Maryam Arefmanesh

Chemo Enzymatic Modification of Lignin for Resin Applications

Maryam is currently working towards her PhD under the supervision of Professor Emma Master.

Chemo enzymatic modification of lignin for resin applications

Lignin is the most abundant natural aromatic polymer and is obtained as a byproduct of biofuel and pulp and paper industries. Lignin has been viewed as a low value byproduct of these industries and its primary usage is to be burned for local power generation. Increasingly, however, there is interest to develop a broader range of products from lignin that also provide environmentally friendly and cost effective alternatives to petroleum derived compounds.
Lignin structure and chemistry differs depending on the botanical source and extraction method. Therefore, full characterization of lignin is required before determining which lignin is better for which application.
Lignin has both phenolic and hydroxyl groups in its structure and depending on the ratio of these groups the properties of lignin can be different. A major limiting factor to broader use of lignin is inaccessibility of hydroxyl functional groups embedded in the lignin structure. Accordingly, lignin modification is necessary to increase the number of hydroxyl groups or to make them more accessible. For example, chemo-enzymatic modification of lignin can generate good candidates for applications in polyurethane and phenolic resins. In addition, by using enzymatic pathways we can link the modified lignin with bifunctional oligosaccharides to make 100% bio based resins.

Amir Arellano

Structural and Molecular Insights of the Evolution of Parasitic Receptors of Striga hermonthica

Amir is a PhD student from the Cell and Systems Biology Department, currently working in the Savchenko Lab where he performs X-ray Crystallography and large scale protein purification for enzymatic analyses of parasitic receptors.

Studies of strigolactone receptors.

Striga spp. (Witchweed) is an obligate parasitic plant that attaches to the host roots to deplete them of nutrients. In Africa, the most destructive Striga species S. hermonthica parasitizes the major food crops sorghum, rice, millet and maize with yield losses ranging from 30 -100%. Presently, the most common method of Striga control is hand weeding, which is time-consuming, labour-intensive and disproportionately carried out by women and children.
There are a number of potential solutions to the Striga problem, and breeding approaches are presently the most economically sought method of parasitic weed control. There are examples of improved genetic resistance to Striga in rice, sorghum, cowpea, and maize, however, complete resistance has not been attained (Afr J Plant Sci 8, 492, 2014). This is most likely due to the high genetic variability that is a consequence of the obligate outbreeding mating system of Striga hermonthica. This genetic variation not only results in resistance breakdown but almost certainly determines local host range and virulence. Thus, crop resistance across different parasitic plant populations will be difficult to achieve (Heredity 108, 96, 2012). This genetic complexity indicates innovative approaches will be required to eradicate Striga that exploit weaknesses in its lifecycle and an understanding of how this parasite evolves and adapts. However, such approaches also require a mechanistic molecular understanding of the Striga lifecycle.
The overarching goal of this project is a complete understanding of Striga hermonthica SL receptors from the fundamental mechanism of perception to their evolution and population biology. Longer term, the information will be exploited to eradicate Striga from the Sub-Sahara Africa.
Genetic variation most certainly contributes to SL perception and sensitivity and understanding how this mechanistically occurs is paramount to perturbing the system in ways in which the parasite will not be able to adapt. Biochemical and structural analysis of the Arabidopsis and the Striga receptors gives us some understanding of which amino acids may be important in determining SL receptors perception (Science, 349, 864 (2015), Science, 350, 203, (2015)). Comparisons of the low sensitive Arabidopsis SL receptor (AtHTL) and the highly sensitive Striga ShHTL7 receptor binding pockets identifies 8-10 key amino acids that most likely contribute to SL specificity and responsiveness.

Sofia Bonilla

Maximizing value in forest products and other processes

Sofia works under the supervision of Grant Allen to identify native enzymes present in pulp and paper biosolids, and to develop an enzyme-based process to improve sludge dewatering.

Proteins for enhancing biosludge dewaterability

Sludge processing and disposal is a challenge due to the high moisture content of biosludge. Chemical
conditioners are commonly used to enhance sludge dewaterability, facilitating processing and reducing its volume and thus, disposal costs. However, the chemical conditioners used are petroleum-derived and can be toxic to aquatic systems. Therefore, there is interest in finding more environmentally-friendly conditioners to improve biosludge dewaterability. There have been limited studies on the potential use of enzymes for enhancing the dewaterability of sludge but little is know about the mechanisms for such enhancement. The objective of this project is to better understand how enzymes affect sludge and
how these changes affect its dewaterability.

After screening several enzymes, we found that lysozyme improved biosludge dewatering properties. Surprisingly, this effect had nothing to do with the catalytic activity of lysozyme. The positive charge on the surface of lysozyme neutralizes the negative charge of sludge particles leading to larger particles and better dewatering properties. This finding led us to investigate other proteins with a net positive charge and asses their potential for improving sludge dewatering. Also importantly, these proteins could improve other liquid-solid separations. We found that another protein, protamine, can improve sludge dewaterability. Lysozyme and protamine can also improve other liquid-solid separations such algal cultures, kaolin and powdered activated carbon suspensions.

Cationic proteins are biodegradable and they can potentially be extracted from waste. Therefore, they offer clear advantages over current chemical or energy-intensive treatments. Furthermore, we have now a better understanding of how to use proteins and enzymes for addressing sludge-related challenges and while doing so, we have developed methodologies to properly evaluate enzymatic treatment of biomass.

Xu (Charlie) Chen

Correlative Microscopy in Biological Samples

Charlie’s research involves electron microscopy sample preparation protocol developing and correlative microscopy study on mixed microbial culture. A novel biological electron microscopy sample preparation protocol by using Ionic liquid to maintain the “wet” sample in its nature state has been developed. In the future, the use of fluoresces in situ hybridization (FISH) can help to identify the different species and correlate to SEM morphology so that make it available to study the interaction between the different species in the mixed culture.

Correlative Microscopy in Biological Samples

Biological samples such as biosludge and microbial culture contain a large amount of different components. It is a mixture of live and dead microorganisms, extracellular polymeric substances (EPS), fibers and inorganic matters. The efficiency of relative treatments such as sludge dewatering and contaminates degradation highly depend on the contributions of the different compositions. The size of those components various from tens of nanometers to hundreds of micrometers. The visualization study of these components need be carried by optical and electron microscope. The correlative microscopy combines two techniques enabling the use both optical and electronic analysis techniques on the same sample. For example, optical techniques, such as fluorescence staining, fluorescence in situ hybridization (FISH) could in principle be used with SEM and energy dispersive spectroscopy (EDS). However, current conventional sample preparation method cannot suit to both microscopy. Also, the involved dehydration and drying processes for the biological EM sample preparation make it enable to present the nature state of sample.

In order to solve these problem, I use a novel correlative sample preparation method ionic liquid exchanging. The ionic liquid is a conductive \\\\\\\\\\\\\\liquid salt\\\\\\\\\\\\\\ at ambient temperature and also has very low vapor pressure which makes it very stable in the vacuum EM chamber. These properties enable a wet sample can be observed in its nature state and also makes it very easy to switch between different microscopy. Currently, an appropriate protocol has been developed and the further study of mixed culture species identification and interaction will be carried on.

Yee Kei (Kiki) Chan

Nutrient Enhancement Using Moringa oleifera

Kiki works under the supervision of Professors Levente Diosady and Yu-Ling Cheng. Her research focuses on identifying potential uses of Moringa oleifera, a plant abundantly found in the tropical and subtropical regions, as a nutrient enhancement or supplement.

Nutrients Extraction and Fortification Using Moringa oleifera

Nutrient deficiencies are widespread in populations from both developed and developing nations and have destructive economic impacts as it significantly reduces productivity across the population. Food fortification is used as a method to alleviate nutrient deficiencies. Nutrients are extracted from a nutrient source and added to staple foods to increase the nutrient content of such foods. Research efforts are ongoing in determining suitable fortificants for specific nutrients and food vehicles. Ideally, the fortified product must have the appropriate nutrient dosage, a reliable cost-efficient production process (extraction and fortification), adequate stability, and satisfactory user acceptability based on its colour, taste and smell.

Moringa oleifera is a plant indigenous to tropical and subtropical regions and is densely packed with nutrients such as iron, calcium and magnesium. It appears to be a promising nutrient source as it grows abundantly and is used in local culinary dishes in its native regions. Recent studies have attempted to add M. oleifera to bread, cereal gruel, biscuits, yogurts and cheese using powder from ground dried leaves at varying concentrations. At the desired nutrient concentrations, the chief complaint of these fortified products is that they are not as appealing as their unfortified counterpart based on sensory characteristics (i.e. colour, taste, texture and smell). Therefore, there exists an opportunity to extract only the effective nutrient compounds from M. oleifera and use them as a fortificant in order to maintain sensory qualities of the fortified foods.

Aqueous and oil extraction techniques for extracting nutrients from plants are well developed and will likely be used in this project. The ideal commercialized production process must be cost effective for developing countries and have relatively simple operations that may be managed by local food producers.

This research will explore the means to reduce common nutrient deficiencies by utilizing a readily available plant, M. oleifera, as a source of food fortificants.

Samantha Cheung

Manipulation of algal biofilm communities through attachment surface and physiochemical conditions

Microalgae can be used to make a variety of materials including biofiuels. With fast growth rates and minimal growth requirements, they are a promising alternative to fossil fuels. In order to lower processing costs of algae biofuels, algae can be grown as biofilms in photobioreactors. My work aims to maximize product yield from algal biofilms by manipulating physiochemical conditions and attachment surface in order to select for species that are abundant in the product of interest. Maximizing yields and lowering production costs is a necessary step in order for algal biofuels to become commercially viable in the future.

Attachment surface as a means of manipulating algal biofilm species composition

Microalgae have become a popular area of research for the production of biofuels and bioproducts because of their potential to compete with conventional sources of fuels and materials in a low-cost and environmentally friendly way. In order to be cost competitive with inexpensive products such as fossil fuels, inexpensive feedstocks such as wastewater and flue gas must be utilized in algal systems. Growing microalgae as a biofilm can utilize these inexpensive feedstocks while also reducing the cost of dewatering when compared to planktonic systems. However, non-sterile biofilms contain a variety of different organisms, some of which, may not produce the desired products. In order for algal biofilms to be economically competitive, there needs to be a better understanding of the communities and how to control them. The objective of my research is to determine whether species in algal biofilms can be manipulated by their attachment surface. Molecular biology techniques will be used to quantify biofilm species. This will contribute to the development of genetic techniques and fundamental knowledge in this emerging area of research. Additionally, this will be the first attempt at manipulating biofilm community composition through material. This research has incredible potential for the optimization of algal biofilm product yields where algal species with desired products can be selectively grown over other biofilm organisms. Algal biofilm species selection and control is a necessary step for algal biofuels to become an economically viable source of biofuels and bioproducts.

Zahra Choolaei

Enzymatic treatment of wastewater

Zahra is working on inding new enzymes that improve the digestibility of pulp and paper wastewater. This is done by purifying new microbial hydrolytic enzymes and screening them for improved digestibility of biosludge. She works under the supervision of Alexander Yakunin and Elizabeth Edwards.

Enzymatic treatment of pulp and paper mill biosludge

The amount of biosludge generated by wastewater treatment facilities is steadily increasing. In addition, its disposal by landfilling or incineration is very costly and hazardous for the environment. Therefore, it is important to find environmentally friendly and cost effective ways to decrease the ultimate amount of biosludge solids sent for disposal.
Anaerobic digestion can decrease wastewater treatment costs and reduce the amount of produced biosludge to half. It also leads to the production of biogas, which is an alternative energy source to fossil fuels. However, the rate-limiting step of anaerobic digestion is known to be the hydrolysis of organic particulate substances.
Although anaerobic digestion has been widely used for the treatment of wastewater in other industries, it is just recently becoming popular for the treatment of pulp & paper mill effluents. This is due to the presence of some components that slow down the treatment process, such as lignin that is known to be not biodegradable under anaerobic conditions.
To address this issue, pretreatments could be applied to biosludge prior to anaerobic digestion to enhance its digestibility. Our focus in this study is on the enzymatic pretreatment of pulp and paper mill biosludge in advance of anaerobic digestion. Hence, by revealing the problematic constituents of pulp and paper mill biosludge and applying appropriate enzymes on it, we are attempting to improve its anaerobic digestibility.

Kevin Correia

Modeling and analysis of biological systems

Kevin works under the supervision of Radhakrishnan Mahadevan in the area of metabolic modeling of Pichia stipitis. The goal of his research is to maximize the production of a high value product using optimization.

Evolution of metabolism in yeasts

My research seeks to understand the molecular basis of yeast metabolism using bioinformatics, metabolic modeling, comparative genetics with CRISPR-Cas9, and fermentation.

Elisa D’Arcangelo

Invasion across the cancer – stroma tissue boundary

Elisa has developed a tool for visualizing how cancer cells mix with surrounding tissue cells in vitro in real time. This approach is used to both, understand the dynamics of invasive mixing behaviours and as a platform for performing targeted compound screens. Elisa works under the supervision of Alison McGuigan.

constructing compartmentalized cancer tissues to mimic the tumor edge

Elisa has developed a tool for visualizing how cancer cells mix with surrounding tissue cells in vitro in real time. This approach is used to both, understand the dynamics of invasive mixing behaviours and as a platform for performing targeted compound screens. Elisa works under the supervision of Alison McGuigan.

Teresa Dean

Validation of a HNSCC/CAF Co-culture Model of Invasion in the TRACER Platform

Head and neck cancer (HNSCC) is the sixth most common malignancy in the world, and has a high propensity for local invasion and metastasis. The assessment of drug effectiveness in cancer often takes place in two-dimensional cell culture models, due to ease of analysis and cost efficiency. However, 2D cultures do not recapitulate the heterogenous tumor microenvironment or three dimensional architecture of tumors, both of which have been demonstrated to have an effect on tumor cell behaviour and phenotype. In order to recapitulate these characteristics in a simple in vitro model, our lab has developed a novel three dimensional cell culture platform (TRACER). Tumor complexity will be recreated in this model by incorporating both an extracellular matrix component and a cancer stromal cell type, cancer associated fibroblasts (CAFs). CAFs as well as extracellular matrix stiffness have been implicated in the progression to an invasive phenotype in tumour cells; We will investigate the interplay of these factors and how they affect the invasiveness of HNSCC cells in our 3D tumour model. Furthermore, we will investigate the ability of an inhibitory compound to abrogate the invasive ability of HNSCC in this model.

The effect of cancer associated fibroblasts on tumour cells

In my research project, I use a novel 3D culture platform to investigate the ability of a stromal cell type to promote tumour progression. The assessment of drug effectiveness in cancer often takes place in 2D cell culture models, due to ease of analysis and cost efficiency. However, 2D cultures do not recapitulate the heterogenous tumor microenvironment or 3D architecture of tumours, both of which have an effect on tumour cell behavior. In order to recapitulate these characteristics in a simple in vitro model, our lab has developed a novel 3D cell culture platform. Tumor complexity is recreated in this model by incorporating both an extracellular matrix (ECM) component and a cancer stromal cell type (cancer associated fibroblasts). Both the presence of cancer associated fibroblasts (CAFs) as well as an increasing ECM stiffness have been implicated in the progression to an invasive phenotype in tumour cells; My research investigates the interplay of these factors.

Christian Euler

Dynamic Control of Metabolism Using Post-Translational Modifications

Protein-Level Control of Metabolism

I am primarily interested in examining native biological design principles for metabolic regulation at the protein level (i.e. allosteric regulation) to develop optimization tools and techniques for metabolic engineering. Ultimately, my aim is to build fast, continuous control systems for the rational redirection of metabolic flux toward valuable products in microbial cell factories. I do this in two ways. On the fundamental side, I use bioinformatic tools to construct and analyze protein-level regulatory networks to elucidate the design rules for metabolic control. On the applied side, I build model allosteric proteins and characterize their dynamics in metabolic systems relative to analogous transcriptional control systems.

Ph.D. Student

Julie-Anne Gandier

Bioengineering for a sustainable forestry industry

Julie-Anne’s work focuses on the engineering and characterization of fungal hydrophobins for surface modification. Hydrophobins are a class of highly surface active proteins produced by some filamentous fungi, whose prominent characteristic is their ability to self-assemble at interfaces and form a monolayer. Julie-Anne intends to harness this property for the immobilization of enzymes into organized films, with the goal of functionalizing surfaces of materials such as wood fibers. She works under the supervision of Emma Master and is the recipient of a Canada Graduate Scholarship from NSERC.

The spectrum-wide characterization of hydrophobin proteins: interface-active proteins with industrial potential

Hydrophobins are secreted non-catalytic fungal proteins that act at interfaces. Their functions range from self-assembling to form a fungal raincoat on the fruiting body, to recruiting enzyme activities to surfaces. Such properties have been harnessed in a wide-range of applications from the stabilization of nanoparticles for drug-delivery usages, to coating surfaces for sensing applications and the stabilization of food foams.

My doctoral work has refined the classification of hydrophobins, thus contributing to more predictable structure-function relationships to inform sequence selections for applications purposes. Hydrophobins have been traditionally subdivided into two classes (I and II); however, based on the alignment of hydrophobin sequences predicted from over 200 fungal genomes, we have identified a new subdivision of this protein family: a high-identity group of Class I basidiomycota sequences. To experimentally validate this subdivision, the structure of one of its members, the Schizophyllum commune hydrophobin SC16 (hyd1), was solved by solution-NMR. While sharing the core hydrophobin structure, elements believed to be necessary to amyloid-fibre formation, a characteristic of Class I proteins, was absent. We demonstrated, however, that SC16 is capable of forming such structures. This finding allows future studies to address a more general mechanism for assembly.

To further experimentally characterize hydrophobin properties, I tailored techniques such as flow field-flow fractionation to their particularities. While techniques such as small angle x-ray scattering can be applied to determine the average dimensions of assemblies in solution, an upstream separation method is essential to identify multiple oligomeric states and determine their population distribution. Traditionally, size exclusion chromatography is used, however, hydrophobins interact with the separating matrix. Flow field-flow fractionation offers a matrix-free approach to the separation of different oligomeric states that may be present in solution as it uses a perpendicular hydrodynamic force to separate molecules based on geometry and size.

CAPTION OF RESEARCH DIAGRAM:Hydrophobins stabilize foams as can be seen in the superior phase of the solution in the cuvette. The yellow colour in the solution is the dye Thioflavin T used to detect amyloid-fibres. These fibres are an assembled protein state adopted by class I hydrophobins at interfaces.

Adriana Gaona Gomez

Modeling and analysis of bioreactors for enzymatic hydrolysis

Adriana is investigating the fluid behaviour in high-solids lignocellulosic enzymatic hydrolysis to increase the conversion of fermentable sugars by combining an experimental and a computational fluid dynamics component. The optimization of biomass conversion at high solids can reduce costs and improve the production of biofuels and bioproducts. Adriana is working under the supervision of Dr. Brad Saville and Yuri Lawryshyn.

Exploring the Fluid Behaviour in Hhigh-solids Lignocellulosic Enzymatic

Ethanol fuel obtained from cellulosic biomass feedstocks has the potential to reduce dependence on fossil fuel. An enzymatic hydrolysis process transforms the biomass into liquid slurry composed of five-carbon and six-carbon sugars, which is then fermented to obtain ethanol fuel. To increase the conversion of fermentable sugars, a high biomass loading in the enzymatic hydrolysis process is required. However, as the solids concentration is increased, the viscosity of the slurry increases, yielding inadequate mixing in the process. As a consequence, an industrial scale-up of the process operating at high-solids loading is not yet economically viable.

There has been increased interest to optimize the enzymatic hydrolysis process by studying the rheological properties of various cellulosic biomass feedstocks and conducting experiments in different bioreactor scales to analyze the slurry flow based on empirical correlations. In spite of these efforts, the rheological results are specific to the characteristics of the system
studied and are difficult to be implemented to different systems.

This limitation can be overcome by employing computational fluid dynamics, in which the biomass slurry flow can be studied by tuning solids load, and bioreactor dimensions and parameters, such as type and number of impellers, as well as rotational speed. I plan to develop a computational fluid dynamics model combined with experimental work to understand the biomass fluid behaviour at high-solids loadings.

Ultimately, we expect that the results of this research will shed light on the relationship between rheological behaviour of the lignocellulosic slurry and the reactor design parameters, in order to promote mixing and optimize the hydrolysis process.

M.Eng. Student

Nishil Gosalia

Nigel Guilford

Anaerobic treatment of waste and contaminants

Nigel brings a wealth of experience in the commercial deployment of environmental technology to BioZone, especially in the area of waste management. He has joined Elizabeth Edwards’ research group to work on the optimization of a novel two-stage process for the anaerobic digestion of organic waste from residential, commercial and industrial sources. The principal objectives are to accelerate the reaction rate, maximize biogas production, assess performance with different feedstocks, stabilize by-products and increase versatility.

Anaerobic Digestion of Organic Wastes of Variable Composition

Problem Statement
More than 13 million tonnes of solid waste is landfilled every year in Canada, where it decomposes anaerobically giving rise to 20 Mt of CO2eq./year of GHG emissions. In theory most of this organic waste could be anaerobically digested under controlled conditions thereby obviating the emissions problem. In practice, using conventional anaerobic digestion technology, this is prohibitively expensive, largely because of the complex and costly pre-processing required. My research seeks to address this problem.
Knowledge Gaps
The anaerobic digestion of food waste, and the organic fraction of municipal solid waste, have been extensively studied. But this represents only 35% of all solid waste; the remaining 65%, from commercial industrial sources, has been largely ignored. The co-digestion of food waste with other waste products like municipal sludges and crop residues has also been studied but the co-digestion of food waste with hard-to-digest paper products (cardboard, boxboard, newsprint, office paper) has barely been touched.
Addressing the Questions
A novel system for anaerobically digesting the organic component of commercial/industrial solid waste, one which involves very little pre-treatment, has been designed and constructed on a lab scale; it has been operating continuously for 500 days to demonstrate the versatility and robustness of the technology under a range of operating conditions. Biogas production and system temperatures are measure continuously; feedstock and digestate are analysed on a regular basis; operating conditions within the digester are analysed four times a week. A powerful synergistic effect of food waste on the digestion of lingo-cellulosic wastes has been found, possibly resulting from enzymatic activity induced by the presence of food waste.
Commercial Prospects
Because of the simplicity of the design, the results obtained in the laboratory, and an initial financial analysis, the technology has good commercial prospects. A demonstration scale version is planned as part of a large commercial waste processing facility planned for Ottawa. The single most important variable is the solids retention time in the reactor.
Role of my Research
The most important contributions of my research are proof of concept, demonstration of robustness and stability under conditions of changing feed stock and identification of key variables to maintain stable operations.

Mahbod Hajighasemi

Protein and enzyme production and characterization

Mahbod’s research focuses on enzymatic degradation of bio-plastics including Poly Lactic Acid, Poly Hydroxybutyrate and their co-polymers. Bio-plastics from renewable sources are green alternatives to current petroleum-derived plastics. Under the supervision of Elizabeth Edwards and Alexander Yakunin, Mahbod performs functional screening of different metagenomic libraries as well as several purified proteins to identify new enzymes capable of depolymerizing bio-plastics. Heterologous expression, molecular and biochemical characterization as well as enzyme-polymer interactions, degradation intermediates and, finally, recycling processes are also part of his research.

Enzymatic Depolymerization of Biodegradable Polyesters

In response to the increase in social awareness about environmental issues, biodegradable plastics are getting more popular. Several types of biodegradable polyesters with different physical properties have been emerged to replace traditional petroleum-based polymers. However, there is no efficient recycling strategy in place for most of these plastics. As a solution, weve developed a patent-pending, enzyme-based technology that not only enables efficient degradation of polyesters as a recycling approach, but may also serves as a platform to convert post-consumer waste to different value-added chemicals by genetically engineered bacteria. As opposed to composting process, enzymatic hydrolysis of bioplastics generates carboxylic acids instead of CO2 as the final product.

In our work, over 250 uncharacterized ?/?-hydrolases from sequenced microbial genomes and metagenomic libraries were recombinantly expressed in E. coli and purified using metal (Ni) chelate chromatography. Purified proteins were screened for hydrolytic activity against polylactic acid (PLA), polycaprolactone (PCL), and a model polyethylene terephthalate substrate (3PET). Multiple rounds of screening yielded 40 active polyester-degrading enzymes, 14 of which were biochemically characterized. In parallel, the crystal structures of three polyester hydrolases were determined and the active site residues critical for polyester hydrolysis were identified using structure-based, site-directed mutagenesis. The product analyses revealed that polyesters were completely hydrolyzed to water soluble oligomeric species and eventually to monomers. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

Alternatively, polyester-hydrolyzing enzymes can be used for surface functionalization of bioplastics. Enzymatic hydrolysis of biodegradable plastics creates hydroxyl and carboxyl groups on the surface of polyester material without modifying the properties of the core polymer. The charged groups on the surface contribute to wettability of the polyester and therefore make it more biocompatible for medical and pharmaceutical applications.

Hedieh Hashtroudi

Investigating the Role of Select Fruit Waste Enzymes (Kiwi and Pineapple) on Anaerobic Digestion of Municipal Sludge

Anaerobic Digester (AD) is the efficient way to reduce the municipal sludge and produce biogas (approximately 70% methane and 30% CO2) that can be used to generate energy. However, due to rate limiting hydrolysis process, sludge pre-treatment is commonly applied to enhance the efficiency of the overall process. Conditioning sludge with enzymes, is one of such pre-treatments assist COD solubilisation and improve the overall performance of the AD process.

Hence this research, consisting of a simulated anaerobic digester process, is carried out to evaluate the performance of AD by adding fruit enzyme (Kiwi, pineapple, papaya) from waste, and also assess the yield of biogas production.

Spencer Imbrogno

Spencer works under the supervision of Professor Emma Master.

Chemo-enzymatic Synthesis of Oligosaccharide Building Blocks

Bio-based, value-added products offer the potential of utilizing renewable and waste resources to replace unsustainable fossil fuel-derived chemicals. Currently, the mainly utilized component of plants is cellulose, which only represents 40 55% of this resource. Other useful feedstocks can be extracted from the remaining wood materials and can be modified for use as renewable chemicals to either replace petrochemicals or access new markets due to novel properties. My project aims to develop enzymatic and chemical approaches to utilize these underused renewable wood resources. Specifically I will be focusing on the modification of neutral oligosaccharides obtained from wood in a single step for subsequent conversion into bio-based macromonomers. This process will involve a dual enzyme oxidation in a single step which will result in increased efficiency and possibly allow for new reactor configurations (i.e. through enzyme immobilization) for improved throughput.

Fundamental research will also be performed on the redox enzymes used in this study. Redox potential measurements of the carbohydrate-active enzymes will be made in order to draw connections between substrate specificity and ideal reaction conditions. This approach can also be used to determine the unique characteristics and assess the potential of engineered enzyme mutants.

Parnian Jadidian

Kaustubh Kadam

Mathematical model for an externally tunable bacterial band pass filter


Ph.D. Student

Kart Kanger

Antimicrobial Resistance in Environmental Metagenomes

Kärt is a visiting Ph.D. student from the University of Tartu, Estonia working under the supervision of Professor Elizabeth A. Edwards. She studies the abundance and diversity of antibiotic resistance genes in various environmental and human-influenced metagenomes. Her project in BioZone focuses on the effect of anaerobic digestion on antibiotic resistance genes in solid organic waste.

Antimicrobial Resistance in Environmental Metagenomes

Antimicrobial resistance can be defined as the ability of a microorganism to withstand the effects of an antimicrobial agent such as an antibiotic drug. The extensive anthropogenic use of antimicrobials has triggered the spread and evolution of antimicrobial resistance mechanisms in microbial communities to the extent that the World Health Organization has recognized antimicrobial resistance as one of todays greatest public health risks worldwide.

Most of the research has focused on antimicrobial resistance in medical environments, such as fighting human pathogenic bacteria and treating bacterial infections. Less attention has been paid to studying the environmental reservoir of antimicrobial resistant bacteria. Due to the natural origin of antimicrobial resistance, antimicrobial resistance genes can be detected in various natural environments. Yet, anthropogenic impact can greatly increase the abundance of these genes and trigger the spread of resistance from natural microbial communities to human pathogenic organisms. A better understanding of the environmental resistome is needed to limit the dissemination of antimicrobial resistance.

Next generation whole genome sequencing can provide a wealth of information about complex environmental metagenomes. The aim of my research is to compare the abundance and diversity of antimicrobial resistance genes in various environmental and human-influenced metagenomes. Among others, the samples include the microbial communities of anaerobic digestion plant, moose and beaver digestive systems and anaerobic benzene degrading enrichment cultures.

Characterizing antimicrobial resistance patterns between different environments provides a better understanding of natural resistome and its role in the dissemination of antimicrobial resistance. The broad spectrum identification and quantification of antimicrobial resistance genes from metagenomes can serve as a potential input for antimicrobial risk assessment and development of control strategies worldwide.

Masood Khaksartoroghi

Masood is currently working towards his PhD under the supervision of Professor Radhakrishna Mahadevan.

Taeho Kim

Metabolic engineering

Taeho is co-supervised by Alexander Yakunin and Radhakrishnan Mahadevan and is focused on metabolic engineering, screening and characterization of enzymes for the production of high-value chemicals or biofuels.

Enzymatic approach in metabolic pathway optimization

Development of sustainable energy and chemical generation is a crucial step towards solutions for global challenges including alleviation of climate changes and dependence on fossil fuels. Photosynthetic chemical and biofuel synthesis in cyanobacteria has emerged as a promising biotechnology for sustainable production of value-added compounds due to several advantages for bio-industrial processes, such as CO2 mitigation, simple input requirements, rapid and simple genetics, and carbon-neutral applications. In this study, a target cyanobacterium strain, Synechococcus elongatus PCC 7942, is engineered to produce 1,3-butanediol (1,3BDO), whose biosynthesis in cyanobacteria has never been demonstrated. 1,3BDO is a direct precursor to catalytically synthesize 1,3-butadiene, which is used in manufacture of synthetic rubber and latex. For cyanobacterial synthesis of 1,3BDO, novel pathways are proposed in this study advantageous in application in cyanobacterial system in terms of driving force in metabolism and cofactor requirement. The proposed 1,3BDO pathway utilizes pyruvate as a starting metabolite and consists of three enzymes: pyruvate decarboxylase, aldolase, and aldo-keto reductase. Introduction of the heterologous 1,3BDO pathway genes was conducted with the target cyanobacterium strain. Also, the platform of cyanobacteria engineering is established to be further used in cyanobacteria engineering applications. A novel method of product detection as well as characterization of mutant cyanobacteria strains will be developed and used for successful demonstration of the unprecedented photosynthetic bioconversion of CO2 into 1,3BDO.

Suzana Kraus

Aerobic and anaerobic degradation of a complex mixture of chlorinated compounds at an industry site in Brazil

Suzana works under the supervision of Professor Elizabeth Edwards.

Aerobic and anaerobic microbial degradation

This research is part of a larger research program that aims to identify the best remediation technique at a highly contaminated industrial site in Brazil. Among various techniques that are being studied, this one aims to evaluate natural and enhanced aerobic and anaerobic degradation processes using groundwater and soil samples. This site has over 30 different compound in its plume, but the compounds of interest for this research are mainly dichlorobenzene (DCB), chloroaniline (CA), dichloroaniline (DCA) and dicholoronitrobenze (DCNB).

To do so, soil and groundwater samples were shipped from Brazil to Canada in 2015 in order to start the experiments in the Biozone Lab. Due to this complex mixture of contaminants at the site, 92 different microcosms had to be set up, according to the division below:
For the aerobic ones, the bottles were divided in:
a. Sterile controls: with sodium azide and mercuric chloride;
b. Active controls: no amendments;
c. Vitamin amended: added some vitamins, N and P.

For the anaerobic microcosms, there were set up as follows:
a. Sterile controls: with sodium azide and mercuric chloride;
b. Active controls: no amendments;
c. Donor amendment: ethanol and lactate were added, 100 mg/L each;
d. Sulfate amendment: 2mM sulfate added;
e. Nitrate amendment: 2mM nitrate added.

After that, the microcosms were analyzed in both HPLC and GC to understand how the contaminants were behaving in each of the above-mentioned situations. Also, some qPCR analysis were performed in some of the bottles.

Rachel Kwan

Protein engineering and discovery of carbohydrate oxidases (Class: AA5 and AA7)

Rachel is working with her team to characterize carbohydrate oxidases for polymer modifying applications. Currently, she is demonstrating the potential of in vitro protein synthesis for rapid mining of novel carbohydrate oxidases with improved substrate profiles.
Since completing her BSc in Biochemistry at the University of Alberta, Rachel has kept her interests for enzymology, proteomics, and synthetic biology. Now under supervision of Prof. Emma Master, Rachel is using her knowledge to develop carbohydrate oxidases for biosensors and additives in the food industry and the pulp and paper industry. Through her project, she has analyzed data from HPLC chromatograms, activity assays, and various DNA manipulations.

In vitro synthesis and screening of carbohydrate oxidases

Oligosaccharide oxidases from the Auxiliary Activity family 7 (AA7; have been proposed for varied industrial applications, ranging from alternatives to glucose oxidase (GO) in baking applications, to synthesis of bifunctional macromolecules used as bio-based cross-linking molecules. In order to expand the range of available carbohydrate oxidases with different substrate profiles,, additional AA7 enzymes will be selected from uncharacterized subfamilies identified through phylogenetic analysis of the AA7 sequences. We are optimizing methods of in vitro protein synthesis in an effort to rapidly map the protein phylogeny. Resulting enzymes will be screened for activity through colorimetric detection of H2O2; the reduction potential of synthesized enzymes will also be measured and correlated to substrate preference. This approach will be used to identify clusters within the AA7 phylogeny that should be targeted for recombinant protein production and detailed characterization.

Figure 1. The cell-free in vitro protein synthesis versus in vivo synthesis.

Peter (Hyun Woo) Lee

Characterization of the Microbial Community of an Anaerobic Digester Treating Solid Waste

Peter works on a project that characterizes the microbial community of an sequentially fed anaerobic digester treating solid organic waste (simulated commercial waste) to analyze how microbial communities react to feeding compositions and to optimize system stability and performance.

Characterization of the microbial community inside an anaerobic digester treating solid organic waste

Large portion of Canadas waste end up in landfill. Landfills account for 20% of methane emission in Canada. Methane is a well-known greenhouse gas (GHG). A measure to decrease wastes diverted to landfill is needed. Anaerobic digestion has the ability to treat solid organic waste as well as produce biogas as a potential energy source.
Anaerobic digestion has been researched since the early 1920s. However, no research has been done on a sequentially fed anaerobic digester inoculated with pulp mill sludge, treating solid organic waste. We need a better understanding of the microbial community inside the system and how the microbial community reacts to different feed compositions.
Leachate and digestate samples are collected periodically to monitor the change in microbial community. DNA are extracted for sequencing and quantification through Q-PCR. The data will help us correlate the behaviors of the microbial community to changing feed compositions. Furthermore, predict changes in microbial community to stabilize the anaerobic digestion system and enhance system performance. This research can be applied in the waste industry to improve efficiency of anaerobic digestion systems.

Sofia Lemak

Protein and enzyme production and characterization

The Clustered Short Palindromic Repeats (CRISPR) immunity system utilizes a wide range of effector complexes in order to target and destroy invading nucleic acids. In the Type I systems, all are similar multi-subunit complexes, with varying composition. The CRISPR-associated complex for antiviral defense (Cascade) from the Type I-E system of Escherichia coli has been shown to be a versatile tool for bacteria to protect themselves against invading generic elements during CRISPR immunity.

Type I is the most abundant CRISPR type in all organisms studied and several Type I Cascade complexes have shown versatility in complex composition. While the structure of the E.coli Cascade has been solved, the precise mechanism of action is not completely understood. Working both in the Interference and Adaptation stages of immunity, the role of Cascade has been shown to vary, and the specifics of the interactions between protein, RNA and DNA have not been described. By analyzing the proteins and substrate sequences in their native state we can eliminate unnecessary components and create a more efficient complex.

In order to better understand the specificity and flexibility of the Cascade complex, my project goal is to characterize both the crRNA and protein components of the E.coli Cascade with respect to assembly and function. Elucidating the minimal requirements for each of these components that are still able to efficiently bind target DNA will lead to a clearer understanding of the mechanisms involved as well as the creation of a more flexible system that can be applied to several aspects of biology.

Zheng Lu

Investigating the Enzymatic Effects of Fruit Waste (Kiwi, Pineapple and Papayas) on Sludge Hydrolysis in Anaerobic Digestion

Zheng is a MEng student in Dr. Grant Allen’s lab.

enzyme application on biosludge

Wastes are generated daily from various sources, including agriculture, industry and households. These waste will eventually turn into biosludge with the enrichment of microorganisms. Disposing the biosludge is expensive. The traditional biosludge treatment of land filling or incineration is still considered to be not very effective. Anaerobic digestion was developed as a better alternative for the biosludge treatment as it can reduce the solid content and, at the same time, harvest biogas that is mainly composed of methane as a renewable energy. The overall process of anaerobic digestion consists of disintegration, hydrolysis, acidogenesis, acetogenesis and methanogenesis. However, the retention time for the whole process is very long due to that the process of hydrolysis limits the overall rate of the anaerobic digestion. It is because most of biomass substrates are in the polymeric form, which cannot be readily used by the microorganisms. During hydrolysis, macromolecules are degraded by the hydrolytic enzymes that are released from the microbial cells, which then are fermentable for these organisms. The methods of thermal, mechanical, chemical, enzymatic, sonic biosludge pre-treatment have been developed to show that they can insist the hydrolysis process to various degrees. However, we will focus on using the exogenous enzymes to enhance the reaction rate.

There are many advantages of using external enzymes in the treatment. Enzymes are environmentally friendly, mild, quick, simple to implement and more specific in its action and therefore efficient as a pre-treatment for sludge biomass. Enzymes are capable of acting in the presence of various toxic and recalcitrant substances and under a wide range of environmental conditions, such pH, temperature, and salinity. Enzymes are able to act in a large range of environmental conditions and remain active even if these conditions quickly change. Enzymes themselves are biodegradable proteins can perform the same function as many harsher chemicals, but at neutral pH, moderate temperature, and without production of hazardous waste (Parawira, 2012). Enzymes will not be interfered by microorganisms, predators, and inhibitors of microbial metabolism.

However, the cost of using commercial enzymes is high. To reduce the cost, we are proposing using the enzymes from the fruit waste into the treatment. Enzymes from the fruit waste are cheaper, which has the potential to economically use in large-scale and more readily available enzymes. Reuse of waste would be more environmentally friendly. Fruit wastes contain a variety of enzyme activities capable of numerous catalytic functions for them to be useful in heterogeneous substrates like sludge biomass. Specifically, our research goal is to apply crude extracted enzymes from fruit waste of kiwi, pineapple and papayas, which are known to have proteolytic functions, onto the different kinds of sludges. And then, we will see how that affects hydrolysis and biogas production at the downstream.

Roman Malekzai

Hydrophobin-lignocellulose interactions and impact on enzyme action

Hydrophobin-lignocellulose interactions and impact on enzyme action
As one of the most abundant renewable carbon sources on the planet, lignocellulose offers a logical feedstock for conversion to biofuels. However, the challenge of converting this polymer into soluble sugars remains. My research will explore the use of hydrophobins to increase the efficiency and yield of enzymatic processes by enhancing specific binding and suppressing non-specific binding of enzymes to the polymer surface.

Hydrophobin Interactions with Lignocellulose and Effects on Enzyme Action

Hydrophobins are small, secreted proteins found in filamentous fungi. In solution, they readily assemble at interfaces (liquid-liquid or solid-liquid) and fungi utilise this in nature to lower the surface tension of water, alter the hydrophobicity of the environment, or protect spores from immune detection.

My study concentrates on the so-called class I hydrophobins, which are encoded by Basidiomycete fungi and assemble as stable rodlets at interfaces. My overall goal is to correlate HFB sequence and structure to affinity to the chemistry and structure of main lignocellulose fractions (i.e., cellulose, hemicelluloses, and lignins). Moreover, I will investigate the impact of HFB binding to lignocellulose on the activity of carbohydrate active enzymes (CAZy). It is anticipated that this study will uncover HFB candidates for use as bio-based coatings for cellulose fibre, stabilizers of hemicellulose-derived emulsions, and stabilizers of lignin nanoparticle suspensions. By investigating the impact of HFBs on CAZy activities, this study may also establish HFB tools to promote or deter enzymatic degradation of lignocellulose materials.

Elisse Magnuson

Bioremediation of Benzene

Benzene is a common contaminant of soil and groundwater, and is a highly toxic and persistent carcinogen. My research aims to investigate benzene degradation by bacterial communities under nitrate-reducing conditions by studying the functional characterization of community members and through analysis of metabolite exchange. This will help to identify the key community members and their roles in benzene attenuation. A better understanding of this process is important to optimizing the commercial opportunities for benzene-degrading bacterial culture in hazardous site remediation.

Bioremediation of Benzene

Benzene is a common contaminant of soil and groundwater, and is a highly toxic and persistent carcinogen. As such, remediation of benzene from contaminated sites and groundwater, particularly in or near sources of drinking water, is of great concern for public and environmental health. Benzene degradation under aerobic conditions occurs readily via microbial consumption, and this process is already well characterized. However, groundwater and other environments contaminated with benzene are often anaerobic or become anaerobic as oxygen is depleted rapidly through aerobic degradation. Degradation of benzene by microbial communities under anaerobic conditions is a slow and relatively uncharacterized process. It can occur naturally under different electron acceptor conditions, such as methanogenic, nitrate-reducing, and iron-reducing. My research aims to investigate benzene degradation by bacterial communities under nitrate-reducing conditions by studying the functional characterization of community members and through analysis of metabolite exchange. This will help to identify the key community members and their roles in benzene attenuation. A better understanding of this process is important to optimizing the commercial opportunities for benzene-degrading bacterial culture in hazardous site remediation.

Elisa McGee

Microencapsulation of food ingredients and nutraceuticals

Elisa is part of Levente Diosady’s food engineering group and is working on the fortification of salt with micronutrients (folic acid, iron, iodine).

Fortification of Tea with Iron

The fortification of black tea with iron has the potential to reduce the prevalence of iron deficiency in the developing world. Tea is an ideal vehicle for food fortification because it is centrally processed, is the most consumed beverage globally (aside from water), and is consumed in regular quantities by those of all socioeconomic status in many regions. Unfortunately there is a prominent technical difficulty because polyphenolic compounds present in tea, which are responsible for colour and flavour, form complexes with iron which reduce the bioavailability of both compounds and cause strong colour development. The objective of this study is to develop a fortification method such that these complexes do not form.

A spectrophotometric method was developed for the quantification of iron-polyphenol complex formation. Iron-polyphenol complex formation was further investigated with a variety of iron sources, temperatures, and polyphenol concentrations using a gallic acid model system and tea extract. Analysis and modeling of iron-polyphenol complex formation prompted the investigation of predicted inhibitors, specifically reducing and chelating agents. Many of these were tested in the presence of iron and black tea extract at pH levels relevant to brewed tea and iron absorption into the body. At the completion of this project a process for effective iron fortification of tea based on our laboratory technique will be ready for in-vivo evaluation of its effectiveness and pilot scale testing.

Oluwasegun Modupe

Process Development for Quadruple Fortification of Salt

Although, micronutrients are required in small amount, their deficiencies remain a scourge to the human race. The consequences of micronutrients deficiencies ranges from mild weight loss to death. Their coexistent with infection and diseases further worsen these consequences. With over 30% of the world population affected, its contribution to the global burden of disease cannot be overestimated.
Given these consequences, WHO has suggested three strategies for combating micronutrients deficiencies. These are; dietary diversification, micronutrients supplementation, and food fortification. Of these strategies, food fortification is the best in terms of economics and ease of implementation.
In line with this, our laboratory has developed technology for double fortification of salt. The choice of salt is due to absolute necessity for it, irrespective of socioeconomic status. Folic acid was added to the ‘tray’ of micronutrients added to salt due to WHO’s call for multiple micronutrient fortification as an effective means of combating multiple micronutrient deficiency (triple fortification of salt). The process developed for triple fortification of salt needs optimization, as a result of low Iodine retention in the salt. Even if the process is optimized, the metabolic interaction of folic acid and vitamin B12 calls for addition of vitamin B12 the ‘tray’ of micronutrients added to salt.
Multiple nutrient fortification has a lot of challenges. These include; interaction among the micronutrients and organoleptic changes. These affect not just stability of the micronutrients in the salt but also acceptability of the fortified salt. An effective, yet a simple technology will be developed in this research work to prevent interaction among these micronutrients. This will improve the stability of the micronutrients in the salt and acceptability of the salt.

Quadruple fortification of Salt

The world, especially the developing countries, is currently struggling with meeting up with the nutritional requirements of her growing populations; with micronutrients taking the central stage, given their roles in human growth, development, and functions. Fortification has been proposed as one of the ways of combating these micronutrient deficiencies. With the success made with iodized salt, it is becomes imperative to build other fortification model around iodized salt. Aside the challenge of making fortification of salt economical and acceptable, the nutrients in food fortified must be stable.
My research focuses on solving world micronutrient deficiency by adding some of the micronutrients with high deficiency prevalence and irreversible devastating effect on humans into salt. The micronutrients of interest are iron, iodine, folic acid and vitamin B12. The chemistry of these micronutrients may cause interactions among the micronutrients. An effective and low cost technology to prevent these interactions will be devised. The research will also focus on ways to reduce organoleptic effect the fortificants may have on salt. The prevention of interaction among the micronutrients and organoleptic effect will be ensured by microencapsulation. Reactive fortificants will be encapsulated while others will be dissolved in appropriate solvent, and sprayed on salt. The addition of the micronutrients is expected to have no big effect on the cost of salt.

Olivia Molenda

Anaerobic treatment of soil and groundwater contaminants

Olivia is characterizing reductive dehalogenase enzymes from mixed microbial cultures used for bioremediation. Her research is focused on substrate characterization using Blue-native PAGE and protein purification for structural characterization. She works under the supervision of Elizabeth Edwards.

Metagenomic investigation of Dehalococcoidetes used for bioremediation of chlorinated ethenes and ethanes in groundwater and soil

Chlorinated ethenes and ethanes are toxic, widespread and recalcitrant groundwater and soil contaminants. While currently there are strategies in place to reduce use and avoid release of these compounds, many sites remain contaminated due to historical military and industrial use. Bioaugmentation using a mixed microbial culture is an effective remediation strategy for these compounds. While the degradation pathways of certain contaminants such as perchloroethene, are already well established, further research is required for optimization of this strategy and for the discovery of novel pathways.
One of the most successful organisms capable of complete anaerobic reductive dechlorination, are the Dehalococcoides mccartyi. D. mccartyi have small genomes (~1.3Mbp) specialized for dechlorination each harbouring many different reductive dehalogenase genes. One of the more interesting features of their small streamlined genomes is the presence of high plasticity regions (HPRs) which contain multiple putative mobile elements, genomic islands, integration elements, prophages and show evidence of recombination. The HPRs account for the major differences between strains including their dechlorinating ability since the majority of reductive dehalogenases are found in HPRs. The mechanisms for recombination in HPRs are not yet well understood, and only a handful of reductive dehalogenases have been characterized to date.
Using a combination of metagenomic and amplicon sequencing we closed eight different strains of D. mccartyi from KB-1, a mixed microbial culture used for bioaugmentation. Combining bioinformatical analyses with molecular techniques such as qPCR, PCR and blue-native polyacrylamide gel electrophoresis/liquid chromatography/mass spectrometry we discover new enzymes, new biological pathways and begin to shed light on gene transfer, recombination and invading pathogenic DNA in D. mccartyi. We hope results can be used to improve bioaugmentation strategies and broaden the range of compounds which can be degraded by D. mccartyi.

Nadia Morson

Chlorinated Solvent Biodegradation

Nadia’s research involves treatment of contaminated groundwater by anaerobic reductive dechlorination. She works under the supervision of Professor Elizabeth Edwards.

Horizontal Gene Transfer in Anaerobic Dechlorinating Cultures

Chlorinated solvents are common groundwater and soil contaminants, as a result of poor disposal practices from industry. Bioaugmentation provides a solution, as microorganisms can anaerobically metabolize these toxic compounds. Dechlorinating microbes, such as Dehalococcoides mccartyi, are able to degrade highly chlorinated compounds, such as perchloroethene and trichloroethene, into ethene, a non-toxic end-product. KB-1, a mixed microbial culture used for bioaugmentation, contains multiple strains of D. mccartyi which allow for successful degradation of PCE. In collaboration with Cornell University, we have received a sample of a mixed dechlorinating culture called Donna II, that contains only D. mccartyi strain 195.
Putative mobile elements or genomic islands have been identified using bioinformatics in D. mccartyi genomes. One of these islands contains vcrA, a gene that encodes a reductive dehalogenase catalysing hydrogenolysis of vinyl chloride to ethene. It is unknown if and how these genomic islands are mobilized within microbial communities.
In an attempt to observe horizontal gene transfer (HGT) among different strains of D. mccartyi, the KB-1 culture was mixed with Donna II to create a culture called DKB, and grown under different conditions. The absence of ammonia is being used as an environmental stressor to stimulate HGT. D. mccartyi 195 from Donna II is capable of fixing nitrogen, however does not have a vcrA gene. At the same time, D. mccartyi from KB-1 do have vcrA but cannot fix nitrogen, creating a controlled environment which would favour a D. mccartyi with both traits.
One of the ways in which this HGT event can be determined is through development of D. mccartyi strain-specific biomarkers and bioinformatical analysis. This task is challenging in that most D. mccartyi strains have >98% 16S rRNA gene sequence similarity.
Determining D. mccartyi biomarkers will be a novel discovery, and will allow for the quantification and further study of this organism in mixed culture with possible in-field applications. Additionally, understanding the role of this vcrA mobile element in HGT may provide better insight to the community evolution of these bioaugmentation cultures, and open the possibility of naturally improving substrate range of D. mccartyi strains used for bioremediation.

Fakhria Muhammad Razeq

Biochemical Characterization of New CAZymes from PULs and Metagenome Sequences

Fakhria is an MASc candidate working with Professor Emma Master. The specific aim of her thesis is to identify and characterize unchartered carbohydrate active enzymes and proteins of unknown function that can be used to fine-tune the chemistry of hemicelluloses to expand their utility and improve their performance in biomaterials.

Biochemical characterization of new CAZymes from polysaccharide utilization loci and metagenome sequences

Hemicelluloses, including xylans and glucomannans, comprise a major fraction of underutilized lignocellulosic biomass, which have a potential for use in high-value biomaterials, such as bioplastics, adhesives, and various packaging materials. However, efficient utilization of hemicelluloses for production of biomaterials is hindered by the structural diversity and complexity of these biopolymers and often requires a large repertoire of different carbohydrate active enzymes (CAZymes).
The overall aim of my graduate thesis is to discover and characterize enzymes that are able to efficiently and selectively modify hemicelluloses, allowing us to fine-tune their chemistry, expand their utility and improve their performance in biomaterials. Specifically, I am looking at identifying and characterizing uncharted CAZymes from polysaccharide utilization loci (PULs) and metagenomic samples.
PULs are set of physically linked and functionally related genes that have evolved to work together to breakdown a specific polysaccharide (Fig1). While function of some of CAZymes on PULs are known or can be predicted, there are some proteins that have no known function. Discovery and characterization of these proteins of unknown function not only will help us to understand how all proteins on a given PUL work together, but also help us to find new CAZymes with novel functions that can be used for efficient utilization of plant polysaccharides.
Metagenomic sequences from anaerobic granules, beaver droppings and moose rumen microbial cultures enriched on cellulose and poplar hydrolysate available in our laboratory serve as another great source for discovery of new CAZymes with potentially novel activities. Together, PULs and metagenomic sequences, serve as a rich source for discovery and characterization of new and novel CAZymes, which can help to produce high-value, specialized biomaterials and chemicals from wood fibre and agricultural residues.

Richard Ndubuisi

Hydrothermal Conversion of Canola oil into Green Diesel

Richard is working on the hydrothermal production of diesel from triglycerides over supported metal catalyst. The ultimate goal of his research is to synthesize an integrated process for producing renewable diesel and isolating protein from canola oil. Richard is in the Diosady group and works in collaboration with Professor Cathy Chin’s lab.

Catalytic Hydrothermal Production of Renewable Diesel and Potential Integration with Protein Isolation

Traditionally, the production of edible oil is based on solvent extraction using hexane. However, the toxicity and flammability of the organic solvent prompts the search for a safer and more sustainable process. Accordingly, a green approach, the aqueous extraction process (AEP) has been proposed as an alternative. This technique is based on the principle of immiscibility of water with the oil. Based on this method, an aqueous solution is contacted with the pulverized oil seeds and the resulting mixture fractionated into oil, solids and aqueous phases via centrifugation. The solids can be used as animal feed whereas food-grade protein can be isolated from the aqueous phase. However, low oil recovery due to the formation of protein-stabilized emulsion with the aqueous phase and excessive energy requirement for drying are challenges to commercialization.

In an original work, we demonstrated that renewable hydrocarbons within the diesel boiling range can be directly produced from the resulting AEP emulsion without the need for water removal. Typically, the production of renewable diesel, also known as green diesel is based on the use of prohibitively expensive catalysts based on precious metals such as Pt in an organic medium. This approach generally results in better yields but requires disproportionate utilization of hydrogen to realize appreciable selectivity to desired alkanes. In our work, however, we used cheaper, lab-made catalysts in a hydrothermal medium and realized good yields and selectivity (both >70%) at 305 o C and minimal initial hydrogen input. My work involved four main stages. The first was re-engineering the high-pressure reactors and adapting to the high temperature, high-pressure (HTHP) decarboxylation reaction. The pressure vessels were previously used for low temperature hydrogenation of oil, i.e., hardening. The second involved the synthesis of the process catalyst whose activity towards the direct conversion of the miscella into fuel was tested in the third stage. Finally, relevant analytical methods were developed for quantitation. By using a hydrothermal medium and working under conditions relevant for aqueous reforming of glycerol into hydrogen, the initial hydrogen pressure in the batch reactor was limited. We have shown that it is possible to form an integrated, sustainable process (AEP-Decarboxylation/decarbonylation, i.e., AEP-DCOx) wherein renewable diesel production is coupled with food-grade protein isolation. Protein has a higher value than fuel so the process could become commercially viable. Moreover, renewable diesel has better fuel properties and it is completely fungible with current hydrocarbon infrastructures and will likely become a major surrogate for biodiesel in fuel blends for compression ignition (CI) engines. Conclusively, the results shed light on the solution to the main issues associated with AEP and lay the groundwork for further exploring this green process integration towards commercialization.

Kayla Nemr

Modeling and analysis of biological systems

Kayla is part of Radhakrishnan Mahadevan’s metabolic engineering research group and is working on screening a library of enzymes to identify candidates for the production of useful chemicals or intermediates from renewable feedstocks, with the ultimate goal of introducing the discovered enzymes into engineered microbes.

Designing aldolase-based biosynthetic pathways for biochemical production in Escherichia coli

Carbon-carbon bond formation is the cornerstone of many processes to synthesize longer, complex compounds from simple building blocks. However, aldol reactions lack specificity and stereo-control. As such, aldolase-based biosynthetic pathways present a promising alternative for the production of a wide range of chemicals through stereo-controlled synthesis under milder conditions. While many biosynthetic pathways have been demonstrated to produce a variety of chemicals, some pathways are complex and require a large number of enzymes. We are designing alternative, simpler pathways via carbon-carbon bond formation using aldolases, which have not been widely explored for their potential to increase the repertoire of chemicals synthesized by microbial cell factories. Our workflow involves: (1) designing potential aldolase-based pathways for a variety of chemicals, (2) screening a library of aldolases with novel activity on a wide range of substrates, (3) modular platform strain engineering, and (3) demonstrating this strategy for 1,3-butanediol production in E. coli.

Using our design-build-test workflow, exploring aldolase-based pathways that require aldehyde intermediates can help guide improved strain engineering strategies, with the ultimate goal of designing robust and scalable microbial cell factories for producing a wider variety of biofuels and biochemical.

Mehdi Nouraei

Microencapsulation of food ingredients and nutraceuticals

Mehdi is part of Levente Diosady’s food engineering group, where he previously completed his M.A.Sc. He is currently working on developing delivery systems for micronutrients, using microencapsulation, self-microemulsification and organogel technologies.

Self-Micro emulsifying Delivery Systems

Many newly discovered drugs and nutraceuticals are hydrophobic in nature. When these drugs and nutraceuticals are ingested, their low aqueous solubility limits their apsorptrion. To overcome this limitation, the active ingeedinet needs to be in a solubilized state in a delivery system. Formulating a food-grade fully dilutable is a challenge. We have developed and evaluated a pltform for a Self Micro Emulasifying Delivery System (SMEDS) that upon exposure to intestinal fluids, for nano-sized droplets (microemulsion) loaded with active ingredient. The in vivo experiments showed that this system increases the bioavailability to 3 folds. Furthermore, a solid version (powder) and semisolid version (gel) of the this delivery systems are developed with delayed release and extended release profiles. The platform is also efficient in extracting lipophilic components from plant material, including fruit peals and vegetables. This novel process can turn agri-food wastes/by- products into highly value-added products without using organic solvents.

Jon Obnamia

Sustainable production of biofuels

Jon is working on a kinetics-based enzymatic hydrolysis model for the optimization of hydrolysis process parameters and prediction of sugar concentration profiles. He is part of Bradley Saville’s research group.

Environmental Impacts of Transportation Biofuels

The transportation sector contributes 22-27% to worldwide annual greenhouse gas (GHG) emissions from the combustion of fossil fuels. From annual global transportation GHG emissions, about 75% typically comes from road transport while 10% comes from aviation transport. Emissions from the transportation sector, and both road and aviation transport are projected to increase, with continued fossil fuel use. To combat this upward trend, less carbon intensive biofuels such as ethanol from lignocellulosic biomass and jet fuel from oilseeds are being developed for commercial production. However, full life cycle assessment is necessary to critically evaluate the environmental impacts of biofuels and determine the likelihood for these biofuels to achieve their environmental objectives.

Kylie O’Donnell

Bioengineering for maximizing value in forest products

Kylie is part of Emma Master’s research group and is studying the effect of surface chemistry on hydrophobin assembly.

Breaking Down Barriers to Hydrophobin-Mediated Surface Functionalization

Hydrophobins (HFBs) are small (7-20 kDa), self-assembling, surface-active fungal proteins that assemble at hydrophobic-hydrophilic interfaces to form highly stable films. HFBs have been described as the most surface-active proteins known and represent non-immunogenic proteins with the capacity to change the property of surfaces via self-assembly as monolayers at liquid-solid interfaces. HFBs represent an attractive surface modification technology that can be paired with a variety of fusion partners to increase their versatility as surface modifiers. The utilization of HFBs is a recent trend in industrial applications such as coatings, various dispersion applications, stabilizing emulsions, personal care products, separation technologies, biosensors and electrodes, biomaterials, etc. We have looked at the effect of linker length and chemistry between a HFB and a functional tag in an attempt to improve upon the application of HFBs utilized in bio-sensing applications. While many attractive potential applications of HFBs exist, little is known about the impact of differentiating physical and environmental effects on their biochemical characterization. Additionally, there are significant differences in how individual HFBs function and many HFB characteristics are dependent on the experimental apparatus used. Therefore, we are also systematically characterizing a set of HFBs over various environmental parameters utilizing uniform characterization methods. This will provide the basis to have the ability to tune HFB assembly to surfaces based on chosen environmental parameters.

Ph.D. Student

Vik Aditya Pandit

Modeling and analysis of biological systems

Vik is currently looking at the electron transport chain of Geobacter sulfurreducens and is trying to incorporate the specific elements that allow for respiration on iron into the Escherichia coli metabolism. This would allow electrodes to serve as the electron donor for biosynthesis processes. This work is being done in silico and experimentally validated, under the supervision of Radhakrishnan Mahadevan.

Engineering carbon fixation metabolic pathways

Amidst the many mechanisms present in nature, two routes present themselves from the outset as being the most viable for heterologous demonstration of microbial electrosynthesis in E. coli. The first is the assimilation of electrical energy by external mediators including neutral red to aid the direct assimilation of CO2 by metabolic pathways. The second is the assimilation of reduced carbon species, which are derived from electrochemical sources, by metabolic pathways. These two routes are the primary focus of my research.

Successful demonstration would allow bioprocesses to become efficient at producing chemicals and fuels directly from CO2. This would address some of the economic challenges facing current bioprocesses that are struggling with high feedstock costs and low cost of oil which makes them uncompetitive.

James Poon

Tissue engineering

James works in Alison McGuigan’s laboratory where he designs scaffolds for tissue-engineering applications. Using PEG-based substrates, James is assessing the effect of mechanical and biochemical signals on the ability of human tracheal epithelial cells to polarize and differentiate.

Manipulating micro-physical cues for tissue engineering

There are currently no acceptable treatments for injuries affecting long segments of the trachea. Tissue-engineered scaffolds are a promising alternative but have not yet been optimized to incorporate functional epithelium. Airway epithelium contains multiciliated cells that coordinate their beating to eliminate mucus and foreign particles. Currently, air-liquid-interface (ALI) culture is used to generate artificial adult airway epithelium in vitro. This culture system provides the apical-basal polarization necessary for differentiation of progenitors into their terminal cell types. However, ALI culture does not produce planar polarized epithelium with correct alignment and beating of motile cilia.

We are particularly interested in using the physical microenvironment to direct cell function. Airway epithelium rests on a basement membrane of aligned collagen fibres that provide topographical cues at the scale of the individual cells. Using elastomeric and hydrogel substrates, we are assessing the ability of primary human tracheal epithelial cells to polarize and differentiate into mature epithelial cells, under ALI, on defined microgrooved substrates of varying pitch and depth. Super-resolution microscopy combined with computational analysis techniques are utilized to study cilia orientation. Our goal is to produce epithelial tissue with concerted cilia function using substrates with optimized grooved features.

Understanding topographical requirements will improve clinical impact, by providing the basis for generating gel coatings with specific properties for lining the lumens of engineered airway replacements to guide epithelial organization.

Scott Proulx

Adipic acid production by E. coli

Production of high-value chemicals has been rooted in the petrochemical industry for the entirety of the 20th century until present day. As fossil fuel reserves begin to decline and as a result of the pressing issue of climate change, alternative processes are desired. Such a process should produce the desired chemical using sustainable, non-toxic materials without compromising economic efficiency. Adipic acid, which is a precursor for nylon production, presents such a challenge.

The use of microbial organisms such as yeast and bacteria as biocatalysts to produce chemicals is a solution that would use sustainable feedstocks such as glucose, and may be operated at moderate reaction conditions with minimal toxic byproducts. However, organisms will not normally produce these chemicals in high enough quantities on their own. By engineering the metabolism of a microorganism, the enzymes that are expressed may be configured to optimally produce a product. The chemical that my research will focus on is adipic acid produced in Escherichia coli.

I began my MASc in January, so I do not yet know many of the fine details surrounding this project. Despite this, it is clear that an ideal strain of E. coli would produce adipic acid at high yield, titer and productivity. Although initial research may begin using glucose as a carbon source, the eventual use of alternative feedstocks such as ethylene glycol is desired.

Luz Adriana Puentes Jacome

Anaerobic treatment of soil and groundwater contaminants

Luz is part of Elizabeth Edwards’ bioremediation research group and is investigating some of the challenges and inhibitions faced by dechlorinating microbial communities such as KB-1 at field sites. These include low pH conditions, the presence of co-contaminants, and the addition of zero-valent iron, which is also intended to promote dehalogentation of VOCs in groundwater, along with emulsifying agents.

Anaerobic biodegradation of chlorobenzenes and Lindane

Chlorinated organic compounds such as chlorobenzenes and lindane, the ? isomer of hexachlorocyclohexane (?-HCH), are environmentally-regulated persistent organic pollutants. Chlorobenzenes are used as intermediates for the synthesis of various chemicals and pesticides. They are found in the environment as a result of uncontrolled industrial discharges and the transformation of other chlorobenzenes or pesticides. In fact, lindane, historically used as an insecticide in agricultural crops, can be transformed and partially dechlorinated by microorganisms into a mixture of different chlorobenzenes. Lindane has been classified by the International Agency for Cancer Research as carcinogenic. Chlorobenzenes and lindane are somewhat hydrophobic. They are found as soil, sediment, and groundwater contaminants in anaerobic environments where they may be reductively dechlorinated by bacteria. Currently, little is known about the physiology of the bacteria and the associated enzymes (reductive dehalogenases or RDases) carrying out this process. Also, engineers need to develop field site remediation strategies that will accommodate their hydrophobic nature and limited bioavailability. This project is providing insight into these engineering and scientific questions by studying three dechlorinating microbial enrichment cultures: KB-1, MCB/Benzene, and GT1. KB-1, commercially used at contaminated sites, transforms aliphatic chlorinated compounds into non-toxic ethene gas. The MCB/Benzene culture biodegrades monochlorobenzene (MCB) and benzene into methane and carbon dioxide. The GT1 culture degrades lindane to MCB and benzene. We evaluate the dechlorination potential of these mixed cultures against chlorobenzenes and lindane while monitoring growth and changes in the microbial populations. Our specific goals are to: (i) study the reductive dechlorination of chlorobenzenes by the KB-1 culture; (ii) investigate the bioconversion of lindane by a mixed consortium of GT1 and MCB/Benzene-degrading organisms; (iii) identify unknown RDase(s); and (iv) explore the use of activated carbon to deliver a lindane-degrading consortium to lindane-contaminated sediment.

Wenjing Qiao

Anaerobic degradation of chlorobenzenes and benzene

Wenjing is a visiting Ph.D. student in Prof. Elizabeth A. Edwards lab from Nanjing University, China.

Anaerobic degradation of chlorobenzenes and benzene

Chlorobenzenes are widespread contaminants at many industrial sites, posing a threat to human health and the environment. Under anaerobic conditions, these compounds can be completely degraded via natural biological processes.
However, achieving complete anaerobic degradation of chlorobenzenes to non-toxic compounds is challenging and the microorganisms involved in these processes are not fully characterized. An investigation at a chlorobenzene contaminated site indicated that indigenous microorganisms capable of degrading chlorobenzenes were present. To verify this hypothesis, a corresponding laboratory microcosms study was carried out to confirm the biological activities of chlorobenzenes in soil samples from the site. As anticipated, anaerobic biotransformation of chlorobenzenes and benzenes is also occurring in the lab microcosms. I am now attempting to characterize the microorganisms responsible dechlorination and create enrichment cultures to further study the mechanisms of transformation.This research approach of combining site investigation with a laboratory study is transferable to any other contaminated sites of concern. Lab microcosms study can support the selection of a site remediation strategy and authenticate site investigation results, making the site remediation strategy much more specific and cost-effective.

Darren Rodenhizer

Darren’s goal in the McGuigan Lab is to develop smart-data-acquisition phenotypic screening platforms for cancer drug discovery. His approach is to use tissue engineering principles to create artificial tissues with properties that mimic the complex and dynamic microenvironments found in real tumours in patients. Cancer cells growing in these tissues are more predictive of therapeutic response compared to other test systems, thus their use reduces the time and cost of getting new treatments to market by reducing the number of “destined to fail” compounds in clinical trials.

Tissue engineered tumour models

The tumour microenvironment is heterogeneous and consists of multiple cell types, variable extracellular matrix (ECM) composition, and contains cell-defined gradients of small molecules, oxygen, nutrients and waste. Emerging in vitro cell culture systems that attempt to replicate these features often fail to incorporate design strategies to facilitate efficient data collection and stratification. By combing cutting-edge tissue engineering principles and device design ingenuity, my work strives to create smart-data-acquisition tissues for use in drug screening platforms. Our first solution the tissue roll for analysis of celluar environment and response (TRACER) is a 3D tissue that can be rapidly taken apart for analysis. TRACER enabled our team to spatially map cell metabolism in concert with cell phenotype in a 3D tissue; for the first time. We envision this technology will provide a platform to create complex, yet controlled tumour microenvironments that can be easily disassembled for snapshot analysis of cell phenotype and response to therapy in relation to microenvironment properties.

Fawzi Salama

Fawzi’s research involves metabolic modeling of microbial communities. He works under the supervision of Professor Radhakrishnan Mahadevan.

Dynamic Metabolic Modeling of Microbial Communities

Microbial communities, assemblies of more than one interacting microbial species, are essential to human and environmental heath. Communities in the soil provide nutrients to plants, communities in the ocean help regulate the planets carbon balance, and communities in the human body aid in the digestion of fibers and provide essential short-chain fatty acids. A simplified representation of this process is shown in the figure below. Typically, however, microbial communities are composed of hundreds or thousands of members. While individual species can be isolated and studied in vitro, communities composed of a large number of species are difficult to study using currently available experimental techniques. Mathematical modeling can aid in this task.

Using the sequenced genome and other biochemical data, the network of metabolic reactions in a species can be constructed, and flux balance analysis (FBA) can be used construct predictive models for a single species [1]. Furthermore, Dynamic FBA (dFBA), an extension of FBA, can be used to account for dynamic behaviours [2]. The goal of my research is to apply this technique and others to the modeling of microbial communities by improving, extending and generalizing previous efforts in this regard [3]. This research will aid in the study of natural microbial communities. It will also aid in the exploitation of synthetic communities for industrial applications, in which there has been growing interest in recent years.

1. Orth, J. D., Thiele, I. and Palsson, B. (2010). What is flux balance analysis? Nature Biotechnology, 28:245248.
2. Mahadevan, R., Edwards, J. S. and Doyle, F. (2002). Dynamic flux balance analysis of diauxic growth in Escherichia coli. Biophysical Journal, 83:13311340.
3. Zhuang, K. et al. (2011). Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments. ISME Journal, 5:30516.

Benjamin Slater

Tissue engineering

Benjamin is working under the supervision of Alison McGuigan, to try to better characterize the motility of epithelial cells in confluent sheets in vitro. More specifically his research focuses on the mechanical signalling and the cellular geometric changes that drive the process.

Ph.D. Candidate

John Soleas

Engineering cell orientation and growth

John’s research involves tissue engineering human tracheal epithelium in a bioreactor while altering substrate stiffness, surface topography and air flow. He is part of Alison McGuigan’s research group.

Architecture can manipulate the differentiation of lung progenitor cells

Mechanical forces are essential for normal lung development. During epithelial differentiation in vivo, the proximal (SOX2+), pseudostratified airway epithelium develops in tube diameters larger than those found in the distal (SOX9+), squamous epithelium. We hypothesized that lung progenitor cell grown in cylinders smaller than 100?m will guide cells towards a distal fate. Our aim was to expose lung progenitors, derived from embryonic stem cells, to a defined architecture, in the form of hollow, three-dimensional cylinders of differing diameters to create cell lined cavities reminiscent of the developing lung tubule and to study how this geometric configuration affects cell fate choice. Using photolithographic and moulding technologies we have created hollow cylinders of 40-400m in diameter with a height of 180m in either 15% gelatin or polydimethylsiloxane. NKX2.1+FOXA2+SOX2+SOX9+ lung progenitors successfully form hollow cavities following 8 days of culture in both materials. Our data suggests that at a 100m diameter distal fate choice SOX9+ (84.656.99%) is favoured over proximal SOX2+ (8.147.60%) or dual positive SOX2+SOX9 (11.587.14%) in gelatin hydrogels. Similar trends were seen in 80m and 40m diameter tubes. In flat control conditions only 2.152.49% SOX9+, 1.001.11% SOX2+ were seen and 95.00 5.07% of cells remained dual positive. To assess whether local or paracrine signalling was affecting fate choice we grew our cells on positive tubular architecture (posts) and found a similar affect. This observation suggests that curvature itself is guiding fate choice. Utilizing a tissue engineering approach to apply architectural cues to heterogeneous lung progenitors, we have guided cell fate choice.
CIHR Training Programme in Regenerative Medicine. McLaughlin Centre. The Henry White Kinnear Foundation.

Shyam Srinivasan

Modeling and analysis of biological systems

Shyam is working in under the supervision of Radhakrishnan Mahadevan on large scale models that can be used to engineer organisms like E. coli and yeast to use them for the production of specific metabolites. The model organisms can help in building models for complicated organisms at a later stage and optimizing metabolite production.

Assessing the impact of bistability in metabolic networks for metabolic engineering design

In metabolic engineering, microorganisms are designed as cell factories to produce various target chemicals. However, bistability in the metabolic network causes bistability in cell factory production. I am looking at alleviating this occurrence by studying and accounting for various factors causing bistability in the design of metabolism for metabolic engineering applications.

Ph.D. Candidate

Dylan Valleau

Characterisation of molecular determinants of bacteria-host interactions

Dylan’s research aims to elucidate how gram-negative bacterial pathogens colonize their host through injection of effector proteins into host cells. Under supervision of Alexei Savchenko, his task is to characterize the role of ubiquitin protein ligase (E3 enzymes) effectors from pathogenic E. coli, Salmonella, and Shigella, through co-immunoprecipitation and mass spectrometry, structural characterization, and in vivo expression analysis in human cell lines.

Azadeh Vatandoust

Azadeh is currently working towards her PhD under the supervision of Professor Levente Diosady.

Naveen Venayak

Modeling and analysis of biological systems

Working under the supervision of Radhakrishnan Mahadevan, Naveen is investigating a promising alternative method for producing chemicals using microbial metabolism of renewable feedstocks rather than chemical synthesis from petroleum. His project involves improving the production of such compounds in model organisms such as Escherichia coli and Saccharomyces cerevisiae.

Applied laboratory automation and synthetic biology to engineer dynamically controlled microorganisms.

Microorganisms show promise in solving many societal concerns in areas such as health, sustainability, energy and consumer products. One area which has received particular attention over the past 30 years is the use of microbes to produce high-value chemicals and fuels. Microbial metabolism provides a diverse network of chemicals, many of which have commercial applications, and many others which can be converted to useful compounds with additional biochemical conversions. However, the complexity of metabolism brings challenges in developing organisms for robust, scalable and economical bioprocesses. To overcome some of these limitation, significant efforts have been made to develop metabolic models for these organisms and to improve the throughput of experiments and data analysis. My work focuses on developing synthetic circuits to dynamically control microbes throughout the duration of a fermentation process, allowing finer control and significantly improved performance. In parallel, we have developed automated liquid handling methods and data analysis pipelines to expedite this process. This work has broad applicability to metabolically engineered organisms, as most are not dynamically controlled and suffer poor performance as a result.

Kaushik Raj Venkatesan

Enhancing the performance of synthetic biological circuits

My research aims at improving the robustness and response speed of genetic circuits in synthetic biology. Specifically, I am studying methods to enhance the performance of the genetic toggle switch, by examining natural switching circuits such as the lambda phage switch. By improving understanding the methods to improve these devices, I aim to make efficient synthetic switches that can be used in metabolic engineering and other applications.

Enhancing Genetic Circuit Performance in Synthetic Biology

Microorganisms have been used to produce several chemicals of industrial and pharmaceutical importance over the past few decades. The advent of metabolic engineering has helped fuel an improvement in the production yields of these chemicals. However, several metabolic engineering strategies result in a growth impediment of the host organism and this results in strains that are not suitable for industrial scale production due to their low productivity.
Dynamic control of metabolism through input regulated switching of gene expression has been proposed as a potential solution to this problem. Advancements in synthetic biology have led to the creation of a device the genetic toggle switch which is able to switch the cells phenotype between stable states based on an input. Implementation of toggle switches however, has been limited due to a lack of robustness or very slow switching speed.
We hypothesize that in any synthetic biological circuit, there is an inherent tradeoff between the robustness to protein production rates and the response speed of the circuit. My work aims to examine the presence of such a tradeoff that a synthetic circuit poses on a cell through experimental evaluation using E.coli as a model host and will result in a deeper understanding of the reasons for failure of synthetic circuits. Design guidelines for synthetic biological circuits to minimize the probability of circuit failure will also be proposed.

Po-Hsiang (Tommy) Wang

Anaerobic treatment of soil and groundwater contaminants

Po Hsiang is part of the Edwards research group. He is investigating anaerobic organochlorine bioremediation, identifying the essential nutrients transferred in microbial consortia, especially corrinoids.

Interspecies nutrient transfer in anaerobic dechlorinating microbial communities

Most microorganisms in the environment live in close association with one another. The importance of studying microbial communities and their interactions is becoming apparent in many fields, including agriculture, bioremediation, human health, waste treatment and industrial biotechnology. Microbes in mixed communities function very differently from microbes in isolated culture. Complex, yet specific interspecies interactions result in emergent phenotypes not present in pure cultures, and thus microbial communities need to be studied as a whole. Moreover, because of these complex interdependencies, it is often challenging to isolate all the dominating microbes from the communities. Genome-scale metabolic models have been used to gain insights into the molecular mechanisms of individual organisms whose genomes and growth characteristics are known. They are now increasingly being used to help elucidate metabolic interactions between microorganisms at the community level. Thus my research utilizes a poorly characterized anaerobic subsurface microbial community used for bioremediation of chlorinated solvents, referred to as ACT-3, as the model. The genome of the dechlorinating microbe in ACT-3, Dehalobacter restrictus strain CF, has been assembled and annotated via the incorporation of comparative genomic and functional genomic data. However, bioinformatic analyses, while powerful, are often insufficient to reliably predict many metabolic features of an organism because of the considerable number of mis-annotations and hypothetical genes in every genome. Thus strain CF was isolated and grown in defined medium to study the specific genomic predictions, particularly where annotations were uncertain or inconsistent with the observed phenotypes. The results of the experimental verifications reveal two aspects of the interpretation of genome annotation that can improve the correspondence between bioinformatic predictions and the reality: (i) cofactor availability for corresponding metabolic reactions and (ii) the potential for enzyme promiscuity to rescue apparently missing pathways, as well as identify essential nutrient interdependencies in anaerobic dechlorinating microbial communities.

Mabel Ting Wong

Maximizing value in forest products and other processes

Mabel’s work focuses on the discovery of lignocellulose active enzymes through metagenomic analysis of moose and beaver digestive systems. Working under the supervision of Emma Master, she is using various omics techniques to identify new enzyme activities relevant to lignocellulose processing in anaerobic enrichments. Metagenome and metatranscriptome sequences are used to identify enzyme candidates for recombinant expression and biochemical characterization.

Bioprospecting lignocellulases

My research focuses on the discovery of lignocellulose active enzymes through metagenomic analysis of pulp mill anaerobic granules, as well as the digestive systems of North American moose and Canadian beaver. Various omics techniques will be applied to identify new enzyme activites encoded by lignocellulose-degrading microbial cultures that are maintained anaerobically over long term (5+ years). Metagenome sequences and secreted proteins are analyzed to identify enzyme candidates for biochemical characterization for efficient wood-processing in the industry.

Zi (Johnny) Xiao

Heterologous functional expression using Clostridium

Many putative genes that could have important bioremediation applications fail to express well in traditional heterologous hosts such as E. coli. These genes are often from gram-positive anaerobes, which lack a representative host. Johnny’s work focuses on the development of Clostridium acetobutylicum as a host to express an elusive benzene carboxylase isolated from a benzene degrading, nitrate-reducing culture. He is under the supervision of Elizabeth Edwards.

Heterologous expression in Clostridium acetobutylicum

Recent advances in DNA sequencing technology and metagenomics have resulted in a large number of gene sequences that lack functional annotation. The dependency on traditional heterologous expression hosts such as E. coli has confined the progress of functional annotation from phylogenetically distant species, since the physiological differences between organisms in distant phyla often fail to express the protein in a functional form. One such protein is a putative anaerobic benzene carboxylase that has been identified in a benzene-degrading Peptococcaceae sp., where heterologous expression using E. coli was attempted, but failed to yield a sufficiently soluble fraction for biochemical characterization. Clostridium acetobutylicum is proposed as an alternative expression host because of its robustness, rapid doubling time, and its phylogenetic proximity to Peptococcaceae, thus being more likely to contain the necessary molecular chaperones and cofactors for functional expression of the putative benzene carboxylase. Although systems for genetically modified C. acetobuylicum have been developed, work on functional expression of putative genes remains limited. Currently, the putative benzene carboxylase genes have been cloned into several E. coliClostridium shuttle vectors, but optimized protocols for C. acetobuylicum growth, transformation, and expression required for functional expression still need to be developed. Success in this work will provide a benzene carboxylase for functional characterization, and validate the gene as a biomarker in the monitoring of benzene-contaminated sites. The optimized Clostridium gene expression system would serve as a convenient expression platform for enzymes naturally expressed by obligate Gram-positive anaerobes and expand the functional annotation of metagenomes from anaerobic environments.

M.A.Sc. Student

Jaehoon (Jason) Ya

Jason investigates the process of P&P biosolids using electro-dewatering (electro-osmosis) technique.

Electro-dewatering of P&P Mill Biosludge

The dewatering of biosludge, the waste generated from the secondary aeration tank of waste treatment processes, has been a major challenge for many pulp and paper mills. The conventional mechanical pressure dewatering method implemented by many industrial mills can only increase the dry solids content up to 4-16% with pure secondary biosludge; thus, the biosludge is usually mixed with primary sludge to noticeably enhance the dewaterability. However, many mills are trying to reduce the release of primary sludge for mainly economic reasons, thereby making the mixing of primary and secondary sludge a less sustainable option in the future.

Electro-dewatering technology based on electro-osmosis can significantly increase the dry solids content of the secondary biosludge. Under the application of an electrical field, the charged ions in the electrical double layer of the sludge particles are drawn towards an electrode, resulting in electro-osmotic flow of water. This flow can enhance the water removal from the biosludge thereby increasing the dry solids content.

Many researchers have studied the electro-dewatering technology on municipal and industrial biosludge; however, only a few studies have examined the effect of the technology on the biosludge produced from pulp and paper mills and similar wood residues. My research, which focuses on the application of electro-dewatering technology on the biosludge produced from pulp and paper mills, will investigate the extent of the dewaterability of electro-dewatering. Other important characteristics of the electro-dewatering technology such as the mixture of different sludges, the energy consumption rate, and the effect of supplementary conditioners will be examined as well.

Ruoyu Yan

Characterization of novel GH115 alpha-glucuronidases for enzymatic tailoring of xylans

Xylans represent the second most abundant polysaccharide in plant cell walls. Whereas this abundant polysaccharide can be used for the production of renewable energy, chemicals, and materials, it remains comparatively underutilized, mainly due to the complexity of corresponding molecules, whose chemistry depends on plant source as well as process technologies used to separate these components from other cell wall components. Accordingly, the overall objective of my doctoral research project is to harness enzyme selectivity to specify and fine-tune xylan chemistry, to enable broader application of xylans in coatings and as nutraceuticals in food and feed. A main aim is to characterize poorly studied clans of glycoside hydrolase (GH) family 115 α-glucuronidase phylogenies, since GH115 enzymes can selectively remove glucuronic acid/4-O-methyl-D-glucuronic acid (GlcpA/MeGlcpA) side groups from xylans with high molecular weight.

Characterized two novel GH115 alpha-glucuronidases.
Identified and investigated synergism among accessory xylanases.
Developed a LC-MS/MS method for characterization of oligomeric sugars.

Xylans represent the second most abundant polysaccharide in plant cell walls. Whereas this abundant polysaccharide can be used for the production of renewable energy, chemicals, and materials, it remains comparatively underutilized, mainly due to the complexity of corresponding molecules, whose chemistry depends on plant source as well as process technologies used to separate these components from other cell wall components. Accordingly, the overall objective of my doctoral research project is to harness enzyme selectivity to specify and fine-tune xylan chemistry, to enable broader application of xylans in coatings and as nutraceuticals in food and feed. A main aim is to characterize poorly studied clans of glycoside hydrolase (GH) family 115 ?-glucuronidase phylogenies, since GH115 enzymes can selectively remove glucuronic acid/4-O-methyl-D-glucuronic acid (GlcA/MeGlcA) side groups from xylans with high molecular weight.

Mitchell Zak

Applying Algal Biofilms for the Recovery of Rare Earth Metals

Mitchell works on analyzing potential applications for algal biofilms utilizing the waveguide photobioreactor. Currently he is looking at using algal biofilms for the separation and concentration of Rare Earth metals from mining effluents.

Applications of Algal Biofilms

Algae possess a variety of potential applications, ranging from biofuel production to wastewater treatment. However, algae cultures have a very low density and therefore most of these applications are economically unfeasible due to high cost of separating water and algae. A possible solution to this problem is to grow algae in the form of a film resulting in a much higher density and reducing separation costs. For this purpose a new type of biofilm photobioreactor called a waveguide, which leaks out light allowing for illumination of the bottom side of the biofilm, was developed.

While the knowledge of algae grown in planktonic cultures is quite extensive, there is less information available on their behaviour when they are grown in biofilms. Furthermore, given the new nature of the waveguide there is little information regarding the applications that it is best suited for. Therefore, it becomes necessary to research the effectiveness of the waveguide as a photobioreactor and how best to utilize it.

For this reason we are currently looking at applying the waveguide in opaque waters where algae cannot typically grow as light cannot penetrate through. One example of this is in the removal of heavy metals from water by biosorption via algae. Also, the removal of nutrients from dark effluents, like in processes such as anaerobic digestion, is another area of interest.

Ultimately, the results of this research should determine which application the waveguide system should be implemented for, allowing for further development and optimization of waveguide based photobioreactor.