Optimization of Moist and Oven-Dried Bacterial Cellulose Production for Functional Properties

Abstract

Bacterial cellulose (BC) is a natural polymer with properties suitable for tissue engineering and possible applications in scaffold production. However, current procedures have limitations in obtaining BC pellicles with the desired structural, physical, and mechanical properties. Thus, this study analyzed the optimal culture conditions of BC membranes and two types of processing: draining and oven-drying. The aim was to obtain BC membranes with properties suitable for a wound dressing material. Two studies were carried out. In the preliminary study, the medium (100 mL) was inoculated with varying volumes (1, 2, 3, 4, and 5 mL) and incubated statically for different periods (3, 6, 9, 12, and 18 days), using a full factorial experimental design. Thickness, uniformity, weight, and yield were evaluated. In the optimization study, a Box-Behnken design was used. Two independent variables were used: inoculum volume (X₁: 1, 3, and 5 mL) and fermentation period (X₂: 6, 12, and 18 d) to determine the target response variables: thickness, swelling ratio, drug release, fiber diameter, tensile strength, and Young's modulus for both dry and moist BC membranes. The mathematical modelling of the effect of the two independent variables was performed by response surface methodology (RSM). The obtained models were validated with new experimental values and confirmed for all tested properties, except Young's modulus of oven-dried BC. Thus, the optimal properties in terms of a scaffold material of the moist BC were obtained with an inoculum volume of 5% (v/v) and 16 d of fermentation. While, for the oven-dried membranes, optimal properties were obtained with a 4% (v/v) and 14 d of fermentation.

Keywords: SEM; TEM; drug-release; swelling ratio; thickness.

Figure 1

TEM images of Gluconacetobacter xylinus cellulose forming bacteria in pristine bacterial cellulose membranes. (a)—rod-shaped bacteria; (b)—ellipsoidal bacteria; (c)—cell structure and wall aspect of G. xylinus.

Figure 2

The response surface plot of thickness performed on the factorial design with 5 levels.

Figure 3

FTIR spectra of bacterial cellulose and filter paper on 650–3600 cm⁻¹ domain. PF—filter paper, i—inoculum, d—harvest day, M—moist membrane, D—dry membrane; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days.

Figure 4

Swelling ratio of dry and moist bacterial cellulose (BC) membranes over 24 h. (a)—moist BC membrane; (b)—dry BC membrane; i—inoculum, d—harvest day; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days.

Figure 5

Moisture content of dry and moist BC membranes over 24 h. (a)—moist BC membrane; (b)—dry BC membrane; i—inoculum, d—harvest day; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days.

Figure 6

Drug release activity of BCd and BCm membranes over 72 h; i—inoculum, d—harvest day, M—moist membrane, D—dry membrane; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days.

Figure 7

Kruskal–Wallis graphic test showing fiber diameter differences among BC dry and moist membranes. i—inoculum, d—harvest day, M—moist membrane, D—dry membrane; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days; the “*” indicates a statistical significance at the level indicated by p.

Figure 8

SEM images of moist BC. (a)—i1d6; (b)—i5d6; (c)—i1d18; (d)—i5d18; (e)—i3d12; i—inoculum, d—harvest day; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days (electron high tension (EHT) = 10.00 kV; magnitude (Mag) = 10.00 KX; working distance (WD) = 7.0 mm; Signal A = SE1).

Figure 9

SEM images of dry BC. (a)—i1d6; (b)—i5d6; (c)—i1d18; (d)—i5d18; (e)—i3d12; i—inoculum, d—harvest day; the number following “i” stands for inoculum volume 1, 3, 5 mL and the number following “d” represents the harvest day 6, 12, 18 days. (electron high tension (EHT) = 10.00 kV; magnitude (Mag) = 10.00 KX; working distance (WD) = 7.0 mm; Signal A = SE1).

Figure 10

Response surface methodology RSM contour plots of drug half-release time of moist (a) and dried (b) bacterial cellulose (BC); Young’s modulus of moist (c) and dried (d) BC; fiber diameter of moist (e) and dried (f) BC.