-A06, showed that all three isolated produced peroxidases and peroxidases but no laccase. Substrate specificity studies revealed that the most suitable substrates to determine peroxidase and polyphenol oxidase activity on these isolates were catechol for polyphenol oxidase, 2,4-dichlorophenol for peroxidases and veratryl alcohol for lignin peroxidases. Previous studies have indicated that peroxidases and polyphenol oxidases are produced in Actinomycetes during the primary stage of growth. This was the case with RU-AOI , RU-A03 and RU-A06. Growth rates were higher that other Actinomycetes, with maxImum biomass being reached at 36 hours for the isolates RU-AOI and RU-A06 and 48 hours for isolate RUA03. pH studies showed that the three isolates were adaptable and could grow over a broad pH range. Catabolism studies of phenolic model compounds showed that the three isolates were capable of catabolizing the model phenolic compounds within a period of 24 hours. Further studies were carried out to determine the effect of these microbes and their enzymes in whole cell and enzyme-based systems on a model phenolic waste, graoe waste consisting of compressed grape skins, pips and stalks. Whole cell studies showed that the isolates were capable of bioconverting the waste at a maximum concentration of 30% grape waste (vol:vol). Peroxidase and polyphenol oxidase activity increased indicating induction of these enzymes in the presence of phenolic compounds, with a maximum increase of up to 15.9 fold increase in extracellular lignin peroxidase activity in RU-AO1. HPLC and phenolic determination assays indicated that bioconversion of the phenolic grape waste had occurred in the presence of the three isolates. Attempts were made to isolate and identify a peroxidase or phenol oxidase gene from one the isolates. As bacteria, Actinomycetes are amendable to gene manipulation making them suitable candidates for methods such as site directed evolution in comparison to fungi. Two clones were selected for sequencing based on positive activity results when assayed for peroxidase activity. However the resultant sequences did not identify a functional gene sequence. Southern Blotting was then carried out to determine the nature of the peroxidase gene. Previous studies have been focused on the catalase-peroxidase gene (CalC gene) found Actinomycetes and other bacteria. A probe was developed from the CalC gene. No hybridization occurred with any of the enzyme restricted DNA from the three isolates. The implications of these results are that the peroxidase genets in the three isolates are in fact lignin peroxidase in nature. This project has the potential in the bioconversion of phenolic wastes and is the first description of the use of thermophilic Actinomycetes in the bioconversion of an industrial phenolic waste.

As a significant component of lignocellulosic biomass, lignin represents a potential source of value-added aromatic chemicals. In this thesis, catalytic systems with earth-abundant metal catalysts such as molybdenum(VI) and hypervalent iodine complexes were developed to selectively break down lignin models into lower molecular weight chemicals under mild conditions. Due to the complexity of lignin, simple lignin model substrates (A to E), representing common linkages in lignin, were used to investigate the catalytic activity/selectivity of these catalysts. With the molybdenum catalysts [7– 11]/SPC/Adogen®464 system (SPC = sodium percarbonate), oxidation of simple β-1 model compound A in acetonitrile showed primarily C-H bond cleavage to form the ketone product, benzoin methyl ether, whereas the Cα-Cβ bond cleavage product, methyl benzoate, was obtained by switching the reaction solvent to benzonitrile. Preference for generating the Cα-Cβ bond cleavage product, i.e. benzaldehyde, can also be achieved with other early to middle transition metal catalysts using H2O2(aq) as the terminal oxidant. Stoichiometric amounts of hypervalent iodine/Lewis acid systems [20a-c] were able to selectively cleave Cα-Cβ bonds to aldehydes with both simple β-1 model compound A and β-O-4 model compound C. In contrast, other lignin model compounds with different linkages were unable to be oxidized to a great extent using these Mo- or iodine-based complexes. The catalytic activity and selectivity of the reported vanadium complexes, copper salts and non-metal system 1-5 on non-volatile organosolv (NVO) lignin was investigated under basic condition. Details of the depolymerisation of lignin were determined by using Gel Permeation Chromatography (GPC) and the two-dimensional NMR technique, quantitative HSQC (q-HSQC) spectroscopy. Vanadium [2] and copper systems were found to be the most active for depolymerization of NVO lignin.

Three dilignol rhamnosides were isolated in 0.15 to 0.40% yield from the ethyl acetate solubles of a methyl alcohol extract of western red cedar (Thuja plicata Donn) leaves using silicic acid and Sephadex LH-20 pressure column chromatography. One of the three dilignol rhamnosides was characterized as l-(3'-methoxy-4'-hydroxyphenyl)-2-0-l"-[2"-hydroxy-4"-(propane-3"'-α-L-rhamnoside)phenyl]-propane-l,3 diol, using NMR and chemical degradation in conjunction with mass spectral techniques on the compound and its derivatives. The other dilignol rhamnosides were not completely characterized, but were shown to be chemically related to the dilignol identified. Based upon NMR, chemical and mass spectral data, the uncharacterized dilignol rhamnosides are speculated to contain phenylcoumaran and guaiacyl benzdioxane structures. The characterized dilignol rhamnoside represents the first reported occurrence of a free dilignol glycoside in plant tissues. The unusual α-L-rhamnose moiety of the dilignol occurs in a previously unreported linkage to the n-propyl hydroxyl group uncommon in lignin. The rhamnoside also displays the previously unreported guaiacylglycerol-catechol-β-aryl ether structure rather than the commonly observed guaiacylglycerol-guaiacyl-β-aryl ether structure. A new combustion-absorption technique was developed and validated which allows high efficiency evaluation of low activity radioactive products separated on thin layer cellulose chromatography plates. The technique was applied to an analysis of the anabolic products of an infusion feeding of U- (14)C--L-phenylalanine to western red cedar leaves. Facile imbibition of U-(14) C-L-phenylalanine occurs within ten hours. Maximum incorporation of 0.30%. and 0.40% of the available radioactivity occurs in the characterized dilignol rhamnoside, and its suspected phenylcoumaran homolog respectively, at the three to five hour period of the infusion feeding. The incorporation results indicate the participation of the dilignol rhamnosides in aromatic metabolism in the leaves of western red cedar. This feeding experiment is preliminary to future detailed biosynthetic studies in the leaf tissue. The combustion-absorption technique is limited to combustible sample weights of 7 mg. / Forestry, Faculty of / Graduate

As global energy demands increase, there is a requirement to decrease our dependency on fossil fuels due to their finite supply and negative environmental impacts. Alternative sources of energy are required that offer sustainability, reduced cost and environmental benefits. Second generation biofuels remove the ‘food vs fuel’ drawback of the first generation. They utilise lignocellulosic biomass, providing cheap and abundant starting materials for energy production. The major biotechnological challenge associated with lignocellulosic processing is the natural recalcitrance of the substrate to sugar conversion (saccharification). This recalcitrance is largely associated with lignin, an aromatic heteropolymer that encases cellulose. To improve bioethanol yields, there is a need for cost effective and environmentally friendly pretreatment methods that can remove lignin. An enzymatic pretreatment strategy was investigated using laccase from the fungus Trametes versicolor (TvL). Expansion of the laccase substrate range towards non-phenolic substrates was explored by screening of a panel of synthetic and naturally derived phenolic compounds as potential redox mediators with laccase. Both groups enabled decolourisation of the recalcitrant dye (RB-5) to varying degrees, which laccase alone was unable to achieve. In the case veratryl alcohol, a lignin model substrate, synthetic compounds 1-HBT, ABTS and violuric acid proved effective laccase mediators. On this basis, TvL with 1-HBT was selected as the most successful laccase mediator system (LMS) and was further explored as a biomass pretreatment method. The effects of LMS treatments towards the saccharification of acid hydrolysed wheat straw were extensively investigated. Optimisation revealed that when both TvL and a TvL LMS of synthetic origin (1-HBT, violuric acid) were applied, saccharification was improved. The observed increase in glucose release was only detected when a second lignin removing technique was applied in succession. Both alkaline-peroxide and organosolv extractions were successfully used to demonstrate the role of laccase/LMS in saccharification improvement, with improvements reaching up to 44.6%. The effect was further demonstrated with additional substrates (corn and sorghum stover) and additional laccases (Pleurotus ostreatus, Agaricus bisporus and Rhus vernicifera). Further studies using β-O-4 structures, py-GC/MS and FTIR analyses provide further information on the structural actions of an LMS towards lignin, including strong evidence for Cα-Cβ cleavage and Cα hydroxyl oxidation mechanisms.

Changes in wood chemistry could have significant impact on both environmental and economic aspects of the pulp and paper industry. Consequently, a considerable amount of effort has been devoted to altering lignin content and/or modifing lignin monomer composition, a cell wall component whose removal is a major part of the chemical pulping process. Analysis of poplar transformed with a cinnamate 4-hydroxylase (C4H):ferulate5-hydroxylase (F5H) construct confirmed significant increases in the mole percent syringyl lignin in transgenic lines. Further, this study demonstrated significant increases in pulping efficiency from greenhouse grown transgenic trees. Compared to wild-type pulp, decreases of 23 kappa units (residual lignin) and increases of >20 ISO brightness units were observed in tree lines exhibiting high syringyl monomer concentrations (93.5% mol S). These changes were associated with no significant change in total lignin content or observed phenotypic differences in the trees. Additionally, pulp yields were not affected by the enhanced removal of lignin.. Furthermore, transgenic lines exhibit reduced fibre coarseness and increased cellulose viscosity. These results suggest that C4H-F5H transformed trees could be used to produce pulp for paper with substantially less severe delignification conditions (lower chemical loading or less energy), and that the pulp produced is of comparable quality to that of the wild-type poplar. Consequently, the ecological footprint left on the environment, measured by the amount of deleterious pulping by-products released into the environment may be significantly reduced. / Forestry, Faculty of / Graduate

Granular water-insoluble lignins were isolated from a series of aqueous organic solvent (organosols) cooks designed for pulping/sac-charification of Douglas-fir sawdust. Among the factors affecting yield and characteristics of the isolated organosolv lignins, only cooking time (5-20 minutes) and concentration of acid catalyst (0-0. IN HC1) were investigated as cooking variables. Cooking temperature (200°C) and solvent composition (acetone/water=60:40) were held constant. It was learned that the acidified organosolv cooking system is far more efficient in delignification and saccharification than a-queous acid hydrolysis under identical conditions. In organosolv cooking, simultaneous dissolution of lignin and sugars occurs in the cooking liquor, allowing continued and total dissolution of the wood constituents. In the present study, only the water-insoluble lignin fraction was isolated and analyzed. An almost quantitative recovery of the precipitable lignin was accomplished by evaporation of the organic solvent from the spent liquor, followed by removal of sugars dissolved in the aqueous solution and reprecipitation of the crude lignin into water. To eliminate the interference from hydrogen bonding and unconjugated carbonyl group in the isolated organosolv lignins, acetylation or reduction was carried out before the lignin samples were characterized. The resulting lignin samples were found to be completely free of cabohydrate contaminants. Both cooking time and acid concentration were found to have a profound effect on the yield of lignin fractions, and chemical and macromolecular properties of the lignin molecules due to two competing reactions, hydrolytic depolymerization and recondensation. These reactions take place simultaneously in the cooking liquor during organosolv cooking. The balance between these two reactions is believed to be responsible for not only the content of functional groups, as revealed by nuclear magnetic resonance, infrared and ultraviolet spectral analyses, but also the size of lignin molecules, as measured by gel permeation chromatographic and scanning electron microscopic analyses of the isolated organosolv lignins. The functional group contents, determined by elemental and spectral analyses, were found to be 0.86-0.97 methoxyl, 0.20-0.49 aromatic hydroxyl and 0.68-0.99 aliphatic hydroxyl groups per C₉-unit of the organosolv lignin molecules. It was also noted that 63-68% of aromatic nuclei have condensed forms with carbon-carbon linkages, having only two hydrogens on each guaiacyl nucleus. The organosolv lignins were found to have much lower molecular weights than those of protoligniri in wood. Typical values of the number average molecular weight of the isolated lignins ranged from 823 to 1,144. The low molecular weight values are due to degradation reactions during the cooking by cleavage of aryl-alkyl linkages of lignin molecules. The particle size of the spherical precipitated lignins ranged from 25 to 500 nm. / Forestry, Faculty of / Graduate

Lignin is the most abundant natural aromatic polymer on the earth. In this work, lignin properties were studied in order to explore its use as a low cost carbon precursor for graphene production. Initial studies focused on kinetics of lignin pyrolysis using ‘Kissinger method’ and ‘ASTM E 1641’ using thermogravimetry. The values obtained for kinetic parameters varied for the two methods and activation energy increased with increase in lignin purity. Lignin was solvent fractionated, using three organic solvents to extract the high molecular weight fraction suitable for the production of highly ordered graphene nano platelets. Acetone and Methanol were successful in sequential fractionation. Finally, polycrystalline graphene was produced using Protobind 1000 and lignosulfonate lignins by carbonization. The acid purified graphene had relatively less catalytic material remaining, and nitric acid purification was successful compared to HCl purification. However, HNO3 purification introduced minor structural damages to the sample.

No description available.

No description available.

No description available.