With the global rise in food costs and increased efforts to reduce single-use plastics, there is an urgent need for a sustainable food preservation solution to curb food waste and increase access to fresh fruits and vegetables (1). With Cellucoat, our vision is to propel this shift toward sustainable packaging for a more sustainable future. The only way to achieve this goal was to integrate our stakeholders and end users, every step of the way.
We used an iterative design process throughout the development of our project to shape a solution that is, at its core, human-centred. This is the story of the individuals who inspired and shaped Cellucoat into the project it is today: a sustainable packaging solution, and a step towards a less wasteful future.
Understanding the Problem
When Cellucoat was conceived, we knew that current food packaging was simply not cutting it. Personal experiences with prematurely spoiling produce and ever-increasing costs sparked the development of this project. However, before moving forward in the lab, there were questions we needed to better understand. What is the extent of waste, and what are its contributors? What are the limitations and key characteristics of current packaging solutions? Is there demand for a sustainable alternative?
To address these questions, we considered all parts of the produce pipeline. From farms to distribution facilities, and from grocery stores to the consumer’s home, we wanted to understand the problem from all angles.
Christopher Clark: Star Produce
Christopher Clark is the packing manager at Star Produce, one of Canada’s preeminent distributors of fresh fruits and vegetables. His expertise lies in the produce packaging and distribution pipeline.
From our conversations with Chris, we learned that the entire process - from growing to processing to shipping - is carefully controlled to preserve fruits and vegetables. Unless it is mouldy, any product that isn’t sold to retailers is redirected into other products. However, despite these measures, spoilage remains an issue. While this is a universal concern, the problem of preservation is unique in the context of the local market. Unlike other regions, where produce is usually sold on the same day it is harvested, the product we see on the grocery store shelf in North America has been harvested, packed, trucked, passed through a warehouse, and then been transported to the retailer to be sold. This process can take up to two weeks, and can be extended even further by shipping delays. This issue is exacerbated by unique consumer behaviour. Whereas their European and Asian counterparts tend to shop at most several days ahead, consumers in North America shop for a week or two of groceries at a time. As such, any signs of decay or damage are undesirable. Together, these factors boil down to a simple conclusion: shelf life is very important.
Current packages generally serve two purposes: first, they separate produce into the specific “catch weights” - or amounts - specified by retailers, and second, they provide physical protection against bruising and damage. This is especially important for softer products, such as stone fruits and berries.
Finally, plastic use is an important consideration for distribution companies. In general, packers walk a fine line between using an appropriately minimalistic amount of plastic to reduce excess waste, while still using enough to prevent damage or a reduced shelf life. According to Chris, the vast majority of current packaging, especially for fruits and vegetables, is still made out of plastic. This is largely due to a lack of viable alternatives.
Chris Messent: CFP
Chris Messent is a category manager at Consolidated Fruit Packers (CFP), a company which markets and exports a variety of fruits from British Columbia. The CFP is also the berry procurement specialist of Star Produce. We learned about the berry harvesting, processing and shipping pipeline. Berries are picked and transported to packing houses, where they are cooled and sorted: some are packed right into consumer clamshells, and others are placed in trays. Once transported into a packing line, the berries are filtered and inspected to remove debris, leaves, and soft or discoloured berries. The filled clamshells are packed into mastercases, and then stacked in pallets to be transported from distribution centres to retailers. Beyond understanding the produce pipeline, this information gave us important insights on the characteristics of a viable package.
CJ is a produce manager at Save-On-Foods, and shared expertise about food and plastic waste at the retail level. Despite careful planning, losses due to excess and unsatisfactory produce represent 4-5% of the store’s weekly revenue. The food waste problem is complex: since consumers shop on a weekly or bi-weekly basis, they choose products that are most likely to last long in their homes. The preference for spotless and slightly unripe fruit means that entire crates of objectively sellable produce often end up being thrown away. For example, when we met with CJ, he showed us palettes of ripe bananas that would be discarded if they weren’t sold by the end of the day (Figure 1). Although Save-On-Foods has a closed-loop program that sends so-called “shrinkage”, or unsellable products, to local farms as feedstock, such initiatives are not widespread. Based on his experience as a produce manager at other stores, CJ shared that common practice is to send this waste to the landfill. Importantly, mould and spoilage is also a significant contributor to waste. For example, berries in clamshells are very vulnerable to going bad, and even a single rotten berry makes the entire package unsafe to eat. This highlighted the need to reduce spoilage, in the hopes of combatting this waste issue.
Ripe and whole bananas, which would be disposed of if unsold on the day the picture was taken.
Although the strawberry crates were cardboard, the product inside was packaged in plastic.
Plastic waste was also prolific. CJ showed us the crates that produce arrives in at the store: while most of the palettes were cardboard, they were full of plastic packaging (Figure 1 and 2). Approximately 300lbs of plastic packaging were thrown out weekly. Despite efforts to reduce the extent of packaging in recent years, the amount of plastic used in the store increased in the pandemic, as consumers associated packaging with health and safety. This plastic is not biodegradable, and a viable alternative is vital. The Save-On-Foods we visited also generously donated spoiled fruits for us to swab.
Consumers and Farmers
As part of our effort to understand the consumer perspective on food waste and packaging, we conducted interviews at the Calgary Farmer’s Market. We asked two questions to validate whether they experienced high levels of food waste, and whether plastic use was excessive and an alternative would be appropriate. We learned that, while some shoppers had developed strategies for reducing food waste in light of diminished shelf lives, others had not. Moreover, 83% of respondents indicated displeasure in the amount of plastic packaging they encountered, and cited the need for an alternative.
This information validated the need to create a solution that both addressed food spoilage to reduce food waste, and the overabundance of plastic in produce packaging. Beyond customer discovery, we also interviewed vendors at the Calgary Farmer’s Market to understand the issue of waste from their perspective. We learned that they, too were impacted by food spoilage: when sending their produce to retailers, spoiled or unsatisfactory product was shipped back to them, and they had to shoulder the burden of these revenue losses. Although they had systems in place to reuse the unsold food at the market level, these systems were small in scale and did not extend to produce sent back by retailers.
The Indigenous Perspective
Food accessibility is an especially critical issue in remote communities, where long transport distances reduce the quality and increase the costs of fresh food. This issue is exacerbated in remote Indigenous communities. As such, we wanted to explore the potential to cater our solution to the specific needs of these communities. To ensure our solution was appropriate and reflective of their needs and interests, there were several key questions we needed to consider. What factors would we need to consider to develop a meaningful, trusting partnership? How could we ensure our solution and research methods were reflective of Indigenous values and interests? How would we go about finding a community interested in pursuing this partnership?
To take the first step in understanding these questions, we met with Nicole Ritchie from the University of Calgary’s Indigeneous Research Support Team (IRST) and Gordon Munroe, a biotechnologist at the Global Institute for Food Insecurity.
Gordon Munroe: Global Institute for Food Insecurity
Gordon Munroe is a biotechnologist, and a researcher at the Global Institute for Food Insecurity (GIFS). We reached out to Gordon for a perspective on the feasibility of a collaboration with an Indigenous community. Though he emphasised that he cannot speak for all Indigenous people, he reiterated the importance of developing trust. In the context of historically inappropriate research within such communities, this process requires time and careful curating. When approaching these communities, it was very critical that we avoid an attitude of white saviorism, and instead propose an equal partnership. Moreover, we learned that environmental stewardship is a widespread value in Indigenous culture, and this would have to be reflected in our project design. Such a partnership would have to balance the value of spirituality with the desire for innovation and developmental opportunities.
Beyond a partnership, Gordon also suggested that we could reciprocate any contributions by sharing our experiences as students in sciences, engineering, technology and engineering (STEM) with students in the community.
Research Grant Officer: Indigenous Research Support Team, University of Calgary
The Indigenous Research Support Team (IRST) at the University of Calgary is a body that serves as a point of contact for university researchers aiming to work within Indigenous communities. Our goal was to understand what the process might look like if we wanted to move forward with an Indigenous research partnership.
The IRST emphasised several important points during our meeting. First, we were reminded that Research Ethics Board (REB) approval was critical for any such projects. To develop a meaningful collaboration, we would need to build a relationship rooted in trust, which takes time to develop. In this process, we would need careful mentorship and guidance to ensure our approach and communication was culturally sensitive and reflected the specific and unique needs of the community we were interacting with. Importantly, the best approach for developing this relationship would be to reach out to a specific community we would be interested in working with, and, depending on their interest, work collaboratively with them to shape our project into a solution that reflected their values and needs. This relationship can take several years to develop.
Feedback on a Partnership with Indigenous Communities
Our conversations with the IRST and Gordon Munroe illuminated several key considerations for moving forward with a long-term collaboration with an Indigenous community to develop our solution. Critically, they both highlighted the developing trust and a long-term, meaningful connection with a specific community. This rapport needs to be carefully curated with a select community through communication with elders and band leaders, and developed over time.
Unfortunately, based on the limited time frame of our research period and the size of our team, we determined that we would not have the resources to pursue this avenue in a meaningful way. Although our focus shifted upstream to a more general packaging solution, we hope its downstream effects will also benefit these communities, and we are open to the idea of catering our solution to Indigenous communities in the future. During this iGEM season, however, we still had the opportunity to support an event hosted by IndigeSTEAM, an organisation dedicated to increasing Indigenous representation in STEM and arts, architecture and agricultural education. Read more about our education and communication initiatives here.
What Else Did We Discover?
Through our discussions with industry stakeholders and end users, we learned that food waste is a significant issue, and a viable alternative to single-use plastics is highly sought after.
Unlike other markets, produce in North America faces the unique challenge of having to endure up to two weeks of transport before it reaches consumers, which heightens the need for a means to increase shelf life. The fruits and vegetables grown by farmers are sorted, and any unsatisfactory produce is either repurposed into other products or thrown away. Once sorted, the produce is packed at distribution centres, stacked into pallets and shipped to grocery store warehouses. At the store, the product is displayed: any defective produce is either returned and footed by farmers, or sent to the landfill. Finally, by the time the product reaches the customer, it sometimes only lasts for several days before being thrown away. The key takeaways? Produce waste is extensive: it travels weeks from the fields to people’s fridges, and there are significant losses in every part of the pipeline because it perishes too quickly.
Importantly, all of the stakeholders we spoke to were unsatisfied with the extent of plastic in the produce pipeline. While farmers generally rely on reusable palettes, the consumer packaging that holds produce for the remainder of its lifetime is overwhelmingly made of single use plastics. Finally, existing biodegradable alternatives have yet to find a foothold in the industry, mostly due to physical properties that simply do not measure up to plastic.
Designing a Solution
After identifying the current state of produce waste and packaging, we began to shape our solution around the input of our key stakeholders.
Our design process began with the selection of our packaging material. While plastic is detrimental to the environment, our research on conventional biodegradable alternatives such as sea-weed or paper-based polymers proved disappointing: these materials have poor barrier strength and lose structural integrity when exposed to moisture (2). However, after a review of literature, we came across bacterial cellulose (BC). This material is a bacterially produced, biodegradable polymer with high mechanical strength and foodsafe (3). Importantly, BC can be functionalized with antimicrobial elements (4).
Spoilage is one of the primary factors that contributes to the reduced shelf life of produce. Our stakeholders had highlighted the losses - both in product and in profit - that occur throughout the produce pipeline. To address this, we decided to functionalize BC with lysozyme, a naturally antimicrobial peptide which is active against mainly Gram-positive bacteria (5). Lysozyme is food safe, and stable at a wide range of pHs and temperatures (5).
Our Original Antimicrobial Design
To produce large amounts of purified lysozyme, we designed a potential expression system of lysozyme in E. coli. To prevent lysozyme from lysing E. coli as it is produced intracellularly, the construct included a lysozyme inhibitory protein which would be removed prior to use (6). According to our design, the purified lysozyme would then be adsorbed into BC, producing an antimicrobially-active packaging material.
Proposed design for lysozyme production in E. coli. This approach and diagram was presented to judges at the MindFuel Tech Futures Challenge competition.
Finally, to apply the packaging, we initially proposed that the active BC would be wrapped around individual fruits. This approach was designed to maximise the surface area of fruit with antimicrobial coverage.
Although our project was designed with stakeholder needs and concerns in mind, it was important to validate a need for our proposed solution. We identified produce distributors as our consumer, and the people purchasing produce as our end-user. With this in mind, we reached out to both our consumers and end users to verify the need for our project and discuss its implementation in their industries and communities.
Christopher Clark: Star Produce
Beyond supplying fruits and vegetables, companies like Star Produce also value innovation throughout the production pipeline. For example, When speaking with Chris, he emphasised a continual interest within the industry for novel solutions, and the packaging field is no exception. Our proposition of a biodegradable packaging alternative was met with enthusiasm, and Chris was also open to meeting about further iterations of our project.
While our original idea was to create a wrapping or coating, Chris suggested that a box format would have much wider applications. There are very few fruits where the respiration rate is so sensitive that it has to be fully encased, and boxes require less labour to apply, as they can simply be filled rather than needing to be shaped. To put things into perspective, “a box would serve about 95% of produce; a wrapping might apply to 5%”. Finally, while recycled plastics are used for some packaging types, plastic clamshells are almost exclusively made of new plastic, and there are very few systems in place to recycle it efficiently. In terms of prototyping, we learned that most clamshells or boxes come in a range of standard sizes and shapes. By focusing on developing a few specific “die-lines”, or patterns, we could streamline the production process of our packages. Boxes are also more compatible for stacking in shipping crates and containers, which makes transport more efficient and less wasteful: trucks can be completely filled, and the lack of extra air space makes refrigeration more efficient.
In terms of the antimicrobial component of our project, we learned that a material with such properties could be very useful: beyond reducing the general risk of spoilage, this could eliminate the need for the absorption pads placed in the bottom of packages, which serve to absorb moisture and prevent decay or mould.
Building on our previous interview strategy, we also assessed public perspectives on our proposed solution. We learned that consumers are interested in product that could prolong shelf life. Our questions also considered how people would feel about using a sustainable packaging made by bacteria. Although some expressed hesitation, when we emphasised that the BC would be purified and made references to beer and bread, which also rely on microorganisms, their responses were much more receptive.
The feedback we received from our stakeholder interviews was multifaceted. Our customer discovery initiative, where we interviewed shoppers at local grocery stores and farmers markets, were integral in validating both the problem we aimed to address, and the need for a sustainable solution. Customers were dissatisfied with the extent of plastic they encountered while shopping, and expressed a desire for longer lasting produce. Importantly, these interviews showed an openness, and even interest, in a bacterially-produced, biodegradable alternative.
In our second conversation with Christopher Clark, he offered several key perspectives on our project. First, his openness to discussing our biodegradable packaging alternative was, as he shared, reflective of a greater trend in the instrustry towards more sustainable materials. A specific area of improvement is the conventional plastic clamshell. This packaging form is prolific in produce packaging, and is made almost exclusively of “new”, or non-recycled, plastic. Moreover, there is no infrastructure to recycle these on an industrial scale, so many end up in landfills. This information verified a need for a sustainable solution at the distributor level, and inspired us to rethink the way Cellucoat would be applied. Rather than an edible wrapping, we realised that our project had the potential to be much more widely impactful as a clamshell replacement.
Beyond validating the need for our solution, we also sought feedback on the technical aspects of our proposed design. As such, we participated in the project pitch component of the Mindfuel Tech Futures Challenge, where judges raised some important questions about the application of our proposed packaging, and the feasibility of recombinant lysozyme expression. Although BC and our chosen antimicrobial peptide are edible, this may detract from the texture or mouthfeel of the food it encases. This approach would also have to meet strict health and safety regulations. Finally, while lysozyme expression is technically possible with the lysozyme inhibitor, this could overwhelm the E. coli cells and limit production levels.
This feedback informed several major shifts in our initial design. First, we started to explore literature for alternative antimicrobial peptides, and came across nisin. Nisin met all of the requirements to be effective for our intended application: it is temperature and pH stable, food safe, and active against spore formation and Gram-negative bacteria (7). Nisin is not only compatible with BC, but actually demonstrates improved antimicrobial activity and stability when immobilised in BC (7). Finally, nisin can be recombinantly produced in E. coli without an inhibitory protein, which facilitates production (8). Together, these factors solidified nisin as a more appropriate choice for our project. Our final proposed plan was to integrate nisin via a co-culture method, where the nisin secreted from our recombinant E. coli would bind onto BC fibres as they were produced by K. xylinus. To assess the amount of nisin attached to BC, we considered adding a split-green fluorescent protein (GFP) tagging approach, where conformational change of nisin as it bound to BC would induce fluorescence. This could indicate when the packaging was no longer antimicrobial, which would be beneficial if it were reused.
Finally, the hesitations about Cellucoat as an edible film solidified our pivot towards a clamshell-like packaging. Not only would this form provide a potential alternative to a widely used, highly wasteful product, but it would also circumvent concerns about texture, taste, and consequences of ingestion.
With the first iteration of our design complete, we sought input and feedback about the various components of our project.
BC Production and Properties
To begin our experimental process, we needed to understand how to effectively produce and quantify the mechanical properties of BC. In our initial conversations with Dr. Jinguang Hu, we learned that as BC dries, its fibres contract and have less space between them. He cautioned that this can make the in situ incorporation of pure peptides and enzymes difficult, and that this approach has largely been unsuccessful in his experience. Moreover, because of its tight matrix structure, the only functional group exposed on the surface of BC is a hydroxyl group, which limits any approach to adding nisin with a BC binding domain. Based on these insights, we rethought our approach to integrating nisin into BC: rather than secreting nisin, the recombinant E. coli would simply be incorporated directly into the BC matrix as it formed. The BC could be processed ex situ to lyse the cells, leaving nisin secured in the tightly woven BC fibres. With this potential pivot in mind, we sought feedback on the feasibility of this idea.
In terms of testing BC properties, Dr. Hu encouraged the idea of conducting uniaxial tests to assess the tensile strength of BC. Dr. Hu also generously provided us with a starter BC culture, from which we grew all of our test samples and prototypes.
Next, we sought to better visualise what a BC material could look like, and how we might bridge the gap between a material grown in a culture plate and an aesthetic, marketable packaging. Christopher Clark and John Kelly emphasised the fact that customers are visual shoppers, and that some degree of transparency is key to a viable packaging. Colours and labels are also important, as they allow customers to differentiate between brands. At a more practical level, these factors allow employees to easily sort items, which streamlines the shipping process. With these considerations in mind, we met with Juliana Schneider, who demonstrated that BC can be dyed both in situ and ex situ, producing a semi-transparent coloured effect, and can be printed on with regular ink. Together, these properties were promising for the integration of our product into the packaging market.
The samples Juliana showed us were robust despite being several years old, which had promising implications for the durability of BC materials. Finally, we were inspired by Juliana’s production process to grow our BC in much larger containers, which we used to develop our later prototypes.
Dr. Jinguang Hu: University of Calgary
Dr. Jinguang Hu is an engineering professor at the University of Calgary, whose research focuses on sustainable energy, biomass valorization, and bioinspired materials. In our conversations with Dr. Hu, we learned that the physical properties of BC change drastically as it dries: as water evaporates, the cellulose fibres contract and form a tightly-woven matrix. Though he had previously experimented with loading enzymes onto BC, this property had proved problematic. If we did decide to attempt to add nisin with this approach, he advised that we check that its active sites do not get obstructed by the BC fibres.
To produce BC, aside from the appropriate nutrients, we needed to consider the pH of the media. In a co-culture, the secondary bacteria might have different optimal growth conditions, so he recommended we quantify this effect in some way. This inspired our co-culture model.
In terms of BC’s compatibility for protein binding, we learned that BC only has a single functional group exposed on its surface. This represents a significant barrier for attaching a protein to the surface of BC via a BC binding domain.
Finally, Dr. Hu expressed approval about using uniaxial tests to assess the mechanical strength of BC. He also graciously provided us with a starter culture of K. xylinus, and allowed us to access his lab for more complex wetlab tests.
Christopher Clark: Star Produce
When discussing packaging types, Chris emphasised that customers shop with their eyes, so any packaging has to be both visually appealing and transparent to some extent. This allows consumers to assess the quality of the product, which is critical given the desire to buy fruit that will last between dispersed shopping trips. This was an interesting perspective to consider for our material. We also learned that labels on packages are used to distinguish between brands, and include barcodes or nutritional information.
The appearance of a packaging also has logistical implications. When boxes for different fruits are different colours, employees at the shipping level can identify products without having to “break down”, or disassemble, shipping palettes.
As an aside, Chris also emphasised that packaging is generally not reused. The health and safety requirements for food packaging are strict and would require the packages to undergo extensive processing, so they are thrown away by consumers.
John Kelly: Food To Market Inc.
John Kelly is the president of Food To Market Inc., a company specialising in produce marketing and sales. John is also the co-founder and past president of Otter Valley Foods Inc., and currently serves on the executive board of the Food Producers of Canada. Aside from reiterating several limitations of current biodegradable packaging materials, including vulnerability to degradation when exposed to moisture, John shared that aesthetics are key to a successful packaging. Again, customers are visual shoppers, and value a degree of clarity so they can carefully select which fruits they want to buy. John also pointed out that packages are not reusable, as the process is not cost effective and lacks the infrastructure to support it.
Juliana Schneider is a trend researcher and futures designer who creates stories and strategies that inspire us to engage with the challenges of our rapidly changing world and considers the cross-disciplinary application of design as a strategic component to develop systems that can evolve, learn, and respond more effectively to current and future challenges. Juliana developed a bacterial cellulose packaging material for her master’s project, titled GrowPak. Given her interest in biodesign - the incorporation of living material in textiles and everyday life - Juliana was very interested in our proposed packaging material, and optimistic about its implications on plastic use. Since Juliana grew relatively large quantities of kombucha scoby - a material similar to BC - for her own project, we sought advice for scaling up our BC production. We learned that all of the material produced for GrowPak was grown in large glass containers at room temperature from a donated starter culture. After two to four weeks, the cellulose was air dried and assembled into bags. To seal the edges of the bags, Juliana wet the seams and pinched them together. This information was integral to our prototype development.
Juliana had also experimented with colouring cellulose. Using materials such as spirulina powder, cabbage juice and charcoal, she found that both in situ and ex situ dyeing were effective at changing the colour of her product. The cabbage juice gave the most pigmented result. This inspired our BioSculpting and prototyping approaches. While laserjet methods damaged the material, Juliana also shared that she had successfully printed on her cellulose using regular printer ink.
Surprisingly, Juliana was able to show us the original, intact bags from her GrowPak project, despite them being several years old (Figure 1). This spoke volumes to the durability of BC.
Juliana Schneider with one of her original intact cellulose bags, filled with mixed seeds.
With our co-culture integration approach in hand, we met with Dr. Silvina Steitz for feedback on our GFP-detector system. Dr. Steitz emphasised the difficulty of expressing or linking GFP - a relatively small protein - onto the surface of BC. Combined with the reservations expressed by Dr. Hu, and the fact that produce packagings are almost never reused, we decided to pivot away from a detection system.
In terms of our nisin incorporation approach, Dr. Brianne Burkinshaw was optimistic about the idea of using a co-culture and lysing the E. coli cells once incorporated in the BC. However, she highlighted two important considerations: first, we needed to find a way to determine and control the growth rates of the two bacterial strains in the co-culture, and second, that autoclaving our nisin-loaded BC could be a potential means to lyse the E. coli while purifying it.
This feedback inspired the development of our co-culture model and our BC post-production treatments.
Dr. Brianne Burkinshaw: University of Calgary
Dr. Brianne Burkinshaw is a biology and biochemistry professor at the University of Calgary, whose research specialty lies in the modelling, functionalization, and applications of bacterial secretion systems. Dr. Burkinshaw was optimistic about the feasibility of our proposed method for incorporating nisin into BC using a co-culture, and lysing the E. coli to purify the final product. However, we learned that co-cultures can be very complex, and that it is hard to control the growth rates of the two respective bacterial strains in the culture. Since K. xylinus grows relatively slowly as compared to E. coli, she encouraged us to find a way to regulate their growth by modifying the culture conditions. Dr. Burkinshaw also pointed out that, while nisin can be produced in E. coli, it could be cytotoxic if secreted. This confirmed the choice to pursue our aforementioned co-culture and lysis approach.
In terms of lysing the E. coli cells, Dr. Burkinshaw was optimistic that autoclaving the E. coli-loaded BC would be sufficient to sterilise it. Moreover, as nisin has higher thermostability at a lower pH, we learned that we could protect the nisin in the BC from denaturing by autoclaving it in an acidic buffer.
Dr. Silvina Steitz: FREDsense Technologies
Dr. Silvina Steitz is a researcher and development sciences at FREDsense technologies, whose areas of research expertise include bacterial secretion and toxins. Dr. Steitz was intrigued by our proposition of adding nisin through a co-culture, and then lysing the E. coli cells. However, she cautioned that residual bacterial cell debris could affect nisin’s antimicrobial activity. This encouraged us to pursue additional purification methods, and emphasised the importance of having reliable antimicrobial tests to assess our final B C product.
We also shared our idea of using a split GFP protein as a system to detect when the nisin in the BC was no longer active. However, we learned that this approach can be problematic with larger molecules: adding the tag to BC itself would be incredibly difficult, and the tight weaving of BC fibres would also make the GFP-tags inaccessible to the nisin. Alternative detection systems could be developed using an antibody-based test, but these approaches are often expensive, depending on the antibody. We determined that this system would also be harder to integrate into the produce pipeline, as these tests are more involved than a quick fluorescent scan.
Testing Antimicrobial Activity
Distributing nisin evenly and at a high enough concentration to be effective against microbes would be a challenge. First, we needed to understand which microbes are responsible for rotting on produce. A local grocery store, Save-On-Foods, generously donated spoiled fruit and vegetables for us to swab and culture.
John McGinnis shared some expert advice on techniques for microbial swabbing, plating and imaging techniques. Using potato-dextrose agar plates, dye and a microscope provided by Francene Cusack, we grew the cultures from the produce samples. Francene also suggested that we run Kirby Bauer tests with our nisin to quantify its activity against various microbes.
Josh also helped us analyse the microscope images of our stained microbial samples, and identified the Bacillus strain: this informed one of the key targets for our nisin activity assays. Rather than remaining narrowed in on nisin’s antifungal properties, this also encouraged us to take a step back and explore its antimicrobial properties as well.
Francene Cusack: University of Calgary
Francene Cusack is a microbiology technician at the University of Calgary, who works closely with fungi and other microbes. Given Francene’s technical expertise, we sought to understand more about the process of growing, staining and visualising microbial cultures. Francene informed us that potato-dextrose agar (PDA) is ideal for growing fun gal swabs, and graciously provided us with PDA plates for our tests. She also lent us a microscope and stain, which we used to visualise and analyse the colony morphology of our cultures. Moreover, since many of our initial samples were fungal, we learned how to safely work with fungi and minimise the risk of contaminating our lab with spores. This helped us work safely before transferring our samples to the University of Alberta team as part of our partnership.
Josh McGinnis: CEO, Everyman Bio
Josh McGinnis is a scientist and software engineer, and the founder of EverymanBio, an early-stage startup that explores the therapeutic uses of microorganisms. Josh also uses his Instagram page, Youtube channel and podcast to educate his audience about modern scientific exploration. He covers topics such as genetic engineering, current genomic science methods, mushrooms, ecology, and education. In our meeting, Josh shared several important technical insights on how to swab and culture the microbes on our spoiled produce samples. Our initial attempts at culturing microbial samples swabbed from the Save-On-Foods shrinkage yielded lawn growth, and overgrown colonies with little morphological differences. To manipulate single fungal colonies, Josh recommended that we add a general antibiotic to our plates to curb bacterial contamination. Any shiny growth could be attributed to bacteria. Moreover, Josh shared that fungi can be identified to some extent with macroscopic and microscopic observations, or more precisely through DNA sequencing. Josh also looked at the microscope images of our cultures and helped identify the Bacillus bacteria strain. Together, this information informed the targets for antimicrobial tests.
We also learned that a lot of mould spores are airborne, which is how it comes into contact with the surface of fruit. The type of mould will also depend on the environment the fruit is exposed to, so we could be reasonably confident that the cultures collected from the Save-On-Foods samples would be representative of the microbes present during the packaging, shipment and display processes.
Another general consideration brought up in our meeting was the importance of gas exchange in packagings, as the buildup of moisture can accelerate mould growth. This validated our pivot from a produce wrap to a box shape, which gives the produce inside room to breathe.
Finally, Josh shared some insight on effective science communication, and ways to navigate the public perception of microbes in everyday life. One of Josh’s key points was that people are afraid of what they don’t know. However, by comparing our work to more familiar applications of microbes, such as those used to make beer and bread, we could encourage curiosity rather than fear about our project. We kept this advice in mind as we developed our composting tips for the Calgary Garbage Day app, in an effort to reduce food waste and provide accessible information about useful microbes in everyday applications.
At this point in our project development, the feedback from industry stakeholders had exposed a shortcoming in our current solution: on its own, BC is simply not strong enough. Despite its ability to withstand relatively high strain forces, the BC we had produced was friable and relatively brittle. To address this issue, we proposed to incorporate polyhydroxybutyrate (PHB), a bioplastic, into our BC. Specifically, we wanted to integrate this solution into our existing co-culture model, by adding a PHB-producing E. coli strain to the mix. We also aimed to modify the amounts of phasin - a key protein for PHB secretion - produced in this expression system.
With this idea in mind, we returned to Dr. Burkinshaw for feedback on the feasibility of our proposed PHB secretion method. Although her response to our design was largely encouraging, she cautioned that the tag we added to purify and quantify phasin production be small enough that it doesn’t affect phasins ability to be secreted. Based on this advice, we settled on the addition of a FLAGTM tag, which is short and does not interfere with protein folding.
Together, this information was critical to the development and refinement of our PHB subproject, which constituted the protection component of our project.
Dr. Brianne Burkinshaw: University of Calgary
Beyond nisin integration, we also sought Dr. Burkinshaw’s feedback on our phasin and PHB production plan. The underlying principle of incorporating PHB into BC was positively received, and Dr. Burkinshaw approved of the rationale behind altering RBS strengths to modify phasin expression. Moreover, she emphasised the importance of including enough carbon sources for both the BC and the PHB production. For the insertion of the His-tag into the PHB plasmid, Dr. Burkinshaw cautioned that we carefully consider the protein structure, folding and active sites when selecting one of the enzyme intermediates to tag. Otherwise, we risked inhibiting the protein and inhibiting PHB secretion. The same principle applied to the phasin protein: since we wanted to tag phasin for purification and quantification, the tag we added needed to be small enough that phasin’s interactions with the intracellular PHB granules would not be affected.
Mitigating Production Costs
Costs are critical in determining the viability of a packaging material, where the dominating competitor - plastic - only costs several cents to make. Both John Kelly and Josh McGinnis highlighted the importance of being able to determine, and ideally reduce, the cost of producing our active PHB-BC. This inspired the development of our techno-economic analysis (TEA), a competitive cost analysis of our project.
As we developed Cellucoat, we were aware that BC is expensive to produce, largely due to the cost of glucose in the conventional Hestrin-Schramm (HS) growth media for K. xylinus. Inspired by Juliana Schneider, and a variety of literature, we developed a media supplemented with fruit waste.
Josh McGinnis: CEO, Everyman Bio
As the founder of his own start-up, Josh echoed John Kelly’s sentiment about the importance of cost analyses. We needed to ask ourselves: is our product competitive? For large quantities of product, every additional cent can amount to a huge cost for the producer. Josh recommended we run a competitive cost analysis, so we could identify areas for improvement.
John Kelly: Food To Market Inc.
During our conversation, John emphasised that plastic costs very little to make. As such, being aware of the production costs of our product would be integral to developing a competitive cost analysis. This would also allow us to quantify the impacts of our fruit waste media on cost.
Although BC is expensive, we learned that suppliers consider all aspects of the price breakdown, including downstream costs. If our product showcased effective antimicrobial activity, and reduced product losses by inhibiting spoilage, this could help balance out the increased production costs.
In the development of GrowPak, Julianna used blended vegetable scraps into a puree, which she used in the growth media of her kombucha BC. No noticeable issues in BC growth were observed, and her final BC product was robust. She also supplemented the media with additional sugar.
Sustainibility and Biodegradability
Sustainability is key to the identity of Cellucoat. While all of the components of our antimicrobial PHB-BC material are biodegradable, we had a responsibility to evaluate the downstream impacts of our solution in the world.
Natalia Gonzalez from the Calgary Waste and Recycling Services (WRS) informed us that, while bioplastic products are increasingly popular, they are difficult to integrate into industrial composting programs. Portions of the processed compost are returned to citizens, and the presence of any biodegradable plastic that is not finished breaking down can be confused with regular plastic contamination. Moreover, citizens already struggle to adhere to basic composting guidelines, so adding more complex sorting steps is a concern. These points were confirmed by Craig More when we toured a local composting facility to learn more about the process.
Our trip to the Shepard Compost Facility, where we learned about our city's composting process and pipeline!
These concerns are reflective of a need for development beyond the scope of our team. However, we wanted to support this change as much as possible. We worked with the CWM to develop composting tips for their Calgary Garbage Day app, an initiative designed to increase good garbage handling practices in citizens. We also included tips targeted to reducing food waste, to further our key values of reduced waste for greater food access. Finally, we considered Natalia’s concern about perceived contamination: this informed our prototyping, where we designed our material to meet consumer packaging standards without looking exactly like plastic. Our hope is that such a product would be easier for people to associate with biodegradable materials such as paper, and feel more comfortable composting in the future.
The CWM also shared our optimism. Although bioplastics are not currently eligible for industrial composting, the composting facility runs annual product tests with the hope that biodegradable products can be accepted in the future. As such, they generously included our PHB-BC composite samples in their tests. While they did not entirely degrade, they lost significant biomass and turned a dark brown colour. The natural appearance of the composted material is similar to the wood chips and leaf particles that remain in compost and are left to degrade further in soil. We are optimistic that this improves its probability of being accepted for composting in the future.
All together, this marks a step towards integrating Cellucoat into a more sustainable future.
Belinda Li: Co-director of Food Systems Lab
Belinda is the Co-Founder and Director of Food Systems Lab, a research and innovation hub at Simon Fraser University that works on solutions for equitable collaboration for sustainable food systems. She uses a combination of participatory design and data-driven approaches to develop, test, and evaluate solutions to improve food system sustainability from farm to fork. She led the design and implementation of a social innovation lab to work on systemic solutions to the social and environmental challenges of bioplastic food packaging.
In our meeting, Belinda’s background informed our sustainable development goal of responsible consumption and production. She also highlighted how bioplastics contribute to environmental pollution as much as regular plastics. This is because people are encouraged to throw bioplastics away, as composting facilities currently do not have the infrastructure to support the degradation of bioplastics. She indicated that many composting processes take the “if it looks like plastic, it is plastic” approach when separating different materials. Thus, Cellucoat’s cradle-to-cradle design may not work at this point as this problem requires systemic change. This emphasised to our team that composting facilities need to be able to qualitatively tell the difference between Cellucoat and other plastics. This reinforced testing iterations of Cellucoat that involved colouring; which functions to make the packaging more appealing to grocers as well as acting as a way to differentiate between different materials for consumers who are responsible for properly disposing of our packaging and composting facilities which break down Cellucoat.
Belinda also discussed the pertinent issue of how many bioplastics claim to be degradable and compostable, when they actually are not. This contributed to Cellucoat incorporating this design aspect into our development processes through testing the compostability of our various Cellucoat iterations.
Natalia Gonzalez: City of Calgary
Natalia Gonzalez is a waste diversion specialist for the City of Calgary’s Green Cart Program (GCP), a division of the Waste and Recycling Services (WRS) program which provides food and yard waste collection services. Natalia has over 15 years of experience in waste management and environmental consulting, and her field of expertise lies in the handling of residential and commercial waste.
In our meeting, Natalia helped contextualise the extent of food waste in Calgary. Collections from the family sector, once processed, represent approximately 130 million tons of compost.
Aside from biodegradable compost bin liners, we learned that bioplastics are not currently accepted by the GCP. Natalia explained two reasons for this. First, allowing some plastics but not others could confuse citizens and increase the amount of non-biodegradable plastic contamination. Second, as samples of the processed compost are donated to citizens for personal use, the presence of plastic materials could be perceived as contamination and harm the reputation of the composting program. In contrast, natural looking materials, such as wood and paper particles, are considered acceptable and would not raise alarm.
However, Natalia shared that the composting program is optimistic about integrating bioplastic in the future. The facility runs annual product testing, where various materials are bagged and run through a cycle of industrial composting at the Shepherd Compost Facility in Calgary. On behalf of the WRS, Natalia graciously included our BC and PHB-BC composite samples in these tests, which became significantly smaller and took on a dark brown, shrivelled appearance. Based on the public acceptance of natural looking particles, this has promising implications for the future compostability of Cellucoat.
Craig More: City of Calgary
Craig More is the program manager of organics at the City of Calgary, whose expertise includes sustainable design. Craig generously gave us a tour of the Shepherd Compost Facility (SCF), a local, state-of-the-art LEED certified facility which processes approximately 600 million kilograms of waste annually. The facility was completed in 2017, with the goal of reducing the amount of biodegradable waste being sent to the landfill. After the two month treatment from raw waste to compost, the byproduct from the SCF is sold to mitigate facility costs. As Natalia mentioned, 5% of the finished compost is given away to citizens each year. Based on the success of this facility in redirecting organic waste from landfills, we were curious if these benefits could one day extend to packaging.
During our tour, Craig gave an in-depth explanation of the industrial composting process. Products from the GCW are processed for obvious contamination and travel along conveyor belts into composting vessels. In the vessels, air is forced through the shredded raw material to increase oxygen content, and remains at a carefully-maintained humidity and temperature. The final compost is screened for metal or plastic contaminants, cured, and stored. In total, the process takes approximately 40 days. Based on literature, we were optimistic that our BC and PHB-BC samples would also degrade significantly within this time-frame.
In terms of composting bioplastics, Craig affirmed that they are currently not accepted. This is in part because facilities have not been designed to process them. As Craig puts it, “We were off to a rocky start because there was no connection between product manufacturers and the composting industry.” In the meantime, Craig emphasised that we should focus on making our material look more natural than plastic-like. While the industrial composting of bioplastics is possible in the future, this information encouraged us to emphasise and explore the characteristics of the BC aspect of our polymer.
Evaluate and Iterate
Seeking Further Feedback
At this point, the feedback and perspectives shared in our conversations with key stakeholders had evolved our project significantly. To evaluate our latest iterations, we wanted to turn back to the community we aimed to benefit with our project.
For another round of expert input, we invited experts from various fields to consult on the feasibility of our project. Our audience included academic contacts, industry stakeholders, and individuals we had consulted about the sustainability of our project. The feedback about the intent and design of our solution was overwhelmingly positive. One area of improvement was the structure of our presentation, to emphasise the impact of what our solution could do for the consumer, and to clarify what the final product would look like. This inspired us to develop a larger prototype that more clearly reflected our vision for a plastic clamshell replacement. To clearly illustrate the ability to delay spoilage and maintain produce quality for longer, we applied our antimicrobially active BC to grapes and filmed a time-lapse video. The faculty talk also allowed us to organise the work we had done into a story that reflected both our vision and our efforts. Based on the notes from the audience about the structure of our presentation, we made changes to maximise clarity and impact.
Cellucoat's Faculty Talk in August, where we presented our project to professors from the University of Calgary along with several of our HP contacts.
Additional Expert Input
For another expert evaluation of our project, we presented our work to Robert Mayall. Beyond sharing important insights for how to bring our narrative together, Robert suggested some final experiments to tie together the threads woven into our project. Based on his feedback, we developed a GFP colour grader to produce quantitative support for the even distribution of E. coli in BC with our co-culture method. Robert’s advice on our delivery also informed edits to our presentation to ensure our story came across in the way we envisioned.
Robert Mayall: FREDsense Technologies
Robert Mayall is the CTO and co-founder of FREDsense Technologies. Beyond his expertise in the field of synthetic biology, Robert is also an iGEM alum and a former mentor the University of Calgary’s iGEM team.
After our presentation, Robert suggested some technical improvements for our script and presentation style. For example, we were encouraged to emphasise the implications of our project at a global scale, and to ensure we were presenting our work as a future solution, rather than an immediate fix. We incorporated these in our edits to perfect our pitch for the iGEM Giant Jamboree.
Robert also recommended we present quantitative results for our antimicrobial tests, to accurately reflect the work we had done in the lab. Finally, he suggested that we quantify our results showing the even distribution of GFP in E. coli, since it was an important aspect for our proof-of-concept. To do so, we decided to revisit a GFP-colour grading software that we had started to develop earlier in the summer.
A Final Reflection
As we entered the final stretch of preparations for our presentation at the Giant Jamboree, we realised we still had a few questions that had been left unanswered. While the technical elements of our project had fallen into place, we still weren’t sure whether our results reflected a viable packaging solution with the actual potential to impact food and plastic waste. Was our BC strong enough? Was the cost of Cellucoat still too high? To answer these questions, we returned once again to Christopher Clark, whose perspectives had shaped many of the original aspects of our project design. Despite our concerns, his perspectives on the future of our project were overwhelmingly optimistic. According to Chris, our packaging would not need to be as strong as plastic: it simply needed to be able to withstand stacking and filling without bulging open. Moreover, impending plastic bans are being taken seriously, so while our BC might still be expensive, these costs are generally accepted as companies continue to invest in sustainable materials. Overall, our project had the potential to represent an important step towards a future of sustainable packaging and reduced waste.
Christopher Clark: Star Produce
Our final presentation was met with enthusiasm. According to Chris, current clamshells are much stronger than they need to be: the only packages that really offer structural support to produce are bags, and large tray containers. While our uniaxial tests had used PET plastic as a reference material, our future tests could simply assess whether Cellucoat could withstand stacking and would not bulge open when filled.
In terms of costs, there is a general understanding and acceptance within the industry that sustainable materials will cost more. The structure of plastic ban legislation has left the food packaging industry several years to come up with alternatives, but the majority of the options available - including paper-based products - are two to three times more expensive to produce than plastic. We learned that companies already foot these costs at a smaller scale to pack organic products, and such materials will become increasingly widespread.
Chris offered an additional insight on cardboard- and paper-based materials. Paper is a limited resource, and the rising global demand for packaging boxes has led to shortages. This was evident during the COVID-19 pandemic. Additionally, since paper absorbs moisture, the insides of paper-based produce packages are coated with wax derivatives to prevent deterioration. This is an issue: to be accepted for recycling, the coating cannot make up more than 2-3% of the product. In comparison, BC is composed of similar cellulosic fibres to paper, so only PHB would be considered a foreign material by recycling companies. We also demonstrated that BC can be made from food waste, which, as Chris stated, “will always be around”. Together, these comprise several compelling arguments for the future implementation of a BC-based material.
International Fresh Produce Association (IFPA)
The IFPA is a trade association that represents companies from every segment of the global fresh produce supply chain. This organisation drives changes within the industry based on international strategies to increase nutrition. Christopher Clark emphasised that this organisation is chaired by leaders in the industry, and would play an integral role in determining the future of produce packaging, both globally and in Canada. We aim to connect with the IFPA to discuss the future of synthetic biology solutions such as Cellucoat, and how we might meet the growing needs for a sustainable packaging alternative while working to reduce food spoilage. In this vein, our team is also exploring the opportunity to attend the IFPA’s Global Produce & Floral Show at the end of October.
Our goal is to improve Cellucoat beyond the 2022 Giant Jamboree, with the hopes that our project could enter the produce packaging market. As such, there are several realms of future research for us to explore. First, to more easily integrate Cellucoat into industrial production lines, we aim to test the compatibility of PHB-BC with hot press shaping technology. This would greatly facilitate its shaping into boxes.
Food waste is endemic to fruit and vegetable production. However, Cellucoat is designed to leverage these byproducts as a means to produce BC and reduce cost, which eliminates the risk of feedstock shortages. As growing seasons are variable between product types, we aim to reach out to Canadian grower’s associations to establish a source of fruit waste. This includes the BC Fruit Grower’s Association and the BC Tree Fruits Cooperative.
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