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Item Open Access Functional properties of Cactus Pear Mucilage: gel formation, edible coatings, films and spherification(University of the Free State, 2023) Mushanganyisi, Dembe Dynne; de Wit, Maryna; du Toit, Alba; Hugo, ArnoMucilage is a hydrocolloid, a gelatinous slimy substance that contains polysaccharides and proteins. Due to mucilage’s ability to absorb large amounts of water, which result in modified viscosity, it can be used as a food additive in the food industry as a hydrocolloid to modify food texture. It is used as an emulsifier, to form gels and as a natural edible coating. The aim of the study was to investigate the functional properties of mucilage and to potentially develop mucilage into a commercially viable food ingredient (hydrocolloid). Cladodes from four cultivars (and two species) were investigated. These include three cultivars from Opuntia Ficus-indica, namely Nepgen, Algerian and Ficus-Indice as well as one cultivar from the O.robusta spp. namely Robusta. Mucilage was extracted, and the yield was determined in percentage, with Algerian at 53.70%, Nepgen at 50.96%, Robusta at 48.40% and Ficus-indice at 37.75%. The mucilage was dried in two ways, namely, freeze-drying, and hot-air dehydration and colour difference was investigated. The drying impacted the colour of mucilage powder, with freeze-dried remaining green and the hot-air dried turned brown, colour difference was observed between the freeze-dried and hot-air dried mucilage a* value. a* value represents the scale from green (negative value) to red (positive value). It is evident that the freeze-dried samples had a green colour (negative values) while the hot air-dried samples had a reddish colour seen by a positive a* value. For example, freeze-dried Robusta had a value of -7.87 as compared to that of hot-air dried at 1. The viscosity of both native liquid and dried mucilage were investigated. Robusta and Nepgen native liquid’s mucilage is viscous more than that of Algerian and Ficus-indice. The reconstituted mucilage powder is thicker than the native liquid powder. Gelling capacity was tested. For spherification or bead formation, mucilage was used to replace the usual gelling agents in both direct and reversed spherification. During gelling ability tests, mucilage from Robusta showed gel-like ability as compared to the other three cultivars. However, it was concluded that mucilage does not form gels on its own, but rather improves gel formation with other hydrocolloids. When mucilage was used to replace the sodium alginate in spherification, it did not form true spheres, but formed a temporary gel-like membrane when incorporated with xanthan and agar. Ultimately, mucilage may be a replacement for food hydrocolloids. It can be used as an emulsifying agent and an edible coating, although different cultivars will give different results.Item Open Access Purification strategies for equine Chorionic Gonadotropin(University of the Free State, 2022) Boneschans, Martie; O'Neill, F. H.; Opperman, D. J.Equine Chorionic Gonadotropin (eCG) is a glycoprotein hormone secreted by the endometrial cups of pregnant mares during days 37 to 120 of gestation. In equids it exhibits luteinizing hormone (LH) activity, while in non-equine species eCG exhibits both LH - and follicle stimulating hormone (FSH) like activity. Due to the high sialic acid content of eCG the 60 kD protein possess a long half-life in mammalian plasma. This, together with its dual hormonal activity (LH and FSH) makes eCG an extremely effective hormone for use in animal reproduction. Recombinant production of the hormone is still in its initial phases, therefore eCG, for commercial use, is still primarily purified from the serum of pregnant mares. Equine serum albumin remains the biggest challenge in the purification of eCG due to its relative abundance in the serum and similar molecular weight (70 kDa). This study investigated some of the strategies involved in the isolation of eCG in order to identify possible areas for improvement. We compared three different acid precipitation steps as an initial phase in the isolation of eCG from eCG-spiked equine serum. Precipitation with metaphosphoric acid (MPA) showed the highest purification factor compared to precipitation with 15% (v/v) –and 25% (v/v) trichloroacetic acid (TCA) but resulted in high eCG losses. We also investigated the use of albumin affinity chromatography in the purification process but found that both albumin and eCG bound to the albumin affinity resin, raising our suspicions about possible interaction between the proteins. Ultrafiltration, using filtration units with a MWCO of 30 kDa, proved an ineffective method in the isolation of eCG as we found that eCG exhibits an affinity for the porous membrane of the filtration unit. During cation exchange chromatography we found that when eCG was mixed with BSA, the proteins exhibited changes in their column binding properties. This was confirmed with anion exchange where neither a continuous salt – nor pH gradient could separate eCG and albumin. This further supports our notion of a possible interaction between the two proteins as albumin is widely known as a carrier protein for thyroid hormones and other molecules. We found that Lectin affinity (LAC) - and hydrophobic interaction chromatography (HIC) did succeed in separating eCG and albumin but we could not elute most of the eCG from the LAC column, therefore future work should include further optimization of the method. HIC was performed on small scale and future work should look in to using higher concentrations of protein.Item Open Access The effect of oleic acid supplementation on lipid droplet production, betaoxidation and rotavirus replication(University of the Free State, 2022) Thobane, Tshegofatso Benedict; O'Neill, H. G.; Sander, W. J.Rotavirus (RV) is the most common cause of severe acute gastroenteritis in infants and young children globally. Rotavirus infections induce cytoplasmic inclusion bodies called viroplasms serving as a site of RV genome replication and assembly. Viroplasms recruit lipid droplets (LDs), which play major roles in energy homeostasis and is a site for lipid storage. The successful formation of viroplasm-LD complexes is essential for RV propagation. The extracellular supply of oleic acid (OA, C18:1) to host cells induce LD biogenesis and modulate cellular fatty acid (FA) metabolism. Rotavirus modulates the host cell lipidome and influence cell LD biogenesis and FA metabolism by β-oxidation for successful viral propagation. Previous studies have shown that chemical fragmentation of LDs by treatment with 3-isobutyl-1-methylxanthine (IBMX) and isoproterenol (ISP) to be deleterious for RV infection. In addition, the chemical modulation of FA β-oxidation by etomoxir (ETO) treatment has been shown to affect the propagation of other viruses including hepatitis C virus (HCV). In this study, we sought to investigate (1) the effect of OA supplementation on LDs during RV infection, (2) the effect of OA supplementation on FA β- oxidation during RV infection and lastly, (3) the effect of LD and FA β-oxidation modulation on RV infection. Briefly, HEK293 cells were supplemented with OA in the presence and absence of IBMX and ISP as well as ETO treatment during RV infection (MOI=5) for 6 h post infection (p.i). Control cells were not treated with OA in the presence and absence of RV infection in which they were also maintained in parallel with the test samples. At the end of each timepoint p.i, LDs where isolated for triacylglyceride (TAG) quantification (LD quantification) while the test samples treated with the same conditions were analyzed for changes in oxygen consumption rate (OCR) (FA β-oxidation quantification) in HEK293 cells. For viral replication kinetics, HEK293 cells were again supplemented with OA and treated with IBMX, ISP and ETO during RV infection as previously describe for 0 h, 2 h, 6 h, 12 h and 16 h p.i, respectively. At the end of each time point, RV was harvested and quantified using tissue culture infection doses 50 (TCID₅₀). Our study shows that supplementation of HEK293 cells with OA increase the overall TAG content in isolated LDs, while the treatment of HEK293 cells with IBMX and ISP reduce TAG content of isolated LDs. We further show an increase in total TAG content of isolated LDs when HEK293 cells are treated with ETO. Rotavirus infection increased the total TAG content of isolated LDs. The supplementation of HEK293 cells with OA increased HEK293 cell OCR, while the treatment with IBMX, ISP and ETO reduced the overall host cell OCR. Treatment of OA-supplemented HEK293 cells with IBMX, ISP and ETO slightly increased the overall OCR of the treated HEK293 cells compared to the OA unsupplemented HEK293 treated with the chemical inhibitors. It is also important to note that RV infection reduced the overall OCR of HEK293 cells. Analysis of viral kinetics showed that OA supplementation increases RV replication over time, while the treatment of HEK293 cells with IBMX, ISP and ETO in the presence or absence of OA supplementation reduce the overall replication of RV over time. The OA supplementation of RV infected HEK293 cells with IBMX and ISP rescues viral propagation over time, which was not observed when HEK293 cells were treated with ETO. The data thus show that OA supplementation increases total TAG content in isolated LDs as well as the overall host OCR while promoting RV infection over time. Rotavirus infection promotes enrichment of LDs by increasing the overall TAG content of isolated LDs while reducing the overall OCR of host cells. It was shown that the chemical modulation of both LD biogenesis and FA β- oxidation is deleterious to RV propagation over time.Item Open Access The evaluation of supramolecular self-associating amphiphiles as novel anti-biofilm compounds(University of the Free State, 2023) Steyn, Hendrik Jacobus Frederik; Pohl-Albertyn, C. H.; Hiscock, J. R.The ability of naturally occurring substances to inhibit bacterial growth has proven to be one of mankind's cornerstone discoveries (Fleming 1929; Whitehead 1933). The potential of these substances was highlighted when Alexander Fleming noticed the inhibition and lysis of Staphylococcus colonies when cultured in the presence of ascomycetous fungus of the genus Penicillium (Fleming 1929). The ability of Penicillium to inhibit bacterial growth was attributed to the production of a substance termed penicillin (Fleming 1929). A characteristic property of antibacterial substances is their selective action; some substances are effective against either Gram-negative or Gram-positive bacteria and in some instances, both (Waksman 1944). When considering that these substances differ in chemical composition, it is evident that the selective action of antibacterial substances directly relates to their mechanism of action. Additionally, the qualitative characteristic of varying concentrations directly influences the substance's efficacy. It is established that higher concentrations of antibacterial substances result in exaggerated inhibition (Waksman 1944). From these findings, the use and application of antibiotic substances have enabled the treatment of various bacterial infections and revolutionised the healthcare industry (Espinel-Ingroff 2003; Tomson and Vlad 2014). The discovery and application of antibacterial substances promoted the search for antifungal substances (Espinel-Ingroff 1997). The efforts of Elizabeth Hazen and Rachel Brown lead to the discovery of an antifungal substance produced by soil actinomycetes. The substance termed fungicidin or nystatin indicated fungistatic and fungicidal activity (Hazen and Brown 1951). From these findings, other cultures were screened for antifungal substances and inhibitory potential. In 1953, Steinberg and co-workers identified and isolated the antifungal substances amphotericin A and B from a Streptomycete culture (Steinberg et al. 1953, Dutcher 1968). A report by Milton Sloane supported the efficacy of antifungal substances when used in the treatment of candidiasis; proving the use of nystatin to be effective (Sloane 1955).Item Open Access Classification and spoilage characteristics of Chryseobacterium isolates from fish(University of the Free State, 2019) Gavu, Lydia; Hugo, C. J.; Hitzeroth, A. C.; Gryzenhout, M.The genus Chryseobacterium was initially reported to have only six species and the number has increased rapidly over the years. The genus is represented by a total of 113 species at present. Species belonging to the genus Chryseobacterium are widespread in nature. They have been reported from clinical, environmental, industrial and food sources. Their spoilage characteristics have been well defined in food products including poultry, meat, milk and milk products and fish. Development of unpleasant odours in food contaminated by Chryseobacterium species usually results from the activity of proteolytic enzymes. The first aim of this study was to classify 11 potential Chryseobacterium fish isolates from a previous study. The methods that were used included 16S rRNA gene sequence analysis, conventional phenotypic methods and the BIOLOGTM Omnilog identification system. Chryseobacterium balustinum, C. gleum and C. piscium were used as reference strains throughout the whole study. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the 11 Chryseobacterium isolates represented members of the genus Chryseobacterium. The highest similarity were obtained for C. piscium by strains SH 23-4 (98.89%), SH 28-3 (100%) and SH 30-1 (100%) and it was therefore concluded that these three strains could be other strains of C. piscium. This finding was confirmed by the phylogenetic treeing methods used in this study because the three strains clustered closely with C. piscium. Strains SH 11-3(a), SH 11-3(b), SH 20-4, SH 25-4, SH 40-3, SH 19-2(b) and SH 11-4(b) had sequence similarity values lower than 98.7%, it is therefore a possibility that these strains might be novel species of Chryseobacterium, however, further investigations need to be performed for confirmation. The BIOLOGTM Omnilog Gen III identification system and conventional phenotypic methods were also useful in classification. The BIOLOGTM Omnilog was also able to identify strains SH 23-4, SH 28-3 and SH 30-1 as possible strains of C. piscium. The other strains could not be identified because of its limited database. The second aim of this study was to estimate the spoilage potential of the 11 Chryseobacterium isolates by determining their spoilage potential in terms of substrate oxidation by the BIOLOGTM Omnilog system. The results indicated that all the isolates could be potential fish spoilers, however, strains SH 11-4(b) and SH 23-4 were able to oxidise the most of the carbon sources (25/31) and could be regarded as being able to cause the most spoilage. Sensory analysis of inoculated FJB samples did not show significant differences in terms of odour production, however, odours such as smelly feet, cabbage-like, fruity, sour and putrid sewage were noted for the 11 Chryseobacterium isolates and their reference strains. Chryseobacterium species were able to produce a total of 87 volatile compounds in fish juice broth (FJB) at 4 and 25 °C. The composition of the volatile compounds detected was, however, slightly higher for samples that were incubated at 25 °C than at 4 °C. The frequently detected compounds occured in FJB samples containing C. gleum and strain SH 30-1 and the least detected compounds occurred in FJB samples containing strains SH 19-2(b) and SH 11-4(b). The frequently produced compounds with more pronounced odours were identified as 2-ethyl-1-hexanol, indole, dimethyl disulphide and 2-phenylethanol. FJB samples inoculated with strains SH 30-1, SH 28-3, SH 25-4 had the majority of volatile compounds associated with unpleasant odours, such as fishy from trimethylamine; spoiled, putrid from dimethyl sulphide; and faecal, nauseating from indole. It was concluded that 7/11 Chryseobacterium strains in this study could be novel Chryseobacterium species. Sensory analysis and Gas-Chromatrography/Mass-Spectrometry were useful in estimating the spoilage potential of the Chryseobacterium species through odour production. The BIOLOGTM system can be used as an effective screening method for identifying the carbon sources utilised by the Chryseobacterium species which could then be investigated further for the potential of these starins to cause food spoilage. Chryseobacerium species used in this study have the potential to spoil fish and/other fish products because of their psychrotolerant and proteolytic nature.Item Open Access The development of a CRISPR-Cas9 gene editing system for Cryptococcus deneoformans(University of the Free State, 2019-01) Du Plooy, Lukas Marthinus; Albertyn, J.; Sebolai, O. M.; Pohl, C. H.The pathogenic yeasts, Cryptococcus neoformans and C. deneoformans, are responsible for potentially fatal meningoencephalitis in immunocompromised individuals, most notably in patients who have AIDS. Only three drugs are commonly administered to treat cryptococcal infections and most are not readily available in developing countries most affected by the AIDS pandemic. Cheaper and more widely available drugs are therefore needed. Developing molecular methods to disable genes encoding virulence factors could help to elucidate the mechanism of action of potential drugs against these fungal pathogens. Previously, researchers mostly relied on biolistic transformation to deliver DNA into cells for homologous integration into the targeted site. Recent developments with CRISPR-Cas9-based systems for gene targeting made it possible to utilise another transformation technique, electroporation, to knock genes out. In this study, a one-step CRISPR- Cas9 system was developed to be delivered into cells with electroporation. This system consists out of two plasmids, carrying a nourseothricin and G418 resistance marker respectively, as well as a CAS9 gene. A third plasmid was used to construct guide DNA, which was then amplified and cloned into the two CRISPR-Cas9 plasmids carrying the CAS9 gene. The plasmids carrying the CRISPR-Cas9 components were maintained transiently for expression of the CRISPR-Cas9 genes before these constructs were degraded by the cells. Donor DNA was also constructed to remove parts of the biosynthetic genes ADE2 and HIS3 to obtain adenine and histidine auxotrophic mutants with visually discernible phenotypes. A second round of transformation can then introduce new donor DNA to repair these auxotrophic genes whilst disrupting virulence genes for virulence studies. Electroporation proved to be very inefficient in this study and gene targeting was unsuccessful. Using large amounts of plasmid and donor DNA yielded the best results, although no more than 8 colonies were seen on a few selective media agar plates. Inefficient transformation could be due to old and faulty electroporation equipment or ineffective delivery of the electrical current to cells. In the future, other transformation methods will be employed to deliver the plasmids constructed in this study into C. deneoformans cells. This system can then be used to remove virulence genes to study their role in infection, which could help to elucidate the mechanism of action behind potential drugs. For instance, a capsule-less mutant could reveal what effect a drug targeting capsule synthesis will have on the ability of these yeasts to cause disease.Item Open Access Studying the regulation of immune signalling molecules related to immunity during Avibacterium paragallinarum infection(University of the Free State, 2019-06) Jawallapersand, Poojah; Boucher, C. E.; Janse van Rensburg, W. J.; Van der Westhuizen, W. A.Infectious coryza is a contagious and acute upper respiratory poultry disease caused by Avibacterium paragallinarum, that plagues predominantly layers but also affects broiler breeds. The chicken’s immune response to Av. paragallinarum serovar C-3 (SA-3) infection (reported to be most virulent in South Africa), and the underlying genetic mechanisms involved, are poorly understood and not well documented. The aim of the study is to understand the complexity of the regulation of immune functions by identifying the molecules that are expressed during Av. paragallinarum serovar C-3 (SA-3 strain) infection. In this study, chickens (control versus experimental groups) were directly challenged via infraorbital injection with Av. paragallinarum serovar C-3 (SA-3 strain) and the immune response was monitored. The mean disease score and mean daily egg production score were recorded and calculated. Blood and sera were obtained for blood microscopy, leukocyte population profiling by flow cytometry analysis, and antibody/cytokine screening with ELISA assays. Finally, control and experimental chickens were sacrificed based on the clinical scores obtained (0, 1, 2 or 3). Post-mortem examination was conducted, and organs were harvested for immunohistochemistry staining for identification of distinct immune cell populations. The in vivo results obtained from the experimental studies in combination with the in silico results obtained from bioinformatics tools for the generation of immune signalling pathway maps may provide insight and a birds-eye view into the immune mechanisms between host-pathogen interactions for this disease. Results from our study could potentially assist with diagnostic tests for serovar C-3 and provide insight towards more efficient vaccine development. Hence, if vaccine practices are improved this will limit importations of birds from huge global markets, thus preventing carry-over poultry diseases and zoonosis as well as maintaining a safe and sustainable economy.Item Open Access The development of a wide range CRISPR-Cas9 gene editing system(University of the Free State, 2019-01) Bisschoff, Eduvan; Albertyn, J.; Pohl-Albertyn, C. H.CRISPR is a revolutionary method to effectively and efficiently alter the genomic make-up of an organism. Unlike any other genetic engineering tool or technique, CRISPR is remarkably cheaper, simpler and faster to perform. In biotechnology, the best eukaryotic organism for research is yeast, due to their fast growth rate and ease of manipulation compared to multicellular organisms. Hence the aim of the study was the development of a wide range CRISPR-Cas9 system for a wide variety of different yeasts for easy and fast gene editing. The system were validated in Saccharomyces cerevisiae and six other non-conventional yeast. System construction began with the incorporation (separately) of three different optimized CAS9 (optimized for expression in Pichia pastoris, Candida albicans and Homo sapiens) genes into the wide range pKM180 vector. The three different CAS9 construct were then tested for correct expression of the Cas9 protein and the effects thereof in all the yeasts. Through western blot analysis it was observed that all three of the different Cas9 proteins were expressed successfully in the different yeasts. However, all of the Cas9 proteins had a negative effect on the growth of the yeast. For the completion of the CRISPR-Cas9 system, a Ribozyme‐gRNA‐Ribozyme cassette was incorporated into the wide range CAS9 vector, containing the C. albicans optimized CAS9. The system was then validated with successful disruption of the ADE2 gene in all of the yeasts. This proved that the wide range CRISPR-Cas9 system was applicable in a wide variety of different yeasts, thus allowing for rapid, cost-effective genetic manipulation of biotechnologically relevant yeast strains.Item Open Access Antibody fragments as a possible therapeutic treatment for infectious bronchitis in poultry(University of the Free State, 2018-09) Coetzee, Janetta Magrieta; Bragg, R. R.; Boucher, C. E.; Van der Westhuizen, W. A.Infectious bronchitis virus (IBV), a coronavirus, is the etiological agent for infectious bronchitis (IB), an acute respiratory disease of poultry. Infectious bronchitis is a notifiable disease, and taking in consideration that poultry is the second most consumed meat within South Africa, it highlights the importance of monitoring IBV outbreaks. Recombinant single chain variable fragments (scFv) have been used in therapeutic treatments for various human and veterinarian viruses, including other coronaviruses, such as SARS-CoV and MERS-CoV. The study aims to select scFv against the IBV antigen, with the use of phage display technology and commercial ELISA plates coated with the most prevalent IBV strains namely, H120 and M41. It has been proven that the S1 protein induces the binding of neutralising antibodies which provides protection against lethal CoV infections, thus, indicating a possible application for a therapeutic treatment. In this study, phage clones were selected from a human domain (dAb) library (Source BioScience, Australia). Panning was repeated three times using commercial ELISA plates coated with M41 and H120 IBV strains. Positive monoclonal phage clones were retrieved from the polyclonal mix by a sandwich ELISA and sequenced. The selected scFvs were expressed by Isopropyl β-D-1 thiogalactopyranoside induction and purified by immunoprecipitation with the Pierce Anti-c-Myc Agarose kit (Thermo Scientific, USA). After purification, binding ability of the scFvs were determined by means of a direct competitive ELISA. The neutralising ability of the scFvs was then determined by a virus neutralisation assay in ovo with the Avipro IBV H120 strain (Lohmann Animal Health Gmbh, Germany). This was performed in 9-day old SPF eggs over a time-period of six days. One set of eggs were injected with a dilution range of the IBV H120 strain and another set by a mixture of 2 μg/ml scFv with the IBV H120 strain. The end-point titres were determined and compared by the Spearman-Karber method (Spearman, 1908). Statistical analysis was performed using the student t-test with a p-value of 0.05. Round 1 of panning resulted in a total of 1.2 x 106 phages/ml and after round 3 a total of 3.0 x 1010 phages/ml were obtained. A total of 96 phage clones were manually selected from which only 12.5% showed a positive result during the sandwich ELISA. The 12 positive clones were sequenced and analysed based on nucleotide and amino acid composition. A total of five scFvs contained a complete variable heavy (VH) chain sequence, two of which was identical. This resulted in four unique and complete scFv sequences. These sequences showed a high variance in the nucleotide composition through- out the sequence. However, variation in the amino acid composition was only observed in the third complementary determining region. The scFvs were expressed and resulted in concentrations ranging from 204.38 μg/ml to 265.07 μg/ml. Detection of IBV antigen binding ability of the purified scFvs was conducted by a direct competitive ELISA. However, no statistical difference in absorbance values were observed, indicating insufficient binding of the scFvs. The in ovo virus neutralisation assay resulted in a one log reduction of the end-point titres. Statistical analysis proved one of the reductions to be statistically significant with a p- value less than 0.05, resulting in a partial neutralisation effect from the scFv. In conclusion, the selection process showed a progressive enrichment of antigen specific clones from the dAb library. The scFvs were successfully expressed, purified and characterised in terms of binding ability.Item Open Access Alcohol dehydrogenase mediated lactonization of 1,6-hexanediol(University of the Free State, 2018-01) Dithugoe, Choaro David; Opperman, D. J.; Smit, M. S.Alcohol dehydrogenases (ADHs) are oxidoreductases that catalyse the interconversion between alcohols and ketones. These are biotechnologically interesting enzymes due to their ability to produce optical active alcohols. Alternatively, lactonization of diols to corresponding lactones has been explored by researchers. The aim of this study was to screen ADHs for the lactonization of 1,6-hexanediol to Ɛ-caprolactone by evaluating a single ADHs enzyme system and a combinatorial ADHs enzyme system. Saccharomyces cerevisiae ADH 1 and ADH 6 together with Alcanivorax dieselolei B5 ADH 1 to ADH 3 genes were PCR amplified and subcloned into pET28b(+) expression vector. An additional 8 ADHs genes were synthesised and cloned into pET28b(+) by Genescript. The pET28b(+) ADHs were screened using cell-free extract (CFE), however, only four ADHs showed activity towards 1,6-hexanediol of which Aedes aegypti Farnesol dehydrogenase (AaSDR-1), a newly discovered ADH, showed activity towards 1,6-hexanediol. From the CFE reactions, an unknown side product was detected. Therefore, purified AaSDR-1 with Streptococcus mutant NADH oxidase 2 (SmNOX) for cofactor regeneration was used to study the reaction, however the same unknown side product also formed. Thereafter, it was speculated that tris buffer react with the products from 1,6-hexanediol. With NMR and GC-MS analysis, the product was identified to be a tricyclic-tris adduct. Different buffering systems, including sodium phosphate buffer, decreases the tricyclic-tris adduct. The AaSDR-1 and Equus caballus ADH (HLADH) were compared with low concentration of SmNOX. It was observed that at low SmNOX concentrations NADH regeneration is favoured, while, at high concentration NADPH regeneration is favoured. However, the single ADH enzyme system had limitation and a combinatorial ADH enzyme system was proposed to screen for the best primary and secondary ADH. From the combinatorial reaction, the combination of Thermus sp. ATN1 ADH (TADH)-AaSDR-1 gave the best activity. Unfortunately, during the single AaSDR-1 enzyme system and combinatorial TADH-AaSDR-1 system, a white-precipitate ‘plastic-like’ structure was formed over time. The precipitate could not be extracted and quantified. The precipitate could potentially be from 6-hydroxyhexanoic acid and therefore the Ɛ-caprolactone concentration might be underestimated. Chemical, photochemical regeneration and bi-convergent cascade systems were tested as alternatives to the SmNOX system. The FMN light driven reaction required high concentration of AaSDR-1 and also produced the white-precipitate ‘plastic-like’ structure. Although the bi-convergent system of NADPH-dependent Cyclohexanone monooxygenase (CHMO) and NADP+-dependent AaSDR-1 can produce Ɛ-caprolactone, the AaSDR-1 competes with the CHMO for substrate and cofactors resulting in low Ɛ-caprolactone yields.Item Open Access Changes in microbial ecology during poultry production(University of the Free State, 2000-03) Schreuder, Johanna Catharina; Viljoen, B. C.; Von Holy, A.; Cox, J.Microorganisms, especially pathogens, play an important role in the deterioration of poultry meat and its products causing spoilage or food poisoning. The meat renders an ideal medium for the growth and progression of microorganisms originating from the environment on the farm (eggs and broiler), transport to the abattoir as well as the abattoir. As far as we know, no attempts were undertaken to determine the total microbial spectrum, including bacterial and yeast populations from the egg unto the chicken carcass after slaughtering. A historical review of the incidence and extent of microorganisms associated with poultry and it's environment is given in Chapter 1. The background of poultry production and contribution of microorganisms (for example pathogens and yeasts) are highlighted. In Chapter 2 a survey was undertaken to determine the incidence, extent and serotypes of different pathogens on and in freshly laid eggs as well as incubated eggs in the breeder broiler hatchery. In addition, the number of viable cells of bacteria and yeasts were also determined. Microbial counts on the dirty eggs predominated. A major decline in microbial numbers on the egg shells, however, were observed after 18 days in the incubator. The deduction in bacterial populations present on the egg shells after 18 days in the incubator, is ascribed to the constant fogging of the environment inside the incubator with clinafarm. The irregular gathering of eggs as well as the dirty hands of the egg collectors could have been the major contributors of the high counts on these eggs. The only pathogens obtained were Listeria and E. coli type 1.In Chapter 3 the microbial populations associated with the caecum and liver of broilers as well as with the environment were determined. Anaerobic plate counts reflecting the dominance of bacterial populations were constantly the highest ascribed to the contents of the gut. Salmonella, Staphylococcus aureus and E. coli type 1 were isolated from the caecum, however no Listeria were isolated. Salmonella and E. coli type 1 were isolated from the liver. The high incidence of pathogens associated with the broilers is an indication of the pathogens that enter the abattoir. These levels of pathogens, further increased during transport to the abattoir.In Chapter 4 the incidence and extent of microbial populations associated with broiler meat and the environment in the abattoir were evaluated. All the pathogens present on the farm were also observed in the abattoir. The main reasons for the similarity in the incidence of pathogens on the farm and the abattoir were ascribed to the transport from the farm to the abattoir, the process of slaughtering, equipment surfaces in the abattoir and people handling the meat. Aerobic plate counts predominated on the neckskin samples, equipment surfaces as well as in the water and air of the abattoir. E. coli type 1 clearly predominated on the neckskin samples as well as on the equipment surfaces, followed by Staphylococcus (U/reus and that by Salmonella. No Listeria isolates was, however, isolated from either the broiler farm or the abattoir, but was indeed observed on the egg shells from the breeder farm. Although a pattern reflecting the incidence of pathogens could be establish between the broiler farm and the abattoir, no comparison could be made with the eggs from the breeder farm and hatchery. This may be blamed on the sampling of insufficient number of eggs, or the lack of multiple repetitions. Despite the inability to detect the primary sources of pathogens the distinct possibility remained that the broiler farm was the main contributor towards microbial contamination and infection. Therefore, more effort is needed to control the diseases and infections on broiler farms, because in an abattoir you only get out what you put in.Item Open Access The utilisation of used and other fats by fungi(University of the Free State, 2000-09) Bareetseng, Sechaba; Christov, L.; Kock, J. L. F.English: In 1997, Jeffery and co-workers discovered that when Mucor circinelloides t. circinelloides CBS 108.16 was cultivated on 30g/1 sunflower oil and 10g/1 sodium acetate, an improved utilisation of the oil, doubling of the biomass production and enhancement of the intracellular polyunsaturated y-linolenic acid (GLA) content occurred as compared to when this fungus was cultivated on only 40g/1 sunflower oil as sole carbon source. Consequently, the aim of this study became to further explore this phenomenon (hypothesis) in selected members of the zygomycotan fungi as well as yeasts when cultivated on various fat and oil substrates in the presence and absence of acetate. The ultimate aim was to identify those taxa that can be further explored for the transformation of edible and tall oils to high value lipids in the presence and absence of acetate. In this study, similar trends were observed in Mucor circinelloides f circinelloides CBS 108.16 as that found by Jeffery et al when cultivated on sunflower oil in the presence and absence of acetate. A similar pattern was also found when this fungus was grown on used cooking oil and tall oil. The enhancing effect of acetate was not generally observed in the other fungi and on some other fats and oils tested. Most fungi, including the yeasts, could grow on fats and oils provided in the presence or absence of acetate. Exceptions to the rule were MorfierelIa alpina MUFS Mo058 and Lipomyces starkeyi CBS 1807 T that were unable to grow. Schizosaccharomyces pombe var. pombe CBS 0356 T could also not grow on linseed oil. The presence of acetate had in many cases a stimulatory effect on growth. In some cases, the addition of acetate had an inhibitory effect on growth. With a few exceptions, most mucoralean fungi, when cultivated on various fats and oils, became oleaginous (i.e. contain ≥ 20% lipids) in the presence or absence of acetate. However, when these fungi were cultivated on linseed ail in the presence or absence of acetate they were unable to accumulate more than 20% lipids according to biomass. Less yeasts became oleaginous when cultivated on various fat and oil substrates when compared to the mucoralean fungi tested. The non-oleaginous yeasts i.e. Kluyveromyces, Saccharomyces, Schizosaccharomyces, Schwanniomyces and Yarrowia became oleaginous when cultivated on various fats and oils in the presence of acetate. In most mucoralean fungi and yeasts, the presence of acetate had an effect on cellular lipid content. The enhancing effect of acetate addition on cellular lipid content was experienced especially on GongronelIa, Mucor circinelloides t. circinelloides CBS 108.16 and Thamnostylum when cultivated on various fats and oils. On the other hand, the addition of acetate had a negative effect on cellular lipid accumulation in some mucoralean fungi and yeasts. In general, both the mucoralean fungi and yeasts utilised different fats and oils [(i.e. containing saturated fatty acids (FAs) and polyunsaturated fatty acids (PUFAs)] as carbon sources in the presence or absence of acetate. MortierelIa and Lipomyces could not utilise any of these fats and oils. The presence of acetate had a positive effect on fat and oil utilisation by most mucaralean fungi and some yeasts. In most cases, a pH increase was observed probably due to acetic acid utilisation during cultivation. In the presence or absence of acetate, most mucoralean fungi and yeasts showed a high preference towards the utilisation of PUFAs present in the residual oil fractions. The addition of acetate had both a positive and negative effect on the degree of preference towards PUFAs in the residual lipids. In most cases (in both mucoralean fungi and yeasts) lower amounts of PUFAs in the cellular lipid fractions in the presence or absence of acetate were found when compared to the oil substrate fed. This is probably due to the utilisation of these FAs through β-oxidation for the production of energy. No general pattern was observed regarding the effect of acetate addition on cellular PUFA content. Many mucoralean fungi when cultivated on various fats and oils (except soap skimmings) could produce GLA in the presence or absence of acetate. All the yeasts in this study could not produce GLA when cultivated on any lipid substrate in the presence or absence of acetate. This is probably due to the lack of a ∆6 desaturase enzyme. The addition of acetate improved GLA production in most mucoralean fungi when cultivated on sunflower oil and used cooking oil. Those fungi capable of producing GLA in this study should now be explored in the transformation of edible fats and oils to high value lipids containing GLA.Item Open Access Mucoralean fungi present in soil from arid regions in South Africa(University of the Free State, 1999-11) Seabi, Buti Oscar; Botha, A.; Viljoen, B. C.English: The aim of the first part of the study was to investigate the ecological niche of mucoralean fungi in arid soil, with specific reference to the position these fungi occupy in the biogeochemical cycle of nitrogen. Consequently, selected mucoralean taxa occurring frequently in soil habitats, including strains from culture collections, as well as isolates obtained from a soil sample from arid Upper Nama Karoo, were used to evaluate in vitro growth to determine nitrogen sources and aw tolerances. Nine mucoralean fungal genera including 18 species were examined for the ability to utilise a series of nitrogen containing compounds and to grow at an aw of 0.955 on solid media. The nitrogen concentration in the media was 0.1 g.r1 and the series of nitrogen containing compounds were ammonium chloride, asparagine, sodium glutamate, sodium nitrite and potassium nitrate. The genera were Actinomucor Schostak., Backusella Hesselt. & J.J. Ellis, Cunningha'fnella Matr., GongronelIa Ribaldi, MortierelIa Coem., Mucor Fresen., Rhizomucor Lucet & Costantin., Rhizopus Ehrenb. and Thamnostylum Arx & H. P. Upadhyay. Thirty-nine fungal strains obtained from culture collections (CBS, MUFS and PPRI), as well as 12 soil isolates from the Karoo, were tested. All the species and strains tested in this study were able to utilise asparagine and glutamate. Strains belonging to CunninghamelIa, Mucor racemosus Fresen., Rhizopus microsporus Tiegh. and Rhizopus stolonifer (Ehrenb.: Fr.) VuilI. were unable to utilise ammonium chloride. Strains of CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus microsporus and Rhizopus stolonifer were unable to grow on nitrate as sole nitrogen source. Nitrite was found to be toxic to species belonging to CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus and Thamnostylum. Members of GongronelIa, MortierelIa, Mucor racemosus, Rhizomucor and Thamnostylum were unable to grow at an a, of 0.955. The aim of the second part of the study was firstly to get an indication whether the mucoralean diversity of the Karoo, as observed in the first part of the study and in the records obtainable from literature, differs from data on mucoralean diversity from other arid regions. The latter included data from literature and what could be found in a soil sample taken from Kimberley Thorn Bushveld. Secondly, the aim was to test the isolates obtained from the Kimberley Thorn Bushveld soil sample in order to further explore the ability of mucoralean fungi to utilise the above mentioned series of nitrogen sources and to grow at an a, of 0.955. In addition, selected mucoralean taxa occurring frequently in soil habitats were tested for the ability to survive elevated temperatures in soil. It was found that the following species of the Mucorales may be encountered in the arid soil of the Karoo; Actinomucor elegans, CunninghamelIa echinulata, MortierelIa isabellina, Mucor circinelloides, Rhizomucor species, Rhizopus oryzae Went. Prins. Geerl. and Rhizopus stolonifer. Future surveys would reveal if genera like Absidia, GongronelIa and Zygorrhynchus, which have been isolated from arid regions, also occur in Karoo soil. Representatives of mucoralean taxa occurring in arid Karoo soil were able to utilise organic as well as inorganic oxidised nitrogen sources. However, at the concentration tested in this study, nitrite was found to be toxic to representatives of CunninghamelIa, Mortierell8, Rhizomucor and Rhizopus. Nitrate could not be utilised by CunninghamelIa, MortierelIa, Rhizomucor and Rhizopus stolonifer. Whether this inability to utilise inorganic nitrogen sources would prevail 'during oligotrophic growth in soil, remains a question to be addressed by future research. Representatives of the above mucoralean taxa occurring in arid soil were able to survive 55°C for 14 h in soil.Item Open Access Ophiostoma species from hardwood sources in South Africa(University of the Free State, 2001-10) De Beer, Zacharias Wilhelmus; Wingfield, M. J.; Wingfield, B. D.English: The ophiostomatoid fungi are an economically important group of fungi, known for their ability to stain sapwood and cause tree diseases. In recent years, certain species in the group have also been considered as potential biological control agents in the pulp and paper industry. White mutants of species like Ophiostoma piliferum utilize pitch, which can cause problems in the pulping process, in freshly cut pulpwood chips. At the same time, other degrading fungi are out-competed. The first chapter of the thesis reviews the development, application, benefits and possible problems, of these biological control products. The possible application of such products in the South African pulp industry is also considered. One of the major concerns for the application of a biological control product such O. piliferum in South Africa, is the fact that it consists of a living fungus originating in the USA. A survey was, therefore, conducted to determine whether the fungus occurs in South Africa. The typical niche for O. piliferum is stained logs, lumber, and pulpwood chips. Isolates resembling O. piliferum were obtained from both exotic and indigenous wood sources. Based on morphology, these isolates could be separated into three groups, which resembled the descriptions of O. stenoceras, O. pluriannulatum, and O. piceae, respectively. For correct identification, the South African isolates had to be compared with herbarium material and authentic isolates from other parts of the world. The comparative taxonomic studies for the three groups of fungi form the basis of Chapters 4, 5 and 6 of this thesis. The taxonomic history of the genera Ophiostoma and Ceratocystis is complicated and confused. Published literature on the two genera, with the emphasis on Ophiostoma and its associated anamorph genera, is reviewed in Chapter 3. This serves as a background for the four following chapters of the thesis. Ribosomal DNA sequencing confirmed that one group of South African isolates is the same as O. stenoceras isolates from the Northern Hemisphere. Similar isolates from Colombia, Uruguay, and Kenya, were also included in the study and represent the first reports of O. stenoceras from these countries. Ophiostoma albidum, O. abietinum and O. nigrocarpum, all closely resemble O. stenoceras morphologically. Our sequence data show that O. albidum should be considered a synonym of O. stenoceras, and that O. abietinum is a synonym of O. nigrocarpum, which is a species distinct from O. stenoceras. For the past three decades, O. stenoceras has been considered the teleomorph of Sporothrix schenckii, the human pathogen. Our results, however, showed that rDNA sequences of the two species are significantly different, confirming that S. schenckii is a distinct species. The group of South African isolates resembling O. pluriannulatum, differed from this Northern Hemisphere species in that isolates have light brown perithecial bases and clubshaped ornamental hyphae on the perithecial bases. Similar isolates were obtained from Equador and Indonesia. Ribosomal DNA sequence data made it possible to distinguish between the two groups, and the Southern Hemisphere fungus is, therefore, described as a new species, Ophiostoma tropieale. Ophiostoma piceae and O. querei are virtually indistinguishable based on morphology, but hosts, rDNA sequences, and mating compatibility, can be used to separate the two species. By applying these criteria, the South African isolates resembling O. piceae obtained in the survey (Chapter 2), grouped with O. querei. Also included in the O. querei group were isolates from Brazil and Japan. One South African isolate, however, were identified as O. floccosum, representing the first report of this fungus from South Africa. The presence and distribution of species of the O. piceae complex in the Southern Hemisphere, are also discussed in Chapter 6. In recent literature, some confusion has emerged regarding the use of the name O. querei as opposed to 0. quercus. The last chapter of the thesis presents a brief review of the Latin and nomenclatural guidelines applicable in this particular case. The conclusion is that both names are grammatically acceptable. However, following the Code of Botanical Nomenclature, O. querei should be given preference. The work presented in this thesis contributes significantly to our understanding of the ophiostomatoid fungi, and in the greater context, biodiversity, in South Africa. Ribosomal DNA sequencing was successfully applied in conjunction with traditional taxonomic criteria to distinguish between species. However, many new questions arose from these results, especially regarding the phylogeny of Ophiostoma spp. with Sporothrix anamorphs. The results obtained from this study, will serve as the foundation for future research addressing these questions.Item Open Access The growth kinetic characterisation of Saccharomyces cerevisiae strains transformed with amylase genes(University of the Free State, 2002-03) Knox, Alison Margaret; Du Preez, J. C.; Kilian, S. G.English: The direct fermentation of starch to ethanol using an amylase-producing yeast is of interest as an alternative to the conventional fermentation processes, which utilise commercial amylases. Starch is an abundant renewable biopolymer, comprising two major components, namely amylose (α-I,4-linked D-glucose residues) and amylopectin (α-I,4 and α-I,6-linked D-glucose residues), typically constituting 80 % of cereal starches. Efficient starch degradation, therefore, necessitates the use of α- and glucoamylases, together with an α-I,6 debranching activity. Naturally amylolytic yeasts are not suited to fermentations, whereas Saccharomyces cerevisiae, known for its strong fermentation capacity, lacks amylolytic activity (with the exception of S. cerevisiae var. diastaticus, which has weak glucoamylase activity). Consequently, the diploid "Sigma" strain of S. cerevisiae was transformed with different combinations of amylase genes from Lipomyces spencermartinsiae (with the PGK] promoter), and Saccharomycopsis fibuligera (with its natural promoters) using an integrating plasmid in a single copy form. These recombinant strains, provided by the University of Stellenbosch, were evaluated in respect of their ability to ferment starch to ethanol. Recombinant strains of S. cerevisiae containing the S. fibuligera amylase genes with non-integrating plasmids in a multi-copy and a single copy form, provided by the University of the Free State, were also evaluated. Notable differences in the hydrolysis zones on starch agar plates indicated that the type of starch medium used and the amylase produced exerted a significant effect. The dimensions of these hydrolysis zones were a poor indicator of the performance of the strains in liquid starch media. Anoxic cultivations in shake flasks and in 2-1 bioreactors containing a 2 % starch medium yielded less than 109 ethanol.l-1 over a 200 h incubation period. Aerobic growth yielded more biomass and, therefore, higher amylase values and higher rates of starch hydrolysis, but with no detectable amounts of ethanol. Initial evaluations indicated poor amylase activity, particularly with strains containing the S. fibuligera amylase genes. The limited amylase production by a strain containing the S. fibuligera amylase genes was not due to proteolytic activity or intracellular enzyme . accumulation. Strains containing the L. spencermartinsiae amylase genes gave the best overall results. However, a strain containing a combination of both a-amylase and . glucoamylase yielded disappointing results. The curious multi-phasic growth profile obtained with this strain, accompanied by a delay in amylase production, suggested regulation of the PGKl promoter by glucose. However, Northern blot analyses indicated very low levels of glucoamylase mRNA, with no clear indication of induction of the PGKl promoter. A recombinant strain (strain steIl7) with both glucoamylase genes (LKAll from L. spencermartinsiae and GLUI from S. fibuligera) proved to be the most prormsmg. Further evaluation in 15-1 bioreactors resulted in the production of ea. 21 g ethanol.l-1 from 55 g starch.l-1 by strain stell7. Despite a relatively high α.-amylase activity of 500 U.l-1 after 150 h, the slow rate of enzyme production remained the rate-limiting step. Although some of these recombinant strains were capable of complete starch hydrolysis, the slow rate of starch saccharification and the concomitant low ethanol productivity rendered these strains unattractive for commercial application.Item Open Access Determination of the malting and milling performance of sorghum cultivars(University of the Free State, 2001-05) Van Loggerenberg, Magdalena; Osthoff, G.; Pretorius, A. J.English: Consumer demands for sorghum food acceptability stress the need for quality evaluation of milled and malted products. The aim of this study was to determine the milling and malting quality of sorghum cultivars with different physical and chemical qualities. The malting, physical and milling qualities of 24 sorghum cultivars were investigated over 2 seasons at 3 localities per season. The most important malting quality parameters that were identified with the canonical variate analysis were free amino nitrogen, diastatic power, germinative energy and vigour, compared to polyphenols, malting loss and water absorption that was identified as less important. From the canonical variate analysis, the most important physical qualities measured were sieve . fraction large, hundred kernel weight, water absorption and moisture content and were used to predict milling quality, while sieve fraction medium and small and meal fractions were found to be less important. Food quality properties were determined from roller and abrasive decortication milled meal. The abrasive hardness index, dehulling index and viscosity were important abrasive decortication milling parameters, with break 2 bran, break 1 bran, L-values, extraction, break 2 meal, break 1 meal and break 2 grits being important roller milling parameters. The best malting cultivars were APN 881, NK 286, PAN 8446, PAN 8660, SNK 3443, SNK 3337, SNK 3663, SNK 3939, SNK 3975, PAN 8564, NS 5655, SNK 3860 and NS 5511. Cultivars with the best physical properties were SNK 3939, SNK 3663, PAN 8061, PAN 8564, PAN 8171, SNK 3443, PAN 8446, SNK 3337, NK 286, APN 881, SNK 3883, SNK 3975, SNK 3567, PAN 8272 and PAN 8370. Good abrasive decortication milling property cultivars were APN 881, SNK 3567, SNK 3863, NK 286, ADV 5010, PAN 8564, SNK 3443, PAN 8370 and PAN 8660. Good roller milling cultivars were SNK 3883, SNK 3939, PAN 8564, NK 286 and SNK 3975. Cultivars with multi-functions were NK 286, PAN 8564 APN 881, SNK 3443, PAN 8660, SNK 3939, NS 5655 and SNK 3975 (good malting and milling).Item Open Access Fruit quality of South African cactus pear cultivars(University of the Free State, 2010-06) Rothman, Anna Maria Petronella; De Wit, M.; Bothma, C.English: The cultivation of cactus pears requires low input and it has been grown widely in drier areas of South-Africa as fodder crop, particularly for times of serious drought. Cactus pears also serve as a source of inexpensive nutritious food for lower income groups Sugar is the main determent of taste of the cactus pear cultivar and content value range from 10 °Brix to 17 °Brix. Glucose is the predominant sugar with fructose as the second sugar, thus the fruit pulp is very sweet. The pulp of the cactus pear cultivar consists of a high pH value (> 4.5) and low acidity level (0.03-0.12 % ), (SaenzHernandez, 1985; Brutch, 1993; Piga, 2004; Salim, 2009). The aim of this study was to determine the fruit quality of cactus pear fruit. Physical/chemical and sensory quality attributes were evaluated for two agricultural seasons ( 2007 and 2008). The influence of factors such as rainfall and temperature on quality was determined. Furthermore, sensory analysis was used to distinguish among the available 33 cultivars, not only for their taste, but also to establish the cultivar most stable to varying environmental conditions. This study determined whether sensory quality of cactus pear fruit was influenced by the physical/chemical parameters by correlating the physical/chemical data with the sensory analysis. There were highly significant differences observed in terms of physical/chemical composition (p < 0.001) among 33 different cactus pear cultivars in South Africa, for seasons 2007 and 2008. This finding indicated that genetic differences among cultivars as well as seasonal changes have a significant influence on fruit quality. It was evident from this study that not only the cultivar and agricultural season, but also the interaction between the cultivar and season had significant influences on fruit quality. The best preferred cultivar, regarding physical/chemical fruit quality attributes, was Nudosa, performing the best regarding fruit mass and pulp glucose. Messina performed the best regarding 0 Bx. Nudosa and Messina performed the best regarding pulp fructose content, while Blue Motto had the best acidity levels (pH and TA). Sensory analysis, used to determine whether the consumer could distinguish among the available 33 cultivars was done by using the FCP technique and it was clear that the consumers could only successfully distinguish between the two seasons (77.72 %) , but not among the 33 different cactus pear cultivars. The fodder cultivar, Robusta, was an exemption and could be clearly distinguished from the other 32 cultivars. The sensory quality of cactus pear fruit obtained from the consumers was indeed influenced by the physical/chemical parameters. Cultivars like Robusta, Fresno and Nudosa had been significantly influenced by seasonal differences. The physical/chemical data and the sensory attributes were correlated.Item Open Access Exploring horizontal gene transfer and phage infections in a South African deep subsurface bacterial population(University of the Free State, 2017-06) Mlandu, Cumisa Manzikazi; Van Heerden, E.; Cason, E. D.Viruses (more specifically bacteriophages which infect both bacteria and archaea) are the most abundant microorganisms in the ecosystem. In extreme environments such as the deep subsurface, bacteriophages play an important role in the altering of biogeochemical cycles and in the evolvability and survival of their hosts. Unfortunately even in their abundance, little is known about phages and their interactions with their hosts and this is due to the fact that 99% of the microorganisms in the environment (especially in the deep subsurface) cannot be cultured. Instead the use of culture-independent techniques such as microscopy and next generation sequencing based techniques. Recent research done on microbial diversities in deep subsurface environments prefers the use of metagenome platforms that have 100% sequence coverage as it allows for comprehensive sequencing as such one is able to identify and assemble genomes of species that are present at low frequencies in environmental samples. These techniques also allow for the detection of phage genes and HGT events within the genomes of the host, thereby giving insight into the host-phage interactions in these extreme environments. Access to the microbial matter in deep terrestrial subsurface is through mines and several in South African deep mines have identified phage genes and HGT events within genomes of indigenous bacteria. Microscopic techniques such as EFM and TEM have been used to detect and morphologically characterize phages (respectively). The characterization of phages in extreme environments using TEM has only been published for deep marine environments, hot springs and solfataric fields and not for the deep mine fissure water. Therefore, phages in the deep terrestrial environment have only been studied through the use of metagenomic sequencing techniques. In this study the main objectives were to identify phage genes and the presence of horizontally acquired genes and their effect on the deep subsurface bacterial communities in terms of evolution and survival. Sampling and concentration (using tangential flow filtration) of fracture water from Star Diamonds mine, Fronteirs mining resulted in samples eligible for microscopic analysis (using SEM, EFM and TEM) and sequencing using whole metagenome sequencing to identify the phage related genes. SEM analysis revealed most of the bacterial communities are in biofilm structures and this is expected due to the unfavourable conditions of the environment. The free phages in the viral fraction fracture water were detected using EFM and the TEM analysis identified phages belonging to the Myoviridae and Podoviridae families which have been previously studied and found in the marine environments. This study is the first to identify free viral particles in the fissure water of deep subsurface South African mines. It is also the first study to detect free viral particles in the South African diamond mine. Whole metagenome sequencing of fracture water from Star Diamonds mine, Fronteir mining was done in order to achieve the above stated main objectives. All three domains of life (Bacteria, Archaea and Eukarya) were present in the fracture water and dominated by bacteria from the phylum Proteobacteria. The phages detected in the metagenome belonged to the order Caudovirales with the Siphoviridae family being the most abundant. The presence of the Myoviridae and Podoviridae families further confirmed the results from the phage characterization using TEM. Previous studies in deep subsurface samples from South Africa have discovered viral infections mostly in Firmicutes, which dominate older fracture water at deeper depths. In this study the majority of the phage sequences identified using VirSorter were from phages that infect hosts from the phylum Proteobacteria which is the most abundant phyla in the fracture water. This study therefore provides valuable insight into other host microorganisms such as the Proteobacteria which generally dominate in younger fracture water at shallower depths. Partially complete prophages were detected and annotated and the presence of prophages in South African deep mines has been previously identified by two researchers. The presence of prophages suggests that phages in this environment can be both lytic (as observed with the detection of free phages using TEM) and lysogenic, but the low abundance of phages detected using TEM and EFM suggests that they prefer lysogenic infections. The CRISPRs, mobile/transposable elements, transposase and retrons detected within the binned metgenome data are suggested to be markers for possible phage mediated HGT events by previous researchers. This study further identified genes suggested to be products of HGT events that were part of the nitrogen fixation, cobalamin synthesis and sulfide reduction pathways and motility and sporulation. These genes conferred novel capabilities to the host (that they were transduced into via HGTs) for survivability and evolution in the extreme deep subsurface environment. This is the first study to specifically look at the bacteria-phage interactions within the subsurface mines of South Africa and it is also the first to find viral infections within the Star Diamonds mine. As future research, phylogenetic analyses would need to be done in order to further confirm the identified HGT events.Item Open Access The effect of irradiation and elevated temperature on the ripening of cheddar cheese(University of the Free State, 2001-11) Seisa, Dipuo Pascalina; Osthoff, G.English: Proteolysis and lipolysis are important biochemical events in the ripening of cheese to contribute to the development of flavour and texture. The characteristic proteolysis of each type of cheese is brought about by the enzymes used in the manufacturing process, e. g. rennet, as well as enzymes from the specific microbial cultures used in each cheese type. The microbial and chemical development can be manipulated by external conditions such as ripening temperature and irradiation. In the current study the effect of temperature together with irradiation on the ripening of Cheddar cheese was investigated. Cheddar cheeses were irradiated after 4 days of ripening at 8 °c and 16°C at a dose of 4kGy and ripening continued up to 12 weeks with unirradiated cheeses ripened at 8 °c and 16°C as controls. In asensorical comparative study, the cheeses were evaluated as not being different in taste. There were no significant differences in the free fatty acid content of the cheeses but the irradiated cheeses and cheese ripened at 16°C had higher TBA-values. The WSNfTotal N (water-soluble nitrogen /total nitrogen) of the cheese ripened at 8 °c was significantly different than that of the other cheeses after 2 weeks, but then reached the same levels as the treated cheeses after 6-12 weeks. The Urea-polyacrylamide gel electrophoresis of the WISF indicated that the cheese ripened at high temperature matures faster than the cheese ripened at low temperature for both the unirradiated and the irradiated cheeses, whereas the irradiated cheeses showed different peptide development over the same period of ripening. The RP-HPLC of the cheeses indicated a difference in peptide profiles of the unirradiated and irradiated cheese ripened at 16°C, and the irradiated cheese ripened at 8 °c from the unirradiated cheese ripened at 8°C.Item Open Access A comparative study of proteolysis in cheddar cheese and yeast-inoculated cheddar cheese during ripening(University of the Free State, 2000-05) Botma, Maryna; Osthoff, G.; Viljoen, B.C.English: Proteolysis is regarded as the most important event in the ripening of Cheddar cheese to contribute to the development of flavour. The characteristic proteolysis of each type of cheese is brought about by the enzymes used in the manufacturing process, e.g. rennet, as well as enzymes from the specific microbial cultures used in each cheese type. The effect of an inoculated yeast, Oebaryomyces hansenii, and its enzymes on proteolysis in Cheddar cheese was investigated. Proteolysis and development of peptides of the yeast-inoculated Cheddar cheese was followed throughout the ripening process and compared to the proteolysis in a standard Cheddar cheese. In a sensorical comparative study, no difference was found by a consumer panel between the two types of cheese, nor was any of the two significantly preferred. An expert panel however, judged the yeast-inoculated Cheddar cheese to be bitter. The proteins and peptides from the cheeses were extracted and fractionated by virtue of differences in solubility and molecular size. Yeast-inoculated Cheddar cheese contained less water-soluble nitrogen indicating a difference in proteolysis between the two types of cheese. Urea-polyacrylamide gel electrophoresis of the waterinsoluble fraction indicated that in the yeast-inoculated cheese rennet hydrolysis of o.si-casein was increased with faster formation of one primary peptide, o.s1-1as well as (o.s1-CN(f102-.)), with little further hydrolysis. The ~-casein was hydrolyzed slowly but with several additional peptides occurring. Reversed-phase high performance liquid chromatography of the watersoluble fraction indicated that a different peptide profile was formed, with at least five unique peptides at high amounts.