# Biofabricating Three-Dimensional Bacterial Cellulose Composites Using Waste-Derived Scaffolds
## 4 Discussion
### SCOBY Growth in Scaffold Materials  
In dip-in samples, SCOBY growth occurred within the reinforcement above the LAI, indicating vertical growth. Unlike conventional setups, SCOBYs did not form a uniform, dense film. Instead, membranes grew within the fibres of the scaffolds, extending up to 3 cm in DipShe and 5 cm in DipCo after the first week. This difference might be due to varying wicking capacities. This variation may result from the materials' specific wicking abilities and capacities attributed to their fibre morphologies. Cotton fibres are hydrophilic [42]), while wool fibres have hydrophobic surfaces [43]. Differences in textile structures (felt vs. woven fabric) may also play a role and warrant further investigation through wicking tests. However, as the resulting membrane morphology differed from initial expectations and appeared more suitable for applications that work with membranes in the nanoscale, such as filtration or biomedical scaffolding, no further mechanical testing was conducted.  
The BC membranes formed in DipShe and DipCo show different characteristics (compare Figures 5 and 7). Membranes in DipShe are smooth and fully closed. There are fewermembranes after the rupture due to remoistening, but the morphology has not changed. In contrast, DipCo displays a non-closed, bulky membrane even before the dynamic moistening process. The different fibre orientations could have also led to different gaps, which might have influenced the difference in membrane structure between DipShe and DipCo potentially influencing the significant differences in yield distributions. Instead, it is unlikely that this is the reason for the nanonetting since gaps are of a different size and scale than the nanocellulose molecules produced.  
The dynamic remoistening process initiated thin, web-like fibres enclosing cellulose spheres in DipCo, a phenomenon observed in agitated fermentation, which typically involves stirring or shaking [44]. However, in this study, the only agitation occurred during incubation at the beginning and after three weeks of growth. Any other agitation could be caused by a liquid stream due to gravity or capillary force, suggesting this must have happened a short time after the eruption until a force balance adapted. The reason for the formation of spheres in the cotton fabric alone remains unknown.  
In DipShe, air pockets within the felt likely stimulated bacterial cellulose production around air bubbles, resulting in membranes distributed throughout the cross-section. In contrast, DipCo exhibited membranes attached only to the outermost fibres, suggesting a closed water surface limiting SCOBY growth to the outer LAI. The growth within the felt and the closed membrane in DipShe explains the higher yield than in DipCo. Although the same method was used, very different materials resulted from the two fabrics. SCOBY shows a high feasibility of growing in cotton with the ability to vary the membrane morphology.  
This study reveals a new form of BC growth, which might be helpful for more technical applications such as biomedical or electrical materials, e.g., usable for filtration systems or energy storage. While waste streams might not suit these applications, alternative, pure, sterile scaffold materials such as porous ceramics or polymer fabrics should be considered.  
Despite low yields due to a homegrown weak culture and limited trend data, this method demonstrated effectiveness within one week, highlighting its economic potential. The aim was to apply the \"waste to value\" principle. With the outcome showing microscale membranes instead of the typical strong biofilms built by SCOBYs, the application field and this kind of waste stream should be reconsidered. This approach produced membranes within the microscale, creating a high surface area. The membranes will be built wherever there is an LAI, meaning membranes will be formed even in three-dimensional scaffolds. The characteristics of the formed membrane depend on the scaffold and need further investigation.