PRISM :: Browsing by Author "Gieg, Lisa"

Browsing by Author "Gieg, Lisa"

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Open Access
Adverse Effects of Remediated and Unremediated Oil Sands Process-Affected Water on Zebrafish Embryo Morphological Development
(2016) Toth, Cameron Graham Andrew; Habibi, Hamid; Chua, Gordon; Gieg, Lisa; Smits, Judit
Oil sands process-affected water (OSPW) generated from bitumen extraction in the Alberta oil sands is accumulating in on-site tailings ponds. The organic fraction of OSPW is a primary target for remediation as it is toxic to aquatic organisms. Here, robust morphometric biomarkers were identified in developing zebrafish (Danio rerio) embryos for determining the acute toxicities of the acid extracted organics (AEOs) and diluted OSPW. AEOs/OSPW exposures reduced survival and increased cardiovascular, growth, swim bladder and behavioural abnormalities in zebrafish embryos. Phosphate biostimulation of indigenous algal species in OSPW reduced the C5 – C12 cluster of naphthenic acids (NAs) abundance in the AEO fraction by ≥ 50%. Following phosphate biostimulation, embryonic zebrafish endpoints previously sensitive to AEO/OSPW constituents were ameliorated. These findings implicate the acute toxicity of C5 – C12 NAs on zebrafish development and highlight the potential of algal-based NA biodegradation for reducing the impact of OSPW to aquatic organisms.
Open Access
Aerobic and Anaerobic Naphthenic Acid Biodegradation by Indigenous Tailings Pond Microorganisms
(2015-01-07) Clothier, Lindsay Nicole; Gieg, Lisa
Surface mining of bitumen extraction from Alberta’s oil sands generates large volumes of oil sands process-affected water (OSPW) that is stored in tailings ponds. Naphthenic acids (NAs) are toxic, corrosive, and complex, cyclic carboxylic acids that accumulate in tailings ponds over time and must be removed for effective reclamation. This work has examined microbial NA biodegradation under aerobic and anaerobic conditions as a possible approach for reclamation. Phosphate stimulation of oxic OSPW resulted in growth of algae (Scenedesmus, Chlorella) and bacteria (Porphyrobacter, Planctomyces) capable of biodegrading low molecular weight NAs resulting in small decreases in acute toxicity. Several new NA-biodegrading bacteria were isolated from OSPW and identified. Additionally, this work has now shown that NA can be biodegraded under nitrate-, iron-, sulfate-reducing and methanogenic conditions and has identified microbial community members likely contributing to NA biodegradation. These findings show that the use of microorganisms for NA remediation may be possible.
Open Access
Aerobic Hydrocarbon-degrading Microbial Communities in Oilsands Tailings Ponds
(2016) Rochman, Fauziah; Dunfield, Peter F.; Voordouw, Gerrit; Gieg, Lisa; Hettiaratchi, Patrick
Oilsands process-affected water (OSPW), produced by the surface-mining oilsands industry in Alberta, Canada, is alkaline and contains salts, various metals, and hydrocarbon compounds. In this thesis, aerobic communities involved in several key biogeochemical processes in OSPW were studied. Degradation of several key hydrocarbons was analyzed in depth. Benzene and naphthalene were used as models for aromatic hydrocarbons, in which their oxidation rates, degrading communities, and degradation pathways in OSPW were researched. The potential oxidation rates were 36.7 μmol L-1 day-1 for benzene and 85.4 μmol L-1 day-1 for naphthalene. Via stable isotope probing (SIP), and high-throughput sequencing of 16S rRNA gene amplicons, it was discovered that strains of the genera Methyloversatilis and Zavarzinia were the main benzene degraders, while Thiococcus and Pseudomonas were the main naphthalene degraders. Cultivated strains of Zavarzinia and Pseudomonas were shown to be growing on benzene and naphthalene. Metagenomics analysis revealed genes encoding oxygenases active against aromatic compounds, as well as catechol oxidases. Although these belonged to many phylogenetically diverse bacteria, only few bacteria were predominant in the SIP experiments. A highly divergent pmoA-like gene was also detected in the metagenome data. Here, the possibility of this gene allowing growth on short alkanes (C1 to C3) was examined. This gene was investigated via SIP and quantitative PCR. Results showed that the monooxygenase encoded by the gene has high affinity toward ethane and mostly propane. For the study of lighter hydrocarbons, methane, ethane, and propane were chosen as model compounds. OSPW was capable of supporting methane oxidation with a rate of 108.2 μmol of CH4 L−1 OSPW d−1, ethane oxidation with a rate of 83.2 μmol of C2H6 L−1 OSPW d−1, and propane oxidation with a rate of 58.6 μmol of C3H8 L−1 OSPW d−1. SIP analysis uncovered Methyloparacoccus to be predominant in methane-incubated samples, whereas Methyloversatilis was predominant in ethane and propane-incubated samples. SIP technique was also employed to study photosynthetic bacterial communities and indigenous aerobic bacterial communities that assimilate methanol, acetate, and protein extracts. All OSPW photosynthetic ‘heavy-DNA’ samples were dominated by unidentified Planctomycetes. Predominant groups in methanol, acetate, and protein extract-SIPs were Betaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Finally, via a modified cultivation technique, a novel Verrucomicrobia was isolated from OSPW. The aerobic bacterium was named Oleiharenicola alkalitolerans gen. nov., sp. nov., and it was studied in depth via phylogenetic, chemotaxonomic and whole-genome sequencing techniques.
Open Access
Characterization and Control of Halophilic Sulfate-Reducing and Methanogenic Microbial Communities in Shale Oil and Gas Systems
(2017) An, Biwen Annie; Voordouw, Gerrit; Gieg, Lisa; Harrison, Joe; Mayer, Bernhard; Mouser, Paula
In recent years, the oil and gas industry has been revolutionized by the expansion of shale oil and shale gas operations due to the advancement of hydraulic fracturing and high economic benefits compared to other unconventional resources. Several mechanisms have been proposed for the possible roles of microorganisms in shale gas fields. However, there remains a lack of knowledge regarding the key microbial players shared by all shale reservoirs and their involvements in the operations. Three case studies were conducted using samples obtained from various shale oil and shale gas reservoirs on the possibility of reservoir souring, microbiologically influenced corrosion (MIC) and microbially enhanced oil recovery (MEOR) through both culture-dependent and independent approaches. Case study I, for the Bakken shale oil field in Saskatchewan Canada, showed that continuous injection of low salinity source water can alter the microbial and geochemical conditions of the reservoir. There is a dominance of halophilic microbial community in the saline shale formations, in which Halanaerobium can synthesize the substrates used by halophilic sulfate-reducing bacteria (SRB) for sulfate-reduction, and halophilic nitrate-reducing bacteria (NRB) can inhibit the growth of SRB through nitrite production. At low salinity, the microbial community is much more diverse involving Halanaerobium, Desulfovibrio, Thiomicrospira, Dethiosulfatibacter and Arcobacter, with high potential for souring and MIC. In addition, at low salinity, nitrate-mediated souring control cannot be as easily achieved as nitrite is further reduced to N2. Case studies II and III were for the Duvernay shale gas field and Montney shale oil field in Alberta, Canada, which showed high MIC potential involving similar halophilic taxa as found in the Bakken field. Finally, samples from all three case studies indicated higher salinities were associated with higher ammonium concentrations, which is a product of methylotrophic methanogenesis using methylamines. The addition of methylamines in the hydraulic fracturing process can facilitate the possible interactions between the key players identified in all three case studies. Through this work, mitigation and monitoring technologies targeting recurring taxa involved in shale reservoirs on souring and MIC can be developed, which is highly beneficial for the environment and the economy.
Open Access
Characterizing and Accelerating Methanogenic Hydrocarbon Biodegradation
(2017) Toth, Courtney; Gieg, Lisa; Dunfield, Peter; Voordouw, Gerrit
Microbial transformation of hydrocarbons to methane is an environmentally relevant but slow process taking place in a wide variety of electron acceptor-depleted environments, from oil reservoirs and coal deposits, to contaminated groundwater and deep sediments. Despite the prevalence of chemical evidence demonstrating methanogenic hydrocarbon metabolism in field investigations, there are significant gaps in our understanding of the anaerobic activation mechanisms of model substrates (particularly monoaromatic and polycyclic aromatic hydrocarbons, PAHs) and whole crude oil, as well as the degradation pathways and microorganisms governing oil transformation to methane. By studying the chemical and functional responses of methanogenic consortia to enrichment on model and mixed hydrocarbon substrates, we can gain a more complete understanding of the fate of hydrocarbon components in electron acceptor-depleted environments. In this dissertation, we sought to characterize the biodegradation of an expanded range of hydrocarbon substrates using a series of chemical and molecular approaches. We also explored cultivation-based strategies for optimizing rates of methanogenic hydrocarbon utilization, of which the most successful methods were adopted for future cultivation studies described here. Members of the Desulfosporosinus genus, known to catalyze methanogenic toluene biodegradation, were also found to co-metabolize other alkylbenzene substrates. Other members of the Firmicutes phylum, such as Desulfotomaculum, were shown to be functionally capable of activating toluene by addition to fumarate in a crude oil-degrading produced water consortium, and are proposed to play a key role in the formation of heavy oil in petroleum reservoirs. Microbial community sequencing, DNA-based stable isotope probing, and metagenomic surveys of previously established and novel methanogenic PAH-degrading cultures suggest that Clostridium may be important for degrading larger aromatic structures by an unknown mechanism. Experimental evidence of a hypothetical energy conservation mechanism in Syntrophus was detected during alkylbenzene biodegradation, suggesting this organism plays a vital role in coordinating syntrophic hydrocarbon biodegradation in a bioenergetically favourable manner. In all, this research has gleaned new insights into the microorganisms and metabolic processes regulating methanogenic hydrocarbon biodegradation, and has produced a wealth of new research questions to be explored in future investigations.
Open Access
Chemical, Physiological and Metabolic Interactions between Pseudomonas, Metals and Environmental Nutrients
(2017) Booth, Sean C.; Turner, Raymond J.; Weljie, Aalim; Gieg, Lisa; Turner, Raymond J.; Eltis, Lindsay; DeVinney, Rebekah
Environmental pollution is one of the major problems facing humanity. Bacteria are capable of removing pollutants from the environment through their metabolic activities. This works for organic pollutants, but metals inhibit the degradation process. Pseudomonas pseudoalcaligenes KF707 is a bacterium that is resistant to metals and is able to degrade pollutants such as polychlorinated biphenyls. In this thesis I present how interactions between the bacterium, its environment and metals affect the bacterium’s physiology and metabolism of biphenyl. Chemical interactions with environmental components affect the toxicity of metals towards bacteria. By examining the tolerance of Pseudomonas species towards copper and aluminium in different media compositions I found that metal bioavailability and carbon source quality had a strong influence on the amount of metal they could withstand. Building on these data, I used metabolomics to understand how metals interfere with organic pollutant degradation. By quantifying the small molecules used and produced by the bacterial cell I was able to determine that metal toxicity is exacerbated by the oxidative stress of metabolizing an organic pollutant. P. pseudoalcaligenes KF707 can swim towards biphenyl but it was unknown how. By deleting genes that were expected to be involved in energy-taxis, a process that allows bacteria to swim to metabolizable carbon sources, I found that this was not how KF707 swims towards biphenyl. I did discover that some unexpected genes were involved in energy-taxis and also that the primary gene for this behavior, Aer, is actually a family of receptors with variable phylogenetic distribution in the genus Pseudomonas. These results provide new insight into the interactions between a bacterium and the nutrients and stressors in their environment.
Open Access
Comparative Evaluation of Coated and Non-Coated Carbon Electrodes in a Microbial Fuel Cell for Treatment of Municipal Sludge
(MDPI AG, 2019-03-16) Nandy, Arpita; Sharma, Mohita; Venkatesan, Senthil Velan; Taylor, Nicole; Gieg, Lisa; Thangadurai, Venkataraman
This study aims to provide insight into the cost-effective catalyst on power generation in a microbial fuel cell (MFC) for treatment of municipal sludge. Power production from MFCs with carbon, Fe2O3, and Pt electrodes were compared. The MFC with no coating on carbon generated the least power density (6.72 mW·m?2) while the MFC with Fe2O3-coating on carbon anodes and carbon cathodes generated a 78% higher power output (30.18 mW·m?2). The third MFC with Fe2O3-coated carbon anodes and Pt on carbon as the cathode catalyst generated the highest power density (73.16 mW·m?2) at room temperature. Although the power generated with a conventional Pt catalyst was more than two-fold higher than Fe2O3, this study suggests that Fe2O3 can be investigated further as an efficient, low-cost, and alternative catalyst of Pt, which can be optimized for improving performance of MFCs. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) results demonstrated reduced resistance of MFCs and better charge transfer between biofilm and electrodes containing coated anodes compared to non-coated anodes. Scanning electron microscopy (SEM) was used to analyze biofilm morphology and microbial community analysis was performed using 16S rRNA gene sequencing, which revealed the presence of known anaerobic fermenters and methanogens that may play a key role in energy generation in the MFCs.
Open Access
Comparing methods for recovering genomic RNA from SARS-CoV-2 in municipal wastewater for molecular analysis
(2023-12-04) McCalder, Janine Alison; Hubert, Casey; Parkins, Michael; Corcoran, Jennifer; Gieg, Lisa; Hynes, Michael
SARS-CoV-2, an enveloped virus with a single stranded positive sense RNA genome (Baltimore class 4), is shed in the excreta (e.g., feces, sputum, urine) of people infected with the virus, resulting in the presence of viral genetic fragments in municipal wastewater. This RNA is quantifiable using wastewater-based surveillance (WBS) molecular biology and genomic methods. RNA extracted from wastewater can be assessed using RT-qPCR to track disease trends within populations of various sizes (e.g., whole city, neighbourhoods, hospitals, etc) in a non-invasive, unbiased, and highly inclusive way. Despite accelerated development and utilisation of WBS during the COVID-19 pandemic, many unknowns and challenges still confront this burgeoning field. Fundamental structural differences between enveloped SARS-CoV-2 and the majority of viruses in wastewater (e.g., non-enveloped enteric viruses) raise important questions about how previously established methods perform for this novel coronavirus. Establishing a reliable workflow for wastewater-based monitoring of SARS-CoV-2 requires a series of connected methods, including viral recovery from wastewater and downstream molecular and/or genomic analyses. Selecting the best possible components of an overall workflow requires method comparison and optimisation. In this study, several wastewater processing methods were assessed for the recovery of a bovine coronavirus surrogate, which has a similar structure to that of SARS-CoV-2, to establish an effective method for viral recovery from wastewater. This comparison revealed that ultrafiltration and direct extraction performed better than electronegative membrane filtration, which exhibited RT-qPCR gene abundance estimates an order of magnitude lower than the other two methods. Consequently, two validated workflows implementing ultrafiltration and direct extraction were then compared during a 29-month longitudinal analysis of SARS-CoV-2 quantification using RT-qPCR. Despite each workflow representing a distinct method for viral recovery, trends were similar with both methods indicating that consistent application of a given workflow, and not necessarily which workflow is used, is paramount in WBS. RNA extraction methods were furthermore compared using an amplicon-based sequencing strategy to assess the SARS-CoV-2 variant profiles in municipal wastewater samples. This comparison did not reveal significant differences in sequencing results between the two extraction strategies, but rather underscored the importance of wastewater sample handling and storage prior to genomic assessment.
Open Access
Control of Microbial Sulfide Production in Low and High Temperature Oil Field with Nitrate and Perchlorate
(2017) Okpala, Gloria; Voordouw, Gerrit; Coates, John; Ryan, Cathryn; Gieg, Lisa; Hubert, Casey
The activity of sulfate reducing bacteria (SRB), which produce sulfide, in low and high temperature oilfields, poses a severe challenge for oil and gas industries. Nitrate injection is used to limit the growth of SRB, through stimulation of nitrate reducing bacteria (NRB) that reduce nitrate to nitrite and subsequently to N2. Data from temperature dependent studies done in this work reveal thermophilic nitrate reducing bacteria (tNRB) isolated from low and high temperature oilfields reduce nitrate to nitrite and not further at 50°C or above. This observation is especially important for nitrate-mediated control of sulfide production in high temperature oil fields, because nitrite is a strong SRB inhibitor. To better understand how nitrate injection works in a seawater flooded high temperature reservoir, dual temperature bioreactors and multi-temperature microcosms were used in monitoring sulfate reduction by mesophilic and thermophilic NRB and SRB. The results indicated that nitrate may be ineffective when injected into a cold zone (<45°C) and that preventing emergence of such a zone by injecting hot produced water may be an effective way to control souring with nitrate. Control of souring with perchlorate under low temperature conditions in batch incubations and bioreactors containing heavy oil was also tested. Perchlorate caused a delay in the onset of sulfate reduction in batch incubations. However, its reduction with oil was not seen in batch culture incubations or bioreactors. Chlorite was more effective at inhibiting SRB activity under these conditions. The research in this thesis thus contributes to improved management of sulfide production in oil fields.
Open Access
Control of SRB – Mediated Microbially Influenced Corrosion in Flowing Systems
(2016) Pinnock, Tijan; Voordouw, Gerrit; Hubert, Casey; Gieg, Lisa
Corrosion is the major threat to oil and gas production and transportation infrastructure around the world. It is now accepted that microorganisms, especially sulfate-reducing bacteria, may play a significant role in the corrosion mechanism in many of the corrosion scenarios in the oil and gas industry. Therefore, an understanding of how to control sulfate-reducing bacteria mediated microbially influenced corrosion is key to controlling microbially influenced corrosion in the oil and gas industry. The biocorrosion threat to a steam-assisted gravity drainage operation was assessed and found to be low. The control of sulfate-reducing bacteria mediated microbially influenced corrosion in model systems involving carbon steel beads under flow was accomplished with biocides and corrosion inhibitors. While significant corrosion control was observed with biocides, oil soluble corrosion inhibitors reduced the corrosion rate by as much ninety-nine percent. These control methods had different effects on the microbial communities involved in the corrosion process.
Open Access
Evaluating the Metal Tolerance Capacity of Oil Sands Tailings Microbes, and their Ability to Degrade Naphthenic Acids While Attached to Biochar
(2016) Frankel, Mathew; Turner, Raymond J.; Gieg, Lisa; Layzell, David
Alberta’s oil sands process water (OSPW) was used herein as a case to investigate co-contaminated wastewater treatment; naphthenic acids (NAs, implicated for OSPW toxicity) and metals have been reported in OSPW. This thesis sought to evaluate the metal tolerances of a native OSPW microbial community, and the effect of OSPW-specific metals on their ability to degrade NAs. Furthermore, this work focused on enhancing OSPW treatment by growing these NA-degrading communities on an engineered adsorbent, biochar, under the hypothesis that combining the effects of biochar with attached OSPW microbes would be more effective at contaminant removal than either independent approach. Metal susceptibility experiments with the OSPW community demonstrated elevated tolerances to OSPW metals. Microbial-biochar work showed biochar NA removal was enhanced by microbial degradation, with mixed results in the effects of metal co-contamination on biodegradation with each biochar. Metal immobilization was improved by microbial attachment to adsorbents, regardless of biochar assayed.
Open Access
Exploration of Multidrug Resistance Efflux Pumps (MDREPs) in Tolerance to Biocides
(2022-06-14) Brown, Damon Craig; Turner, Raymond J; Gieg, Lisa; Chu, Angus; Hubert, Casey Russell James; Kumar, Ayush
Biocides are broad-spectrum antiseptics used in industry and increasingly in domestic environments to control microbial growth. Microbial tolerance towards biocides is a well documented phenomenon in industries such as medical, water treatment and food processing but has not been well described in the oil and gas industry. A key form of acquired biocide tolerance is the acquisition of multidrug resistance efflux pumps (MDREPs) through horizontal gene transfer. In this project, I set out to determine if biocide tolerance could be determined and monitored using DNA-based quantitative PCR (qPCR) to facilitate the testing of samples from oil and gas pipelines where biocides are used to control microbiologically influenced corrosion. I first had to design primers for use in qPCR capable of detecting these poorly conserved genes using the annotated genomes of six species (Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica) chosen to represent metabolic clades frequently identified in microbiologically influenced corrosion environments. During the creation of the model community, a surprising knowledge gap was identified correlating different microbial growth methods, so the decision was made to use optical density, ATP measurements, DNA concentrations and qPCR targeting 16S rRNA and determine how well these methods agreed. A mixed community of D. vulgaris, G. subterraneus, P. putida and T. aromatica was successfully grown reproducibly in bioreactors allowing for sessile and planktonic sampling following exposure to sublethal concentrations of biocides. Using the developed primers, the MDREP genes were quantified and ratioed to 16S rRNA copy numbers to track changes in the relative abundance. These results provide a proof-of-concept for the creation of a monitoring program aimed at targeting genetic markers and assessing microbial tolerance to biocides without costly sequencing.
Open Access
From genomic data to metabolism of an uncultured thermophilic bacterium S2R-29 and a hydrocarbon degrader Solimonas aquatica
(2022-12-07) Tran, Triet M; Dunfield, Peter; Hynes, Michael F; Gieg, Lisa
This thesis investigated metabolic functions through in-depth analyses of two bacterial genomes from two distinct environments: an uncultured thermophilic bacterium from Dewar Creek geothermal spring in British Columbia, Canada and Solimonas aquatica from freshwater in Taiwan.Twenty-two SAGs (Single-celled amplified genomes) of S2R-29 were retrieved from the source of Dewar Creek hot springs. S2R-29 is known to have extremely low GC content (24.23%) and predicted to be a facultative anaerobic bacterium. Based on phylogenetic trees constructed from full-length 16S rRNA gene sequences, the closest relatives to S2R-29 are members of the phyla Candidatus Calescamantes and Candidatus Fervidibacter. Metabolic analysis revealed 4 hypothesized energy sources for S2R-29: Cellobiose, glycerol, proteins, and DNA. Low GC content in S2R-29 might be directly related to the DNA degradation process and immediate environment surrounding the bacteria. S2R-29 is most likely a facultative anaerobe capable of twitching motility. The optimal growth temperature of S2R-29 was predicted to be at 65 – 70oC and appears to be almost exclusively present in Dewar Creek hot springs.Copper membrane monooxygenases are usually encoded by three genes found in xmoCAB operon, which is also known as pmoCAB in methanotrophs. Four sets of primers were designed to amplify the members of two phylogenetic clusters named tailing group 1 and 3. Three out of four primer pairs successfully amplified xmoA genes. Solimonas aquatica is the only xmoA-carrying bacterium from tailing group 1 with a cultured representative. Culture-based studies were therefore performed on Solimonas aquatica, which carries two different copies of xmoCAB. Solimonas aquatica cultures were grown and monitored for 30 to 60 days under various carbon sources including glucose, methane, propane, and butane in medium 10D. Gas chromatography and optical density were able to confirm the growth of Solimonas aquatica onglucose and butane only. Optical density of Solimonas aquatica cultures did not increase when cultured using butane-related intermediates aside from 1-butanol, in which the bacteria showed almost similar trend compared to butane-grown culture. However, based on the presence of genes encoding copper membrane monooxygenases and soluble di-iron monooxygenases, the exact mechanism and the enzyme responsible for butane-related oxidation is still unknown.
Open Access
Gaseous BTEX Biofiltration: Experimental and Numerical Study of Dynamics, Substrate Interaction and Multiple Steady States
(2018-01) Süß, Michael; De Visscher, Alex; Sen, Arindom; Siegler, Hector; Gieg, Lisa
Air pollution has a global impact on the environment and human health. In recent decades growing consciousness of air pollutants has led to a substantial decline in hazardous emissions. Nevertheless, air quality problems persist. A group of pollutants of particular concern are benzene, toluene, ethylbenzene and xylene, commonly referred to as BTEX. BTEX are known for their adverse effects on human health such as the carcinogenicity of benzene among others. Continuous development, improvement and exploring of new innovative control technologies are of great importance and striven for by researchers and industry. Biological methods such as biofilters are considered to be a sustainable and environmentally friendly technology. Hence, the present dissertation investigated the employment of a promising microorganism, Nocardia sp., to treat BTEX in a biofilter as well as the experimental and computational study of different steady states. At an empty-bed residence time (EBRT) of 1.5 min and an inlet concentration between 0.05 – 0.14 g m- 3 single benzene, toluene, ethylbenzene and m-xylene were removed with an efficiency of 100%, 93%, 96% and 87% respectively. With increasing inlet concentration, the removal efficiency (RE) declined, however an increase of EBRT generally resulted in higher RE. A similar trend was observed when BTEX were treated as a mixture and highest RE were achieved at low concentrations. In addition, the determination of kinetic parameters of the microorganism were carried out and the threshold substrate concentration for benzene and m-xylene were estimated. The exploration of a possible jump of steady states were numerically examined by considering only the biofilm. Therefore, two independent computer simulations were developed, which includes diffusion limitation and substrate degradation following Haldane kinetics. Results clearly indicate a jump of steady states in a very small range of inlet concentration and a distortion of prevailing Haldane kinetics. A further development of one model was carried out and aforementioned determined kinetic parameters were applied. This model correctly described the jump of steady states in an actual biofilter at a concentration change of 0.272 g m-3. Obtained results are supported by experimental validation.
Open Access
Genomic and Metabolic Studies of Two Candidate Phyla of Bacteria
(2017) Jones, Gareth Mathew; Dunfield, Peter; Gieg, Lisa; Grasby, Stephen; Storey, Douglas
Candidate phyla (or candidate divisions) are deeply rooted groups of Bacteria or Archaea that currently lack cultivated representatives. By combining traditional enrichment and cultivation practices with modern metagenomic based techniques, this work describes two candidate phyla more thoroughly than could have been produced by focusing on one aspect alone. Members of the candidate phylum OP11 were enriched from two geographically distinct geothermal springs: one from Ngatamariki, Waikato, New Zealand and the other from Lakelse, B.C., Canada. The organisms were grown under thermophilic and anaerobic conditions, using cellulose as the sole substrate. Stable isotope probing was also used to confirm carbon flow from cellulose to OP11 in both sites. Continued studies on the Canadian spring demonstrated that the OP11 organism was possibly utilizing H2 and a degradation byproduct of cellulose under fermentative conditions as its energy source. Additionally, this work provides evidence for symbiosis between the OP11 and a member of the Chloroflexi phylum; a result that has not been demonstrated previously. Metagenomic analysis of the two organisms further described the relationship, where the Chloroflexi was likely acting as the primary cellulolytic organism, and OP11 was consuming degradation byproducts. Additionally, a natural FeSO4 spring site, located in Kootenay National Park, B.C., Canada was also studied for the prevalence of the WPS-2 candidate phylum. The work includes an environmental survey using 16S rRNA gene based community analysis to demonstrate the candidate phylum’s distribution throughout the site. High-throughput metagenomic sequencing and microfluidic cell sorting coupled with whole genome sequencing were used to create a genomic reconstruction of a member of the WPS-2 candidate phylum. This represents the first genomic reconstruction from this group of organisms. Through this work, substantial progress was made in further describing several deeply rooted, uncultured groups of bacteria. It presents a thorough exploration of these largely unstudied organisms, and presents a discussion on combining modern metagenomics based techniques with more traditional tools of environmental microbiology in order to describe currently uncultivated organisms.
Open Access
Identification of genes and interactions involved in syntrophic biodegradation under methanogenic conditions
(2023-07) Goldsmith, Ciara Ayame; Gieg, Lisa; Hubert, Casey; Chua, Gordon
Biodegradation, or the breakdown of pollutants by microorganisms, occurs in many global environments. Hydrocarbon biodegradation can be carried out by a diverse range of microorganisms, such as sulfate reducing microorganisms (SRM), and methanogens. Syntrophy, or the partnership of two species to metabolize a substrate despite an energetic barrier, is a dominant metabolic process in many environments where hydrocarbon biodegradation occurs. Understanding the syntrophic interactions that occur within microbial communities is important, since there is a possibility of uncovering novel microbial mechanisms and devise potential industrial applications. There is still much to be discovered about the specific interactions that occur within syntrophic partnerships, which is what this thesis project aimed to investigate. Metatranscriptomics was used to determine how gene expression changed when a bacterium maintained a syntrophic lifestyle, compared to an independent lifestyle in a pure culture. Co-cultures of the marine SRM Desulfatibacillum alkenivorans strain AK01 and either the methanogen Methanospirillum hungatei strain JF-1, or the marine methanogen Methanococcus maripaludis strain S2 were established. The differential gene expression analysis revealed that certain genes believed to be associated with syntrophic metabolic pathways and energy conservation were upregulated in both lactate-amended co-cultures. An additional contaminant organism within the cultures, Anaerovirgula multivorans, also may act as a syntroph based on gene expression and chemical activity data. Additionally, a co-occurrence network analysis was performed on eight mixed methanogenic hydrocarbon degrading consortia amended with different substrates. Multiple networks containing potential syntrophic organisms and methanogens were present, which could be used to establish future co-culture experiments.
Open Access
An Integrated Approach to Improving Efficiency in Photosynthetic Microbial Systems
(2021-05-26) Zorz, Jacqueline; Strous, Marc; Mayer, Bernhard; Larter, Steve; Welch, Greg; Gieg, Lisa; Hallam, Steven
Cyanobacteria-based biotechnology is regarded as a promising opportunity for renewable bioenergy and bioproducts. As cyanobacteria are photosynthetic microorganisms, they only require sunlight, carbon dioxide, nutrients, and water to grow, and can be cultivated using non-arable land and non-potable water. These characteristics, along with their rapid growth rates and amenability to genetic modifications, merit research of cyanobacteria for roles in mitigating greenhouse gas emissions and carbon capture and sequestration. Despite these favourable attributes, cyanobacterial bioenergy has yet to become successful at an industrial scale. This thesis explores, through use of metagenomics, metaproteomics, growth experiments, and modelling, fundamental and applied strategies to improve the productivity and feasibility of cyanobacteria in biotechnology. A photosynthetic microbial mat, sourced from highly productive haloalkaline soda lakes, was previously used as inoculum for enrichment of a mono-cyanobacterial microbial consortium. In this thesis, the microbial composition and function of the productive haloalkaline lakes of origin were analyzed using metagenomics and metaproteomics (Chapter 2). This analysis showed high diversity and functional redundancy within the mat community, and suggested approaches for niche differentiation between phototrophic species, as well as mechanisms for lateral gene transfer and biogeographic dispersal. In Chapter 3, the cyanobacterial enrichment culture was used to conduct growth experiments in conjunction with red light transmitting filters, composed of organic semiconducting materials with the potential to produce electricity. These growth experiments were used to model photosynthesis and to determine under which conditions electricity-producing light filters could be advantageous to photosynthetic growth and overall energy output. Lastly, in Chapter 4, the cyanobacterial enrichment culture underwent a prolonged dark and anoxic incubation, similar to what might be experienced in their natural lake habitat. This incubation resulted in the lysis of cyanobacterial cells and release of a highly valued pigment compound, phycocyanin. The molecular mechanism behind the lytic process was investigated using metagenomics and metaproteomics. In conclusion, this body of work examined fundamental microbiological and ecological processes in a highly productive photosynthetic mat and used biological principles to facilitate improvement of cyanobacterial biotechnology systems.
Open Access
Marine microbial communities capable of hydrocarbon biodegradation along shipping routes in the Kivalliq region of the Canadian Arctic
(2022-06) Ji, Meng; Hubert, Casey; Gieg, Lisa; Harrison, Joe; Else, Brent
The extreme cold and icy marine environment in the Canadian Arctic is not well understood regarding its potential for biodegradation of oil spills. Reduced ice cover due to climate change has led to increased human activities, with attendant risks of oil and fuel spills associated with shipping traffic. This threatens the marine ecosystem and well-being of Canadians living in Arctic communities that rely on the ocean for food and cultural livelihood. Naturally occurring hydrocarbon-degrading bacteria within the marine microbiome have the potential to catalyze biodegradation of crude oil compounds, with previous studies showing cold-adapted oil-degrading bacteria inhabiting different marine biomes in the Canadian Arctic. However, the Kivalliq region in Nunavut, Canada, which has been impacted by increased vessel traffic in Hudson Bay in recent decades, has not been investigated in this regard. Determination of the baseline microbiomes in pristine ice, seawater and surface sediment was complemented by mock oil spill microcosms in seawater and sediment to assess biodegradation potential in the Kivalliq marine environment. Incubations of seawater or sediment amended with crude oil were monitored over a 21-week period using 16S rRNA gene amplicon sequencing, metagenomics, cell counting and hydrocarbon geochemistry. Analysis of microbial baselines showed little variability in diversity or taxa in similar marine biomes apart from sites with fresher water. Seawater microcosms demonstrated growth from putative hydrocarbon-degrading organisms corresponding to losses in alkane hydrocarbons. Alkane and polycyclic aromatic hydrocarbon losses and detection of associated genes for hydrocarbon degradation corresponded with appearances of putative hydrocarbonoclastic taxa in sediment microcosms. The Kivalliq marine microbiome’s potential to mitigate pollution effects associated with oil spills suggests that incorporating microbial diversity and microbiome assessments into monitoring environmental change will lead to improved efficacy of spill bioremediation strategies and preparation measures in the Arctic.
Open Access
Metabolite-Sensing Transcription Factors for Developing Whole Cell Naphthenic Acid Biosensor Technology
(2022-09) Bookout, Tyson; Gieg, Lisa; Lewenza, Shawn; Chua, Gordon; Dunfield, Peter
Naphthenic Acids (NA) are a complex group of acyclic and cyclic alkyl-substituted carboxylic acids. These compounds are naturally produced during the degradation of petroleum and are present in high concentrations in the waste produced during the bitumen extraction process. NAs are very toxic, and monitoring and treating the oil sands process-affected water (OSPW) is costly and time-consuming. Whole cell biosensors are a proven technology holding the potential for a rapid and low-cost method of monitoring NA in tailings ponds. We are exploiting the sensitive and reliable detection mechanisms present in bacterial cells for inducing appropriate responses to environmental changes, by engineering bacterial strains to produce a simple and quantitative output in proportion to NA. Using bacterial genomics and synthetic biology, we designed transcription factor-based biosensors using a novel Pseudomonas species isolated from the OSPW, by examining the upregulated genes in the presence of different NA mixtures. The promoters of these genes are then synthesized and cloned directly upstream from a bacterial lux operon, that is used as a bioluminescent reporter. We are particularly interested in those involved in transcriptional regulation, antibiotic efflux, or bioremediation. We have identified three strong promoters for NA-detection: a promoter driving the expression of a hypothetical gene, a divergent NA-inducible promoter from the atu operon regulated by a TetR family repressor, and a promoter for a toxic RND efflux system regulated by a MarR family regulator. These transcriptional regulators bind within the promoter sequence to repress gene expression, unless in the presence of a target analyte. Upon analyte binding, a confirmational change occurs to release the protein from the promoter region and allow transcription. Using electrophoretic mobility shift assays, this mechanism of promoter repression and NA detection was confirmed for both AtuR and MarR regulators. Through lux expression experiments, the hypothetical promoter, divergent atu promoter, and MarR regulated promoter were confirmed to sensitively respond in a dose-dependent manner to a custom mixture of NA compounds, a mixture of primarily acyclic NAs, and the complex OSPW mixture, respectively. These biosensors are sensitive within 2-30 parts per million, and the capability of these biosensors to uniquely respond to NA compounds and not other hydrocarbons such as alkanes and BTEX, demonstrates the potential for using a panel of these biosensors for detecting NA toxicity in the environment.
Open Access
Methanogenic and Aerobic Biodegradation of Model Polycyclic Aromatic Compounds Associated with Canadian Oil Sands
(2017) Montoya, Oscar; Gieg, Lisa; Hubert, Casey; Voordouw, Gerrit; Larter, Stephen; Strous, Marc
Polycyclic aromatic compounds (PACs) are ubiquitous molecules that can be of high importance to remediate due to their potential negative health and environmental effects. The present study used Canadian Oil Sands-derived microbial consortia established methanogenically or aerobically and amended with phenanthrene, dibenzothiophene (DBT), or 2,6-dimethylnaphthalene (2,6-diMN) as sole carbon and energy sources under three salinities. Methane formation was statistically higher in PAC-amended treatments relative to unamended controls under brackish conditions. A fumarate addition metabolite was tentatively detected in incubations amended with 2,6-diMN. DBT was degraded with concomitant methane formation, the first report of this metabolism. 16S rRNA gene sequencing revealed the dominance of methanogens and known PACs degraders. Genus Smithella was only detected in DBT-amended incubations in relatively high abundances suggesting its role in DBT degradation anaerobically. Aerobic degradation of PHEN and 2,6-diMN was also observed. Time-course experiments showed faster PHEN degradation under saline conditions and that the microbial communities were dominated by Janibacter sp, which was also isolated. This research shows that microbial communities from bitumen-impacted environments have the natural ability to degrade PACs, with potential applications in bioremediation.