3D bacterial cellulose biofilms formed by foam templating

Abstract

Bacterial cellulose is a remarkable fibrous structural component of biofilms, as it forms a mechanically strong hydrogel with high water adsorption capabilities. Additionally, bacterial cellulose is biocompatible and therefore of potential interest for skin regeneration and wound healing applications. However, bacterial cellulose produced through conventional production processes at water-air interfaces lack macroporosity control, which is crucial for regenerative tissue applications. Here we demonstrate a straightforward and efficient approach to form a macroporous bacterial cellulose foam by foaming a mannitol-based media with a bacterial suspension of Gluconoacetobacter xylinus. The bacterial suspension foam is stabilized with Cremodan as a surfactant and viscosified with Xanthan preventing water drainage. Further foam stabilization occurs through cellulose formation across the foam network. As bacterial cellulose formation is influenced by the viscosity of the growth media, we fine-tuned the concentration of Xanthan to allow for bacterial cellulose formation while avoiding water drainage caused by gravity. With this simple approach, we were able to design 3D bacterial cellulose foams without any additional processing steps. We argue that this templating approach can further be used to design foamy biofilms for biotechnological approaches, increasing the surface area and therefore the yield by improving the exchange of nutrients and metabolic products.

Fig. 1

Schematic of the bacterial cellulose foam formation process. G. xylinus extrudes bacterial cellulose as a function of oxygen and migrates toward the air–water interface. To construct a bacterial cellulose foam, a suspension of G. xylinus in growth media is foamed. The air bubbles are stabilized through interfacial stabilization by Cremodan. To avoid water drainage and to enhance stability of the foam Xanthan is added as a thickener. After bacterial growth, the foam was increasingly stabilized by BC formation leading to stable cellulose foam structures after 4 days

Fig. 2

Foamability and stability of Cremodan and Cremodan–Xanthan mannitol-based media. a Foam overrun and b foam height h and initial foam height h₀ for 0.1–4 wt% Cremodan in mannitol-based media. c Foam overrun and d foam height h and initial foam height h₀ for 3 wt% Cremodan and 0.1–1.5 wt% Xanthan in mannitol-based media

Fig. 3

Bacterial cellulose growth and its rheology and structure. a Interfacial rheology of mannitol-based media with 1% inoculated G. xylinus at room temperature. The biofilm formation is measured as a time sweep at a constant deformation γ of 0.1% and a frequency ω of 1 rad/s. b Formation of a biofilm at the water–air interface by G. xylinus after 5 days and c SEM image of a dried bacterial cellulose pellicle (scale bar = 4 μm). d Foam stability of 3–5 wt% Cremodan and 0.2 wt% Xanthan solutions with 1% inoculated G. xylinus. e Foam formed by a 0.5 wt% Xanthan and 3 wt% Cremodan mannitol-based solution with inoculated bacteria after 7 days. f SEM of an air dried foam lamellae of a 0.5 wt% Xanthan and 3 wt% Cremodan foam (scale bar = 200 μm)

Fig. 4

Operational window of foam templating for 3D bacterial cellulose biofilms as a function of Xanthan and Cremodan concentration. Foams of mannitol-based media are stable above a Cremodan concentration of 3 wt% and a concentration of 0.5 wt% Xanthan. However, bacterial cellulose growth is inhibited at high viscosities (above 1 wt% Xanthan). At low viscosities and therefore low Xanthan concentrations, stable foams can be formed with Cremodan concentrations above 4 wt%. However, due to water drainage, dry foams are formed which inhibit a complete bacterial cellulose network