# Biofabricating Three-Dimensional Bacterial Cellulose Composites Using Waste-Derived Scaffolds
## 2 Materials and Methods
### Growing Using Scaffolds  
The dipping-in approach was used to examine if BC forms in vertical fibrous scaffolds. In this approach, scaffolds were vertically hung in a 50 mL tube (Falcon\({}^{\text{\textregistered}}\), Corning, NY,  
Figure 1: BC and SCOBY as a material have mainly been used to create design prototypes in fashion shows and exhibitions rather than in mass production. The primary application appears to be using them as substitutes for leather or textiles. (**A**): Jacket by [PERSON] used BC material CELIUM\({}^{\text{\textregistered}}\) by POLY-BION\({}^{\text{\textregistered}}\) ([[https://www.polybion.bio/contact/](https://www.polybion.bio/contact/)]([https://www.polybion.bio/contact/](https://www.polybion.bio/contact/))) (accessed on 9 September 2024) [23]; (**B**): Shoe made out of SCOBY [24]; (**C**): Shoe made out of self-dyeing shape-grown BC [25]; (**D**): lampshade made from BC material [26]; (**E**): shoe out of BC reinforced with nonwoven fabrics [27]; (**F**): biodegradable, self-gluing packaging material [28]. USA) with only the bottom part of the scaffold dipped in the inoculated SCOBY and incubated at room temperature (see Figure 2). To create the scaffolds, textile samples (sheep wool felt and cotton fabric) were cut into 10 \(\times\) 2.5 cm strips, sewn on the top edge using cotton yarn, leaving the thread edges (see Figure 2B) to suspend the scaffolds in 50 mL Falcon tubes with a tape (see Figure 2C). The cotton fibre fabric was sourced from the material collection at the School of Product Design, University of Canterbury, New Zealand, and waste sheep wool felt was obtained from discarded packaging waste (Woolpack by Planet Protector, Auckland, New Zealand). The cotton fabric was a plain weave with a thickness of 0.689 mm, a roughened surface, a density of 0.248 g/cm\({}^{3}\), and a fibre diameter of 16.13 \(\upmu\)m. The sheep wool felt has a thickness of 8.003 mm, a density of 0.048 g/cm\({}^{3}\), and a fibre diameter of 34.65 \(\upmu\)m. With an absorption capacity of 436.31% for water and 441.06% for kombucha infusion in cotton and 426.09% for water and 405.38% for kombucha infusion in wool felt, no significant difference in absorption capacity was observed between the two materials. These samples will be referred to as DipCo and DipShe, indicating the reinforcement (Co = cotton; She = sheep wool) and a number indicating the week of incubation. Scaffolds were autoclaved in a front-loading benchtop autoclave (3870 ELD-C, 85 L, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 120 \({}^{\circ}\)C for 15 min prior to addition of infusion inoculate. All fermentation processes were carried out under sterile conditions in a laminar flow hood. A total of 7.5 mL of the infusion was added to the cotton samples and shaken until they were soaked with liquid completely. The Falcon tubes were filled to the top and left for 5 min to ensure complete wetting of the sheep wool samples placed in them. The tubes were stored vertically in racks with the lids on top but unsealed for measurements in the period of time from 1 to 6 weeks. After three weeks, 5 mL sweetened tea prepared as the starter culture (5 g/L tea, 100 g/L sugar) was added to each sample and the Falcon tubes were turned around to ensure the whole fabric was wet. This step allowed the reinforcement materials to reabsorb moisture, ensuring uniform hydration while also revealing the effects of shear forces and liquid movement on growth. All samples were weighed dry before and after the fermentation using a precision balance (FZ-500i, 520 g \(\times\) 0.001 g, A&D Co., Tokyo, Japan) to analyse the extent of SCOBY growth. The growth was assumed to be the  
Figure 2: **(A)**: Schematic concept of experimental setup displaying SCOBY in orange and scaffold fibres in grey showing the growth after 2 days ((**A**), **left**), and the resulting growth after 1–6 weeks ((**A**), **right**).