Integrated Human Practices

Real people, real solutions

Human Centred Design

Catering Our Solution to People's Needs

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

Stakeholder Interviews

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.

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.

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.

Figure 1. 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.

Verifying Need

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.

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.

Preliminary Feedback

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.

Design solutions

Expert Consultations

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.

Antimicrobial Integration

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.

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.

Strengthening BC

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.

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.

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.

Figure 2. 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.

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.

Faculty Talk

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.

Figure 3. 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.

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.

Future Directions

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.

Further Applications

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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