The overall aim of this study was to eliminate the root cause of the ‘cheese flavour’ in spread caused by four key microbes Bacillus spp, Staphylococcus spp, yeasts and moulds. The major sources of these bacteria were in the product ingredients mainly sweet cream buttermilk and skimmed milk along with environmental aerosols. The causative organisms were present in about 63% of the products and mainly ‘feed’ on the oil element of the recipe, containing high level C12 that generates the distinctive cheese flavour when broken down by bacteria. The key hurdle factor in spread preventing microbial growth is water droplet size. The spread showing cheese off flavour had a droplet size distribution of 95% <10 micron. To achieve finer droplet size distribution, trial products were made in the Scrape Surface Heat Exchanger (SSHE) over the current churning method with a distribution of droplet size 98% <5 micron. The trial product showed a 50% reduction in the generation of the ‘cheese flavour’ methyl ketones. The Staphylococcus spp cross contamination source where from personnel with direct food contact processing area. Further education on personal hygiene helped to reduce the level of Staphylococcus spp contamination in the product. The trial product from the SSHE was further challenge tested with Listeria monocytogenes over a 10 week shelf life period to evaluate product robustness against microbial growth and spoilage. The organism did not show any growth over the period of time. The liquid phase of the emulsion was further modified with various salts at different concentrations and challenged with L. monocytogenes isolated from various parts of the dairy environment. It was observed that a pH range of 5.5 or lower with added 0.063% potassium sorbate showed significant antibacterial affect compared to the nutrient enriched MPC-broth and the unsalted liquid phase of the emulsion with no added potassium sorbate. To understand L. monocytogenes survival within a dairy process, the organism was further challenged by exposure to pasteurisation heat treatments and the standard CIP cycle of acid and caustic treatment. No recovery rate of the organism was observed. Therefore it could be concluded that the contamination within the industry is more likely to be post process or environmental contamination rather than survival through the plant itself as per RASFF alert of Listeria spp outbreak in dairy. Therefore, reducing the available water in the liquid phase of the spread and achieving a <5 μm droplet size and a finer distribution within the product will be limiting factors to microbial growth. An air purifier system BAXX has reduced the level of environmental contaminants, especially yeast and mould.

Calcium has been shown to play important roles in the control of tip growth in a wide range of cell types. This thesis sets out to test the effects of exogenous Ca2+ on various morphological events during the life cycle of the human pathogenic fungus Aspergillus fumigatus. The Ca2+ chelating agent EGTA induced marked morphological effects on colonies of A.fumigatus. That is radial growth rate decreased and branching frequency increased with increasing concentration of EGTA. Ion substitution experiments indicated that these effects were due to the chelation of Ca2+ and not Mg2+, Zn 2+, Cu2+ or Fe2+. However, Mn2+ was able to substitute for Ca2+. Calcium ions were required for germ tube emergence but not for the preceding period of spherical growth in spores of A.fumigatus. That is the rate of spherical growth was independent of exogenous [Ca2+ ] but germ tube emergence was retarded and spores became more swollen as exogenous Ca2+ decreased. The effect of applied electrical fields on spores of A.fumigatus was investigated. Germ tubes were anodotropic and polarisation increased with increasing field strength. The galvanotropic response was dependent on Ca2+ and polarisation decreased with exogenous [Ca2+ ]. The effect of exogenous [Ca2+] on growth kinetics of colonies of A.fumigatus growing on solid medium was investigated. When colonies were incubated on medium buffered at pCa 4-8 (10-4-10 -8 M) hyphal growth unit length and mean hyphal extension rate decreased linearly with the log of Ca2+ concentration. In contrast the specific growth rate remained constant over the range pCa 4-7 and was only reduced when colonies were incubated in medium buffered at pCa 8. This suggests that exogenous Ca2+ acts on processes that govern apical growth and branching but do not affect growth per se. The effect of a broad range of Ca2+-channel blockers and calmodulin antagonists on the growth and morphology of colonies of A.fumigatus were also investigated.

No description available.

In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced. / Graduation date: 2000

Bioremediation in the vadose zone is unpredictable because of poor understanding of factors influencing microbial growth in this environment. A lab-scale experimental system was developed to examine, noninvasively, interactions between microbial growth, water flow, and solute transport in unsaturated porous media. Measurements of microbial colonization, and its impact on hydrology, were facilitated by using the luxCDABE-containing reporter bacterium Pseudomonas fluorescens HK44 and digital CCD imaging. Experiments were conducted in glass-walled two-dimensional flow cells (45 x 50 x 1 cm) packed with silica sand. Several bioengineering problems associated with chamber design and function required solution before microbial experiments were successful. These included: choice of materials for chamber components; development of sterilization, packing, and inoculation protocols; and development of procedures for data collection and chamber maintenance during experiments lasting several days. Bacterial growth was mapped daily by quantifying development of salicylate-induced bioluminescence. A model relating the rate of increase in light emission after induction successfully predicted microbial densities over four orders of magnitude (R��=0.95) provided that sufficient oxygen for the bioluminescence reaction was available. Total model-predicted growth during a one-week experiment agreed with potential growth calculated from the mass-balance of the system and previously established kinetic parameters (predicted, 1.2x10���� cells; calculated, 1.7x10���� cells). Although the rate of expansion of the colonized zone (and predicted populations in newly colonized regions) remained relatively constant, the proportion of the daily potential growth remaining within the chamber declined over time. Monitoring of bioluminescence revealed the development of an (hypothesized) anaerobic zone associated with microbial growth in the unsaturated porous media. Water content and flow streams were measured using light transmission. Accumulation of microbial growth modified the hydrologic properties of the sand causing up to 50% decrease in saturation within the colonized zone, diversion of flow around the colonized zone, and lowering (5 cm) of the capillary fringe height. Apparent solute velocity through the colonized region was reduced from 0.39 cm min����� (R��=0.99) to 0.25 cm min����� (R��=0.99). These experiments provide proof-of-concept for combining light transmission and bioluminescence technologies to study interactions between microbial growth and hydrology in unsaturated porous media. / Graduation date: 2002

Stable carbon isotope fractionation during methanogenesis is affected by the availability of substrates. The effects of different substrates on methanogen biomass, total lipid extract, biomarkers and methane under both abundant and limiting substrate conditions were studied. Methanosarcina barkeri was grown with methanol, acetate, trimethylamine (TMA) and H2/CO2, and carbon isotope fractionation in methane production was greatest with methanol, followed by H2/CO2, TMA and acetate. In contrast, biomass was isotopically lightest in M.barkeri grown on methanol, followed by TMA, H2/CO2 and acetate. Generally, fractionation was greater in cultures grown with abundant substrate availability as compared to those supplied with limiting substrate. During autotrophic growth, fractionation was greatest during slower growth for both methane and biomass production. The results of these fractionation studies under controlled laboratory conditions can be applied to the interpretation of isotopic signatures for methane and methanogen biomarkers, and ecological processes, in marine environments. Several hypersaline springs off the western shore of Lake Winnipegosis, MB support unique microbial mat communities. These low temperature springs contain water with a mean salinity as high as 6.1%. Studies were undertaken to contrast the anaerobic microbial communities of these springs, specifically the methanogens and sulphate-reducing bacteria (SRB), and their contributions to biogeochemical cycling in these mats. Comparisons of lipid profiles revealed changes in the proportions of the dominant fatty acids related to the amount of mat growth. Cultures of SRB and methanogens were established with six different substrates. Methanogenic cultures grew best on TMA and methanol, but could use formate, H2/CO2 and glycine betaine as well. In contrast, H2/CO2 was the preferred substrate of the SRB enrichment cultures, which were also able to use formate, but not TMA, the breakdown product of the compatible solute glycine betaine. Maximum methane production occurred at 5% salinity. The lipid composition of the mats, including methanogen biomarkers, and the results of the enrichments on different substrates and at different salinities, suggest that methanogenesis in these springs is supported by compatible solutes whereas sulphate reduction is linked to availability of hydrogen and formate. / February 2005

A major objective of biodiversity-ecosystem functioning (BDEF) research is to determine the consequences of species loss, caused both naturally and anthropogenically, on the functioning of ecosystems. The impact of plant species loss on the soil microbial community has not received much attention even though soil microbes influence many important ecosystem functions such as decomposition and nutrient cycling. The objective of this research was to investigate how the functional group composition of the aboveground plant community influenced the belowground microbial community. Plant functional groups (graminoids, legumes and non-leguminous forbs) were removed from a northern grassland system in the Yukon Territory, Canada. One metre square plots had one of the three functional groups removed or left intact as a control and this was crossed with a fertilizer treatment and a fungicide treatment that targeted mycorrhizal fungi. After five seasons (2003-07) of implementing treatments the soil microbial community was analyzed using substrate-induced respiration (SIR, a measure of metabolic diversity) and phospholipid fatty acid analysis (PLFA, a measure of community composition). Plant functional group removal had almost no effect on the soil microbial community. The only response detected was an increase in stress (indicated by the PLFA stress ratio of cy19:0 to 18:1ω7c) which occurred when legumes were removed and fertilizer was not added, indicating that legumes had a positive effect on the nutrient status of microbes. Likewise, soil properties (total carbon, pH, moisture and nutrients) showed limited response to plant removals. Fertilization decreased the metabolic diversity of the soil microbial community. We detected no soil microbial or plant biomass response to the fungicide indicating that mycorrhizae had little influence in this system. Based on the low-productivity of the grassland, and the lack of response in both the soil properties and the microbial community, we hypothesize that the main determinants of the microbial community may be litter input. When litter decomposition rates are slow, such as in this northern system, five growing seasons may not be sufficient to detect the impact of a changing plant community on the soil microbes.

Lysine is one of the more limiting amino acids in protein sources for chickens. Since lysine is also an essential amino acid for animals, it is an important component of animal dietary formulation. Therefore, an accurate pre-determination of bioavailable lysine in feedstuffs is important. An optical density (OD) based microbiological assay for lysine determination using E. coli lysine auxotroph has been previously developed. However, because the assay is based on bacterial growth response to extracellular lysine measured as OD, it can be relatively time consuming (10-12h). Therefore, more rapid assays are needed if pre-formulation estimates are required. In this dissertation whole cell fluorescent biosensors for the quantification of bioavailable and total lysine in feed protein sources were developed. The biosensor for quantification of bioavailable lysine was based on the growth response of E. coli to an external source of lysine and lysinecontaining small peptides. Green fluorescent protein (GFP) was inserted in the genome of E. coli lysine auxotroph as a part of a mini-Tn5- transposon by conjugation. Bacterial growth response to external lysine and small peptides was monitored and recorded by measuring the fluorescence emitted by GFP. The second type biosensor developed was designed for the quantification of total lysine. It was based on the measurement of a promoter activity, which was induced and modulated by extracellular concentration of lysine. Cad promoter was amplified from E. coli K-12 genome and was cloned into promoterless gfp plasmid. The construct was electroporated into electrocompetent E. coli cells. The promoter activity was induced under the conditions of low pH and graded concentrations of lysine. Lysine-dose response was measured by the fluorescence of GFP. Both methods were characterized as having a high potential for practical application.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.