Unraveling Chemical Compositional Changes of Biodegraded Crude Oil using Novel Chromatographic and Mass Spectrometric Techniques

Crude oil is an immensely complex mixture that contains thousands of distinct elemental compositions with hydrogen, carbon, oxygen, nitrogen, and sulfur atoms. The chemical fingerprint of a crude oil varies depending on its origin and degree of weathering, a phenomenon which includes biodegradation. This dissertation focuses on chemical composition of crude oil and its biodegradation with an emphasis on biosouring, an enzymatic process in which Sulfate Reducing microbial Communities (SRCs) reduce sulfate, thiosulfate, and elemental sulfur to sulfide. Biosouring in crude oil reservoirs results in hydrogen sulfide production, precipitation of metal sulfide complexes, increased industrial costs of petroleum production, and exposure issues for personnel. Potential treatment strategies for biosouring include the injection of nitrate or perchlorate anions into crude oil reservoirs. The objectives of this dissertation include development of new analytical techniques and their application to characterize the chemical composition of crude oil, and how it changes during biosouring and treatments, including substrate consumption and product formation. Chapter 1 introduces the motivations for the research and analytical methods applied, including single and multi-dimensional gas chromatography with vacuum ultraviolet ionization and time-of-flight mass spectrometry (GC-VUV-TOF and GCxGC-VUV-TOF), gas chromatography combined with variable ionization time-of-flight-mass spectrometry (GC-Select-eV-TOF-MS), and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with electrospray ionization. Chapter 1 then describes the experiments done examining biotransformations under different reducing environments and with different crude oils. The ability to structurally characterize and isomerically quantify crude oil hydrocarbons relevant to refined fuels such as motor oil, diesel, and gasoline represents an extreme challenge for chromatographic and mass spectrometric techniques. In Chapter 2, GC×GC-VUV-TOF is applied using a tunable vacuum ultraviolet soft photoionization source, the Chemical Dynamics Beamline 9.0.2 of the Advanced Light Source at the Lawrence Berkeley National Laboratory, to directly characterize and isomerically sum the contributions of aromatic and aliphatic species to hydrocarbon classes of four crude oils. When the VUV beam is tuned to 10.5 ± 0.2 eV, both aromatic and aliphatic crude oil hydrocarbons are ionized to reveal the complete chemical abundance of C9–C30 hydrocarbons. When the VUV beam is tuned to 9.0 ± 0.2 eV only aromatic hydrocarbons are ionized, allowing separation of the aliphatic and aromatic fractions of the crude oil hydrocarbon chemical classes in an efficient manner while maintaining isomeric quantification. This technique provides an effective tool to determine the isomerically summed aromatic and aliphatic hydrocarbon compositions of crude oil, providing information that goes beyond typical GC×GC separations of the most dominant hydrocarbon isomers. A comprehensive analysis of changes in crude oil chemical composition during biosouring and experimental treatments is presented in Chapter 3. Analyses using GC-VUV-TOF and FT-ICR MS combined with electrospray ionization were applied in this chapter to identify hydrocarbon degradation patterns and product formations in crude oil samples from biosoured, nitrate-treated, and perchlorate-treated bioreactor column experiments. Crude oil hydrocarbons were selectively transformed based on molecular weight and compound class in the biosouring control environment. Both the nitrate and the perchlorate treatments significantly reduced sulfide production; however, the nitrate treatment enhanced crude oil biotransformation, while the perchlorate treatment inhibited crude oil biotransformation. Nitrogen- and oxygen-containing biodegradation products, particularly with chemical formulas consistent with monocarboxylic and dicarboxylic acids containing 10−60 carbon atoms, were observed in the oil samples from both the souring control and the nitrate-treated columns but were not observed in the oil samples from the perchlorate-treated column. These results demonstrate that hydrocarbon degradation and product formation of crude oil can span hydrocarbon isomers and molecular weights up to C60 and double-bond equivalent classes ranging from straight-chain alkanes to polycyclic aromatic hydrocarbons. These results also strongly suggest that perchlorate injections may provide a preferred strategy to treat biosouring through inhibition of biotransformation. Microbial catabolism is a natural biological process that can sour crude oil reservoirs, transform spilled oil, and is an engineering tool in hydrocarbon pollution remediation strategies. In Chapter 4, differences in hydrocarbon structure (such as degree of branching or position of alkyl substituents) are examined to determine how they affect relative catabolic degradation of alkanes, monocycloalkanes, and monoaromatic hydrocarbons in order to determine which compounds are altered most by these processes and which are recalcitrant. GC-Select-eV-TOF-MS at 14 eV was used to characterize catabolic patterns of hydrocarbon isomers in crude oil from four different oil fields exposed to a single complex microbial community. Changes in alkanes, monocycloalkanes, and monoaromatic hydrocarbons are comprehensively characterized to identify differences in biodegradation as a function of degree of branching and alkyl position. Hydrocarbon isomers with terminal alkyl branches were more prone to biodegradation than those with methyl groups further from the terminal position. Compounds with alkyl branches adjacent to each other experience less biodegradation than isomers with nonadjacent methyl groups. These patterns were consistent regardless of hydrocarbon structural class or oil composition, implying that isomeric structure, including the shape of the carbon chain and the alkyl position, dictates patterns of catabolism of hydrocarbons of a particular molecular class. The patterns identified in this chapter demonstrate that microbial metabolism leaves a distinct signature from other transformation processes occurring in spilled oil, such as atmospheric oxidation or evaporation, allowing the effects of biodegradation to be distinguished from those of other processes acting upon weathered oil. A summary of research implications and ideas for further research directions, including incorporation of isotope labeled tracers to track a compound’s specific biodegradation pathway and characterization of volatile organic compounds (VOCs) from biodegrading microbial communities, are provided in Chapter 5