Guelph iGEM

Hardware

Introduction

The SeQUESTer filter medium and apparatus facilitate the testing and implementation of microorganisms in water biofiltration applications by ensuring biocontainment, facilitating growth, and improving treatment performance.

The filter medium’s high porosity allows for higher cell proliferation and better treatment efficacy than existing methods of microbial immobilization used in water treatment such as alginate beads. Its adaptability and scalability support its safe integration into existing water treatment infrastructure and compatibility with a variety of microorganisms.

The 3D-printable filter apparatus simulates standard filtration environments, providing researchers with an accessible low-cost method of testing the performance of microorganisms in biofiltration applications.

Implementation of Microorganisms in Water Bioremediation Applications

Lab Scale Testing of Microorganisms in Filtration Systems▶

Although many researchers have genetically engineered microorganisms for water bioremediation applications, their implementation is hindered by biosafety concerns and lack of infrastructure for integration into existing water treatment systems. The developed filter medium addresses these concerns, supporting the implementation of synthetic biology research in the real world.

Scalability and Adaptability
The shape and size of the filter medium is highly adaptable, which supports its easy scalability for integration into water filtration systems of various sizes. Filter media can be stacked in series or lined up in parallel to accommodate the rates of filtration required in different systems. These properties make it possible to adapt the filter medium to the needs of existing water treatment systems, rather than having to redesign existing infrastructure to incorporate the biofiltration system. This is highly advantageous to users and makes it more likely that they will integrate the microorganism into their water treatment process.

Structural Stability and Inertness
The structural stability of the filter medium makes it capable of withstanding mechanical forces applied by flowing water, supporting its longevity in water filtration applications. Its structural properties also make it easy to handle during replacement.

Biocompatibility
The agarose-based medium is water insoluble, inert, non-toxic, biocompatible, and can be easily modified to meet the needs of specific microorganisms by altering the nutrients incorporated [1]. This makes the filter medium widely applicable to pure and co-cultures of various microorganisms used in biofiltration.

Performance Improvement
The microstructure of the filter medium is highly porous and composed of many interconnected channels in which microorganisms can proliferate and water can flow through. Its high porosity gives the filter medium a very high surface area, supporting the establishment of high cell biomass in the filter which can participate in filtration. The small pores slow down the flow of water, allowing for good control over the amount of time that water is in contact with the microorganisms by adjusting the flow rate of water into the filter. This is important because time is required for microorganisms to treat the water and the duration of treatment required differs depending on the microorganism and contaminant used. The small diameter of the pores also promotes contact between the adhered microorganisms and the water being filtered, improving their ability to effectively treat water in comparison to culturing in solution or using alginate beads.

Biocontainment
Biocontainment is a significant concern when using microorganisms in any system, especially when they come into contact with water intended for consumption. The composition of the filter medium has been engineered to support the adhesion of microorganisms, adsorption of microbial metabolites, and the establishment of a microenvironment within the filter medium that limits cell growth to the filter’s interior. The filter medium’s compositional adaptability makes it well suited for use with microorganisms that are genetically modified to require a nutrient present in the filter medium for survival. The nutrient could be incorporated into the filter medium, limiting their existence to the apparatus itself. 0.2 um filter paper was also incorporated into the filter apparatus to ensure biosafety during testing.

Lab Scale Testing of Microorganisms in Filtration Systems▶

The filter apparatus provides an accessible method for testing the performance of microorganisms in real world filtration systems. The 3D printable system is inexpensive, accessible, and easy to use and modify. Its simple modular design makes it easy to modify dimensions and shapes to meet the individual needs of researchers.

Implementation of Microorganisms in Biosensors▶

The adaptability and other features of the developed filter medium mentioned above make it well suited for supporting the integration of genetically engineered microorganisms into biosensors designed to detect compounds in aqueous solutions.

How it Works

How it works graphic

1. S. cerevisiae cells colonize the interior surface of the rehydrated filter medium, which provides the nutrients required for growth. Zeolite incorporated into the filter medium supports cell adhesion, preventing cells from washing off when water is applied [2], [3].

2. Water is passed through the filter medium at a flow rate that provides enough time for S. cerevisiae cells to uptake lead from the water. The filter medium’s highly porous structure provides ample surface area for cell proliferation while forcing water to come in close contact with the cells, facilitating lead uptake.

3. During treatment, the water supplies dissolved oxygen to the S. cerevisiae cells and facilitates the transfer of metabolites.

4. Zeolite incorporated into the filter medium adsorbs metabolites produced by S. cerevisiae, preventing them from contaminating the treated water.

5. Once the lead adsorption capacity of the S. cerevisiae cells has been reached, the filter medium is removed and replaced. The used filter medium can be further processed to remove and lyse the cells, allowing for the recycling of the absorbed lead.

6. When used in the filter apparatus, the treated water is filtered through a 0.2 um filter to ensure that no lead-containing cells are present in the filtered water.

Components▶


Agarose and Nutrients
Agarose is a polysaccharide that is soluble in water at high temperatures then forms a gel composed of flexible interconnected polymer chains as it cools [1]. It is a purified version of agar which can be used as a standard cell culture medium and is accessible to researchers due to its use in synthetic biology experiments such as gels [1], [4]. Agarose is ideal for biofiltration because it is insoluble in water at room temperature, biocompatible, inert, and non-toxic, and its mechanical properties including structural stability, permeability, and porosity are controllable [1], [4], [5]. The molecular purity found in agarose allows for greater consistency in pore shape and size across the entire structure, with the ability for fine tuning of pore size based on agarose concentration and freeze drying parameters.

Bioavailable carbon sources and amino acids present in the filter medium aid in maintaining inoculated yeast cultures by providing nutrients required for growth. Though typically unicellular, S. cerevisiae cells are able increase expression of cohesive and/or adhesive surface proteins under the correct environmental cues [6]. Presenting as floc (submerged), flor (floating), filament, or biofilm (substrate-bound) colonies, such cues include limiting macronutrients such as nitrogen, or culturing on structured substrates such as agarose to induce a multicellular phenotype [6], [7].

Zeolite
Zeolite is a non-toxic, environmentally friendly, highly porous, hydrophobic, negatively charged mineral used in water filtration applications [3], [8], [9]. Its porous and highly hydrophilic microstructure supports cell adhesion and the adsorption of metabolites produced by S. cerevisiae [2], [3].

How it’s Made

How its made graphic

The filter medium consists of a freeze casted agarose cryogel. The medium itself consists of standard yeast tryptone glucose (YTG) media for optimal Saccharomyces cerevisiae growth, with the addition of 5% w/v agarose. The YTG media used contains the same composition as the one used by the iGEM Guelph Wet Lab team (1% w/v yeast extract, 2% w/v tryptone, 2% w/v glucose) and is a close alternative to standard yeast peptone dextrose (YPD) media used widely as growth medium for microorganisms such as S. cerevisiae. Zeolite was added because it has been found to increase microorganism immobilization and adsorb metabolites [2]. Since the zeolite could not be autoclaved due to its tendency to crystallize at high temperatures, it was subjected to UV treatment in a biosafety cabinet (BSC).

For a circular dish with a height of 1.6 cm and a diameter of 7.1 cm, 30 mL of medium is carefully poured in, making sure no air bubbles are present similarly to pouring agar plates. Ensuring no bubbles creates a uniform structure that will be more evenly freeze-dried, creating consistent pores throughout the medium.

How its made graphic

After media solidification, the filter medium is placed in a -20°C refrigerator for 3-4 days to ensure that it is completely frozen. Once the filter medium is completely frozen and all the water has become ice, it is freeze dried. During the freeze-drying process, the ice is sublimated and all the water in the medium is removed. This increases the porosity of the agarose filter media and purifies its composition to only include the nutrients (yeast extract, tryptone, glucose) and agarose.

How its made graphic

Due to the discrepancies between temperature requirements for the agarose freeze drying process and the optimal temperature range of S. cerevisiae, the yeast was inoculated onto the scaffold after freeze-drying. Due to S. cerevisiae’s preference for a wet environment, the filter medium was rehydrated for 1-2 days before inoculation with yeast culture. After the filter medium is fully saturated, S. cerevisiae cultured in YTG broth is pipetted onto the surface. The filter medium is then incubated to allow the yeast to permeate into the filter and colonize the medium.

Filter Apparatus Assembly Instructions▶

Materials
- 3D printed parts: base, inlet, outlet, outlet guide
- 0.2 um bacterial filter
- Freeze-dried filter media
Assembly

3d printed apparatus
Figure 1: 3D printed filter apparatus.
To assemble the water filter device, follow the steps below:
  1. Identify the base piece which has the text ‘iGEM Guelph’ on it.
  2. Insert 1 dried filter medium into the base piece until it sits against the inner lip.
  3. Insert up to 2 more dried filter media until they lay flush against each other.
  4. Rehydrate the filter media.
  5. Attach the inlet piece with the small, centered nozzle by screwing it onto the left thread of the base piece until it is tight.
  6. Attach the outlet piece with the larger hole by screwing it on to the right thread of the base piece until it is tight.
  7. Hold a filter sheet against the hole of the outlet.
  8. Thread the outlet guide onto the small outlet thread to ensure the filter sheet is in place.

3D Printed Parts Files
Use .STL files to 3D print parts.

Use .STL files to 3D print parts.

Proof of Concept

The filter medium was successfully fabricated and shown to support the growth and adhesion of Saccharomyces cerevisiae in conditions relevant to continuous flow applications. Visual and microscopic analyses as presented in the Filter Permeability experiment confirmed the presence of yeast cells throughout the porous interior of the medium. This verified that the structure can sustain biological activity necessary for use in bioaccumulation-based water bioremediation applications.

S. cerevisiae cells
Figure 2: Phase contrast microscopy image of filter medium cross section showing S. cerevisiae cells in and around a micropore.
In the same experiment, it was demonstrated that water can flow through the rehydrated filter medium at a controllable rate. This allows users to control the contact time between the water and the yeast cells, which is necessary for effective water treatment. Additionally, the final 3D printed filter apparatus displayed no leakage or damage, confirming its watertight integrity and compatibility with the filter medium.

Dyed water
Figure 3: Dyed water permeating through rehydrated filter medium.
The freeze casted filter media were reproducible and demonstrated good structural properties which allow it to retain its structural integrity and shape when subjected to a continuous flow of water.

Medium replicates
Figure 4: Freeze casted filter medium replicates.
These results demonstrated the feasibility of the system’s biological, material, and mechanical components, confirming that they can function together as an integrated unit suitable for further development in continuous flow water filtration applications. For detailed experimental methods and results, refer to the Experiments Page. .

Feasibility and User Testing

Experts from microbiology, engineering, and water treatment backgrounds in academia and industry including Stephanie Willerth, Guneet Kaur, and Everett Horner were consulted to ensure that the developed hardware meets the needs of microorganisms and can be realistically implemented in water treatment infrastructure. Throughout its development, the filter medium was tested with S. cerevisiae from the iGEM Guelph Wet Lab team to validate the design and gain user feedback. This iterative approach to development is described in the Hardware Engineering Cycle.

Experimental Results

Experimental results can be found on the Hardware Experiments page.

Next Steps

1. Optimize the filter medium
Determine the filter medium composition that results in optimal growth and adhesion of S. cerevisiae cells.

2. Modify filter medium to support other microorganisms such as Escherichia coli
The current filter medium can easily accommodate other microorganisms by substituting existing nutrients in the filter medium (yeast extract, tryptone, glucose) for the nutrients needed by the new microorganism.

3. Determine the lifespan of the filter medium
Perform growth experiments with inoculated filter media to determine how long the cells can survive on the filter medium when a continuous flow of water is applied.

4. Test performance of genetically engineered S. cerevisiae
Once the iGEM Guelph Wet Lab team produces genetically engineered S. cerevisiae capable of absorbing lead, experiments can be conducted to assess its performance in water filtration applications and determine critical parameters such as water flow rate and contact time required for optimal treatment.

Documentation

A detailed protocol for how to make a filter medium and adapt it for use with other microorganisms can be found on the Hardware Protocols page.

Filter apparatus documentation including 3D printable parts files and assembly instructions can be found in How It's Made.

References

[1] Millipore Sigma. (n.d.). Agarose: Properties and Research Applications. Millipore Sigma. Retrieved May 5, 2025, from https://www.sigmaaldrich.com/CA/en/products/chemistry-and-biochemicals/biochemicals/agarose?srsltid=AfmBOor0-clvHaNQABFQvAFMHQvZe6ia9ek_M-hBtjlM1gj3PMx43-jv

[2] Emami Moghaddam, S. A., Harun, R., Mokhtar, M. N., & Zakaria, R. (2018). Potential of Zeolite and Algae in Biomass Immobilization. BioMed Research International, 2018, 6563196. https://doi.org/10.1155/2018/6563196

[3] Zarrintaj, P., Mahmodi, G., Manouchehri, S., Mashhadzadeh, A. H., Khodadadi, M., Servatan, M., Ganjali, M. R., Azambre, B., Kim, S., Ramsey, J. D., Habibzadeh, S., Saeb, M. R., & Mozafari, M. (2020). Zeolite in tissue engineering: Opportunities and challenges. MedComm, 1(1), 5–34. https://doi.org/10.1002/mco2.5

[4] Guastaferro, M., Baldino, L., Reverchon, E., & Cardea, S. (2021). Production of Porous Agarose-Based Structures: Freeze-Drying vs. Supercritical CO2 Drying. Gels, 7(4), 198. https://doi.org/10.3390/gels7040198

[5] Balgude, A. (2001). Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures. Biomaterials, 22(10), 1077–1084. https://doi.org/10.1016/S0142-9612(00)00350-1

[6] Willaert, R. G. (2018). Adhesins of Yeasts: Protein Structure and Interactions. Journal of Fungi, 4(4), 119. https://doi.org/10.3390/jof4040119

[7] Palecek, S. P., Parikh, A. S., Huh, J. H., & Kron, S. J. (2002). Depression of Saccharomyces cerevisiae invasive growth on non‐glucose carbon sources requires the Snf1 kinase. Molecular Microbiology, 45(2), 453–469. https://doi.org/10.1046/j.1365-2958.2002.03024.x

[8] Pfeifer, A., Škerget, M., & Čolnik, M. (2021). Removal of iron, copper, and lead from aqueous solutions with zeolite, bentonite, and steel slag. Separation Science and Technology, 56(17), 2989–3000. https://doi.org/10.1080/01496395.2020.1866607

[9] Keiken. (2025, March 21). What are the Benefits of Zeolite for Water Filtration Purposes? Keiken. https://www.keiken-engineering.com/news/what-are-the-benefits-of-zeolite-for-water-filtration-purposes#:~:text=Zeolite%20is%20a%20non%2Dtoxic,water%20filtration%20and%20treating%20wastewater.