Colouring

Overview

Bacterial cellulose (BC) is a powerful biomaterial used in a variety of industrial and medical contexts around the world. Its usage as a packaging, however, has been historically limited due to a variety of factors. Though some of these factors involve its structural and chemical properties, significant attention has been drawn to its unconventional appearance, naturally unappealing colours, and lack of transparency. The colouring subproject was created to mitigate many of these concerns and to develop solutions relating to the customization and colouring of our BC product.

Background and Existing Packaging Techniques

In a meeting with Chris Clark, Category Director of Star Produce, we learned that humans are very visual shoppers, especially when shopping for perishables like fruits and vegetables. To cater to this demand, packaging companies have created transparent or translucent packaging, allowing consumers to get a preview of the product they intend to purchase. Furthermore, packaging companies may also incorporate a variety of colours and styles into their products. This allows for a number of benefits for the produce companies purchasing these packages; firstly, colour is an important part of marketing a product, and being able to utilise that as part of a company’s image is a valuable part in ensuring repeat customers; secondly, colouring the packaging also allows for fruit companies to hide slight visual defects that would otherwise render a perfectly edible fruit unsellable.

Though it may seem as if these options are somewhat at odds, the dual goals allows for flexibility when producing the BC; not all packaging needs transparency and not all packaging needs to be coloured. By developing both options, we allow for the choice to be made on a per-product basis, helping us to fulfil the same role in the market as plastic packaging.

These two directions, transparency and colouring, quickly became the primary goals of this subproject, and research into existing literature and techniques for achieving this with BC began.

Transparency

Our work towards transparency focussed heavily on the usage of various post-autoclave treatments on the BC. We initially began with the usage of 0.1M NaOH solution to purify our BC in. This resulted in a slight lightening of the surface, but little overall change in either colour or transparency. We soon moved to using NaHCO3 solution, which yielded significantly better results. The transparency of the BC was improved, the visual homogeneity improved, and the solution bleached our BC into a light, white colour, allowing it to serve better as a base for its later dyeing. NaHCO3 also features additional functionalization, helping to resist the degradation of BC to cellulases. We also experimented with the usage of peroxide to produce even whiter BC, but ultimately decided the loss of functionalization outweighed the benefits to visual appeal.

Bacterial Cellulose Dyeing

Our initial project design was shaped heavily by the work of Suzanne Lee’s BioCouture (1) and by trend researcher Juliana Schneider’s GrowPak (2). BioCouture served as the initial inspiration for the subproject, opening our eyes to the possibility of dying BC into a variety of colours. Juliana’s work, however, provided a much more detailed look into the initial conditions and methods necessary to colour BC, and subsequent meetings with her served as the framework to begin our own experiments. Using her work in conjunction with existing literature (3) (4), we identified three different methods for the incorporation of dye into our BC; first, in-situ dying through the incorporation of dye in the growth media; secondly, ex-situ dry incorporation, putting dried BC into a dye solution and allowing its natural absorbance to incorporate the dye; and lastly, ex-situ wet incorporation, putting wet BC samples into a dye solution and allowing the dye to transfer through the movement of water.

Choice of Dye

Our dye of choice for incorporation into BC was the dye extracted from the leaves of red (purple) cabbage. Our choice of red cabbage dye (RCD) was a decision inspired by a few factors. First, one of our main considerations was a dye’s naturalness. In order to not compromise on sustainability, nor potentially cause issues with compostability, we wanted a dye that was both natural and biodegradable. This immediately disqualified the vast majority of dyes used in existing packaging in industrial settings. Another consideration was historical usage, and our meetings with Juliana revealed that she had conducted work with RCD that resulted in more effective colour saturation, especially relative to other natural dyes. This supplements literature that has used RCD as a fabric dye in the past (5). Finally, one of the last considerations was its effectiveness as a prototype. RCD is incredibly easy to make, and its properties as a natural pH indicator allow for rapid prototyping of multiple colours without needing to extract multiple different dyes from different substances. These considerations made RCD an obvious choice for our work.

In-Situ Media Dyeing (ISMD)

The colouring of the media was a good first step in testing the methods of dye incorporation. This solution was based on the observation that BC mimics the colour of its growth medium: BC grown in HS Media came out lighter than BC grown in amber, kombucha-style growth mediums. This led to the development of RCD-HS Media, where the water in our normal solution was substituted with our RCD solution. The RCD-HS was then autoclaved to prepare it for the growth of BC plates.

This autoclaving process, however, was devastating on the media. The anthocyanins present in solution degraded heavily as a result of the high temperatures (6) and pressures, and the solution changed from a lovely purple to a reddish-brown colour. Samples were still prepared from this solution at various ratios of RCD-HS to normal HS, but, once produced, the second round of autoclaving removed most of the difference in the between the samples, leading to inconclusive results.

Ex-Situ Dry Dyeing (ESDD)

The second method explored was the usage of ex-situ dry dyeing. This involved taking post-autoclave BC and allowing it to air dry fully before placing it in a solution of RCD in order to absorb the colour. By allowing the solution to permeate into the BC rather than incorporate it during growth, we are able to avoid many of the challenges autoclaving brings while allowing for the development of a variety of beautiful colours. Though we started off small, we eventually worked towards creating a palette of colours through the manipulation of the RCD pH. By adding a few drops of NaOH and HCl, we were able to change the purple into blues, pinks, greens, and yellows, and dyed our BC accordingly.

The BC dyed in this manner retained colour well and was able to withstand slight rinsing and massaging without losing all of the colouration. The colours produced were also stable over time, with the colour of the dyed BC remaining the same over weeks and months.

Ex-Situ Wet Dyeing (ESWD)

Our final method of dyeing utilised ex-situ wet dyeing. This is very similar to the ESDD method, but the BC is directly processed from purification into colouration. This method relies on the diffusion of the dye particles throughout the solution, into the BC. The colours produced in this method were similar to those in ESDD due to the near-identical conditions of the solution.

After the dyeing was complete, the samples were left to dry. Compared to ESDD, the samples appeared to have taken up the dye more effectively, with the colours showing more vibrancy relative to the already-saturated hues of the previous method. Though the colourfastness was lessened as a result, the ESWD method remains viable for those who desire stronger tones in the packaging. However, the colours, even within the same batch, were more inconsistent than seen in previous ESDD experiments.

Conclusion and Future Directions

The results from our subproject support the usage of ex-situ methods when attempting to dye BC with RCD. The choice between ex-situ wet and ex-situ dry dyeing methods is a choice of saturation, as well as its ability to incorporate well within existing workflows. Some may prefer the time savings of not having to let the BC dry prior to colouration in ESWD over the more consistent hues produced by ESDD. At the same time, teams whose workflows allow for BC to dry already may favour the less variable colouration method.

RCD is an imperfect dye in many ways, but it served as a good jack-of-all-trades for the purposes of this subproject. However, there are undoubtedly better dyes that beat RCD in vibracy, coulourfastness, or colour variety. For future directions, preparing an arsenal of dyes and colour samples will allow for an freedom of choice and an abundance of properties to select from, creating purpose-built packaging.

Though RCD is not fit for the purpose, in-situ dyeing methods may remain an option (and may result in better overall colouration) depending on the dye being used. If dyes with higher thermal stability are acquired, in-situ dyeing may very quickly become the most time-efficient method for producing coloured BC. It may be an option, even, to produce a thermally-stable dye using a genetically engineered bacteria in a co-culture with the K. xylinus, allowing for the near autonomous production of coloured BC.

References

1. Fairs M. Microbes are "the factories of the future" says Suzanne Lee [Internet]. Dezeen. 2014. Available from: https://www.dezeen.com/2014/02/12/movie-biocouture-microbes-clothing-wearable-futures/
2. Schneider JJ. Growpak – A step towards closing the loop [Internet]. Juliana J. Schneider. Available from: https://julianajschneider.com/growpak
3. Shim E, Kim HR. Coloration of bacterial cellulose using in situ and Ex situ methods. Textile Research Journal. 2018Apr22;89(7):1297–310.
4. Costa AF, de Amorim JDP, Almeida FC, de Lima ID, de Paiva SC, Rocha MA, et al. Dyeing of bacterial cellulose films using plant-based natural dyes. International Journal of Biological Macromolecules. 2019Jan;121:580–7.
5. Yadav P, Grover E, Fatima N. Printing of silk with natural red cabbage dye. Textile Trends [Internet]. 2010Jul24;53:36–40. Available from: https://www.researchgate.net/publication/292901130_Printing_of_silk_with_natural_red_cabbage_dye
6. Dyrby M, Westergaard N, Stapelfeldt H. Light and heat sensitivity of red cabbage extract in soft drink model systems. Food Chemistry. 2001;72(4):431–7.