CHAPTER II

REVIEW OF RELATED LITERATURE

This chapter presents the conceptual and related literature, both local and foreign that

is fundamental for better understanding of the study.

CONCEPTUAL LITERATURE

Bacterial Nanocellulose (BNC)

Cellulose, a naturally occurring polymer, is produced by almost all groups of living

organisms and is considered to be the most abundant biomacromolecule in nature.

Chemically, it is a homopolymer composed of β-(1,4) glucose. It is also known as the main

component of cell wall in plants. It contributes to the cell’s growth and mechanical strength

of the cell wall of the cells of the organism. Cellulose has microfibrils that are composed of

amorphous and crystalline region. These microfibrils are formed from strands of

nanocellulose. Nanocellulose is known for its non-toxicity, low density and high

biodegradability.

Studies on the properties and applications of nanocellulose are mostly focused on

plants since they are the main producers of cellulose. At the same time, another significant

knowledge has found with the study of bacteria. Cellulose produced by bacteria is the purest

form of cellulose. Bacterial strains belonging to the genera Acetobacter, Agrobacterium,

Rhizobium, and Sarcina can be used to produce cellulose. Non-wood nanocellulose like fruit

wastes can be obtained by environmentally friendly fermentation process using

Komagataeibacter xylinus bacteria from genera Acetobacter. The nanocellulose produced is

usually called bacterial nanocellulose (BNC).

1. History

2. Bacterial nanocellulose (BNC) is a linear polysaccharide

3. composed of b-
4. D-glucopyranose monomers linked by
5. b-1,4-glycosidic linkages. The repeating unit is the
6. disaccharide cellobiose (McNamara et al., 2015). Bacte-
7. rial nanocellulose was first described in 1886 by A. J.
8. Bacterial nanocellulose (BNC) is a linear polysaccharide
9. composed of b-
10. D-glucopyranose monomers linked by
11. b-1,4-glycosidic linkages. The repeating unit is the
12. disaccharide cellobiose (McNamara et al., 2015). Bacte-
13. rial nanocellulose was first described in 1886 by A. J.
The production of cellulose by Acetobacter xylinum cells in the presence of glucose

and oxygen was observed by A.J. Brown who first described the bacterial nanocellulose in

1886 (Brown,1886). BNC is a linear polysaccharide that is composed b-D-glucopyranose

monomers linked by β-(1,4)-glycosidic linkages. Disaccharide cellobiose is the repeating unit

(McNamara et al., 2015). From Acetobacter, the classification of acetic acid bacteria has

changed and reclassified as Gluconacetobacter, which has recently further moved to a new

type of Komagataeibacter. The current generic name, Komagataeibacter, comes from the

Japanese microbiologist’s name, Kazuo Komagata. This was proposed by Yuzo Yamaba in

2012 (Yamada et al., 2012).

2. Structure

Bacterial nanocellulose (BNC) has properties and structural elements of a well-known

plant cellulose with special features of nanoscale materials. The molecular formula of BNC is

shown in Figure 1 which has a repeated connection of dextrose or D-glucose building blocks.

The remarkably functionalized, linear rigid chain homopolymer-polymeric dextrose-of the

hydroxyl group is characterized by large chemical modification ability, high hydrophilicity

and sufficient biocompatibility. Initiated by the high donor reactivity of the OH groups, the
molecular structure imparts cellulose with its usual properties such as degradability, chirality,

and broad chemical changeability (Helenius, G. et al., and Czaja, W. et al).

It has ordered and disordered regions in which the ordered domains are mostly

crystalline. On the other hand, in a very fine fibril or fiber-like strand called microfibril, the

disordered molecules maintain a privileged path parallel to the chains and form surface

disorder on the microfibrils. Large networks of hydrogen bonding deliver a plurality of

partially crystalline fiber structures and morphologies to cellulose (Lanyon Y.H. et al.).

Figure 1. Molecular Structure of Cellulose (n= degree of polymerization) (Stanisławska,

A., 2016)

Scanning Electron Microscopy has often been used to characterize BNC and bacterial

cellulose nanocomposite’s morphology and microstructure. BNC's back-scattered electron

imaging allows the structure of the membrane network, porosity determination, morphology,

and dimension of fibers to be defined. Figure 2 displays BNC's SEM photograph of a

coherent 3D cellulose fibre network. Various functional nanocomposites are obtained after

chemical modification of bacterial cellulose that leads to the attachment of small molecules,

inorganic nanoparticles or nanowires and other polymers. The BNC composition is most

frequently modified by the combination of different chemical components. The composition

and homogeneity of the bacterial nanocellulose content, the presence of the embedded

product, and the potential orientation of the cellulose crystals are determined by SEM (Sabu,

T. et al, 2012).
Figure 2. SEM Micrograph of a Bacterial Nanocellulose Sample (Kondo, T. et al., 2016)

In the study of Nogi and Tano, (2009) and Klemm D. et al., (2006), the structure of

the nanofibers of BNC is around 100 nm in diameter and 100 µm in length. These are made

up of packs of cellulose microfibrils which has a diameter of 2 to 4 nm. BNC is a water-

swollen network of cellulose nanofiber. A native BNC network is shown in the SEM image

from Figure 3. Grande et al. (2008) measured the morphological properties of dried BNC

networks using image analysis. The segments and the x-axis of the nanofibers formed an

average angle of 85.64 ± 0.56° while the average distance between junction points is 0.523 ±

0.273 µm.
 Figure 3. SEM micrograph of a bacterial cellulose sample showing a coherent 3D

network formed by cellulose fibers (Stanisławska, A., 2016)

3. Difference between bacterial nanocellulose and plant cellulose

Cellulose is frequently isolated from plants as the main reinforcing component of their

cell walls, which is generally used as raw materials for paper and fibres industries (Moon et

al., 2011). Cotton linens and wood pulp are the most common commercial sources of

cellulose. However, cellulose obtained from plant sources is still contaminated with non-

cellulosic materials/polysaccharides, such as hemicellulose and lignin (Abeer et al., 2014).

The removal of such materials can be achieved using chemical and mechanical processes,

which may end up weakening the structure and other important properties. This process is

therefore, environmentally not suitable for cellulose production (Jozala et al., 2016). Thus, it

is necessary to find other alternative sources for obtaining high purity cellulose (Lestari et al.,

2014a). With the new development and the emergence of nanotechnology, cellulose has

attracted more attention in the new form of ‘‘nanocellulose’’ to be used as a novel and

advanced material in modern biotechnology (Islam et al., 2017).

Bacterial nanocellulose (BNC), a pure form of cellulose, devoid of pectin, lignin and

hemicelluloses or other compounds present in plant pulp possesses a high degree of purity,

crystallinity index, and biocompatibility. The ultra-fine structure of the BNC possesses higher

level of surface area, higher mechanical properties, higher degree of polymerization, high

merits of crystallinity, high elasticity, and high porosity and water content. These properties

of BNC make it a superior choice to the cellulose that are plant-based. Moreover, the BNC is

highly superior when it comes to readiness for modification compared to plant cellulose.
During the fermentation period, BNC could be shaped to devise tubes, spheres, layers of thin

fibers or membranes according to the needs of the functional materials for numerous

applications with one-step synthesis (Khan et al.,2016).

The abundance of hydroxyl groups (multi-hydroxyl molecular structure) in the BNC

facilitates its functionalization with other reinforcing compounds that leads to BNC’s new

properties such as electro-conductivity, anti-microbial activity, and multi-functional BNC

composites. BNC can also be applied in different sectors including biomedical,

bioprocessing, and pharmaceutical applications, electro-conductive materials, food industry,

packaging and wastewater treatment. In addition, BNC-based materials that are

functionalized are utilized in numerous fields, including oil adsorption, biomedical materials,

anti-counterfeiting labels, bioimaging, UV screening, chemical sensing and fuel cells.

The productivity of cellulose from plants and microorganisms was compared in the

study of Donini et al. (2010). In their study, they compared the cellulose production from 1

ha of eucalyptus with an MAI or mean annual increment of 50 m3 with a basic density of 500

kg/m3. An MAI of 25 t/ha/year was generated. They came up with the process of planting to

cultivation for seven years. This yield for about 45 % cellulose contents. The process yields

about 80 t of cellulose/ha after cultivation (7 years). The researchers found out that with

bacteria, same production could be achieved to a hypothetical yield of 15 g/L in 50 h of

culture with an average of 0.3 g/h in a 500 m3 bioreactor in approximately 22 days. This

production method is more efficient since it yielded pure and ecologically sustainable product

which is bacterial nanocellulose.

4. Properties

4.1. Thickness
 The shape and thickness of the resulting nanocellulose hydrogel can be controlled by

selecting appropriate synthesis conditions. The first of these is the type of bacterial culture. In

1989 Williams and Cannon discovered a change in thickness of the flat BNC by the use of

different Gluconacetobacter strains. Further control of the thickness and size of BNC can be

achieved by variation of the volume of the culture medium (Udhardt, 2004), the cultivation

time (Borzani and De Souza 1995), and the cultivation vessel (Levy et al. 2004; Tamura et al.

1999). Table 1 illustrates the selected BNC-producing bacterial strains and the desired culture

for each condition.

Table 1
Selected bacterial strains BNC producing and the conditions needed for the desired
culture
Microorganism Carbon Additive Cultivation Efficiency (g/l)
source time
A.Xylinum BRCS glucose ethanol, oxygen 50 h 15.30
Acetobacter sp. V6 glucose ethanol 8d 4.16
Acetobacter sp. A9 glucose ethanol 8d 15.20
Gluconoacetobacter glucose lignosulphonate 7d 10.10
xylinus
IFO 13773
Lactobacillus mali saccharose - 72 h 4.20
JCM1116
Acetobacter xylinum glucose sodium alginate 5d 6.0
NUST4.1
A.xylinum BPR2001 fructose agar 56 h 12.00
(Rama K.V and Singh L.)

4.2. Porosity

Bacterial nanocellulose membranes are promising three-dimensional nanostructured

matrices and have been used for innumerable applications because of their unique green

nature along with sustainable and cost-efficient production methods. However, the tight pore

structures present in the native membrane restrict their practical applications. Therefore, to

tailor the pore size, shape and volume of original nanocellulose membranes, different types of

carbon sources in the growth culture were applied with all other conditions being constant.
 The experiments of Ashrafi, Z. et al (2019) were able to demonstrate that feeding the

bacteria with different types of carbon sources under static culture conditions alters the

morphology of the bacterial nanocellulose membrane at both macro- and micro-scales. The

carbon sources used in the experiment are as follows:

Table 2
Brunauer, Emmett, Teller (BET) surface area, pore volume, and pore size of the BC
membrane structures cultivated with five different carbon sources
Carbon source BET Surface Area Pore Volume Average Pore
(m2g-1) (cm3g-1) Diameter (Å)
Mannitol 32 ± 0.35 0.12 82
Fructose 65 ± 0.45 0.25 82
Sucrose 31 ± 0.16 0.15 85
Glycerol 52 ± 0.52 0.23 88
Glucose 22 ± 0.48 0.11 85
(Ashrafi, Z. et al., 2019)

Among five different types of applied carbon sources, glycerol- and fructose-fed

samples result in the most porous structure with the highest pore surface area. The higher

inner surface area is a critical feature of the bacterial nanocellulose membranes which adjusts

many fundamental properties such as mechanical and thermal stability, fluid permeability,

pliability and compatibility, sorption properties and even the lifecycle of materials. This work

offers a simple effective method to induce porosity in the native BC membrane without the

need for chemically or mechanically destroying the original unique structure and crystallinity

of these natural masterpieces.

4.3. Surface Morphology

The macroscopic morphology of bacterial cellulose relies solely on the conditions of

the culture. Bacteria accumulate cellulose mats on the surface of the nutrient broth, at the

oxygen-rich air-liquid interface, under static conditions (S-BC). At the surface of the bacterial

cell, the cellulose sub fibrils are continually extruded from linearly ordered pores, crystallized
into microfibrils and forced bons, creating parallel but disorganized planes. The adjacent S-

BC strands branch and interconnect less frequently than these in BC produced in agitated

culture (A-BC), in a form of irregular granules, stellate and fibrous strands, well-dispersed in

culture broth. The strands of reticulated A-BC interconnect to form a gridlock pattern, and

have both roughly perpendicular and roughly parallel orientations (Bielecki et al., 2005).

5. Applications

The properties of bacterial nanocellulose such as high degree of crystallinity, high

purity, high density, high water-binding capacity, good shape retention, and larger surface

area may have numerous applications in different areas including paper, textile industry,

waste water, nonwoven cloth, pharmaceuticals, mining, refineries and food (Wu and Liu

2012; Ashjaran et al., 2013; Lin and Dufresne, 2014).

The applications of the bacterial nanocellulose are summarized in the following

categories:

6.1. Food Applications

Since BNC is a pure form of cellulose, it can be used as stabilizing and thickening

agent in processed foods and for the promotion of water binding and gelling agent. In

addition, BNC is a type of dietary fiber and has been classified as a "generally recognized as

safe" or GRAS food, approved by the US Food and Drug Administration (FDA) in 1992 (Shi

et al. 2014).

In the food industry, the traditional dessert, "Nata" was the first bacterial cellulose

product prepared from Acetobacter xylinum. Due to its distinctly soft texture and high fibre

content, it is also commonly used in food processing (Chawla et al., 2008).

 Bacterial nanocellulose has also attracted interest in the food industry as a material for

active packaging, which is defined as packaging able to perform roles other than that of a

barrier to the outside environment. This is mostly due to BNC’s edibility and potential as a

good carrier for a wide range of antimicrobial agents (Cagri et al.). This potential has been

exploited by Nguyen et al., who developed BNC films incorporating nisin as a proof-of-

concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of

vacuum-packaged frankfurters. For that purpose, BNC films were soaked in a nisin solution

under static conditions, followed by drying at 50°C. Nisin-containing BNC films showed

effectiveness in controlling L. monocytogenes and reducing total plate counts on the surface

of frankfurters, thus opening interesting prospects as a new method to improve the safety and

extend the shelf life of processed meats.

6.2. Medicine

The valuable qualities of BNC have contributed to the growth of medicine in the

world. BNC has been found to have no cytotoxic or genotoxic properties; therefore, BNC

materials are popular in medication. The most important BNC-rare applications:

BNC connection with collagen contributes to a more selective reduction of proteases

and interleukins, resulting in the antioxidant activity; examples of bioactive implants:

cartilage for example: septum of the nose, ear, intervertebral discs.The natural BNC has

mechanical properties, including tear resistivity and shape retention, which are better than

many artificial materials. In collation with organic layers, like polypropylene or cellophane,

BNC processed into a film or sheet show excellent mechanical strength. These properties

helped in creating tube-like prototypes of blood vessels with a length of 5-25 cm or longer.

BNC in a form of patches or large lobes are used as dressing materials that allows

wounds to heal, breathe and prevent the formation of scabs and scars and treat skin burns
(Shah, J. and Brown M.R.). Company Biofill® produce BNC for wide applications in surgery

and dental implants. Second and third degree of burns, ulcers and others cases are

successfully treated by Biofill® which is temporary substitute for human skin. Advantages of

Biofill® are: prompt pain relief, close adhesion to the wound, faster healing, reduce post-

surgery trouble, revised exudates retention, facility of wound control (transparency), reduced

infection rate, reduced costs and time of treatment. limited elasticity in areas of great mobility

is the only one disadvantage of BNC (Ślęzak A., et al. 2016).

As indicated in the previously conducted studies, Avery et al studied that the features

of BNC with epithelial tissue specific to the organ in which it is implanted are particularly

important for bioimplants in the circulatory system where inflammation is responsible for the

degenerative changes, and the possibility of covering the body's own tissues can bring a

negligible effect thrombogenicity. BNC are used as surgical implants such as artificial heart

valves, blood vessels (tube) or neurotubes (regeneration of nerves), and tracheotomy tube

(reconstructive surgery).

6.3. Other Applications

The production of durable and flexible paper with high filler content is one of the

potential applications of fragmented BNC. This is used in papermaking as a binding agent

and is ideal for bank note paper (Chawla et al. 2009; Ashjaran et al. 2013). Li et al. (2015)

developed an environmentally friendly paper-based energy storage device using BNC-

polypyrrole nanofibers in combination with multiwalled carbon while BNC-based nano paper

demonstrated by Mautner et al. (2015) was suitable for tight ultrafiltration operations.

Bijak (2016) listed some applications of BNC in various industries including in the

field of cosmetology, fabrics, environmental protection, and science. In the field of

cosmetology, components of artificial nails and emulsion stabilizers such as creams and
tonics are BNC-based product. Textiles, tent and camping equipment and materials with

highly adsorbent are also included in the application of BNC in the field of fabrics. BNC are

used as a sewage treatment, oil absorption, toxins and water filtration for environmental

protection. In the field of science, BNC are applied for chromatography, protein

immobilization and as component of the culture medium.

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