Highly Aligned Bacterial Nanocellulose Films Obtained During Static Biosynthesis in a Reproducible and Straightforward Approach

Abstract

Bacterial nanocellulose (BNC) is usually produced as randomly-organized highly pure cellulose nanofibers films. Its high water-holding capacity, porosity, mechanical strength, and biocompatibility make it unique. Ordered structures are found in nature and the properties appearing upon aligning polymers fibers inspire everyone to achieve highly aligned BNC (A-BNC) films. This work takes advantage of natural bacteria biosynthesis in a reproducible and straightforward approach. Bacteria confined and statically incubated biosynthesized BNC nanofibers in a single direction without entanglement. The obtained film is highly oriented within the total volume confirmed by polarization-resolved second-harmonic generation signal and Small Angle X-ray Scattering. The biosynthesis approach is improved by reusing the bacterial substrates to obtain A-BNC reproducibly and repeatedly. The suitability of A-BNC as cell carriers is confirmed by adhering to and growing fibroblasts in the substrate. Finally, the thermal conductivity is evaluated by two independent approaches, i.e., using the well-known 3ω-method and a recently developed contactless thermoreflectance approach, confirming a thermal conductivity of 1.63 W mK^-1 in the direction of the aligned fibers versus 0.3 W mK^-1 perpendicularly. The fivefold increase in thermal conductivity of BNC in the alignment direction forecasts the potential of BNC-based devices outperforming some other natural polymer and synthetic materials.

Keywords: aligned fibers; anisotropy; bacterial nanocellulose; biofabrication; bioinspiration; hydrogels; nanomaterials; sustainability; thermal conductivity.

Figure 1

Methodologies to align BNC fibers. A,B) Schematics of the reported methods to align bacterial cellulose fibers such as top‐down approaches: uniaxial stretching,^{[ 35} , ³⁶ , ^{37 ]} wet spinning,^{[ 38 ]} and wet‐drawing and wet‐twisting^{[ 39 ]} to name some and bottom‐up such as flow‐induced alignment in bioreactors,^{[ 40 ]} patterned templates,^{[ 42} , ^{43 ]} physical and oxygen gradients.^{[ 42} , ^{43 ]} Bottom‐up methodologies benefit from the use of BNC biosynthesis. C) Illustration of the natural BNC biosynthesis and the structural hierarchy of BNC fibers. K. xylinus, a cellulose‐producing bacteria strain, synthesizes cellulose from the glucose present in the culture media through the enzymes GK (glucokinase), PGM (phosphoglucomutase), UGP (UDPGlc pyrophosphorylase), and CS (cellulose synthase).^{[ 43 ]} Cellulose is first produced as elementary fibrils, a cellulose chain of glucose units bound by β‐1,4 glycosidic bonds then assembled into microfibrils, which are in turn assembled into microfibrils bundles. The last assembly is the cellulose fiber, which in the standard biosynthesis process in static forms a BNC pellicle at the liquid–air interface. The illustration scales are not representative of reality. D = diameter.

Figure 2

Methodology to obtain aligned BNC (A‐BNC) and the reuse of the bacterial substrates. A) Screenshot of the extrusion of BNC fibrils by K. xylinus strain's individual pores when immersed in HS liquid culture media with no restriction of movement. B) K. xylinus colonies grow confluent on the surface of HS solid media (agar). C) Square section of agar with confluent bacterial colonies (marked in blue) is placed at the bottom of the culture tube filled with HS liquid culture media, restricting the bacteria movement. Due to the oxygen availability, bacteria move toward the liquid–air interface to reach higher DO concentrations, leaving aligned BNC (A‐BNC) fibers behind. Hence, A‐BNC is synthesized with directionality toward the liquid–air interface of the culture, forming BNC. D,E) Optical images of the macrostructure of A‐BNC within the length of a A‐BNC and BNC film. F) Reuse of bacterial substrates: agar containing bacterial colonies (marked as blue) and the produced BNC film (marked as green) are reused to grow aligned BNC. G) Reusing the agar square piece, which still maintains bacterial confluence, at the bottom of the vessel of a new culture tube allows obtaining A‐BNC and BNC repeatedly for at least 7 cycles. H) Reusing the produced and not cleaned BNC film (marked in green in F) allowed the “Film‐to‐film” biosynthesis method. I) Scanning electron microscopy (SEM) image of an uncleaned BNC film containing bacteria. J) Reusing uncleaned BNC films in a new culture tube allowed obtaining “Film‐to‐film,” which consist in 2 BNC films bound at the extremes of an A‐BNC film. This process can be repeated multiple times and avoiding bacteria and time waste.

Figure 3

Transparency and chemistry of A‐BNC. A) Pictures of A‐BNC and BNC, respectively. B,C) Pictures of A‐BNC and BNC on top of a micrometer‐mesh, indicating a hazier BNC than A‐BNC. D) Dry A‐BNC shows iridescence effects under the incidence of white light. E) FTIR reveals that A‐BNC and BNC have similar chemical compositions. However, the A‐BNC spectra show more intense transmittance peaks in the OH and CH region.

Figure 4

Morphology and structure analysis. A) Picture of the as‐produced nanocellulose films containing BNC and A‐BNC. The top B,C,D) and bottom E,F,G) rows contain the images obtained by TEM, SEM, and AFM of BNC and A‐BNC. B,E) TEM images and selected area electron diffraction (SAED) patterns of BNC and A‐BNC samples. C,F) SEM micrographs of BNC and A‐BNC samples (Insets: color analysis from the OrientationJ plugin for ImageJ). D,G) AFM images of BNC and A‐BNC samples (Insets: isotropy distribution computed using MountainView8).

Figure 5

Nanoscale density study with SAXS. A,B) Small angle X‐ray scattering (SAXS) scattering signal of BNC and A‐BNC. C) Integration of the spectra as intensity versus azimuthal degree plot, from −90° to 90°, whereas BNC gave no peak. D) Table with the quantitative orientation parameters orientation index (OI), full width at the half‐maximum intensity (FWHM), and Herman's order parameter (S‐parameter) values for BNC and A‐BNC.

Figure 6

Polarization‐resolved second harmonic generation (PSHG) microscopy. Stacks of 18 images (frame/10° light polarization) were analyzed with ImageJ using a heat map and Z projections. A) Maximum and minimum SHG intensity projection of 150 × 150 µm² for BNC and A‐BNC, along with the intensity color map. B) Plot of the SHG intensity for each angle polarization, along with their alignment coherency, obtained with the OrientationJ plugin for ImageJ.

Figure 7

The behavior of A‐BNC and BNC under polarized light. Visualization under a polarized light microscope at 0° and 45° (incident polarized light beam vs sample orientation) of A,D) BNC samples, B,E) A‐BNC samples and C,F) N‐shaped A‐BNC (Insets of whole films in (A)–(C)). Samples are circularly rotated within the same plane, perpendicularly to the incident polarized light beam.

Figure 8

Hydrophilicity and cell substrates evaluation of A‐BNC and BNC. A) Pictures of an A‐BNC film's wetting with a colored water drop. B,C) Measurement of the apparent contact angle (ACA) of A‐BNC and BNC films, respectively. D,E) Optical images of confluent cell cultures at day 7 on A‐BNC and BNC, respectively. F) Orientation coherency plot of A‐BNC, BNC, and the slide (control), computed with the OrientationJ plugin of ImageJ. *** = P ≤ 0.001, **** = P ≤ 0.0001.

Figure 9

Thermal conductivity study of A‐BNC. A) 3ω measurements of A‐BNC at the parallel and perpendicular directions were evaluated, indicating a maximum five‐fold increase of the thermal conductivity in the parallel direction of A‐BNC fibers. B) Thermal conductivity of A‐BNC measured by anisotropic thermoreflectance thermometry, showing similar results as shown in (A). C) Comparison of the obtained measurements to the standard collagen,^{[ 65 ]} PLA,^{[ 70 ]} or PE^{[ 68 ]} used in implants. D) Table of the reported thermal conductivity of polymeric materials.^{[ 62} , ⁶⁵ , ⁶⁸ , ⁷⁰ , ⁷¹ , ⁷² , ^{73 ]}