Browsing by Author "Dunfield, Peter F."

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    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.
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    Biodegradation of Aromatic Hydrocarbons by Methanogenic Consortia and Groundwater-Associated Microbial Communities
    (2021-01-08) Taylor, Nicole; Gieg, Lisa Marie; Hubert, Casey R. J.; Dunfield, Peter F.
    The biodegradation of hydrocarbons is an important environmental process responsible for in situ remediation of crude oil and gas components. Microorganisms of many lineages and redox conditions have been characterized to degrade numerous types of petroleum hydrocarbons, including those with aromatic structures. Alkyl-substituted mono- and polycyclic aromatic hydrocarbons are more chemically reactive than their unsubstituted counterparts, and as such their anaerobic degradation pathways have been studied to varying degrees. Aromatic hydrocarbons require enzymatic functionalization before biodegradation can occur; these activation enzymes and products are often unique to anaerobic reactions, therefore identifying the metabolites produced or the enzymes carrying out these reactions lends evidence to identifying in situ bioremediation of aromatic hydrocarbon contamination. Hydrocarbon biodegradation in the deep subsurface is often associated with methanogenesis. Anaerobic toluene degradation has been extensively studied and has been shown in multiple studies to involve an activation process known as fumarate addition, however methanogenic biodegradation of other alkylbenzenes and polycyclic aromatic hydrocarbons is comparatively poorly understood. In this work, the biodegradation of ethylbenzene and p-xylene was examined in the presence of toluene; p-toluic acid was found as a metabolite of p-xylene biotransformation, but no evidence of fumarate addition to either p-xylene or ethylbenzene were observed. A second methanogenic biodegradation study of naphthalene, 2-methylnaphthalene, and phenanthrene revealed 2-naphthoic acid as the primary metabolite produced by microbial cultures. A third study involved evaluating the use of a trapping device for passively sampling microorganisms from groundwater contaminated with aromatic hydrocarbons; this study showed that the chosen sorptive material did not influence the biodiversity of microbial communities, did not influence the rate of hydrocarbon biodegradation, and the presence of hydrocarbons was correlated to higher biomass recovery.
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    Developing methane biofiltration In oil sands tailings ponds
    (2018-09-21) Zeb, Gulrukhsar; Dunfield, Peter F.; Layzell, David B.; Chua, Gordon
    Oil sands tailings ponds in Alberta are mostly anoxic environments estimated to emit 2.7 Mt of CH4 each year to the atmosphere. Because CH4 is 34 times more potent than CO2 as a greenhouse gas, and the government of Alberta has called for a 45% reduction in CH4 emissions by 2020, this release of CH4 has become a pressing issue. One potential way to combat these large fluxes of CH4 from the tailings ponds is via biofiltration. CH4 biofiltration is the process by which aerobic methane-eating or methanotrophic bacteria are used to oxidize CH4 to CO2, a far less potent greenhouse gas. We have tested the potential for biofilters comprised of biochar to be floated on top of the tailings ponds. Experiments conducted in the lab demonstrate that biochar is an excellent matrix to capture and slow the diffusion of CH4 within small volume pores, and in addition acts as a good matrix for growth of methanotrophs. The CH4 oxidation rate in tailings was faster when biochar was added, compared to controls that did not contain any biochar. Furthermore, a mesocosm was designed that mimicked a real-life tailings ponds with CH4 fed from below and O2 from above. After two months of incubation, the mesocosm was able to oxidize CH4 with over 90% efficiency when supplied at a rate higher than the highest CH4 efflux recorded in any tailings pond to date (26 tonnes ha -1y-1). The biofilter was analyzed for its microbial community via sequencing of the 16S rRNA gene. Results revealed a community dominated by various species of methanotrophs, which suggest that it will be robust to environmental fluctuations. The biofilter was also utilized to monitor the co-degradation of naphthenic acids during methanotrophy, and to test the resilience of the biofilter to environmental changes (e.g. in CH4 supply).
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    Diversity of methane and short chain hydrocarbon degrading bacteria with an emphasis on methane biofilter systems
    (2018-10-03) Khadka, Roshan; Dunfield, Peter F.; Voordouw, Gerrit; Hubert, Casey R. J.
    Methanotrophs house enzymes capable of methane oxidation, act as a sink for atmospheric methane and play a key role in the global carbon cycle. This study conducted multiple studies on methanotrophs, including: examination of the evolutionary history of copper membrane monooxygenases (CuMMOs), application of methanotrophic communities in protocol design for monitoring methane biofilter systems, and the analyses of single cell genomes containing new CuMMO-encoding genes. CuMMOs are encoded by three genes, usually in an operon of xmoCAB, and oxidize ammonia, methane, and short chain alkanes and alkenes. To examine the evolutionary history of CuMMOs, phylogenetic inferences and compositional genome analyses were applied to a set of 66 genomes. Individual phylogeny of all genes xmoA, xmoB, and xmoC closely matched in almost all genomes, indicating this operon evolved as a unit. However in Verrucomicrobia pmoB has a distinct phylogeny from pmoA and pmoC. The gammaproteobacteria AMO (Nitrosococcus spp.), the gammaproteobacterial Pxm, the thaumarcheotal AMO and the NC10 pMMO showed little or no compositional bias in the xmo operon indicating similar compositional biases to its genome. Based on the analysis, possible lateral gene transfer events of xmoCAB genes were predicted. The nitrifying bacterium Nitrosococcus postulated as the donor of pmoCAB to both the alpha- and gammaproteobacterial methanotrophs. To design a monitoring protocol that would allow a simple, cost effective and accurate estimation of whether a methane biofilter is operating efficiently, microcosms using compost as a biofilter material were tested via growth and starvation experiments for long periods. Analysis of 16S rRNA gene sequences suggested that non-methanotrophic methylotrophic bacteria belonging the family Methylophilaceae showed a rapid response to biofilter methane oxidation activity and may be a good monitoring target. A monitoring system based on these “methanotroph-associated methylotrophs” is proposed and a ratio of Methylophilaceae to Methylococcaceae of 0.35 was related to high methane activity and 0.1 to low activity. Novel copper membrane monooxygenase encoding operons (xmoCAB) were detected while screening metagenomes obtained from oil sands environments. Quantitative PCR assays were developed for detection of xmoCAB genes in methane, ethane and propane enrichment cultures from environmental samples. Single cell genomes were sequenced from the xmoCAB positive sorted cells of a propane enrichment culture. Screening the genomes identified Polaromonas and Rhodoferax as containing multiple xmoCAB operons. Potential propane oxidation pathways were predicted based on enzymes present in single cell genomes of these two genera.
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    Methane Cycling and Methanotrophic Bacteria in Base Mine Lake, a Model End-Pit Lake in the Alberta Oilsands
    (2018-08-29) Albakistani, Emad; Dunfield, Peter F.; Voordouw, Gerrit; Gieg, Lisa Marie
    We studied methanotrophic bacteria over three years (2015 - 2018) in Base Mine Lake, Fort McMurray, Canada. The lake represents the first large scale demonstration of end-pit lake technology in the Alberta oilsands. 16S rRNA gene amplicon sequencing and measurement of methanotrophic rates were applied to evaluate the effect of seasonal changes on methanotrophic diversity and activity, and to understand the biogeochemical cycling of methane and oxygen. Based on 16S rRNA gene sequence relative abundance, the predominantly detected methanotrophic genera were Methylobacter/Crenothrix in the winter and Methylocaldum in the summer. Methanotrophs were most abundant in winter throughout the water column, and in summer at the bottom of the lake near the fluid fine tailings interface. Potential methanotrophic rates decreased over three years from 2015-2018.
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    Microbial Taxonomic and Functional Diversity in Canadian Saline and Thermal Springs
    (2019-07-15) Ruhl, Ilona Albertovna; Dunfield, Peter F.; Strous, Marc; Grasby, Stephen E.; Fox, Jeremy W.
    By demonstrating a strong effect of salinity on microbial species diversity, this work expanded the known environmental parameters that influence microbial taxonomic diversity. Additionally, this work showed that environmental stress reduces both taxonomic and functional diversity of microbial communities. pH and temperature are strong drivers of microbial taxonomic diversity, with extreme environments supporting reduced diversity. To examine the effect of another environmental parameter, salinity, on diversity, 16S rRNA and 18S rRNA gene amplicon libraries constructed from 55 sediment samples ranging in conductivity from near-fresh to halite-saturated were sequenced on the Illumina platform. Increasing conductivity, as a proxy for salinity, was found to be correlated with a reduction in bacterial taxonomic diversity in a linear manner, with peak diversity occurring at the lowest salinities examined. Conductivity also influenced the diversity of Archaea and microbial Eukarya, and these trends were explained in terms of energetic costs of salinity tolerance and mechanisms of energy production in these organisms. A small number of studies suggest that functional diversity (all of the different metabolic functions within a community) declines with decreasing taxonomic diversity of microbial communities. This relationship was tested using a dataset of 12 geothermal springs ranging in temperature from 24 to 86 °C. Functional diversity was calculated by quantifying the Pfam (protein family) diversity contained within metagenomic libraries constructed from these samples. Functional diversity was found to decrease with temperature in a similar manner to taxonomic diversity. Lastly, a study of an uncultivated clade of Bacteria, GAL08, dominating microbial communities in a geothermal spring at Dewar Creek, British Columbia, was undertaken. Material from Dewar Creek was subjected to single-cell sorting and sequencing, resulting in the identification of 13 partial single-cell genomes belonging to GAL08. Analysis of the genomes revealed the presence of three species of GAL08 at Dewar Creek. Surveys of sediment from Dewar Creek showed that GAL08 is a persistent and stable member of the microbial community, and is thermophilic, with a temperature preference of 55 to 70 °C. Laboratory enrichment experiments determined that GAL08 is likely microaerophilic. By demonstrating that the effect of salinity on microbial species diversity can be explained in terms of energy constraints, this work expanded our understanding of how environmental parameters may drive microbial diversity. Furthermore, this work represented one of the few thorough investigations into the relationship between functional diversity and environmental stress in microbial communities. Elucidating how microbial taxonomic and functional diversity responds to changes in environmental parameters, such as temperature, is timely considering the ongoing changes in global climate.
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    Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria
    (Nature Publishing Group, 2019-07-01) Rochman, Fauziah F.; Kwon, Miye; Khadka, Roshan; Tamas, Ivica; Lopez-Jauregui, Abraham; Sheremet, Andriy; Smirnova, Angela V.; Malmstrom, Rex; Yoon, Sukhwan; Woyke, Tanja; Dunfield, Peter F.; Verbeke, Tobin J.
    Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived amino acid identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell genomes (SAGs) from propane-enriched samples were therefore constructed and screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed close homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium was capable of catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies non-nitrifying Betaproteobacteria as possessing these enzymes.
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    Phytoplankton ecology in the early years of a boreal oil sands end pit lake
    (2024-01-12) Furgason, Chantel C.; Smirnova, Angela V.; Dacks, Joel B.; Dunfield, Peter F.
    Abstract Background Base Mine Lake (BML) is the first full-scale end pit lake for the oil sands mining industry in Canada. BML sequesters oil sands tailings under a freshwater cap and is intended to develop into a functional ecosystem that can be integrated into the local watershed. The first stage of successful reclamation requires the development of a phytoplankton community supporting a typical boreal lake food web. To assess the diversity and dynamics of the phytoplankton community in BML at this reclamation stage and to set a baseline for future monitoring, we examined the phytoplankton community in BML from 2016 through 2021 using molecular methods (targeting the 23S, 18S, and 16S rRNA genes) and microscopic methods. Nearby water bodies were used as controls for a freshwater environment and an active tailings pond. Results The phytoplankton community was made up of diverse bacteria and eukaryotes typical of a boreal lake. Microscopy and molecular data both identified a phytoplankton community comparable at the phylum level to that of natural boreal lakes, dominated by Chlorophyta, Cryptophyta, and Cyanophyta, with some Bacillariophyta, Ochrophyta, and Euglenophyta. Although many of the same genera were prominent in both BML and the control freshwater reservoir, there were differences at the species or ASV level. Total diversity in BML was also consistently lower than the control freshwater site, but consistently higher than the control tailings pond. The phytoplankton community composition in BML changed over the 5-year study period. Some taxa present in 2016–2019 (e.g., Choricystis) were no longer detected in 2021, while some dinophytes and haptophytes became detectable in small quantities starting in 2019–2021. Different quantification methods (qPCR analysis of 23S rRNA genes, and microscopic estimates of populations and total biomass) did not show a consistent directional trend in total phytoplankton over the 5-year study, nor was there any consistent increase in phytoplankton species diversity. The 5-year period was likely an insufficient time frame for detecting community trends, as phytoplankton communities are highly variable at the genus and species level. Conclusions BML supports a phytoplankton community composition somewhat unique from control sites (active tailings and freshwater lake) and is still changing over time. However, the most abundant genera are typical of natural boreal lakes and have the potential to support a complex aquatic food web, with many of its identified major phytoplankton constituents known to be primary producers in boreal lake environments.
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    Rhizobiophage Functional Genomics
    (2020-08-19) Koswaththa Muhandiramlage, Damitha Gunathilake; Hynes, Michael F.; Yost, Christopher Karl; Dunfield, Peter F.; Strous, Marc
    Rhizobiophages are bacterial viruses that specifically infect nitrogen-fixing, legume-nodulating bacterial group rhizobia. In this study, the whole genome sequences of eight rhizobiophages were generated. These included four Rhizobium leguminosarum myophages (AF3, P9VFCI, RL2RES and RL38JI), two R. leguminosarum siphophages (P11VFA and B1VFA), one R. leguminosarum phage with unknown morphology (V1VFA-S) and one M. japonicum phage (Cp1R7A-A1). Characterization of Cp1R7A-A1 indicated a siphophage morphology with a prolate capsid, a distinct genome, and a close phylogenetic relationship to certain Caulobacter siphophages with similar morphology. The myophages AF3, P9VFCI, RL2RES and RL38JI belong in the ICTV family Ackermanviridae and in a T4-like group of viruses. A recombination dependent replication mechanism and circularly permuted genomes due to a pure headful packaging mechanism were postulated for these phages. B1VFA and V1VFA-S had genomes similar to each other. A theta replication mechanism and phage genome terimini with direct terminal repeats were suggested for these two. P11VFA was similar to the R. leguminosarum phage L338C genome available in databases. Quantitative reverse transcription PCR for selected genes of phages Cp1R7A-A1 and Lo5R7ANS, using RNA extracts from Mesorhizobium japonicum separately infected with these phages, confirmed an early-late type maximum gene expression in Lo5R7ANS. Selected replication genes showed a maximum expression 30 minutes after infection, but structural and packaging gene expression maximized after 90 minutes. Discrete timepoints of maximum expression for any selected gene in Cp1R7A-A1 were not observed during the given incubation periods, probably due to the slow rate of replication and assembly. RNAseq analysis of Cp1R7A-A1 infected M. japonicum indicated the expression of all 237 putative phage genes and gave some insights into host gene expression during phage infection. Attempts were made to engineer R. leguminosarum and M. japonicum to carry a plasmid with a functional CRISPR/Cas9 system. A CRISPR/Cas9 system derived from Streptococcus pyogenes was cloned into the broad-host range vector pRK415. The new vector pRK415Cas9 functioned successfully in E. coli. However, it was not functional in Rhizobium or Mesorhizobium species. Cas9 promoter expression studies and RT-PCR revealed that the Cas9 promoter was expressed in Mesorhizobium. However, whether correct translation occurs has yet to be tested.
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    Seafloor Sediment Bacterial Community Profiling for Baselines and Environmental Effects Monitoring at a Deep-Sea Oil Production Site Offshore Nova Scotia, Canada
    (2019-02-22) Stacey, Deidra Kathryn; Hubert, Casey R. J.; Dunfield, Peter F.; Vamosi, Steven M.; Else, Brent G. T.
    Monitoring effects of environmental pollution is a critical aspect to preserving ecosystem health, but is challenging if baseline conditions are never established. Microorganisms are the first responders in a marine pollution event, hence oil-degrading bacteria can be used to monitor dispersion and biodegradation of oil spills. Deep-water subsurface oil reservoirs are predicted to exist along the Scotian Slope offshore Nova Scotia. Seafloor sediment from 19 Scotian Slope stations spanning a ~70,000 km2 area were used to generate 51 bacterial 16S rRNA gene amplicon libraries (V3-V4 region) to form a DNA baseline. A 300-day-long mock oil spill experiment using Scotian Slope sediment identified potential bacterial bioindicators of pristine and contaminated conditions, relative to baseline, underpinning an environmental monitoring approach that is proposed. This study shows that bacterial rRNA gene amplicon sequencing offers a novel parameter for baselines and environmentally responsible development of offshore deep-water oil drilling in Canada and beyond.
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    The Application of Hybrid media for the Mitigation of Methane Emissions through Biofiltration
    (2018-04-17) La, Helen; Hettiaratchi, Joseph Patrick A.; Achari, Gopal; Dunfield, Peter F.; Chu, Angus; Ponnurangam, Sathish; Heitz, Michèle W.
    The on-going annual increase in global CH4 emissions can be largely attributed to anthropogenic activities. However, as more than half of these emissions are diffuse and possess a concentration less than 3% (v/v), physical-chemical treatments are inefficient as an abatement technology. In this regard, biotechnologies, such as biofiltration using methane-oxidizing bacteria, or methanotrophs, are a cost-effective and efficient means to combat diffuse CH4 emissions. Utilizing a packing material such as biochar that has a high sorption potential may increase the contact time required for improving removal efficiencies of CH4. As CH4 biofiltration requires water addition to maintain microbial activity, the results from the adsorption experiments indicate adsorption capacity is not lost with water addition if biochar is the dominant packing material. Additionally, fixed bed columns of biochar may be a reliable back-up system to a hybrid system for CH4 biofiltration especially for high inlet loads. Generally, increasing the compost content of the packing material, water content, and inlet flow rate together has the most damaging effect on adsorption. The results from batch studies indicate lava rock and biochar can support the growth of methanotrophs for the oxidation of CH4 to CO2. Nitrogen additions of up to 191 g (N)/m3 matrix maximized oxidation activities but inhibited it at concentrations above this value. The methanotrophs likely enter into a starvation phase (or a stationary phase of growth where the population may cease to divide but remain metabolically active) in response to unfavorable nitrogen conditions and their CH4 oxidation activities eventually recover as toxic NH3/NO3 intermediates are further oxidized during nitrogen metabolism. Column biofiltration experiments demonstrated that a 7:1 volumetric mixture of biochar and compost can successfully remove up to 877 g CH4/m3·d with empty-bed residence times of 82.8 minutes. As a biologically-active material, compost served as the sole source of nutrients and inoculum for the biofilters which greatly simplified the operation of the system. Higher elimination capacities may be possible with higher compost content such as a 1:1 ratio of either biochar or lava rock, while maintaining the empty-bed residence time at 82.8 minutes.
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    Transitioning an Alkaliphilic and Photosynthetic Microbial Consortium from Laboratory to Outdoor Demonstration Scale
    (2023-10-05) Haines, Marianne Victoria; Strous, Marc; Strous, Marc; Tutolo, Benjamin M.; Dunfield, Peter F.; De la Hoz Siegler, Hector; Kleinegris, Dorinde M.M.
    The 21st century’s challenges—climate change, growing population, resource decline, habitat and species loss—mean that current practices must be replaced, redesigned, and improved. Phytoplankton, reliant on water, light, nutrients, and CO2, offer versatile applications in nutritional supplements, agricultural feed, bioplastics, wastewater treatment, and bioenergy production. Currently, the most successful commercial ventures center on select taxa like Spirulina and Chlorella and produce high-value products for human consumption. Expanding the scope of viable commercial taxa and their applications hinges on overcoming critical challenges in cultivation, notably biomass productivity, robustness, and resource use. Inspiration can be drawn from natural environments where phytoplankton flourish, like alkaline soda lakes. These lakes are characterized by elevated pH and high carbonate alkalinity. Growing phytoplankton in high pH (10+), high carbonate alkalinity medium (0.5 M) increases the driving force for CO2 capture into solution and helps exclude competitors and predators which can cause biomass instability. This thesis chronicles the transition of biomass from alkaline soda lakes, dominated by the cyanobacterium Sodalinema alkaliphilum, from laboratory to large-scale outdoor demonstration. Chapter 2 explores the microbes inhabiting such lakes and their societal applications. Chapter 3 describes the design, construction, and operation of laboratory photobioreactors with programmable lighting and online growth measurements. Chapter 4 follows outdoor biomass cultivation in a 1,000 L photobioreactor, demonstrating sustained growth at a pH sufficient for CO2 capture from air. In Chapter 5, cultivation in a 3,000 L open raceway pond (ORP) reports long-term medium re-use, water requirements, and CO2 capture from air, although optimisation is necessary. Operational seasons ranged 70–140 days—160 being the maximum possible in Calgary’s temperate climate. Average daily yields were ∼ 3–4 g/m2/day (ash-free) with modeling predicting productivity could reach 6 g/m2/day by reducing biomass density. Finally, Chapter 6 quantifies ORP biomass losses, with stable isotope probing unveiling insights into S. alkaliphilum physiology and ecology. In conclusion, this research explores the feasibility of growing S. alkaliphilum biomass at scale for extended durations and has generated baseline data and operational insights which can be used to inform and refine the sustainability and productivity of future iterations of this technology.