Abstracts

Jasmeen Kaur Parmar

University of Toronto

Growth and stability of chain elongating isolates on lactate-rich digestate 

Transforming wastes into a wide range of valuable chemicals is a central challenge for achieving resource recovery within a circular bioeconomy. Novel anaerobic digestion technologies have the potential to address this need by driving the microbial process away from methane production, which limits the product spectrum, towards higher value chemicals. Medium-chain fatty acids (MCFAs) have garnered interest as a target product for resource recovery due to their high market value, potential to be upgraded to other high-value oleochemicals, and a need to replace existing unsustainable refinery-based production methods. Studies have successfully demonstrated MCFA production from mixed communities, however, these communities result in low product yields due to competing biochemical pathways that produce other end-products (e.g. acetate, butyrate). An alternative approach is to use a two-stage process wherein a mixed community produces lactate from wastewater biosolids and food waste in the first stage, and genetically modified chain elongating isolates produce MCFAs and other chemicals in the second stage. While this approach could result in a broad platform for chemical production from organic wastes, the growth stability and nutritional requirements of chain elongating isolates has not been evaluated. In this study, we screened a panel of seven chain elongating isolates to assess their growth on lactate-rich digestate. Our results reveal key nutritional requirements to grow chain elongating isolates in digestate, including amino acid deficiencies. These deficiencies represent targets for chain elongator engineering or design of synthetic co-cultures with amino acid exchange. We are also assessing long-term stability of these chain elongating isolates under unsterile continuous reactor operations. Our results will inform the scale-up of a two-stage system with in-line extraction for renewable chemical production from organic waste feedstocks.

Hedieh Hashemi

University of Toronto

High-throughput Isolation and Identification of Anaerobic Bacteria from a Lignocellulose Degrading Microbiome

The transition to a circular economy is more than just feasible - it is necessary for the
preservation of our society. Anaerobic digestion of organic waste and/or lignocellulosic biomass to renewable energy and chemicals via microbial metabolism represents an important step towards carbon neutrality. The technology stands to transition the chemical industry away from petroleum and other environmentally impactful feedstocks, like palm oil and coconut oil, by providing an economically advantageous and renewable process alternative.
Anaerobic digesters operated to inhibit methane production can convert lignocellulosic biomass into high value medium chain fatty acids (MCFAs). However, the individual bacterial species within these microbiomes are often unknown. Having access to these specific strains is necessary to study the metabolism of these bacteria in more detail, and opens the door to the design of synthetic consortia that optimizes MCFA production rates and yields.
In our study, we aimed to isolate anaerobic bacteria within enrichment cultures responsible for converting sorghum biomass into MCFAs using high-throughput techniques. Subsequently, we are using 16S rRNA sequencing followed by bioinformatic analysis to identify the taxonomy of the isolated species, followed by physiological growth experiments to learn the growth conditions and metabolic processes of these bacteria. The resulting isolates and physiological characterization will be used to design synthetic consortia for improved MCFA production.

Jinjin Chen

University of Toronto

Genetic Engineering of Acidithiobacillus ferridurans  Using CRISPR/Cas9 System for Enhanced Biomining

Biomining processes utilize microorganisms, such as Acidithiobacillus, to extract valuable metals by producing sulfuric acid and ferric ions that dissolve sulfidic minerals. However, excessive production of these compounds can result in metal structure corrosion and groundwater contamination. Synthetic biology offers a promising solution to improve Acidithiobacillus strains for biomining, but genetic engineering of these slow-growing microorganisms is challenging with current inefficient and time-consuming methods. To address this, we established a CRISPR/dCas9 system for gene knockdown in A. ferridurans JAGS, successfully downregulating the transcriptional levels of two genes involved in sulfur oxidation. Furthermore, we constructed an all-in-one CRISPR/Cas9 system for fast and efficient genome editing in A. ferridurans JAGS, achieving seamless gene deletion (HdrB3), promoter substitution (Prus to Ptac), and exogenous gene (GFP) insertion. Additionally, we created a HdrB-Rus double-edited strain and performed biomining experiments to extract Ni from pyrrhotite tailings. The engineered strain demonstrated a similar Ni recovery rate to wild-type A. ferridurans JAGS, but with significantly lower production of iron ions and sulfuric acid in leachate. These high-efficient CRISPR systems provide a powerful tool for studying gene functions and creating useful recombinants for synthetic biology-assisted biomining applications in the future.

Nazanin Yasoubi

University of Waterloo

Evaluation of pulsed xenon UV irradiation on inactivation of Listeria monocytogenes on stainless-steel surfaces

     UV-C disinfection is a safe and effective method for the inactivation of bacteria, viruses, spores, and other microorganisms. Traditionally, continuous wave low pressure mercury lamps have been used for UV-C disinfection purposes, however, the Minamata Convention and other regulatory bodies around the world are working to disallow the use of mercury instruments to prevent mercury contamination which has well known negative health effects. There are two major alternatives to mercury UV-C lamps, pulsed xenon UV lamps (PX-UV) and UV-C light emitting diodes (LEDs). The use of pulsed lamp (PL) technology for food or food contact surfaces decontamination was approved by U.S. Food and Drug Administration (FDA) in 1996 for xenon flashlamps at dosages below 12 J/cm2. There are a number of possible parameters that can affect the extent of disinfection using UV-C lamps including light intensity, wavelength, exposure time, characteristics of the microorganism, surface/sample properties, light penetration, and shielding effects due to existence of particles which could shield the microorganisms from the UV light.
    Listeriosis is a foodborne disease caused by Listeria monocytogenes. U.S. Food and Drug Administration (FDA) has reported the severity of this disease with high fatality rate of 20 to 30 percent even with sufficient antibiotic treatments and more than 90 percent of those affected require hospitalization. In addition to the public health burden of foodborne diseases, there are significant economic costs associated with outbreaks and recalls. L. monocytogenes can be readily isolated from a broad range of ecological niches such as soil, water, and vegetation making it difficult to prevent contamination of foodstuffs. Thus, Food processing environments are rigorously monitored for L. monocytogenes as it can persist in the facility for a long period of time. It can resist many stressors, persist in cold environments, and grows in protective biofilms. PX-UV could be an appropriate disinfection method since it remains no residues and could be timesaving. Regardless, the seriousness of listeriosis in vulnerable populations makes it imperative to developed improved methods for the inactivation of L. monocytogenes in food processing facilities.  
In this study, the impact of different operational parameters of PX-UV on the inactivation of L. monocytogenes on stainless-steel (SS) was investigated. Since most of the facilities in the food industries are made of stainless steel, the material which were chosen to work on, was stainless steel. To address the lack of information concerning the effect of frequency on disinfection efficacy of PX-UV, a study of the effects of frequency, exposure time, and light angle was undertaken.  Developing a mathematical expression to quantify disinfection can be helpful for sizing the device for various applications or to determine the minimum dosage needed to achieve a certain level of disinfection for a specific process and specific microorganism. Therefore, the UV disinfection kinetics for the PX-UV lamp and L. monocytogenes was quantified. 
The results of the study proved that the log reduction of 5 (99.999%) was achieved by UV dosage of ~100 mJ/cm2 which is much lower than the approved value by FDA. The study on various radiation angles and frequencies at constant dosage showed that the total dosage received at the surface was the most significant factor in achieving the desired disinfection and highlighted the importance of using radiometry on site to determine the actual fluence being applied at the specified location. However, as it was expected, increasing exposure time or frequency can increase the inactivation rate. It should be noted that at high frequencies, the dose per pulse was lower than at low frequencies when designing a device for a specific application. It has been proved that PX-UV could achieve the required dosage for L. monocytogenes inactivation much faster than other UV methods.

Mohamed Nasr

University of Toronto

Towards Upcycling Sustainable Feedstocks into Value-Added Chemicals Using Model and Non-Model Yeasts

Synthetic Biology aims at reprogramming microbes to be used in a multitude of applications. To this end, we are engineering model and non-model yeasts for utilizing sustainable feedstocks to produce chemicals of industrial interest. Feedstocks of relevance to this work are potato starch obtained from food waste streams, in addition to 1-carbon (C1) molecules such as CO2, formate, and methanol. Ongoing work towards this goal includes engineering the model yeast Saccharomyces cerevisiae for starch digestion. Additionally, we are engineering the acid/heat/inhibitor tolerant non-model yeast Pichia kudriavzevii for improved CRISPR-mediated genome editing efficiency, to be followed by the introduction of pathways for C1 molecule utilization. Finally, in an effort to contribute towards a meaningful circular bioeconomy through yeast fermentation, we aim to use our engineered strains to produce industrially relevant molecules, such as the building blocks adipic acid and polyhydroxyalkanoates (market size of ~5 billion and ~80 million USD, respectively) as well as amino acids that could be used for human and animal feed (market size of ~30 billion USD).  

Eileen Bates

University of Waterloo

Engineering Escherichia coli for Lycopene Biosynthesis: Optimizing Isopentenol Utilization Pathway and Cellular Lipid Content for Increasing Yield

Lycopene is a potent antioxidant and an important intermediate in the synthesis of other commercially viable carotenoids. Extraction of carotenoids from natural sources often produces low yields making biosynthesis by microbial hosts a promising route of production. Carotenoid biosynthesis requires the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These are naturally produced in prokaryotes by the 2-methyl-D-erythritol-4-phosphate (MEP) pathway, which is highly regulated and relies on central carbon metabolism, making it difficult to engineer. An alternative isopentenol utilization pathway (IUP) has recently been shown to convert isopentenol into IPP and DMAPP, increasing carotenoid production in Escherichia coli. Increased cellular lipid content is known to increase carotenoid yield in Yarrowia lipoytica through upregulation of DGA1, presumably due to the strains increased ability to dissolve these hydrophobic compounds in their lipid bodies. Increased lipid content in E. coli can potentially be accomplished through expression of a foreign monoglycosyldiacylglycerol synthase (MGS) which forms extensive cytoplasmic vesicles. Strains expressing the IUP pathway and lycopene biosynthesis genes were co-expressed with mgs and evaluated for lycopene content, lipid content, and growth performance. However, while IUP expression resulted in higher lycopene titres, co-expression with MGS showed no increase in lipid content. Future work will focus on validating the bioactivity of MGS by observation of the elongated cell morphology in lycopene expressing strains previously reported.  

Samuel Koo

McMaster University

Integrated Development of High-Throughput Phage Purification Strategies for Use in Human Phage Therapy

Bacteriophages are a promising antimicrobial alternative to modern antibiotics as they can rapidly overcome bacterial resistance. However, the propagation of therapeutic phages in gram-negative bacteria results in proinflammatory contaminants, notably endotoxins (lipopolysaccharides). To meet the strict regulations (0.5 EU/mL or 5 EU/kg/h for intravenous applications) regarding endotoxin levels, the widely accepted method for phage purification is based on polyethylene glycol precipitation followed by cesium chloride density gradient ultracentrifugation. However, this method is very labour-intensive while only producing small quantities of purified phage product. There have been a handful of studies on using membrane adsorbers in this application, however those studies have focused on only removing endotoxins or only recovering phages and thus there is a strong need for further study of removing endotoxins from bacteriophage preparations.

Two phages were isolated, sequenced, and propagated using E. coli plate cultures generating bacteriophage feedstocks with titers of 109 - 1010 PFU/mL containing endotoxin concentrations in the range of 3 000 – 30 000 EU/mL. In some early purification tests with commercial anion exchange membrane absorbers, Mustang E (Pall) and Natrix Q (Millipore), in the standard syringe filter format, we discovered that the separation of phage from endotoxins was very poor. Recognizing the limitations of this sequential approach and the need to study a wide variety of solution conditions (e.g., complexation of endotoxins into various aggregate forms is determined by the presence of detergents/ions), we have since pivoted to using a high-throughput screening approach recently developed at McMaster University.  Also, we are using helium ion microscopy to directly visualize phage binding onto the membrane adsorbers. We anticipate that this approach will elucidate the governing factors for phage binding onto membranes which will be used to develop a universal method of generating clinical-grade phage preparations.

Cho-E Choi

Western University

3D Bioprinting Biomaterial-Nanoparticle Composite for Drug Delivery and Bone Repair Applications

Hydrogels are three-dimensional (3D) water-swellable polymeric matrices that have found extensive use in tissue engineering and drug delivery. Hydrogels can be conformed into any desirable shape using 3D printing, making them suitable for personalized treatment. Among the different 3D printing techniques, digital light processing (DLP)-based printing offers the advantage of quickly fabricating high-resolution structures, which reduces the chances of cell damage during the printing process. Here, we have used DLP to 3D-print biocompatible gelatin methacrylate (GelMA) hydrogel scaffolds intended for bone regeneration. However, GelMA on its own is incapable of promoting this biological process. To overcome this limitation, we introduced the biologically active zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, which contain zinc and can promote bone regeneration. Next, we optimized the composition as well as the printing process to obtain robust scaffolds that could retain shape once printed. In addition, the GelMA/ZIF-8 composite hydrogel showed good mechanical properties, biocompatibility, and sustained drug-release behavior. Furthermore, the composite hydrogel promoted the proliferation and differentiation of bone adipose mesenchymal stem cells in vitro. In conclusion, this study presents a novel DLP printing technique to fabricate GelMA/ZIF-8 composite hydrogel with excellent mechanical properties, biocompatibility, sustained drug release behavior, and enhanced bone regeneration properties. This hydrogel composite has potential applications in bone repair, drug delivery, and implant coating, making it a promising candidate for future clinical translation.
 

Sarah Kefi

University of Quebec at Rimouski (UQAR)

Effect of microalgae culture conditions on the production of compounds preventing the biological colonization of surfaces

Bacterial biofilms are complex biological systems that are difficult to eradicate, as they confer bacteria effective protection against external factors and antimicrobial agents. In many sectors of human activity, this phenomenon causes important problems. In marine transport ships, anti-fouling agents are often incorporated into outer hull paints to prevent the attachment and transport of invasive species. However, many such agents can be toxic to non-targeted organisms in the general environment. Recent legislative requirements and increased environmental awareness thus spurs the development of alternative natural and non-toxic antibiofilm compounds. One potential source of such metabolites is microalgae: these microorganisms are known to compete with bacteria in the natural environment, and have developed various means of defense to this end, such as the production of secondary metabolites. Antimicrobial metabolites produced by microalgae are of various molecular classes and sizes, and yields are highly affected by environmental conditions or stimuli. This project thus aims at identifying the most promising fraction or molecule(s) that would avert the development of biofilms for a wide spectrum of bacterial species, and enhance its production through process optimization and stress induction strategies. The search for the target molecules is a bio-guided fractionation approach, where the extracts will be tested on biofilm forming bacteria.

Nicholas Rasmussen

University of Waterloo

Novel Hydrogel Photobioreactor for Space Cultivation

The prospect of crewed space activities in the future will greatly depend on our ability to provide life supporting resources, like oxygen and food, in a secure and economical way. In situ food production is a promising method to meet these demands as it helps to limit earth resupply dependence. However, the ability to efficiently produce food in a microgravity environment remains a challenge due to mass transfer and phase separation limitations in microgravity. Other constraints include energy usage and available growing space in space environments. Proposed solutions include higher order plants, insects, and microorganisms. There has been considerable interest in microalgae due to its high nutritional density and short growth cycles compared to higher order plants. Traditional photobioreactors have been proposed since they are well studied terrestrially, however considerable modification will be required to work in microgravity due to gas exchange limitations. Additionally, liquid cultures will have considerable launch weight due to high water volume requirements making this technology expensive to launch and therefore impractical. 
A novel hydrogel-based photobioreactor is proposed to grow Chlorella vulgaris to overcome the challenges faced by traditional photobioreactors, and to grow microalgae more efficiently for long duration space activities. The algae will be separated from the liquid culture making harvesting easier. The hydrogel also acts to separate the liquid and gas phases overcoming phase separation challenges in traditional photobioreactors. This proposed study will look to develop a unit model for the bioreactor to model algal growth and resource use over time. The impact of the reactor design will also be evaluated against cell viability and growth kinetics. Different hydrogel materials will also be tested to determine the influence of water diffusivity of microalgae growth.

Elise Heinen

University of Quebec at Rimouski (UQAR)

Optimization of the conditions for extraction and purification of marine secondary metabolites preventing the biological colonization of surfaces

In maritime and aquaculture sectors, bacterial biofilms form a substrate leading to a biological dirtying of surfaces. Such biofouling on material and equipment is often problematic as it may cause a loss of the optical properties, a decrease in heat exchange capacities or an accelerated corrosion for example. In the maritime transport industry, the struggle against biofouling involves the protection of submerged surfaces with antifouling paints. Their mechanism of action is most often based on the release of organic tin derivatives. These paints are extremely toxic to marine ecosystems and since the 80s, the use of paints with Tributiletin (TBT) is forbidden in most countries around the world for boats less than 25 meters. As legislation changes and general ecological awareness of populations increase, the search for new natural and less toxic alternative compounds becomes a necessity. This is where this project intervenes: to find new natural molecules with non-stick but non-toxic properties. The focus is on the secondary metabolites of microalgae endemic to the St. Lawrence ecosystem. The first objective is to optimize the extractions methods to maximize yields. To date, ultrasound assisted extraction methods are very promising. When coupling this method with bead milling, a maximum yield of 52,12% ± 0,54% was attained. The second objective of this project is to study the biological activity of the extracts. Biological tests are conducted to determine the potential non-stick and/or antibacterial effect on different strains of bacteria are carried out. When an effect is demonstrated, the third objective is to purify the extract in an attempt to identify the molecule or the synergistic mechanism responsible for this effect. This innovative project seeks to provide solutions to current issues of biofouling and to successfully develop new bioprocesses to replace conventional polluting processes.

Shane Orgnero

University of Toronto

Engineering Synthetic Microbial Consortia for Carbon-Efficient Waste to Chemicals Production

Achieving a circular economy requires the conversion of societal waste streams into new materials, chemicals, and fuels. Biological processes such as anaerobic fermentation (AF) represent a platform technology that can be used to recycle carbon from wastewater biosolids and food waste into industrially useful chemicals, including medium-chain fatty acids (MCFAs) and alcohols. However, current AF technologies result in 50-60% of the available carbon being lost to CO2, resulting in low carbon conversion efficiencies. In addition, conventional microbiomes used in commercial AF are large and undefined, limiting the ability of researchers to apply cutting-edge synthetic biology tools to improve strain, and microbiome, performance. In this project, we establish a proof-of-concept synthetic microbiome that can convert organic waste into chemical products, including MCFAs and alcohols, at a theoretical efficiency near 100% through a novel CO2 off-gas recycling scheme. In the future, we will employ targeted genetic engineering to improve strain key performance indicators (KPIs) and enhance waste degradation, in addition to adaptive laboratory evolution strategies aiming to improve both single-strain KPIs as well as global microbiome performance. The process has been demonstrated using batch fermentations containing a synthetic waste feed (cellobiose, H2, and CO2) and synthetic consortia of lactic acid bacteria, acetogens, and chain-elongating bacteria. Work is ongoing to optimize the platform microbiome as well as to define genetic engineering targets and adaptive evolution strategies.

Mauricio Garcia Benitez

University of Toronto

Evaluation of the effect of cytosolic and membrane protein expression on acetate excretion in Escherichia coli

In metabolic engineering, protein overexpression can have a significant impact on cellular metabolism. In particular, the expression of membrane proteins has been predicted to affect overflow metabolism due to resource allocation constraints such as limited membrane space or saturation of the translocation machinery in the cell. 
 
In this study, we evaluated the impact of expressing a cytosolic and a membrane protein on acetate excretion in E. coli. We constructed vectors to express different membrane proteins and cytosolic proteins tagged with sfGFP in strains of E. coli using an IPTG inducible system. The strains were characterized using different IPTG levels and with different carbon sources to obtain data at different growth rates.
 
Our results show that a high IPTG level would increase the expression of the protein and cause a significant growth reduction. Specifically, the expression of the membrane protein caused a shift and a higher slope in the plot of acetate excretion rate vs growth rate. These findings suggest that resource allocation constraints for membrane proteins could lead to a higher flux of acetate due to direct competition for membrane space or translocation complexes with other proteins, such as complexes for the respiratory chain, forcing the cell to produce energy by acetate production.
 
Our study contributes to a better understanding of the impact of protein expression on acetate excretion in E. coli and has relevance for metabolic engineering projects, such as increasing tolerance to biofuels by expressing an efflux pump to alleviate the toxic accumulation of the target biofuel inside the cell.
 

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Joel Howard

University of Toronto

Identification and functional characterization of chain elongating bacteria

By distributing metabolic tasks across distinct community members, microbiomes can carry out metabolic pathways that are infeasible for a single microbe, like the conversion of lignocellulosic biomass (LCB) into medium chain fatty acids (MCFAs). The microbial conversion of LCB into MCFAs has recently emerged as a platform for sustainable chemical manufacturing. An important functional guild in this platform are chain elongating bacteria, which synthesize MCFAs through the reverse β-oxidation (RBO) pathway. However, despite their biotechnological importance, few chain elongating bacteria have been isolated and fewer have been functionally characterized. Here we describe the functional characterization of known chain elongators and a genomic strategy for their identification. To functionally characterize chain elongators, we developed an automated high-throughput anaerobic screening platform and used it to measure growth and MCFA production of eight known chain elongators across multiple electron donors. To uncover uncharacterized chain elongators, we aligned core RBO genes to over 300,000 bacterial genomes from the RefSeq database and compared their synteny to known chain elongators. We obtained six candidate chain elongators identified from the genetic analysis and applied our screening platform to validate their chain elongation phenotype. Our results provide valuable insights into chain elongator identification and metabolism, which will improve efforts to engineer chain elongating microbiomes with greater MCFA titers, rates and yields, offsetting our reliance on petrochemicals.

Mara Jezernik

University of Toronto

Making chemicals from CO2: upgrading the product spectrum in microbial electrosynthesis reactors

Microbial electrosynthesis (MES) presents a way to convert CO2 and renewable electricity into organic chemicals for improved long term storage and transport of energy. However, precise control over the production of high-value chemicals from MES remains a challenge when working with mixed microbial cultures. This project explores the benefit of bacterial co-cultures for the specific production of higher value chemicals from CO2, with a focus on medium-chain fatty acids (MCFAs) and their respective alcohols. To this end, we are operating several MES reactors in parallel with pure cultures, synthetic communities or mixed communities to compare the productivity and titers of MCFA production from CO2. This experiment overcomes the problem of comparing results between vastly different reactor designs, which will provide a better understanding of the advantages of co-cultures for MCFA production in MES. Microbial co-cultures have demonstrated benefits over mixed cultures by selectively improving the range and specificity of fermentation products, and this benefit should be explored in the context of MES to further expand the scope of high value products.

Alessandra Castro

University of Toronto

Microbial expansins for bio-based materials engineering

Microbial expansin-related proteins represent an intriguing example of non-enzymatic proteins with potential to tune the physical attributes of lignocellulosic materials and is the target of the proposed research. This research proposes that the impact of microbial expansin-related proteins on lignocellulose structure depends on the biomass source, that microbial expansin-related proteins that act on lignocellulose adopt similar and differentiating molecular structures, and that microbial expansin-related proteins alter the porosity of lignocellulosic fiber.
Accordingly, this will include the production and purification of ten microbial expansins for functional characterization, in which 5 of them were found to have sufficient production for further experimental studies. Functional characterizations will employ biophysical approaches including pull down assays and BET specific surface area measurements to quantify impacts of microbial expansins on cellulose fibre porosity. The study will also use electron microscopy to visualize the impact of microbial expansins on fibre morphology. Although the focus will be treatment of cellulosic materials, those microbial expansins that do not act on cellulose will also be tested on chitin materials to distinguish microbial expansins on the basis of cellulose versus chitin specificity. Preliminary examination of pull-down assays shows some binding to cellulose, while other show higher binding to chitin.
This study is anticipated to generate molecular level insights that advance the fundamental knowledge of microbial expansin-related proteins, including sequence-functional relationships that predict expansin which act on cellulosic materials to demonstrate the applied potential of a new class proteins that increase the dissolution of cellulosic fibre from wood.

Jens Kastenhofer

University of Toronto

The role of heterotrophs in photoprotection in algal biofilms

There is growing interest in the development of algal biofilm-based systems as they offer potential advantages over suspension-based systems by providing highly concentrated biomass attached to a solid substrate. In large scale open photobioreactors and in nature, these phototrophic biofilms exist as consortia of autotrophic algae as well as heterotrophic microorganisms. Enhancing our understanding of the interaction between the members of these consortia will aid the design, control and optimization of bioprocesses that utilize phototrophic biofilms. In this work, we investigate whether the presence of heterotrophic bacteria in Chlorella vulgaris biofilms alleviates photoinhibition by offering a sink for excess electrons generated during high light intensities. We assess growth, expression of RuBisCO and photosystem genes, as well as chlorophyll and xanthophyll content as physiological indicators of light induced stress and compare that to axenic cultures.

Arianna Castro Rojas

Western University

Surveying the Diversity of GIY-YIG Nuclease Domains for Gene Editing

Gene editing involves making specific modifications to an organism's genetic material by introducing targeted double-stranded breaks in DNA using endonucleases. The CRISPR/Cas9 system is most used for this purpose, and GIY-YIG homing endonucleases can be combined with Cas9 to create a dual-cutting targeted nuclease. TevCas9 is a dual endonuclease that includes the nuclease and linker domains of the I-TevI GIY-YIG homing endonuclease and Cas9, which allows for the specificity and programmability of the RNA-guided CRISPR/Cas9 system to be harnessed for cutting target DNA twice, resulting in discrete-length excisions. While the preference of the I-TevI nuclease domain for cleavage sites resembling its cognate motif (5’-CN↑NN↓G-3’) is a key component of TevCas9's targeting and specific activity, it can also limit the diversity of target sequences accessible to editing. To address this limitation, we are investigating the diversity of cleavage motif preferences within the GIY-YIG nuclease domain superfamily to identify domains that can be combined with Cas9 (GIY-Cas9s) and have different preferences compared to the I-TevI nuclease domain. Preliminary activity screening of uncharacterized nuclease domains has identified 17 domains that exhibit strong cleavage activity on a randomized DNA substrate library in vitro, and five of the most active domains have been further investigated and found to have diverse cleavage site preferences. This research aims to expand the versatility of GIY-Cas9 dual endonucleases as genome-editing tools and enable editing of target gene sequences that were previously inaccessible using TevCas9.

Sheida Stephens

University of Toronto

The Standardization of Bacterial Biofilm Quantification by Proxy: An Adaptation of a Common Microplate Assay

Biofilms (or the natural aggregation of cells on process surfaces) is a common nuisance reported in bioprocesses. A process that harnesses this nuisance and turns it into an advantage is a cellular immobilization or biofilm process. A common issue with these types of processes is the lack of methodologies for biofilm quantification. We are proposing a new methodology that expands on a dependable method developed in the 1990s for biofilm quantification using crystal violet and 96 well microplates. 
    Biofilm research in microplates commonly test the effect of different process conditions in each well on biofilm thickness. One of the most reliable methods to measure biofilm thickness uses a quantification method that does not measure cell number or mass but uses stain as a proxy to approximate these. The way this works is that crystal violet, a common and inexpensive stain, is added to empty wells and is taken up by the cells in the biofilm adhered the well walls. The stain in the cells can then be pulled out of the cell walls using ethanol or acetic acid, and the absorbance can be measured spectrophotometrically: a higher absorbance is an indicator of a thicker biofilm inside the microtiter well. A problem with this method is that it is highly dependent on the batch of microplates: the brand, the type of plastic used, the number of wells, etc. There is, to our knowledge, no way to standardize these readings such that they can be compared across different laboratories and different experiments done months apart. The method we are proposing uses Escherichia coli as a means of standardization: by creating a “synthetic biofilm” in a microtube, we can relate the crystal violet absorbance of known cell concentrations to dry cell weight. We validate this method using a hydrolase enzyme known to break down biofilms in K12 strains of E. coli and test it using R. palustris. We expect this method will be used as a supplement to the current microplate assay and will allow researchers increased comparability between experiments and between experimenters.

Saleth Sidharthan

McGill University

Fabricating a perfusable encapsulation device housing beta cells to treat type-1 diabetes

Type-1 diabetes is an autoimmune disorder in which the pancreatic beta cells are destroyed by the host’s inherent immune system which results in insufficient insulin production and subsequent hyperglycemia. So far, life-long insulin injections and constant blood glucose monitoring have been adopted as practicable treatments to mitigate type-1 diabetes. Studies over the past decades have revealed that islet transplantation could be a viable long-term treatment for type-1 diabetes. A major obstacle to this approach is the rejection of the transplanted foreign cells by the recipient’s immune system and it demands lifelong immunosuppression to prevent rejection. This could be overcome by immunoisolating the cells by encapsulating them into polymeric constructs. Although this is a viable strategy, entrapping the cells into the polymer compromises the nutrients and oxygen distribution to the cells which could affect insulin secretion and cause cell death. This limitation could be countered by perfusing the cell-laden polymer constructs with nutrient media. The objective of this project is to fabricate an implantable cell encapsulation device housing beta cells which would allow perfusion while also immunoisolating the cells. The cell-laden device would ensure adequate mass transfer to the cells while immunoprotecting them, thus supporting cell viability and insulin secretion and possibly leading to diabetes reversal. Rodent scale devices have been fabricated and this would serve as a critical tool for demonstrating the preclinical efficacy of the device in diabetic models.

Shirley Wong

University of Waterloo

Mining a compost microbial community for carbon utilization pathways

Over a billion tonnes of food waste is generated annually, most of which ends up in landfills where it massively contributes to the emission of greenhouse gases. To improve global sustainability, this low-value carbon resource can instead be converted into high-value bio-based products. Microbial valorization of food waste has been validated for many applications, from biofuels to bioplastics. However, food waste is a highly variable feedstock. Not all microorganisms currently investigated for biomaterial synthesis can efficiently metabolize the organic constituents of food waste. Pseudomonas alloputida, a highly promising bioplastic producer, can use a wide range of carbon sources, including volatile fatty acids (VFAs) derived from municipal food waste. On the other hand, it is unable to metabolize starch or lactose, which are large constituents of agricultural or dairy industry waste.

To expand the range of substrates for platform organisms such as P. alloputida, exogenous carbon utilization pathways should be identified and integrated. Environmental microbial communities are rich, untapped reservoirs of such genes. Here, we present a preliminary community analysis of a compost microbiome enriched for utilization of carbon sources present in industrial and commercial food waste: starch, lactose, and VFAs. Eventually, functional metagenomic screening of this community will aid in the discovery of new or improved carbon utilization genes. Additionally, we further characterize the ability of engineered P. alloputida to use VFAs in bioplastic production, demonstrating its use in food waste valorization.

Alexa White

Western University

Characterization of the novel TevCas12a dual endonuclease

CRISPR has revolutionized gene editing with its ability to create precise double strand breaks. However, these breaks can be easily repaired by the organism and do not remove sections of DNA. We created a novel dual endonuclease, TevCas12a, to overcome these problems. TevCas12a is a fusion of Acidaminococcus sp Cas12a and the nuclease and linker domain of the I-TevI homing endonuclease. They both produce non-compatible breaks for easy DNA insertion and deletion. However, when tested in vitro, instead of creating a single DSB, we saw a unique property whereby TevCas12a was able to rapidly degrade the entire DNA substrate creating distinct cleavage products. This in vitro inexact cleavage property can quickly degrade both single and double strand, linear and genomic DNA! This phenomenon is seen both with a guide RNA that targets the substrate and a non-target guide. This unique property is only seen when both I-TevI and Cas12a are active. Mutations were made to catalytically inactivate the different nuclease domains, eliminating the inexact total degradation. The degradation was not seen with active I-TevI, inactive Cas12a, nor with inactive I-TevI, active Cas12a, thus, indicating that both I-TevI and Cas12a are required for this property to be seen. Based on preliminary sequencing data, most of the cuts are in the preferred cleavage motif of I-TevI, suggesting that I-TevI play the predominant role in this unique feature. TevCas12a is a guide RNA dependent (but not specific) dual endonuclease capable of cleaving at specific locations but ultimately swiftly degrading all DNA in vitro. This has potential applications for targeting antibiotic resistant bacteria in the microbiome or could have a future in oncology to destroy the DNA of tumors since TevCas12a is able to quickly destroy all types of DNA.

Robert Chen

McGill University

Optimizing the production of alginate microcapsules using microchannel emulsification

Cell encapsulation is a promising method for cultivating cells and transplanting allogeneic cells into hosts for therapeutic purposes. However, there are several challenges to overcome, including limited cell survival, immune response, mass transfer limitation, variability, scalability, and high costs. Although encapsulation shows great potential, scaling up the process remains difficult.
This project focuses on producing consistent alginate microbeads using microchannel emulsification and internal gelation and seeks to improve the design of the current device to increase cell viability and decrease production variability. To achieve this, the project aims to add a continuous flow of acidified oil for consistent bead gelation and develop a modular reaction column to adjust the residence time of the beads in acidified oil. Additionally, the project proposes the addition of a collection reservoir with a buffer to catch the beads and maintain a constant pH.
The proposed improvements in the device design aim to address the challenges associated with cell encapsulation and improve the consistency of bead production. The addition of a continuous flow of acidified oil and a modular reaction column will enable precise control of the gelation process, while the collection reservoir with a buffer will help to maintain a constant pH and enhance cell viability.

Fareed Abuzaid

University of Toronto

Understanding the Impact of Reactor Design and Operation on Biomass Productivity of Algal Biofilm Photobioreactors

Microalgae bioprocessing can benefit the circular bioeconomy by utilizing solar energy, carbon dioxide, and waste to produce valuable products. Current algae production relies on suspended systems which suffer from low biomass densities, making harvest and dewatering an energy-intense process. Algal biofilms have the potential to overcome some of the limitations associated with the traditional suspended algae cultivation methods due to their high biomass concentration and surface attachment, which leads to easier recovery and downstream processing. Although promising, there is still a gap in algal biofilm systems growth models, specifically when considering the relationship between the biofilms and suspension inside algal biofilm photobioreactors. This work aims to explore the interaction between algal biofilms and suspension to help characterize algal growth in continuous algal biofilm photobioreactors. This project will determine the impact of various engineering variables on the growth and productivity of biofilms and suspension in a continuous parallel plate air-lift reactor. Our work will focus on understanding how engineering variables such as hydraulic retention time (HRT) and biofilm surface-area impact biomass productivity in biofilm reactor systems. The results from these studies will aid in developing biofilm photobioreactor models, incorporating both algal biofilms and suspension productivities, that will predict optimal HRT and biofilm surface-area operating conditions.

Zitong Xu

University of Ottawa

Alkaline-modified microporous PVDF membranes for whey and casein protein separation

In this research, the performance of alkaline-treated membranes at different treatment temperatures, 20℃, 30℃, 40℃, 50℃ and 60℃ are investigated. The performance of membranes is carried out by dead-end filtration of two water-soluble polymers, i.e., PAA and PEO, and two proteins, i.e., casein proteins and whey proteins. The chemical composition of modified membranes is analyzed by FTIR. The surface properties of membranes are investigated by the SEM, the contact angle measurement, and the surface Zeta potential measurement. The rejection of PAA, PEO, casein protein, and whey protein of PVDF membranes were 12.6%, 24.3%, 76.4%, and 69.4%, respectively. The filtration results show that the membrane treated at 60℃ has the best performance. The flux of PVDF membranes is 36.1 LMH for PAA, 20.5 for PEO, 17.3 LMH for casein protein, and 17.7 LMH for whey protein. After 60℃ alkaline treatments, the rejection of PAA, PEO, casein protein, and whey protein increased to 33.2%, 46.9%, 88.5%, and 80.4% respectively. Whereas the flux of PAA and PEO filtration were 29.5 and 20.1 LMH, respectively, which were only 18.3% and 2.0% lower than that of the PVDF pristine membrane. For casein and whey protein filtration, the fluxes are 17.5 and 18.4 LMH, respectively, which were even higher than that of the pristine PVDF membranes.

Matthew Edghill

University of Toronto

Adaptive Laboratory Evolution of E. coli for Assimilation of the Recyclable Feedstock Ethylene Glycol

Bioprocess engineering relies on cellulosic-based feedstocks derived from agriculture. However, feedstock production utilizes acid hydrolysis and competes with crop consumption. Thus, to facilitate a sustainable future for the bio-production of chemicals, alternatives must be considered. Ethylene glycol (EG) has been proposed as an alternative feedstock as it can be bio-upcycled from PET plastic. Prior work facilitated the engineering of an E. coli strain which could consume EG. My research aims to optimize this strain’s EG assimilation, to facilitate EG’s usage as a sustainable, yet economically viable feedstock within bio-production.

To do so, I utilized adaptive laboratory evolution (ALE) to generate a strain with highly efficient EG metabolism. The growth kinetics of this evolved strain were assayed and compared to its ancestor, revealing it to have a 58% higher growth rate when EG is the sole carbon source. Interestingly, when the assay was repeated using glucose as the carbon source, this strain grew slower than its ancestor, suggesting an evolutionary trade-off in native metabolism for EG metabolism.

Plasmid sequencing revealed no mutations in the heterologous enzymes directly involved in EG metabolism, suggesting adaptations within native metabolism. Furthermore, HPLC was used to monitor EG consumption in cultures. Analysis indicated that 100% of EG was consumed by the evolved strain within 2 days, while the ancestral strain consumed 43.5% in 2 days. The greater growth and consumption by the evolved strain, illustrates its superior EG assimilation. With further optimization, it may be utilized within industry, promoting the integration of sustainable feedstocks like EG.

Aranksha Thakor

University of Waterloo

Isolating Functional Carbon Source Utilization Pathways for Pseudomonas alloputida from Complex Microbial Communities

Functional metagenomics has the power to access novel pathways and genes from the genetic makeup of complex microbial communities, particularly useful for ones that cannot be cultivated in traditional lab settings. The ability to phenotypically screen metagenomic libraries for carbon source metabolism pathways is a valuable resource for improved bacterial genome engineering. The isolation of carbon source utilization genes from soil-derived metagenomic libraries can be achieved by phenotypic complementation of metagenomic libraries in the strain of interest. Pseudomonas alloputida is a suitable platform for metabolic engineering to produce added value products or serve as a tool for bioremediation. P. alloputida is also a natural producer of Poly-hydroxyalkanoates (PHA), a carbon storage molecule naturally produced by the cell that has the potential to replace petroleum-based plastics. The use of Pseudomonas as a chassis for PHA production is of value, but the native metabolism of P. alloputida does not allow the strain to grow on simple waste carbon sources such as galactose, maltose, and starch. We present here the introduction of novel pathways for galactose and maltose utilization in Pseudomonas alloputida KT2440. We also present here the monomer composition of PHA derived from using maltose as a sole carbon source using various maltose utilization clones. Future work will involve inserting the genes and pathways of interest into the genome of KT2440. 

Huan Wu

University of Ottawa

Potential of green alga C. vulgaris under for bioremediation of water contaminated by Pb(II) near-threshold concentration  

The main objective of this study was to determine the ability of Chlorella vulgaris to adsorb Pb(II) and assess the effect of the heavy metal ion on the growth of C. vulgaris under near-threshold Pb(II) concentration. Lead (Pb) is one of the most common heavy metal contaminating aquatic environment, which may exist in contaminated waterbodies at a concentration that is near the drinking water standard (10 ppb), i.e., the near-threshold concentration. In this study, the effects of near threshold concentration of Pb(II) exposure on the growth of C. vulgaris. Furthermore, the adsorption of Pb(II) on C. vulgaris cells were investigated with either short contact time without cell growth or during algal cultivation with cell growth. The findings of the study indicate that C. vulgaris is able to grow normally and can adsorb Pb(II) efficiently under in Pb(II) concentration range of 10-100 ppb. Furthermore, the FTIR results suggest that the adsorption of Pb(II) onto C. vulgaris cells occurs via binding with proteoglycan molecules on the cell wall. The adsorption process is influenced by the adsorption pH. These findings have important implications for the use of live C. vulgaris in the bioremediation of Pb(II) contaminated water bodies.

William Chau

University of Toronto

Sustainable Chemical Bioproduction Using Ethylene Glycol Utilizing Escherichia coli

High-volume chemicals see extremely broad ranges of usage across multiple industries, but they are also the major contributors from the chemical sector to climate change. High-volume chemicals are dependent on fossil fuels for carbon feedstock and energy, which releases GHG emissions, and their production processes and reaction intermediates often pose serious danger to both worker safety and the environment. The overall aim of my research is to engineer an ethylene glycol (EG) utilizing strain of Escherichia coli (E. coli) to produce a sustainable alternative to high-volume small chemical production to mitigate the aforementioned emissions and risks. The EG utilizing E. coli strain was previously engineered to be able to use EG as its sole carbon source, and EG was chosen as the substrate because of its orthogonality and the fact that it can be produced from electrochemical reduction of carbon dioxide[1] and from microbial breakdown of pE[2], pEG[3], and pET[4] plastics. An aldo-keto reductase and a transaminase were chosen due to their broad substrate specificity and screened for activity on small substrates. The results concluded that these two enzymes can be used to sustainably produce short-chain aldehydes and amines.

Zahra Negahban

University of Waterloo

A HYBRID  dFBA MODEL FOR MEDIA OPTIMIZATION OF MAMMALIAN  CHO CELL CULTURE 

Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's workhorse for producing therapeutic proteins. This is due to their extracellular secretion capabilities, humanlike posttranslational modifications, and high suspension density and titer. As a result, they are frequently used to produce more than 80 % of all monoclonal antibodies, the largest class of commercial recombinant proteins. The mAb market is enjoying a healthy pipeline and is expected to grow at a faster pace , reaching a valuation of US $300 billion by 2025.  Due to the high variability in cell lines, media needs to be optimized for best productivity in each process. Process modeling can help us predict the best possible media while reducing expensive experiments.

Chuchi Chen

University of Waterloo

Recombinant Protein Production in Microalgae

Protein production is a critical area of research in synthetic biology, and microalgae has emerged as a promising alternative expression system due to its eukaryotic cellular mechanism, low-cost culturing conditions, and eco-friendly nature. The lack of molecular tools for genome editing is hampering efforts to use algal biotechnology more extensively. In this study, the use of recombinant virus, the agrobacterium mediate transformation system, and transient gene expression are evaluated in Chlorella vulgaris and Chlorella variabilis. A recombinant PBCV-1 system is being developed to investigate its potential of being a novel platform for protein production in Chlorella variabilis. Chlorella vulgaris has also been stably transformed using Agrobacterium for mGFP production. Furthermore, the growth media and conditions are being optimized for both wild-type Chlorella and transgenic Chlorella to increase recombinant protein production yield. Successful development and optimization of the systems could have a significant impact on the industry, revolutionizing the production of recombinant proteins to be more affordable and versatile.