Tailor-Made Biosystems - Bacterial Cellulose-Based Films with Plant Cell Wall Polysaccharides

Abstract

Bacterial cellulose (BC) is a natural biopolymer metabolized by Gram-negative bacteria strains in carbon- and nitrogen-rich media. Due to its natural purity, biodegradability, biocompatibility and outstanding mechanical properties, it is considered to be an exceptional biomaterial with versatile scientific and commercial applications. The current review presents data concerning the assembly, structural properties and mechanical behavior of specific biosystems based on bacteria-produced cellulose and polysaccharides derived from plant cell walls. Bacterial cellulose, hemicellulose, their binary composites and ternary composites with pectin are described with respect to the mechanical properties of the obtained materials. The properties of one-component and composite films are discussed with respect to the type of polysaccharides used, their origin and applied pretreatments, as well as various methodological approaches to bacterial cellulose synthesis. This work is focused entirely on the relationship between production methods and composition, and the resulting properties of the obtained materials, it constitutes a universal source of knowledge for specialists from various fields of science and industry.

Keywords:

• bacterial cellulose
• hemicelluloses
• pectin
• mechanical properties

1. Introduction

Bacterial cellulose (BC) is a biopolymer produced as a primary metabolite by several strains of bacteria using glucose, fructose, sucrose, mannitol, among others, as carbon sources.^[1,2] In terms of chemical structure, bacterial cellulose is identical to the cellulose produced by plants. BC is a linear polysaccharide composed of β-D-glucopyranose monomers linked by β-1,4-glycosidic linkages, forming molecules of cellobiose.^[3] As is the case with plants, bacterial cellulose exists mainly in cellulose I crystallographic form, with two coexisting sub-allomorphs, triclinic Iα and monoclinic Iβ structures.^[4] Metastable Iα cellulose is the dominant structure, comprising up to 80% of BC.^[5] Compared to plant-produced cellulose, BC has a lower degree of polymerization ranging between 2000-6000 (with 13,000 − 14,000 for plants). BC is produced in the pores located in the cytoplasmic membranes and extruded in the form of protofibrils, 2-4 nm in diameter.^[6–8] Then, protofibrils may assemble, forming nanofibrils with typical widths in range of 5–20 nanometers and micrometer scale lengths or microfibrils up to 100 nm in diameter.^[9] Fibrils, that include both of nano- and microfibrils, may also aggregate, creating fibers – multi-scale structures, composed of well-organized fibrils with diameter up to 50 μm and length in several milimeters.^[10,11]

BC is assembled in the form of highly hydrated (99% of water), high crystallinity (84-90%) and high purity cellulose membranes at the surface of a carbon- and nitrogen-containing medium.^[12–15] The resulting pellicles are constituted by an ultrafine network structure of highly entangled cellulose fibrils.^[5,9,16,17]

Bacterial cellulose-based membranes and films show outstanding mechanical properties and a large specific surface area, comparable with materials obtained with plant-based cellulose. BC is characterized by a high degree of crystallinity, reduced fibril diameter and uniform chemical structure, which lead to stiff fibril connections. Applications have already been found for BC in tissue engineering, biosensor fabrication, antimicrobial and biodegradable film production and as a texture improver.^[18,19] BC does not cause allergic reactions, so its suitability for the role of being used as a temporary skin substitute, artificial blood vessel etc. is already being investigated.^[6,16] BC composites have been used for wound dressing, vessel regeneration.^[20] Also, some approaches involving BC-laser sensitized magnetic particle use were applied for breast cancer treatment.^[21] Kwak and others reported healing acceleration in burned rat skin with the application of BC membranes.^[22] Cellulose fibers, incorporated with hemicelluloses, also demonstrated the stimulation of fibroblast growth and proliferation.^[23] Another potential area for hemicellulose use is in the production of biodegradable and antibacterial films for food storage.^[24] A brief review of BC-based food packaging material was provided by Cazon and Vasques.^[25] They summarized reports concerning the great potential for BC modification with its high degree of porosity, which allows for modifying agents to be immersed in the material.

Among many applications, BC composites with plant cell-wall polysaccharides have gained particular importance in studies concerning the properties of plant cell walls (PCW). This has been attributed to the chemical similarity of BC to natural cellulose, which allows for the creation of what are widely known as plant cell-wall analogues – polysaccharide assemblies which have a composition and structure that mimics those of PCW. The idea of BC use for PCW modeling was firstly proposed in 1982 by Haigler et al.^[26–28] Since then, pectin, xyloglucan, glucomannan, arabinoglucuroxylan and arabinogalactan have been added to the culture medium and their effects on the bacterial cellulose structure as well as their mechanical properties have been shown by several groups.^[29–34]

With the use of precise techniques, such an approach allows one to minimize the limitations of the method used to determine the qualitative composition of PCW analogues, cellulose crystallinity or physical properties of the network (thickness, density, permeability).^[35] The synthesis of bacterial cellulose (BC) in the presence of hemicelluloses and/or pectins allows for the attainment of films, which may be varied in their composition or/and structure, according to research necessities. This synthetic approach allows researchers to modify the cell-wall environment and to study the impact of each specific compound on the properties of the cellulose-based material. Production, handling and storage are more straightforward, as compared to plant cell-wall-extracted materials.^[36] BC-based composites may be considered to be a model object of PCW, this has been confirmed by numerous scientific investigations. A number of papers, dedicated to the determination of PCW structure and mechanical properties have already been published.^[37–39]

Therefore the scope of this paper is focused exclusively on data concerning the structural and mechanical properties of BC films, BC-hemicellulose binary and BC-hemicellulose-pectin ternary composites, thereby providing information that may be useful not only to scientists from the cell wall community, but in general, for researchers concerned with plant polysaccharide-based composites.

2. Structural and mechanical properties of bacterial cellulose

BC-producing bacteria include a number of genera.^[26,40] Komagataeibacter xylinum (previously known as Acetobacter or Gluconacetobacter xylinum) is a widely used bacteria strain for BC biosynthesis, this is due to its plant-like synthesis, high yields, ultrafine structure and chemical purity.^[24,33] In general, the mechanical characteristics of films improve with the growing concentration of BC. Komagataeibacter BC hydrogels with a higher percentage of BC are entirely opaque, compared to low-BC samples, which contribute both opaque and transparent regions. High-BC hydrogels provide superior levels of stress resistance and Young`s modulus, but the maximum strain measurements are not changed significantly. However, low-BC samples showed a higher level of moisture uptake per unit weight.

K. rhaeticus BC films show a more homogeneous distribution of cellulose fibers, as compared to the K. xylinum one, being much closer to the PCW-like structure. Comparing both, K. xylinum films exhibit a higher tensile strength and Young`s modulus in the dried state, whereas elongation is higher for K. rhaeticus samples. This difference may be explained by the higher degree of porosity of the K. rhaeticus film (looser structure, lower degree of hydrogen bonding), lower fibril orientation and decreased crystallinity. The opposite relationship was obtained for BC samples in their wet state, which also indicated that some Komagataeibacter strains are sensitive to post-production treatment.^[41]

Kombucha (Komagataeibacter strains) untreated BC films show a bulky low-crystalline structure. However, NaOH-washed samples showed a sufficient increase (3 to 20 times) in mechanical properties and crystallinity level up to about 47%.^[40,41] Such a tendency was further explained by stronger nanoparticle interconnections and fibril entanglements.^[42]

The classic medium for K. xylinum growth was established in 1954 by Hestrin and Schramm (HS medium), it includes glucose, bactopeptone, yeast extract, agar (for solid medium), with a pH value of 4.5-6 and a temperature of 25-30 °C.^[43–46] Bacteria synthesize cellulose as a primary metabolite. The main sources of carbon and nitrogen for cellulose growth are glucose/fructose/sucrose/mannitol and peptone/yeast extract, respectively, the effect of additives is assumed to be negligible.^[17] Volova et al. reported that fibril shape parameters are closely dependent on the growth medium used, with thin low-crystalline fibrils being obtained from a galactose-enriched medium, while the thickest one were obtained from the sucrose one. The mechanical properties of the film directly depend on fibril diameter growth only to a minor degree.^[47] An informative summary of BC production under different cultural conditions has been provided by Lin et al.^[48]

Since K. xylinum is an aerobic bacteria, growing techniques may include forced aeration. Static conditions allow for the attainment of uniform and smooth films with defined characteristics.^[49] Agitation reduces the degree of polymerization, the cellulose Iα content and total crystallinity. Chemically, the BC produced under agitation, is equivalent to one produced in static conditions, but with curved and entangled fibrils, which results in an enhanced density and water-holding capacity but up to a 20% decrease in Young`s modulus.^[49–51]

Cellulose synthesizing complexes are located on the bacteria surface close to the cell membrane pores. Cellulose is extruded with an approximate speed 2 μm/min and forms a ribbon composed of nearly 46 microfibrils, each containing 18-36 cellulose chains. Ribbons tear from their forming sites possibly due to lateral stress and aggregate into fibrils via glucan chain crystallization. The resulting BC fibril length is approximately 1.6-5.8 μm.^[48]

Generalized data concerning BC film mechanical properties are given in Table 1. Yamanaka et al. provided one of the first reports about BC dedicated to the relationship between its structural and mechanical properties, and determined that the Young`s modulus of the BC film to be higher than 15 GPa. They also reported the presence of structural “three-way branching points” along fibrils, which was indicated by continued cellulose excretion during bacteria cell division. Structurally, BC is deposited in the pellicle in a number of thin fibrillar layers, with a thickness estimated to be approximately 0.1 μm. Such layers are believed to be connected via an interfibril hydrogen bond network, which is quite dense due to its enhanced contact area.^[4]

Table 1. BC film mechanical properties (measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Source	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	References
BC air-dried	K. xylinum	256.0	1.07	16900	65	20	^[^4^]
BC hot-pressed	AJ 12368	91-260	0.8-1.9	16400-17500
BC	K. xylinum	0.33-0.54	17.5-40.0				^[^31^]
BC	K. xylinum	1.57	18.0				^[^29^]
BC dried	K. xylinum	5.21	3.75				^[^52^]
BC swollen		1.56	8.0
BC	K. xylinum ATCC 23769	32.0 ± 5.0	3.4			RT	^[^53^]
BC pinned		28.0 ± 5.0	1.7
BC	K. xylinum ATCC 53524	1.2-2.0	23.0-30.0	10.0-14.2			^[^54^]
BC NaOH treat.		1.2-1.6	21.0-26.0	9.1-13.2
BC	K. xylinum	150 ± 16.0	4.5 ± 0.5	3700 ± 800	30	20	^[^55^]
BC	K. xylinum	1.7	>33.0			25	^[^30^]
	ATCC 53582
Crystalline bacterial cellulose	G. hansenii ATCC 23769	33.0	76.0	5200.0	50	25	^[^42^]
BC	K. xylinum ATCC 1765	194.4	3.4	11400.0	40	23	^[^56^]
BC ATCC 700178	K. xylinum,	0.15 ± 0.08	20.7 ± 8.3	1.1 ± 0.38			^[^26^]
BC ATCC 10245	G. hansenii	0.36 ± 0.08	18.6 ± 8.0	2.87 ± 1.33
BC ATCC 23769		0.12 ± 0.04	18.0 ± 5.0	1.26 ± 0.66
BC NBRC 13693		0.62 ± 0.17	18.7 ± 3.3	3.08 ± 0.66
BC ATCC 53524		0.68 ± 0.13	20.7 ± 6.0	5.56 ± 2.29
BC KTH 5655		0.62 ± 0.16	16.2 ± 2.91	3.83 ± 1.08
BC	K. xylinum B-12068		10.1-15.2	3.7-5.5		RT	^[^47^]
BC untreat.	K.	27.9 ± 1.7	45.2 ± 1.4	43.7			^[^57^]
BC H2O pretreat.	xylinum	75.8 ± 7.8	8.4 ± 2.5	950.0
BCNaOH pretreat.		73.7 ± 11.8	5.5 ± 4.6	1069.1
BC K. xylinus (dry)	K. xylinum,	141 ± 70	4.0	10100 ± 800		RT	^[^41^]
BC K. xylinus (wet)	K.rhaet. TJPU03	0.1 ± 0.01	22.0 ± 1.0	6.1
BC K. rhaet. (dry)		70 ± 6	5.0	5300 ± 400
BC K. rhaet. (wet)		0.16 ± 0.01	27.0 ± 2.0	0.0083
BC diff. H2O act.	K. xylinum	15.5-22.3	1.4-3.7	650-1800			^[^25^]

The isotropic randomized structure of the pellicle was observed for BC produced by K. xylinum. Under the high strain-rate deformation of the pellicles, double stress peaks were observed, this was attributed to the failure of the short and long fibrils. Slow deformation revealed similar stress values with one peak only, indicating the presence of strain-dependent mechanical properties connected with inter-fiber slippage and disentanglement.^[54] Such a fracturing behavior was previously observed by Astley et al. (2003), who reported BC layers sliding past each other during deformation.^[29]

BC pellicles behave as a highly hydrophilic material, holding up to 100 times its dry weight in water.^[43] Sanchavanakit et al. reported water swollen films that show a strain which is twice as high at its breaking point and a Young`s modulus which decreased by a factor of three when compared to the dried films.^[52] Rambo et al. (2008) declared that water swelling decreased inter-fibril hydrogen bonding, thereby degrading the mechanical properties of the film.^[53]

Cybulska et al. (2010) reported the pure BC network to be porous and consist of branched nanofibrils, distributed randomly with an average length and diameter of 1 μm and 10-20 nm, respectively.^[32] Chi and Catchmark also reported porous BC film with ribbons of about 30-60 nm in diameter. Crystalline bacterial cellulose (CBC), which is acid treated BC with removed amorphous regions was observed as rod-shaped particles 0.1-2 μm and 2-14 nm in length and height, respectively.^[42] Bendaoud et al. (2017) reported the attainment of non-porous BC pellicles, which were characterized by linear elastic stress-strain behavior, a low strain at break and exhibiting brittle fracturing. The rigid and brittle behavior of the network was potentially attributed to its vitreous polymer state.^[58]

Wang et al. described a BC film which was processed with wet-stretching and shaking to provide a highly linear structure. Stretching led to the creation of a densely packed aligned cellulose network. During network elongation, BC changed its arrangement from a layered one to fibril needlelike bundles. Denser packaging promoted the formation of an enhanced hydrogen bond network, which resulted in an increased Young`s modulus (4.4 times), tensile strength – (5 times) and strain at break – (1.3 times). Also, the stretched films showed lower water adsorption, improved toughness and strength, which were explained through decreased defect size and the enhanced hydrogen bond network.^[56] Dayal et al. reported that its water binding capacity enhanced the mechanical properties of BC films.^[30]

BC films also demonstrated the non-linear dependence of mechanical properties under different water activities. Young`s modulus and the tensile strength increase with growing water activity, reaching its maximum value at a_w = 0.43. At lower moisture contents, water adsorbs on cellulose, easing the mobility of both crystalline and amorphous cellulose regions, promoting fibril reorganization and improving chain resistance under axial deformation. With further water addition, the hydrogen bond network degrades in conjunction with fibril swelling, the plasticizing of the cellulose film and the enhancement of fibril movement within the network.^[25]

3. Structural and mechanical properties of hemicelluloses

Hemicelluloses are defined as polysaccharides, they can be water- or alkali-extracted from plants^[59], they are able to link cellulose via hydrogen bonding.^[60] Hemicellulose composition may vary between plant species.^[61] Common hemicellulose monosaccharides are D-xylose, L-arabinose, D-glucose, D-galactose, D-mannose, D-glucuronic acid etc.^[59]

In PCW, there are several main types of hemicelluloses – xyloglucan XG, xylans and mannans. XG is a major PCW hemicellulose component, which consists of a β-(1,4)-D-glucan backbone, decorated with xylose sidechains. Xylose residues may have some galactose- and fucose- grafted substitutes.^[62] Compared to other PCW hemicelluloses, the XG structure is highly regular. The majority of higher plants produce repeatable XG of the XXXG-type (X – glucose unit with xylose residue, G – glucose unit), with the X unit prone to further substitutions.^[63] Widely used in experimental studies, tamarind XG is composed of XXXG, XXLG, LXG and XLLG units in a ratio of 1.4:3:1:5.4, respectively.^[64] Analysis of polysaccharide subunits in different plants and tissues shows predominant composition of XXXG, XXLG, XLXG and XXFG, which means attribution of the main chain with β-(1,2)-D-galactopyranoses and α-(1,2)-L-fucopyranoses. Plant cell wall XG is usually fucosylated, whether substitutes with fucosyl attributes also has biological activity effect in PCW.^[40,65] Hoffman et al. (2005) provided comprehensive overview on structures of XG in various plant species according to type of subunit and tissue.^[63] The molecular weight (MW) of XG is in the range of 650-2500 kDa.^[66] In its solid state, XG has a flat, two-fold twisted backbone, similar to cellulose.^[67]

Xylans are linear polymers of β-(1,4)-linked xylose. According to the type of backbone α-(1,2)-substitutes used, several xylan groups, such as glucuronoxylan (GX), methyl glucuronoxylan (MGX), arabinoxylan (AX) are known. AX is the main hemicellulose of commelinid monocots.^[66] It contains O2 and/or O3 arabinose substitutes, irregularly distributed along the backbone. The average MW is 65-5000 kDa. Xylans have various randomly distributed backbone substitutes, which are difficult to determine. Its biosynthetic pathway is complex^[68], but predicted to be similar to XG.^[69]

Mannans are a group of polysaccharides, composed of (1,4)-β-D-mannopyranoses with a low degree of galactose substitution (linear mannan), high-galactose substitutes (galactomannan), glucopyranose (glucomannan) and galactose-glucopyranose (galactoglucomannan GGM) incorporation.^[70] The glucose to mannose ratio for GGM is reported to be 1:1.6.^[71] GGM and glucomannan in small amounts exist in dicot PCW.^[72] Konjac and spruce glucomannans are widely used in experimental studies by acting as a model polysaccharides.^[73] Konjac glucomannan (KGM) contains randomly distributed β-(1,4)-linked D-mannose and β-(1,4)-linked D-glucose in 3:1 ratio with a low degree of α-(1,6) branching and degree of polymerization of 200.^[73,74] Softwood mannans is a low galactosyl substituted glucomannan with degree of polymerization of 510 with a sugar ratio of galactose:glucose:mannose 0.03:1:3.4.^[75] Both Konjac and spruce glucomannan provide structures similar to low-galactosylated GGM, with an ability to be extracted with a reasonable yields, even from residues of papermaking industry.^[70,71,73] High content of mannans in Konjac and softwood allow to reduce mannans degradation by lowering its yield.^[73]

Hemicellulose distribution in the plant cell wall is still an open question, especially from the viewpoint of accepted PCW models. Hemicelluloses are synthesized in Golgi, targeting the plasma membrane for newly synthesized cellulose fibrils.^[59] According to the most recent views developed by researchers, hemicelluloses are recognized as tethers/hotspot linkers, which enhance cellulosic network properties. Nonetheless, some sort of hemicellulose-only networks were observed in pea root cells.^[76] In a similar way, hemicelluloses are reported to form separate microdomains in PCW ^[38], or they form a hydrogen bond network.^[71,72] Networks consisting entirely of hemicellulose are fragile for the most part due to their short polysaccharide backbones and branched substitutes.^[77] Also, it is necessary to declare the role of lignin (copolymers of p-hydroxyphenyl, guaiacyl and syringyl units) on plant cell wall network.^[78] Plant cell wall lignin is suggested to be more associated with xylans, compare to the other cell wall polysaccharides. Higher lignin content in cell wall decrease its mechanical properties due to decreased aggregation of other plant cell wall polysaccharides.^[79] There are relatively few published reports dedicated to this theme. Here, we will evaluate the current data concerning the structural and mechanical properties of hemicellulose films.

3.1. Xyloglucan

Generalized data concerning the mechanical properties of XG are given in Table 2. Kochumalayil et al. (2010) reported that uniformly casted XG films are only produced in negligible quantities.^[80] High-MW XG may form cast films on its own, whereas low-MW XG requires external plasticizing agents. High-MW XG networks show robustness with a smooth surface. Young`s modulus for dry XG films is approximately 6 GPa, which is defined by strong intermolecular connections and low free volume.<sup>[26]</sup> With moisture uptake, the intermolecular bonds of branched XG degrade, however, they are still strong enough to oppose mechanical loads. Bendaoud reported that XG films exhibit linear elastic stress-strain behavior, but have a low strain at break value and brittle fracturing. Cross-sections of XG networks showed a smooth structure with uniform fibril dispersion and few aggregates. Stress at break and Young`s modulus for the XG films were 1.5-1.8 times higher, compared to the BC control.^[58]

Table 2. Mechanical properties of XG films (measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Source	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	Reference
XG	Tamarind	61.0 ± 8.6	4.8 ± 3.6	4300 ± 230	50	RT	[^80]
XG	Tamarind	28.0-114.0	2.7-9.2	1630-5950	50	RT	[^36]
XG	Tamarind	78.0 ± 8.6	5.7 ± 3.6	4597 ± 164	50	RT	[^81]
XG	Tamarind	92.9 ± 5.8	8.9 ± 2.0	4100 ± 150	50	23	[^82]
XG	Tamarind			1500-3500	50	25	[^58]
XG	Tamarind	78.0 ± 8.6	5.7 ± 3.6	4597 ± 164	50	23	[^83]

Pre-sonicated XG solutions form casted films with improved mechanical characteristics, as compared to non-sonicated ones.^[84] It is clear that more dense dispersions of polymer chains in a network enhance its properties via hydrogen bond densification and fibril sticking.

3.2. Xylans

Aspen, birchwood, corncob and grass xylans were shown to have non-network-forming polysaccharides.^[85] Data concerning the mechanical properties of xylan films are summarized in Table 3. A number of xylan networks with lignin/pectin additives have been reported. Under the same network density, xylan concentration is directly proportional to tensile strength, strain at break (increased amount of entrapped water) and Young`s modulus.<sup>[87]</sup> Da Silva Braga et al. reported dense xylan-lignin networks with reduced surface roughness.<sup>[90]</sup> The contiguous glucuronoxylan network with lignin additives, obtained by Bancegul et al. showed a high degree of extensibility with elongations reaching up to 12%. The presence of approximately 1% lignin and pectin impurities improves the xylan network-forming properties due to its dispersibility, thereby increasing Young`s modulus but decreasing strain at break. Lignin improve network elongation properties by creation of intermolecular ester and ether bonds with xylan. Both compounds act as natural plasticizers, and produce an effect similar to increasing the degree of acetylation.^[89]

Table 3. Mechanical properties of xylan films (DS – degree of substitution; DP – degree of polymerization, measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Source	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	Reference
Xylan	Maize bran	10.0-11.0	8.0-38.0	57.0-118.0		19 ± 1	[^86]
Xylan	Cotton stalk	1.1-1.4	45.5-56.7		40 ± 4	20 ± 2	[^87]
Xylan	Bamboo	10.02 ± 2.01	1.28 ± 0.5	780 ± 420			[^88]
Xylan	Birchwood	19.3 ± 1.4	0.8 ± 0.1	2400 ± 200	50	23	[^89]
Xylan	Sugarcane bagasse	9.2-22.3	0.7-1.3	8.9-38.9			[^90]
Glucuronoxylan	Cotton stalks	14.0	12.0				[^91]
AX	Maize bran	26.5 ± 4.1	7.4 ± 2.9	72.4 ± 35.2	54	25	[^92]
AX	Corn hull	53.8 ± 0.4	6.2 ± 1.6	1316 ± 90	54	22	[^93]
AX	Barley husks	50.0	2.5	2930	50	23	[^94]
AX	Barley husks	40.0	1.4	2800	50	23	[^59]
AX	Rye flour	52.4 ± 5.9	4.7 ± 1.8	1750 ± 740	50	23	[^95]
AX (high DS)	Rye flour	58.0 ± 11.0	8.1 ± 3.3	2500 ± 400	50	30	[^96]
AX (low DS)		32.0 ± 3.0	2.1 ± 0.3	2200 ± 200
AX	Rye bran	15.7	6.1	533.0	50	23	[^97]
AX	Rye flour	42.5 ± 6.5	11.9 ± 4.3	2300 ± 200	50 ± 2	23 ± 1	[^98]
AX (high DP, high DS)	Rye flour	63.0	12.4	1800.0	50	23	[^99]
AX (low DP, high DS)		47.0	5.2	1890.0
AX (high DP, low DS)		60.0	12.2	1700.0
AX (low DP, low DS)		36.5	4.0	1900.0
AX	Rye flour	62.0 ± 3.0	4.0 ± 0.9	3100 ± 200	50	23	[^100]
AX	Wheat flour	8.1-98.0	7.1-39.0	325-2475	59	25 ± 1	[^101]
AX	Rye flour	16.0	6.0	550.0	50	23	[^102]
AcAX	Rye flour	65.1 ± 4.2	21.0 ± 3.1	2160 ± 80	50	23	[^103]
AX	Corncob	53.54 ± 12.8	7.08 ± 1.3	1662 ± 268	50	25	[^104]
AX	Wheat flour	61.0-131.0	22.0-46.0		59	20	[^105]
AX	Oat spelt	25.0 ± 4.1	6.0 ± 0.8	626 ± 126	50	23	[^106]
AX	Sorghum bagasse	32.0-34.0	2.0-2.6	2299-3400	50	23	[^107]

As far as AX film-forming properties are concerned, there are far more promising candidates than glucuronoxylan, a number of studies were conducted in this field. Pure AX films were reported to be smooth with uniformly distributed arabinoxylan chains.^[93,99,100] The AX branched structure enhanced plastification through the use of substitutes such as xylitol and sorbitol. Water was also reported to be a good AX plasticizer.^[59]

Pure alkali-extracted AX films were cohesive, but also brittle with minor insoluble particles of denaturated proteins.^[95,98] The authors determined the mechanical properties of AX films to be highly dependent on AX extraction methods.^[95] The same authors reported that mild acid and heat treatment partially degrades arabinose sidechains.^[94,95] Low-arabinose AX films showed a cracked surface, in contrast to amorphous high-arabinose samples (arabinose substitutes prohibit xylan backbone interactions). Non-treated high-arabinose AX films showed enhanced mechanical properties. Being amorphous, xylan chains disentangle and become orientated in the direction of the applied force. The decreased degree of arabinosylation resulted in crystallinity growth, which positively contributed to the desirable mechanical behavior of the film. With further AX dearabinosylation, the film softens due to increased moisture uptake.^[95] Stevanic et al. reported arabinoxylan debranching to increase crystallinity by about 20% and provide excessive embrittlement. Debranching also resulted in a decrease in strain at break by 45%.^[100]

Sarossy showed that co-extracted β-glucan improves the mechanical behavior of AX film. Enhancement occurs due to the increase in the density of the composite structure. After β-glucan removal, the authors reported a 50% drop in tensile strength, a 40% drop in strain at break and 20% lower values of Young`s modulus.<sup>[98]</sup> Being a natural polymer, AX films are highly hydrophobic.<sup>[92]</sup> Mikkonen et al. reported that the storage modulus of AX films decreases with growing relative humidity, which indicates moisture-induced plasticization. The higher number of AX substituents degrades the hydrogen bond network, which results in enhanced composite flexibility.<sup>[96]</sup> An increase in both the degree of polymerization and in the degree of substitution of AX resulted in an increase in tensile strength and Young`s modulus, with the first parameter being affected the most.^[99] Changes in the degree of substitution of AX did not affect the elongation at break. An increase in the number of unsubstituted xylopyranoses also lead to an increase in AX crystallinity.^[106]

3.3. Glucomannan

As with AX, glucomannan forms films without any additives. Generalized data concerning KGM mechanical properties are given in Table 4. Pure KGM films are reported to be mainly smooth, with small pores and cracks visible in cross-sections and obvious crystalline regions and numerous aggregates in its body, formed by high-MW glucomannan fractions.^{[108,109,111,114]} The films showed mechanical behavior characteristic of brittle materials. As for all polysaccharide chains, a number of factors affect its mechanical properties, such as the flexibility and mobility of the backbone, degree of entanglement, segmental motion and antiplasticization of low-MW tailings.

Table 4. Mechanical properties of Konjac glucomannan films (measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Source	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	Reference
KGM	Konjac	10.1	7.7		47	5	[^108]
KGM	Konjac	20.1	8.0		63	RT	[^109]
KGM	Konjac	2.3-3.4	7.8-12.2		0-100	RT	[^110]
KGM	Konjac	8.2	1.8				[^101]
KGM	Konjac	57.0	11.0	1914.0	54		[^111]
KGM	Konjac	40.0	7.0		50		[^112]
KGM deacetylated	Konjac	36.2	9.0		67	25	[^113]
KGM	Konjac	30.2 ± 1.1	32.0 ± 3.1		50		[^114]
KGM	Konjac	61.0 ± 3.0	25.0 ± 0.5	1200.0	50	20	[^115]
KGM	Konjac	41.47 ± 1.79	7.46 ± 0.75		50	25	[^116]

Extensive backbone entanglements cause fragmentary chain alignment, which may increase tensile strength and decrease elongation with decreasing MW. However, it may go the other way, as low-MW chains limit entangling abilities. Antiplasticization with low-MW chains/water enhance the desirable mechanical behavior of the film, possibly acting as a slippage area for long chains or this may be due to structure compaction.^[110]

4. Structural and mechanical properties of binary BC-hemicellulose composites

Structural and mechanical properties of BC-hemicellulose composites differ significantly, according to the method of composite production. The most frequently used methods of BC-hemicellulose composite production are medium growth and casting. Medium growth method is the same as for the pure BC growth, with the difference that corresponding quantities of hemicellulose are diluted in growth medium before autoclaving and bacteria inoculation.^[34]

BC-hemicellulose composites, produced by casting are often called reconstituted or regenerated composites, which refers to the production method.^[96,99,117] Generally, scraps of never-dried BC are mechanically treated and dispersed in ultrapure water, with further addition of hemicellulose solution. Also, instead of BC, commercial cellulose fibrils are often used. Additional treatment may allow vacuum filtration of pulp and degassing by ultrasonication in vacuo.^[96,118] On the last step, pulp is deposited on sterile Petri dishes and dried for 23-60 °C from 4 h to several days.^[99,117,118]

As it will be shown in the next chapters, casted BC-hemicellulose composites show improved mechanical properties, compared to medium grown, due to better hemicellulose dispersion, denser film structure and improved fiber-to-fiber interconnectivity.

4.1. BC-xyloglucan

For approximately 50 years, XG was thought to be the main hemicellulose to form a cellulose-hemicellulose network in PCW, causing 20-40 nm links to form between contiguous fibrils. A number of in vitro and in situ cellulose-XG interactions were observed, such as cellulose tethering^[119], fibrils coverage^[120] or the formation of covalent links between them.^[121] The mechanical properties of BC-XG composites are provided in Table 5.

Table 5. Mechanical properties of BC-XG composites (measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Hemicellulose source	Fabrication method	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	Reference
BC-XG	Tamarind	Grown in medium	0.14-0.33	38.0-58.0				[^31]
BC-XG	Tamarind	Grown in medium	0.18	48.0				[^122]
BC-XG	Tamarind	Grown in medium	0.09	42.0				[^29]
BC-XG (high-MW)	Tamarind	Grown in medium	0.18	48.0				[^123]
BC-XG (Gal-depleted)			0.09-0.15	16.0-25.0
BC (reconstituted)-XG	Tamarind	Casted	92.0-130.0	1.6-2.6	8300-11000	50	RT	[^117]
BC-XG	Tamarind	Grown in medium	18.2 ± 2.7	0.29 ± 0.1	4300 ± 1400	>33	35	[^124]
BC-XG	Tamarind	Grown in medium	2.7-37.7	>25.0			25	[^125]
BC (reconstituted)-XG	Guibourtia hymenifolia	Casted	59.0 ± 11.0	2.4 ± 0.55	4750 ± 230	50 ± 5	RT	[^118]
BC-XG	Tamarind	Grown in medium	3.1	37.0	7.5			[^34]
CBC-XG	Tamarind	Casted	52.0	77.0	9700			[^42]
Cellulose cryst.-XG	Tamarind	Casted			2650-4750	50	25	[^58]

Structurally, XG appears in binary composite form as a thin ladder rung-like structure, 30-50 nm long. Also, some substantial links longer than 70 nm occur between diverged cellulose ribbons.^[119] Measurements of XG link lengths showed that they may link three or more cellulose fibrils together. As for the incorporation level, several different studies reported a BC:XG ratio in the binary network ranging from 1:0.1 to 1:0.7.^{[29,32,106,108]} Gu et al. reported several differences between BC-XG composite and pure BC, like thicker BC fibrils, a denser pellicle structure and lower porosity.^[109,114] Chain length, sidechain pattern and the degree of XG acetylation influence its bonding to CBC. The XG binding rate increases with its length, with a minimum of 12 glucosyl residues, which are necessary for bonding. The degree of XG acetylation does not have any impact on binding with cellulose, while the presence of fucosyl third saccharide substitutes improves XG flexibility thereby increasing its exposure to the cellulose surface.^[126]

XG provides interfibril adhesion to form a binary casted composite with cellulose.^[117] XG chains are reported to obstruct nanofibril aggregation, which may be explained through its adsorption on the fibril surface. However, XG may have some issues with adhesion on the BC surface, this is related to its diffusion abilities (presence of side branches) and hydrogen binding with BC.^[127]

The presence of high-MW XG in its binary cellulose-based composite causes the formation of highly organized cross-linked structures. Addition of low-MW XG to the growth medium leads to a higher level of incorporation into the cellulose network with almost no lateral organization of cellulose fibrils and abundant XG cross-bridges. XG may participate in the formation of more than one cross-link, with the lengths of the cross-links and aligned regions having similar values.^[40]

BC which is synthesized in the presence of hemicelluloses is more accessible to enzymatic hydrolysis. This effect is related to the structure of fibrils packed into ribbons or bundles, and to the general structure of the composite, where fibrils synthesized in hemicellulose-containing medium aggregate less tightly as compared to pure BC.^[128] A comparison between the measured cellulose:XG ratio and the number of cross-bridges, suggested that only a small fraction of XG is involved in bridge formation with the majority of it presumably becoming aligned with cellulose.^[119] Other authors reported that XG is partially responsible for creating cellulose interchain bindings and also partially responsible for binding cellulose during its synthesis through reactions with inner hydroxyl groups.^[129]

The addition of any kind of XG to the growth medium decrease cellulose crystallinity in general by lowering Iα BC and increasing Iβ content at the early stage of BC-XG composite growth.^[119] An increased level of Iβ cellulose was also indicated for CBC-XG films, as compared to pure CBC.^[42] The tendency for cellulose Iβ formation in the presence of XG was explained by decreases in shear stress, which normally occurs during ribbon twisting when fibrils assemble.^[127]

Binary 1:1 BC:XG networks showed enhanced mechanical properties, compared to pure polysaccharide films. Based on XG content, networks may be classified into several structural types – continuous BC networks (<40% XG content), co-continuous networks (≈40-50% XG) and continuous XG ones (>50% XG). In continuous BC networks, the amorphous XG is well dispersed, thereby reinforcing it and increasing its Young`s modulus. In the case of BC dispersion in the XG network, mechanical properties are dominated by the XG component. The mechanical behavior of the co-continuous network may be explained through the lubricating action of XG, increasing stress and strain at break. Such an effect was also observed for the BC-xylan co-continuous matrix.^[58]

Compared to pure BC, lower failure stresses and higher failure strains were observed for BC-XG networks, grown in XG-containing medium.^[31] Stress-strain tests on the BC-XG network showed that at both low and high compressive strain-rates, the XG network was stronger and provided a significantly more robust mechanical response as compared to the BC sample. Both the viscoelastic and elastic properties were observed for the XG material at low and high-strain deformation rates.^[125]

The extensibility of the BC-XG composite grown in hemicellulose-containing medium was reported to increase by up to 300% as compared to pure BC.^[27,106] High-MW XG promotes the greater extensibility of the composite under uniaxial and biaxial tensions. This has contributed to the alignment of cross-linked domains with tensile direction, resulting in the restraint of cellulose-XG knots at greater strains.^[29,116]

Xyloglucan composites with bacterial cellulose prepared by casting showed higher tensile strength, compared to composites grown in the liquid medium (Table 5). This effect was explained by better fibril dispersion and better fiber-to-fiber interconnectivity, in case of casted films.^[117,118] However, the scarcity of data makes it impossible to draw decisive conclusions on the remaining mechanical parameters.

4.2. BC-xylans

The mechanical properties of BC-xylan composites are summarized in Table 6. Xylans are known to form intertwined networks with BC. BC-xylan composites exhibit increased fibril length, with a lower level of cellulose crystallinity and a lower average diameter of fibril at the same time, compared to pure BC. It was hypothesized that xylan and XG may convert localized amorphous cellulose regions into more uniformly distributed ones, resulting in a higher resistance to complete hydrolysis.^[42] Xylans may act as fillers of BC in a binary composite, by mainly aggregating on the cellulose surface.^[131] Linder et al. discovered that xylan aggregates may adsorb on a cellulose surface via unsubstituted regions of both chains.^[132] Apart from the random uniform distribution of xylans and fibril bundles in films, xylans were observed to interact mainly with amorphous cellulose.^[130,133]

Table 6. Mechanical properties of BC-xylan composites (AGX - arabino-4-O-methyl glucuronoxylan; GX – glucuronoxylan; DS – degree of substitution; DP – degree of polymerization, measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Hemicellulose source	Fabrication method	Tensile strength (MPa)	Strain at break (%)	Young`s modulus (MPa)	RH (%)	T (°C)	Reference
BC-AX (high DS)	Rye endosperm	Casted	53.0-68.0	3.3-8.3	2700-3200	50	30	[^96]
BC-AX (low DS)			46.0-49.0	2.8 ± 1.3	3100-3700
CMF-AX (high DP, high DS)	Rye endosperm	Casted	90.0	10.9	2520.0	50	23	[^99]
CMF-AX (high DP, low DS)			83.0	11.0	1900.0
CMF-AX (low DP, high DS)			57.0	3.7	2680.0
CMF-AX (low DP, low DS)			42.0	4.2	1780.0
CNF-AX	Rye endosperm	Casted	108-143	6.0-7.2	4800-7300	50	23	[^100]
CNF-AcAX	Rye endosperm	Casted	72.5-90.1	10.5-19.2	2390-3330	50	23	[^103]
BC-AX	Wheat flour	Grown in medium	0.98-10.2	18.0			25	[^125]
CNF-xylan	Birchwood	Casted	23.0-57.0	0.6-1.1	5200-7600	50	23	[^130]
CBC-xylan	Beechwood	Grown in medium	42.0	71.0	7300	50	25	[^42]
CNF-xylan	Beechwood	Casted	45.0-100.0	1.2 ± 0.4	5800-10100			[^131]
Cellulose (regenerated)-AGX	Spruce wood	Casted	19.0-75.0	2.8-24.8	400-2150	30-90	25	[^99]
BC-AGX	Softwood	Grown in medium	0.67	100.0	0.4 ± 0.1			[^73]
BC-GX (acetylated)			0.82	100.0	0.7 ± 0.1

The Young`s modulus of BC-xylan composite decreases with increasing xylan content. This effect contributed to weakened cellulose interfibril interactions.<sup>[131]</sup> An improvement in mechanical properties was reported with the increase in cellulose nanofibril (CNF) content in casted xylan films. The best mechanical performance was reported for xylan films with 5-10% vol. CNF. The further addition of CNF resulted in diminished mechanical properties for the casted composites. Cellulose acetylation improves mechanical behavior by increasing tensile strength and Young`s modulus, but decreases tensile strain with increases in cellulose concentration.^[134]

However some researchers have reported that CBC film of BC grown in xylan presence show 27% and 40% higher tensile strength and Young`s modulus, respectively, as compared to pure BC. The enhanced mechanical properties were explained in this case by the enlarged hydrogen bond network of BCNC and flexible long-chained xylans (nanoreinforcing fillers).^[42]

The structural and mechanical properties of BC-AX composite, cultivated in Hestrin-Schramm medium with AX presence, indicate different types of AX-cellulose interactions, as compared to XG or pectin. Binary AX-BC composites were shown to have diminished mechanical properties as compared to pure cellulose samples. AX was shown to form microscale grains, closely bonded to cellulose.^[125] Such AX domains existing on the BC surface, were reported to interact via nonspecific adsorption mechanisms.^[135]

Köhnke et al. reported that AX adsorbs irreversibly on the cellulose surface according to a substitution pattern. The adsorption and affinity of AX to BC only increases with a decreasing Ara:Xyl ratio. However, further reductions in the of Ara:Xyl ratio will decrease AX solubility, which may lead to composite phase separation. Also, substitution patterns have an impact on the viscoelastic properties of samples with lower amounts of arabinan. Reports have shown that AX adsorption on crystalline regions of cellulose is irreversible, and depends on the AX concentration in solution and the degree of arabinose substitution.^[99]

Adsorption is enhanced with increases in the MW of AX, as well as in the presence of lignin. AX slightly decreases the crystallinity of BC, with lignin intensifying this process. It has been shown that lignin can promote AX aggregation in ternary composites, thereby intensifying AX adsorption, while improving the incorporation of AX at low quantities.^[136]

Egues et al. showed that the presence of lignin and arabinose-xylose enhances composite formation in alkaline-extracted hemicelluloses. For BC films, grown with addition of alkaline extracted hemicelluloses with lignin impurities, lignin promotes the formation of thicker composites, with a dense structure and a reduced number of cracks. Lignin promotes the formation of thicker composites, with a dense structure and a reduced number of cracks.^[137] Upon attachment to cellulose, branched AX may rearrange itself into extended conformations, thereby entrapping another AX.^[86] In an AX-continuous matrix, reinforcement of cellulose nanofibrils increases film surface roughness. Cellulose nanofibrils addition leads to the formation of heterogeneous lamellar CNF structures coated by thin pore-clogging AX layers.^[100]

The AX presence in growth medium does not affect the level of Iβ cellulose to the same extent as compared to BC (38 and 40%, respectively).^[125] Debranching of arabinoxylan causes an increase in the degree of crystallinity. Also, only backbone residues of xylans may take part in the crystallization and show co-crystallizing properties under conditions of relatively low arabinose concentration.^[96]

The BC-AX composite shows both viscoelastic and elastic properties at slow strain-rates and elastic properties at high-strain rates.^[125] Stevanic et al. demonstrated that BC additives enhance the mechanical behavior of AX films.^[96] This effect was also observed for AX-CNF casted films.^[100,103] For cellulose microfibrils (CMF) a significant increase in tensile strength was only reported for mixtures with a high content of AX with high degree of polymerization.^[99] In general, stiffer, stronger and more ductile films were obtained for cellulose-AX composites, as compared to pure AX films. Moisture sorption and the associated moisture-induced softening were both reduced with increased cellulose reinforcement content. AX debranching decreased the strength and breaking strain, resulting in more brittle films.^[96] BC-arabinoglucuronoxylan (AGX) composites, produced by casting technique, demonstrated a smooth structure with a multilayered porous cross section. AGX may be adsorbed on the cellulose network as well as agglomerating in empty voids. Blended BC-AGX composites showed a higher degree of stiffness as compared to pure BC, which may be explained by cellulose-AGX hydrogen bonding.^[138]

Cellulose-xylan composites prepared by casting, showed higher tensile strength and Young`s modulus, compared to the composites grown in medium (Table 6). Better mechanical properties were contributed to the extended hydrogen bond network of fibrillated cellulose and flexible long-chained xylans that act as nano-reinforcing fillers.^[42] However, casted films were less extensible, showing lower strain at break, than composites grown in medium. Degree of AX polymerization showed positive correlation with composite mechanical properties. This effect was attributed to increased interconnectivity between xylan chains, which enabled longer chains to slide across each other.^[96,99] An increase in relative humidity decreased mechanical properties of cellulose-xylan composites, as a result of moisture-induced plasticization.^[99]

4.3. BC-glucomannan

Studies concerning BC-mannans co-crystallization have suggested that mannans bind to BC via weak interactions.^[139] The mechanical properties of BC-glucomannan composites are provided in Table 7. Berglund has shown that intercalated acetylated galactoglucomannan (acGGM) and GGM is incorporated into the BC network.^[73] GGM shows a good affinity to cellulose nanofibrils by forming rigid layers on its surface.

Table 7. Mechanical properties of BC-glucomannan composites (measurement conditions: RH –relative humidity of air, T – air temperature).

Composition	Hemicellulose source	Fabrication method	Tensile strength(MPa)	Strain at break(%)	Young`s modulus(MPa)	RH (%)	T(°C)	Reference
CNF-KGM	Konjac	Casted	73.0-118.0	6.7-10.8		50	23	[^131]
BC-GGM	Softwood	Grown in medium	0.6	91.0	0.7 ± 0.2			[^73]
BC-AcGGM			0.97	53.0	2.4 ± 0.3
CNF-GGM	Spruce wood	Hot-pressed	50.8 ± 7.2		400.0 ± 45.0	50	23	[^140]
CNF-KGM	Konjac	Casted	95.0-135.0	5.9 ± 2.0	6400-7700			[^131]

Also, the similarity between the cellulose and GGM surface charges resulted in a smooth lubricating effect for the fibrils coated with GGM.^[140] However, low-MW (20-40 kDa) glucomannans are not capable of coating or tethering cellulose fibrils due to their relatively short chains.^[141] A reduction in the galactose content of galactomannan inversely relates to the level of incorporation into the BC network. Due to the structural features of galactomannans, kinetic (entanglement of BC-BC bonding region fibrils) and thermodynamic (lowest free energy state) controls are proposed to explain mannan impact on fibril association. It has been reported that for low-Gal mannans, fibril annealing resulted in BC-mannans-BC bonding.^[142]

Pure BC, produced in a Haigler-Benziman medium, formed a twisted-ribbon-like structure, whereas BC, grown in the presence of acetylated glucomannans resulted in the formation of interwined fibrils, gathered in loose bundles.^[28] Tokoh et al. discovered that acetylated glucomannan in the medium prevents the assembly of BC fibrils.^[143]

A lower number of acetate substitutes stimulate adsorption, water solubility and thermal degradability, whereas close substitutions may result in stronger interactions.^[73] The adsorption of mannans, as well as that of all of hemicelluloses, is highly dependent on its concentration and on the composition of cellulose nanofibrils.^[144] GGM adsorbs on the nanofibril surface in a rigid conformation with a tight association due to the lower water content in the GGM layer. It has been shown that the affinity of GGM to cellulose does not correlate with the presence of xylan, moreover, the opposite relationship with xylan was observed.^[86]

Carob and Konjac glucomannan adsorption on crystalline regions of cellulose decreases the accessibility of cellulases to cellulose.^[145] A higher Gal:Man ratio may lower the adsorption of mannans, but branched mannans result in steric hindrances for cellulases.^[146]

In casted binary composites, KGM acts as a filler, intertwining with BC fibrils. At relatively low concentrations, KGM mixes well with BC, forming multilayered smooth structures without visible aggregates.^[131] The content of KGM in the binary composite has an inverse relationship with material phase separation. However, with increasing concentrations of KGM, a number of egg-like structures on the composite surface were reported, they were possibly formed by agglomerations of high-MW KGM.

As with XG and xylan, mannans in the composite decrease BC crystallinity. The structural differences between mannans and cellulose hinder the cellulose association processes and decrease its crystal size, thereby limiting the changes occurring in the crystalline lattice of the cellulose.^[147] Whitney showed that KGM and low-galactose galactomannan reduce cellulose crystallinity in the BC-mannans composite, cultured in mannans presence, from 80-85% to 25%.^[142] After that, Tokoh et al. reported that BC grown in mannan-containing medium resulted in a significantly reduced Iα cellulose level, as compared to the control samples.^[148]

The presence of mannans had a significant effect on the mechanical properties of their binary composites with BC. The plasticity of hemicellulose films is enhanced by the high degree of substitution of GGM.^[86] GGM addition improves the BC composite strength in its wet state. An increase in tensile strength and toughness by 170% was reported after the addition of 2% wt. GGM. A moderate increase in Young’s modulus (33%) was observed after the addition of mannan polysaccharides.^[140] Acetylated GGM increased Young`s modulus due to its tendency to co-crystallize with BC.^[73] The mechanical behavior of composites made from regenerated cellulose-KGM showed a minor decrease in mechanical properties with the increase in KGM concentration. It was observed that mainly low-MW chains of both cellulose and KGM form the continuous matrix base. KGM additives improved the mechanical behavior of the CNF-KGM casted composite, which was attributed to the favorable dispersibility of KGM in cellulose fibrils.^[131]

Composites prepared with casting method and fibrillated cellulose showed improved mechanical properties, compared to the composites, grown in medium (Table 7). Studies showed that glucomannans better penetrate into fibril network in casted films, due to glucomannan-mediated interactions facilitating uniform fibril distribution during composite formation.^[140]

5. Structural and mechanical properties of BC-hemicellulose-pectin ternary composites

Most recent studies suggest that pectin forms its own wall network with plasticizing and water-binding properties, providing physical strength to PCW.^[138,139] The crosslinking of pectin polysaccharides with other wall components provides additional structural and functional complexity to the wall.^[145] Susanne E. Broxterman provided a comprehensive overview of most reported cross-links between cellulose, hemicellulose and pectins. To date, several types of these have been recognized in the cellulose-hemicellulose-pectin system, such as covalent linkages, adsorption by H-bonds, diferulic bridges, calcium bridges, borate-diol ester linkages etc.^[149]

At high polysaccharide concentrations, XG and pectin do not mix, but rather, form micro domains. In vitro adsorption experiments onto cellulose fibrils have shown that XG and pectin bind to cellulose both simultaneously and competitively.^[38] Haas et al. suggested the double-role of pectin – both as a minor cellulose bonding component in XG-rich walls and as a prime cellulose association mediating component in XG-poor walls.^[150] In the second scenario, in areas of PCW lacking XG, the pectin establishes contacts between the fibrils, and takes over the tethering role of XG.^{[149,151,152]} Covalent linkages of cellulose and galactose/arabinose rhamnogalacturonan I (RGI) sidechains were observed for carrot samples. It was proposed that RGI and xylans were present in covalently linked complexes of some fruit species.^[149] However, the pectin-cellulose supramolecular complexes have yet to be determined.^[153]

Structural studies on binary/ternary PCW analogues prepared by addition of XG and/or pectin to growth medium showed that the BC fibrils in the BC-pectin composite are drastically thinner (110 ± 33 nm, 45 ± 9 nm and 123 ± 29 nm for pure BC, BC-pectin and BC-pectin-XG, respectively). Studies have shown that pectin and some hemicelluloses act as inhibitors of microfibril association, while XG promotes their association through surface coating and the establishment of interfibril linkages.^[154]

Several polysaccharide ratios in the ternary BC-XG-pectin composite were tested, such as 26% BC, 44% pectin and 20% XG^[155]; 63% cellulose, 22% pectin and 15% XG.^[122] Irrespective of the component ratio, binary BC-pectin composites were generally thicker than ternary composites, suggesting the existence of stronger bonds of cellulose XG to cellulose.^[155]

According to the research of Gu and Catchmark, pectins show a 2.5 times lower adsorption rate, compared to xylans, both being non-comparable with XG. Due to structural differences (shorter sidechains) pectin binds more easily to porous cellulose, occupying a lower surface area.^[156] In the BC-XG-pectin composite, grown in medium with 0.5% w/v addition of XG and pectin, XG may interact with all of pre-crystallized, crystallized and preferably amorphous BC, while pectin preferentially binds with crystalline regions.^[33]

The effect of the composition of BC-XG-pectin composites on BC crystallinity is unclear. Some studies concerning BC-XG-pectin composites grown in Hestrin-Schramm medium with polysaccharide additives have shown that BC crystallinity has dropped significantly in the presence of XG and pectin additives. However, the ternary composites of other studies showed a higher degree of cellulose crystallinity, compared to pure BC. Researchers have hypothesized that the pectin network may contribute to simultaneous coating of the fibrils by XG.^[157] Raman and FT-IR spectra have shown that the content of Iβ cellulose increases with XG content (up to 73%) and decreases with pectin concentration in the composite.^[34] The XG:pectin ratio in BC-XG-pectin ternary composites grown in liquid medium with polysaccharide addition was shown to have an influence on the mechanical properties of the composite. The lower XG:pectin ratio resulted in a thinning of the cellulose fibrils and an increase in Young`s modulus. Studies have also shown that the removal of pectin significantly reduces composite tensile strength, which was not observed in the case of XG removal. Experimental results indicated that PCW stiffness is dependent on the XG:pectin ratio, in which pectin play a dominant role in the strengthening of ternary composites.^[34] Introducing xyloglucan into a cellulose/pectin composite led to very compliant materials characterized by time-dependent creep behavior and high failure strains.^[122]

6. Conclusion

Plant-like synthesis, high yields, an ultrafine structure and chemical purity have resulted in bacteria-produced cellulose being widely used in many scientific fields. The structural and chemical similarity with plant-based cellulose allows for the production of so-called plant cell-wall analogues and the study of the role of individual polysaccharides in PCW. Studies have shown that the materials produced are sensitive to the conditions of bacteria growth and require a carefully controlled growth process. It has also been demonstrated that BC synthesis in the presence of other plant-based polysaccharides such as hemicelluloses or pectin induces structural and chemical modifications in final composites. Structural changes induced by composition and growth conditions were directly reflected in the mechanical and thermal properties of the films, thereby providing a set of versatile materials. As they are relatively easy to modify, BC-based composites have a great potential in finely-tuned biosystems, the properties of which are ideally suited to targeted applications.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by the National Science Center, Poland (Project no. 2019/35/D/NZ9/00555).