Infrared spectra of microbial cells are highly specific, fingerprint-like signatures which can be used to differentiate microbial species and strains from each other. In this study, the potential applicability of Fourier transform infrared (FTIR) spectroscopy for the classification of yeast strains in terms of their biological taxonomy, their use in the production of wine, beer, and bread, and their sensitivity to killer yeast strains was investigated. Sample preparation, spectral data preprocessing methods and spectral classification techniques were also investigated. All yeast strains were grown on a single growth medium. The FTIR spectra were baseline corrected and the second derivative spectra were computed and employed in spectral analysis. The classification accuracy was improved when the principal component spectra (calculated from the second derivative spectra) were employed rather than the second derivative spectra or raw spectra alone. Artificial neural network (ANN) with 10 units in the input layer and 12 units in the hidden layer produced a robust prediction model for the identification of yeasts. Cluster analysis was employed for the classification of yeast strains in terms of their use in the production of wine, beer, and bread and in terms of their sensitivity to killer yeast strains. The optimum region for the classification in the former case was found to be between 1300 and 800 cm-1 in the infrared spectrum whereas the optimum region for the classification of yeast strains in terms of their sensitivity was between 900 and 800 cm-1 . The results of this work demonstrated that FTIR spectroscopy could be successfully employed for the classification and identification of yeast strains with minimal sample preparation.

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Environmental and food spoilage fungi cause billions of dollars in damage in North America alone each year, in the form of rotted wood and crops, spoiled food, and human and animal illness. Each of these threats could be drastically reduced if early and more rapid detection processes are developed to replace the serological methods that are currently in practice. The current North American protocol for establishing identification of contaminating fungi both in environment and food have a time frame of approximately one week to twenty-two days. The use of a Fourier transform infrared (FTIR) spectrometer, coupled with a focal-plan-array (FPA) detector, can theoretically shorten the time (analysis within minutes after obtaining a pure culture) it takes to identify and classify a fungal cell. FPA-FTIR spectroscopy is advantageous as little to no sample preparation is required and results are obtained in less than one minute per sample. The fungal subset chosen for this study includes representatives from five phyla, including Zygomycota (Mucor heimalis), Ascomycota (Neurospora crassa, Ophiostoma minor, Chaetomium globosporum, Alternaria brassicicola), Basidiomycota (Schizophyllum commune, Chaetomium globosporum), Deutromycota (Aspergillus niger, Penicillium notatum, Aureobasidium pullulans) and the Mycetozoa (dictyostelium discoideum, physarum polycephalum). Different variables were tested and evaluated, including variability in growth parameters, wet deposition of fungi versus dry smearing of fungi, optimal absorbance range, and spectral processing parameters as well as discrepancies from one instrument to another, as well as spectral reproducibility from one instrument to another. By following the experimental protocol developed, reproducible spectra were attained, and differentiation of the fungi within the set selected for this study was achieved. The results of this work demonstrate that FPA-FTIR spectroscopy can potentially be employed for the accurate identification of environmental and food spoilage fungi.

The activated sludge process comprises a complex and enriched culture of a mixture of generalist and specialist organisms. The lack of knowledge on species diversity of microbial communities is due to the simplicity of bacterial morphology and the phenotypic characters, and the unculturable portion of microbial cells in natural habitats. Although a wide range of bacteria can be isolated using conventional microbiological techniques of sample dilution and spread plate inoculation, many well-known activated sludge bacteria can not be isolated using them. The individual microbial cells in activated sludge grow in aggregates that consist of floc-forming organisms together with filamentous microorganisms that form the backbone of the activated sludge floes. Overgrowth of these filamentous microorganisms often causes settling problems called bulking and foaming. These problems consist of slow settling, poor compaction of solids and foam overflow into the effluent. Although methods for the isolation of filamentous bacteria from mixed liquor samples have been investigated, the attempts have been largely unsuccessful. In this study we investigated bulking and foaming activated sludge to identify the dominant filamentous organisms using microscopy and molecular techniques. Using microscopy, the dominant filament associated with the foaming sample was "Microthrix parvicella" and in the bulking sample was Nocardia spp. The foaming sample was investigated using molecular techniques that involved 165 rDNA sequencing. Although some of the clones isolated from the sludge foam were associated with filamentous bacteria causing foam, no positive identification could be made. In the part of the study that was conducted in Australia, a rRNA-targeted oligonucleotide probe was designed for the identification of a filamentous organism occurring in activated sludge foam. This organism resembled Eikelboom Type 0041 and was classified in the candidate bacterial division TM7. The discrepancy that the sequence data did not indicate the dominant filamentous organisms observed by microscopy, highlights the fact that natural microbial communities need to be studied using a combination of techniques since none of the techniques available are sufficient to determine the complete community structure of complex communities such as activated sludge. / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

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Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in