Introduction

A good seed coating is biodegradable, biocompatible, and colourful. Moreover, they can be used to enhance crop yield in many ways. Certain seed coatings improve germination by attracting water in dry soils¹, or provide nutrients to the germinating seed². Farmers considered the colour and the delivery of active compounds as the most important properties of seed coatings. Active compounds help protect the plant during its earliest developmental phases. This strong establishment phase leads to the development of a sturdy crop with increased yields. During the development of BCoated, we demonstrated that bacterial cellulose (BC) is not just biodegradable³, but also biocompatible. We realised the applicability by developing a novel coating procedure. Additionally, we demonstrated that BCoated coatings fulfil all criteria needed for seed coatings, and can do more.

Coating approaches

Seed coatings are traditionally powdered, solubilised and then applied to the seed⁴. This method is theoretically easier to scale, but in the lab we lacked the required machinery. We couldn’t mill the BC, which makes it more difficult to dissolve. We assume milled BC can be dissolved in N-methyl morpholine-N-oxide. This solvent no longer dissolves cellulose when sufficient water is supplied, which will prevent damage to the cellulose in the seeds^5,6. On a small scale, we have coated seeds with BC dissolved in water with 20% glycerol. This process is also interesting on a large scale, as the BC needs less treatment after its production. This means that its properties, as induced during the functionalisation, are maintained better.

We also worked on a second coating method, the in situ coating procedure. With this procedure, seeds were coated inside the culture medium while the BC is produced⁷ (Figure 1). This method turned out to be a successful way to coat seeds. A pre-treatment step is added to the coating protocol to increase the coating efficiency. Seeds are treated with polydopamine, meant to affix bacteria to the seed surface, and/or mechanical scarification, meant to increase surface area for BC attachment. With pre-treatment, the coating efficiency reached 80% (Figure 2).

Biocompatibility test

Following successful coating, the effect of the coating on germination efficacy was investigated. BC is a generally biocompatible material³, but we had to establish that the coatings do not negatively impact the seeds. In a germination assay, we compared the germination rates of seeds with and without coating. To prevent germination during the coating step, seeds were osmoprimed beforehand. Osmoprimed and coated seeds germinate well, reaching higher germination rates than untreated seeds (Figure 3). From this data it can be concluded that BCoated coatings will not harm germination.

Tailored coatings

In the functionalisation phase of our project, we investigated biological methods to change the properties of BC. We changed the water holding capacity of BC through an enzymatic treatment. By establishing a conjugation method, we opened the door for genetic modifications of BC-producer K. sucrofermentans, which can be used to alter properties such as porosity and biodegradability. These properties are important to fine-tune the delivery of active compounds⁸. In the use case about Striga, we demonstrated that BC is a suitable material for the slow release of active components.

Through our human practices, we identified a gap in the availability of active compounds. This drove us to add proteins to our BC matrix. We tested the incorporation of the insecticidal protein Cry3Aa to protect seeds from the wireworm, a major cause of crop losses in Europe. We designed and produced proteins with a cellulose binding domain (CBD) in the lab. We mixed the proteins with BC and tested the binding affinity before and after washing. Finally, we tested the effect of adding Cry3Aa to mealworm feed on their viability. The parts developed for these experiments are the first part of the part collection.

Protein production

No successful protein secretion in BC producing bacteria has been reported⁹. This is one of the motivations to develop a consortium for BC production. This consortium also contains S. cerevisiae, which is highly efficient in protein production and secretion¹⁰. In our production platform, we developed a consortium of K. sucrofermentans and S. cerevisiae. Our wet and dry lab efforts both show that the two organisms can live together. This allows for the production of proteins directly in the bioreactor.

Protein binding

An important challenge is ensuring that the proteins secreted by S. cerevisiae bind to the BC matrix. In this context, CBD can be fused to the proteins, allowing, through non-covalent bonds, a more efficient attachment to the BC¹¹.
CBD binding affinity was tested by fusing it to a green fluorescent protein (GFP). The fusion protein was produced in the lab and incubated with BC to allow binding. The fluorescence was tested directly after incubation, again after washing, and a third time after freeze-drying and re-solubilising. BC with CBD-GFP showed a significantly higher fluorescence than BC treated with normal GFP in all three measurements (Figure 4). With this we demonstrated that we can sucessfully bind proteis to BC for additional functionalities.

Insecticide coating effect

To test the effect of Cry3Aa on the mealworm, we used CBD-Cry3Aa to coat wheat bran. Wheat bran is a common food source for the mealworm, and is rich in cellulose¹². We therefore hypothesised that the CBD also will allow Cry3Aa binding to bran and act as a proof-of-principle. This method could be performed in a higher-throughput manner. The wheat bran is incubated with CBD-Cry3Aa, washed and freeze-dried. The treated bran was fed to the mealworms, and growth was measured after one week (Figure 5). The bran coated with the insecticidal protein caused a significant starvation effect in mealworms. Due to their taxonomic group, this will likely be translatable to the wireworms and other coleopteran larvae that threaten agricultural yields.

Colour

As users see the colour of seed coatings as a factor to determine quality, we also set out to change the colour of the seed coatings. Initially, we coloured the BC post-production with natural colourants. The BC can be made red, pink or green with red beet powder, freeze-dried strawberry powder or spirulina (Figure 6). After this, we also attempted to colour the BC with the addition of proteins. We made fusion proteins that contain a CBD, a blue or yellow chromoprotein and a secretion tag. Similar fusion proteins with a fluorescent protein instead of a chromoprotein have also been made for simpler detection. These fusion proteins are part of our part collection, with proteins that add functionalities to BC. The proteins have been produced in S. cerevisiae. The fluorescent proteins have also been detected in the supernatant, which shows that the proteins are indeed secreted. This means that the chromoproteins can be used to colour the BC simultaneously with the production.

Conclusion

BC’s ability to carry proteins for entirely new applications of seed coatings was confirmed. We additionally showed that the protein retained its activity after washing and freeze-drying. The coating has a custom water holding capacity to control germination. Active ingredients can be incorporated and released, the rate of which can be altered in the future with biodegradability and porosity. Lastly, by adding colour we comply to industry standards without a need for chemical colourants. Although our initial goal was to show this effect on actual seeds, due to time constraints, we were not able to develop a high-throughput seed coating protocol in time. However, we believe that these experiments put together show that our solution has the potential to make tailored seed coatings.

Future focus

To achieve a functional production process, a few steps still have to be taken. All parts of the process have been developed in different microbial cultures. All the achievements will have to be combined into the same microbial production consortium. Additionally, new coating properties can continuously be developed to assist arable farmers and see companies in the many struggles they face. The modularity of BCoated is then still its strength, allowing for flexibility to move with the developments in agriculture. All the while providing tailored coatings for tomorrow’s crops.