Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl esterase from Aureobasidium pullulans were set as the primary objectives of this study. A further objective was to investigate a possible co-operative effect with other selected lignocellulolytic enzymes on substrates relevant to industry. In a comprehensive review, feruloyl esterases from various micro-organisms were compared both functionally and with regard to their primary structure, where applicable. Feruloyl esterases show intriguing differences in substrate specificity and sequence structure. Enzymes that are closely related regarding their amino acid sequence exhibit different substrate specificities. Sequence similarities can be found with a range of other enzyme families, including serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl hydrolases and xylanases. More data on the three dimensional structure of feruloyl esterases as well as an examination of all available feruloyl esterases with the same substrates is necessary before structure-function relationships can be established and before the feruloyl esterases can be organized into discrete families based on ancestral origins. The highest production levels of feruloyl esterase by A. pullulans are achieved when grown on birchwood xylan. Expression was not repressed when glucose or xylose was present in the medium. However, free ferulic acid supplemented to the medium affected fungal growth and therefore did not increase feruloyl esterase activity. It is also suggested that the synthesis of feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as naphthol AS-D chloroacetate as substrates. Feruloyl esterase from A. pullulans was purified to homogeneity using ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic interaction and ultimately gel filtration chromatography. With a molecular weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported to date. Kinetic data was produced using both synthetic and natural substrates. A. pullulans feruloyl esterase shows properties similar to other fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal sequence of A. pullulans feruloyl esterase was identified, but no similarities to known enzyme families were found. Peptide mass mapping did not reveal structural information. In an effort to evaluate the significance of feruloyl esterase from A. pullulans in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse were selectively treated using feruloyl esterase and hemicellulolytic enzymes. The enzymatic degradation reaction was monitored using microdialysis sampling, anion exchange chromatography, online desalting and mass spectrometry. It has been shown, that feruloyl esterase activity together with xylanase activity releases monosaccharides from both substrates. Sugars of higher degree of polymerisation were not released, giving evidence for the recalcitrance of the material. The fibre architecture of the substrates was apparently not accessible to the enzymes and therefore complete hydrolysis was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant is, bestaan. Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt van funksie en primêre struktuur omvattend met mekaar vergelyk, waar toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur van feruloïel esterase kan waargeneem word. Ensieme wat nou aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase, lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n analise van al die beskikbare feruloïel esterase met dieselfde substrate is nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat die feruloïel esterases in eie families op die grond van huloorsprong georganiseer kan word. Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3- naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is die ensiem die grootste van die feruloïel esterases tot dusver beskryf. Kinetiese data is met behulp van sintetiese en natuurlike substrate geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting gee nie. Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel esterase en lignosellulitiese ensieme behandel om die belang van feruloïel esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie, oplyn ontsouting en massaspektrometrie gemonitor. Wanneer die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele hidroliese verhinder.

No description available.

Environmental concerns about urban air quality, global climate change, energy security and economic considerations motivate a growing interest in alternative fuels for the transportation sector. Ethanol, a fermentation-derived fuel, can be produced by bioconversion of renewable materials, such as wood, grass, and waste. Combustion of ethanol fuel, in both neat and blended form, can improve the engine efficiency, and lower the emission of CO, NO[subscript x], and volatile organic compounds (VOC), hence reducing the urban ozone level. Moreover, enhanced agricultural activities for production and collection of lignocellulosic feedstocks and industrial developments for production of ethanol will help the economic growth by creating new jobs and new income sources. Bioconversion of lignocellulosic feedstocks into ethanol requires a pretreatment process to increase the digestibility of cellulose by cellulolytic enzymes. The dilute-sulfuric acid pretreatment can hydrolyze hemicelluloses (xylan), disrupt lignin structure, and increase the yield of ethanol production from fermentation of monomeric units of cellulose (glucose). In this study, herbaceous (corn stover and switchgrass) and woody (poplar chips) feedstocks were pretreated with dilute sulfuric acid (0.6, 0.9, and 1.2% w/w) in a batch reactor at relatively high temperatures (140, 160 and 180��C). A unifying kinetic model including reaction time, temperature and acid concentration was developed, and pertinent kinetic parameters were determined. This model can predict the percentages of xylan remaining in the pretreated solids, net xylose yield in the liquid prehydrolysate, and xylose loss after pretreatment of a feedstock at a certain set of reaction conditions. Using this model, four optimum reaction conditions for obtaining maximum net xylose yield in the liquid prehydrolysate were identified. The yield and rate of ethanol production from the optimum prehydrolysates by the pentose fermenting yeast, Pichia stipitis, were determined. It was found that pretreating the selected feedstocks at 170-180��C with 1.0-1.2% sulfuric acid for 1-3 min resulted in the recovery of 80-85% of the original xylan in the liquid prehyrolysate. It was also found that feedstocks with higher neutralizing capacity (e.g., corn stover) produced lower sugar yields as a result of acid neutralization. Pretreatment of feedstocks at conditions beyond the optimum reaction conditions would increase the extent of xylose degradation, and lower the yield and rate of ethanol production due to loss of fermentable sugars and formation of toxic byproducts. The optimum prehydrolysates of corn stover produced the highest yields of ethanol (0.39-0.47 g ethanol/g xylose) followed by switchgrass (0.36-0.45) and poplar (0.26-0.44). The inhibitory effects of byproducts (e.g., acetate) was more pronounced in poplar prehydrolysates. / Graduation date: 1997

Lignocellulose is composed of lignin, hemicellulose, and cellulose. Lignocellulose waste is a sustainable and renewable resource available for use in biotechnological applications. Efficient enzyme production and enzymes with high catalytic activity are needed for the use of lignocellulose. The study of cellulases and xylanases that degrade cellulose and xylan into constituent monosaccharides is required to advance industrial application of these enzymes. The use of a traditional pure culture approach to discover and characterize cellulases and xylanases from novel actinomycete isolates and the use of metagenomics to uncover previously unidentified cellulase genes was undertaken. Actinomycetes were cultivated from soil samples and the isolate with the best cellulase and xylanase activity was subjected to strain improvement through protoplast fusion. Enhanced enzymatic activity was found in one fusant. Differential release of sugars from xylan was observed through gas chromatographic analysis between the parental and fusant cultures. Genome shuffling was observed in 16S rRNA genes after protoplast fusion. Finally, one putative endo-β-1,4-glucanase was discovered in a metagenomic library created from cellulose-enriched potting soil. / UOIT

Thèse de doctorat : Génie des procédés et des produits : INPL : 2007. / Titre provenant de l'écran-titre. Bibliogr.

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

The study deals with the chemical and flame retardant (FR) treatment of woven flax fabric and preparation and characterization of flax reinforced phenolic composites. Sheets of flax fabric were subjected to chemical treatments using NaOH and silane coupling agents. A phosphate-based flame retardant (DAP) was applied to decrease the flammability of the flax fabric. The effect of the chemical treatments and FR treatments on the thermal and flammability properties of the fabric and composites was investigated using thermogravimetric analysis (TGA), vertical flame resistance test and cone calorimeter. The mechanical properties of the flax fabric and composites, before and after environmental ageing, were investigated. Ageing studies were carried out by exposing the samples in an environmental chamber at specified conditions for two weeks. TGA results showed that the treatment of the fabric with FR shifts the decomposition temperature to lower level and increases the char residue. Vertical flame resistance testing showed that FR treatment of the flax fabric improved the flammability properties. There was no after flame and afterglow observed for FR treated flax fabric due to self-extinguishment after removal of the flame. Flax Fabric without FR burned completely and there was no result for the char length due to the complete destruction of the fabric. The FR treatment, however, seemed to have had a negative effect on the tensile strength of the flax fabric. This was further intensified upon exposure of FR treated flax fabric to high temperatures during ageing studies which drastically reduced the tensile strength by more than 90%, and the flax fabric were found to be brittle and darker in visual appearance. TGA results of flax/phenolic composites showed that for composites containing chemically treated and FR treated flax fabric the decomposition temperatures shifted to lower temperatures; however there was no significant difference in the amount of char residue. Untreated flax/phenolic composites exhibited the lowest char residue. Cone calorimeter results showed that the peak heat release rate (PHRR), smoke production rate (SPR) and carbon dioxide (CO2) emission rate was reduced for the flax/phenolic composite produced using FR treated flax fabric. The tensile strength of these composites was reduced while there was an increase in modulus value. Exposure of the FR treated composites to high temperatures further reduced the tensile strength and increased the E-modulus. Both FR treated and untreated composites changed in colour and the FR treated composites were found to be brittle after exposure to high temperatures.

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mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} --><!--[endif]--></p><p>The aim of the project was to develop a method to investigate differences in adhesion of microorganisms to materials that contains lignocellulose. The method was tested on a gram-positive (<em>Micrococcus lutea) </em>and one gram-negative (<em>E-coliJM109</em>) bacteria.</p><p>The study was begun by cultivation of the two microorganisms. The cultivation was done to calculate the generation times of the bacteria and to obtain growth curves. Cells from these cultivations were also frozen (-70ºC) and later used for inoculation.</p><p>At STFI-Packforsk AB the total charge on the mass was measured and later a conductivity titration on the mass was executed as well, all to find out more about the different properties of the mass. Properties that in a later part of this study could possibly be linked to the adhesion of cells to the pulp. The adhesion experiments that were executed gave poor results. The adhesion experiment with <em>M. lutea</em> was the only experiment that gave a reproducible result. In this experiment <em>M. lutea </em>was contacted with bleached leaf. A reduction of cells was observed in all of the dilutions where <em>M. lutea</em> had been in contact with the mass. The number of colony forming units of the culture was 1,2×10⁷ before the adhesion and 2×10⁶ subsequently.</p><p> </p>

No description available.

Current research efforts are focused on 'second generation biofuels', which includes biofuels produced from lignocellulosic material. Lignocellulosic material is primarily composed of cellulose, a glucose polymer, xylose rich hemicellulose and non-fermentable lignin. Saccharomyces cerevisiae is widely used on an industrial scale for the production of ethanol from glucose; however, native S. cerevisiae does not contain the genes required for fermentation of xylose into ethanol. Others have sequentially expressed trans-genes from xylose fermenting organisms to engineer strains of S. cerevisiae capable of fermenting this pentose. The goal of this thesis was to generate a single cassette of 9 genes which have been shown to ferment xylose and arabinose. The 17 kb DNA fragment harboring all the genes necessary was introduced into the yeast genome using one-step homologous recombination based transformation. Expression of this cassette was verified by demonstrating that the first and last genes on this cassette were transcribed. The modified strain exhibited xylose utilization under microaerobic fermentation conditions. Further genetic and process engineering methods may be employed to improve the yield. The experiments described here demonstrate that generating a functional cassette of pentose fermenting genes is still achievable.