Browsing by Author "Strous, Marc"
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Item Open Access An Alkaliphilic Cyanobacterial Consortium for Air-Capture and Conversion of Carbon dioxide(2022-08) Ataeian, Maryam; Strous, Marc; Cey, Edwin; Larter, Stephen R; Dunfield, Peter F; Hug, Laura ACyanobacteria encompass a diverse group of photosynthetic Bacteria with important roles in nature and biotechnology. The ability of cyanobacteria to fix carbon dioxide (CO2) through oxygenic photosynthesis, with minimal nutritional requirements and non-potable water, has led to their adoption for bio-production of high-value nutraceuticals. While cyanobacteria-based biotechnologies hold much potential for the production of bio-products, there remain several challenges for efficient and cost-effective implementation of this technology at scale. This thesis explores multiple innovative approaches to overcome those challenges using a cyanobacterial consortium, enriched from phototrophic microbial mats of alkaline soda lakes in British Columbia, Canada. First, the cyanobacterial consortium was investigated as an alkaline capture and conversion system (chapter 2). This study demonstrated successful cyanobacterial growth at a pH above 11, with high and robust biomass productivity. And for the first time, feasibility of direct capture of CO2 from the atmosphere into the spent medium was shown. In chapter 3, a combination of Nanopore and Illumina sequencing was used to obtain a complete genome of the abundant cyanobacterium of the consortium, provisionally named Candidatus “Phormidium alkaliphilum”. Evolutionary changes in the genome during three years of laboratory cultivation were investigated and differences in gene content between alkaliphilic and neutral pH Phormidium species were studied. Favourable genomic factors contributing to the ecological success of this genus in both alkaline soda lakes and in photobioreactors were discussed. Lastly, in chapter 4, the community composition of the consortium’s heterotrophic members and their ecological interactions with Ca P. alkaliphilum were analyzed using metagenomics, metaproteomics, and stable isotope probing/proteomics. Adaptation to different photobioreactors (tubular and stirred) at different pH and with different nitrogen sources (ammonium, urea, and nitrate) was explored. Genome information from each heterotrophic population was investigated for six ecological niches created by cyanobacterial metabolism and one niche for phototrophy, explaining the robustness of the system. In conclusion, this work explored innovative solutions based on biological and chemical mechanisms to improve the feasibility of cyanobacterial biotechnology and expanded current knowledge on ecological interactions of the genus Phormidium in its natural environment.Item Open Access An Exploration of Cell Death and Lysis in an Alkaliphilic Cyanobacterial Consortium using Multi-'Omics Approaches(2023-12-01) Khot, Varada Milind; Strous, Marc; Hynes, Michael Francis; de Koning, Jason; Ryan, Cathryn; Petersen, JillianCyanobacteria are important primary producers in many ecosystems, converting light into chemical energy. Their death plays a vital role in releasing organic carbon and nitrogen into the environment, supporting the life of other, heterotrophic organisms, and facilitating carbon sequestration in sediments. With warming aquatic systems, algal blooms are becoming more frequent, persistent, and larger in size, highlighting the need to understand the mechanisms of their death. Cyanobacterial death, whether via viral predation or other mechanisms such as programmed cell death, remains an underexplored topic in non-marine aquatic ecosystems. In this thesis, cell lysis and death of an alkaliphilic Cyanobacterium, Candidatus Sodalinema alkaliphilum, is explored primarily from the perspective of viral infections. Ca. Sodalinema alkaliphilum was enriched, along with its associated heterotrophic microbial community, from alkaline soda lakes on Cariboo Plateau in British Columbia, Canada. This robust cyanobacterial consortium has also been the focus of biotechnological research aimed at producing sustainable bioproducts. Robustness of this consortium can be defined as its capacity to maintain phototrophic function in spite of (a)biotic disturbances. First, bioinformatic techniques to investigate the viral ecology and dynamics of microbial communities in silico were reviewed in Chapter 2. These included identifying viral sequences, classifying their taxonomy, performing phylogenomic analyses, and predicting their hosts. Methods from Chapter 2 were subsequently applied to characterize viral populations in the alkaliphilic cyanobacterial consortium and to investigate viral dynamics by comparing bacterial defence mechanisms across multiple years. This study was the first metagenomic survey of viruses in an alkaliphilic phototrophic consortium and provided valuable insight into the viral ecology of a phototrophic enrichment culture. Finally, based on findings from Chapter 3, the same cyanobacterial consortium was challenged with threats from its natural environment, the alkaline soda lakes. In Chapter 4, the trigger and mechanism of a sudden and rapid cell lysis of the Ca. Sodalinema alkaliphilum was explored using metagenomic, metatranscriptomics analysis, and transmission electron microscopy. This work enhanced our understanding of viral dynamics within an alkaliphilic phototrophic microbial community and highlighted the complexity of cell death in the natural environment.Item Open Access Assimilation of carbon and nitrogen by microbial mats from alkaline soda lakes(2020-11-19) Liu, Yihua; Strous, Marc; Hubert, Casey R J; Tutolo, Benjamin M; Hu, JinguangBackground:Soda lakes are extreme terrestrial ecosystems characterized by high pH, alkalinity, and sodium carbonate concentration. Despite the extreme environment, soda lakes host diverse microbial communities with high primary productivity, carried out by fast-growing phototrophic microbes such as cyanobacteria. In Goodenough Lake, a soda lake on the Cariboo Plateau in BC Canada, carbon isotope analysis indicated that the photosynthetic rate but not bicarbonate availability controlled carbon dioxide assimilation. However, the roles of individual cyanobacteria populations in carbon fixation remain unknown. Despite the rapid growth of microbial mat communities, common nitrogen sources, ammonium and nitrate, were detected only occasionally and in trace amounts in lake water. Mat communities may use alternative nitrogen sources like urea and dinitrogen gas as enzymes for urea assimilation and dinitrogen fixation were highly expressed.Objective:The objective is to measure carbon and nitrogen assimilation by microbial populations in mat communities.Approaches:Incubation of microbial mats from Goodenough Lake with heavy stable isotope labelled bicarbonate, and nitrogen sources, followed by isotope ratio mass spectrometry and proteomics.Results and conclusions:Over 90 different microbial populations were detected in microbial mat communities using proteomics. The sampled mat microbial communities were different from each other, even if samples were close together, but the most abundant populations were the same across samples. The two most abundant cyanobacterial populations exhibited different carbon fixation dynamics, and their abundance was negatively correlated, suggesting that they occupy different ecological niches. Among nitrogen sources, urea was consumed at the highest rate, followed by ammonia. The nitrate consumption rate was much lower, and the fixation of nitrogen was not detected. Urea was consumed mainly during the day. Rates for nitrate and ammonia consumption were similar during the day and the night.Item Open Access Autofermentation of alkaline cyanobacterial biomass to enable biorefinery approach(2023-04-08) Demirkaya, Cigdem; Vadlamani, Agasteswar; Tervahauta, Taina; Strous, Marc; De la Hoz Siegler, HectorAbstract Background Carbon capture using alkaliphilic cyanobacteria can be an energy-efficient and environmentally friendly process for producing bioenergy and bioproducts. The inefficiency of current harvesting and downstream processes, however, hinders large-scale feasibility. The high alkalinity of the biomass also introduces extra challenges, such as potential corrosion, inhibitory effects, or contamination of the final products. Thus, it is critical to identify low cost and energy-efficient downstream processes. Results Autofermentation was investigated as an energy-efficient and low-cost biomass pre-treatment method to reduce pH to levels suitable for downstream processes, enabling the conversion of cyanobacterial biomass into hydrogen and organic acids using cyanobacteria’s own fermentative pathways. Temperature, initial biomass concentration, and oxygen presence were found to affect yield and distribution of organic acids. Autofermentation of alkaline cyanobacterial biomass was found to be a viable approach to produce hydrogen and organic acids simultaneously, while enabling the successful conversion of biomass to biogas. Between 5.8 and 60% of the initial carbon was converted into organic acids, 8.7–25% was obtained as soluble protein, and 16–72% stayed in the biomass. Interestingly, we found that extensive dewatering is not needed to effectively process the alkaline cyanobacterial biomass. Using natural settling as the only harvesting and dewatering method resulted in a slurry with relatively low biomass concentration. Nevertheless, autofermentation of this slurry led to the maximum total organic acid yield (60% C mol/C mol biomass) and hydrogen yield (326.1 µmol/g AFDM). Conclusion Autofermentation is a simple, but highly effective pretreatment that can play a significant role within a cyanobacterial-based biorefinery platform by enabling the conversion of alkaline cyanobacterial biomass into organic acids, hydrogen, and methane via anaerobic digestion without the addition of energy or chemicals.Item Open Access Helminth Regulation of the Colonic Microbiome: Implications for the Control of Colitis(2020-11-18) Shute, Adam; McKay, Derek Mark; Buret, André Gerald; Sharkey, Keith A.; Strous, MarcThe mammalian gut is a dynamic and complex organ composed of host cells, bacteria, fungi and viruses; while parasitic protozoa and helminths can be transient residents in the intestine. Through the advancements in high throughput sequencing, an increasing number of studies have catalogued taxonomic compositional changes in the gut microbiota following infection with parasitic helminths. However, the functional implications of such helminth-microbiota interactions on the host are not well understood, especially in the context of controlling inflammation. The McKay laboratory continuously demonstrates that mice infected with the rat tapeworm, Hymenolepis diminuta, are protected from dinitrobenzene sulfonic acid (DNBS)-induced colitis. My thesis seeks to determine if H. diminuta is dependent on the intestinal microbiota to protect from colonic inflammation and to determine the underlying mechanisms that are responsible for this anti-inflammatory event. Although H. diminuta does not require the microbiota to infect or be recognized/expelled by the murine host’s immune system, distinct compositional changes to the colonic microbiota occur during infection with H. diminuta. Removing microbes by using germ-free mice or disrupting the intestinal microbiota through broad-spectrum antibiotic use inhibits the anti-inflammatory mechanism of H. diminuta towards DNBS-colitis. In addition to characterizing the compositional shift in the intestinal microbiota of mice infected with H. diminuta, we demonstrated a significant increase in fecal short chain fatty acids (SCFA), in particular acetate and butyrate. Infecting free fatty receptor 2 (ffar2)-knockout mice with H. diminuta inhibited the beneficial effect towards DNBS, and in parallel to this, treating mice with neutralizing IL-10 antibodies also blocked the protective effect of H. diminuta when challenged with DNBS. Immunostaining for IL-10Rα demonstrated increased reactivity in colonic tissues of mice that were either treated with butyrate enemas or infected with H. diminuta, as compared to tissue from naive mice. The data in this thesis provides proof-of-principle evidence that H. diminuta modulates both the gut microbiota and the immune system of its host to protect from chemically induced colitis. Where the removal of these constituents, inhibits H. diminuta’s ability to protect the host from colitis.Item Open Access The Impact of Community-Driven Methane Oxidation on the Fate of Fugitive Methane in Shallow Groundwater(2019-09-10) Kuloyo, Olukayode Olakunle; Mayer, Bernd; Strous, Marc; Stein, Lisa Y.; Hubert, Casey R. J.; Cey, Edwin E.; Ryan, M. Cathryn; Larter, Stephen R.Petroleum development from unconventional reservoirs has increased global concerns over the impacts of fugitive methane on freshwater aquifers and the climate. Although methane leakage from wells is well-documented, information regarding the consequences on groundwater is limited. To address these concerns, a comprehensive, multidisciplinary investigation of the migration and fate, and oxidation of fugitive methane in shallow aquifers was conducted. Ten weeks of methane injection and thirty-six weeks of monitoring using surface efflux and ground-penetrating radar measurements, aqueous and isotope geochemistry, 16S rRNA amplicon sequencing and microbial cell counts, was conducted at the CFB research aquifer in Borden, Ontario, Canada. The results showed that a significant fraction of methane vented to the atmosphere while an equal portion persisted in the aquifer despite active growth of methanotrophic bacteria. Methane oxidation in the aquifer was relatively ineffective due to oxygen-limitation. Furthermore, this study demonstrated that even small-volume releases of methane gas can cause extensive and persistent free phase and solute plumes emanating from leaks, that are detectable only by contaminant hydrogeology monitoring at high resolution. The influence of methane, oxygen and alternate electron acceptors such as nitrate on methane oxidation and microbial communities was investigated further using sand-packed, continuous culture mesocosms. The mesocosms were inoculated with groundwater from the methane injection experiments and maintained for thirty-six weeks with varying methane and oxygen concentrations. Methane oxidation was found to be strictly dependent on oxygen and led to the enrichment of 13C in residual methane. Nitrate stimulated the growth of nitrate-reducing bacteria but did not significantly impact methane oxidation. Methylotrophic populations were shown to persist for many weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Amplicon and shotgun metagenome sequences of samples from the mesocosms revealed complex bacterial communities consisting of non-methylotrophs alongside Alphaproteobacterial and Gammaproteobacterial methylotrophs. Metaproteome analyses and biomass delta 13C values determined from the metaproteome demonstrated communal methane oxidation in the mesocosms by identifying several non-methylotrophic populations that co-metabolized methane-derived C1 substrates such as methanol, formaldehyde and formate. Altogether, this work expands current knowledge on the migration and fate of methane in shallow aquifers, showing that effective detection and monitoring of methane in groundwater requires high-resolution multidisciplinary approaches, with potential implications for minimizing the impacts of gas exploration from unconventional reservoirs.Item 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, StevenCyanobacteria-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.Item Open Access Inversion Modelling of Copper Transport in Saccharomyces Cerevisiae(2020-05-12) Wilkins, Aaron Francis; Wieser, Michael E.; Karchewski, Brandon; Dettmer, Jan; Strous, MarcCopper is an essential nutrient but the uptake into cells is poorly understood. This dissertation summarizes the development of a mathematical system of equations to model the transport of copper in S. cerevisiae. Yeast is a model organism for studying the copper transport in human hepatic cells because the chaperone proteins and structures are well conserved between the species. An experiment is performed to investigate the transport between the growth media and the cells to model the process behind this important pathway. Transport mechanisms for this process are presented, mathematically modelled, and evaluated. Rate limited diffusion did not appear to be adequate in modelling the transport, but a term including a target copper concentration which cells actively maintain was introduced, and with a delayed activation, fit the data much more effectively. With this model, a framework is established for incorporating organelles to eventually model the intracellular copper transport and analyze the copper isotope distributions in the future. This work contributes to a larger initiative to incorporate copper isotope analysis as an innovative medical diagnostic tool in assessing human cellular pathology.Item Open Access Long-Term Electromethane Production in Continuous Flow Alkaline Microbial Electrolysis(2018-09-14) Salehi, Vajiheh; Strous, Marc; Birss, Viola; Hubert, Casey R. J.; Larter, S. R.Microbial Power to Gas (P2G) is a promising technology for storing renewable energy in the form of natural gas (methane). Energy storage is necessary because renewable energy is often produced at times when it is not demanded. Methane can be used as a transportation fuel in combustion engines due to its low energy density. Microorganisms can produce methane in a single compartment microbial electrolytic cell at room temperature and neutral pH. However, this technology faces several challenges, including anode corrosion, membrane failure, and the fact that the final product is a mixture of methane, hydrogen and CO2. Here, the performance of a continuous-flow MEC (without a membrane separator) was studied for microbial P2G, while monitoring hydrogen and methane gas production at the cathode, as well as microbial community changes over time, all in a pH 10 medium. A steel cathode was found to be preferred over various carbons, as the carbons changed their morphology and surface chemistry with time. Platinized titanium mesh was developed for oxygen evolution reaction (OER) and the hydrogen evolution reaction (HER) carried out on stainless steel cathode in order to produce hydrogen necessary for biologically produce methane. The results showed that this alkaline environment is a superior alternative to neutral one for methane production. High rate of hydrogen production was observed in bicarbonate buffer with 97% columbic efficiency. Methane generation reached up to 17 µL/L reactor/day in 1.0 M bicarbonate buffer solution (BBS). Methanobacters a hydrogenotrophic methanogen along with Delta proteobacteria, and Archobacter, an aerobic sulfide, formate and acetate oxidizer, were significantly enriched in MEC. These results showed in this study indicates that inoculation and enrichment procedures are necessary to the initial success of larger-scale systems.Item Open Access Methane oxidation and methylotroph population dynamics in groundwater mesocosms(Society for Applied Microbiology and John Wiley & Sons Ltd., 2020-01) Kuloyo, Olukayode; Ruff, S Emil; Cahill, Aaron; Connors, Liam; Zorz, Jackie K; Hrabe de Angelis, Isabella; Nightingale, Michael; Mayer, Bernhard; Strous, MarcExtraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand-packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty-five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of 13 C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty-nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.Item 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, MarcPolycyclic 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.Item Open Access Microbial Ecology of Subsurface Oil Sands Deposits in Northern Alberta, Canada(2018-08-09) Ridley, Christina M.; Gieg, Lisa Marie; Voordouw, Gerrit; Strous, Marc; Biddle, Jennifer; Hubert, Casey R. J.The subsurface Athabasca Oil Sands in northern Alberta, Canada, is an important yet understudied microbial habitat. Over geological time, progressive biodegradation of low molecular weight hydrocarbons by indigenous microorganisms has resulted in an enrichment of high molecular weight hydrocarbons known as bitumen. This is an extreme environment characterized by low water and nutrient availability, thereby resulting in low microbial biomass. The objective of this study was to characterize the microbial ecology of this unique ecosystem, which could ultimately provide a basis for the development of microbially enhanced oil recovery (MEOR) technologies. To that end, pristine core samples were collected from 220 to 320 meters below the surface during 3 annual field trips. Due to the high bitumen and low biomass content of the core samples, extensive method development and validation was performed to identify a robust molecular biological workflow that would allow for consistently successful extraction, amplification and sequencing of genomic DNA from core samples. This molecular biological method was then used to characterize the prokaryotic and eukaryotic microbial communities of the subsurface oil sands through 16S and 18S rRNA gene sequencing. Results revealed a surprising predominance of aerobic and facultative microorganisms in an environment that is traditionally considered anoxic. The genera Pseudomonas, Acinetobacter and Fontibacter were the dominant prokaryotes in all core samples (average relative sequence abundance >5%). Methanoculleus was the only methanogen detected at an average relative sequence abundance >1%. Fungi from the family Trichocomaceae and the order Hypocreales were the prevailing Eukaryotes. Core sample incubations were used to determine the effects of atmosphere and temperature on microbial community succession. Microcosms were established under aerobic, microaerobic and anaerobic atmospheres at reservoir (8°C), mesophilic (33°C) and thermophilic (60°C) temperatures. Results indicate that the predominant community members remained similar to the controls; however, some microcosms incubated at 60°C experienced enrichment of thermophiles, including the genera Thermus, Geobacillus and Thermanaerothrix. Many of the dominant taxa observed in this study could have potential MEOR applications, including hydrocarbon degradation, biosurfactant production and methanogenesis. Thus, the information revealed in this study could be used as the basis for MEOR technologies.Item Open Access 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.Item Open Access Mobility and persistence of methane in groundwater in a controlled-release field experiment(Nature Publishing Group, 2017-04) Cahill, Aaron G.; Steelman, Colby M.; Forde, Olenka; Kuloyo, Olukayode; Emil Ruff, S.; Mayer, Bernhard; Ulrich Mayer, K.; Strous, Marc; Cathryn Ryan, M.; Cherry, John A.; Parker, Beth L.Expansion of shale gas extraction has fuelled global concern about the potential impact of fugitive methane on groundwater and climate. Although methane leakage from wells is well documented, the consequences on groundwater remain sparsely studied and are thought by some to be minor. Here we present the results of a 72-day methane gas injection experiment into a shallow, flat-lying sand aquifer. In our experiment, although a significant fraction of methane vented to the atmosphere, an equal portion remained in the groundwater. We find that methane migration in the aquifer was governed by subtle grain-scale bedding that impeded buoyant free-phase gas flow and led to episodic releases of free-phase gas. The result was lateral migration of gas beyond that expected by groundwater advection alone. Methane persisted in the groundwater zone despite active growth of methanotrophic bacteria, although much of the methane that vented into the vadose zone was oxidized. Our findings demonstrate that even small-volume releases of methane gas can cause extensive and persistent free phase and solute plumes emanating from leaks that are detectable only by contaminant hydrogeology monitoring at high resolution.Item Open Access Rhizobiophage Functional Genomics(2020-08-19) Koswaththa Muhandiramlage, Damitha Gunathilake; Hynes, Michael F.; Yost, Christopher Karl; Dunfield, Peter F.; Strous, MarcRhizobiophages 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.Item Open Access Robust, high-productivity phototrophic carbon capture at high pH and alkalinity using natural microbial communities(2017-03-29) Sharp, Christine E; Urschel, Sydney; Dong, Xiaoli; Brady, Allyson L; Slater, Greg F; Strous, MarcAbstract Background Bioenergy with carbon capture and storage (BECCS) has come to be seen as one of the most viable technologies to provide the negative carbon dioxide emissions needed to constrain global temperatures. In practice, algal biotechnology is the only form of BECCS that could be realized at scale without compromising food production. Current axenic algae cultivation systems lack robustness, are expensive and generally have marginal energy returns. Results Here it is shown that microbial communities sampled from alkaline soda lakes, grown as biofilms at high pH (up to 10) and high alkalinity (up to 0.5 kmol m−3 NaHCO3 and NaCO3) display excellent (>1.0 kg m−3 day−1) and robust (>80 days) biomass productivity, at low projected overall costs. The most productive biofilms contained >100 different species and were dominated by a cyanobacterium closely related to Phormidium kuetzingianum (>60%). Conclusion Frequent harvesting and red light were the key factors that governed the assembly of a stable and productive microbial community.Item Open Access Transcriptome Profiling in Ephedra sinica and Catha edulis Reveals Enzymes Putatively Involved in Ephedrine Alkaloid Biosynthesis(2016-01-14) Groves, Ryan A; Facchini, Peter; Moorhead, Gregory; Ro, Dae-Kyun; Strous, Marc; Vogel, HansAmphetamine analogs are a class of medicinal compounds produced by Ephedra sinica and Catha edulis. Two commonly used members of this class of compounds include the cough suppressant (1S-2S)-pseudoephedrine, and the stimulant (1R-2S)-ephedrine. These secondary metabolites are produced in planta through a L-phenylalanine derived multi-step pathway. Despite the importance of this biosynthetic pathway, only one enzyme in the pathway has been characterized at the molecular level. Recently, two new transcriptomes have been created for E. sinica and C. edulis with the hopes of facilitating future gene discovery in this untapped pathway. In this investigation, I outlined an efficient transcriptomics based approach to potential gene candidate selection for all putative stages of the amphetamine analog biosynthetic pathway. This investigation also describes a complete approach to molecular cloning, protein expression, and assaying of gene candidates, resulting in the discovery of a key biosynthetic enzyme involved in amphetamine analog biosynthesis in E. sinica.Item Open Access 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.Item Open Access Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes(2023-02-09) Kleiner, Manuel; Kouris, Angela; Violette, Marlene; D’Angelo, Grace; Liu, Yihua; Korenek, Abigail; Tolić, Nikola; Sachsenberg, Timo; McCalder, Janine; Lipton, Mary S.; Strous, MarcAbstract Background Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. Results Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software ( https://sourceforge.net/projects/calis-p/ ). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. Conclusions We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.