Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen Streptococcus mutans.

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.

Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase.

N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling.

Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection.

A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.

Growth kinetics of Hyphomicrobium and Thiobacillus spp. in mixed cultures degrading dimethyl sulfide and methanol.

The growth kinetics of Hyphomicrobium spp. and Thiobacillus spp. on dimethyl sulfide (DMS) and methanol (in the case of Hyphomicrobium spp.) in an enrichment culture created from a biofilter cotreating DMS and methanol were studied. Specific growth rates of 0.099 h(-1) and 0.11 h(-1) were determined for Hyphomicrobium spp. and Thiobacillus spp., respectively, growing on DMS at pH 7. These specific growth rates are double the highest maximum specific growth rate for bacterial growth on DMS reported to date in the literature. When the pH of the medium was decreased from pH 7 to pH 5, the specific growth rate of Hyphomicrobium spp. decreased by 85%, with a near 100-fold decline in the yield of Hyphomicrobium 16S rRNA gene copies in the mixed culture. Through the same pH shift, the specific growth rate and 16S rRNA gene yield of Thiobacillus spp. remained similar. When methanol was used as a substrate, the specific growth rate of Hyphomicrobium spp. declined much less over the same pH range (up to 30%) while the yield of 16S rRNA gene copies declined by only 50%. Switching from an NH(4)(+)-N-based source to a NO(3)(-)-N-based source resulted in the same trends for the specific growth rate of these microorganisms with respect to pH. This suggests that pH has far more impact on the growth kinetics of these microorganisms than the nitrogen source. The results of these mixed-culture batch experiments indicate that the increased DMS removal rates observed in previous studies of biofilters cotreating DMS and methanol are due to the proliferation of DMS-degrading Hyphomicrobium spp. on methanol at pH levels not conducive to high growth rates on DMS alone.

Linking performance to microbiology in biofilters treating dimethyl sulphide in the presence and absence of methanol.

The performance and microbiology of two inorganic biofilters treating dimethyl sulphide (DMS) in the presence and absence of methanol was investigated. Addition of methanol was shown to result in an increase in DMS removal for methanol loadings below 90 g MeOH per cubic metre per hour with the optimal methanol loading around 10-15 g MeOH per cubic metre per hour for a DMS loading of 3.4 g DMS per cubic metre per hour, a fivefold increase in the DMS removal rate compared to the biofilter treating DMS alone. Microbial community analysis revealed that the addition of methanol led to a significant increase of up to an order of magnitude in the abundance of Hyphomicrobium spp. in the biofilter co-treating DMS and methanol compared to the biofilter treating DMS alone, whilst there was no significant difference in the abundance of Thiobacillus spp. between the two biofilters. Given the behaviour of the biofilter co-treating DMS and methanol, the magnitude of the increase in Hyphomicrobium spp. in the biofilter co-treating DMS and methanol and the ability of Hyphomicrobium spp. to use both methanol and DMS as growth substrates, it was concluded that Hyphomicrobium spp. were the microorganisms responsible for the bulk of the DMS degradation in the biofilter co-treating DMS and methanol.

Effects of Two Manure Additives on Methane Emissions from Dairy Manure.

Liquid manure is a significant source of methane (CH4), a greenhouse gas. Many livestock farms use manure additives for practical and agronomic purposes, but the effect on CH4 emissions is unknown. To address this gap, two lab studies were conducted, evaluating the CH4 produced from liquid dairy manure with Penergetic-g® (12 mg/L, 42 mg/L, and 420 mg/L) or AgrimestMix® (30.3 mL/L). In the first study, cellulose produced 378 mL CH4/g volatile solids (VS) at 38 °C and there was no significant difference with Penergetic-g® at 12 mg/L or 42 mg/L. At the same temperature, dairy manure produced 254 mL CH4/g VS and was not significantly different from 42 mg/L Penergetic-g®. In the second lab study, the dairy manure control produced 187 mL CH4/g VS at 37 °C and 164 mL CH4/g VS at 20 °C, and there was no significant difference with AgrimestMix (30.3 mL/L) or Penergetic-g® (420 mg/L) at either temperature. Comparisons of manure composition before and after incubation indicated that the additives had no effect on pH or VS, and small and inconsistent effects on other constituents. Overall, neither additive affected CH4 production in the lab. The results suggest that farms using these additives are likely to have normal CH4 emissions from stored manure.

Techno-economic evaluation of a tandem dry batch, garage-style digestion-compost process for remote work camp environments.

The extraction of natural resources often involves housing workers in remote work camps far from population centres. These camps are prevalent in northern Alberta where they house approximately 40,000 workers involved in oil sands processing. The central, full-service cafeterias at these camps produce a significant quantity of food and cardboard waste. Due to their remote nature, these camps face high waste disposal costs associated with trucking waste long distances to the landfill. In this study, we investigated the techno-economic feasibility of on-site treatment of food and cardboard waste in a tandem dry batch, garage-style anaerobic digestion-compost process in which the waste material is converted into renewable energy used to heat the camp water supply and a nutrient-rich soil amendment for local land reclamation projects. Dry batch digestion and windrow composting pilot trials were performed on a simulated work camp waste in order to assess technical performance. The quality of the final compost was found to meet regulatory standards. A complete mass balance was then developed for a facility treating 3000 tonnes food waste and 435 tonnes waste cardboard annually. An economic assessment of such a facility was performed and, depending on the level of capital support and recognition of carbon credits for landfill methane mitigation, would require waste disposal costs to be between $115 and $195 CAD per tonne to meet financial criteria for project selection in Alberta's oil and gas industry.