Received: 6 October 2023 Revised: 11 December 2023 Accepted: 12 December 2023

DOI: 10.1002/app.55163

REVIEW

Bacterial cellulose: A comprehensive review

Vincent-Daniel Girard 1,2 | Jérémie Chaussé 1,2 | Patrick Vermette 1

1
Laboratoire de bio-ingénierie et de
biophysique de l'Université de Abstract
Sherbrooke, Department of Chemical and This review article provides a comprehensive overview of bacterial cellulose
Biotechnological Engineering, Université
(BC), focusing on its physicochemical properties, production methods, purifi-
de Sherbrooke, Sherbrooke, Québec,
Canada cation techniques, and applications. It aims at providing a nuanced under-
2
AxCell Laboratories, Sherbrooke, standing of the current state of knowledge in those fields and bridging the gap
Québec, Canada between academic research and industrial applications. The physicochemical
Correspondence
properties of BC, including its chemical structure, morphology, thermal prop-
Patrick Vermette, Department of erties, mechanical properties (such as tensile and compression properties), spe-
Chemical and Biotechnological cific surface area, cytotoxicity, and biocompatibility, are discussed. Production
Engineering, Université de Sherbrooke,
2500 boul. de l'Université, Sherbrooke, QC methods of BC, including microorganism comparison, culture conditions, and
J1K 2R1, Canada. vessel types, are thoroughly explored. Purification methods and sterilization
Email: [email protected]
techniques for BC are also addressed. Furthermore, the review highlights
Funding information industries and applications that have shown interest in BC along with com-
Mitacs, Grant/Award Number: Project mercially available products, including medical, cosmetic, textile, food ingredi-
IT25748
ents, and scaffolds in cell culture. A conclusion summarizes key findings and
potential future directions in BC research and development.

KEYWORDS
bacterial cellulose, biocompatibility, industrial applications, physicochemical properties,
production, purification

1 | INTRODUCTION applications. Its unique characteristics, including its

chemical structure, morphology, thermal stability,
Cellulose, the most abundant biopolymer on Earth, is a mechanical strength, biocompatibility, and versatility,
fundamental component of plant cell walls and can also have garnered significant interest in both industrial and
be found in certain bacteria and archaea.1 When synthe- biomedical fields.
sized by bacteria, it is commonly referred to as “bacterial Although the characterization of bacterial cellulose
cellulose” (BC) and is under the form of an extremely dates back to 1886, a deeper understanding of its proper-
hydrated biofilm exempt of lignin, hemicellulose, and ties gained significant attention around 2000, leading to a
pectin. Various bacterial species, including those belong- surge in the number of articles published on the subject5,6
ing to the genus Komagataeibacter, Acetobacter, Agrobac- (See also Data S1). This appeal is also backed by many
terium, Pseudomonas, Sarcina, and others, are known for review articles covering BC properties and potential appli-
their ability to produce BC.2–4 BC has emerged as a cations in various fields.3,7–26 Among the organisms recog-
highly promising biomaterial with exceptional physio- nized as bacterial cellulose producers, Komagataeibacter
chemical properties and a wide range of potential xylinus (formerly known as Gluconacetobacter xylinus or

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2024 The Authors. Journal of Applied Polymer Science published by Wiley Periodicals LLC.

1 of 35 wileyonlinelibrary.com/journal/app J Appl Polym Sci. 2024;141:e55163.

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GIRARD ET AL. 2 of 35

Acetobacter xylinum) stands out with their higher yields. the choice of vessels or bioreactors significantly influence
The produced membrane consists of a woven, three- BC production. Optimizing production parameters is
dimensional network of highly pure cellulose nanofi- essential for achieving efficient and cost-effective produc-
brils.27–29 tion of BC materials.
The structure of BC exhibits two distinct regions: Purification methods are crucial to remove impurities
crystalline and amorphous. Crystalline regions consist of and ensure safety and biocompatibility of BC-based mate-
highly ordered, parallel cellulose strands, while amor- rials. Lipopolysaccharides (LPS), components of bacterial
phous regions are comprised of randomly oriented outer membranes, can induce immune responses and
fibers.30 The prevalence of crystalline regions contributes pose potential risks in biomedical applications. Therefore,
to the exceptional tensile strength of BC. Indeed, a single special attention is given in in vivo studies that remove
filament of BC is estimated to have a Young's modulus of LPS during purification. Techniques, including washing,
up to 115 GPa.31–33 BC also possesses a high-water reten- bleaching, and chemical treatments, have been employed
tion capacity, ranging from 95% to 98%, facilitating the to eliminate impurities and obtain highly pure BC.
infusion of water-soluble substances.34 Additionally, Sterilization is a vital step in the production of BC
chemical grafting enables the modification of BC's struc- materials to ensure their sterility and safety. Autoclave
ture, texture, stiffness, and chemical properties by intro- sterilization, utilizing high-pressure steam, is a com-
ducing other molecules into the cellulose matrix.15,29 monly used method that maintains the integrity and
The physiochemical properties of BC justify its multi- properties of BC while achieving sterility. Alternatively,
faceted applications. At the molecular level, BC shares a electron beam (E-beam) sterilization offers advantages
chemical structure similar to plant cellulose, comprising such as shorter processing time and a lower risk of ther-
β-1,4-glycosidic linked glucose units. However, BC fibers mal degradation. Other methods, including gamma irra-
exhibit exceptional purity and form a dense, intercon- diation and chemical sterilization, have also been
nected network, resulting in superior mechanical proper- documented. The choice of sterilization method should
ties compared to other forms of cellulose. BC depend on the specific application requirements and the
demonstrates outstanding thermal stability, allowing characteristics of BC materials.
exposure to high temperatures without significant degra- Various industries have recognized the immense
dation. Moreover, BC thermal properties can be tailored potential of BC. In the medical field, BC finds its most
through modifications in the production process, offering utility in wound dressings, tissue engineering scaffolds,
opportunities for optimizing its performance in specific and artificial blood vessels. The cosmetic industry utilizes
applications. BC for face masks due to its high water-holding capacity
Biocompatibility is a critical aspect of biomaterials, and low environmental impact. In the textile sector, BC
particularly in the field of medical applications. BC serves as a sustainable and biodegradable material for
exhibits excellent biocompatibility and low cytotoxicity, fabric production. Additionally, BC has found applica-
making it highly suitable for biomedical applications. tions as food ingredients, such as stabilizers, thickeners,
Extensive studies have demonstrated that BC materials and emulsifiers, owing to its excellent gelling and film-
do not induce significant inflammatory responses or tox- forming properties.
icity when in contact with living tissues. These biocom-
patible properties make BC an attractive candidate for
applications such as wound healing and tissue engineer- 2 | BACTERIAL C ELLULOSE (BC)
ing. BC also supports cell adhesion, proliferation, and dif- PH YSIC OCH EMIC AL P RO PERTIES
ferentiation, further enhancing its potential in
regenerative medicine. 2.1 | Chemical structure and
The production method of BC plays a pivotal role in morphology
its quality and scalability. Various parameters and prop-
erties, including crystallinity index, porosity, cellulose Bacterial cellulose (BC) consists of long chains of β-D-
concentration, thermal resistance, and mechanical resis- glucopyranose units linked together by β1-4 glycosidic
tance, are impacted by the methodology from production bonds, which result in the presence of a free hydroxyl
to purification. Cultivating specific bacterial strains, such group at the surface area.20,29,35–37 The degree of BC poly-
as Komagataeibacter xylinus, Gluconacetobacter hansenii, merization typically ranges from 2000 to 6000, while veg-
and Gluconacetobacter xylinus, in a suitable culture etal cellulose has a higher degree of polymerization,
medium under controlled conditions is crucial for obtain- ranging from 13,000 to 14,000.20,38,39 Although both BC
ing high BC yields with desired characteristics. Factors and vegetal cellulose share the same molecular formula
such as temperature, pH, dissolved oxygen, agitation, and (C6H10O5)n, they exhibit significant differences in their
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
3 of 35 GIRARD ET AL.

physiochemical properties. BC displays a higher crystal- At the macroscopic level, BC accumulates at the air-
line index, greater water-absorbing capacity, and higher liquid interface of the culture medium. Once the first
purity.20,37,39–42 layer is saturated, daughter cells anchor themselves verti-
Cellulose has also demonstrated an isoelectric point cally to access nutrients and oxygen since they require
lower than 2.8, resulting in a generally negative charge.43 aerobic conditions.41 Daughter cells continue producing
A positive charge can be induced in BC through the cellulose and start building the second layer. This cycle
cross-linking of glycidyl trimethylammonium chloride continues until the exhaustion of nutrients. Thus, previ-
(GTMAC), while the negative charge can be enhanced by ous cellulose layers are pushed down in the media by the
oxidizing BC with 2,2,6,6-tetramethylpiperidine 1-oxyl new layer.37,45
(TEMPO).44 These chemical modifications have mainly Structural morphology of BC biofilms is influenced by
been studied in the field of cell culture to enhance cell various culture conditions, including agitation, growth
adhesion to BC scaffolds. medium, and the bacterial strain. Agitated culture condi-
The synthesis of BC occurs through metabolic path- tions result in a lower degree of polymerization, lower
ways and the action of a cellulose synthase enzyme com- crystallinity index, and a sphere-like shape compared to
plex located on the outer membrane of bacteria membrane-like shape.35,45,48 Variation in the growth
belonging to the Gluconacetobacter genus.36,42 As seen in medium and bacterial strain also affects the structure and
Figure 1, when the glucan chains are secreted outside of crystallinity index of the produced BC material.37,39
the cells through a linear row of pores also located on the Crystallinity is an essential parameter as it impacts
outer membrane, the cellulose synthase synthesizes 3–15 various physiochemical properties. BC is a semicrystal-
of those glucan chains (also called BC fibrils) into a subfi- line biopolymer with a variable crystallinity index influ-
bril (also called elementary fibril or mini-crystal) which enced by the previously mentioned factors. BC crystalline
are about 1.5 nm thick.29,36,37,39,45 Following this, the index has been documented to range between 60% and
subfibrils merge into microfibrils through the action of 81%.20,37,39,41,45,48 Hydrogen bonds and van der Waals
hydrogen bonds, and the microfibrils then form cellulose forces play significant roles in the organization of the
ribbons.37 Each cellulose ribbon comprises around 1000 crystalline regions, contributing to the material robust-
glucan chains and can reach lengths of 1–9 μm and a ness and elasticity.29,35,36
thickness of 3–4 nm.38 At this point, the ribbons from dif- Hydrogen bonds are also responsible for water mole-
ferent cells crystallize into stackable sheets which com- cules binding within the BC structure. Apart from the
bine to form the BC biofilm consisting of a pure and cellulose content (2% wt.) and minimal amounts of pro-
thick cellulose biofilm composed of an ultrafine network teins and nucleic acids, the majority (98% wt.) of BC is
of uniaxially 3D-oriented nanofibers stabilized by hydro- water.29,40 Approximately 10% of this water is considered
gen bonds.29,36,39,46,47 free bulk water capable of easily penetrating and leaving

F I G U R E 1 BC assembly from the bacterial cell to the stacked sheets forming the resulting biofilm. An example of sheet is marked by a
blue circle. [Color figure can be viewed at wileyonlinelibrary.com]
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 4 of 35

F I G U R E 2 Chemical structure of
BC and its organization with free and
bounded water.34 This figure is a
reproduction from Figure 5 in
“Dehydration of bacterial cellulose and
the water content effects on its
viscoelastic and electrochemical
properties” by R. Rebelo et al. and is
permitted within the terms and
conditions of the Creative Commons
Attribution (CC BY) license. [Color
figure can be viewed at
wileyonlinelibrary.com]

the BC molecular structure, while the remaining water is exposing its cellulose fibrils and facilitating acid hydroly-
bonded through hydrogen bonds or linked to the numer- sis. This led to the development of many alternative treat-
ous hydroxyl groups available (Figure 2).29,38 ments to improve its transformation to bacterial cellulose-
derived nanocrystals. For instance, in a study by Niédja
Fittipaldi Vasconcelos et al., bacterial cellulose-derived
2.1.1 | Nanocrystals nanocrystals produced with 34% wt/wt HCl and 24% wt/
wt H2SO4 showed good properties: a zeta potential of
Cellulose nanocrystals (CNCs) are a partially degraded
43.9 mV, a crystallinity index of 83% and a L/D ratio of
form of cellulose which are of colloidal size and highly 21.1 (note that L/D ratios are simply the length of the
crystalline. Cellulose nanocrystals have first been crystals divided by their thickness; L/D ratios higher than
described in 1959 by Rånby and Ribi when investigating 13 yield anisotropic phases within the material, resulting
acid-mediated hydrolysis of cellulose fibers from wood in improved reinforcement properties).55 Many other
and cotton.49 Contrary to native cellulose, cellulose nano- studies investigated different BC treatments following
crystals form fully homogeneous solutions when stabi- hydrolysis or post-treatment. Those treatments include
lized in aqueous solutions. The synthesis of such crystals drying or freeze-drying,56 coupling the bacterial cellulose-
is now commonly performed using concentrated sulfuric derived nanocrystals with cellulose acetate butyrate,57 or
acid.50 The acid breaks down the long chains of cellulose varying available sulfate groups during hydrolysis.58
into smaller fragments about 5 nm wide and 100 nm to
several micrometers long. Cellulose nanocrystals are then
washed thoroughly with deionized water, centrifuged at 2.2 | Thermal properties
25,000g and resuspended in deionized water in various
concentrations.50–53 Thermal properties, including the degradation tempera-
While plant cellulose is still the most common way of ture of the different components of bacterial cellulose
making cellulose nanocrystals, bacterial cellulose-derived (BC), as well as its glass transition and melting tempera-
nanocrystals possess a whole array of unique applications tures, should be considered if cellulose undergoes ther-
in medicine, health, and catalysis. Examples include drug mal stress or is exposed to temperature variation during
carriers, controlled drug release, and reinforcements for production or processing.59,60 These properties also pro-
nanomaterials.54 vide information about the purity and composition of the
BC offers a unique opportunity in making cellulose resulting BC product.
nanocrystals as it is naturally devoid of many impurities BC thermal degradation involves three processes
found in vegetal cellulose. BC is also very porous, accompanied by mass losses. Firstly, before reaching
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
5 of 35 GIRARD ET AL.

200 C, the mass loss is attributed to the evaporation of a single filament or microfibril of BC has been estimated
residual water or moisture left in the matrix, especially in to 130–140 GPa.64 Hence, researchers aiming to enhance
the amorphous regions.59,61 Although removal of this BC mechanical properties should focus on increasing the
water affects hydrogen bonding, it has minimal impact concentration of fibers.
on the material structure. Another phenomenon occurs One way to manipulate cellulose concentration is
in this temperature range, where residual proteinaceous through culture conditions and strain used. Culture
matter and culture medium ingredients left over from fer- medium used for BC production influences the concen-
mentation can degrade around 159 C, as documented by tration of cellulose fibers, thus affecting mechanical prop-
Johnsy George.62–64 Through differential scanning calo- erties and macroscopic morphology of BC, as well as its
rimetry (DSC) analysis, they showed that this phenome- physicochemical properties.71,76 Addition of various addi-
non could be diminished or even eliminated after NaOH tives, such as carboxymethyl cellulose (CMC), pectin, gel-
purification. atin, cornstarch, and corn steep liquor, to the
The second process is related to the degradation of fermentation media can modify the BC produced, alter
BC fibers and accounts for 70%–80%65,66 of the mass loss its chemical structure, increase solid concentration, and
documented by TGA. This process involves depolymeri- enhance its mechanical properties.77 BC derived from sta-
zation, which eliminates crystalline regions and resulting tionary cultures generally exhibits a higher Young's mod-
in dehydration (of bound water) as well as decomposition ulus, but lower water-holding capacity compared to
of the glycosyl units.59,65,67,68 This process can be acceler- agitated cultures due to variations in cellulose
ated through direct catalysis or esterification of the concentration.78
hydroxyl groups. For example, and as previously men- The exceptional BC mechanical properties can also be
tioned, Roman et Winter have documented a method attributed to its nano- and micro-scale morphology. The
using H2SO4 to hydrolyse cellulose.50–53,58 Once degrada- presence of nano-fibrils contributes to the mechanical
tion is complete, the next step is the formation of a strength, but it is primarily the micro-level structure of
charred residue through oxidation.63 The residual carbo- the compressed sheets, characterized by high planar ori-
naceous matter is then broken down, and gaseous low- entation of ribbon-like fibers, that accounts for BC
molecular-weight products are formed. impressive mechanical properties.71–73 The crystal struc-
The decomposition temperature is a key criterion for ture of cellulose, particularly Cellulose I and Cellulose II,
investigating the thermal stability of a material.65 For BC, significantly influences mechanical properties due to var-
it is known to be affected by structural parameters such iations in binding forces.
as molecular weight, crystallinity, and fiber orienta- The transformation of BC from Cellulose I to Cellu-
tion.61,66,68 Many studies have documented its variation, lose II, accompanied by conformational changes and the
which is easily influenced by the production process (e.g., breaking of hydrogen bonds, results in a decrease of
temperature, agitation, microorganism, and culture the mechanical properties.72 Alkaline purification of BC
media), purification process, and modification of the removes impurities within the gel network, enabling
material composition.65 For example, the addition of col- more effective fibril interactions, thereby enhancing
lagen between cellulosic fibers or a coupling with algi- mechanical properties. However, the concentration of
nate has been shown to increase the temperature NaOH should not exceed ≈ 2.5%, as higher concentra-
required for degradation, corresponding to an increase in tions can trigger a reaction leading to the formation of
thermal stability of the resulting material.19,69 Table 1 Cellulose II from Cellulose I.64
lists temperature-dependant properties that have been Overall, BC mechanical properties are predominantly
documented through TGA or DSC analysis of BC. influenced by cellulose concentration and fibril network
morphology, which, in turn, are influenced by the BC-
producing strain, culture medium composition, purifica-
2.3 | Mechanical properties tion process, drying methods, and sample preparation
prior to mechanical testing.79 These factors collectively
BC is renowned for its remarkable mechanical properties, make BC a fascinating and versatile material with excep-
particularly its high tensile strength, compression modu- tional mechanical characteristics.
lus, and surface area, which can be attributed to its
unique structural characteristics.71–73 In fact, BC exhibits
a higher Young's modulus, greater water uptake capacity, 2.3.1 | Tensile properties
and a higher aspect ratio of fibers compared to plant cel-
lulose.17 BC mechanical properties, in addition to its fibril Variations in tensile properties of BC have been observed,
network morphology, are highly dependent on cellulose likely attributed to structural variations within the net-
concentration.74,75 For instance, the Young's modulus of work. Indeed, BC hydrogels exhibit anisotropic features
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 6 of 35

T A B L E 1 Thermal properties of BC-based materials including degradation temperature (Td), glass transition temperature (Tg), melting
temperature (Tm), crystalline melting temperature (Tcm), crystallization temperature (Tc) and enthalpy of melting (ΔHm) obtained through
TGA (with DTG analysis unless specified otherwise) and DSC analyses.

Material Thermal properties Supplementary information Reference


Bacterial cellulose-derived Td: 379 C (TGA) George et al.63
nanocrystals (enzyme-treated BC Tg: 66.7 C (DSC)
to form BC nanocellulose crystals) Tm: 203.3 C (DSC)
ΔHm: 83.2 J/g (DSC)
Bacterial cellulose-derived Td: 184 C (TGA) George et al.63
nanocrystals (H2SO4-treated BC to
form BC nanocellulose crystals)
Non-purified BC Td: 158.68 C (TGA) First Td is associated to the George et al.62
Td: 312.23 C (TGA) degradation of proteinaceous matter.
Tg: 13.94 C (DSC)
Tcm: 120.47 C (DSC)
Purified BC NaOH-purified: No peak associated to proteinaceous George et al.62
Td: 350.95 C (TGA) matter was identified and Tcm were
Tg: 41.41 C (DSC) inferior to 120.47 C implying
KOH-purified: changes in the crystalline region due
Td: 354.44 C (TGA) to the purification. The superior Td
Tg: 44.24 C (DSC) are explained by an efficient removal
of low molecular weight impurities
Na2CO3-purified:
which affected the orientation of BC
Td: 355.56 C (TGA)
fibrils and crystalline phase. The
Tg: 47.41 C (DSC)
heating process during purification
K2CO3-purified: also transforms the metastable 1α
Td: 355.85 C (TGA) phase into the thermally stable 1β
Tg: 48.82 C (DSC) phase.
BC from agitated culture Td: 288.88 C (TGA) Mohite and
(NaOH-purified) Tg: 44.28 C (DSC) Patil61
Tcm: 84.44 C (DSC)
Tc: 343.06 C (DSC)
BC (NaOH-purified) Td: 290 C (TGA, degradation Revin et al.70
reaction begins)
BC (NaOH-purified) Td: 328.36 C (TGA) BC and chitosan composite showed Jia et al.67
lower Td which implies the
crystallinity was weakened by the
chitosan chloride in the growth
media.
Non-purified BC Td: 289 C (TGA) Gea et al.64

BC (NaOH-purified) Td: 322 C (TGA) The purification treatment is designed Gea et al.64
BC (NaOH purified and bleached Td: 359 C (TGA) to prevent polymorphic crystal Gea et al.64
with NaOCl) transformation from cellulose 1 to
cellulose 2 since it is detrimental to
mechanical properties.
BC (assume NaOH-purified, but is Td: 311 C (TGA) Barud et al.68
not clarified) Td: 330 C (DSC)
Tg:
16 C (Thermo-Mechanical
analysis)

due to their laminated architecture on the length scale of polymer network configuration and polymer-water inter-
10–100 μm, comprising layers rich in cellulose ribbons actions. Consequently, the layers align parallel to the ten-
and ribbon-depleted gaps between the layers.74 sion direction until the ribbons straighten and eventually
Chen et al. reported that during tensile testing of BC, fractures. It has been demonstrated that ordered fibers
the initial stretching process causes changes in the alignment reduces resistance to deformations. Thus,
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
7 of 35 GIRARD ET AL.

cellulose concentration and fiber orientation/structural phase is not a plateau due to water being expelled from
morphology are expected to be the most influential struc- the BC sample as its volume decreases under applied
tural factors affecting BC tensile properties.74,78 stress. The resistance offered by the incompressible water
Costa et al. reported additional variations in results leaving the sample is expected to significantly influence
when comparing hydrated and dry samples. According to the compression behavior, as water is in part bound to
Almeida et al. and Gao et al., water and waste by-prod- the fibers and its removal enhances stiffness. Moreover,
ucts present in the culture medium act as plasticizers, the second phase, characterized by fiber entanglement
thereby altering the mechanical performance of the bio- and increased network density, was documented to occur
material.77,80,81 In hydrated BC, the presence of water at approximately 50% strain, while the third and final
reduces the amount of hydrogen bonds between mole- phase associated with material densification was docu-
cules and weakens intermolecular forces. This is causing mented at around 75% strain.74
greater elongation and lower tensile strength and Young's The compression modulus was determined by calcu-
modulus compared to dry BC.72 Indeed, significant plas- lating stress divided by strain within the linear region.77
tic deformation of the material is observed in the form of Table 3 lists the elastic compression modulus of various
irreversible ductile fracture before rupture in the case of types of BC and BC formulations, with meticulous atten-
hydrated BC.76 tion given to sample preparation.
Table 2 presents the ultimate tensile strength (UTS),
elongation at break, and Young's modulus (E) from
numerous studies, for different types of BC, BC formula- 2.3.3 | Specific surface area
tions (including culture medium), material states, and
methodologies, with careful attention given to sample Numerous studies, as identified by Guo & Catchmark,
preparation.76 For instance, strain affects cellulose con- have investigated the specific surface area of cellulose
centration and structural morphology, which subse- materials using Brunauer–Emmett–Teller (BET) theory
quently impact tensile properties.72,74 The choice of with N2 adsorption. These studies revealed variations in
sample shape, such as dumbbell or rectangular, also the range of 0.58–200 m2/g, indicating that the surface
influences the results.72 Furthermore, lower water con- area is influenced by factors such as sample selection,
tent tends to enhance toughness, and the drying method pre-treatment, and drying methods.85 Once again, cellu-
affects the structural morphology.72 For example, freeze- lose concentration and fiber morphology significantly
drying preserves the structure, while oven drying leads to impact the specific surface area. In comparison, activated
densification or even collapse of the structure.79 As a charcoal has shown a specific surface area of 1734 m2/g
rule, increasing cellulose concentration raises UTS and and polycaprolactone-based 3D cell scaffolds, 86.5 m2/
Young's modulus, while reducing elongation at break. g.86,87
Table 4 presents specific surface area of different
types of BC and BC formulations, with careful attention
2.3.2 | Compression properties given to sample preparation.75

Studying BC compression properties is important as it

yields valuable insights into its structural integrity, 2.4 | Cytotoxicity
mechanical stability, and potential applications. BC stiff-
ness is primarily influenced by both cellulose concentra- Cytotoxicity testing is an essential initial step in evaluat-
tion and arrangement of its fibril network, both in dried ing biocompatibility of new biomaterials before proceed-
and hydrated states.74 ing to animal and clinical studies. While there is no
When subjected to compressive stress, Ferreira et al. single protocol that allows the evaluation of cytotoxicity
identified that freeze-dried cellulose materials exhibit a for all materials, the International Organization for Stan-
distinct compression curve characterized by three dardization (ISO) has established the norm ISO-10993-5
regimes. Initially, in the linear regime (<5%), stress (Biological evaluation of medical devices) providing gen-
increases linearly due to elastic compression. This is fol- eral guidelines for the preparation, execution, and analy-
lowed by a plateau regime (10%–50%), where pore col- sis of such a protocol. Given that bacterial cellulose
lapse and compression of cellulose within the structure (BC) comes in various forms, including membrane,
result in nearly constant stress. Finally, an exponential hydrogel, dry powder, and can be coupled with other
increase in stress occurs as the pores are fully collapsed materials that alter its texture and physicochemical prop-
and cellulose fibers deform and come closer together.75 erties, cytotoxicity protocols can vary significantly from
Similarly, hydrated BC demonstrates a three-phase one article to another. However, researchers commonly
behavior, with some variations. In this case, the second use two main methods to evaluate BC cytotoxicity: 1. by
 T A B L E 2 Tensile properties of BC including UTS (in MPa), Elongation at break (in %) and Young's modulus (in MPa) with careful attention given to the methodology and the state of the
tested material.

UTS Elongation at Young's

GIRARD ET AL.

Methodology Material state Sample variation (MPa) break (%) modulus (MPa) References
Dumbbell shape with cross-section Hydrated ATCC 700178 0.15 ± 0.08 20.72 ± 8.32 1.10 ± 0.38 Chen et al.74
dimensions of 2 mm  10 mm (ISO 37-4) ATCC 10245 0.36 ± 0.08 18.60 ± 8.03 2.87 ± 1.33
ATCC 23769 0.12 ± 0.04 17.97 ± 5.04 1.26 ± 0.66
NBRC 13693 0.62 ± 0.17 18.69 ± 3.25 3.08 ± 0.66
ATCC 53524 0.68 ± 0.13 20.72 ± 6.03 5.56 ± 2.29
KTH 5655 0.62 ± 0.16 16.20 ± 2.91 3.83 ± 1.08
BC produced by A. xylinum. Dried Not purified (native) 88.9 ± 9.3 1.24 ± 0.9 7600 ± 1200 Gea et al.64
Rectangular strips (30 mm  5 mm) 2.5 wt.% NaOH overnight 139.1 1.34 ± 0.6 14,100 ± 1600
± 12.6
2.5 wt.% NaOH overnight & 2.5 wt.% 207.0 1.59 ± 0.2 18,800 ± 2000
NaCl overnight ± 13.8
N.A. Dried Native 20–200 N.A. Sheet: 20000 Single fiber: Wang et al.17
130000
G. hansenii CGMCC 3917 with samples cut Hydrated - 0.26 ± 0.02 15.55 ± 1.30 0.005 ± 0.0003 Feng et al.79
in cuboid shape Freeze-dried - 11.94 2.78 ± 0.25 6.65 ± 0.16
± 1.15
Oven-dried - 328.79 4.31 ± 0.12 18.12 ± 0.68
± 26.96
BC produced by A. xylinum Dried Stationary fermentor 92.0 N.A. 2700 Krystynowicz
Rotating disc fermentor 22.9 N.A. 300 et al.78

BC produced by A. aceti AJ 12368 and Dried - N.A. N.A. 15,000 Yamanaka et al.82
samples cut in rectangular shape
BC produced by BRP2001 strain. Samples Hydrated - 0.88 63.7 2.11 Nishi et al.31
were cut in rectangular strips (60 mm 10% cellulose - 11.23 55.8 27.69 Scionti et al.72
 10 mm) Yamanaka et al.82
20% cellulose - 23.24 50.1 98.91
30% cellulose - 59.64 39.8 359.18
8 of 35

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 TABLE 2 (Continued)
9 of 35

UTS Elongation at Young's

Methodology Material state Sample variation (MPa) break (%) modulus (MPa) References
35% cellulose - 76.86 30.7 528.28

40% cellulose - 142.14 29.3 738.28

Dried - 239.63 2.72 10515.25
(92% cellulose)
N.A. Hot-pressed - 241.42 8.21 ± 3.01 6860 ± 320 Torres et al.73
(dried) ± 21.86 Grande et al.83
Different additives in culture medium, - BC - - 7.55 ± 0.33 Dayal and
alkaline washed, pressed into a flat piece - BC + 1% carboxymethyl cellulose (CMC) - - 5.93 ± 1.03 Catchmark77
at 25 N and cut in rectangular strips
- BC + 3% CMC - - 4.03 ± 0.17
(20 mm  3–4 mm)
- BC + 5% CMC - - 8.45 ± 2.17
- BC + 1% pectin - - 16.70 ± 1.91
- BC + 3% pectin - - 9.09 ± 3.60
- BC + 5% pectin - - 5.06 ± 1.03
- BC + 1% gelatin - - 21.94 ± 2.01
- BC + 3% gelatin - - 3.67 ± 1.92
- BC + 5% gelatin - - 4.14 ± 1.70
- BC + 1% corn steep liquor - - 8.54 ± 0.69
- BC + 3% corn steep liquor - - 6.05 ± 1.37
- BC + 5% corn steep liquor - - 5.62 ± 0.48
- BC + 1% cornstarch - - 3.84 ± 1.01
- BC + 3% cornstarch - - 3.24 ± 1.60
- BC + 5% cornstarch - - 3.44 ± 0.93
GIRARD ET AL.

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GIRARD ET AL. 10 of 35

TABLE 3 Compressive elastic modulus of BC (in kPa) based on sample preparation, formulation and hydrated or dry state of the tested
material.

Material Compressive elastic

Sample preparation state Sample formulation modulus (kPa) References
Sample had an 8 mm diameter and a Dry BC 9510 ± 230 Li et al.84
6 mm height, for hydrated, samples BC + 1.0% wt. chitosan 8040 ± 470
were immersed in PBS for 24 h at
BC + 1.5% wt. chitosan 9130 ± 400
37 C to simulate in vivo conditions
BC + 2.0% wt. chitosan 4740 ± 1000
Hydrated BC 37,320 ± 1070
BC + 1.0% wt. chitosan 580 ± 170
BC + 1.5% wt. chitosan 820 ± 150
BC + 2.0% wt. chitosan 9640 ± 460
Different additives in culture medium, - BC 7.43 ± 0.70 Dayal &
alkaline washed, samples were taken - BC + 1% carboxymethyl 107.75 ± 5.16 Catchmark77
from the pellicle and were 20 mm cellulose (CMC)
 20 mm. They were compressed to
- BC + 3% CMC 21.60 ± 6.79
a force of 25 N.
- BC + 5% CMC 25.55 ± 4.88
- BC + 1% pectin 80.03 ± 4.49
- BC + 3% pectin 142.30 ± 2.12
- BC + 5% pectin 129.27 ± 8.43
- BC + 1% gelatin 60.75 ± 5.73
- BC + 3% gelatin 80.40 ± 9.33
- BC + 5% gelatin 83.05 ± 2.19
- BC + 1% corn steep liquor 26.70 ± 2.81
- BC + 3% corn steep liquor 46.10 ± 6.64
- BC + 5% corn steep liquor 46.87 ± 4.07
- BC + 1% cornstarch 81.58 ± 5.40
- BC + 3% cornstarch 82.24 ± 4.77
- BC + 5% cornstarch 87.10 ± 1.84

seeding cells directly on top of it and/or 2. by incubating cells exhibited no cytotoxicity effects (close to 100% via-
cells with soluble BC, or BC nanofibers in suspension. bility over 7 days) when seeding cells directly over BC.89
Cytotoxicity is usually measured using assays such as The absence of BC cytotoxicity has been confirmed by
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- several studies with similar protocols.90-95
lium bromide) or neutral red, which evaluates cells meta- Studies evaluating BC composites with surgical
bolic activity and viability. The ISO 10993-5 norm meshes (such as BC-hydroxyapatite, BC-titanium, and
indicates that any viability lower than 70% is associated BC-polytetrafluoroethylene) also found no cytotoxicity.
with potential cytotoxic effects. While this might not One study using osteoblasts and fibroblasts found no
always hold true, most studies agree that low viability is cytotoxicity when cells were seeded on a BC-coated surgi-
associated with cytotoxicity, albeit with varying thresh- cal mesh.96 Another study using rabbit bone marrow
olds. In this review, we use 70% as the threshold to deter- mesenchymal stem cells found a viability rate of 94%
mine cytotoxicity. when seeded on BC.97 In both studies, surgical meshes
Several studies evaluated BC cytotoxicity using differ- were fully covered by BC, meaning that cells were not in
ent cell lines and testing methods. For instance, studies direct contact with the implants but rather with the BC.
that tested cells directly over BC found no cytotoxicity Studies investigating BC or BC-derived materials in
effects. One such study conducted by Volova et al. solution also found no cytotoxicity. In one study, CNCs
reported that NIH 3 T3 mouse fibroblasts had a viability were dispersed with concentrations ranging between
of 140% over 7 days compared to polystyrene flask.88 10 and 100 μg/mL, and these solutions were tested for
Another study found that triple-negative breast cancer 24 h against RAW-Blue macrophages for cytotoxic effects.
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
11 of 35 GIRARD ET AL.

In all samples, no significant change in viability was

observed.98 In another study, BC powder was prepared

Catchmark85
by lyophilizing hydrated BC and then pulverizing it. This

References
powder was then resuspended at various concentrations

Li et al.84
between 1 and 1000 μg/mL over peripheral blood mono-

Guo &
nuclear cells and incubated for 5 days. Similarly to the
previously described article, no statistical reduction in
viability could be found in all samples, although a lower
viability with a value of 90% was observed at the highest
Specific surface area

BC concentration of 1000 μg/mL.99 These results are also

corroborated by Lopes et al.,100 which tested cellulose
nanofibrils with concentrations between 50 and 500 μg/
11.9 ± 0.2

16.2 ± 2.9

39.2 ± 1.3
29.1 ± 0.7
mL over HDF cells, MRC-5 cells, and THP-1 differenti-
(m2/g)

ated macrophages. After 24-h incubation, no cytotoxic

22.06
19.85
29.5
201

effect could be observed for any of the cell types men-

tioned earlier, supporting the notion that BC and BC-
derived materials are generally safe and biocompatible
Lower fraction of a cellulose nanowhiskers suspension
Upper fraction of a cellulose nanowhiskers suspension

for use in medical devices. Table 5 compiles various cyto-

toxicity studies performed on BC and BC composites.
obtained from BC hydrolysis by HCl

obtained from BC hydrolysis by HCl

2.5 | Biocompatibility
Specific surface area of BC (in m2/g) based on sample preparation, variation, and drying method.

One critical aspect of utilizing BC in medical applications

is understanding its biocompatibility in vivo. Biocompati-
BC from agitated culture
BC + 1.0% wt. chitosan
BC + 1.5% wt. chitosan
BC + 2.0% wt. chitosan

BC from static culture

bility refers to the ability of a material to perform its

Sample variation

desired function with an acceptable adverse response in

the host organism.
Extensive research has been conducted to investigate
BC biocompatibility in different animal models, provid-
ing valuable insights into its performance as an implant-
BC

able material. BC products were implanted in host

animals, and parameters such as implantation time,
Freeze-dried

Freeze-dried

inflammation response, and other observations were

recorded to assess the biocompatibility profile.
method
Drying

Table 6 provides a comprehensive summary of BC

products, host animals used, implantation time, inflam-
mation results, and other notable observations reported
by authors. This table serves as a valuable reference to
vacuum at 10 mmHg. Standard N2 adsorption
Samples were degassed at 30 C for 4–8 h under

understand findings and trends observed across different

adsorption was performed at 77 K using BET

research efforts.
procedure done at 77 K using BET theory
Samples were degassed at 25 C for 5 h, N2

In vivo studies consistently demonstrate the favorable

biocompatibility of BC. Upon implantation, BC scaffolds
or membranes are generally well tolerated by the host tis-


sues without significant adverse reactions. Inflammation

responses, a common indicator of biocompatibility, were
found to be minimal or within acceptable levels in most
Sample preparation

cases. The implantation time varied across studies, rang-

ing from a few days to several weeks, allowing for the
evaluation of biocompatibility both short- and long
TABLE 4

theory

terms.
Several notable observations emerge from these stud-
ies. Firstly, BC has been shown to possess excellent tissue
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 12 of 35

TABLE 5 In vitro cytotoxicity of bacterial cellulose-derived materials based on cell types used, cytotoxicity assays and culture times.

Materials Cells Assay Culture time Cytotoxicity References

BC NIH 3 T3 fibroblasts MTT 7 days No Volova et al.88
BC/Gelatin MDA-MB-231 cells MTT 7 days No Wang et al.89
Compressed BC Canine bone marrow Hoechst 33342/PI 10 days No Mendes et al.90
BC-HA Human primary chondrocytes Prestoblue 8 days No Kumbhar et al.91
BC-GAG Human primary chondrocytes Prestoblue 8 days No Kumbhar et al.91
BC-Exos L929 fibroblasts CCK-8 + Live/Dead 5 days No Wang et al.92
EI-BCMs NIH 3 T3 fibroblasts CCK-8 7 days No An et al.93
BC EqMSCs CellTiter 96® 14 days No Favi et al.101
BC hESCs Live/dead 6 days No Lou et al.94
CNFs/PEGDA NIH 3 T3 fibroblasts MTT 7 days No Tang et al.95
Ti6Al7Nb U2-OS osteoblasts/L929 fibroblasts Neutral Red 2 days No Dydak et al.96
Surgical mesh-BC L929 fibroblasts Neutral red 2 days No Dydak et al.97
TEMPO+Amine BC RAW-Blue macrophages MTT 1 day No Weiss et al.98
BC MC3T3-E1 cells FE-SEM analysis 1 day No Khattak et al.99

Abbreviations: CNFs/PEGDA, cellulose nanofibrils/polyethylene glycol diacrylate; EI-BCMs, electron irradiated bacterial cellulose membranes; EqMSCs,
equine-derived bone marrow mesenchymal stem cells; Exos, umbilical cord exosomes; FE-SEM, field emission scanning electron microscope; GAG,
glycosaminoglycan; HA, hydroxyapatite; hESCs, human embryonic stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TEMPO,
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl; Ti6Al7Nb, scaffold implants coated with gentamycin-saturated bacterial cellulose.

integration properties. It promotes cellular adhesion, pro- minimal inflammation responses, promotes tissue inte-
liferation, and extracellular matrix production, facilitat- gration, supports cellular viability, and shows potential
ing the formation of a functional interface between the for gradual biodegradation. These findings underscore
BC material and the host tissue. This property is particu- the potential of BC as a promising biomaterial for various
larly advantageous for applications such as wound heal- biomedical applications.
ing and tissue regeneration.
Secondly, BC's porosity and interconnected structure
allow for efficient nutrient and oxygen exchange, 3 | PRODUCTION
enabling cell survival and growth within the BC matrix.
This feature is crucial for tissue engineering applications 3.1 | Microorganism comparison /
where the implanted material must support cell viability sourcing
and tissue regeneration.
Thirdly, BC exhibits minimal foreign body reactions. Although multiple bacteria can synthesize cellulose,
Foreign body reactions occur when the host's immune research is often mainly focused on the bacterium Gluco-
system responds to the presence of a foreign material, nacetobacter xylinus (GX), formerly known as Acetobacter
potentially leading to chronic inflammation. However, xylinum. This bacterium is found on fruit skins and tea
in vivo studies consistently demonstrate that BC evokes leaves, along with many other cellulose-synthesizing bac-
minimal foreign body reactions, suggesting its compati- teria. In popular culture, GX is used to produce the fizzy,
bility with the host immune system. probiotic drink Kombucha. Kombucha Gluconacetobacter
Moreover, it is worth noting that BC does elicit fibro- is accompanied by a wide range of other bacteria and
sis in most long-term biocompatibility studies. Encapsu- yeasts. For instance, Sarcina and Agrobacterium have
lation can be found in thin, micrometer range, or in also been confirmed to produce cellulose.122 BC produced
larger bands that fully surround the implant. Fibrosis is, during kombucha fermentation is generally referred to as
however, not generally considered as an inflammatory SCOBY (symbiotic culture of bacteria and yeast), as well
marker if cells in the fibrous layer remain ordered and if as “kombucha mushroom”, even though no mushroom is
there is no immune recruitment surrounding the present in the culture.
encapsulation band. Researchers have been looking at ways of acquiring
In conclusion, in vivo studies conducted on BC dem- increasingly productive strains of bacteria for BC. In gen-
onstrate its favorable biocompatibility profile. BC exhibits eral, the Gluconacetobacter genus remains predominant
 TABLE 6 In vivo biocompatibility of bacterial cellulose-derived materials given the surgical application tested, the host animal and the maximum implantation duration.
13 of 35

Surgical Host
application Host animal Maximum Cell response/inflammation/
tested Product animal type duration tissue response Concerns References
Subcutaneous BC j BC-HA Rat Wistar 60 days No significant inflammation of Massari et al.102
all materials. Fibrous
encapsulation around all
materials.
Subcutaneous BC-Urine-derived stem cells Mouse Athymic 1 month USC cells differentiated and Athymic mice are highly Bodin et al.103
(USC) expressed SMC markers. immunodeficient, making
tissue response analysis
skewed.
Subcutaneous BC coated with bone Rat Sprague– 4 weeks Novel bone formation around Qin Shi et al.104
morphogenic protein-2 Dawley the scaffold.
(BMP-2)
Tooth peri-implant Electron-beam irradiated BC Dog Beagle 8 weeks Mild signs of inflammation such Only 2 dogs were used for this Lee et al.105
(EI-BCMs) as edema and exudate. No experiment.
foreign body reaction.
Subcutaneous BC functionalized with Arg- Sheep Merino 32 weeks Mildly inflammatory compared Weak images for the 32-week Andrade et al.106
Gly-Asp (RGD) peptide to expanded PTFE. Fibrous mark.
encapsulation around the
implants.
Aortocoronary BC-cobalt-chromium mesh Pig Unspecified 4 weeks No signs of inflammation. No control groups. Deborah Fusco
bypass et al.107
Artificial blood BC Sheep Unspecified 13 months Confluent endothelial cells No control groups. Skewed Carl Johan Malm
vessel surrounding the implants. Of conclusions about patency of et al.108
8 sheep, 4 died within the first BC as artificial blood vessels.
day, showing acute
thrombosis. Another died
within 2 weeks with
seemingly unrelated causes.
Another sheep was sacrificed
after 8 months due to
unilateral occlusion. Minimal
inflammation on the
remaining 2 sheep.
Artificial carotid BC Pig White, 3 months Mild and chronic inflammation Jens Wippermann
arteries domestic around BC particles. et al.109
Subcutaneous Bilayer BC scaffold Mouse Athymic 8 weeks Thin encapsulation around the Only 2 mice were used. No Héctor Martínez
implants, suggesting normal control groups. Athymic mice Ávila et al.110
are highly immunodeficient,
GIRARD ET AL.

(Continues)

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 TABLE 6 (Continued)

Surgical Host
application Host animal Maximum Cell response/inflammation/
GIRARD ET AL.

tested Product animal type duration tissue response Concerns References

non-pathological foreign body making tissue response
reaction. analysis skewed.
Nerve-protecting Self-patented BC product Rat Wistar 180 days No visible signs of Karolina Kowalska-
tubes inflammation. Ludwicka et al.111
Subcutaneous BC Rat Wistar 12 weeks No signs of inflammation. Gisela Helenius
et al.112
Artificial carotid BC Sheep Texel 12 weeks Low inflammation, slight Scherner et al.113
arteries foreign body reaction. Fibrotic
encapsulation of the grafts.
Signs of cellular ingrowth.
Dural replacement BC Rabbit White, 360 days Early signs of inflammation Xu et al.114
New which declined over time.
Zealand
Subcutaneous BC/Exosomes Rabbit White, 1 year Low inflammation that did not Wang et al.92
New penetrate the implant.
Zealand
Intramuscular MBC Rat Wistar 3 months Low inflammation with the Piasecka-Zelga
presence of encapsulation et al.115
around the implants.
Subcutaneous BC Mouse BALB/c 12 months Mild and benign inflammatory Pértile et al.116
reaction that decreased along
time and did not elicit a
foreign body reaction.
Dental canal paper BC Rat Sprague– 8 weeks Lower inflammation was Aya Yoshino et al.117
point Dawley reported with BC than with
regular PPs.
Subcutaneous BC Rabbit Chinchilla 1 week Minimal inflammation and Héctor Martínez
bastards foreign body reaction. Ávila et al.118
Dural replacement BC Dog Mongrel 270 days Predominant fibrotic structures Mello Lr et al.119
around the implants. Low
inflammation. Low foreign
body reaction.
Subcutaneous BC-PS Dog Unspecified 1 week No macroscopic signs of No control groups. Jingxuan Yang
inflammation. et al.120
14 of 35

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15 of 35 GIRARD ET AL.

as a source of high-yield BC production. However, some

Abbreviations: GAG, glycosaminoglycans; HA, hydroxyapatite; MBC, bacterial cellulose mesh modified with chitosan; PPs, paper points; PS, potato starch; PTFE, polytetrafluoroethylene; SMC,
species and subspecies thereof have vastly different

Dieter Schumann

Dieter Schumann
Kumbhar et al.91
production capabilities. Table 7 lists some known BC pro-

Mendes et al.90
References
ducing bacterial strains alongside with their productivity.

et al.121

et al.121
3.2 | Culture conditions

3.2.1 | Culture medium

Gluconacetobacter xylinus culture is relatively straightfor-

No control groups.

ward, requiring carbon and nitrogen sources and other

macronutrients. Various carbon sources have been inves-
tigated, including sugars such as glucose, fructose, and
Concerns

simple or complex sugars. However, it is worth noting

that fermenting with complex sugars like starch may pre-
sent some challenges. While glucose is generally accepted
without foreign body reaction.
Cell response/inflammation/

as a cheap and effective carbon source, other carbohy-

implant. No inflammation of
the synovial membranes and
Fibrous tissue surrounding the

Mild inflammatory responses.

Mild inflammatory responses.

Mild inflammatory responses

drates have been investigated. In particular, date syrup,

sucrose and mannitol may also be used to increase BC
yield.65,76,88,131
other joint tissues.

When it comes to nitrogen sources, commonly used

tissue response

options include yeast extract and peptone, which have

proven to be effective in supporting GX growth and BC
production.76,132 Alternatively, hydrolyzed proteins, free
amino acids or supplements can also be used.30,133,134
Macronutrients are essential for bacterial growth, for
Maximum

which yeast extract is the main source. Moreover, various

duration

3 months
90 days

90 days

forms of food waste can sufficiently meet the bacterium

1 year

demand for macronutrients.30,99,134-137

Other additives include ethanol, polymers such as
domestic

polyethylene glycol (PEG), agar, agarose, pectin, gelatin

Albino
animal

and many others.138-142

White,
Wistar

Wistar
Swiss
Host

type

Among culture medium formulations, the Hestrin

and Schramm (HS) medium has been widely recognized
animal

as favorable for BC production, providing optimal condi-

Mouse
Host

tions for fermentation.143,144 HS medium has been exten-

Rat

Rat

Pig

sively studied and demonstrated to promote BC

production.145
Moreover, GX has shown its versatility in utilizing
agricultural waste as a complete culture medium.
Researchers have explored the fermentation of GX in var-
BC-HA j BC-GAG

ious agrifood residues such as coconut pulp, coconut

water, and even non-saleable fruit juices like pineapple
Product

juice.30,99,134-137
BC

BC

BC
(Continued)

3.2.2 | Temperature
smooth muscle cells.
cartilage (knee

Artificial carotid

Artificial carotid
Artificial bone

Subcutaneous
application

Although optimal temperature varies among GX subspe-

TABLE 6

arteries

arteries
Surgical

joint)

cies, it is generally accepted that the best BC yield can be

tested

achieved with incubation temperatures between 28 and

30 C.23,35,76,138,146-148 That said, BC production can be
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GIRARD ET AL. 16 of 35

TABLE 7 Yield of bacterial cellulose for bacterial strains, culture media, and cultivation methods.35

Incubation
Medium Bacterial strain time (days) BC yield (g/L) References
a
Glycerol Gluconacetobacter sp. RKY5 6 4.59 Kim et al.123
b
5.63
Glucose yeast extract broth Acetobacter xylinum K086 7 0.14–0.39a Singhsa
Acetobacter xylinum K975 1.11–1.55 a et al.124

Acetobacter xylinum K428 0.09–0.22a

Acetobacter xylinum K1011 0.57–1.46a
Acetobacter xylinum KX 1.14–1.84a
Glycerol Acetobacter sp. V6 7 4.98b Jung et al.125
Molasse Komagataeibacter sucrofernentans 14 8.2 ± 0.2a, 8.1 ± 0.2a Revin et al.126
H110
Stillage Komagataeibacter hansenii C110 9.5 ± 0.1a, 9.2 ± 0.1a
Citrus waste solution Gluconacetobacter intermedius 8 7.2a Hu et al.127
HS media CIs26 2.1a
Citrus waste-modified HS 5.7a
Glucose Gluconacetobacter hansenii 2 1.33b Jung et al.128
Glucose (modified HS 14 14.72a Hodel et al.129
medium)
Mannitol (modified HS 20
medium)
Pineapple peel juice Gluconacetobacter swingsii 13 2.8a Castro
et al.130

Note: This table has been adapted from the Table 1 in “Bacterial Cellulose: Production, Characterization, and Application as Antimicrobial Agent” by Lahiri et al.
and is permitted within the terms and conditions of the Creative Commons Attribution (CC BY) license.
Abbreviations: HS, hestrin and schramm media.
a
Static cultivation.
b
Agitated cultivation.

sustained in a much wider range of temperatures. For pH at the beginning, conditions are then suitable for the
instance, it has been documented that BC can be pro- subsequent fermentation process.
duced with incubation temperatures between 20 and The second strategy revolves around controlling the
35 C.148 Low temperatures generally slow down the bac- pH during fermentation. Researchers have found that
teria metabolism, thereby slowing the rate of BC produc- actively maintaining a pH of 5 throughout the fermenta-
tion. High temperatures cause cell death and media tion period significantly enhances BC production com-
degradation, which inhibit BC formation. pared to cultures with uncontrolled pH levels. This
approach involves continuously monitoring and adjust-
ing the pH to ensure it remains at the desired level, pro-
3.2.3 | pH moting efficient conversion of glucose into bacterial
cellulose.150 In a separate study, the authors focused on a
When it comes to achieving optimal BC production, the specific approach where they initially controlled the pH
behavior of acetic acid bacteria presents several challenges at a value of 4. This deliberate pH adjustment aimed to
regarding pH control. In this regard, two main strategies drive the conversion of glucose into gluconic acid. At this
are used in the field, each with its own approach. pH level, the production of bacterial cellulose (BC) was
The first strategy involves setting the pH before inoc- almost negligible, but cells were still capable of multiply-
ulation. Researchers have discovered that achieving a pH ing. Once all the available glucose was consumed, the
between 5 and 5.5 prior to introducing the bacteria gener- authors discontinued pH control, allowing cells to utilize
ally yields the best results.23,76,138,149 By establishing this the accumulated gluconic acid for BC production. As a
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
17 of 35 GIRARD ET AL.

result of this utilization process, the pH naturally The following section describes in further details how
increased to 5.5. Notably, compared to cultures with different methods of production affect BC yield, shape,
uncontrolled pH conditions, this approach led to a signif- and properties.
icant twofold increase in BC yield.151

3.2.6 | Vessels and bioreactors

3.2.4 | Dissolved oxygen
Production of BC is most often conducted in bioreactors,
Dissolved oxygen (DO) concentration is an important as they provide controlled culture parameters such as
parameter to monitor for BC production. GX is a strict temperature, pH, agitation, and feed. Although some
aerobic bacterium. This implies that all fermentations types of reactors are more common than others, Figure 3
performed with GX must have some passive or active illustrates the six types of bioreactors most referenced in
supply of oxygen. As seen in the Vessels and bioreactors literature: the static reactor, the stirred-tank reactor, the
section, many designs revolve around maximizing oxygen aerosol reactor, the rotary reactor, the airlift reactor, and
transfer through agitation or enhanced effective air- the biofilm reactor.
media surface area. For instance, the airlift reactor pro- Static reactors, which are simply trays in which
vides an active supply of oxygen to the bacterial culture; growth medium is inoculated with GX, currently domi-
and even, it has been reported that enriching oxygen con- nate most industrial scale-ups of BC production. While
tent into the gas feed still improves BC yield.152 In most these may not be the most performant units, they are cer-
studies, the reported optimal relative dissolved oxygen tainly extremely straightforward to design and scale and
concentration for BC production is 10%, for which are inexpensive. Technically, any tray can serve as a static
reactor if the temperature and oxygen demands are
CL met.158-161
DO ¼
C Stirred-tank reactors are very common when it comes
to scaling up bacterial fermentation processes. They con-
and CL is the dissolved oxygen concentration (mol/L) sist of a simple stainless-steel vessel with a motorized
and C is the saturated dissolved oxygen impeller at its center. As previously mentioned, yields in
concentration.151,153,154 agitated cultures highly depend on the strain of bacteria
used. Based on personal experience, the authors have
noticed that strains of GX isolated from kombucha seem
3.2.5 | Agitation to prefer static conditions over agitated ones. Agitated
cultures also tend to disturb the formation of contiguous
The production of bacterial cellulose, while straightfor- BC membranes, which lets BC pellets float freely inside
ward, does not behave like any other bacterial culture. To the medium. In industrial processes, stirred-tank reactors
that extent, it is now known that BC forms at the inter- remain a competitive means of producing BC in high
face between the culture medium and the air. When yields.35,153,158,162-164
undisturbed, BC slowly grows as a thin film above the Aerosol reactors are inspired by spray reactors, as they
culture medium and thickens over time. Any agitation use pulverized growth medium to make BC. Here, the
can prevent the formation of a homogeneous BC film. main advantage is provided when small droplets of
This means that one must work in a quasi-static environ- medium interact with the surrounding air, increasing the
ment to produce BC membranes that are thick and effective liquid-air surface area. Another major advantage
contiguous. to using this method is it can be considered as a static
Obviously, there are also advantages to agitated BC bioreactor, forming large and thick BC sheets. This is
culture. It is in fact reported that while membranes pro- because the bacterial cellulose is formed at the bottom of
duced in agitated environments are smaller, the yield is the spray chamber and is essentially static, allowing for
in fact generally similar than in non-agitated cultures. undisturbed BC synthesis. The first installment of an
This is because agitation introduces oxygen into the aerosol reactor for BC production dates to 2007 when a
medium, which boosts the metabolism of GX bacteria. group of researchers from Germany were trying to opti-
For instance, in agitated cultures, reported yields include: mize BC production in surface cultures. The group pub-
10 g/L in 3 days155; 2.0 g/L in 1 day156; 3.8 g/L in lished three papers in which the last one suggested a
7 days.124 For non-agitated cultures, reported yields novel aerosol reactor working on a fed-batch principle.
include: 4.0 g/L in 1 day156; 1.9 g in 7 days124; 4.8 g/L in The unit was able to produce 7-cm-thick BC over the
5 days.157 course of 40 days, at which point they had to stop
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 18 of 35

F I G U R E 3 Bioreactors currently used to produce BC (Static, Stirred-tank, Aerosol, Rotary disk, Airlift, Biofilm) and their operating
mechanism. [Color figure can be viewed at wileyonlinelibrary.com]

prematurely because of an unexpected contamination. rotates, carrying some media and exposing it to the air,
They also found that the BC produced from this bioreac- effectively increasing medium-air interface areas. In a
tor was less polymerized, but had more than twice the first study by Norhayati et al., eight 7-cm-diameter disks
tensile strength as regular BC produced in surface cul- were submerged in the medium so that 39% of the disk
ture.154,165,166 In 2019, another research group found diameter would be underwater. With a shaft angular
aerosol reactors also allowed for the deposition of nano- speed of 7–11 rpm, a fivefold increase in dry-mass BC
scale building blocks throughout the fermentation pro- yield was observed.168 Following this study, many other
cess. By mixing carbon nanotubes with fresh medium articles were published to optimize some of the operating
into the sprayed inlet, they achieved BC-carbon nano- parameters of the rotary disk reactor. In three separate
tubes composites, which demonstrated high tensile articles, the best conditions for BC production were
strength and conductivity.167 found to be achieved with disk rotation speeds of 7 rpm,
Rotary disk reactor is a type of bioreactor equipped at a pH of 5.169-171
with a rotating biological contactor connected to a rotat- Airlift reactor is a type of stirred tank reactor in which
ing stainless-steel shaft. The contactor has poly(methyl no moving parts are used for mixing the contained bacte-
methacrylate) (PMMA) disks partially immersed in the rial culture. The basic structure of an airlift reactor con-
culture medium. During the fermentation, the shaft sists of a vertical column divided into two interconnected
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
19 of 35 GIRARD ET AL.

zones: a riser and a downcomer. Small air bubbles are fed proteins, by-products like acetic acid, and other cellular
through the riser column, promoting flow in which remnants such as lipopolysaccharides (LPSs).98,176-178
medium goes up in the riser and down in the downco- The medium used during the fermentation also dif-
mer. The air provides mixing capabilities, but most fuses into the BC and introduces additional impurities
importantly increases dissolved oxygen concentration into the final product.179 The medium may contain resid-
within the growth medium.152,172 This is a very important ual nutrients, salts, or other compounds that can become
aspect since oxygen catalyzes BC production within the embedded within the BC structure.180 Furthermore,
medium. Airlift reactors produce suspended pellets depending on the specific fermentation protocols used,
which have a distinct oval shape.152,173 While the base various additives may be incorporated, leading to the
design of the airlift may provide some advantage towards presence of even more impurities in unpurified
BC synthesis, specific requirements towards BC produc- BC. Examples of such additives include pH regulators,
tion necessitates some modifications of the reactor. For stabilizers, and organic matter such as plant debris from
instance, one research group developed an external-loop food waste.134,136,181-183
airlift bioreactor in which they were able to achieve 200x While removal of impurities is not mandatory in cer-
higher cellular density than that of classical inoculum tain applications within the food, agricultural, or textile
preparations.174 In another modified version of the airlift industries, it is crucial for most medical applications.
reactor, the authors inserted porous plates inside a tubu- Medical devices, such as implants or scaffolds, as well as
lar reactor to diffuse an enriched oxygen supply. The wound dressings, require exceptionally high levels of
research group found that the BC produced with this purity to ensure biocompatibility and minimize the risk
method had improved water-retention capabilities and of adverse reactions. Impurities in these medical applica-
enhanced Young's modulus.173 tions could potentially trigger immune responses, inflam-
In Biofilm reactors, solid matrix supports made from mation, or infections, compromising their safety and
composite or natural materials such as polypropylene are efficacy.
fully submerged into the bioreactor. As such, one could
for example submerge a 3D printed scaffold matrix in BC
growth medium. If the matrix is suitable for BC growth, 4.2 | NaOH purification
it is expected that the support be filled with BC after fer-
mentation. The growth medium is usually agitated to pre- Purification of BC involves impurity removal, which is
vent BC from forming at the liquid-air interface, but typically achieved by treating it with a concentrated and
rather on the support themselves. In one study conducted heated solution of sodium hydroxide (NaOH) for a dura-
in 2009, plastic composite supports enriched with dried tion exceeding 1 h. The commonly employed concentra-
soy hulls, defatted soybean flour, yeast extract, tion of NaOH is 0.5 M (20 g/L). The use of NaOH serves
dried bovine red blood cells and mineral salts provided three purposes. Firstly, it hydrolyzes proteins and cell
the best conditions for BC production. BC grown on such debris, breaking them down into smaller components,
solid supports had higher crystallinity (93%) and similar facilitating their removal through diffusion in the purifi-
crystal size (5.2 nm) compared to BC synthesized in a cation medium. Secondly, NaOH solubilizes amphiphilic
regular stirred tank reactor.175 As mentioned above, some substances, including fatty acids, lipopolysaccharides
natural materials may also be used as solid support for (LPS), and other compounds produced by the hydrolysis
biofilm reactors. This has been shown in a study where of NaOH. Lastly, NaOH helps in minimizing the bacterial
luffa sponges (Luffa aegyptiaca) were used as a matrix for load. Theoretically, all microorganisms should be eradi-
BC production. Although the authors did not declare any cated during this process, but some spores may survive,
inherent benefits using this specific material for BC yield which usually leads to an additional sterilization step.
and/or properties, the study definitely opens up possibili- NaOH concentration, temperature, treatment dura-
ties regarding complex 3D matrices as solid scaffolds for tion, and the number of treatments all influence the final
biofilm reactors.149 purity and inherent properties of the BC product. For
instance, treating BC with of 2.5% w/v NaOH, followed
by a treatment with 2.5% w/v sodium hypochlorite
4 | PURIFI CATI ON (NaOCl) at 80 C, resulted in a twofold increase in the
Young's modulus of BC compared to untreated BC.64 Fur-
4.1 | Impurities thermore, it has been observed that NaOH concentra-
tions higher than 6% w/v lead to the conversion of BC's
Native unpurified BC is known to contain an array of cellulose I polymer into cellulose II, causing a reduction
impurities, originating primarily from cellular debris of the in crystallinity and a decline in mechanical
bacteria involved in its synthesis. Impurities comprise strength.184,185
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 20 of 35

4.3 | LPS removal This means that often, custom setups are required to
achieve full BC depyrogenation. To summarize current
Endotoxins are a major component of the outer cell wall knowledge on the subject, Table 8 reports depyrogena-
in Gram-negative bacteria. The most common endotoxin tion methods (both from scientific literature and patents)
found from the outer bacterial membranes are lipopoly- to remove endotoxins in BC materials. The table also lists
saccharides (LPS). In general, the relative abundance of test details, results, as well as some concerns raised
LPS compared to total endotoxins makes it so that the regarding techniques that may be incomplete or less
terms “endotoxin” and “lipopolysaccharide” are often aligned with established literature. Some of those con-
used interchangeably. LPS are glycolipids that contain cerns are explained in the following paragraphs.
three main regions: a fatty region which consists of non- To regulate the quantity of LPS in medical devices,
polar acyl chains, called lipid A; a polysaccharide region the Food and Drug Administration (FDA) has established
and an antigen O which is made from repeating patterns guidelines specifying permissible limits, often reported in
of oligosaccharides. In particular, the lipid A is strongly endotoxin units (EU/mL). One EU/mL is approximately
agonistic and elicits strong immune reactions. These equivalent to 0.1–0.2 ng/mL of LPS, and medical devices
amphiphilic molecules can be found on most surfaces, on should not exceed 20 EU/device or 0.5 EU/mL. Common
the skin, in the gut, and on food (especially gram- fer- techniques for LPS detection include the Limulus amebo-
mented foods such as miso, kombucha, sauerkraut). LPS cyte lysate (LAL) test, TLR4 reporter cell assay, and the
do not usually pose a problem when ingested.186 How- rabbit pyrogen test.193,194
ever, when LPS are part of implantable medical devices, However, due to the 3D structure of BC, soluble test
they become highly problematic. Upon recognition by the assays cannot uniformly penetrate inside the membrane,
mTLR4 receptors of the innate immune system, LPS can making robust readings almost impossible. In addition,
provoke heavy immune reactions, even in low doses.187 LPS bind strongly to BC nanofibers, making their extrac-
In fact, doses lower than 1 ng/mL can elicit immune reac- tion quite challenging. To that extent, direct testing of
tions, leading to typical symptoms such as fever, diarrhea, native BC hydrogels for endotoxins is not possible. How-
and, in severe cases, anaphylactic shock and death.188 ever, solutions of BC, such as with cellulose nano crystals
It is important to remember that bacterial cellulose (CNCs), can be directly tested, albeit with the use of spe-
(BC) is produced by a Gram-negative bacterium called cial techniques such as ultrasonication.98 Indirect
Gluconacetobacter xylinus. Consequently, after fermenta- methods utilizing medium extraction are commonly used
tion, native BC contains high concentrations of endo- to evaluate LPS content in BC.
toxins. Despite this, native BC finds applications in As in 2023, only one extraction method is widely used
cosmetics, textiles, and foodstuff, as LPS generally do not to quantify LPS content in medical devices. The protocol
pose a significant problem unless they are implanted. is presented in the ISO 10993-12 (Sample Preparation
However, in the case of medical devices made from BC and Reference Materials) norm. The document specifies
or utilizing BC as a component, LPS removal becomes retrieval techniques of extractable components within
essential. The challenge lies in the strong adherence of medical devices. The norm does not specify what extract-
LPS to BC fibers,189,190 making their removal particularly able is targeted by the protocol, meaning it can be any-
difficult.178,188,191 For medical devices, various depyro- thing, up to reasonable extractability limits. The ISO
genation techniques, such as dry heat depyrogenation, 10993-12 states that for hydrogels (such as BC), 0.1 g of
organic solvent washing (e.g., butane), and the use of the medical device should be weighed and transferred in
ion-exchange chromatography columns, have been 1 mL of non-pyrogenic water at 37 C for 72 h. Samples
explored.192 However, these standard methods often face should be vigorously shaken throughout the incubation
challenges due to the complex nanofibrillar structure of period, but not as to damage the devices.195 While using
BC, as well as its sensitivity to acids and strong bases the ISO 10993-12 method facilitates testing, it does not
(higher than 1.0 M). guarantee complete extraction of BC endotoxin content.
One of the depyrogenation methods that have been They remain indirect tests, and with LPS strongly bonded
documented is using sodium hydroxide. BC is boiled in a to BC nanofibrils, it is likely that endotoxins do not fully
solution of caustic soda with concentrations usually rang- diffuse into the extraction medium. With unknown LPS
ing from 0.25 to 1.0 M. This treatment is typically fol- concentration in BC, it is likely that researchers testing
lowed by a series of rinses, sometimes involving non- BC biocompatibility in vivo may obtain contradictory
pyrogenic water, aimed at stabilizing pH and/or remov- results. For example, Pértile et al.116 and Gisela Helenius
ing endotoxins. However, concentration, time, and et al.112 conducted similar long-term biocompatibility
amount of sodium hydroxide and water rinses differ sig- studies on bacterial cellulose. Both also used similar puri-
nificantly across studies and patents, leading to a lack of fication techniques. However, Gisela Helenius et al. did
standardization in the approach. not report any inflammation, while Pértile et al. stated
 TABLE 8 Final endotoxin concentration in bacterial cellulose-derived materials given depyrogenation methods, detection techniques used and tested components.
21 of 35

Final
endotoxin
Product Depyrogenation methods Test used Tested component concentration Concerns Reference
CNCs in solution Multiple ultrasonication HEK mTLR4 CNCs solubilized at 10–100 Not fully Weiss et al.98
treatments and washes in reporter ug/mL specified < 5
non-pyrogenic water. EU/mL
Densified BC membranes Multiple supercritical CO2 LAL test Following ISO 10993-12 (76 h, 27 EU/mL Tested extraction media Pigaleva et al.197
treatments and washes in 37 C)
non-pyrogenic water.
Densified BC membranes Sodium hydroxide perfusion PyroGene Following ISO 10993-12 (76 h, 0.1 EU/mL Tested extraction media Héctor Martínez Ávila
system and non-pyrogenic recombinant 37 C) et al.118
water washes over 42 days. Factor C assay
Bilayered densified BC Sodium hydroxide perfusion PyroGene Following ISO 10993-12 (76 h, 0.15 EU/mL Tested extraction media Héctor Martínez Ávila
membranes system and non-pyrogenic recombinant 37 C) et al.110
water washes over 42 days. Factor C assay
Porous BC membranes Unspecified purification method LAL test Following FDA guidelines on <0.1 EU/mL • Unspecified purification Bodin et al.103
that was also used to remove validation of LAL test method for paraffin removal
paraffin microbeads, followed (1987) • Tested extraction media—
by autoclaving in non- Use of withdrawn FDA
pyrogenic water. Guidelines on Validation of
LAL Test198
Oxidized BC Sodium hydroxide and Not specified Not specified Not specified • Documentation for treatment Patent #CA2645764C199
hydrogen peroxide with aqueous hydrogen
treatments. peroxide scarce
• Unspecified tested
components
BC membranes with Sodium hydroxide and Not specified Not specified Not specified • Documentation for treatment Patent #CA2524184C200
polyhexamethylene hydrogen peroxide with aqueous hydrogen
biguanide treatments. peroxide scarce
• Unspecified tested
components
BC membranes Sodium hydroxide treatment. LAL test Not specified <0.06 EU/ml Patent #CA2536523C201
BC membranes Sodium hydroxide treatment. Not specified Not specified Not specified Unspecified tested component Patent #US8951551B2202
BC membranes Series of freeze-thawing. Not specified Not specified Not specified Unspecified tested component Patent
#AU2004266155B2203
BC membranes Alternating sodium hydroxide Not specified Not specified <8.84 EU/mL Unspecified tested component Patent
treatments, ethanol soaking, #CN113957738A204
and polysulfone resin polymer
adsorption.

Abbreviations: CNCs, cellulose nanocrystals; HEK, human embryonic kidney cells; LAL, limulus amebocyte lysate.
GIRARD ET AL.

10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 22 of 35

that implants caused a mild inflammatory reaction with bonds between every glucose monomers.93 This partial
a tendency to calcify over time. However, one direct test- degradation can have both advantages and disadvantages.
ing method has been documented in the patent On the positive side, in two 2017 studies, it led to
#EP4133278A1.196 In the filing, the inventors describe improved cell adhesion in vitro and biodegradability
the use of the cellulase enzyme to degrade BC nanofibers in vivo when implanted for guided bone regenera-
into glucose monomers. This allows for a full recovery of tion.93,105 This enhanced biodegradability may be advan-
LPS trapped inside the BC scaffolds and gives more accu- tageous for many more biomedical applications or
rate endotoxin quantification measurements. environmental uses in the future.
In summary, the utilization of depyrogenation tech- On the other hand, partial degradation of C-O-C
niques for BC and the quantification of LPS content in bonds in BC can result in a decrease in its mechanical
BC present certain drawbacks that warrant attention. It is strength and structural integrity. This may limit its use in
crucial to approach BC endotoxin removal and quantifi- applications where strength and durability are critical.
cation with utmost caution due to these limitations. Furthermore, the extent and control of the degradation
These considerations highlight the importance of imple- need to be carefully considered, as excessive degradation
menting rigorous protocols and methodologies to ensure can compromise the desired properties of BC.
accurate and reliable results with implantable medical
devices made from hydrogels.
5.3 | Other sterilization methods

5 | STERILIZATION Although less popular, some other methods of steriliza-

tion have been used, mainly in the medical field, to steril-
5.1 | Autoclave ize BC. For instance, a study showed that gamma
irradiation with a dose of 25 kGy was able to preserve BC
Autoclave is the most common way of sterilizing internal structure, with only a small decrease in thermal
BC. Bacterial cellulose is placed in an autoclave and is stability, while effectively getting rid of all microorgan-
subjected to high pressure steam at temperatures ranging isms inside.207
from 121 to 134 C for 20 min. This process effectively The use of dispersions of preservatives (antiseptic
kills all bacteria, fungi, and spores without altering BC compounds) with different hydrophilicities has also been
structure. This process is useful both for dry and hydrated investigated as a potential chemical method for steriliza-
BC. Limitations often occur when BC is coupled with a tion. In a recent study, dehydroacetic acid, methylene
heat-sensitive materials such as collagen, chitosan, or gel- bisthiocyanate, methyl parahydroxybenzoate (MPHB),
atin, for example. propyl parahydroxybenzoate (PPHB) and benzalkonium
chloride (BAC) have been mixed in various concentra-
tions with hydrated BC and placed in a growth medium
5.2 | E-beam to investigate if growth would occur. All samples exhib-
ited sterility for more than two weeks except in a specific
BC sterilization using electron beam (e-beam) irradiation mix of MPHB and PPHB.208 This finding might be useful
is a promising method for achieving high levels of micro- for shelf preservation of BC in food products. It is not
bial inactivation. E-beam sterilization involves subjecting expected that such a sterilization is usable in any medical
BC to a beam of high-energy electrons generated by an application, as it is risky and uses chemical compounds
electron accelerator.205 This beam penetrates the mate- that might interact with the body and cause inflamma-
rial, damaging the DNA of microorganisms and render- tion or necrosis around BC.
ing them unable to reproduce.206
One of the significant advantages of e-beam steriliza-
tion for BC is its ability to provide rapid and effective 6 | INDUSTRIES WITH INTEREST,
sterilization without the need for high temperatures. This A P P L I C A T I O N S A N D AV A I L A B L E
makes it suitable for heat-sensitive mixes of BC and com- PRO D U C T S
pounds, as it eliminates the risk of thermal degradation
or material alteration. E-beam sterilization also does not Industrial applications of BC vary greatly from field to
leave behind any chemical residues or by-products, mak- field. In this review, we identified four main categories
ing it particularly suitable for BC products that come into for which BC has been used: the medical industry, the
contact with sensitive biological systems or food. cosmetics industry, the textile industry, and the foodstuff
However, e-beam irradiation may cause partial degra- industry. An overview of these applications is available in
dation of the BC structure, particularly targeting C-O-C Figure 4 and the specifics are detailed below.
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
23 of 35 GIRARD ET AL.

FIGURE 4 Main uses of bacterial cellulose within various industries. [Color figure can be viewed at wileyonlinelibrary.com]

6.1 | Medical performing a craniectomy. It prevents additional cerebro-

spinal leakage while adding a protective layer. It is
In the last 20 years, BC has seen an exponential increase developed and commercialized by DePuy Synthes (Head-
in the number of publications regarding its use in medi- quarters in Raynham, USA), the orthopedic company of
cine. Applications targeted by current developments J&J.214 Additional information identified included a gen-
include wound dressing, cartilage tissue engineering, eral recall by the FDA initiated in 2015 due to the pack-
bone tissue engineering, soft tissue engineering, dental aging being misleading. Indeed, it was not clear for
implants, artificial blood vessels or vascular grafts, ure- surgeons that the product was designed to be left
thral implants, artificial cornea and retina, nerve implanted and not explanted.215 The packaging was cor-
implants, drug delivery and tympanic mem- rected, and the product was placed back into circulation.
branes.7,19,25,209-211 In 2023, this represents a microbial Membracel is a wound dressing designed for the treat-
cellulose market estimated at 570 million USD (with ment of burns, pressure injuries, diabetic wounds, surgi-
CAGR (compound annual growth rate) of 14.8%) only in cal wounds, ulcers, skin graft donor wounds and injuries
America, mainly associated to the medical industry.25 from epidermolysis bullosa. The dressing is said to con-
Thus, this review will focus on the currently commercial- tribute to the formation of granulation tissue through
ized BC in the medical industry and their accessibility. favorable gas exchange and absorption of the exudate. It
Nanoskin is a BC membrane containing silver ions as is developed and commercialized by Vuelo Pharma in
an antibacterial wound dressing. It is developed and com- Curitiba, Brazil. On their website,216 it seems to be only
mercialized by Innovatec in Sao Carlos, Brazil.19 available in Brazil.19,25,217-219
Suprasorb X and Suprasorb X + PHMB are BC wound KKF polymers is a company who was previously
dressings with possible drug-activated properties when named Jenpolymer as a start-up. The company was
incorporated with polyhexamethylene biguanide founded from a group of more than 60 Ph.D. from the
(PHMB). It is developed and commercialized by Loh- University of Jena, Germany. The company's expertise is
mann & Rauscher (Headquarters in Rengsdorf, Germany) focused on the development of products from synthetic
and is available online through the company and natural polymers. The company produces BC in the
website.25,212 forms of coatings, balls, membranes and especially tubes.
Nanoderm is also a wound dressing with antimicro- BC tubes are designed for vessels replacement in coro-
bial properties, due to silver particles integrated in the nary artery bypass and protected under the name
BC. It is developed and commercialized by Axcelon Bio- BASYC-Tech-Tubes.217,220,221
polymers Corp, located in London (Ontario, Canada).213 Located in Linköping, Sweden, S2M medical222 have
Synthecel Dura Repair is a BC membrane used by focused their expertise on wound healing. Epiprotect 2117
neurosurgeons to repair/seal the dura mater after and Epiprotect ulcer are their 2 wound dressings made of
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 24 of 35

BC.25 Dressings are used as a protective healing barrier Gengiflex, also linked to Fibrocel, is a dental implant
for wounds that can go as deep as breaching the dermis used to reduce inflammation and stimulate regrowth of
(notably burns and ulcers but also pre-surgical wounds). periodontal tissues. Studies evaluating Gengiflex (dental),
They worked as a scaffold for the regeneration, adhering BioFill (burn wounds) and BioProcess (ulcers) go as early
to the wound during the healing period. Once this period as 1997 and 1993.131,231
is complete, the BC dressing can be peeled off. In a clini-
cal study dealing with burnt wounds, Epiprotect was com-
pared to silver sulphadiazine cream (10 mg/g). The 6.2 | Cosmetics
results showed that Epiprotect dressing needed to be
changed only once before complete healing of the The global cosmetics market reached a value of US
wounds. Patients with the Epiprotect dressing spent 6.3 $262.21 billion in 2022 and is estimated to maintain a
fewer days in the hospital.223 In the case of a clinical CAGR of 4.2% until 2030.232 The emergence of new chal-
study regarding the efficiency of the dressing towards lenges in the cosmetic industry has spurred the develop-
ulcers (in 8 patients), the mean healing time was 43 ment of biobased cosmetics contributing to the market
± 6 days and the mean number of dressings used expansion.233
was 1.7 ± 0.2.224 BC has shown significant potential for the cosmetic
CelMat Face is a mask-shaped dressing for facial industry, particularly in addressing environmental con-
wounds and burn. There is also CelMat Wound Dressing, cerns related to the sourcing of non-degradable materials
intended for wounds in different anatomical location. As that bioaccumulate in nature after being used. BC is a
a wound dressing, BC is known to absorbs exudates, and sustainable and biodegradable material that is being
due to its high porosity, it maintains gas exchange accel- extensively studied as a viable alternative to common
erating regeneration.29,209 Those products are developed toxic polymers like polyacrylamide, nylon, and polyethyl-
and commercialized through BOWIL biotech225 located in ene, which are commonly used in the cosmetic indus-
Wladyslawowo, Poland. The company states to be the try.9,10,233 Furthermore, BC has the capability to replace
first GMP (Good Manufacturing Practices)-compliant BC other environmentally problematic substances in cos-
production plant for class IIb and III medical devices. metics, such as plastic microbeads and silicone, serving
Another company that is well present in review arti- as decorative or exfoliating agents and emulsifiers,
cles for BC used in the medical industry is a company respectively. The use of BC could potentially offer a solu-
named Xylos Corporation located in Langhorne, tion to those additional environmental issues in the near
United States.19,25,217,218,226 Their wound dressing XCell future.233
has been clinically studied regarding its ability to autoly- BC exhibits distinctive dermo-pharmacological prop-
tically remove non-viable tissue and create a healthy erties attributed to its unique morphology, characterized
wound bed.227 In a review by Ludwicka et al., the authors by its arrangement of nanofibers with a diameter smaller
mention XCell was purchased from Xylos Corporation by than 100 nm and a highly porous structure. This mor-
Lohmann & Rauscher and re-commercialized under the phology enables BC to deliver active ingredients or small
name Suprasorb.226 Another one of their products is molecules without interfering with their functionality.
called Securian. It was developed as a tissue reinforce- Notably, the presence of nanofibers in BC with a diame-
ment matrix.217 It was possible to identify a 510 ter inferior to 100 nm. This is even smaller than the pores
(k) premarket approval (dated 2009) for this product, in of human skin (250–500 μm234), suggesting the potential
which it was classified as a surgical mesh (device ID: for deeper penetration of various active ingredients.226
K083823).228 BC possesses several desirable properties, including high
BioCelltrix is said to be a surgical mesh made of BC mechanical strength, purity, water absorption, and reten-
developed and commercialized by BC Genesis tion. It also demonstrates high permeability to liquids
(USA).226 229 and gases, along with a lack of toxicity or cytotoxicity. BC
Finally, many other products have been described in exhibits excellent biocompatibility with the skin, is non-
other reviews, they include Dermafill, NexFill, BioProcess, allergenic, and adheres well to the skin. Furthermore, BC
BioFill and Gengiflex.230 Dermafill, NexFill, BioProcess and has the capability to replace synthetic petroleum-derived
BioFill seem to come from the same company Fibrocel and non-degradable cosmetic ingredients.9,10,233,235,236
Produtos Biotechnologies LTDA (Fibrocel, previously Bio- The unique properties of BC have garnered significant
fill Industrias226) and were just different names. Their interest in various cosmetic applications, particularly in
wound dressings are recommended for skin tear, diabetic the field of skincare. Our skin undergoes changes
wounds, second degrees burn, skin graft donor sites and throughout our lifetime due to various factors, such as a
traumatic lacerations.19,25,217,218 decrease in fibroblastic and collagen function, exposure
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
25 of 35 GIRARD ET AL.

to ultraviolet radiation, smoking, stress, and inadequate BC membranes were used to incorporate vitamin B as
hygiene practices. To counteract the effects of these fac- an active ingredient to test its potential as a delivery
tors on the skin, cosmetic solutions often focus on incor- matrix. The results indicated no toxicity, allowing con-
porating antioxidants and moisturizing agents, as well as cluding that BC matrices are safe for skincare use.9,10,241
other active ingredients.9 Additionally, BC was successfully tested with epilobium
A notable example of a skincare product that is gain- angustifolium extract, a natural antioxidant. BC exhibited
ing popularity is the face mask. Face masks are typically successful delivery of the antioxidant while maintaining
composed of materials such as cotton, non-woven fabrics, its non-cytotoxic properties.10
and poly-vinyl alcohol.233 Among these materials, BC has Lastly, BC used as a face mask showed no irrita-
emerged as a biobased replacement material in the cos- tion.10,239 In a 2014 clinical study by Almeida et al., the
metic industry. Face masks serve as a delivery system for skin irritation, and moisturizing effects of BC membranes
various active ingredients known for their benefits in with and without glycerin were tested. Results demon-
skincare, such as anti-aging effects, lifting and firming strated minimal-to-no immune reaction, and the addition
properties, cell renewal stimulation, hydration, wrinkle of glycerin increased the moisturizing effect, which is
reduction, reduction of brown spots, evening out skin particularly relevant for treating dry skin conditions.9,242
tone, exfoliation, support for the skin's collagen and elas- BC presents another potential application in the cos-
tin matrix, reduction of fine lines, and enhancement of metic industry as an emulsion stabilizer, thickener, and
light reflection, among others.226,237 texture modifier. It can effectively serve as an emulsion
Face masks can be formulated with or without active stabilizer for water and oil emulsions without the need
ingredients. In the case of BC face masks without for additional surfactants. Additionally, BC rheological
active ingredients, they have demonstrated the ability to and structural properties make it suitable for use as a
enhance skin hydration.9,233,235-240 Importantly, the water thickener, stabilizer, and texture modifier.9,233,235
transferred to the skin from BC masks does not evaporate Studies have explored the use of BC as an oil-in-water
as quickly as free water, leading to prolonged hydra- emulsion, demonstrating its compatibility with sensitive
tion.236 Notably, when monitoring various skin parame- skin and lack of harm.9,235 Furthermore, research has
ters such as dullness, texture, elasticity, sebum content, shown that a dry formulation containing BC and carbox-
moisture content, and desquamation levels, no other ymethyl cellulose at a concentration of 0.50% was capable
effects on the skin were observed solely from the use of of stabilizing the oil–water emulsion for up to 90 days.233
BC face masks.235 BC has also garnered significant interest for its poten-
Moreover, BC face masks can serve as an effective tial use in scrubbing and exfoliating applications, mainly
delivery matrix for hydrophilic and small molecular due to its gentle effect on the skin. This is attributed to
active ingredients.9,236 Numerous studies have documen- BC soft, hydrated texture and its small fiber size.233,235
ted this behavior. For instance, a clinical study compared For instance, a facial scrub formulation incorporated
a BC placebo face mask with three BC face masks con- powdered BC as the primary ingredient, along with olive
taining different active ingredient formulations (anti- oil, ascorbic acid, aloe vera extract, and powdered gluti-
aging, lifting, and cell renewal) over a period of 4– nous rice.242,243 When compared to a commercial scrub-
8 weeks. The results demonstrated that BC masks were ber, the BC scrubber exhibited higher viscosity at lower
well tolerated with no irritation. The anti-aging formula- shear rates and comparable viscosity at higher shear
tion showed a reduction in skin roughness and wrinkles, rates. This characteristic ensures that the BC scrubber is
along with improved dermal homogeneity and firmness safe for the skin without the inclusion of non-degradable
after 2 months. The lifting formulation improved skin ingredients.
firmness and elasticity after 1 month. The cell renewal Enzymes have long been used in the cosmetic indus-
formulation facilitated the production of new skin cells try, particularly for skin peeling, smoothing (such as bro-
through its exfoliating action.237 melain and papain), and anti-aging purposes. However, a
Another clinical study examined BC face masks with current challenge faced is the limited stability of enzymes
formulations containing vegetable extract and propolis at room temperature, mainly due to the surfactants and
extract. It revealed that the vegetable extract formulation oils used for their preservation. BC presents a potential
improved skin moisture content, while the propolis solution for immobilizing and stabilizing these enzymes,
extract formulation led to decreased skin hydration. Both although further research is needed in this area.233
results were as expected, highlighting BC efficiency in Plastic microbeads are commonly used as decorative
delivering active ingredients without compromising their or exfoliating agents in various daily hygiene and cos-
intended effects due to degradation or native BC metic products, including shampoos, toothpastes, scrubs,
properties.239 and soaps. However, there is growing concern about the
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 26 of 35

environmental impact of these microbeads. Joonwon, BC for cosmetic applications, it is anticipated that the uti-
P. has patented (WO patent NO: WO2019004520A1) a lization of BC in this market will experience a significant
method for producing BC powder with BC particles that rise. However, the current scarcity of commercial prod-
have the potential to replace microbeads. These BC parti- ucts, their limited application primarily focused on face
cles possess a small size, low residual water content masks, and the limited scientific literature available still
(15%), and are unable to absorb water, thereby maintain- necessitate further extensive research in this field to dem-
ing their granularity.233 onstrate clear beneficial effects accepted by specialists.
When compared to the medical industry, the process
of bringing a cosmetics application to the market requires
significantly less research and certification. This leads to 6.3 | Textile
faster and more cost-effective development of cosmetic
applications compared to medical applications. As a As mentioned before, BC possesses remarkable mechani-
result, several companies have successfully introduced cal properties making it suitable for a wide range of
BC face masks to the market. Table 9 lists the current applications. Particularly, the textile industry has drawn
state of BC commercialization in the cosmetic considerable attention from researchers. BC, when dried,
industry.226 demonstrates high tensile strength, and forms a non-
Considering the exponential growth of the research woven or nano-spun fabric, making it moldable and bio-
conducted on the subject and the favorable properties of degradable. Additionally, BC remains remarkably thin,

TABLE 9 List of available facial mask products made from bacterial cellulose for cosmetic purposes.

Cost (if
Product name Producing company Reported Particularities found)
DHC bio cellulose mask DHC (USA—Japan)244 Hydrates, soothes, and brightens with 10 $
neem leaves, shell ginger and vitamin
C.
Fresh eyes rejuvenating eye masks (Box Bel Mondo (USA)245 Reduce swelling and puffiness. 35 $
of 6) Transfers niacinamide, alpha arbutin, 60 $
Brightening facial sheet masks (Box of ascorbyl palmitate and plant extracts 60 $
4) for skin tone.
Anti-aging facial sheet mask (Box of 4) Hydrates while transferring peptides and
antioxidants.
SUPERSTART probiotic boost skin Elizabeth Arden (USA)246 Skin regeneration with sodium 67 $
renewal biocellulose mask hyaluronate and glycerin.
Giorgio Armani luminessence bright Giorgio Armani (USA)247 Hydrates and brighten skin. 175 $
infusion bio-cellulose mask (Box of 6) Dimethicone, glycerin, beech tree
extract, licorice root extract and
salicylic acid.
Y theorem bio cellulose facial mask 111SKIN (UK)248 Rejuvenate, strengthen, and hydrate the 185 $
(Box of 5) skin. 215 $
Anti-Blemish bio cellulose facial mask Combats blemishes and inflammation.
(Box of 5)
Bio-cellulose sculpting masque (Box of Luzern (Switzerland/USA)249 Increase hydration and firmness while 150 $
5) diminishing lines and wrinkles.
Blanc expert second skin whitening Lancome Focused on hydration and whitening 133 $
bio-cellulose mask (Box of 6) (Singapore–Japan–France)250 with brown algae extract and vitamin
C.
Moisturizing—soothing biocellulose Tech nature (France)251 Fermented coconut water. Contains -
mask hyaluronic acid, fucogel and aloe vera.
Healthy glow anti-pollution
biocellulose mask
Eye enhancers biocellulose patches
Energizing anti-aging biocellulose
mask
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
27 of 35 GIRARD ET AL.

positioning it as a promising candidate for lightweight ModernSynthesis specializes in the development of

leather alternatives. Nonetheless, several factors influ- nonwoven BC textiles reinforced with other natural tex-
ence the usability of BC in the fashion and textile sectors. tiles. The company proposes that their material has the
Notably, challenges such as wettability/hydrophobicity, potential to replace various textiles, fabrics, and leathers.
wear resistance, elasticity, breathability, dyability, and The BC used by ModernSynthesis is derived from agricul-
overall comfort while wearing the product are crucial in tural waste media and fermented using Komagataeibacter
determining the viability of BC adoption.18,252 rhaeticus. ModernSynthesis secured a seed funding of
Previous studies have explored the potential of BC as $4.1 million in April 2022.269
an ecological substitute for leather. Initial investigations Polybion manufactures BC leather known as Celium.
involving the simple drying of BC revealed that the In 2022, the company raised $4.4 million in a Series A
resulting textile became brittle, lost breathability within a financing round to construct the world's largest bacterial
few weeks, and exhibited inadequate hydrophobicity. cellulose biomanufacturing facility. Notably, from Janu-
However, BC retained its promise as a material for textile ary 2023 to July 2023, the Danish fashion brand Ganni
applications.253-257 showcased a leather blazer made entirely from Celium at
Subsequent research focused on overcoming the limi- their headquarters flagship store in Copenhagen. Poly-
tations of basic BC fabric, leading to the development of bion seems to operate with two divisions—one in Mexico
improved products. For example, entrapping BC with and one in Spain.270
50% mushroom proteins and incorporating glycerol
yielded a fabric exhibiting superior water resistance, ten-
sile strength, flexibility, and crease recovery compared to 6.4 | Food ingredients
cowhide leather.258 In 2019, Kami nski et al. discovered
that BC treated with glycerol demonstrated enhanced BC finds numerous uses in the food industry due to its
resistance to abrasion and improved elongation at unique properties and versatility. As BC is primarily com-
break.259 This finding was further supported by a similar posed of cellulose, it is generally recognized as safe
study conducted by Harmon et al., in 2020.260 Other (GRAS) by the Food and Drug Administration (FDA),
research groups explored the use of emulsified acrylated ensuring its suitability for a wide range of food applica-
epoxidized soybean oil (AESO) with polyethylene glycol tions.11,271,272 The most popular use of BC in the food sec-
(PEG), polydimethylsiloxane (PDMS), and perfluoro- tor is in the form of nata de coco, a desert originating
carbon-based polymers to enhance the structural integ- from the Philippines. Coconut water is fermented using
rity of BC leather. Although the overall tensile strength Acetobacter xylinum bacteria and forms a 1 cm-thick film
diminished, the elongation at break increased five-fold, of BC which is then infused with more fresh coconut
and hydrophobicity improved.261 Impregnating BC solely water. The nata de coco BC is then cut into 1 cm  1 cm
with PDMS and perfluorocarbons also resulted in  1 cm cubes and eaten with agave or cane syrup. While
improved mechanical properties compared to simple BC nata de coco is more popular in eastern countries, it has
fabrics.262 Additional strategies investigated to enhance slowly made its way in Europe and the Americas where
the chemical and physical properties of BC fabrics it's chewiness and tangy flavor have become greatly
include incorporating lauryl gallate oligomers,263 cou- appreciated in fruit salads. Although BC can be eaten by
pling BC with electro-spun cellulose,264 and bleaching.265 itself as an exotic desert, its uses become exponential
Another crucial parameter often overlooked in novel when applied to food ingredients and technologies. For
textiles is their dyeability. Given BC composition, numer- example, BC can serve as a thickening and stabilizing
ous viable methods exist for dyeing the polymer. Some agent,273 enhancing the texture and mouthfeel of sauces,
potential candidates for dyeing BC includes immobilized dressings, and desserts. Its emulsifying properties could
laccase266 and in situ chemical dyes such as acid blue make it useful in creating smooth and homogeneous mix-
62, reactive blue 19, and direct blue 78.267 tures in products like slow-melting ice creams,274 thick
Several companies are currently involved in the dressings,275 creams,276 and high-humidity doughs.277
development and sale of textiles made from Moreover, BC acts as a dietary fiber, adding bulk to the
BC. Nanollose Ltd produces Nularbor, a brand encom- diet, promoting digestive health, and aiding in regular
passing fiber, yarn, fabric, and garments. Nularbor fabric bowel movements.278 Its fibrous nature also contributes
stands out as one of the rare woven BC textiles, offering a to increased satiety,275 making it a potentially beneficial
regular lightweight fabric option that mitigates most of ingredient for weight management.279 BC can encapsu-
the drawbacks associated with nonwoven BC textiles. late bioactive compounds, such as antioxidants, vitamins,
Nanollose, headquartered in Perth, Australia, seems fully and flavors, protecting them from degradation and
operational as of 2023.268 improving their stability in food products.35,271,280
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
GIRARD ET AL. 28 of 35

Additionally, BC can be formed into edible films and 7 | CONCLUSIONS

coatings, acting as a barrier to preserve the freshness and
extend the shelf life of perishable products like fruits, This review article presented a comprehensive overview
vegetables, and meats.281,282 The versatility of BC also of bacterial cellulose (BC), highlighting its physicochemi-
makes it suitable for the development of health and func- cal properties, production methods, purification tech-
tional foods, allowing for the incorporation of specific niques, and diverse applications in various industries.
health benefits such as cholesterol reduction, prebiotic Building upon the existing knowledge, we can identify
effects, and blood glucose regulation.283-285 several promising areas for future research and
development.
Firstly, the intrinsic properties of BC, such as its bio-
6.5 | Scaffolds in cell culture compatibility and low cytotoxicity, make it an ideal can-
didate for various medical applications. However, further
Scaffolding materials have gained significant attention advancements are necessary to bridge the gap between
for their ability to sustain 3D cell cultures. Within this preclinical studies and human trials. The refinement of
context, BC has attracted considerable interest as a scaf- production methods, purification techniques, and optimi-
folding material, particularly within the medical industry. zation of culture parameters are crucial steps towards
While this article has already discussed the medical sec- achieving safe and effective BC-based therapies for
tor's keenness towards BC, let us reiterate that BC scaf- human use.
folds can facilitate three-dimensional colonization and Furthermore, the food industry holds great promise
penetration of cells within its structure. This is achieved for the future of BC. While current applications are pri-
by providing nanoscale anchor points, which help cells marily limited to nata de coco, the unique properties of
migrating into the scaffold. This makes it suitable for BC, such as its high water-holding capacity and biode-
many specific applications in regenerative medicine. gradability, make it an attractive ingredient for various
However, it is worth noting that current BC products for food products such as thick dressings, slow-melting ice
healthcare are not marketed explicitly as general scaffold- creams, and dietary fiber. Further research and develop-
ing materials, as their intended purpose is primarily ment in this area can unlock new opportunities for incor-
focused on targeted medical applications. porating BC into functional foods, packaging materials,
The commercialization of BC as a pure scaffolding and encapsulated bioactive compounds. For now,
material remains uncommon across various industries. improvements on BC yields and scale up are the main
One sector where BC remains suitable as a pure scaffold- limiting factors hindering its use in such a large industry.
ing material is the cultivated meat industry. BC has been Scaffolds, both in the medical field and the emerging
recognized as an excellent scaffold, promoting cell attach- cultivated meat industry, represent another area of
ment and proliferation. Numerous articles have immense promise. BC biocompatibility, porous structure,
highlighted BC potential as a scaffolding material for and mechanical strength make BC an excellent candidate
diverse cell types, including fibroblasts, human mesen- for tissue engineering and regenerative medicine applica-
chymal stem cells (MSCs), chondrocytes, and osteo- tions. Additionally, BC-based scaffolds have shown
blasts.286-293 Recent studies have also explored BC's potential in the cultivation of meat, offering a sustainable
applicability as a scaffold for cultivated meat. For exam- and environmentally friendly alternative to traditional
ple, Rybchyn et al. demonstrated that BC provided favor- animal agriculture. Future research should focus on
able surface properties for the formation of anchor points refining scaffold design, enhancing biodegradability, and
necessary for the development of mature myotubes.294 investigating the compatibility of BC scaffolds with differ-
Thus, bacterial cellulose could enable the growth of mus- ent cell types for various applications.
cle tissue. Regarding BC production, one crucial aspect requir-
BNC Scaffold is a division of Cass Materials Pty Ltd, ing attention is the improvement of lipopolysaccharide
an Australian company headquartered in Perth. They (LPS) removal during the purification process. LPS is a
produce BC in the form of dried sheets, flocks, and potent endotoxin present in BC, and its presence can
fleeces, which could be suitable for cultivating animal adversely affect the biocompatibility and safety of BC-
cells in bioreactors. The company continues its opera- based products. In this review, we addressed some of the
tions and operates a branch in Örnsköldsvik, Sweden.295 challenges towards endotoxin removal within BC prod-
AxCell Labs, a biotechnology company based in Que- ucts. However, we still believe that standardization of
bec, Canada, specializes in BC scaffolds for protecting LPS removal protocols is a crucial step in allowing the
subcutaneous implants. While primarily focused on the propulsion of effective and safe medical devices.
medical sector, the company has also confirmed the effi- Finally, scaling up BC production in specialized bio-
ciency of their BC for in vitro scaffolding needs.296 reactors is essential for meeting the growing demand
 10974628, 2024, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/app.55163 by Nat Prov Indonesia, Wiley Online Library on [03/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
29 of 35 GIRARD ET AL.

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35 of 35 GIRARD ET AL.

AUTHOR BIOGRAPHIES
revolutionize the field of medicine. His work aims to
uncover new possibilities for medical devices that
Vincent-Daniel Girard, B.ing, have yet to be considered.
Vincent-Daniel Girard completed
his undergraduate degree in bio- Patrick Vermette, ing., M.Sc.A.,
technological engineering in 2020 at PhD, Full Professor, Faculty of
the Université de Sherbrooke, Sher- Engineering, Université de Sher-
brooke, QC, Canada. He is doing brooke. He was trained first as
his master's degree in chemical chemical engineer but, rapidly he
engineering in Patrick Vermette's has acquired a recognized expertise
group, which is oriented towards on the development and validation
biotechnological engineering and tissue engineering. of various biomaterials, technolo-
Vincent-Daniel is interested in agri-food biotechnol- gies, and medical devices covering
ogy and has experience in bacterial, yeast and fungal both medical needs and regenerative medicine. He is
fermentation. Over time, he expanded his repertoire involved in the development of complex techniques
to include mammalian cell culture and experimental for both mammalian and microbial cell cultures. In
protocols on live models. Since 2019, Vincent-Daniel addition, for more than 20 years, Patrick has
has been truly passionate about bacterial cellulose designed numerous bioreactors and unit operations
(for medical applications), which has been his pri- linked to various types of biomanufacturing
mary field of research. approaches. Patrick is also an entrepreneur and is
coaching others to derisk their technology to add
Jérémie Chaussé, B.ing, Jérémie value.
Chaussé completed his undergradu-
ate degree in biotechnological engi-
neering in 2020 at the Université de
Sherbrooke, located in Sherbrooke, SU PP O R TI N G I N F O RMA TI O N
Quebec, Canada. He is currently Additional supporting information can be found online
pursuing a master's degree in chem- in the Supporting Information section at the end of this
ical engineering as part of Patrick article.
Vermette's research group, which
specializes in the fields of biotechnological engineer-
ing and tissue engineering. Jérémie's research is cen- How to cite this article: V.-D. Girard, J. Chaussé,
tered around exploring the natural physicochemical P. Vermette, J. Appl. Polym. Sci. 2024, 141(15),
properties of bacterial cellulose, with a specific focus e55163. https://doi.org/10.1002/app.55163
on how biomaterials have the potential to