Glycosphingolipids (GSLs), composed of a polar saccharide head and a lipophilic ceramide tail, are ubiquitous components of the plasma membrane of eukaryotic cells. They serve in many regulatory capacities and have antigenic properties towards natural killer T (NKT) cells of the innate immune system. Critical to the recognition of glycosylceramides by NKT cells are antigen presenting cells (APC), such as dendritic cells, which are responsible for binding, processing, and delivery of ligands to these lymphocytes. This event is mediated by CD1d, a major histocompatibility complex-like protein expressed on the surface of APCs, which binds GSL antigens by the ceramide moiety and presents the polar group to the T cell receptors of CD1d-restricted cells. The subsequent immune response involves NKT cell proliferation and emission of numerous cytokines, such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), resulting in the stimulation of the innate and adaptive immune systems through maturation of APCs, activation of T cells, and secretion of antibodies by B cells. To understand the structure-activity relationship between GSLs and NKT cell activity and the requirements for intracellular processing of antigens, analogs of the model compound alphaGalCer (KRN-7000) have been synthesized. These include fluorophore-appended 6”-amino-α-galactosylceramides and N-alkenoyl GSLs, such as PBS-57, a potent alphaGalCer surrogate useful in NKT cell stimulation studies. A nonantigenic beta-C-galactosylceramide has also been prepared as an inhibitor of these innate lymphocytes. To probe the potential for using NKT cells to bias the immune system between the proinflammatory TH1 response or the immunomodulatory TH2 mode, versions of alphaGalCer with shortened ceramides have been created. One of these truncated analogs, PBS-25, has successfully been cocrystallized with CD1d and the binary complex structure solved by X-ray crystallography. Synthetic glycosphingolipids derived from Novosphingobium capsulatum and Sphingomonas paucimobilis have also been made. In assays with classical Valpha14i/Valpha24i NKT cell lines, these Gram-negative bacterial antigens were recognized directly and specifically by host immune systems through CD1d-restriction, unlike GSL-deficient microbes (e.g., Salmonella typhimurium). A search for other GSL-bearing alpha-proteobacteria led to the discovery of another natural glycosphingolipid, an N-alkenoylphytosphingoid-alpha-galactoside, isolated from the outer membrane of Ehrlichia muris.

The hypothesis for this study was that phenanthrene degrading bacterial inoculants, in combination with grass species able to tolerate petroleum hydrocarbon contamination, will result in increased degradation, as compared to natural rates of hydrocarbon degradation, or to rates of degradation attributed to bacteria or plants alone. Three experiments were performed to examine this hypothesis: i) assessment of the effect of phenanthrene degrading bacteria (<i>Sphingomonas yanoikuyae</i>, <i>Rahnella aquatilis</i>, and <i>Arthrobacter globiformis</i>) on seed germination, location of attachment on seeds and roots, and inoculant survival on selected grass species, ii) determination of the inoculant survival in contaminated soil in the absence of plants and the ability to degrade target compounds, and iii) degradation potential and survival of selected grass species and bacterial inoculants in soil. In general, all applied inoculants were able to effectively colonize the seeds and had a neutral or positive effect on seed germination and seedling growth. Possible plant and bacteria pairs were chosen based on positive influence of the inoculant and are as follows: perennial ryegrass (<i>Lolium perenne</i>) or creeping red fescue (<i>Festuca rubra</i>) with <i>A. globiformis</i> or <i>S. yanoikuyae</i>, or slender wheatgrass (<i>Elymus trachycaulus</i>) with <i>A. globiformis</i> or <i>R. aquatilis</i>. Soil-based assessment of the survival and degradation of hydrocarbons by the selected inoculants was examined with or without a manure nutrient amendment. The addition of the inoculants had a positive impact on the efficacy of hydrocarbon removal in the soil. The manure-amended soil, or <i>A. globiformis</i> inoculated non-amended soil treatments reduced total petroleum hydrocarbon concentration by ~45%, whereas the non-amended control only resulted in a ~20% reduction. When soils were amended with manure and inoculated with any of the phenanthrene degrading bacteria, contaminant concentration decreased in soil by ~33%. <i>Sphingomonas yanoikuyae</i> survived the longest in soil in the absence of plants. A growth chamber experiment was conducted to determine the efficacy of plant and bacteria pairs for hydrocarbon removal in recalcitrant contamination found in soil from Bruderheim, Alberta. Additional replicates containing this soil were spiked with hexadecane, phenanthrene, and pyrene so the effectiveness of the plant and bacteria pairs at higher levels of fresh contamination could be assessed. In the spiked treatment, inoculation with <i>S. yanoikuyae</i> increased creeping red fescue root biomass. In the non-spiked treatment, <i>S. yanoikuyae</i> application increased creeping red fescue root and shoot biomass. Perennial ryegrass root and shoot biomass did not increase when inoculated with <i>S. yanoikuyae</i>, although root biomass values were observably higher in non-spiked soils. Creeping red fescue inoculated with <i>S. yanoikuyae</i> resulted in the greatest decrease in hydrocarbon concentration as compared to other treatments (~61%). The perennial ryegrass treatment, when inoculated with <i>S. yanoikuyae</i> increased percent hydrocarbon removal (~10%) above that obtained with perennial ryegrass alone. The addition of plants and <i>S. yanoikuyae</i> increased hydrocarbon degradation relative to control soils, although the addition of vegetation alone had a comparable effect. A critical benefit of inoculation was the increase in creeping red fescue root biomass at higher concentrations of contamination. This is important because the larger the root biomass the larger the volume of soil that can be remediated. The results indicate that the use of specific plant-bacterial inoculants can enhance remediation of hydrocarbon contaminated soils.

The hypothesis for this study was that phenanthrene degrading bacterial inoculants, in combination with grass species able to tolerate petroleum hydrocarbon contamination, will result in increased degradation, as compared to natural rates of hydrocarbon degradation, or to rates of degradation attributed to bacteria or plants alone. Three experiments were performed to examine this hypothesis: i) assessment of the effect of phenanthrene degrading bacteria (<i>Sphingomonas yanoikuyae</i>, <i>Rahnella aquatilis</i>, and <i>Arthrobacter globiformis</i>) on seed germination, location of attachment on seeds and roots, and inoculant survival on selected grass species, ii) determination of the inoculant survival in contaminated soil in the absence of plants and the ability to degrade target compounds, and iii) degradation potential and survival of selected grass species and bacterial inoculants in soil. In general, all applied inoculants were able to effectively colonize the seeds and had a neutral or positive effect on seed germination and seedling growth. Possible plant and bacteria pairs were chosen based on positive influence of the inoculant and are as follows: perennial ryegrass (<i>Lolium perenne</i>) or creeping red fescue (<i>Festuca rubra</i>) with <i>A. globiformis</i> or <i>S. yanoikuyae</i>, or slender wheatgrass (<i>Elymus trachycaulus</i>) with <i>A. globiformis</i> or <i>R. aquatilis</i>. Soil-based assessment of the survival and degradation of hydrocarbons by the selected inoculants was examined with or without a manure nutrient amendment. The addition of the inoculants had a positive impact on the efficacy of hydrocarbon removal in the soil. The manure-amended soil, or <i>A. globiformis</i> inoculated non-amended soil treatments reduced total petroleum hydrocarbon concentration by ~45%, whereas the non-amended control only resulted in a ~20% reduction. When soils were amended with manure and inoculated with any of the phenanthrene degrading bacteria, contaminant concentration decreased in soil by ~33%. <i>Sphingomonas yanoikuyae</i> survived the longest in soil in the absence of plants. A growth chamber experiment was conducted to determine the efficacy of plant and bacteria pairs for hydrocarbon removal in recalcitrant contamination found in soil from Bruderheim, Alberta. Additional replicates containing this soil were spiked with hexadecane, phenanthrene, and pyrene so the effectiveness of the plant and bacteria pairs at higher levels of fresh contamination could be assessed. In the spiked treatment, inoculation with <i>S. yanoikuyae</i> increased creeping red fescue root biomass. In the non-spiked treatment, <i>S. yanoikuyae</i> application increased creeping red fescue root and shoot biomass. Perennial ryegrass root and shoot biomass did not increase when inoculated with <i>S. yanoikuyae</i>, although root biomass values were observably higher in non-spiked soils. Creeping red fescue inoculated with <i>S. yanoikuyae</i> resulted in the greatest decrease in hydrocarbon concentration as compared to other treatments (~61%). The perennial ryegrass treatment, when inoculated with <i>S. yanoikuyae</i> increased percent hydrocarbon removal (~10%) above that obtained with perennial ryegrass alone. The addition of plants and <i>S. yanoikuyae</i> increased hydrocarbon degradation relative to control soils, although the addition of vegetation alone had a comparable effect. A critical benefit of inoculation was the increase in creeping red fescue root biomass at higher concentrations of contamination. This is important because the larger the root biomass the larger the volume of soil that can be remediated. The results indicate that the use of specific plant-bacterial inoculants can enhance remediation of hydrocarbon contaminated soils.