Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)

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Open Access
Utilisation of edible oils and GLA production by Mucor in the presence of acetate
(University of the Free State, 1998-10) Badenhorst, Jacqueline; Kock, J. L. F.
English: Surveys launched across South Africa indicate that many frying establishments abuse their frying oils and fats during the frying process, resulting in degradation and concomitant production of potentially toxic oxidation products. Some of these compounds have been shown to be toxic to animals and in human in vitro studies. Consequently, strict regulations under the Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act 54 of 1972) were published on 16 August 1996. It is now an offense to use or sell used cooking oil or fat for human consumption containing high levels of these degradation products. Since frying establishments are not allowed to discard their used oils and fats by selling to the public for consumption or dumping into municipal drainage systems, it is important that these oils and fats are collected for re-use in another form. Consequently, the aim of this study was the biotransformation of used oil wastes (containing no toxic substances) to high value lipids containing gamma-linolenic acid (GLA). This polyunsaturated fatty acid is prescribed for the treatment of eczema. In order to achieve this, Mucor circinelloides f. circinelloides CBS 108.16 was first grown on 40 gil unused sunflower oil and, as expected, produced neutral lipids (NL) similar in fatty acyl composition to the original oil. The apparent repression of the Á6 fatty acid desaturation was partially reversed when cells were grown on oil (30 g/l) and sodium acetate (10 gil) as mixed substrates resulting in an increase in GLA content. Furthermore, a three-fold increase in oil substrate utilisation and doubling of biomass production to 19.1 gil occurred when sodium acetate was added to the oil substrate. When sodium acetate (10 gil) was added to a growth medium containing used cooking oil (UCO) similar results were obtained. This experimental procedure was repeated for seven additional Mucor strains and again the stimulatory effect of sodium acetate in combination with UCO was obvious. Next, the effect of different UCO concentrations in the presence of 10 g sodium acetatell on biomass and lipid production was investigated in Mucor circinelloides CBS 108.16. According to our results, a maximum biomass concentration of 48 gIl consisting of 82 % oil yielding about 35 g NUl and up to 900 mg GLNI was achieved. The addition of 30 g UCO/I in combination with 10 g sodium acetate/l proved to be the optimum UCO concentration in order to obtain maximum GLA yield. Similar results with this strain were obtained when UCO was replaced with fresh unused cooking oil. When these experiments were repeated with linseed oil and sodium acetate as sole carbon sources, much less GLA was produced (351 mg GLNI). According to bioreactor studies, the effects of sodium acetate addition can be attributed to the change in pH of the medium during cell growth in the presence and absence of acetate. In the absence of sodium acetate the pH decreased to 2.2, whereas in its presence it increased to about pH 8.0. During metabolism of sunflower oil in the presence of sodium acetate, the percentage of saturated fatty acids in the extracellular lipids increased, suggesting a higher specificity of the fungal lipase for unsaturated fatty acids. When the sodium acetate was omitted from the medium and the pH gradually increased according to a pH profile mimicking the natural increase in pH found in the medium containing sodium acetate, similar results as in the presence of sodium acetate were obtained. This observation indicated that the pH increase alone during cultivation was responsible for the increased sunflower oil utilisation, biomass and GLA production.
Open Access
Diseases of Acacia mearnsii in South Africa, with particular reference to ceratocystis wilt
(University of the Free State, 1998-12) Roux, Jolanda; Wingfield, M. J.
English: The Acacia mearnsii industry is a relatively small, though very profitable industry in South Africa. Wood derived from A. mearnsii is currently in greater demand than that of either pine or eucalyptus in South Africa. Despite the importance of this industry, very little attention has been given to the genetic improvement, disease tolerance or general improvement of A. meamsii as a forestry species. The result has been that, during the last few decades, pathogens have become adapted to, and spread through plantations of this tree. Although relatively little research has been conducted on the impact of pathogens on A. mearnsii, this situation has changed during the past nine years, and particularly since the identification of Ceratocystis wilt. The planting of exotics has many advantages over native plants. In South Africa, exotic forestry species, such as Eucalyptus spp., Pinus spp. and A. mearnsii were introduced to halt the uncontrolled logging of native forests. These native forests were logged mainly for furniture and building material, but also for fuel wood, resulting in the near complete destruction of South Africa's native forests. The introduced exotics prevented the further destruction of these forests and soon became a large industry. This was particularly due to the fact that it was found that they also had a superior growth rate when compared to native species. This accelerated growth rate brought rapid results from breeding trials and, thus, a relatively rapid improvement of the material planted. Because they had been separated from their natural enemies, these trees were also initially disease free. The A. mearnsii industry has, and will continue, to face many problems and challenges from pests and diseases. After the initial phase in which the tree was removed from the pathogens affecting it in it's native range, it faced attacks by native South African pests and diseases. These can spread from native Acacia species, or from any other native plants in the same, or even different families. Exotic, mono culture industries are also constantly under threat from the introduction of pathogens from other countries, including the country of origin. This can be done by the introduction of new germ plasm or on any other plant species or plant material brought into a country. Because A. mearnsii is now planted as a monoculture, in contrast to it's native situation, diseases and pests can potentially be much more severe and will spread more rapidly and widely throughout even aged and genetically uniform stands. Propagation of A. mearnsii has, recently, advanced considerably and this is concurrent with increased demand for this wood on world markets. Lessons learned from eucalypt and pine forestry need, however, to be heeded to save unnecessary losses and time. With the advent of vegetative propagation of A. mearnsii in South Africa, it is important to include disease screening trials at the early stages of the development of clones. In order to do this, a knowledge of all possible pathogens of A. mearnsii is needed. This includes pathogens known in South Africa and those that occur beyond the borders of the country. It is also necessary to have a detailed knowledge of the biology and population structure of these pathogens in order to gain an impression of the possible success of control measures. This thesis is a compilation of work conducted on some of the known pathogens of A. mearnsii in South Africa. It also includes a large component dealing with the identification and clarification of previously unknown pathogens of A. mearnsii. It, therefore, does not focus only on diseases of A. mearnsii, but includes a chapter on a disease of Eucalyptus. The causal agent of this disease has, however, also recently been found on A. mearnsii in South Africa and this chapter aims at elucidating the possible origin of the isolates from South Africa. It also illustrates the potential threat of this pathogen to the A. mearnsii industry. South Africa is a semi-arid country that regularly suffers from severe drought. Forestry activities in the country are also mainly restricted to areas with poorer soil and where agriculture cannot be pursued on a profitable basis. Factors such as drought, hail, frost and sub-optimal soil conditions can all contribute to increased stress on trees. Under these conditions, many fungi can act as opportunistic pathogens, causing large scale losses. They often live as endophytes within their hosts, not causing any negative affect until the onset of stress. At this stage, they spread throughout trees, preventing them from recovering from the stress condition and leading to cankers and tree death. Careful management, particularly site/species matching, is required to minimise losses caused by these pathogens. This thesis provides a basis for future research on the development of management strategies to control diseases of A. mearnsii in South Africa. Information, however, also provide valuable knowledge for forestry industries outside South Africa by highlighting the threat of exotic pathogens and the importance of strict quarantine measures to prevent the spread of pathogens. This is true for the movement of not only A. mearnsii material, but as was seen here, the movement of any forestry products, since many pathogens have a wide host range. Although the thesis is comprised of a series of individual entities, these all provide information regarding the hygiene of A. mearnsii plantations. This thesis thus aims at identifying future focus points for intensive research, while at the same time focusing on those pathogens that have been known to the South African industry for a longer period of time. Chapter one provides a review of the available literature on diseases affecting not only A. mearnsii, but also other Acacia spp. important to the forestry industry, world wide. It also highlights some of the uses of these species in the countries where they are planted. The multi-purpose use of Acacia spp. is an important aspect emerging from this review. In many countries, Acacia spp. are not only planted as forestry species but are also used for soil reclamation, nitrogen fixation and fodder. The main focus of the chapter, however, is on the A. mearnsii industry in South Africa, with a brief discussion on all the diseases currently known to occur in the country. It is concluded that much research is still needed to reduce the impact of these diseases and to ensure that the Industry functions optimally. Ceratocystis albofundus must be considered as one of the most important pathogens of Acacia spp., world-wide. Currently this pathogen occurs only in South Africa, but if it is to spread to other countries, large scale losses will be incurred. It may also affect, not only A. mearnsii, but most likely many other plant species. Breeding programmes for A. mearnsii in South Africa focus strongly on this pathogen. In Chapter two, the population diversity of C. albofundus was investigated and compared with data for other Ceratocystis spp., using nuclear and mitochondrial DNA fingerprinting. It was found that the C. albofundus population has a greater genetic diversity than any of the species with which it was compared. This will thus mean that intensive breeding programmes will be necessary to ensure durability of disease tolerance. It also supports previous hypotheses that C. albofundus is native to South Africa and may be a temperate species, not found in tropical areas where its close relative, C. fimbriata, commonly occurs. The first unequivocal report of C. fimbriata and Ch. elegans from A. mearnsii is presented in Chapter three. Both these fungi were isolated from dying trees with typical symptoms of Ceratocystis wilt caused by C. albofundus. Both were shown to be capable of causing disease to seedlings under green house conditions. It was, however, found that C. albofundus is more virulent than either Ch. elegans or C. fimbriata. Both isolates were identified using molecular and morphological approaches. Unfortunately only one isolate of each exists and surveys to obtain additional samples continue to be a priority. The first report of a wilt disease of Eucalyptus, caused by Ceratocystis fimbriata in the Republic of the Congo in West Africa is recorded in Chapter four. This is not only the first report of C. fimbriata as a pathogen of Eucalyptus in Africa but is also one of the few unequivocal reports of this fungus from the continent. Pathogenicity of C. fimbriata on Eucalyptus spp. was confirmed in glass house tests. In this Chapter, C. fimbriata and C. albofundus from A. mearnsii, and C. fimbriata from Eucalyptus in Brazil were also compared to the C. fimbriata from the Congo. Comparison of the lTS region of the rRNA operon showed that isolates from all three areas grouped together in a clade of C. fimbriata, separate from European isolates. Sequence data showed that C. fimbriata from A. mearnsii in South Africa is nearly identical to the fungi from Eucalyptus in Brazil and Congo, suggesting that they may have a common origin. These findings stress the importance of sound quarantine measures to prevent the introduction of potentially devastating pathogens to South Africa. It is not yet known why C. fimbriata has not caused more diseases on A. mearnsii or Eucalyptus spp. in the country, but the situation will need to be monitored closely. Apart from C. albofundus, there are many other fungi that cause disease of A. mearnsii in South Africa. Chapter five reports on a species of Seiridium that was isolated from stem cankers on A. mearnsii. Morphological and molecular comparisons, as well as pathogenicity studies have shown that the species from A. mearnsii is similar to those species responsible for Cypress canker in many parts of the world. It also confirms previous reports that the taxonomy of the three Seiridium spp. causing cypress canker needs re-evaluation, since molecular data support the view that the three species, represent a single taxon. Pathogenicity trials on mature Cuppressus lusitanica and on A. mearnsii trees showed that both the cypress and A. mearnsii isolates are capable of causing lesions on both hosts. Many of the fungi isolated from diseased A. mearnsii during the current and previous studies of diseases resulted in the isolation of fungi, commonly found as latent pathogens on other forest trees. Chapter six encompassed a survey of the endophytic fungi of A. mearnsii, with the specific aim of identifying possible pathogens. Thirty different fungal taxa were found as endophytes of the xylem and rachi. These included F. graminearum and Botryosphaeria dothidea, which are known pathogens. During periods of environmental stress, these fungi can apparently cause disease. This is especially true because A. mearnsii is often planted on marginal sites in South Africa. Chapter seven represents the first report of Fusarium graminearum from A. mearnsii and presents evidence for the fungus being involved in disease of A. mearnsii. This pathogen was first isolated during 1994-95 disease surveys, but was not identified due to the fact that cultures on artificial media did not sporulate. In the current study, additional isolates were obtained from stem cankers and die-back symptoms and the fungus was identified based on β-tubulin gene sequences. Field inoculations using F. graminearum showed extensive lesion formation in the xylem. Previously, this Fusarium sp. was known only as a pathogen of maize and wheat in various parts of the world. Results of this study are, therefore, enigmatic and intriguing.
Open Access
Molecular taxonomy and mating type genes in Ceratocystis sensu stricto
(University of the Free State, 1999-01) Witthuhn (née Strydom), Regina Cornelia; Wingfield, B. D.; Wingfield, M. J.; Harrington, T. C.
English: Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants. In the research presented in this thesis, I have developed a rapid and reliable PCR-based RFLP identification method for species of Ceratocystis. A 1.6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small and large sub-unit rRNA genes, the 5.8S rRNA gene and the internal transcribed spaeers 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (AluI, DraI, HaeIII and RsaI) produced RFLPs that separated the species of Ceratocystis. The 1.6 kb PCR products, from the better known species of Ceratocystis, were sequenced and phylogenetically analyzed. The delimitation of taxa was consistent with results of previous studies using isozymes and rDNA sequence analysis. Ceratocystis coerulescens is a well-known cause of blue stain in spruce and pine. It was shown that C. coerulescens encompasses at least five morphological types. I compared isolates of C. coerulescens sensu lato and morphologically similar species on the basis of lTS DNA sequences. A 600 bp fragment within the ribosomal DNA operon, including the 5.8S rRNA gene and ITS 1 and 2, was amplified, sequenced and analyzed using PAUP. The five morphological types previously known as C. coerulescens, and the two other taxa from conifers, formed a strongly supported monophyletic group that includes all the Ceratocystis species occurring primarily on conifers. The species from hardwood trees, C. eucalypti, Ch. australis and Ch. neocaledoniae, also formed a monophyletic group, sister to the conifer group. The fourth species from hardwoods, C. virescens, formed a group basal to the two sister groups. The phylogeny of species in the C. coerulescens complex based on ITS DNA sequences were compared to the phylogeny based on the MAT-2 HMG box DNA sequences. A 210 bp PCR fragment of part of the MA T-2 HMG box of species in the C. coerulescens complex was amplified. C.fimbriata was used as the outgroup taxon and was distinct from the other Ceratocystis species studied. The species from conifers formed a single clade, sister to the clade formed by the species from hardwoods. Phylogenetic analysis of the MAT-2 HMG box DNA sequences differed slightly from the phylogenetic analysis based on ITS DNA sequences. Based on ITS DNA sequences C. vireseens was basal to the other species in the C. coerulescens complex, while C. laricicola and C. polonica could not be separated from each other. Differences in the MAT-2 HMG box DNA sequences for the latter two species clearly showed them to be as distinct from each other. Recent mating studies on C. coerulescens have prompted a study of the expression of mating type genes in species of Ceratocystis sensu stricto. C. eucalypti is strictly heterothallic. Most other Ceratocystis species, including C. virescens, C. coerulescens and C. pinicola are homothallic. The MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. Part of the MAT-2 idiomorph in C. eucalypti, C. vireseens and C. pinicola was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA binding motif. The expected ~300 bp PCR products were cloned and sequenced. Specific primers were designed that amplified a 210 bp fragment only in MAT-2 isolates of C. eucalypti, C. vireseens and C. pinicola. This fragment was absent from the self-sterile (MAT -1) progeny of C. vireseens and C. pinicola, confirming the deletion of MAT-2 during uni-directional mating type switching. The known DNA sequence data for the C. eucalypti MAT-2 mating type idiomorph was increased from 280 bp to 1 371 bp, using TAIL-peR and uneven PCR.
Open Access
Determining the canning quality of small seeded white beans (Phaseolus vulgaris L.)
(University of the Free State, 1999-05) De Lange, Anna Francina; Osthoff, G; Labuschagne, M. T.
English: It is important to the producers, processors and consumers in south Africa to have dry bean cultivars with acceptable canning quality. Therefore, dry bean breeders needs sui table screening methods to evaluate the various lines at an early stage (F4) when only small amounts of seed are available. A micro-canning method to evaluate canning beans .in tomato sauce has been developed and compared to the commercial processing procedures with comparable results. External factors that influenced the canning quality like the water quality were investigated. This micro-canning method could therefore be used to investigate the effect of genotype and environment interactions that significantly influenced the canning quality. The objectives of this study were to: o Evaluate different qeriet i.c material for use as parents in the breeding program. $ To obtain a better understanding of canning quality characteristics of beans and to ensure that the most important characteristics are evaluated and the component interrelationships. • Determine the genotypic, environmental and genotype x environment interactions that influenced the canning quality. • To ascertain the patterns of interrelationships of the canning quality parameters and chemical analysis. • To investigate the patterns and relationships between standard and choice grade cultivars. e To investigate stability of locali ties between seasons as well as the clustering of different environments and seasons. Small seeded white beans, carioca and yellow haricot beans were used to determine variability in canning quality but as a result of a lower canning quality of coloured beans, only small seeded white beans were used for further investigations. As result of the investigations of different a characteristics, only the seed size, water absorption during soaking and canning, the texture and subjective visual appearance evaluations were used to determine canning quality. These characteristics were interrelated but single no parameter could explain variation' in canning quality. Canonical correlation analysis was used to determine to what extent variation of chemical components was responsible for differences in canning quality and these results indicated that mainly potassium and calcium would influence the water absorption and texture, respectively. Canonical variate analysis was used to determine the difference between unacceptable, standard and choice grade cultivars. A model was described from these analysis that could be applied to independent data sets that results in coordinates that differentiates the lines or cultivars as unacceptable, standard and choice grade. Significant interactions between genotype, environmental and seasonal effects for canning quality traits indicated that cultivar responses to variation in localities and seasons differ. Environmental effects resulted in inconsistent quality measurements since trait expression is strongly influenced by genotype x environment interactions. Results from this study suggested that the difference between cultivars could therefore be due to a complex interaction of the chemical and structural composition, which is genetically determined and influenced by the anv i ronmen t . as well as the changes that occur during processing. The Additive Main Effects and Multiplicative Interaction (AMMI) model mainly grouped KwaZulu-Natal separately as a region with poor canning quality. The rest of South Africa's localities grouped different for each season. Resulting from this investigation, several recommendations can be made: c Breeding material should be tested fo~ more than one season in order to select superior and more stable lines. o Elimination of canning quality evaluations of KwaZulu-Natal could improve the genetic progress. • Exploitation of the interactions by breeding for specific adaptation in a region of homogeneous area. Demarcating one or two areas in South Africa for the exclusive production of small white canning beans could improve the overall canning quality of the small white bean production in South Africa.
Open Access
Utilization of wood-decay fungi for biokraft pulping of softwood
(University of the Free State, 1999-05) Wolfaardt, Jacobus Francois; Rabie, C. J.; Wingfield, M. J.
English: The forest products industry is one of the most important earners of foreign exchange for South Africa. The major focus of the industry is the production of pulp with an annual capacity of 2,4 million tons. Wood from plantations of exotic trees is the most important source of fibre, but other fibre sources are also used. Biotechnology can play a significant role in the industry to produce high value products at lower cost and could reduce the environmental impact of conventional processes. Biopulping is potentially the most important of these biotechnological processes, because it can influence all downstream operations. The aim of this study was, therefore, to develop a biopulping process for the treatment of softwood at the Sappi Ngodwana kraft mill in Mpumalanga. Initially, 278 strains of wood-decay fungi were collected from various natural habitats. This collection represents a diversity of fungal families and included species that have not previously been recorded from South Africa. The first step in selecting suitable fungal strains for biopulping was to characterize different groups on the basis of the enzymes that they produce and their oxidase reactions. The suitability of these strains for the pre-treatment of softwood chips prior to kraft pulping was subsequently assessed by evaluating their influence on kappa number, yield and strength properties of pulp. Seven of the strains tested were able to reduce the kappa number of pulp significantly, without having a significant influence on the pulp yield. These strains were more efficient than strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora that have been patented for other biopulping applications. Treatment of wood with strains of Peniophora sp., an unidentified specie as well as two strains of Stereum hirsutum resulted in pulp with improved strength properties. The envisaged biopulping process aimed at treating wood chips in outside chip storage with a biopulping fungus. The aim of one study was to investigate conditions such as temperature, moisture, CO2 and microbial populations that develop in a chip pile, and to determine the suitability of the chip pile for colonization by biopulping fungi. High temperatures and high moisture levels were observed in some areas of the chip pile, which suggested that part of the pile was unsuitable for colonization by mesophilic fungi. It will, therefore, be necessary to manage the chip pile to maintain a suitable environment for biopulping. Problems were experienced with poor colonization of freshly chipped softwood by biopulping fungi. The effect of contaminating microbes and inhibitory compounds present in wood was, therefore, studied. It was found that the inhibition of biopulping fungi by α-pinene and by contaminating microbes were both very important. The inhibition by microbes, as well as by extractives, was mitigated by a short steam treatment of wood chips. Steaming for ten minutes under atmospheric pressure could be an economical method to improve colonization by biopulping fungi. Alternatively biopulping fungi with good competitive ability and tolerance to monoterpenes could be selected. Pinus patula wood chips were pre-treated with a selected strain of Stereum hirsutum to determine the optimal conditions for the kraft pulping of pre-treated softwood and to do an economic evaluation of the process. Chips were pulped on a small scale under various pulping conditions. Lignin content, yield, and viscosity of the pulp as well as alkali consumption were evaluated. The results were used to develop models for biokraft pulping. This study showed that biopulping can reduce the kappa number of pulp or reduce the pulping time. Pulp yield losses were relatively small when pulps with low kappa number were produced. Increased alkali consumption was, however, an important factor in the economic evaluation.
Open Access
Factors associated with coniothyrium canker of Eucalyptus in South Africa
(University of the Free State, 1999-05) Van Zyl, Leonel Merwe; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.
English: English: In chapter one of this thesis, the literature pertaining to the genus Coniothyrium and its importance in plant pathology, is reviewed. Special attention is given to Coniothyrium species associated with Eucalyptus but the focus is on Eucalyptus stem canker pathogen, C. zuluense. Coniothyrium zuluense is an important pathogen in South Africa and has, since its discovery, become widespread throughout plantation areas of KwaZulu-Natal. The current means for reducing the impact of this disease is to plant disease resistant species and clones of Eucalyptus. It is evident from this review that very little information is available pertaining to the biology, reproductive system, or the population structure of C. zuluense. Such information is essential for managing the disease successfully in the future. The strategy currently used to reduce the impact of Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Results of the study represented in chapter two showed that there is considerable variation in colony colour and pathogenicity of a large collection (344) of C. zuluense isolates. Conidial morphology and growth requirements are, however, similar for all isolates tested. The considerable variation in pathogenicity indicates that C. zuluense has been present in South Africa for an extended period of time, or that virulence is changing rapidly due to strong directional selection pressure. Results of the taxonomic and pathogenicity studies in chapter two, suggest that the C. zuluense population is well established. In chapter three, the population diversity of 108 C. zuluense isolates, differing in their pathogenicity to a susceptible Eucalyptus clone, was investigated using Amplified Fragment Length Polymorphism (AFLP) technology. Results indicated that the level of genetic diversity is relatively low, but higher than expected for an asexually reproducing pathogen. Genetic similarity values also indicated a significant population differentiation between different plantation regions (subpopulations), suggesting that gene flow, together with selection, might be responsible for most of the gene diversity. New epidemics would, therefore, not be as a result of the emergence of new aggressive strains, but would rather be due to the introduction of susceptible Eucalyptus species, together with environmental conditions favouring disease development. A Coniothyrium species associated with similar symptoms to those associated with C. zuluense in South Africa was observed on E. camaldulensis in Thailand in 1996. It was previously thought that C. zuluense was restricted to South Africa. In chapter four, I show using morphological and molecular comparisons, as well as pathogenicity studies, that C. zuluense and the Coniothyrium sp. from Thailand are the same organism. This is, thus, the first record of this important Eucalyptus stem canker pathogen, C. zuluense, outside South Africa. Bacteria commonly exude from necrotic cankers on severely infected Eucalyptus clones in plantations. In chapter five, it was shown that bacteria associated with Coniothyrium canker in the field are species of the genus Pantoea. These species were identified based on 16S rDNA sequence data as P. ananatis pv. ananatis and a species closely related to P. stewartii subsp. stewartii. It was also shown that a synergistic interaction between C. zuluense and both Pantoea species exists. Inoculation studies, using both Pantoea species together with C. zuluense isolates, resulted in a significant increase in pathogenicity as opposed to inoculations where the bacterial and fungal isolates were used alone. Future studies should consider the presence or absence of both bacteria species in disease development in Thailand. During plant-pathogen interactions, pathogens are known to produce cell wall degrading enzymes, in particular pectin degrading enzymes. Polygalacturonase (PG) is the first enzyme produced during such interactions and is known to be a determining factor in pathogenicity. Chapter six showed that C. zuluense isolates and both Pantoea species, P. ananatis pv. ananatis and an unknown Pantoea sp., produce PG. Experimental assays show that levels of PG activity for both Pantoea spp. are significantly higher than those obtained for C. zuluense isolates. As PG is the first enzyme produced during disease development it is hypothesised that the two Pantoea species might play a significant role in the development of Coniothyrium canker. Production of PG could also be used as an assay to evaluate pathogenicity in different isolates of C. zuluense. Pathogen-produced cell wall-degrading enzymes play a key role in activating plant defence responses. Most inducible defence responses are the result of transcriptional activation of genes. Various plant resistance (R) genes, as well as pathogenesis-related proteins, such as polygalacturonase inhibiting proteins (PGIPs), have been linked with resistance to various fungal and bacterial pathogens. In chapter seven, a partially sequenced resistance gene from disease resistant E. grandis clone, TAG 5, was shown to be similar to a gene associated with a disease resistance gene in Arabidopsis thaliana. The most exciting aspect of this study was, however, the discovery of a shift in reading frame of this gene for the susceptible Eucalyptus clone, ZG 14. The complete sequence of this gene should provide a more complete view of its importance in disease resistance. Screening for similar interruptions in the open reading frame of various commercially available Eucalyptus clones could significantly speed up breeding programmes aimed at producing improved disease resistant clones.
Open Access
Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins
(University of the Free State, 1999-06) Botes, Adriana Leonora; Smit, M. S.; Litthauer, D.
English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).
Open Access
Mycotoxin contamination of maize in relation to insect infestation, agricultural practices and agroecology in the Republic of Cameroon
(University of the Free State, 1999-09) Ngoko, Zachee; Marasas, W. F. O.; Wingfield, M. J.; Cardwell, K. F.
English: Maize (Zea mays L.), the staple food crop of the majority of the population of Cameroon, is damaged by insects and diseases from the fields to the stores. As a result, the quantity and the quality of harvested grain is reduced. This study was undertaken to identify constraints associated with the production and post-harvest losses of this commodity in two ecological zones ofCameroon from 1995 to 1997. Farmers' perceptions of diseases and pests play an important role in their acceptance of new pest management technologies. From the survey conducted to assess their perceptions, farmers reported that borers (Busseola fusca) were the main constraint to maize production in the Humid Forest and Western Highlands. Locusts (Zonecerus variegatus) and rodents were the second most important limiting factor in the Humid Forest. The storage weevil (Sitophilus zeamais) was the most damaging storage insect. Diseases were not generally known by farmers who could only recognize smuts and ear rots by the visible damage caused by them. While the period of the outbreaks of insect infestation was not reported with precision, most farmers reported that diseases occurred at the mid-season. Control practices were not well established. Disease surveys conducted from 1995 to 1997, revealed that lowland blight (Bipolaris maydis, Diplodia leaf spot (Stenocarpella macrospora) and sheath blight (Rhizoctonia solani) were the most important maize diseases in the Humid Forest, while highland blight (Exserohilum turcicum) and grey leaf spot (Cercospora zeae-maydis) prevailed in the Western Highlands. Phaeosphearia leaf spot (Phaeosphaeria maydis) was specific to the Western Highlands with a negative relationship with grey leaf spot. Busseola fusca infested maize plants at all stages of growth with high prevalence in the Humid Forest. The identification of factors -affecting maize yield demonstrated that diseases, insects and their interactions with soil infertility, soil texture, weeds, and maize varieties were responsible for the reduction of maize production. Yield reductions were 30% and 33.6% respectively, in the Humid Forest in 1995 and 1996 due to Stenocarpella macrospora, Puccinia polysora and Rhizoctonia solani. In the Western Highlands, Cercospora zeaemaydis, Busseola fusca, stem diseases, and physiological spot caused yield reductions of 51.2%, and 37.9% in 1996 and 1997, respectively. Mycological and chemical analyses of maize grain collected from 72 farmers' stores showed that several pathogens were associated with grain quality deterioration. Nigrospora spp. were the most frequently isolated fungi on kernels, followed by Fusarium moniliforme and Fusarium graminearum. Aspergillus spp. were rare in both zones. Fumonisin B1, deoxynivalenol and zearalenone were detected in maize samples at levels ranging from 300 to 26,000ng/g, 100 to 1300 ng/g, and 50 to 110 ng/g, respectively. This is the first report on the natural occurrence of these Fusarium mycotoxins in maize in Cameroon. Surveys conducted to identify the biological and physical factors that enhanced the infection of maize kernels by fungi and the contamination with fumonisin , identified several agricultural techniques related to grain quality in the Western Highlands. Harvesting in June (11.1 %) or July (23.6%), sorting right from the field (16.7%), drying over the fireplace with husk (19.4) or without husk (33.3%) and storing shelled grain in bags (19.4%) or boxes (9.7%) reduced fumonisin contamination. Continuous production of maize on the same field, harvesting in August, and the infestation by the weevil Sitophilus zeamais were factors that increased fumonisin contamination. Crop rotation, sorting maize during all the post-harvest processes and the treatment of maize grain with appropriate insecticides should decrease the risk of contamination by fumonisin. Continuing collaborative research should aim at understanding farmers' needs and priorities, investigating the epidemiology of maize diseases, screening for resistance to the most important maize diseases and improving harvesting, sorting, drying and storing methods in Cameroon.
Open Access
The production of 3-hydroxy fatty acids by the yeast Dipodascopsis uninucleata and its implications
(University of the Free State, 1999-11) Venter, Pierre; Kock, J. L. F.; Coetzee, D. J.
English: In 1991, a novel eicosanoid namely 3-hydroxy-5, 8, 11, 14-eicosatetraenoic acid (3-HETE) was uncovered in the yeast Dipodascopsis uninucleata by van Dyk and co-workers. Strikingly, the production of this compound was found to be sensitive to low concentrations of aspirin and indomethacin. With this as background, a study was conducted that unveiled the possible biochemical pathway used by this yeast for the production of 3-HETE. Here, various fatty acids were fed to D. uninucleata, and the extracted samples analysed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography - mass spectrometry. It was found that 3-hydroxylation of fatty acids in D. uninucleata requires a 5Z, BZ - diene system either directly or following initial incomplete l3-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy - 5Z, BZ, 11Z, 14Z - eicosatetraenoic acid (3R - HETE), which rules out normal l3-oxidation as a biosynthetic route. Consequently, studies on the biological dynamics and distribution of 3-hydroxy oxylipins in D. uninucleata followed. The occurrence of oxylipins was mapped by immunofluorescence microscopy (IF) in fixed cells, with or without cell walls, using an antibody raised against 3R-HETE. This antibody turned out to cross-react with other 3-hydroxy oxylipins. These compounds were detected in situ in gametangia, asci, as well as between released aggregating ascospores. Aspirin (1mM), which is known to suppress the formation of 3-hydroxy oxylipins from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as cell aggregation, suggesting a prominent regulatory role of 3-hydroxy oxylipins for the latter. Since these oxylipins are associated with the aggregation of sexual cells in D. uninuc/eata, the next step was to screen for the presence of these compounds in aggregating cells of other yeasts such as the biotechnological important Saccharomyces cerevisiae. It was found that oxylipins such as 3-hydroxy-8:0 and 3-hydroxy-10:0 are produced over the growth cycle of the flocculating yeast Saccharomyces cerevisiae ATCC 26602. Using oxylipin specific antibodies in IF studies, it was demonstrated that these compounds are synthesized continuously from an early stage of growth and are associated with the cell wall and are present between flocculating cells. Similar results were obtained with a NewFlo phenotype flocculent brewing yeast strain. This implicated the involvement of oxylipins in cell aggregation. Further investigations using scanning- and transmission electronmicroscopy, indicated that changes in the depositing of lipid rich osmiophilic layers in the yeast followed the same pattern as the IF results. Immunogold studies verified the presence of oxylipins in these osmiophilic layers. It was uncovered that the oxylipin containing osmiophilic layers play an important role in cell aggregation. Surprisingly, further investigations implicated the presence of aspirin sensitive 3-hydroxy oxylipins in the LPS layer of the Gram-negative bacterium Escherichia co/i. In this study it was found that aspirin, at moderate concentrations, influences the biosynthesis of the endotoxic 3-hydroxylated myristic acid in the Lipid A of Gram-negative bacteria. This discovery therefore suggests an important role for aspirin as a therapeutic agent in the treatment of LPS mediated diseases
Open Access
Evaluation of the growth and survival of probiotic microorganisms in commercial bio-yogurt
(University of the Free State, 2000-11) Hattingh, Analie; Viljoen, B. C.
A review of the literature highlighting the importance of the 'therapeutic minimum' and the survival of the probiotic bacteria in fermented milk bioproducts is given in Chapter 2. Special reference is made to the historical background of probiotics, its therapeutic value and the survival through passage in the gastrointestinal tract. In addition, technology of bio-yogurt, factors affecting the survival of probiotic bacteria in yogurt, and the media for the differential enumeration of these microorganisms in dairy products are discussed. In Chapter 3 existing media proposed for the selective enumeration of starter cultures employed in the manufacture of bio-yogurt are compared and evaluated. It is essential for comparison reasons to standardize enumeration methods for microbial analyses in order to study the incidence of the probiotic bacteria in the presence of the conventional starter cultures. The media proposed by Chr. Hansen's laboratory proved to be the most suitable for the enumeration of the different cultures. It is essential that bio-yogurts meet the criteria of a minimum of 106 cfu/rnl of probiotic bacteria until the expiry date to induce any potential therapeutic advantages for the consumer. Consequently, in Chapter 4 we evaluated samples of AB-yogurt obtained from supermarket outlets statistically based on the enumeration of viable probiotic cultures, Lactobacillus acidophilus and Bifidobacterium bifidum; as well as conventional yogurt starter cultures, Streptococcus thermophilus and Lactobacillus bulgaricus and the maintenance with respect to the 'therapeutic minimum'. Based on the data obtained, the AB-yogurts examined comply with the criteria regarding the number of viable cells of L. acidophilus, but the consumer would not have received sufficient numbers of B. bifidum cells at the time of consumption. In Chapter 5, we monitored the survival of viable cells of the probiotic cultures and starter cultures present in bio-yogurt at frequent intervals from day 1 until the expiry date at day 31 stored at 4eC and loec. B.bifidum never exceeded counts of 106 cfuZgin any of the samples and a constant decline in its numbers was observed. L. acidophilus, despite maintaining counts higher than 106 cfu/ g in the yogurt samples, also exhibited a substantial decrease in its numbers during storage. Due to the poor survival of probiotic cultures in yogurt, we incorporated a probiotic yeast species, S. boulardii as part of the starter culture in Chapter 6 and monitored its progression and survival in yogurt and milk products. Despite good growth and the survival of the yeast species until the expiry date, excessive gas and alcohol production proved, however, to be major constraints. In order to further study the effect of yeast growth on the survival of probiotic bacteria in bio-yogurt, pure cultures of Kluyveromyces marxianus, Issatchenkia orienialis, Debaryomyces harisenii and Yarrotoia lipolytica were inoculated into commercial AB-yogurt, sterile milk and pasteurised sweetened yogurt in Chapter 7. The yeast species were able to progress in the bio-yogurt reaching maximum counts exceeding 107 cfu/ g. Despite the inability of some species to utilise lactose, the yeast species utilised available organic acids, galactose and glucose derived from bacterial metabolism of the milk lactose, as well as possible free fatty acids or free amino acids present in the dairy products and thereby sufficiently contributed to the retention or enhancing of the pH values. The production of excessive gas and alcohol was major constraints in implementing Kluyveromyces marxianus, Issatchenkia orienialis. The inclusion of Y. lipolytica and D.hansenii in AB-yogurts, therefore, seemed the most promising in controlling the pH to assure the viability of the pro-biotic microorganisms. In Chapter 8, Y. lipolytica and D.hansenii cultures were inoculated into commercial plain and fruit AB-yogurt at moderate (105 -106 cfujml) and low level (10² - 10³ cfujml), directly after manufacture and with the ABT-starter before fermentation, to compare the effects that the yeast will have on viability of probiotic bacteria. Viable bifidobacteria counts remained virtually the same in the bio-yogurt inoculated with the yeast cultures during the refrigerated storage period. A rapid decrease in L. acidophilus occurred after 2 weeks storage (2-4 log cycles) in the yeast-inoculated bio-yogurt suggesting possible antagonistic action of the yeast against L. acidophilus. Addition of the yeast primarily encouraged the growth of streptococci, which had an influence on the pH of the yogurt environment. A gradual decrease in the pH of all the bioyogurt products was observed. pH was affected by enhanced growth of streptococci, utilization of organic acids by the yeasts and the fact that L. bulgaricus was excluded form the yogurt starter culture. The yogurt inoculated with D. hanserui was still acceptable and had a pleasant taste compared to the control yogurt after 30 days storage. Inclusion of yeast as part of the starter culture for bio-yogurts seems promising. In Chapter 9, possible enhancement of the growth and survival of Bifidobacteria in bio-yogurt by the addition of a prebiotic was investigated. Commercial AB-yogurt was fortified with 1, 2 and 3% of the fructooligosaccharide, neokestose, and growth and survival of bifidobacteria as well as L.acidophilus and S. thermophilus were monitored during storage at SoC over 30 days. With the addition of neokestose the viable bifidobacteria count remained significantly higher in all the yogurts when compared to traditional yogurts without the addition of neokestose. L. acidophilus reduction in viable counts did not exceed a 30% reduction; therefore neokestose also had a better survival effect on L. acidophilus. The addition of neokestose had no affect on the survival of S. thermophilus.
Open Access
Biology of botryosphaeria dothidea and sphaeropsis sapinea as endophytes of eucalypts and pines in South Africa
(University of the Free State, 2001-12) Smith, Hendrik; Wingfield, M. J.; Coutinho, T. A.; Crous, P.W.
English: Botryosphaeria dothidea and Sphaeropsis sapinea both very important pathogens in the South African forestry context. These fungi are well established in the country and contribute substantially to annual losses incurred. Currently very little can be done to control the fungi and the damage they cause. The understanding of their respective disease etiologies is thus of great importance to develop relevant counter measures. The overall aim of this dissertation was to investigate various poorly understood aspects of these two fungi and to try and relate the results to practical contributions towards controlling the impact the two pathogens have. Studies have been conducted during the course of five years and each study represents an independent research investigation. The introductory chapter presents a review of the literature pertaining to all aspects of biology, history and taxonomy of B. dothidea and S. sapinea. The two fungi are clearly very similar in all these aspects and perhaps the only clear difference is that S. sapinea is restricted to pines in South Africa. Many other similarities and some differences between these two important pathogens are highlighted and many of these have provided the background for further investigations. In chapter two the presence of B. dothidea and S. sapinea lS demonstrated as symptomless endophytes in healthy, pine and eucalypt tissue. Botryosphaeria dothidea was found to be common in all the Eucalyptus spp. tested, occurring at high percentages in symptomless leaves of Eucalptus smithii, E. camaldulensis, E. grandis and E. nitens. Sphaeropsis sapinea was, in contrast, only present in young, green Pinus patuIa and P. radiata cones, but virtually absent from the cones of P. elliottii and P. taeda. Botryosphaeria dothidea is associated with die-back and canker diseases of eucalypts in South Africa. Despite this fact, little is known about the infection process. The fungus is known to occur endophytically in leaves of various Eucalyptus species in South Africa. In chapter three I consider the ability of B. dothidea to infect apparently healthy Eucalyptus leaves and the subsequent location and structure of these infections once inside leaf tissue. Scanning electron microscopy revealed that conidia of B. dothidea can infect healthy leaves through stomata. These infections ultimately reside amongst mesophyll cells and constitute a number of individual infections per leaf. Two morphologically similar fungi are associated with die-back and canker of eucalypts in South Africa. The one was identified as part of the Botryosphaeria dothidea-complex. In chapter four, the identity of the second fungus was determined by comparing morphology, pathogenicity and DNA sequence analysis of isolates of both taxa. Based on results obtained, Botryosphaeria eucalyptorum, and its anamorph Fusicoccum eucalyptorum, are described as a new species. I found that the teleomorph is morphologically similar to other taxa in the B. dothidea-complex, but conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal transcribed spaeers ITS 1 and ITS2. As is the case with B. doth idea , the fungus is pathogenic to Eucalyptus, there do not, however, appear to be differences in pathogenicity between the two. Sphaeropsis sapinea is the most important pathogen of pines in South Africa. The fungus, which reproduces only asexually, occurs only on exotic pines. In chapter five, I investigated the diversity of the S. sapinea population in South Africa and compared it with a population from Northern Sumatra. Both populations were obtained from exotic P. patuIa plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percentage maximum genotypic diversity, based on Stoddard and Taylor's index, for the South African population was much higher than the Northern Sumatran population, thus indicating that the South African S. sapinea population was more diverse than the Northern Sumatran population. These results support the hypothesis that the population of S. sapinea in South Africa has been introduced from various parts of the world, during the last century. In chapter six, I investigated the role that latent S. sapinea infections in seed cones of P. patuIa, play in post-hail associated die-back. Pinus patuIa seed cones were found to be infected during the second year of development, with extensive colonization only occurring m the third year when cones mature, prior to seed discharge. Vegetative compatibility tests revealed that the presence of S. sapinea in individual third year seed cones is confined to a single genetic entity. Sphaeropsis sapinea colonisation of third year seed cones thus, apparently results from a single successful infection per cone. The probable role of latent infections by S. sapinea indicated that tree age and by implication, increased numbers of attached seed cones, contributes to more severe die-back after hail damage. The control of damage caused by S. sapinea is highly dependant on a dynamic hybridisation programme. Alternative species of pines is thus constantly evaluated for potential. In chapter seven, 65 families representing both the northern and southern populations of P. greggii were evaluated for their tolerance to infection and subsequent die-back caused by S. sapinea. Families were evaluated following natural infection after hail damage, as well as by artificial inoculation. Variation in tolerance occurred and was highly significant between the two provenances, with the northern provenance proving to be very tolerant. Pinus greggii trees of the southern provenances were comparable with P. patula. The potential of the families from northern origins has to be investigated further. Cultures of Cytospora isolated from Eucalyptus trees in South Africa, Congo, Thailand, Venezuela, Mexico, Uganda and Australia, as well as Cytospora-like isolates from Indonesia were compared in chapter eight. Comparisons were based on the homology of the internal transcribed spaeer regions and the 5.8S ribosomal DNA of the nuclear ribosomal DNA repeat unit. Isolates clustered into at least three unrelated groupings, with a fourth grouping that included isolates that morphologically resembled Cytospora. Results from this chapter indicated that the current description of Valsa ceratosperma encompasses several distinctly different species and needs to be further refined. Botryosphaeria dothidea and S. sapinea are two of the most important pathogens of eucalypts and pines in South Africa. The fact that they exist as symptom less endophytes in trees has added a fascinating aspect to our understanding of their role in tree diseases. In the past, they have generally been considered to be wound infecting opportunistic fungi. Results of these studies have shown that this is not so and that they are clearly able to infect healthy trees. They are unlikely to be able to infect dead or moribund tissue. The investigations presented in this dissertation have added considerable knowledge to our understanding of B. dothidea and S. sapinea and will also promote efforts to reduce disease caused by them. However, there are many questions that remain to be answered pertaining to them and it is my hope that this study will provide a foundation and stimulus for further work.
Open Access
Bioremediation of a bleach plant effluent from the pulp and paper industry
(University of the Free State, 2003-11) Van Driessel, Brian; Christopher, L.
English: Bleach plant effluent was characterised by physico-chemical methods. The chemistry of the bleach plant effluent was examined to devise effective treatment methods. Effluent contained trace amounts of nitrogen as well as carbohydrates and no ortho phosphate could be detected in the wastewater. The best decolourisation activities were obtained using adsorption as treatment method, with activated carbon removing > 99% colour from effluent. Chitosan (81%) and chitin (77%) could remove appreciable levels of colour from bleach plant effluent, followed by biomass from Rhizomucor pusillus, a mucoraelean fungus (71%). Chitosan and chitin from the cell wall of R. pusillus might be involved in the fungus decolourisation ability. Effluent pH was inversely related to effluent decolourisation when R. pusillus, chitosan or chitin was used as adsorbents. This might in part be due to acid catalysis during nucleophilic addition reactions, where amino groups of chitin/chitosan react with carbonyl groups in Eo-effluent. Also, chitin and chitosan amino groups can be protonated under acidic conditions and acquire positive charges that can interact with the chromophores found in Eo-effluent. However, pH exerted no significant effect on decolourisation when activated carbon was employed as adsorbent of effluent colour. Decolourisation employing commercial adsorbents seemed to be mainly due to chemisorption. Adsorption experiments conducted at various ionic strengths indicated that coulombic interactions are responsible for a fraction of the decolourisation activity of chitosan and chitin. Nevertheless, decolourisation obtained with RM7 and activated carbon was unaffected by the ionic strength. Flocculation of coloured compounds from Eo-effluent by chitosan containing solutions resulted in a maximum decolourisation of 75%. Anion-exchange treatment removed 96% colour from Eo-effluent. Ultraviolet irradiation could decolourise the Eo-effluent by about 42 to 43%. Decolourisation using organic solvent extraction proved ineffective with a highest colour removal efficiency of only 21% being achieved. Biological methods used for effluent remediation were: 1) Trickling filters, 2) Activated sludge reactors and 3) Rotating biological contactor reactors (RBC). Treatment using one biological system was followed by treatment in another system With trickling filters containing immobilised white-rot fungi, the highest decolourisation (61%) was obtained with Coriolus versicolor. This fungus required a co-substrate to efficiently decolourise the effluent. Effluent treatment in an activated sludge reactor reduced toxicity, COD and chlorophenol levels. However, colour and high molecular mass compounds were not affected significantly by this method of treatment. Decolourisation was studied in a RBC using immobilised C. versicolor and R. pusillus, respectively. The decolourisation rate by both fungi was proportional to initial colour intensities. Decolourisation was not adversely affected by colour intensity, except at the lowest level tested. Decolourisation of 53 to 74% could be attained using a hydraulic retention time of 23 h. Rhizomucor pusillus, removed 55% of AOX compared to a 40% AOX reduction by C. versicolor. Treatment employing R. pusillus and C. versicolor, respectively, rendered the effluent essentially non-toxic. Addition of nutrients to the decolourisation media stimulated colour removal by C. versicolor, but not significantly in the case of R. pusillus. Ligninolytic enzymes (manganese peroxidase and laccase) were only detected in effluent treated by C. versicolor. Decolourisation mechanisms were investigated using gel permeation chromatography. Rhizomucor pusillus decolourised the effluent by adsorption and C. versicolor removed effluent colour by adsorption as well as by biodegradation. Coriolus versicolor could decolourise the effluent for a period of 34 d whereas R. pusillus decolourised the effluent up to 54 d. Further improvements in effluent quality could be attained when treatment using one system was followed by treatment in another system, possibly because of toxicity reduction in the pre-treatment steps.
Open Access
The survival of yeasts and probiotics as adjunct starters in cheese
(University of the Free State, 2003-11) Kotze, Annie Dorethea; Viljoen, B. C.
A literature review dealing with various aspects of cheesemaking and ripening is given in Chapter 1. With respect to starter cultures, the criteria for strain selection as well as the taxonomy of different starter strains were discussed. Cheese ripening is a complex system that involves numerous chemical, physical and bacteriological changes and can be accelerated by various techniques. Special reference was made to yeasts associated with dairy products, their characteristics as well as their contribution in the dairy industry. Yeasts play a very important role in the ripening and manufacturing of a wide variety of cheeses and can cause spoilage or have beneficial effects on cheese ripening. Furthermore, the properties of probiotic bacteria were discussed including their therapeutic value, their survival in bio-products and the possible expansion of the probiotic product range. Cheese offers certain advantages as a delivery system for live probiotic bacteria to the human gastro-intestinal tract. Debaryomyces hansenii and Yarrowia lipolytica are typical foodborne yeast species frequently associated with dairy products and capable of predominating the yeast composition in such systems. The two species fulfil a number of criteria to be regarded as co-starters for cheese making. They are known for their proteolytic and lipolytic activity as well as their compatibility and stimulating action with the lactic acid starter cultures when co-inoculated. Recent studies indicated that yeasts could be included as part of starter cultures for the manufacturing of cheese, enhancing flavour development during the maturation. The potential of D. hansenii and Y. lipolytica as agents for accelerated ripening of matured Cheddar cheese has been evaluated during four cheese treatments. The interaction between the two yeast species and the lactic acid bacteria was surveyed incorporating (i) D. hansenii, (ii) Y. lipolytica and (iii) both species as adjuncts to the starter culture and (iv) a control cheese without any additions for the production of matured Cheddar cheese. The physical and chemical properties of the cheeses were monitored in order to evaluate the contribution of the yeasts to cheese maturation. The yeasts grew in association with the lactic acid bacteria without any inhibition. An enhanced viability of the lactic acid starter bacteria was observed. The yeasts species when individually added contributed to the development of bitter and fruity flavours despite accelerated development of strong Cheddar flavours. When both species were incorporated as part of the starter culture, the cheese, however, had a good strong flavour after a reduced ripening period. The cheese retained the good flavour and aroma for nine months after production. Furthermore, the significant differences in the fatty acid contents of the model and control cheeses designated enhanced enzymatic activities in the model cheese, attributed to the presence of the two yeast species in the model cheese. The simultaneous application of D. hansenii and Y. lipolytica as part of the starter culture for the production of matured Cheddar cheese is proposed. The health benefits of probiotic-containing products are becoming a key factor affecting consumer choice, and therefore the existing limited range of such products needs to be expanded. Cheese may offer certain advantages as a carrier system for live probiotic organisms to the human gastro-intestinal tract. The possibility of introducing strains of Bifidobacterium bifidum and Lactobacillus acidophilus into South African commercial Cheddar and Gouda cheese were explored. The organisms viability during long-term ripening and storage, their effect on cheese flavour and texture as well as the chemical properties of the cheese were determined. L. acidophilus remained highly viable for at least 120 days of ripening, while the numbers of B. bifidum remained above the therapeutic minimum for 70 days in both cheeses, satisfying the criteria for probiotic foods in Gouda cheese with its shorter shelf life. The viability profile of the lactic acid starter bacteria was not affected and a normal good cheese texture and appearance were retained. Both Cheddar and Gouda cheese offer potential to be effective vehicles for the delivery of probiotic strains to the consumer despite the development of sour and bitter tastes. Additional strains need to be selected and evaluated to prevent these constraints.
Open Access
Development and application of a small-scale canning procedure for the evaluation of small white beans (phaseolus vulgaris)
(University of the Free State, 2004) Van Loggerenberg, Magdalena; Osthoff, G.; Pretorius, A. J.
English: Laboratory canning and evaluation of dry beans are common practices for testing canning quality of cultivars before commercial release to canning industries. Suitable laboratory canning and evaluation procedures for small white beans in tomato sauce were identified. Standard values for choice and standard grade beans for laboratory evaluation of canning quality were defined, using four small white bean cultivars from nine localities during the 2000/01 season. The cultivar Teebus was used as reference standard for choice grade beans and its canning quality complied with international guidelines when the modified canning technique (MCT) was used. From the laboratory and modified canning evaluation procedures hydration coefficient, percentage washed drained weight, visual appearance (scale 1 to 10), splits (scale 1 to 10), texture (kg.100 g -1 .12 s -1 ), size, clumping, L-values, aL-values and bL-values were identified as suitable canning parameters for small scale evaluation of beans. Beans canned with the MCT were also canned and evaluated industrially and results compared. The interpretation of the different canning parameters with laboratory and industrial canning were simplified by the use of canonical variate analysis (CVA). Canonical variate analysis indicated the same groupings for cultivars according to choice and standard grade canning quality for laboratory and industrial canned beans. Laboratory canning and evaluation could be used in the evaluation of the canning quality of beans intended for industrial canning. Canning quality of seven small white bean cultivars from 33 localities and two seasons was determined with the MCT and CVA. Cultivars with acceptable and unacceptable canning quality were identified using laboratory evaluation and CVA. The CVA resulted in a prediction model for canonical variates 1 and 2 (CV 1 and CV 2) by identifying two discriminative equations for CV 1 and CV 2 scores. The CVA for environments identified differences in the canning quality of beans from different regions, while also indicating seasonal differences. The canning quality of dry bean cultivars from different environments can be determined using CVA. The model equations for the prediction of the canning quality of small white beans were validated on four cultivar samples from four regions (2000/01 season) and 24 breeding samples from three localities (2002/03 season) that were not included in the development of the model. The CVA and the model identified the same entries from breeding trials over localities not to be significantly different from Teebus (P > 0.05) in canning quality, but were unable to group cultivars statistically correct according to choice grade. The model was however capable of grouping standard and choice grade cultivars separately. The model could be applied to identify breeding trial entries as choice grade and to identify entry x locality interactions. The use of small-scale canning and evaluation procedures in combination with CVA could be employed to classify cultivar canning quality as either choice- or standard grade and to determine environmental canning quality. These techniques could be used, with the assistance of the prediction model to compare samples from a breeding program with a reference standard.
Open Access
Lipids and ascospore morphology in yeasts
(University of the Free State, 2004-10) Bareetseng, Andries Sechaba; Kock, J. L. F.; Pohl, C. H.; van Wyk, P. W. J.
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 oxylipin’s 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.
Open Access
Yeasts from Lesotho: their classification and possible applications
(University of the Free State, 2004-11) Tarr, Shahida; Kock, J. L. F.; Albertyn, J.; Smit, M. S.
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.
Open Access
The role of lipids in the flocculation of Saccharomyces cerevisiae
(University of the Free State, 2005-11) Strauss, Catharina J; Kock, J. L. F.; Van Wyk, P. W. J.; Lodolo, E. J.
English: Although beer production is one of the oldest biotechnologies in the world, a major constraint in brewing remains controlling flocculation. Evidence points towards a possible role of lipids, associated with the cell surfaces, as a major factor responsible for flocculation. Therefore, the aim in this study became to evaluate the contribution of lipids, especially oxylipins, in the flocculation of Saccharomyces cerevisiae UOFS Y-2330. Saccharomyces cerevisiae UOFS Y-2330 was selected as a model, since it was found to demonstrate both Flo1 and NewFlo phenotype flocculation behaviour, when cultivated in different media. In a defined medium with glucose as a sole carbon source, this strain immediately flocculated strongly and lost this ability before stationary phase was reached. In a complex medium containing glucose, this yeast strongly flocculated towards the stationary growth phase without losing this ability during this phase. This inverse pattern may be ascribed to a switch in sensitivity of the yeast to flocculate in the presence of glucose as well as pH level, which may, in turn, influence the availability of calcium ions. In both media, matured cells produced protuberances upon flocculation as observed by electron and immunofluorescence microscopy, which may be involved in cell adhesion. This was followed by further investigations into the role of lipids over the growth cycle of this yeast. Here, it was uncovered that Sacch. cerevisiae UOFS Y-2330 does not only demonstrate inverse flocculation, but is also characterised by two different lipid turnover patterns. During Flo1 phenotype flocculation, this yeast showed two neutral lipid accumulating stages (i.e. at 8 h and from 12 h). This is probably triggered by flocculation, which may be regarded as a survival mechanism where cells accumulate especially neutral lipids as reserve energy source - a similar mechanism is probably operative when cells enter stationary growth. Contrary to Flo1 behaviour, this strain in NewFlo phenotype mode demonstrates only a single lipid accumulation phase i.e. when cells enter stationary growth, which coincides with the increase in flocculation. In addition, an increase in phospholipids was experienced during active growth in both flocculation behaviours, probably as a result of active membrane production. These results prompted us to investigate the possible role of oxylipins present on the cell surfaces during the flocculation process. It was found that some strains of Sacch. cerevisiae (include strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When aspirin was added to cultures of Sacch. cerevisiae UOFS Y-2330, flocculation was significantly inhibited as well as the production of 3-hydroxy (OH) 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3-OH 8:0 could be detected before flocculation onset in this yeast. Next, the involvement of these oxylipins in co-flocculation was assessed. According to the lectin-theory, the yeast Schizosaccharomyces pombe lacks the specific receptors necessary to facilitate co-flocculation with Sacch. cerevisiae species. In this study we demonstrate oxylipin associated co-flocculation between Sacch. cerevisiae UOFS Y-2330 and S. pombe strains using differential cell staining, immunofluorescence and ultrastructural studies. Using a 3-OH oxylipin specific antibody coupled to a fluorescing compound, 3-OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3-OH oxylipins was confirmed using gas chromatography-mass spectrometry. Whether these 3- OH oxylipins play a role in affecting co-flocculation of Sacch. cerevisiae with S. pombe cells through possibly entropic-based hydrophobic interactions and/or hydrogen bonds still needs to be verified. Studies on the physiological, genetic as well as colloidal aspects of flocculation using this model strain may lead to important new insights in this fascinating phenomenon as well as applications in industry.
Open Access
Evaluation of Yarrowia lipolytica as a host for cytochrome P450 monooxygenase expression
(University of the Free State, 2006-06) Obiero, George Ogello; Smit, M. S.
Biohydroxylation reactions are catalyzed by various types of hydroxylating enzymes (Ayala and Torres, 2004) which include dioxygenases, lipooxygenases as well as CYP450 monooxygenases. These particular hydroxylation reactions have several advantages over chemical synthesis. Several microorganisms including yeasts have the ability to hydroxylate various substrates. The exploitation of microbial hydroxylations for the production of industrially useful products such as pharmaceuticals is a more recent development (Holland et al., 2000). Yeasts from the genera Schizosaccharomyces, Pichia, Saccharomyces and Yarrowia have all been used to express foreign CYP450 genes (Mukarami et al., 1990; Nthangeni et al., 2004) since they offer an advantage especially when a eukaryotic environment is required for the functional expression of the heterologous gene (Blanquet et al., 2003). A recent evaluation of several yeasts revealed that Y. lipolytica is, a highly attractive alternative host for secretion and expression cloning (Muller et al., 1998; Juretzek et al., 2001). However, a literature search on successful expression of CYP450s in Y. lipolytica yielded only six cases. Three of these were done in our laboratory. In most of the reported cases, the recombinant CYP450 activities were never evaluated in terms of whole cell biotransformations. It was therefore the aim of this study to evaluate Y. lipolytica as a recombinant whole-cell biocatalyst for hydroxylation reactions by using available Y. lipolytica strains overexpressing different CYP450s which were (i) CYP1A1 coding for polyaromatic hydrocarbon hydroxylase (ii) CYP53B1 coding for benzoate para-hydroxylase (iii) CYP52F1 coding for alkane hydroxylase and (iv) CYP557A1 coding for putative fatty acid hydroxylase. Hydroxylase activities of the genetically engineered strains were compared with activities in wild type yeasts expressing the relevant CYP450s. A variety of substrates used for biotransformation reactions included, acetanilide, benzoic acid , phenylnonane, trans-cinnamic acid, 4-nitrophenyl octyl ether and 4-nonyloxybenzoic acid. Experiments using Y. lipolytica overexpressing CYP1A1 illustrated the limitation of using Y. lipolytica for the biotransformation of substrates such as AA since the endogenous enzymes degraded this substrate within only 12 h after substrate addition. In an attempt to distinguish the activities of the putative fatty acid hydroxylase and the alkane hydroxylase overexpressed in Y. lipolytica from the endogenous CYP450s, 4-nitrophenyl octyl ether, 4-nonyloxybenzoic acid and phenylnonane were used as substrates. 4-nitrophenyl octyl ether proved to be expensive and less sensitive to TLC, GC and GC-MS analyses. It has been used in other studies because it yielded p-nitrophenol which can be assayed colourimetrically by measuring absorbance at 420 nm. However, in our experiments, intermediates accumulated that were not completely transformed into p-nitrophenol. Further biotransformation experiments were carried out using 4- nonyloxybenzoic acid as the substrate. Biotransformation experiments were done using Y. lipolytica strains with intact and partially disrupted -oxidation pathway overexpressing CYP52F1 and CYP557A1. Additional experiments were carried out using wild type Y. lipolytica W29, R. minuta and R. retinophila strains. The results demonstrated that, the wild type Y. lipolytica W29 demonstrated the highest specific hydroxylase activity when 4-nonyloxybenzoic acid was used as the substrate. The main limitation here was the inability to selectively induce the overexpressed CYP450 genes alone without the background endogenous CYP450 activity. Due to the limitations above, the next strain used was Y. lipolytica TVN91 (overexpressing benzoate-para hydroxylase from R. minuta). In this case, the host strain lacked the specific CYP450 to perform the same hydroxylation reaction. The substrate used here was BA. Different growth and induction conditions were evaluated to optimize benzoate-para-hydroxylase activities. A comparison of the hydroxylase activities indicated that the activity of the recombinant Y. lipolytica strain overexpressing the CYP53B1 was about 30 times slower than that of the wild type R. minuta from which the gene was cloned. Continuous addition of stearic acid resulted in the best activity with Y. lipolytica TVN91, because the hydroxylase activity was maintained for a longer duration. When PN and CA were used to evaluate substrate transport limitation, the results demonstrated that substrate transport was not limiting and the specific hydroxylase activity was not increased. PN was initially converted to BA before hydroxylation to form pHBA. These results further demonstrated that hydroxylase activity of PN was much faster than that of BA. The results from the bioreactor study demonstrated that an improved aeration and mixing led to an increase in the benzoate para-hydroxylase activity. The possibility of using a chemically defined media (YNB) supplemented with yeast extract and casamino acid for biotransformation was also demonstrated. The results of this study also demonstrated that it is possible to use harvested recombinant cells for biotransformation without significant loss of activity. This makes it possible to study in detail the kinetics of the overexpressed CYP450s. It was, however apparent that the hydroxylase activities were significantly increased by both aeration and cell concentration.
Open Access
Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism Saccharomyces cerevisiae
(University of the Free State, 2007-05) De Smidt, Olga; Albertyn, J.; Du Preez, J. C.
English: When Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose, the fermentative alcohol dehydrogenase, ADH I , catalyses the regeneration of NAD+ from NADH and produces ethanol from acetaldehyde. When the fermentable carbon source is depleted, a variety of other enzymes are derepressed in order to utilise the previously excreted ethanol via oxidative respiration and gluconeogenesis . To provide both the carbon source and energy for this system, the yeast cell requires an efficient method for oxidising this previously excreted ethanol. ADH II is a catabolite repressible isoenzyme which primarily functions in the cell to oxidise ethanol to acetaldehyde, which can be metabolised via the tricarboxylic acid cycle or act as intermediate product in gluconeogenesis. ADH III is a mitochondrial isoenzyme participating in the respiratory metabolism by forming part of the ethanol-acetaldehyde shuttle that is important for shuttling mitochondrial reducing equivalents to the cytosol under anaerobic conditions. The physiological roles and regulation of ADH1, ADH2, ADH3, ADH4 and ADH5 were investigated by monitoring transcription levels in chemostat and batch cultivations with Northern blotting and real-time RT-PCR. ADH I was shown to be the key enzyme in the reduction of acetaldehyde to ethanol and also demonstrated ample ability to oxidise ethanol. ADH2 transcription was inhibited by glucose and ethanol in chemostat cultures pulsed with both these carbon sources, but only glucose repression was evident in batch cultures. Northern blot analysis showed that the ADH3 gene was induced during the ethanol phase of the pulses suggested that the mitochondrial ADH III enzyme could also be involved in the first step in ethanol utilisation. The growth kinetics of a strain expressing only ADH III demonstrated that the ADH3 gene product could fulfil the same function as ADH II. ADH4 transcription was detected for the first time in batch cultures and was shown not to be involved in the production or assimilation of ethanol. ADH5 transcription was also demonstrated for the first time and data suggest that ADH V is not involved in ethanol production in a adh1-adh4 deletion mutant.
Open Access
Oxylipins in Cryptococcus neoformans and related yeasts
(University of the Free State, 2007-11) Sebolai, Olihile Moses; Kock, J. L. F.; Van Wyk, P. W. J.; Pohl, C. H.
English: Literature shows that Cryptococcus neoformans is an important human pathogen responsible for many deaths worldwide. To compound this, treatment of cryptococcal infections has over the years been difficult. This is largely due to the widespread use of antifungals, leading to the emergence of drug resistant strains. The capsule (with glucuronoxylomannan as major polysaccharide) is the principal virulence factor of this pathogen, and can influence the hosts’ immune response. Moreover, recent studies have identified novel bioactive compounds, which can also contribute to the virulence of pathogens such as Cryptococcus neoformans and Candida albicans. These include compounds such as oxylipins (oxidized fatty acids), which have been reported to modulate the hosts’ immune response during infections. This exposes new targets for antifungal action. In this study, the 3-hydroxy fatty acid, 3-OH 9:1, has been discovered in Cryptococcus neoformans var. neoformans UOFS Y-1378 using gas chromatographymass spectrometry. Immunofluorescence confocal laser scanning microscopy and immunogold transmission electron microscopy revealed that this 3-OH oxylipin accumulates in capsules, where it is released as hydrophobic droplets through protuberances (each about 30 nm x 400 nm) into the extracellular environment. This discovery further expands our knowledge of the known spectrum of biologically active compounds associated with this main virulence factor of Cryptococcus neoformans. 3-OH 9:1 is produced in yeast mitochondria probably through β-oxidation or fatty acid synthesis pathway type II (FAS II). Evidence supporting this statement, was provided after mapping the migration of 3-OH oxylipin-containing osmiophilic material during ultrastructural studies. Here, osmiophilic material was shown to originate in mitochondria and is deposited inside the yeast cell wall, from where it is released into the surrounding medium, along capsule protuberances or through capsule detachment. When acetylsalicylic acid (ASA, an inhibitor of mitochondrial function – including 3-OH oxylipin production) was added, the migration of the osmiophilic material as well as capsule detachment from cell walls and hence oxylipin release was abrogated. This data is in accordance with literature, where a novel release mechanism for the major virulence factor of Cryptococcus neoformans is reported. Here, virulent polysaccharide packaged lipid vesicles are reported to cross the cell wall and the capsule into the surrounding environment. This Ph.D. study implicates the lipid vesicles to contain 3-OH oxylipins. It was also demonstrated that 3-OH oxylipins are widely distributed in other members of the pathogenic yeast genus Cryptococcus, following immunofluorescence confocal laser scanning microscopy (using antibodies directed towards 3-OH oxylipins) and gas chromatography-mass spectrometry. In the examined strains these compounds were mainly associated with cell wall surfaces, protuberances, appendages and collarettes. According to literature, yeasts that are dependent only on mitochondrialaerobic respiration for growth, are more sensitive to ASA compared to yeasts that possess both energy production pathways i.e. aerobic respiration and fermentation. In this study, in vitro data corroborate this hypothesis. Here, the growth of all nonfermenting Cryptococcus species was much more sensitive to ASA compared to the fermentative yeast, Saccharomyces cerevisiae (which could tolerate as much as 5 mM ASA). Already at an ASA concentration of 2 mM, a decrease in growth of most Cryptococcus species was evident, and at 3 mM ASA, the growth of all Cryptococcus species was significantly inhibited. The observed ASA effect may be due to inhibition of mitochondrial function, which includes inhibition of oxidative phosphorylation and respiratory electron transport chain – functions important for energy generation. These data suggest that ASA can be used as an antimitochondrial antifungal agent to combat growth of these pathogenic yeasts. This discovery should now be further researched in vivo taking into account the toxicity of ASA and other non steroidal anti-inflammatory drugs.