# Biofabricating Three-Dimensional Bacterial Cellulose Composites Using Waste-Derived Scaffolds
## 3 Results  
The dip-in tests were aimed to evaluate whether a SCOBY grows vertically along the liquid-air interface (LAI). The setup ensured that the fabrics were thoroughly saturated with the infusion at the start of the experiment and stayed moist throughout the incubation time. Microscopy was used to evaluate the growth morphology of BC membranes and assess the feasibility of vertical SCOBY growth among the fibres of scaffold materials. Samples incubated for one to six weeks were tested periodically to determine an optimal growth period.  
From week one, the growth of BC membranes was confirmed on the LAI, indicating that the culture was healthy and that the consortia could produce nanocellulose. Optical microscopy at different heights above the LAI of the samples revealed biofilm growth in the form of thin membranes between the fibres as early as week 1 in the sheep wool and cotton samples, already proving vertical growth (see Figure 3).  
DipShe showed membranes up to 3 cm above and even below the LAI. The Shapiro-Wilk test for DipShe confirmed that all yield values, except for week 4, were normally distributed. A linear regression resulted in \(R2=0.14\) and \(p=0.083\), indicating no significant linear trend. The [PERSON]'s post hoc test only found a significant difference between week 3 and 5 (adjusted \(p=0.000603\)). This absence of a trend is also visible in Figure 4 and is further evident when comparing the growth and yield from week 1 to week 6 (see Table S1). Notably, the highest yield was observed after three weeks.  
In DipCo, membranes were observed from week one, extending across the samples up to 5 cm above the LAI. By the third week of fermentation, these membranes extended up to 7 cm above the LAI. However, this phenomenon was no longer observed after remoistening at week four's beginning. Like DipShe, the boxplots show high variability and no significant trend in yield, as confirmed by the linear regression with \(R2=0.011\) and \(p=0.64\). The highest yield was found after two weeks of fermentation (see Figure 4), which was significantly different from week 1 (adjusted \(p=0.00212\)). Additionally, a trend toward significant differences was observed between week 2 and all subsequent weeks. The yield in the sheep wool samples appears higher than in the cotton samples and the Mann-Whitney U Test showed a significant difference between the distributions of DipShe and DipCo with \(p=0.000000388\).  
Figure 3: Analysis of SCOBY growth in dip-in samples using light microscopy. DipShe1 and DipCo1: Dried sample exemplars fermented for one week (with no differences observed from one to six weeks) showing the LAI. The sheep wool samples at the top had double the volume of liquid, raising the LAI. Microscopy images showing the growth observed (in cm) over the LAI according to the scale on the right. Membrane growth is shown in colour (yellow), while shaded areas mark a lack of BC membrane. In the sheep wool samples, no growth is observed higher than 3 cm over the LAI, while in cotton samples (bottom), growth is observed at 5 cm over the LAI.