Introduction to BC

While cellulose is the most abundant natural polymer on Earth, the variety synthesised by bacteria differs from the more well-known plant-derived counterpart (Figure 1). Unlike plant cellulose (PC), non-cellulose components including hemicellulose, lignin, and pectin, resulting in a material of exceptional purity¹.

Chemically, PC and BC are both linear homo-polysaccharides made of repeating β-D-glucopyranose units linked by β-1,4-glycosidic bonds (Figure 1). Due to the presence of multiple hydroxyl (-OH) groups in their structure, extensive hydrogen bonding occurs between the hydroxyl and oxygen atoms of glucose units. Together with van der Waals forces, these interactions promote the parallel alignment of polymer chains into crystalline nanofibers. These nanofibers further assemble into microfibrils with a hierarchical structure, giving the material its excellent mechanical strength¹.

In addition to its high purity, BC differs from PC by having higher crystallinity and greater degree of polymerisation. Its ribbon-like cellulose nanofibers weave into a three-dimensional reticulated network, with high surface area and porosity. This unique structure provides BC with uniquely high water holding capacity and prolonged water retention. Together with these remarkable mechanical properties, including elasticity and durability, BC’s non-toxic nature, biocompatibility and biodegradability make it suitable for agricultural applications³.

Seed coatings serve as a multifunctional platform, delivering nutrients, pesticides, or other bioactive compounds that can improve germination and protect against biotic and abiotic environmental stressors⁴. However, commercial seed coating formulations often rely on synthetic polymers, which pose environmental risks and underscore the need for more sustainable alternatives.

Bacterial cellulose (BC) is a promising alternative, possessing inherent properties that align well with the general requirements for modern seed coating applications. However, these properties alone do not always meet the specific demands of all seed coating types. Therefore, the BCoated project set out to develop strategies to functionalise BC, tailoring seed coatings to customer needs through a modular production platform (Figure 2).

Functionalisation

To broaden the applicability of BC, we utilised cellulose-binding domains (CBDs) to anchor proteins onto the BC matrix, thereby functionalising it with bioactive compounds relevant to seed coating applications. In parallel, to further optimise the performance of BC, we focused on tuning three key properties: water holding capacity (WHC), porosity and biodegradability.

To functionalise BC with proteins, we engineered Saccharomyces cerevisiae, a member of our consortium co-cultured with Komagataeibacter sucrofermentans, to CBD-fused proteins during BC production. These secreted proteins bind to the BC matrix as it forms during biosynthesis via the CBD.

Learn more about the consortium used in our production platform

To alter the WHC of BC, we employed enzymes that selectively modify its surface chemistry by introducing new functional groups. These modifications can either increase or decrease the hydrophilicity of BC, thereby directly influencing its WHC. Using enzymes also reduces the need for harsh chemicals and extreme conditions, making the process more sustainable.

To fine-tune the biodegradability and porosity of BC, we engineered our BC-producing strain, K. sucrofermentans. By introducing heterologous genes or overexpressing native genes under inducible promoters, BC’s structure can be modulated during biosynthesis. This results in the production of BC with controlled levels of porosity and biodegradability, tailored to meet the specific requirements of the desired seed coating application.

Protein Functionalisation

Bacterial cellulose (BC) can be functionalised by immobilising proteins onto its matrix. We leveraged the presence of S. cerevisiae in the consortium used in our production platform, and engineered it to secrete recombinant proteins directly into the culture medium. Previous efforts to engineer the native BC-producing strain K. rhaeticus to complete this task proved to be inefficient, largely due to the burden imposed on the host⁵. Therefore, S. cerevisiae offers a more genetically accessible and efficient platform for protein production and secretion, making it an ideal candidate for this purpose⁶.

A number of proteins were selected to either alter the physical properties or visual appearance of BC. Furthermore, detection proteins were used to quantify the effect of proteins chosen for functionalisation. To facilitate protein attachment to the BC matrix, cellulose-binding domains (CBDs), short peptides (36–185 amino acids) located at the C- or N-terminus of proteins, were employed (Figure 3). These were fused to the protein to promote more efficient non-covalent binding to BC fibrils, increasing protein adhesion. Furthermore, CBDs have also been shown to act as a thermostabilising domain for some proteins, which is advantageous for seed coating applications where protein stability is vital⁷.

There is a variety of CBDs, each exhibiting different affinities depending on the structure of cellulose. As BC is characterised by high crystallinity (>60–90%), CBDs with an affinity for crystallised structures were chosen^8,9.

Discover the key findings in our experiments and results section

Water-holding capacity

The natural state of BC is hydrophilic, making it very appealing for agricultural applications. However, its surface chemistry is primarily dominated by hydroxyl (–OH) groups. Modifying these functional groups can either enhance or reduce hydrophilicity, thereby directly affecting its water-holding capacity (WHC). Tailoring this nature and ultimately the WHC of BC could enable the development of a polymer capable of regulating seed hydration and, in turn, either delaying or promoting germination depending on the environmental conditions.

BC is often modified through conventional chemical methods, but these rely on harsh conditions or toxic reagents that either compromise the biological properties of BC or pose risks to the environment. A promising alternative is the use of enzymes. These biocatalysts can alter the surface chemistry of BC by introducing new reactive functional groups and ultimately change its properties, especially the WHC². Therefore, apart from synthetic biology we also experiment with greener chemistry to tune BC’s properties.

The enzymes most often employed for this purpose include EC1 oxidoreductases such as laccases and lytic polysaccharide monooxygenases or EC3 hydrolases including cellulases and lipases². Here, we experimented with Laccase and Lipase in enzymatic treatments of BC.

We employed the Laccase/TEMPO-mediated catalytic system to target the –OH sites in BC chains to introduce carboxyl (−COOH) or aldehyde (−CHO) groups groups, (Figure 4). The introduction of surface-active aldehyde and carboxyl groups could increase the hydrophilicity of BC even more, due to the addition of more polar groups. This in turn could increase WHC and potentially enhance its tensile strength in the wet state¹¹. This modification is particularly ideal for coatings tailored to promote germination by enhancing water uptake in drought-prone areas.

To achieve the opposite effect, lipases were employed (Figure 5). These enzymes catalyse an esterification reaction, for which palmitic acid, a soil-compatible long-chain organic acid, was chosen as the substrate¹². As the hydrophilic groups in BC could be substituted with less polar groups, its water resistance could increase, thus reducing the WHC. Such a modification is especially beneficial for hydrophobic coatings, as they provide a protective barrier around the seed, preventing premature germination by repelling moisture.

To determine whether modifications were made to the surface chemistry of BC after direct enzymatic treatment, Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy was utilised. Finally, the water holding capacity protocol was used to assess whether these enzymatic modifications could alter the WHC of BC.

Discover the key findings in our experiments and results section

Biodegradability

Biodegradability is a key property that, along with the materials thickness, influences the controlled release of compounds from seed coatings. By tuning the biodegradability of bacterial cellulose (BC), it is possible to tailor the decomposition rate and compound release to suit a variety of seed coating needs (Figure 6).

Previous findings demonstrate that heterologous expression of crdS, a curdlan synthase gene from Agrobacterium sp., in Komagataeibacter xylinus modified its native cellulose nanofiber-producing system. This enabled the co-synthesis of curdlan (β-1,3-glucan) alongside cellulose nanofibers, which improved the biodegradability of the BC¹⁶. Hence, this approach was also adopted by BCoated and implemented in K. sucrofermentans.

Conversely, to reduce biodegradability, bslA, a gene that naturally encodes a hydrophobic protein expressed by Bacillus subtilis during biofilm formation, can be heterologously expressed. The presence of the protein gives biofilms a water-repellent characteristic. Researchers found that its heterologous expression led to structural and mechanical changes in the BC fiber network, producing less brittle and more hydrophobic material, thereby lowering its biodegradability¹⁷.

The biodegradability of BC produced by both wild-type and engineered K. sucrofermentans was evaluated through monitoring the breakdown of BC using the enzyme cellulase, which catalyses the hydrolysis of cellulose.

Discover the key findings in our experiments and results section

Porosity

Another crucial property that can impact BC’s performance as a seed coating material is its porosity, which particularly affects the embedding and controlled release of beneficial compounds. Higher porosity can facilitate the loading of compounds¹⁸, whereas lower porosity can delay their release during long-term storage, minimising premature loss before reaching the soil. As different seed coating applications require distinct levels of porosity, we used synthetic biology to seek to accomplish this.

Research has shown that overexpression of certain native genes in Komagataeibacter strains can either increase or decrease porosity. One such gene is galU, which plays a crucial role in regulating carbon metabolic flux between BC biosynthesis and the pentose phosphate (PP) pathway. Its overexpression results in BC with decreased porosity¹⁹.

To achieve the opposite effect and increase porosity, we targeted genes associated with cell motility, namely motA and motB. Previous studies noted that cell motility correlated with the synthesis and extrusion of bacterial nanocellulose microfibrils²⁰. In K. sucrofermentans, both motA and motB were native genes, targeted for overexpression, to enlarge pore size and increase porosity.

Efforts were made to characterise porosity, including the use of the mass-density protocol and Scanning Electron Microscopy (SEM). High-resolution images of BC’s topography and composition were obtained, making SEM ideal for visualising the porosity of BC.

Discover the key findings in our experiments and results section

Inducible promoters

After introducing different genes into K. sucrofermentans, these genes can be expressed through inducible promoters to fine-tune the properties of bacterial cellulose (BC). A promoter in the Komagataeibacter toolbox is pLux, which is induced by N-Acyl homoserine lactone (AHL) and was tested in the Komagataeibacter rhaeticus iGEM strain. It was found to be strong and not leaky²². However, this inducer is expensive and not suitable for biomanufacturing purposes. Therefore, we searched for an alternative inducible promoter and selected hpdR/phpdH for testing. This promoter was previously tested in Pseudomonas putida, and its inducers, levulinic acid or 3-hydroxypropionic acid, are low-cost²³.

Production Platform

To initiate bacterial cellulose (BC) production, a tailored production platform was first established. This platform needs to fulfil two main criteria: maximising BC output and enabling controllable BC properties. Achieving these objectives involved enhancing the growth and metabolic efficiency of K. sucrofermentans, while also introducing S. cerevisiae into the system. Within this microbial consortium, S. cerevisiae contributes to higher production yields, expands the range of utilisable sugars, and adds versatility by enabling the incorporation of various sugar monomers or by secreting proteins that can interact with the cellulose matrix.

K. sucrofermentans as a chassis for BC production

A large variety of BC producing organisms occur naturally in the soil, consisting of bacteria, archaea and eukaryotes²⁴. The species differ in their efficiency of BC production. The main BC producing chassis belong to the acetic acid bacteria. Acetic acid bacteria are rod-shaped, gram-negative obligate aerobes. They metabolise carbon sources by oxidative fermentation to obtain ethanol or acetate. Some acetic acid bacteria species form cellulose fibres, which constitute the basis of the biofilm in which they live. This defensive layer protects the organisms from lack of water, radiation and other unfavourable conditions. Additionally, it gives an advantage by supporting attachment and allowing the accumulation of substrates²⁵.

Within the acetic acid bacteria family, species from the genus Komagataeibacter have been found to give the highest yields and are therefore most extensively studied²⁶. K. sucrofermentans is one of the most efficient BC producers²⁷. In addition, this species has been demonstrated to grow on waste from the confectionary and biodiesel industries, such as thin tillage and whey^28,29.

With this unmodified K. sucrofermentans strain our first BC is produced. BC is typically formed at the water-air interface in liquid cultures. Changing the agitation during cultivation can have a large impact on BC production. Static cultures obtain the highest crystallinity and lowest water-holding capacity compared to agitated reactors. Agitated cultures show an increase in productivity for BC production over static cultures, but result in more irregular and spherical BC products³⁰. Temperature and pH also influence the BC yield. The optimum temperature for Komagataeibacter species is around 30°C and the optimum pH is around pH 5.5³¹. Since the optima differ between strains, the optimum pH was experimentally determined. In addition, the effect of different cultivation conditions on the characteristics of the produced cellulose were determined. Produced dry weight and macro structure of the BC produced under different circumstances were compared. The BC produced by the unmodified K. sucrofermentans functioned as a baseline material, against which other BC batches were compared.

Consortium design to increase yield and functionalisation possibilities

The scalability of biotechnological production of cellulose is largely dependent on the titre, rate and yield (TRY). At this time, K. sucrofermentans’ BC yield has not been found sufficient for industrial purposes^33,34. The yield can be increased through co-culture³⁵, genetic engineering of K. sucrofermentans³⁶ and the addition of chemical additives such as ethanol³⁷. In this iGEM project, two approaches were combined to enable the high yield, scalable production of cellulose.

In nature, you hardly ever find a culture consisting of only one species. Soil samples, for example, can contain over 50,000 different microbial species³⁸. Also SCOBY (Symbiotic Culture of Bacteria and Yeast), a culture commonly used to ferment kombucha, contains an array of different species. Amongst those species are several of the genus Komagataeibacter, responsible for the production of BC³⁹ and Saccharomyces spp.⁴⁰. The approach of using a consortium to increase cellulose production has been explored before. This can increase productivity and allow the use of waste streams as media components⁴¹, both of which make production more economically feasible.

The high costs of BC production are mainly due to medium expenses⁴². Waste streams are used as a strategy to decrease medium costs⁴². When using waste streams as media, sugars have to be hydrolysed into monomers before they can be consumed by Komagataeibacter, and even then, not all sugars can be consumed⁴³. The addition of a second microorganism in the co-culture that hydrolyses disaccharides with exo-enzymes, such as S. cerevisiae, increases sugar availability in waste stream-based media^44–46.

We hypothesised that the organisms can additionally complement each other’s metabolism. Previously, it has been shown that ethanol supplementation increases BC yield in K. sucrofermentans. Ethanol is used for energy generation, as the conversion of ethanol to acetaldehyde and acetic acid releases electrons. Through the cofactor PQQ, this is used to create a proton motive force for energy generation. It also decreases the generation of gluconic acid from glucose, which requires the same cofactor. Therefore, a larger part of the glucose flux can go to BC³⁷. In addition, ethanol can be used for assimilation (Figure 8). To decouple glucose from energy and biomass generation, the medium was therefore supplemented with ethanol-producing S. cerevisiae.

Komagataeibacter produces organic acids such as acetate, which can inhibit cellulose production through acidification of the medium, which inhibits cell growth^37,43. In the consortium designed for our production platform, S. cerevisiae is used for medium supplementation and removal of inhibitors: it produces ethanol and consumes acetate in a crossfeeding design (Figure 8).

The consortium was designed with S. cerevisiae strain IMX1812 from the Industrial Biotechnology Department of Delf University. This strain has no glucose transporters, therefore there is no glucose competition for cellulose production. The yeast will grow on maltose, which is present in many waste streams and is not consumed by K. sucrofermentans. The experimental design was started by creating a respiration deficient S. cerevisiae strain. This ensured that all growth and energy generation is coupled to fermentation and therefore ethanol production, which is beneficial for cellulose production. Also in natural SCOBY cultures cellulose accumulates in less oxygenated areas, where S. cerevisiae can ferment productively⁴⁷.

It is important to create antagonism in a consortium to prevent one of the species from overgrowing the others. One of these approaches is to create interdependence through secreted biomolecules⁴⁸. That approach was taken in this consortium. K. sucrofermentans growth is dependent on ethanol, which is produced by S. cerevisiae. On the other side, S. cerevisiae growth can be made dependent on K. sucrofermentans through acetate. It was found in literature that knocking out the glycerol-3-phosphate dehydrogenases gpd1Δ gpd2Δ and expressing mhpF gene from E. coli heterologously led to an anaerobic yeast fermenting acetate to ethanol⁴⁹. This is integrated into future design plans to further enhance the dependency while mitigating the acidification in the medium. The ethanol homeostasis circuit in the next section will also limit S. cerevisiae growth based on K. sucrofermentans growth, further enhancing the antagonism. The K. sucrofermentans strain is not further modified for the consortium.

During initial experiments, the consortium was balanced by the availability of sugars in the medium and the inoculation concentrations for the respective microbes. Through experimental trials, the optimal conditions to grow K. sucrofermentans and S. cerevisiae were determined. Experiments were performed on varying sugar sources and concentrations to test which strains can grow on which sugars. Additionally, the species were grown together on different inoculum concentrations to learn about the stability of the consortium. Aside from wet-lab experiments, a digital twin of the consortium is made in the dry-lab. This is used to explain experimental data and find future design plans.

Besides the increase in yield, it was proposed that the yeast can also be exploited to manipulate the structural and functional properties of the synthesised cellulose in situ and allow the control of community dynamics through engineered circuits. Moreover, S. cerevisiae is engineered to express a target protein/molecule in fusion with a cellulose-binding domain (CBDs) or carbohydrate-binding molecules, so that functional proteins to alter the structure of the matrix could be synthesised with the cellulose, or other substrates could be copolymerised⁵⁰.

Discover the key findings in our experiments and results section

Ethanol homeostasis circuit

The addition of ethanol to K. sucrofermentans culture can improve yield, but ethanol concentrations that are too high can inhibit cell growth or cause cell death. The ethanol concentration in the medium should therefore be carefully maintained at a constant level that is optimal for BC production. Ethanol concentrations for optimal BC production are strain-specific and range between 0.5% (v/v) and 2% (v/v), with an optimal concentration of 1% (v/v) for our strain³⁷. Yeast can produce ethanol up to concentrations of around 10%⁵¹, which is much too high for K. sucrofermentans and would result in almost no BC production³⁷. Therefore, a method has to be developed to limit ethanol production by S. cerevisiae, ideally to a constant concentration and not to a constant production rate, to allow for fluctuations in ethanol consumption.

In recent years, genetic circuit-based regulations of metabolic flux have received increasing interest. These synthetic circuits are used to modulate gene expression, mainly to drive the cellular metabolism from growth to production. A synthetic circuit consists of a sensor, signal transduction and an actuator⁵². The signal transduction is usually a genetic controller, while the sensor and actuator can be in different levels of cellular machinery⁵³. In the context of ethanol production, the sensor should detect the ethanol concentration, the signal transducer couples this detection to ethanol production, and the actuator, when activated, inhibits ethanol production.

The design of the ethanol homeostasis circuit is given in (Figure 9). The sensor is an ethanol-inducible promoter designed for this experiment. This promoter regulates the expression of pTEV+, the genetic controller that is the first step of the signal transduction. pTEV+ is a protease that cleaves a specific amino acid sequence. This specificity was exploited in a shielded bidirectional degron created by⁵⁴, which is only activated when pTEV+ cleaves a sequence in the middle, thereby exposing the two degrons on both sides of the cleavage site. The bidirectional degron was fused to PGK1, a key enzyme in ethanol production⁵⁵. When pTEV+ is expressed, this now causes degradation of PGK1 and therefore a decrease in ethanol production, the final actuator of the synthetic circuit. When the ethanol concentration drops as a result of this, the signal transduction also ceases, and the inhibition of ethanol production stops.

The sensor of the synthetic circuit and the signal transducer and actuator were developed separately (Figure 9).The signal transducer and actuator domain is developed in a purely fermentative strain. This strain is created by adding ethidium bromide to the yeast growth medium for three consecutive growth cycles. This causes the rho- mutation to occur, which is a loss of mitochondrial DNA. The signal transducer and actuator were activated by the ꞵ-estradiol inducible synthetic promoter Z3ev. The effect of different induction levels on growth was assessed, which is a direct indicator of ethanol production due to the rho- mutation.

The sensor is based on the KlADH4 promoter, an ethanol-inducible promoter previously used by iGEM12_TU_Munich (BBa_K801020)⁵⁶. This promoter is activated by ethanol through unknown transcription factors and is not glucose dependent⁵⁷. The promoter is originally from the fungus Kluyveromyces lactis, but iGEM12_TU_Munich demonstrated its activity in S. cerevisiae as well. With this part as a basis, multiple promoters were designed to decrease leakiness and extend the dynamic range, which enlarges the design space of the synthetic circuit⁵³. This was done by combining one, two or three repeats of the ethanol-responsive UASE elements with different core promoters. Also, an insulation sequence was added to decrease leakiness. With these design strategies, promoters with several dynamic ranges were developed. As their activity differs for set ethanol concentrations, they have been used to design the circuit to balance at the required ethanol concentration.

Modelling is an effective tool to understand how a synthetic circuit works and where the bottlenecks are. In this project, we therefore not only made the ethanol homeostasis circuit in the wet-lab, but also modelled it in the dry-lab. Design choices were made based on iterative interactions between the model and laboratory data.

Discover the key findings in our Experiments and Results section

Seed coating & application

Seed Coating

After successfully altering the BC to match desired properties and making efforts to allow BC functionalisation to be more accessible through engineering of the consortium, we set out to create a protocol for the coating.

We developed an in situ coating protocol, where seeds were coated directly in the culture with Komagataeibacter sucrofermentans. This was done using a protocol developed by Rühs et al., 2020⁵⁸ which we modified to be applicable to seeds. We also considered the use of an ex situ approach, which instead entails producing BC separately before regenerating the polymer around the seeds. However, the ex situ method was not further developed as solubilising BC turned out to be difficult, possibly due to the high crystallinity of our material.

The in situ coating protocol was further optimised down the line, where the highest success rate was found by pretreating seeds with a polydopamine (PDA) step combined with a mechanical scarification procedure. PDA coatings are black coatings that function by fixing bacteria to the outside of the seed, ensuring that BC is deposited locally to form our coatings (Figure 10).

In addition to this, we found that the addition of a mechanical scarification in combination with the PDA coating enhanced the efficiency of our protocol. This is likely as the scarification increases the surface area for bacterial fixation and BC to form.

We reached a high efficiency seed coating protocol, and investigated the potential of seed osmopriming , a step where uniform germination is induced utilising osmotic pressure⁵⁹. This resulted in a protocol that was able to coat seeds in situ, which were still capable of germination. In the future, downstream processing steps to enhance the throughput of the protocol and ensure the shelf life stability of our final product will be pursued.

Using BCs properties in a seed coating

After we created our high-efficiency coating protocol, we set out to use it to tackle real-world issues, using our functionalised BC. First, we used our CBD domains to attach a Bacillus thuringiensis (Bt)-protein, Cry3Aa, an insecticidal toxin specific to beetles (Coleoptera). Second, we set out to develop a delivery system for an active compound by using our BC, targeting Striga hermonthica, a parasitic weed that causes massive annual cereal losses in sub-Saharan Africa^60-63.

Use case 1: Controlled Delivery of Pesticides

By attaching the insecticidal Bt-protein Cry3Aa to our seed coatings, we set out to reduce seed predation behaviour to reduce crop losses associated with seed eating pests, specifically beetle larvae.

We chose Bt-proteins as a biological alternative to conventional chemical pesticides⁶⁴. Once ingested by insects within the target group, these proteins are activated by proteolytic cleavage and bind to specific receptors to cause their mid-gut cells to rupture (Figure 11). This pesticide approach provides a high degree of specificity while minimising non-target effects.

As a model organism, we used common mealworms, also belonging to the order Coleoptera to study the effect of the incorporation of Bt-protein Cry3Aa into the seed coat.

Although the protein was not able to be incorporated directly into a seed coat due to time constraints, its effect was simulated by direct attachment to wheat bran, which is high in cellulose fibres.

This attachment did not result in increased insect fatality, but instead reduced feeding behaviour and enhanced starvation effects, showing that the incorporation of this protein presents a useful case to reduce seed predation.

Discover the key findings in our experiments and results section

Use case 2: Parasitic weed suppression

Striga, or Witchweed, is a genus of parasitic weeds that causes massive yield losses annually (Figure 12) ^60-63. S. hermonthica typically targets cereal crops, such as sorghum, which was used to develop the coating protocol and for any subsequent experiments. By using the seed coating as a platform, we set out to create a system that enabled the gradual release of a bioactive compound that can inhibit this weed, "compound X". We are not currently able to disclose the exact nature of this compound.

Unfortunately, modified BC could not be used for these assays; however, the compound was included in BC, after which the compound X release rate was independently assessed. This release rate was assessed by using a compound X auxotrophic E. coli strain, Strain A. If the compound was successfully embedded in the BC, it should be able to diffuse out of the BC matrix and support auxotroph growth. We assessed this for both liquid and solid media.

In the future, we will use our coated sorghum seeds to deliver compound X in greenhouse assays. However, due to time constraints, we were not able to complete this for the wiki.

Discover the key findings in our experiments and results section

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