Comparison of BNC production
At first, the effect of the different cellulosic additives upon BNC production by Komagataeibacter sp. SFCB22-18 was investigated. As cellulosic additives, Avicel (microcrystalline cellulose produced by acid hydrolysis of wood pulp) and carboxymethylcellulose (CMC; cellulose derivatives with carboxymethyl groups at some of the hydroxyl groups of glucopyranose) were used. Since these cellulosic additives have been widely used in the food, pharmaceutical, paper, and cosmetic industries due to their unique physical and chemical properties [21, 22], they are good candidate of altering BNC properties [23, 24]. In comparison with the control group (i.e., without additives, 0.4 g/L), Avicel and CMC addition showed slight increase in the BNC production (Fig. 1). With the additions of 0.1% Avicel and CMC, the Komagataeibacter strain produced 0.5 g/L and 0.7 g/L of cellulose pellicles, respectively. As the concentrations of cellulosic additives increased to 1%, BNC production also increased considerably. In particular, the addition of 1% CMC showed the highest BNC production of 2.0 g/L, which is equivalent to 5 times higher than in the control group. This increase might be because of the proper incorporation (or adsorption) of additives and reduction in crystallinity, which is a rate limiting step during BNC production [23, 25]. Meanwhile, Avicel did not show much improvement in BNC production because Avicel is less soluble than CMC [26]. Without optimization of reaction conditions, it was again proved that soluble additives may give more beneficial effects on production of BNC [23, 24, 27].
Morphological and structural properties of modified-BNC
The morphological structures of BNC with and without cellulosic additives were analyzed by SEM (Fig. 2). In the pure BNC, the thread-like parallel stacked cellulose bundles with large clumps by aggregation were observed. However, the clumps were not observable in altered BNC, possibly attributed by adsorption of additives on BCN surfaces [28–30]. This is probably due to the limitations of intermolecular interactions among cellulose fibrils, such as hydrogen bonding, the van der Waals force, and the electrostatic interactions which stabilize the highly ordered BC structure [14]. Accordingly CMC-altered BNC consisted of fibers having slightly further distance than Avicel-altered BNC, because of repulsive force. Therefore, cellulosic additives were efficiently incorporated into BNC, thereby modifying its structural morphology and crystalline properties.
The FT-IR spectra (Supplementary Fig. 1) of pure BNC and altered BNC were showing a broad OH peak stretching in the range of 3,500-3,000 cm− 1 and C-O-C stretching at around 1,160 cm− 1, typical BNC spectrum reported [31, 32]. In pure BNC, additional peaks of 1,160 cm− 1 (C-O-C stretching) and 1,035–1,060 cm− 1 (C-O stretching) were observed [31, 32]. In Avicel- and CMC-altered BNC, the peak intensity at 3,500-3,000 cm− 1 increased in comparison with that in pure BNC. In addition, at Avicel-altered BNC, the peak intensities at around 1,620–1,650 cm− 1 (OH bending) increased, showing stronger adsorption of water molecules to BNC. This may be due to either the inhibition of BNC crystallization by reducing the degree of polymerization (DP) of the modified BNC [33] or the exposure of a many OH groups by disruption of intermolecular bonds in BNC [23]. At CMC-altered BNC, strong absorption peaks at 1,572 cm− 1, corresponding to the carboxyl group [34] was observed, implying that CMC was well incorporated (or adsorbed) on pure BNC.
Degree of crystallinity
The crystallinity of cellulose may indirectly reflect its physical properties such as hardness, elasticity, permeability, and reactivity [35]. According to a previous report, BNC crystallinity is negatively related to polymerization of glucose [36]. Therefore, we analyzed and compared crystalline properties of BNC modified by the addition of cellulosic additives using XRD and crystalline cellulose-binding proteins.
At first, the crystallinity of BNC through the addition of different cellulosic substrates was measured by XRD (Supplementary Fig. 2). The XRD pattern of pure BNC showed strong diffraction peaks at 2θ = 22.7° (principal peak), 14.5°, and 16.8°, representing cellulose Iα, which is naturally produced crystalline cellulose [37]. The Avicel- and CMC-altered BNC showed similar patterns to cellulose I. However, as the concentrations of additives increased, the diffraction peaks at the 2θ angles of 16.8° decreased considerably in comparison with pure BNC. This means that the cellulose I structure was transformed to cellulose allomorph, resulting in the conversion of some crystalline structures into amorphous structures through the addition of cellulosic substrates during BNC production [38, 39]. Furthermore, the crystallinity index of BNC measured by XRD was reduced by the addition of Avicel and CMC (Table 1); for example, while the crystallinity of pure BNC was 77.6%, those of modified BNC were about 69.2–73.4%. Similarly, when calculating the total crystallinity index (TCI, A1375/A2900 from FT-IR), a slight decrease in TCI values was observed after addition of cellulosic substrates. These observation agrees well with the previous studies indicating the cellulosic additives slightly hinder the crystallization of BNC during BNC synthesis, of which results are in accordance with the increase in BNC production or the increase in water retention ability (i.e. OH group exposure at FT-IR).
Table 1
Summary of the crystallinities of BNC incorporating different concentrations of cellulosic additives
| |
Crl
(%)
|
TCI
(%)
|
Relative value of CBM binding (%)
|
|
BC
|
77.6
|
0.994
|
100
|
|
Avicel
|
74.1
|
0.991
|
110.8
|
|
CMC
|
1.2
|
0.999
|
17.5
|
| |
Additive conc.
(%)
|
Crl
(%)
|
TCI
(%)
|
Relative value of CBM binding (%)
|
|
BC + Avicel
|
0.1
|
72.2
|
0.993
|
243.3
|
|
1.0
|
73.4
|
0.969
|
233.0
|
|
BC + CMC
|
0.1
|
69.2
|
0.988
|
177.8
|
|
1.0
|
70.2
|
0.970
|
249.0
|
At second, Surface accessibility of the crystalline region of BNC was observed by using cellulose binding proteins. CBD has been suggested to understand the surface accessibility of cellulosic substrates [16, 40, 41] on the basis of the distinct molecular recognition ability whether the binding region in cellulose is crystalline or amorphous. It has not been previously reported that cellulose-binding proteins can be used to elucidate the morphology of the BNC surface, one of the nano-sized cellulosic materials. Among cellulose-binding proteins, we selected CtCBD3, originating from Clostridium thermocellum ATCC 27405 and belongs to type-A cellulose-binding proteins, which predominantly bind to the crystalline region of cellulose. The amount of bound protein in pure BNC was approximately 1.9 nmol/mg substrate (Fig. 3). The modified-BNC samples showed 1.8–2.5 times higher binding affinities to CtCBD3 than pure BNC. The differences to XRD results might be because cellulose binding protein are generally bound to the surface of the cellulose, while XRD or FT-IR focused on the fiber’s interior structure and bulk properties [16]. Thus, the increase in protein binding in this study indicates that the crystalline regions of BNC were considerably exposed to the surface of modified BNC by cellulosic additives. This may suggest that the existence and stability of various functional groups were on the surface of modified-BNC. Therefore, the addition of cellulosic substrates such as Avicel and CMC during BNC synthesis can efficiently alter the surface crystallinity of the BNC during fermentation. In summary, since Avicel and CMC may attach to microfibrils surface during the production or crystallization of BNC, measuring binding properties using cellulose-binding protein may be a feasible option for identifying the crystalline properties of BNC [30, 33, 42].