| In the first chapter, carbon and hydrogen isotope fractionation of n-alkanes was examined in order to compare aliphatic hydrocarbon biodegradation under oxic and anoxic conditions. Desulfoglaeba alkanexedens, a sulfate-reducing bacterium known to degrade n-alkanes, and Pseudomonas putida strain GPo1, an aerobic alkane degrading bacterium, were used as model organisms for the study. Hexane and octane were used as model substrates. Bulk isotope enrichment factors (epsilon bulk) for carbon and hydrogen under anoxic conditions were respectively, -5.5 +/- 0.2‰ and -43.1 +/- 6.3‰ for hexane, and -5.2 +/- 0.4‰ and - 27.8 +/- 4.2‰ for octane. The epsilon bulk for carbon during aerobic hexane biodegradation was -4.3 +/- 0.3‰, while hydrogen isotope fractionation was too low for quantification. The correlation of n-alkane carbon and hydrogen isotope fractionation (Λ reactive) for sulfate-reducing conditions was between 9.12+/- 1.67 and 6.02+/- 1.37, while the comparable measure for aerobiosis was < 2. We compared our results to carbon and hydrogen isotope values from an oil reservoir and demonstrate how carbon and hydrogen isotope analysis can be used to garner an understanding of in situ microbial processes within oil formations and contaminated environments.;The second chapter characterizes a draft genome for Desulfoglaeba alkanexedens (strain ALDC) and 3,168,086 base pairs encoding a total of 2,856 protein open reading frames (ORFs) were identified. The G+C content of the sequenced genome is 60.12%. This information was used to conduct a differential proteomics study of the strain grown separately on butyrate and n-decane in order to characterize the physiology of the organism with particular regard to anaerobic n-alkane biodegradation. Shotgun proteomics using nano high performance liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer revealed 97 and 75 proteins expressed exclusively under the alkane- and butyrate-growth condition, respectively. Another 217 proteins were common to both growth conditions. The analysis revealed that alkylsuccinate synthase and the proteins associated with the arginine biosynthesis pathway were only expressed in the presence of n-alkanes. Higher rates of sulfate reduction were observed during n-decane mineralization by the bacterium in the presence of citrulline, ornithine, aspartate, and arginine, relative to the amino acid unamended control.;The results from the second chapter were expanded to the third chapter to investigate methanogenic degradation of n-alkanes by a microbial enrichment. The contribution of complete and incomplete hydrocarbon mineralization to overall carbon cycling, as well as the role of different methanogenic pathways to overall methane production in these environments is unclear. Carbon flow in a model methanogenic consortium capable of n-alkane mineralization was investigated using a combination of proteomics, stable isotope probing, and the degree of delta13C incorporation in mineralization end products. Results show that 13C from uniformally labeled substrates was distributed evenly among consortium members in the presence of hydrocarbons, and used by a small portion of the community members when provided only in the form of fatty acids. Therefore, syntrophy plays a larger role during the mineralization of hydrocarbons. In addition, patterns of delta13 C enrichment for methane and CO2 in the presence and absence of hydrocarbons also suggest that complex microbial interactions occur during methanogenic hydrocarbon mineralization. Collectively, this study suggests that each organism in this enrichment culture fills a unique niche, and may contribute to the capacity for hydrocarbon degradation in ways that are previously unrecognized. (Abstract shortened by UMI.)... |
