ISOTOPIC FRACTIONATION AND ANAEROBIC PHYSIOLOGY OF n-ALKANE DEGRADATION BY BACTERIAL ISOLATES AND MIXED COMMUNITIES

Abstract

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 (&#949; 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 &#949; 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 (&#923; 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. In butyrate-grown cells, butyrate is likely activated to its CoA thioester using acetyl-CoA transferase and subsequently metabolized by a typical complement of &#61538;-oxidation proteins. Proteins comprising the flagellar basal body and motor were also differentially detected in butyrate-grown cells. Proteins common to both growth conditions included the sulfate-reducing metabolic machinery, the &#61538;-oxidation components, and essential cell- housekeeping complexes including ATP synthases, H+/Na+-pumping Rnf-type complexes, and heterodisulfide reductases. D. alkanexedens expresses more proteins related to oxygen stress when grown on decane compared to butyrate, apparently reflecting the oxygen-sensitive nature of glycyl-radical enzymes necessary for hydrocarbon metabolism. For example, rubrerythrin and desulfoferredoxin were detected only in alkane-grown cells, and are predicted to have peroxidase and superoxide reductase activities respectively. The combination of shotgun proteomics coupled with draft genome sequence analysis provided greater insight on the general physiology and regulatory controls of in situ metabolic activity in this model n-alkane-using, sulfate-reducing bacterium.

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 &#61540;13C 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 &#61540; 13C enrichment for methane and CO2 in the presence and absence of hydrocarbons also suggest that complex microbial interactions occur during methanogenic hydrocarbon mineralization. For example, the &#61508;&#61540;&#61472;13C of CO2 was statistically identical in all incubations, but the &#61508;&#61540;&#61472;13C for methane was greater in the presence of hydrocarbons compared to fatty acids alone. In all incubations, aceticlastic and hydrogenotrophic methanogens incorporated 13C into their proteomes to an equal extent, suggesting that one pathway is not dominant during methanogenic hydrocarbon decay by the model consortium. Stable isotope probing of proteins identified signature enzymes responsible for methanogenesis from CO2 and acetate labeled with 78.0 ± 4.4 % and 73.3 ± 1.0 % 13C, respectively. A protein capable of catalyzing fumarate addition to a hydrocarbon was not found, despite previous evidence for the presence of genes encoding these enzymes. Five proteins were identified only in the sole presence of hydrocarbons, including a polyphosphate kinase, CheA signal transduction histidine kinase, argininosuccinate lyase, cellulase, and a resolvase-like protein. 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.

In conclusion, this dissertation provides enrichment factors for n-alkane biodegradation and reveals characteristic patterns of compound specific isotope enrichment for mineralized end products during methanogenic hydrocarbon degradation. Both can be used to monitor in situ biodegradation on n-alkanes without previous knowledge of bacterial species or biodegradation processes at a site of interest. Furthermore, information regarding the proteins expressed during anaerobic alkane mineralization by a model SRB provided new insight into the physiology of this process.