Oil sands mining operations in northeastern Alberta are rapidly expanding. Upgrading and extracting the bitumen from the sand requires large volumes of water generating large quantities of oil sands process water/materials (OSPM) which is high in organic content. Some of the major organic components found in OSPM include unrecovered bitumen, polycyclic aromatic compounds (PACs), naphthenic acids (NAs) and humic acids. Concerns of acute and chronic toxicity resulting from OSPM have led to provincial legislation preventing the discharge of OSPM into local water and mandating the reclamation of areas affected by oil sands mining. To date, OSPM is stored on lease in settling basins while the mining companies evaluate reclamation strategies. One of the reclamation strategies involves the use of wetlands constructed with differing amounts of OSPM and organic amendments such as peat. Currently, numerous wetlands, both natural and constructed, are present on oil sands leases. To determine the sustainability of these wetlands for reclamation, the assimilation and flow of carbon and nitrogen within the systems need to be defined. Stable isotope analysis can enhance this understanding. To effectively use stable isotopes in the field, there is the need to determine the changes in stable isotope values occurring from the microbial degradation of organic components such as NAs which contribute a significant portion to the dissolved organic carbon (DOC) in reclamation sites. This study examined the microbial degradation of commercial and oil sands derived NAs by oil sands derived microbial cultures. Changes in stable isotopes values in the biomass (δ13C, δ15N), DOC and dissolved inorganic carbon (DIC) (δ13C) arising from degradation of the DOC were tracked in both static and semi-continuous tests. Utilization of commercial and oil sands derived NAs resulted in minimal change of the DOC stable isotope values. The biomass was 13C enriched for both the commercial (0.3 to 2.9 per mil (‰)) and oil sands derived NAs (3.7 to 8.5 ‰) relative to the DOC stable isotope values. DIC stable isotope values showed higher variability (-5 to +5.5 ‰). The semi-continuous tests showed biomass that was 15N enriched (3.8 to 8.4 ‰) with the assimilation of ammonium. Isotope trends established in the laboratory study provide further understanding into assimilation of carbon and nitrogen compounds in the field. DOC and DIC concentration and carbon stable isotope values were determined for water sampled from 13 oil sands aquatic reclamation sites varying in age, construction and organic material. Both DOC and DIC concentrations were elevated in OSPM affected sites, by an average of 40 mg/L for DOC and 83 mg/L for DIC concentrations. DOC concentrations were also elevated by approximately 10 mg/L at high organic sites. δ13C DOC values were slightly 13C enriched in young sites: 0.6 ‰ compared to δ13C DOC values at the mature sites. Also, from June to July 13C enrichment (0.3 to 1.9 ‰) of the DOC for all sites was seen. Corresponding with the enrichment seen in the DOC, 13C depletion (-8.8 to -0.3 ‰) of the DIC was seen for most sites from June to July. The trends seen from June to July may be a result of the release of readily degradable organics from the spring thaw stimulating the microbial community. The baseline values determined for DOC and DIC may assist future field food web studies.

Oil sands mining operations in northeastern Alberta are rapidly expanding. Upgrading and extracting the bitumen from the sand requires large volumes of water generating large quantities of oil sands process water/materials (OSPM) which is high in organic content. Some of the major organic components found in OSPM include unrecovered bitumen, polycyclic aromatic compounds (PACs), naphthenic acids (NAs) and humic acids. Concerns of acute and chronic toxicity resulting from OSPM have led to provincial legislation preventing the discharge of OSPM into local water and mandating the reclamation of areas affected by oil sands mining. To date, OSPM is stored on lease in settling basins while the mining companies evaluate reclamation strategies. One of the reclamation strategies involves the use of wetlands constructed with differing amounts of OSPM and organic amendments such as peat. Currently, numerous wetlands, both natural and constructed, are present on oil sands leases. To determine the sustainability of these wetlands for reclamation, the assimilation and flow of carbon and nitrogen within the systems need to be defined. Stable isotope analysis can enhance this understanding. To effectively use stable isotopes in the field, there is the need to determine the changes in stable isotope values occurring from the microbial degradation of organic components such as NAs which contribute a significant portion to the dissolved organic carbon (DOC) in reclamation sites. This study examined the microbial degradation of commercial and oil sands derived NAs by oil sands derived microbial cultures. Changes in stable isotopes values in the biomass (δ13C, δ15N), DOC and dissolved inorganic carbon (DIC) (δ13C) arising from degradation of the DOC were tracked in both static and semi-continuous tests. Utilization of commercial and oil sands derived NAs resulted in minimal change of the DOC stable isotope values. The biomass was 13C enriched for both the commercial (0.3 to 2.9 per mil (‰)) and oil sands derived NAs (3.7 to 8.5 ‰) relative to the DOC stable isotope values. DIC stable isotope values showed higher variability (-5 to +5.5 ‰). The semi-continuous tests showed biomass that was 15N enriched (3.8 to 8.4 ‰) with the assimilation of ammonium. Isotope trends established in the laboratory study provide further understanding into assimilation of carbon and nitrogen compounds in the field. DOC and DIC concentration and carbon stable isotope values were determined for water sampled from 13 oil sands aquatic reclamation sites varying in age, construction and organic material. Both DOC and DIC concentrations were elevated in OSPM affected sites, by an average of 40 mg/L for DOC and 83 mg/L for DIC concentrations. DOC concentrations were also elevated by approximately 10 mg/L at high organic sites. δ13C DOC values were slightly 13C enriched in young sites: 0.6 ‰ compared to δ13C DOC values at the mature sites. Also, from June to July 13C enrichment (0.3 to 1.9 ‰) of the DOC for all sites was seen. Corresponding with the enrichment seen in the DOC, 13C depletion (-8.8 to -0.3 ‰) of the DIC was seen for most sites from June to July. The trends seen from June to July may be a result of the release of readily degradable organics from the spring thaw stimulating the microbial community. The baseline values determined for DOC and DIC may assist future field food web studies.

Identification of ignitable liquids in fire debris analysis using pattern recognition is an important step in determining the nature of a suspicious fire. Complex mixtures that make up ignitable liquids are susceptible to microbial degradation when fire debris evidence is presented in the form of soil. Microbial degradation results in a selective metabolism of certain classes of compounds required for identification of an ignitable liquid. Various ignitable liquids that may be used to initiate or propagate a fire contain different classes of organic compounds. These include normal alkanes, branched alkanes, cycloalkanes, aromatics, terpenes, and others. In this work, microbial degradation of nine ignitable liquids in soil was evaluated over a period of twenty-six days. The degradation of aromatic compounds in gasoline was faster with toluene and C2-alkylbenzenes than in C3-alkylbenzenes. However, the overall loss of aromatics made gasoline chromatographically unidentifiable. The complete loss of n-alkanes in medium and petroleum distillates resulted in patterns that resembled naphthenic-paraffinic products. Normal alkanes were more susceptible to microbial degradation than isoalkanes, which was specifically demonstrated in medium and heavy petroleum distillates. In diesel, pristane and phytane remained prominent in comparison to the normally prevalent n-alkanes, which could no longer be detected post-degradation. The degradation of isoalkanes and cycloalkanes was evaluated in a naphthenic-paraffinic product. Isoalkanes were degraded significantly faster than cycloalkanes. The remaining peaks in the naphthenic-paraffinic pattern consisted solely of cycloalkane compounds, and could no longer be classified as a naphthenic-paraffinic product. The terpene compounds in turpentine were also observed to be susceptible to degradation by microorganisms. The loss of !-pinene, limonene, and camphene was significantly noticeable in comparison to other terpene compounds, such as 1,4-cineole. Microbial biodegradation in different soil types was investigated. The difference in soil texture can affect the rate of metabolism of ignitable liquids due to the variance of available oxygen, nutrients and mobility of the microbial population. The degradation of isoalkanes, cycloalkanes, aromatics and heavier normal alkanes was faster in clay, whereas normal alkanes of lower molecular weight were degraded more readily in sand. There has been no explanation of this occurrence within the scientific literature, however it could be hypothesized that the difference in microbial flora and water saturation levels could affect the selective degradation between the two soil types. Fire debris evidence is often stored for long periods of time before analysis due to case backlogs. The storage condition of arson-related soil samples is a sensitive subject. If evidence, containing soil, is stored at room temperature, petroleum compounds in any ignitable liquid residues that are present will be degraded within a week. Therefore, it is important to freeze or refrigerate soil samples. The storage of both refrigerated and frozen soil samples containing gasoline were evaluated over six months. Less than 6% of the aromatic compounds distinctive of gasoline remained when stored at 5 °C, while minimal change was observed in the same compounds when stored at -15 °C. Microbial degradation of petroleum-based ignitable liquids is advantageous from the environmental perspective. However, within the forensic community the effect of microbial action could lead to misclassification or inability to identify the presence of an ignitable liquid in fire debris evidence.

The nuclear waste inventory of the UK comprises large quantities of intermediate level wastes (ILW), which will be immobilised by encapsulation within a cementitious grout in stainless steel containers, followed by disposal in a deep engineered geological disposal facility (GDF) within a suitable geological formation. These wastes contain, in addition to radioactive elements, a heterogeneous mix of organic materials, including plastics, cellulose and rubber. Cellulosic items, such as cloth, tissue, filters, paper and wood, are considered particularly problematic, because they are known to be susceptible to degradation under alkaline conditions, forming small chain organic acids with the ability to complex metals and radionuclides. It is predicted that under alkaline conditions isosaccharinic acid (ISA) will form particularly strong complexes with Ni(II), Am(III), Eu(III), Np(IV), Th(IV), and U(IV). As a result, the presence of ISA could affect the migration behaviour of these elements, by increasing their solubility and reducing sorption, thus enhancing their mobility into the near and far field surrounding a GDF. During site operation and then after closure of a GDF, microbial communities have the potential to colonise the steep biogeochemical gradients, running from highly alkaline in the GDF “near field” to circumneutral pH conditions in the surrounding geosphere. Within these steep pH gradients microbial processes can control the fate of organic compounds, such as ISA, and have therefore been considered as an effective self-attenuating mechanism to remove ISA from the groundwater. This thesis aims to deliver a greater understanding of the microbial processes that can potentially use ISA as a carbon source and electron donor, removing it from solution, and thus having a positive impact on radionuclide mobility under GDF-relevant conditions. A microbial enrichment approach was chosen that approaches GDF-relevant conditions to explore the biodegradation of ISA. Cross-disciplinary analyses of water chemistry (pH, Eh, photospectroscopy, IC, ICP), mineralogy (ESEM, XRD, TEM, XAS) and microbiology (light microscopy, next generation sequencing) have demonstrated the ability of bacteria to degrade ISA over a wide range of biogeochemical conditions. Furthermore, key radionuclides (and their non-active analogues), including Ni(II) and U(VI), were precipitated from the groundwater system during ISA biodegradation. Moreover, in the case of uranium, microbial metabolism led to the reduction of U(VI) to U(IV), which is also less soluble. This study highlights the potential for microbial activity to help remove chelating agents from groundwaters surrounding an ILW GDF, and suggests that safety cases that do not include microbial processes may be overly conservative, over-estimating the impact of ISA on radionuclide transport.

Polychlorinated biphenyls (PCBs) are carcinogenic, persistent, and bioaccumulative contaminants that pose risks to human and environmental health. PCB biodegradation by indigenous microbial communities could be a cost-effective and an environmental-friendly bioremediation strategy for in situ PCB removal. A comprehensive understanding of the microbial PCB degradation at the contaminated site is required for the acceptance and optimization of using microbial PCB degradation as the site clean-up strategy. This thesis describes investigations of the aerobic and anaerobic microbial degradation of PCBs under both field and laboratory conditions. The microbial PCB degradation potential in sediments from Indiana Harbor and Ship Canal (IHSC), a site that was historically contaminated by PCBs, was explored by analyzing the PCB congener distributions and microbial communities in two core sediment samples. PCB congener analysis suggested the possibility of in situ dechlorination in deep sediments. Molecular analysis of biomarker genes revealed the potential of both aerobic and anaerobic PCB degradation in sediments. Microbial communities were characterized by the combination use of terminal restriction fragment length polymorphism (T-RFLP), clone library, and pyrosequencing. These methods elucidated the dominant role of Proteobacteria, especially Acidovorax and Acinetobacter in sediments. To improve the microbial PCB degradation, phytoremediation with switchgrass (Panicum vigratum) was employed under laboratory conditions. Congener analysis showed that both phytoextraction and microbial PCB degradation contributed to the enhanced PCB removal in the presence of switchgrass. Bioaugmentation with Burkholderia xenovorans LB400 was performed to further promote aerobic PCB degradation. The presence of LB400 was associated with improved degradation of PCB 52, but not PCB77 or PCB 153. Increased abundance of the biphenyl dioxygenase gene, which is indicative of aerobic PCB degradation, and its transcript were observed after bioaugmentation, suggesting active aerobic PCB degradation. To promote the anaerobic PCB degradation, redox cycling (alternating flooding and non-flooding) was performed. Redox cycling was found to improve the removal of PCB 153 in unplanted soils and to increase the dechlorinating Chloroflexi population. Characterization of the microbial community by T-RFLP and clone library revealed that Proteobacteria and Acidobacteria were dominant. Species that contain dechlorination potential were identified, including Geobacter and Clostridium, suggesting that their possible role in PCB dechlorination. The research described in this thesis provides scientific knowledge and evidence for the feasibility of employing bioremediation including natural attenuation, phytoremediation, and bioaugmentation to clean up PCB contamination. Such information will be critical in selecting and optimizing remediation strategies for PCB contaminated sites.

<p>Reactive Black 5 and Procion Red MX 5B, an azo and anthraquinone dye repectively were decoulorized by mean of microorganisms growing on wood chips. The process consisted of three reactors, two anaerobic reactors and one aerobic reactor. The anaerobic process was used in order to make it possible to break the nitrogen bond of the azo group, (-N=N-) and the aerobic one to increase the possibility for the degradation of possible intermediates. After pumping wastewater through the system it was shown that mixtures or Reactive Black 5 and Procion Red MX 5B were efficiently decolourised at 50 mg/l as well as 200 mg/l of each of the dyes.</p><p><strong> </strong></p>

Reactive Black 5 and Procion Red MX 5B, an azo and anthraquinone dye repectively were decoulorized by mean of microorganisms growing on wood chips. The process consisted of three reactors, two anaerobic reactors and one aerobic reactor. The anaerobic process was used in order to make it possible to break the nitrogen bond of the azo group, (-N=N-) and the aerobic one to increase the possibility for the degradation of possible intermediates. After pumping wastewater through the system it was shown that mixtures or Reactive Black 5 and Procion Red MX 5B were efficiently decolourised at 50 mg/l as well as 200 mg/l of each of the dyes.

abstract: Decay of plant litter represents an enormous pathway for carbon (C) into the atmosphere but our understanding of the mechanisms driving this process is particularly limited in drylands. While microbes are a dominant driver of litter decay in most ecosystems, their significance in drylands is not well understood and abiotic drivers such as photodegradation are commonly perceived to be more important. I assessed the significance of microbes to the decay of plant litter in the Sonoran Desert. I found that the variation in decay among 16 leaf litter types was correlated with microbial respiration rates (i.e. CO2 emission) from litter, and rates were strongly correlated with water-vapor sorption rates of litter. Water-vapor sorption during high-humidity periods activates microbes and subsequent respiration appears to be a significant decay mechanism. I also found that exposure to sunlight accelerated litter decay (i.e. photodegradation) and enhanced subsequent respiration rates of litter. The abundance of bacteria (but not fungi) on the surface of litter exposed to sunlight was strongly correlated with respiration rates, as well as litter decay, implying that exposure to sunlight facilitated activity of surface bacteria which were responsible for faster decay. I also assessed the response of respiration to temperature and moisture content (MC) of litter, as well as the relationship between relative humidity and MC. There was a peak in respiration rates between 35-40oC, and, unexpectedly, rates increased from 55 to 70oC with the highest peak at 70oC, suggesting the presence of thermophilic microbes or heat-tolerant enzymes. Respiration rates increased exponentially with MC, and MC was strongly correlated with relative humidity. I used these relationships, along with litter microclimate and C loss data to estimate the contribution of this pathway to litter C loss over 34 months. Respiration was responsible for 24% of the total C lost from litter – this represents a substantial pathway for C loss, over twice as large as the combination of thermal and photochemical abiotic emission. My findings elucidate two mechanisms that explain why microbial drivers were more significant than commonly assumed: activation of microbes via water-vapor sorption and high respiration rates at high temperatures. / Dissertation/Thesis / Doctoral Dissertation Biology 2020

After Deepwater Horizon oil reached the Florida coast, oil was buried in Pensacola Beach (PB) sands to ~ 70 cm depth, resulting in Total Petroleum Hydrocarbon (TPH) concentrations up to ~ 2 kg per meter of beach. This study followed the decomposition of the buried oil and the factors influencing its degradation. The abundance of bacteria in oiled sand increased by 2 orders of magnitude within one week after oil burial, while diversity decreased by ~ 50%. Half-lives of aliphatic and aromatic hydrocarbons reached 25 and 22 days, respectively. Aerobic microbial oil decomposition, promoted by tidal pumping, and human cleaning activities effectively removed oil from the beach. After one year, concentrations of GC-amenable hydrocarbons at PB were similar to those in the uncontaminated reference beach at St. George Island/FL, and microbial populations that disappeared after the oil contamination had reestablished. Yet, oxihydrocarbons can be found at PB to the present day.

Ignitable liquids consist of either a single organic compound or a complex organic mixture. In regards to fire debris analysis, the analyst is responsible for determining if an ignitable liquid residue is present. However, when extracted from soil-containing fire debris evidence, chemical degradation from microorganisms is observed to result in the loss of compounds based on chemical structure. It can also happen when the evidence container is stored at room temperature before analysis. This can present a challenge to the fire debris analyst when identifying and classifying the ignitable liquid residue based on the criteria established by standard test methods. The purpose of this research was to observe the microbial degradation of fourteen compounds, at room temperature over a period of time, for possible by-product formation that could coincide with compounds normally present in an ignitable liquid. Additionally, a quantitative assessment was performed to observe and record the loss rate of compounds in a representative simple mixture. Finally, the loss rate from the simple mixture was compared to commercially available ignitable liquids. Degradation studies were conducted to observe the microbial degradation of a representative compounds (individually and in a simple mixture, both weathered and unweathered) and seven ignitable liquids of different ASTM E1618 classifications. Potting soil was spiked with 20 µL of a liquid/compound and was allowed to stand at room temperature for a period of time. The simple mixture was evaporated to 50% and 90% using a steady nitrogen gas flow to compare the degradation process to the unweathered mixture. All samples were extracted and analyzed using passive-headspace concentration and gas chromatography-mass spectrometry. The formation of by-products was not observed when degrading the compounds from the simple mixture individually as seen in other research. The simple mixture, unweathered and 50% weathered, resulted in rapid degradation of their oxygenated compounds. The straight-chained alkanes and toluene were observed to be more susceptible to microbial attack than the highly-substituted aromatics and the branched and cyclic alkanes. The 90% weathered mixture followed the same degradation trend as the unweathered and 50% weathered samples, although it only contained two compounds. The loss rates/half-lives for each simple mixture sample (unweathered, 50% weathered, and 90% weathered) were determined to be approximately 3.5, 3.5, and 0.84 days. The unweathered and 50% weathered sample half-lives were similar due to containing compounds with similar susceptibility to degradation, while the 90% weathered sample contained one compound that was more highly susceptible to degradation. When comparing the 3.5 day half-life to the seven different ASTM class liquids, the isoparaffinic product and the naphthenic-paraffinic product had similar rates of degradation while aromatic solvent and normal alkane classes had the shortest half-lives. When observing the degradation of the gasoline, medium petroleum distillate and the miscellaneous, the constituent compounds were seen to exhibit a range of degradation rates that corresponded to half-lives less than and greater than 3.5 days.