Degradation of Gonometa postica silk, which occurs during bleaching, is not only a problem for the consumer but also for the producer. The purpose of this study was to determine the effect of hydrogen peroxide bleaching on Gonometa postica silk. The specific goal was to determine the influence that temperature, pH and time duration has on the releasing of hydrogen peroxide, colour change, breaking strength, stiffness and shrinkage. A literature study was undertaken to describe the physical properties, chemical and molecular structure of Gonometa postica silk. Research on the degradation effect of hydrogen peroxide on silk was investigated and the factors that influenced the bleaching effect, were identified. An experimental study was carried out to determine the releasing of hydrogen peroxide at 50°C, 60°C, 70°C and 90°C at a pH 8 and pH 10. The Gonometa postica silk was bleached with hydrogen peroxide in a 5 l water bath and dried afterwards. The samples were kept in standard atmosphere conditions of 20 ± 1°C and 65 ± 2% relative humidity before and during testing. The colour change, breaking strength, stiffness and shrinkage of Gonometa postica silk were determined after hydrogen peroxide bleaching. The release of hydrogen peroxide was determined by potassium permanganate titrations during the bleach action, while the colour changes (delta E) of the samples were determined by a colorimeter. The instron tensile tester was used to determine the breaking strength of bleached and unbleached samples according to the SABS method 13934 â 1:1999. The breaking strength was measured in Newton and elongation in mm. The stiffness of the silk was measured by a Shirley stiffness tester. Shrinkage caused during bleaching was determined in grain difference and expressed in percentages. To illustrate the degradation caused by bleaching, scanning electron microscope photos were taken. The results indicated that temperature, pH and time duration plays an important role in the bleaching of Gonometa postica silk. At a pH 10, the colour change (bleaching effect) increased rapidly as the temperature increased. More bleaching (colour change) could be seen at low temperatures at a pH value of 8. High temperatures damaged the fibre, which lowered the breaking strength, and caused an increase in the stiffness of the fibre as the temperature incresed. Hydrogen peroxide released quicker at high temperatures. Different temperatures did not influence shrinkage. The pH-value was the factor with the highest influence on the release of hydrogen peroxide. More bleaching and release of hydrogen peroxide could be seen at a pH value of 8 and better results were obtained in colour change at a pH value of 10 at low temperatures. The pH-value had no significant influence on breaking strength, stiffness and shrinkage. Better results could be seen with an increase in time of bleaching. The results showed that 70°C for 3 hours at a pH value of 8 are the best conditions for bleaching Gonometa postica silk. Althought some damage to the fibres was evident under these treatment conditions, an effective white colour was obtained. The conclusion that can thus be made is that factors, such as temperature and pHvalue, have the biggest influence on colour change, tensile strength and stiffness of Gonometa postica silk.

The overall goal of the study was to evaluate solar baking against conventional retting as decortication methods of Agave americana fibre in terms of textile properties. The study focused mainly on: ⢠Identification of the most cost-effective, efficient and eco-friendly methods of partial degradation of Agave americana leaves to release the textile fibre. ⢠Evaluating the physical structure of Agave americana fibre decorticated by solar baking and conventional retting. ⢠Evaluating the essential textile properties and some secondary textile properties of Agave americana fibre fabric to predict its possible end uses in textiles. Preliminary comparison of ten different leaf partial degradation methods, suggested the feasibility of investigation of solar baking as a partial degradative method for fibre extraction. Conventional retting was chosen to be the control method. The solar baking process was found successful, energy saving, more eco-friendly and faster than conventional retting of the Agave americana leaves. Fibre decortication was entirely done by hand after the leaves were partially degraded. After hand decortication the fibres were then knotted, twined and woven into fabric. Long beautiful fibre with natural look was obtained from Agave americana leaves. Agave americana fibre in its natural condition is coarse, harsh and stiff when dry. Fibre identification tests confirmed that Agave americana react like all other natural cellulosic fibres in burning behaviour, solubility and Shirlastain C identification tests. Microscopic evaluation indicated that the fibre consisted of a number of irregularly sized and shaped individual cells, each with a lumen. The Shirlastain C colour reaction and the crosssectional view of the Agave americana fibre are unique and would be useful to distinguish Agave americana from other natural cellulosic fibres. The physical structure and the length of Agave americana fibre were evaluated while the fibre was in a fibre form. The retted and solar baked Agave americana fibre yarn was evaluated for tensile strength and elongation at break. The thickness, stiffness, dimensional stability, crease recovery, dye ability, moisture regain and water absorption of the Agave americana fabric of the solar baked and retted fibres were evaluated. Agave americana fibre showed adequate tensile strength and elongation at break to be a useful textile fibre. No significant differences were found between the tensile strength of the retted and the solar baked fibre. Agave americana exhibited excellent dimensional stability; it showed relaxation shrinkage with no residual shrinkage. Agave americana showed good water absorption and moisture regain properties. Agave americana accepted the direct dye easily even without bleaching. The Agave americana fibre fabric was found to be relatively stiff. Agave americana exhibited poor crease recovery no significant difference in crease recovery were found between retted and solar baked fibre fabrics, but the warp yarns recovered significantly better from creases than the weft yarns. Agave americana fibre is a promising speciality cellulosic fibre which has a potential of being valuable for current as well as future applications. The research proved that solar baking is an efficient, fast and environment friendly alternative to conventional retting as a partial degradation method for Agave americana fibre decortication.

Escherichia coli that cause diarrhoeal disease are classified into virotypes based on virulence properties, mechanisms of pathogenicity, clinical syndromes and distinct O:H serotypes. These virotypes include enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffuse adhering E. coli (DAEC) and verocytotoxin-producing E. coli (VTEC) also known as Shiga toxin -producing E. coli (STEC) which includes the most virulent subgroup, the enterohaemorrhagic E. coli (EHEC). The most predominant EHEC is E. coli O157:H7. Cattle are the principal reservoir for diarrhoeagenic E. coli (DEC) and ground beef products are the major vehicle for their transmission. The aim of this study was to determine the incidence of DEC in minced beef and boerewors and to investigate the growth, survival and thermal inactivation of E. coli O157:H7 in boerewors. In the first part of this study, a survey was conducted on 30% of butcheries in the Bloemfontein District and isolated virotypes were identified by serotyping. More minced beef samples were positive for DEC (38.10%) compared to boerewors samples (28.57%). Enteropathogenic E. coli were the most frequently isolated virotype while VTEC were the most virulent virotype recovered from both samples, hence highlighting the potential of ground meat products as vehicles of transmission of DEC. An enrichment step enabled optimal recovery of DEC on chromocult⢠chromogenic agar (Merck 1.10426). In the second part of this study, boerewors with and without sulphur dioxide preservative were manufactured following a typical commercial procedure and inoculated with E. coli O157:H7 at low (3.5 log cfu/g) and high (7.5 log cfu/g) inoculum size, and stored at 0 o C, 4 o C and 10 o C followed by evaluation of growth and survival every 48 hours for 10 days. Boerewors with preservative had significantly lower recoveries compared to boerewors without preservative at all the three storage temperatures. The low and high inocula with preservative had significant (p<0.001) reductions in recoveries at 0 o C, declining to below detectable limits at days 8 and 10 respectively. At 4 o C, the low and high inocula with preservative recoveries declined significantly (p<0.001) to below detectable limits and to 1.01 log cfu/g respectively at day 10. At 10 o C, significant (p<0.001) increases of 1.85 log cfu/g and 2.35 log cfu/g respectively were observed from day 0 to day 10. At 0 o C, the low and high inocula without preservative treatments had declines in recoveries of 1.21 log cfu/g and 1.45 log cfu/g respectively. Insignificant (p<0.001) increases of 0.19 log cfu/g and 0.26 log cfu/g respectively were noted at 4 o C while significant (p<0.001) increases of 1.97 log cfu/g and 1.84 log cfu/g respectively were noted at 10 o C from day 0 to day 10. The combination of sulphur dioxide preservative and low temperature (0 o C and 4 o C) demonstrated the best efficacy against the sur vival of E. coli O157:H7. It was established that thermal inactivation end point temperatures of E. coli O157:H7 inoculated into boerewors with preservative at 7.00 log cfu/g, was 60 minutes at 60 o C, 80 seconds at 65 o C and 60 seconds at 70 o C. This study demonstrated that E. coli O157:H7 can survive in boerewors with and without preservative, stored at 0 o C, 4 o C and 10 o C and is more sensitive to heat treatment at 70 o C.

In view of the decline of natural habitats due to urban and industrial development, the need to search for new yeasts is pressing since few natural habitats have been thoroughly investigated for yeast species. Yeasts have not been isolated from Lesotho before despite being an ideal environment for yeasts. In addition, isolation of yeasts able to utilize complex substrates, similar to intermediates in the degradation of chlorophenols would be important in their detoxification. Yeasts with these abilities are usually isolated from polluted environments and their presence from the pristine environment in Lesotho would be unexpected. The species Lipomyces starkeyi and L. tetrasporus were found distributed throughout the various habitats while Debaryomyces hansenii, D. hansenii var. fabryi and D. occidentalis were found in 60% - 70% of the different regions. Debaryomyces polymorphus, Dipodascus spicifer, Galactomyces geotrichum, G. reessii, Kluyveromyces lactis , L. kononenkoae, L. mesembrius, L. spencermartinsiae, Pichia anomala, P. fabianii, P. guilliermondii and Yarrowia lipolytica were site specific. The fatty acid profiles of the isolated lipomycetous yeasts are similar to those reported in literature. This corroborates the value of this phenotypic characteristic in the taxonomy of these yeasts. The PCR products of the ITS region of some of the type strains of the family Lipomycetaceae showed high length variation enabling rapid identification. The type strains, the sub-species and the varieties of the family Lipomycetaceae could be differentiated from each other using RFLP profiles obtained with the restriction enzymes used in combination i.e. Cfo I, HaeIII and MboI. The RFLP profiles for the five Lesotho isolates with atypical carbohydrate patterns could be separated into three groups. The first group, comprising of three isolates (52b, 73, 93), gave similar restriction patterns suggesting that they are the same species. Isolate 58b yielded a distinct profile while isolate 97 had similar sized ITS-PCR (1000bp) to that of L. lipofer and L. tetrasporus . It is important to assess the variation in RFLP profiles between various strains from different habitats of a particular species in order to determine the conserved status of this genotypic character. More strains of a species representing the Lipomycetaceae should be subjected to similar RFLP analysis to further determine its conserved status. The D1/D2 sequence data enabled separation of the five isolates into three groups. The first group, comprising of three isolates, showed 1% nucleotide substitutions to L. starkeyi suggesting that these isolates are probably known Lipomyces spp. The other two isolates yielded sequences that were 99% identical to L. kononenkoae subsp. kononenkoae (isolate 58b) and 100% identical to that of L. tetrasporus (isolate 97) further suggesting that these isolates are probably known Lipomyces species. Isolation of basidiomycetous yeasts from unusual carbon sources has been reported, however, this is the first report of isolation of ascomycetous yeasts able to grow on unusual substrates. The substrate that yielded the highest number of isolates was 1,4 cyclohexanedimethanol. Pichia anomala and Y. lipolytica strains (identity confirmed with D1/D2 sequencing) isolated from 2-chlorobutyric acid could not degrade the 2-chlorobutyric acid probably due to toxicity of low molecular weight organic acids at low pH. The ability of P. anomala isolated from 1,4 cyclohexanedimethanol to grow on dodecane and pristane is unusual and this strain should also be subjected to sequencing to confirm identification. Debaryomyces hansenii, P. anomala, P. fabianii, P. guilliermondii and Y. lipolytica could grow on the cyclohexane derivatives although they preferred a shorter straight chain hydrocarbon as confirmed by higher growth rates in the presence of dodecane. The isolates could not grow in the presence of monoterpenes, although they had been isolated from substrates that were supposed to mimic the structures of monoterpenes and which previously had yielded limonene utilizing Rhodotorula species. The inability of these strains to grow on monoterpenes might be due to toxicity of the monoterpenes, because of their hydrophobicity and lipophilicity resulting in partitioning into the lipid bilayer of cell membranes.

Some yeasts produce sexual spores (ascospores) in a variety of shapes and surface ornamentations. These intriguing structures have hitherto been used only in yeast classification. In this study the likely primary function of ascospore shape and ornamentations with associated lipids in water-driven movement as aiding the dispersal of ascospores from enclosed containers (asci), is proposed. This interpretation might find application in nano-, aero- and hydro -technologies with the re-scaling of these structures. Through sexual reproduction, some yeasts produce microscopic containers (asci) that enclose ascospores of many different shapes and various nano-scale surface ornamentations. Some spores are spherical with an equatorial ledge (like the planet Saturn), or resemble hats with a bole and brim, while others look like corkscrews, walnuts, spindles with whip-like appendages, needles, and hairy or warty balls. Until now, these structures were used to classify yeasts and little thought was apparently given to their possible functional role. From literature and microscopic observations, it was found that the yeasts Dipodascopsis uninucleata and Dipodascus have evolved sophisticated means that enable the dispersal of oxylipin-coated spherical and bean-shaped spores from bottle-shaped containers (asci) without inverting or shaking them. Here, spores are pushed by turgor pressure towards the narrow opening and then ejected. Studies of Dipodascopsis suggest that oxylipin-coated, interlocked hooked ridges on the surfaces and stretching across the length of bean- to ellipsoidal-shaped spores are responsible for the alignment of the latter. Here, spores inside the container are positioned side-by-side in a column of linked clusters with elongated sides attached by interlocking hooked ridges in gear-like fashion and orientated mainly with one end towards the opening. It was concluded that hooked ridges form turbine-like structures at both ends, causing propeller-like rotation when the spores are pushed by water pressure towards the ascus opening. This rotational movement loosens the spores (by the unlocking the hooked-ridges) near the container neck, which is necessary for sliding past each other for eventual release. Eventually, spores are released individually from the bottle-shaped ascus while rotating at about 1200 rpm at approximately 110 length replacements per second. With some species of the genus Dipodascus, compressible oxylipin-coated sheathed surface structures and not gears are used to separate and loosen spherical spores in a similar bottle -shaped container before individual release under turgor pressure. These spores simply slide past each other when pressed towards the opening. It is presumed that more complex mechanics are needed to allow the effective release of bean- to ellipsoidal-shaped ascospores compared to spherical sheathed ascospores, for which alignment and rotation are unnecessary. Using gas chromatography-mass spectrometry, it was discovered that a saturated 3-OH 14:0 (mass fragments:175 [CH3O(CO)-CH2-CHO-TMSi]; 330 [M + ]; 315 [M + -15]) is produced by the yeast Eremothecium ashbyii. In order to map the oxylipinâs location in the yeast, antibodies (against these oxylipins) and immunofluorescence microscopy on cells in sexual mode was employed. The oxylipin was present as part of a V-shaped structure on sickle-shaped spores. With the aid of confocal laser scanning microscopy to observe cells treated with antibody and fluorescine (FITC anti-rabbit IgG), it was concluded that the hydrophobic V-shaped structure was present as a mirror image on both sides at the blunt end of an otherwise hydrophilic spore as indicated by differential ascospore staining. Scanning electron microscopy showed this structure to be fin-like protuberances. Next, the function of these fin-like structures and ascospore shape was addressed. Using microscopy, it was discovered that spores are sometimes forced through the ascus with the spiked tip rupturing the ascus wall. Water pressure caused a boomerang movement when the blunt end is pushed forward with the spike leading the way in a circular motion. This happens only when micron streams of water move across the fins from the blunt end towards the tip of the spore. It is believed that this part of the study has only scratched the surface of water-driven ascospore movement in yeasts on a micrometer scale and that the mechanical implications of many spore shapes with a large number of different hydroxy oxylipin-lubricated, nano-scale surface ornamentations await similar explanation and elaboration. Why did some yeasts evolve peculiar spore movement with the beneficial consequence, so far as we can see, to escape from closed or partially closed containers? Of course, this should be important from a survival point of view since without this ability, yeasts will probably not be able to disperse properly. It is believed that if appropriate ultrastructural studies (using glutadialdehyde and osmium tetroxide as fixatives) are conduc ted on yeasts aimed at exposing ascospore surface ornamentations and not merely membrane structure, conducted in the past, clues can be gained to reveal the mechanics behind the motion of nano-sized particles in fluids. Consequently a further aim of this study became to assess ascospore structure (using above ultrastructural method) especially nano-scale ornamentations with associated lipids especially oxylipins in various unrelated yeasts. These were obtained for the yeasts Eremothecium sinecaudum (ascospores corkscrew-shaped and coated with oxylipins), Dipodascopsis uninucleata var. wickerhamii (smooth surfaces without oxylipins), Lipomyces kononenkoae (smooth ascospore surface with lipid sacs), L. tetrasporus (ridged ascospore surface with lipid sacs), Saturnispora saitoi (Saturn-shaped ascospores covered with oxylipins), Ascoidea africana (hat-shaped ascospores covered with oxylipins), Ambrosiozyma platypodis (double brimmed hat-shaped ascospores), Nadsonia commutata (ascospore surface warty; cells contain oxylipins) and N. fulvescens (ascospore surface hairy-like; cells contain oxylipins). Interesting patterns regarding lipid turnover (i.e. total-, neutral-, phospho-, glycolipids and associated fatty acids) were found when asexual and sexual stages of above yeasts are compared.

Psittacine beak and feather disease (PBFD) is a readily recognizable disease of wild and captive psittacines in Australia but it is also a problem worldwide wherever captive species are bred. The disease caused by beak and feather disease virus (BFDV) is characterized by the progressive development of feather dystrophy and loss. Although the occurrence of PBFD in South Africa has been reported only recently, it is already rampant and threatens the extinction of the endangered Cape parrot and black-cheeked lovebird. Currently no vaccine for PBFD is commercially available but the loss of approximately 10-20% of breeding stocks of psittacines annually in South Africa alone is enough to realise the importance of one. Genetic and antigenic differences in BFDV are significant aspects for production of a vaccine but the lack of a culture system for BFDV has limited studies into its genetics, antigenicity and pathogenicity. The objective of the study thus became to establish techniques that could be used to investigate genetic and antigenic differences that may be present in BFDV in South African psittacines. Molecular investigations involved the testing dried blood samples for BFDV nucleic acid using polymerase chain reaction (PCR). A region within the Rep gene was amplified and digested with HaeIII to yield restriction length fragment polymorphisms (RFLPs). Six RFLPs were identified, cloned, sequenced (UFS 1- 6) and phylogenetically analysed. BFDV was purified from body organs of PBFD- affected birds by cesium chloride density gradient centrifugation and fractions tested by PCR and haemagglutination (HA) assays. BFDV-specific antibodies were raised in two rabbits by inoculation with purified BFDV in Freundâs incomplete adjuvant and tested by haemagglutination inhibition (HI) assays and an enzyme-linked immunosorbent assay (ELISA). HA and HI assays were attempted using erythrocytes from African grey parrots and Brown-headed parrots. HI assays were also used to test parrot sera for the presence of antibodies to BFDV. UFS 1-6 were closely related to known BFDV isolates and UFS 1 may represent a unique genotype in South Africa as it was separated from its closely related isolates by a 90% bootstrap value. UFS 3, 4 and 5 isolated from budgerigars belong to the budgerigar lineage as they clustered with isolate BG3-NZ. Together with three previously identified genotypes, UFS 1 indicates the introduction of BFDV into southern Africa on four separate occasions. Purification of BFDV from organs was successful but yielded low titres possibly because low quantities of virus were present in the organs or because little virus was circulating in the bird upon its death. PCR and HA assays confirmed the presence of BFDV in fractions; the two results correlated well. HA and HI assays were successfully established using erythrocytes from African grey parrots and Brown-headed parrots. Antibodies to BFDV were successfully detected in sera of three parrots by the HI assay. However, the assay could not detect non-psittacine raised antibodies (rabbit-raised) due to non-specific reactions. BFDV-specific antibodies were successfully raised in rabbits and were verified by the use of an ELISA. The high level of genetic diversity observed in the study compels further investigation into the genetics of BFDV as such levels of diversity may become a limiting factor in the applicability of PCR as a diagnostic test. The entire diversity of BFDV has not been studied and future work may lead to the identification of more BFDV strains, an important factor in vaccine development. The rabbit- raised antibodies together with the HA and HI assays and ELISA can be used to study the antigenic differences that may be present in known BFDV isolates that may also lead to the identification of more strains of the virus.

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The dimorphic fungus Yarrowia lipolytica is an n-alkane assimilating yeast. During nalkane oxidation toxic fatty aldehydes are formed that are further oxidized by fatty aldehyde dehydrogenases (FALDH) to carboxylic acids that then enter the β-oxidation pathway. Very little research emphasis has been placed on FALDHs in yeasts and their precise role in n-alkane metabolism. This study aimed at contributing to the limited knowledge of yeast FALDHs and in particular the four putative Y. lipolytica FALDHs (YlFALDH1 - 4) that were recently identified in the fully sequenced genome of Y. lipolytica E150. The contribution made from this study to YlFALDHs was with reference to their promoter expression and subcellular localization. The promoter and terminator of each YlFALDH was initially used to construct reporter cassettes in conjunction with β-galactosidase (lacZ) by utilizing the Sticky-end PCR (SEP) and Enzyme-free cloning methods. These two methods proved to be unsuitable for the expression study. The promoter region of each YlFALDH was then cloned into the pINA781 expression vector containing lacZ to study the expression further. With the aid the pINA781 integrative vector and qualitative plate assays, with 5-bromo-4-chloro-3- indolyl-β-D-galactosidase (X-gal), it was observed that the promoters of YlFALDH1 and 2 were inducible by dodecane and hexadecane but not by oleic acid, glucose or glycerol. The promoter of YlFALDH2 also seemed to display the same level of transcriptional strength as the inducible POX2 promoter. Induction of the YlFALDH3 and 4 promoters was not observed. Localization of the YlFALDH proteins was studied with the aid of green fluorescent protein (GFP) from Aequorea victoria and putative localization sequences (LS) from each YlFALDH isozyme. The putative Y. lipolytica LSs comprised of the last 150 bp of the COOH-terminal of the YlFALDH proteins. These LSs were modeled from Rattus norvegicus FALDH that possesses a 35 amino acid hydrophobic protein anchor at its COOH-terminal. For localization studies, chimerical JMP5 molecules were created with an inducible ICL1 promoter, GFP and putative Y. lipolytica LS from each isozyme. Chimerical molecules were also constructed with a pKOV136 vector that contained a constitutive TEF promoter, a GFP-LS fragment (from a JMP5-chimera) and LIP2 terminator. No fluorescence was observed with epifluorescence or confocal laser microscopy when either of the JMP5- or pKOV136-chimeras were transformed into Y. lipolytica E150 and Po1g respectively. Consequently the subcellular localization could not be identified.

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Acetylsalicylic acid (ASA)-sensitive 3-hydroxy (3-OH) oxylipins were uncovered in 1991 in the yeast Dipodascopsis uninucleata. Since then, various similar oxylipins were found to be widely distributed in fungi. Interestingly, 3-OH oxylipins were reported to play a role in ascospore release from enclosed asci, where they are involved in assisting nano-scale gear-like (D. uninucleata); sliding (Dipodascus); drilling (Eremothecium sinecaudum) and piercing movements (E. ashbyi and E. coryli). In Ascoidea africana, a 3- OH 10:1 oxylipin was found to be associated with hat-shaped ascospores carried inside ellipsoidal asci. However, in this study no function was proposed for this oxylipin. Since only one species representing the genus Ascoidea was studied, it became the aim to further expand this study to also include A. corymbosa and A. rubescens. Using confocal laser scanning microscopy (CLSM) on cells stained with fluorescein-coupled 3-OH oxylipin specific antibodies, this study suggests that oxylipins are specifically associated with ascospores and not vegetative cells of A. corymbosa and A. rubescens. Using gas chromatography - mass spectrometry (GC-MS) the oxylipin, 3-OH 17:0, was identified in A. corymbosa. Here, oxylipin-coated razor sharp ascospore brims may play a role in rupturing the ascus to affect forced release of hat-shaped ascospores. Literature suggests that 3-OH oxylipins are produced by β-oxidation or fatty acid synthesis in mitochondria of yeasts. Since these oxylipins accumulate in sexual cells (asci), increased mitochondrial activity is therefore expected in these structures. Strikingly, this assumption is supported in this study. Using selective fluorescence mitochondrial staining and CLSM, evidence is provided that mitochondrial function is much higher in asci containing increased amounts of 3-OH oxylipins compared to the corresponding asexual vegetative cells. Furthermore, when ASA, a mitochondrial inhibitor, was added in increased concentrations to cultures of Ascoidea, the sexual stage was found to be more sensitive. Ascospore liberation from asci was first inhibited followed by asci formation while some vegetative growth could still be observed.