Dr. Andrew Gibson
Characterizing the microbial degradation of Kraft lignin and lignin-derived compounds
SUPERVISOR: Dr. Lada Malek (Biology)
ABSTRACT: Analytical methods for characterizing the microbial degradation of Kraft lignin and lignin-derived compounds were utilized with the goal of biologically generating demethylated lignin for subsequent industrial applications. Selected ion flow tube mass spectrometry (SIFT-MS) technology was used for the first time with both bacterial and fungal cultures growing on lignin as a sole carbon source. Methanol and other volatile compounds were evaluated using this method and lignin-derived compounds were identified. Methanol oxidation products were found in the headspace of seven microbial cultures, as well as several unknown products not present in the SIFT-MS compound library. An assay was then developed to both confirm the results obtained by the SIFT-MS and help to understand the nature of the microbial demethylation reactions. The Ti(III)-NTA assay was found to be an economical method for rapidly determining the relative degree of lignin demethylation by cultures of microorganisms and their enzymes. Using the Ti(III)-NTA assay, some fungal cultures were found to degrade lignin monomers completely and others to metabolize methanol. Four cultures were then selected for growth optimization; to both maximize vicinal diol generation and methanol formation. By altering variables such as induction day, incubation length, culture agitation, hydrogen peroxide concentration and micronutrient concentrations (known to promote enzyme production), the effect on four fungal species was investigated. Induction with vanillin after 1 week of growth on glucose resulted in the highest demethylation activity. In the final study, culture media from the fungus Absidia cylindrospora and the bacterium Sphingobium sp. SYK-6 were used to partially purify demethylating activity. The fungal enzyme had higher specific activity than the bacterial III enzyme, but was much less abundant. Further research is needed to purify these enzymes responsible for demethylation.
Relating gene expression profiles to behavioural and neurophysiological responses to standard chemosensory cues using wild yellow perch (Perca Flavescens)
SUPERVISOR: Dr. Greg Pyle (formerly with Biology)
ABSTRACT: To play their ecological role in an ecosystem, a fully functioning olfactory system is vital for fishes. A fish's olfactory system is very sensitive to low concentrations of contaminants. A great deal of research has been conducted on the toxicity of metals on the olfactory systems of fish. While tightly-controlled laboratory studies are necessary to reveal the mechanism of toxicity, field-based studies using more realistic conditions are also needed to produce more environmentally relevant data for regulatory needs. This research has four main areas of focus: 1) to examine the effect of copper on gene transcription in olfactory tissues of yellow perch (Perca flavescens); 2) to test if a modified diet can protect olfaction against copper toxicity in rainbow trout (Oncorhynchus mykiss); 3) to examine chemosensory-mediated behaviours and gene transcription profiles in wild yellow perch from metal contaminated lakes; and 4) to investigate the potential recovery of yellow perch with contaminant-induced chemosensory function from metal contaminated lakes. We used gene transcription (a recently developed custom made micro-array for yellow perch and real-time PCR), neurophysiological testing (electro-olfactography (EOG)), and behavioural assays (avoidance from alarm cues). In this research, yellow perch were exposed to elevated concentrations of copper for 3 and 24 hours. While 3 hours of exposure did not influence gene expression, 24-hour exposures to copper elicited a differential expression of 71 genes. Of these 71 genes, differential expression of two subunits of Na/K-ATPase was further explored using real-time PCR in a time-series study. To investigate if increased dietary sodium can protect fish's olfaction against copper-induced olfactory impairment, rainbow trout were fed with diets having elevated concentrations of sodium. While fish exposed to 10 μg/L Cu and fed with a normal sodium diet had an impaired EOG response to standard olfactory cues, olfaction in Cu-exposed fish fed with Na-supplemented food (regardless of low or high levels of supplementation) remained intact. However, subsequent feeding trials found no evidence to support that Cu-exposed fish preferentially chose high-Na food. In addition, in this study olfactory impairment of fish from one clean and two metal contaminated lakes were compared using gene expression, EOG and behavioural I-maze choice experiments. While behavioural testing and EOG confirmed the impairment of olfaction in fish from metal contaminated lakes, the micro-array was not able to detect differential gene expression. To investigate if impaired olfaction of fish from metal contaminated lakes has recovery potential, EOG and behavioural testing methods were employed. Yellow perch from metal contaminated lakes with impaired olfaction kept for 24 hours in water from clean lakes. The results showed that impaired EOG responses of fish from metal contaminated lakes can recover quickly (within 24 hours) in water from a clean lake. When the behavioural testing methods were employed the results showed that olfactory mediated behaviours of fish from a moderately contaminated lake recovered after 24 hours of holding in clean water. However, olfactory mediated behaviours of fish from a severely contaminated lake did not recover after 24 hours of exposure to clean water. The data produced by this thesis significantly improves our knowledge regarding the protection against metal induced olfactory toxicity as well as the recovery potential for impaired olfaction in fish. These results could be used to draft ecologically-relevant regulations that will protect fish inhabiting sensitive fresh water ecosystems.