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The production and characterization of bacterial cellulose pellicles

obtained from oil palm frond juice and their conversion to
nanofibrillated cellulose

Siti Nur Nadhirah Said Azmi , Zainatul ’Asyiqin Samsu ,

Ahmad Syafiq Fauzan Mohd Asnawi , Hidayah Ariffin ,
Sharifah Soplah Syed Abdullah

PII: S2666-8939(23)00048-8
DOI: https://doi.org/10.1016/j.carpta.2023.100327
Reference: CARPTA 100327

To appear in: Carbohydrate Polymer Technologies and Applications

Received date: 13 February 2023

Revised date: 19 May 2023
Accepted date: 26 May 2023

Please cite this article as: Siti Nur Nadhirah Said Azmi , Zainatul ’Asyiqin Samsu ,
Ahmad Syafiq Fauzan Mohd Asnawi , Hidayah Ariffin , Sharifah Soplah Syed Abdullah , The pro-
duction and characterization of bacterial cellulose pellicles obtained from oil palm frond juice and their
conversion to nanofibrillated cellulose, Carbohydrate Polymer Technologies and Applications (2023),
doi: https://doi.org/10.1016/j.carpta.2023.100327

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The production and characterization of bacterial cellulose pellicles obtained from oil

palm frond juice and their conversion to nanofibrillated cellulose

Siti Nur Nadhirah Said Azmi a, Zainatul ’Asyiqin Samsua, Ahmad Syafiq Fauzan Mohd

Asnawi c, Hidayah Ariffin d, e and Sharifah Soplah Syed Abdullah a, b *

a
Section of Bioengineering Technology, b Section of Food Engineering Technology, c Section

of Chemical Engineering, Universiti Kuala Lumpur Branch Campus Malaysian Institute of

Chemical and Bioengineering Technology, Lot 1988 Vendor City Taboh Naning, 78000 Alor

Gajah, Melaka, Malaysia,

d
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular

Sciences, e Laboratory of Biopolymer and Derivatives, Institute of Tropical Forestry and

Forest Products (INTROP), Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia

*Email: sharifahsoplah@unikl.edu.my

Abstract

This study explored the potential of using oil palm frond (OPF) juice as a fermentation

medium for bacterial cellulose (BC) synthesis by Acetobacter xylinum 0416, followed by its

conversion into nanofibrillated cellulose (BNFC). The effect of adding nitrogen sources to

OPF juice on the BC yield and thickness was investigated, with corn steep liquor (CSL)

showing the most promising results, increasing BC yield and thickness by six- and four-fold,

respectively. The resulting BC-OPF+CSL pellicles demonstrated excellent thermal stability,

high crystallinity and a large swelling ratio. The study also investigated the conversion of BC-

OPF+CSL pellicles into BNFC using either homogenization, ultrasonication, or a

combination of both. The combination of ultrasonication and homogenization produced the

1
best results, disintegrating the BC fibers into BNFC with a diameter of 13-21 nm and water

retention value of 6105 %. Low BNFC crystallinity and thermal stability were also observed,

indicating successful BC fibrillation. These findings suggest that OPF juice is a promising

alternative fermentation medium for BC production and that combining ultrasonication and

homogenization is a simple and safe method for converting BC-OPF+CSL pellicles into

BNFC. This research offers a potential eco-friendly solution for BC production and its

conversion into value-added products such as BNFC.

Keywords: Bacterial cellulose (BC); Acetobacter xylinum; oil palm frond (OPF) juice;

bacterial nanofibrillated cellulose (BNFC)

1. Introduction

Cellulose is the most abundant naturally occurring biopolymer found in various organisms,

including plants, animals, and some bacteria. Due to its exceptional mechanical properties,

low density, favourable environmental impact, biodegradability, and plenty availability from

bio resources, the novel biopolymer has found widespread application. Despite having many

benefits, cellulose has a few disadvantages that restrict its use, such as such as high water

absorption capacity and insufficient interfacial adhesion (Liu et al., 2021). As a result of the

aforementioned constraints, it becomes imperative to transform cellulose into nano/micro-

cellulosic forms or blend it with suitable polymers in order to create the desired composite

characteristics.

2
Nanocellulose (NC) is extracted from native cellulose composed of nano-scaled structure

materials and is classified into three types: cellulose nanocrystals (CNC), cellulose nanofibrils

(CNF), and bacterial cellulose (BC). CNC and CNF are usually extracted from lignocellulosic

or plant materials (Mokhena and John, 2020). Meanwhile, BC is synthesized by bacteria from

glucose via a bottom-up method (Nasir et al., 2017; Pradhan et al., 2022). This biopolymer is

of great interest because it is abundant, attractive, and has exceptional properties, including

high aspect ratio, good mechanical properties, renewability and biocompatibility (Trache et

al., 2020). This makes nanocellulose an excellent choice to substitute the synthetic materials

such as steel, Kevlar, poly(vinyl)alcohol (PVA) and polyurethane (PU). Moreover, cellulose-

based materials have been extensively applied in food, pharmaceutical, paper, textile, and

wastewater treatment applications (Azman Mohammad Taib et al., 2022). Through nitration

process, plant derived NC is converted into energetic biomaterials known as nitrocellulose

with enhanced properties and have the potential to be used in advanced composite explosives

and solid propellants (Tarchoun, Sayah, et al., 2022; Tarchoun, Trache, et al., 2022). In

addition, NC when combine with other nanomaterials such as graphene, exhibit great promise

in separation, adsorption, optics, flexible electronics, energy storage, thermal management,

barrier and packaging, and electromagnetic shielding (Azman Mohammad Taib et al., 2022).

However, NC derived from BC pellicles has not yet been extensively studied, and more

research is required in view of its properties and potential applications.

Several bacteria genera, such as Gluconacetobacter (formerly Acetobacter), Agrobacterium,

Aerobacter, Achromobacter, Azotobacter, Rhizobium, Sarcina, and Salmonella, produced BC

extracellularly into nanofibers (Wei Liu et al., 2020; Yee & Abd Razak, 2017). The

production of BC involves a series of enzymatic reactions comprising four key steps. Firstly,

glucose is phosphorylated by glucokinase, resulting in the formation of glucose-6-phosphate.

3
Subsequently, phosphoglucomutase catalyzes the isomerization of glucose-6-phosphate to

glucose-1-phosphate. The third step involves the conversion of glucose-1-phosphate to

uridine diphosphate glucose (UDP-glucose) through the action of UDP-glucose

pyrophosphorylase. Finally, cellulose synthase synthesizes cellulose from UDP-glucose

(Moniri et al., 2017). BC is non-toxic and has shown great promise in many fields since it was

discovered because its structure is much better than plant cellulose (Seddiqi et al., 2021).

Although BC and plant celluloses have the same molecular formula (C6H10O5)n, BC lacks the

lignin, hemicelluloses, and pectin typically found in plant-derived celluloses. As a result, BC

purification is a simple, low-energy process, in contrast to the harsh chemicals that are

typically needed to purify plant celluloses. However, the medium's high production cost (30

% of the total cost) poses a significant obstacle to meeting the demands of commercial

manufacturing for BC (Fernandes et al., 2020; Rahman et al., 2021). In recent years, using

agricultural and industrial wastes for sustainable BC production has emerged as a viable and

affordable replacement for the currently used medium (Raiszadeh-Jahromi et al., 2020; Saleh

et al., 2021). For instance, several studies have shown that BC can be produced from various

fruit juices and agricultural wastes, such as coconut, pineapple, orange, pear, apple and grape

(Kongruang, 2008; Kurosumi et al., 2009). Zahan et al., (2015) reported that fruit juice alone

produced a high yield BC instead of using a high-cost defined medium such as Hestrin and

Schramm (HS). Therefore, we investigated using oil palm frond (OPF) juice, an agricultural waste

extracted from fresh OPF petiole, as the fermentation feedstock for BC production. OPF juice

contains a high amount of sugars (glucose, sucrose, and fructose), minerals, and nutrients

(Abdullah et al., 2015), making it an excellent nutrition source for bacterial growth throughout the

fermentation process. Several studies have indicated OPF's potential as a good fermentation

feedstock for BC production (Mohamad et al., 2022; Supian et al., 2021). Similarly, we reported a

successful high BC yield from yeast extract, peptone-supplemented agricultural waste, an oil palm

4
frond (OPF) juice, i.e., 6-fold higher than Hestrin–Schramm (HS) medium. The yield exhibited

remarkable physical characteristics (Said Azmi et al., 2019). Nevertheless, Kurosumi et al.,

(2009) suggested that nitrogen source supplementation in fruit juices was able to increase the

BC yield. However, present nitrogen sources, such as yeast extract and peptone, are costly.

Therefore, the potential of cheap nitrogen sources to enhance bacterial cellulose production

should be explored.

Although BC is in the NC category, its pellicle form is not in NC structure, causing

insolubility in water. Therefore, BC pellicles require additional modification to broaden their

application in composite synthesis and reinforce material research (Torres et al., 2019).

Kawee et al., (2018) prepared BC suspension into nanofibrils and named it bacterial

nanofibrillated cellulose (BNFC). BNFC is known to have a large specific surface area,

excellent thermomechanical properties, great mechanical properties, hydrophilicity, and able

to form a highly porous mesh (Abraham et al., 2011). The BNFC's chemical composition is

similar to CNF, but it did not have porous mesh for easier adsorption and produced gels in

water with shear-thinning and had thixotropic behavior. The most current research focused on

the CNF from plants, which is not pure cellulose and requires complex pre-treatments. CNFs

were typically extracted from lignocellulosic biomass using mechanical treatment combined

with enzymatic, chemical and mechanical pre-treatments (Pääkko et al., 2007; Saito et al.,

2007). Mechanical treatments by a high-pressure homogenizer, ultrasonicator, microfluidizers

and grinders have been widely used to disintegrate plant cellulose fibre (Osong et al., 2016).

Although plant-derived CNF has several advantages, its use is limited due to chemical use

and extraction process complexity.

Therefore, this research provides a potential substitute for cellulose sourcing that

eliminates the conventional treatment procedures typically employed for lignocellulose in

order to obtain pure cellulose. The effect of nitrogen sources supplementation on the

5
production of BC pellicles was examined by a simple fermentation technique employing oil

palm frond (OPF) juice. The conversion of BC pellicles obtained from OPF juice to BNFC

using ultrasonication and homogenization is the subject of a new study that may serve as an

alternative nanofibrillated cellulose source.

2. Materials and methods

2.1. Preparation of inoculum

A. xylinum 0416 was purchased from the Malaysian Agricultural Research and Development

Institute, Serdang, Malaysia. The Hestrin–Schramm (HS) medium was prepared according to

Faisul Aris et al. (2019), with the following composition (w/v): 4 % glucose, 0.5 % granulated

yeast extract, 0.5 % peptone, 0.27 % Na2HPO4 and 0.115 % citric acid in 100 mL of distilled

water. The medium pH was adjusted to 6.4 by adding either 0.1 M HCl or 0.1 M NaOH

before sterilizing using an autoclave at a temperature of 121°C for 15 minutes. 10 % (v/v) of

A. xylinum 0416 was aseptically transferred into the sterile medium. The fermentation process

was carried out for 3 days at room temperature (±30°C).

2.2. Preparation of OPF juice

50 kg of freshly cut OPF petiole were collected from an oil palm plantation in Alor Gajah,

Melaka, Malaysia. The OPF juice was extracted by pressing the petioles with a conventional

sugarcane press machine and then centrifuged at 3,214 × g for 30 min at 4°C (Eppendorf

Centrifuge 5810 R, Germany) to remove solid materials. The resulting OPF juice was stored

in the freezer at -20°C until further use.

2.3. Effect of nitrogen sources supplementation to OPF juice on BC production

6
The OPF juice was supplemented with five nitrogen sources in order to evaluate its effect on

BC production. The amount of supplemented nitrogen sources added was based on a fixed

C/N ratio of 23.96, similar to HS medium. Carbon and nitrogen content was determined using

an elemental analyzer. Table 1 summarizes the medium composition that included HS

medium as a control media. All media were sterilized at 121°C and 15 psi for 15 minutes. The

sterile media were inoculated with 10 % (v/v) A. xylinum 0416 inoculum before incubation at

room temperature and placed in static conditions for 10 days. The BC pellicles formed were

taken out of the fermentation media, and the remaining glucose was measured. The BC

pellicle purification were carried out by immersion in 0.1 M NaOH at 80°C for 30 minutes to

remove any remaining cells. The BC pellicles were then washed several times under running

water until a neutral pH was obtained, and they were dried at 40°C to a constant weight. The

optimal medium was selected based on the highest BC yield obtained.

Table 1
The composition of each fermentation medium

Types of media
Composition
(% w/v)
HS M1 M2 M3 M4 M5 M6 M7

Glucose 4 - - - - - - -

OPF Juice *- ̷ ̷ ̷ ̷ ̷ ̷ ̷

Citric acid 0.115 - 0.115 0.115 0.115 0.115 0.115 0.115

Na2HPO4 0.27 - 0.27 0.27 0.27 0.27 0.27 0.27

Yeast extract 0.5 - 1.83 - 0.91 - - -

Peptone 0.5 - - 1.54 0.77 - - -

Corn steep - - - - - 2.415 - -

liquor (CSL)
Ammonium - - - - - - 0.914 -
Sulphate

7
Ammonium - - - - - - - 1.067
Phosphate
pH 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4

*Replace with distilled water

2.4. Conversion of BC to bacterial nanofibrillated cellulose (BNFC)

The BC pellicles obtained from static OPF juice fermentation were converted to BNFC using

mechanical methods, i.e., homogenization, ultrasonication and a combination of both.

Morphological characteristics, chemical structure, thermal analysis and crystallinity

characterized the samples obtained. The commercial NFC (produced by supermass colloider)

was provided by CelluloseLab, Canada.

2.4.1. Ultrasonication

The purified and wet BC pellicle was cut into 0.125 mm3 cubes and immersed in distilled

water with approximately 0.1 % w/v of BC to water. The mixture was sonicated using Sonics

Vibra-Cell VCX 750 Sonicator with a 13 mm diameter ultrasonic probe at 750 W, 40 %

amplitude and 20 kHz frequency for 30 minutes. The temperature was kept below 60°C

throughout ultrasonication using an ice bath. After the process was completed, the suspension

was concentrated by centrifugation at 4500 × g for 30 minutes (Eppendorf Centrifuge 5804,

Germany). The colloidal suspension of BNFC was then stored at room temperature before

analysis.

2.4.2. Homogenization

0.1 % w/v of purified BC cubes to water was homogenized (Silent Crusher M) at 10,000 rpm

for 15 minutes. The homogenization process was stopped every 5 minutes to prevent

overheating. The BC suspension obtained was stored at room temperature before analysis.

8
2.4.3. Combination of ultrasonication and homogenization

A combination of ultrasonication followed by homogenization was performed to evaluate the

effectiveness of converting BC pellicle to nanofibrillated size. Then, the same processes were

reversed and the evaluation was repeated. Initially, 0.1 % w/v of BC cubes were ultrasonically

treated at 40 % amplitude for 30 minutes, with pulses on for 30 seconds and off for 30

seconds. The treatment was followed with 15 minutes of homogenization at 10,000 rpm.

Under the same conditions, the second combination treatment involving homogenization and

ultrasonication was carried out. The samples from both combination treatments were stored at

room temperature before analysis.

2.5. Analytical methods and characterization of BC and BNFC

2.5.1. Determination of sugar

1 ml of supernatant from the fermentation broth was taken, and the glucose concentration was

measured using the YSI 2700 Biochemistry Analyzer. The sugar consumption was calculated

by subtracting the initial and final glucose concentration (g/L) in the fermentation broth.

2.5.2. Measurement of BC thickness

The purified BC thickness from static fermentation was measured using a Vernier caliper in

centimeters at five positions, and the averaged values were recorded.

2.5.3. Determination of BC yield

The BC production yield in different types of media (M1-M7) was compared with the yield

obtained from the HS medium. The purified BC pellicle was dried at 40°C until it reached a

constant weight to determine the BC's dry weight from the fermentation medium. Then, dried

9
BC was measured by an electronic balance and kept for further characterization. The BC yield

was estimated using the following equation:

𝑚 𝐵𝐶
𝑌𝑃⁄𝑆 = 𝑆 −𝑆 (1)
𝑖 𝑓

𝑚𝐵𝐶 is the dry mass of BC produced per litre of medium (g/L), 𝑆𝑖 is the initial sugar

concentration (g/L) and 𝑆𝑓 is the final sugar concentration (g/L) in the fermentation broth.

2.5.4. Scanning Electron Microscopy (SEM)

Ultrafine BC fibrils structure was characterized using SEM (Hitachi Model SU3500). The

purified BC films were frozen at –80°C for 24 hours and freeze-dried for 48 hours. Thin

layers of freeze-dried BC were then coated with gold using an ion sputter, examined at an

accelerated voltage of 5 kV, and photographed at 5 k.

2.5.5. Transmission Electron Microscopy (TEM)

TEM was used to determine the dimensional information and BNFC's structural surface

morphology obtained after BC's mechanical treatment. A diluted BNFC suspension (0.1 %

w/v) was dropped on a carbon-coated formvar grid and stained with 1 % uranyl acetate. The

images were scanned by TEM (JEOL JEM 2100F) with an accelerating voltage of 200 kV.

The BNFC fibers diameters were obtained by analyzing the TEM micrographs with ImageJ

software (version 1.52r, NIH, National Institutes of Health, Bethesda, MD, USA). Using the

software, the size distribution histograms of BNFC samples were plotted from the diameters

of at least 50 fibers.

2.5.6. Fourier Transform Infrared Spectroscopy (FTIR) analysis

10
FTIR analysis was carried out on a PerkinElmer Spectrum 400 GladiATR to identify the

molecular structures and chemical bonds of BC membrane. The vibrational modes from the

BC film were measured at wave number region of 4000–800 cm−1 and resolution of 8 cm−1.

2.5.7. Thermal analysis

Thermal stability of BC films was performed using thermogravimetric analyzer (TGA/DSC 1

STAR System, Mettler Toledo). The crucible was loaded with 4-5 mg of dried BC samples

and placed inside the TGA. The scan was operated from room temperature to 600°C with the

heating rate of 10°C min-1 and nitrogen gas flow rate of 20 ml min-1.

2.5.8. X-ray diffraction analysis

The X-ray diffraction (XRD) technique was used to determine the crystallinity of BC

samples. XRD patterns of the freeze-dried BC films were determined using a Bruker D8

Advance diffractometer generated at a voltage of 40 kV at the Cu Kα radiation wavelength (λ

= 1.5406 Å) and filament emission of 40 mA. The samples were scanned between 10° and

80° at 2θ range. The speed was scanned in 0.5°·min−1 with a step size of 0.02°. The

crystallinity index (CrI) was calculated using the XRD deconvolution method.

2.5.9. Water holding capacity (WHC)

Dried BC was cut into 1 cm x1 cm, weighed and placed on a Petri dish containing 5 ml of

distilled water. The sample was taken out after 15 mins, 45 mins, 60 mins, 2 hours, 3 hours, 4

hours and 5 hours, and weighed after being left to dry for 5 mins on a filter paper (wet

sample). WHC was calculated by comparing the samples' dry and wet masses (Pa’e et al.,

2019).

11
 𝑀𝑤 −𝑀𝑑
𝑊𝐻𝐶 = 𝑀𝑑
(2)

𝑀𝑤 is the mass of the wet sample while 𝑀𝑑 is the mass of the dried sample.

2.5.10. Water retention value (WRV)

WRV is an empirical measure of cellulose's water-holding capacity. 5 ml of 0.1 % aqueous

suspensions of BNFC was centrifuged for 30 min at 4500 x g to obtain the pellet in order to

determine the WRV value (Nechyporchuk et al., 2016). The supernatant was discarded, and

the sediment was weighed. Next, the sediment was dried at 60°C for 48 h and stored in a

desiccator for 30 mins to completely remove the water. WRV value was calculated using the

following formula (Nechyporchuk et al., 2016):

𝑊𝑤 −𝑊𝑑
𝑊𝑅𝑉 (%) = 𝑊𝑑
𝑥 100 (3)

Ww is the wet sediment’s weight and Wd is the of the dried sediment’s weight.

3. Results and discussion

3.1. Production of BC from OPF juice supplemented with nitrogen sources

The ideal nitrogen source is essential for cellular metabolism, growth, and formation. This

experiment was conducted to determine the efficacy of each nitrogen source in boosting BC

yield. Fig. 1 depicts the yield and thickness of BC produced from OPF juice supplemented

with various nitrogen sources. In 10 days, the non-supplemented OPF juice produced a

remarkable BC yield of 0.051 g BC/g glucose compared to the control medium (HS) of 0.032

g BC/g glucose. The promising BC yield obtained in this study demonstrated that OPF juice

has sufficient nutrients to support BC synthesis by A. xylinum 0416 and could be employed as

12
a complete fermentation medium for BC production. Although OPF juice contains nitrogen

naturally, adding nitrogen sources to OPF juice increased the BC yield by 49-144 %

compared to the control medium. The highest BC yield (0.196 g/g) was found in OPF juice

supplemented with corn steep liquor (CSL), followed by the combination of yeast extract

(YE) and peptone (P) (0.177 g/g), yeast extract (0.167 g/g), peptone (0.100 g/g), ammonium

sulphate (AS) (0.072 g/g), and ammonium phosphate (AP) (0.053 g/g), as shown in Fig. 1.

Likewise, the BC thickness pattern exhibits a similar trend. OPF supplemented with CSL

produced the thickest BC pellicles at 0.89 cm, followed by a combination of yeast extract and

peptone, yeast extract, peptone, ammonium sulphate and ammonium phosphate that produced

a thickness of 0.83 cm, 0.71 cm, 0.45 cm, 0.36 cm and 0.30 cm, respectively.

Thickness Yield (g/g)

1.00 0.25

0.80 0.20
BC thickness (cm)

BC yield (g/g)
0.60 0.15

0.40 0.10

0.20 0.05

0.00 0.00

Fig. 1. Bacterial cellulose (BC) thickness and yield results after adding nitrogen sources to oil palm

frond (OPF) juice. Data is presented as mean ± SD (n =3).

CSL was the most effective nitrogen source for producing BC when combined with OPF juice

compared to other nitrogen sources tested. CSL is a complex organic nitrogen source derived

13
from corn wet-milling by-product that comprises 16 % nitrogen, 21-45 % protein, 20-26 %

lactic acid, and 4 % mineral elements (Mirza & Mushtaq, 2006). The enhanced cellulose

production by OPF juice supplemented with CSL could be attributed to the CSL's high protein

and nitrogen content. Furthermore, the presence of lactate in CSL, which is missing in other

nitrogen sources, explains why CSL produces the most BC (Matsuoka et al., 1996). Lactate

stimulates cell growth by facilitating the tricarboxylic acid (TCA) cycle and by generating

oxidation energy as lactate is converted to pyruvate, resulting in greater BC synthesis

(Naritomi et al., 1998). Lactate was found to serve for the formation of acetoin and biomass

building blocks (Adler et al., 2014) which reflects the formation of BC pellicles. CSL is also

considered a low-cost substrate and rich in nutrients, which is useful for A. xylinum growth

and supports the stable production of BC in OPF juice (Aswini et al., 2020). In addition to

CSL, yeast extract and peptone are also classified as complex organic nitrogen sources. It is

well known that microorganisms can easily access organic nitrogen sources. OPF juice

supplemented with the yeast extract and peptone mixture significantly increased BC yield

compared to adding yeast extract and peptone separately. Yeast extract comprises 61 %

protein, 26 % carbohydrates, free amino acids, vitamins, and minerals (Zhang et al., 2007).

Meanwhile, peptone has 10-15 % total nitrogen as the only protein source. When these

organic and complex nitrogen sources are dissolved in water, the release of excess nitrogen as

ammonia raises the medium's pH. Along with the nitrogen sources, ammonia provided

growth-promoting elements that improved BC production.

In contrast, inorganic nitrogen sources, such as ammonium sulphate and ammonium

phosphate, were less favourable for BC production. This finding agrees with Yodsuwan et al.

(2012) where using ammonium sulphate as a nitrogen source reduced bacterial growth and

BC yields. This study demonstrated that the yield of BC synthesis by A. xylinum 0416 is

greater when complex organic nitrogen sources are added to OPF juice compared to inorganic

14
nitrogen sources. Moreover, the findings of Embuscado et al. (1994) confirmed that organic

nitrogen sources, except urea, are the best nitrogen source for cellulose production, providing

better yields than inorganic nitrogen sources. It is important to highlight that CSL can be a

substitute for yeast extract and peptone, which are expensive organic nitrogen sources in BC

production by A. xylinum 0416.

3.2 Characteristics of BC

3.2.1 Scanning Electron Microscopy (SEM)

Fig. 2 depicts the BC pellicle characteristics synthesized in HS medium and OPF juice

enriched with CSL. Fig. 2a displays a porous, well-organized, lengthened, larger-width fibril

network of BC generated in HS media. On the other hand, BC from OPF-enriched CSL has

slightly thinner fibrils and a more compact structure. Gündüz & Aşık (2018) and Lima et al.

(2017) observed similar findings for the BC structure produced from sisal and carrot juices.

The BC produced during the black tea broth fermentation (Kombucha) (Goh et al., 2012) and

thin stillage and whey (Revin et al., 2018) also exhibited an ultrafine network structure

comprised of a dense cellulose network structure. Therefore, it can be hypothesized that the

OPF juice fermentation yielded a typical BC structure with porous 3D networks, similar to

other agricultural by-products.

15
 (a)

(b)

Fig. 2. Scanning Electron Microscopy (SEM) images of (a) bacterial cellulose (BC) produced in HS

medium and (b) BC produced in oil palm frond (OPF) juice.

3.2.2 Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy

16
Both samples' FTIR spectra were detected at wavenumbers ranging from 4000 to 800 cm-1.

Fig. 3(A) displays the FTIR spectra for BC pellicles synthesized in HS medium (BC-HS) and

OPF juice enriched with CSL (BC-OPF+CSL). The similarities between their spectra imply

that the cellulose has the same chemical structure. These bands are prevalent in the BC's IR

spectra (Hirai et al., 1998; Yamamoto et al., 1996). The moderate intensity peaks at 3343 cm-1

and 3292 cm-1 for BC-HS and BC-OPF+CSL are attributed to the stretching vibration of

hydroxyl groups (–OH) involved in the inter- and intra-molecular hydrogen bonds (Dima et

al., 2017). The transmittance at 2893 cm-1 and 2920 cm-1 for BC-HS and BC-OPF+CSL,

respectively, were assigned for C-H stretching. Weak bands were detected at 2847 cm-1 and

2850 cm-1 due to the symmetric stretching vibration of methyl (–CH3), whereas typical

cellulose bands were seen between 1630 cm-1 and 900 cm-1 (Rosa et al., 2010). The peaks

located at 1629 cm-1 correspond to the vibration of water molecules absorbed in cellulose,

which agrees with Saikia and Parthasarathy (2010). BC-OPF+CSL exhibited the typical

cellulose I band at 1543 cm-1 (CH2 symmetrical bending) and 1035 cm-1 (C-O bond

stretching). Meanwhile, peaks at 1572 cm-1 of BC-HS suggested a weak intensity of aromatic

compounds with functional groups of C-H bending. Peaks at 1053 cm-1 indicated strong C–

O–C and C–O–H stretching vibrations of the sugar ring.

3.2.3 Thermogravimetric analysis (TGA)

Fig. 3(B) illustrates the TGA analysis of BC-HS and BC-OPF+CS. The initial weight loss

stage occurs at temperatures ranging from 40 to 85°C and is caused by water evaporation

from the BC pellicles. This phase contributes 9 % and 11 % BC-HS and BC-OPF+CSL

weight loss, respectively. A thicker BC-OPF+CSL can store a significant amount of water in

its pores, resulting in a greater weight loss due to water evaporation from the BC pellicle.

17
Most cellulose degradation occurs between 200 and 340°C, resulting in a weight loss of 60 %

and 44 % for BC-HS and BC-OPF+CSL, respectively. This was associated with cellulose

deterioration, including polymerization, dehydration, and disintegration of glucose units.

Based on these findings, BC-OPF+CSL required a higher temperature to degrade the sample

than BC-HS, suggesting that the BC generated from OPF juice had better thermal stability.

The maximum rate for BC's degradation percentage or weight loss of BC occurred at 300 -

360°C (Barud et al., 2008; Pa'e et al., 2019). In light of this study, BC can handle

temperatures above 300°C and are stable at high temperatures. This is a required

characteristic for applications that use high temperatures, such as the food industry's

pasteurization process. Moreover, using agricultural waste in BC production minimizes

environmental effects. It lowers production costs and makes the BC pellicle more valuable

from a business point of view.

3.2.4 X-ray diffraction (XRD)

Fig. 3(C) illustrates the X-ray diffraction patterns of BC-HS and BC-OPF+CSL. The

crystallinity index values of BC-OPF+CSL (84 %) were slightly higher than BC-HS (81 %),

which is similar to that reported in a previous study (Revin et al., 2018; Shezad et al., 2010).

The appearance of the three distinctive peaks, 14.5°, 17° and 23°, indicates the crystalline

structure in both BC. The variation in relative peak intensity was attributed to the small

differences in chain orientation among the three samples. The XRD profiles of the three main

peaks at 2ϴ recorded from the samples are similar to those obtained by Dórame-Miranda et

al. (2019) and Revin et al. (2018).

The higher crystallinity obtained by BC-OPF+CSL is related to the thick texture of the BC

pellicle produced, which became rigid after drying. The X-ray patterns revealed the same

cellulose chemical structure, yet different diffraction intensity was identified. BC from OPF

18
juice supplemented with CSL had a more defined crystalline region with a denser fibril

network than BC-HS, providing rigidity and strengthening the BC sheet. This finding was

consistent with the findings of Revin et al. (2018), who stated that morphological changes

could influence various BC properties and microstructures, including crystallinity.

BC-OPF+CSL

2920
Transmittance (a.u.)

3292
1644 1543 1035
BC-HS

2893 1646
1571
3343

1053

4000 3200 2400 1600 800

Wavenumber (cm-1)

19
 DTG

BC-OPF+CSL BC-HS
Intensity (a.u.)

12 16 20 24 28
2ϴ(°)
C

20
 500

400

300
WHC (%)

200

100
BC-HS BC-OPF+CSL
0

-100
15min 45min 60min 2hr 3hr 4hr 5hr
Time
D

Fig. 3. Properties of BC-HS and BC OPF+CSL. Fourier Transform Infrared Spectroscopy (FTIR)

spectra (A), thermogravimetric analysis (TGA) curves (B), X-ray diffraction (XRD) pattern (C) and

water holding capacity (WHC) analysis (D).

3.2.5 Water holding capacity (WHC)

WHC is the cellulose's ability to prevent water release from its structure (Cai & Kim, 2010),

which is one of the most significant physical characteristics of BC pellicles, given its potential

as wound dressing material in the biomedical application (Portela et al., 2019). Fig. 3(D)

shows similar water absorption trends in BC-HS and BC OPF+CSL pellicles. Within two to

three hours of soaking, BC OPF+CSL reached a higher WHC (390 %) compared to BC-HS

(197 %), and then it began to decline. Generally, BC pellicles generated in static fermentation

represent approximately 1 % of the total weight, with the rest being water (Portela et al.,

2019). Lin et al. (2014) reported that WHC of wet BC from WBY hydrolysates by G.

hansenii CGMCC 3917 is 104 times its dry weight.

BC from OPF+CSL medium has a thinner fibril width and thicker structure, allowing it to

hold a huge amount of water and retain it in the BC matrix (Fig. 2). The fibrils thickness,

polymerization degree, and crystallinity influence BC's water retention. Due to the higher

21
surface area provided by the thinner and longer strands, more water may be retained. The

water molecules are sandwiched between the thick fibrils pores, and these fibrils act as a

shield for water molecules, resulting in high water-holding capacity and quick water-

absorbing ability (D. Lin et al., 2014).

3.3. Conversion of BC to nanofibrillated cellulose (BNFC)

3.3.1 Transmission Electron Microscopy (TEM)

Fig. 4 shows TEM images of bacterial nanofibrillated cellulose (BNFC) produced from

different mechanical treatments. Fig. 4(b), 4(c) and 4(d) show very distinct cellulose fiber

bundles compared to BC pellicles in Fig. 2(b). This observation suggests that ultrasonication,

or combining ultrasonication and homogenization, effectively disintegrated the BC fiber

networks into smaller fibrils and separated them into individual cellulose fibers. However,

homogenization treatment failed to break BC into nanofibrils. The BC fibers were still not

fragmented and completely exfoliated to a single cellulose fiber, as shown in Fig. 4(a).

200 nm 100 nm

(a) (b)

22
 100 nm
100 nm

(c) (d)

(e)

Fig. 4. Transmission Electron Microscopy (TEM) images of fibrillated bacterial cellulose (BC) under

different mechanical treatments; a) Fibrillated BC produced by homogenization process, b) Fibrillated

BC produced by ultrasonication process, c) Fibrillated BC produced by homogenization and followed

by ultrasonication process, d) Fibrillated BC produced by ultrasonication and followed by

homogenization, e) Commercial plant nanofibrillated cellulose (NFC).

The BNFC's diameters distribution was determined using the ImageJ software, and the mean

diameter of fibrillated cellulose is shown in Table 2. For each mechanical treatment, 50 fiber

measurements were acquired from five TEM images to assure data reproducibility when

measuring fiber size distributions. Nanocellulose is defined as cellulose with a size of less

than 100 nm in at least one dimension (Lin & Dufresne, 2014). The fibrillated cellulose

23
generated through ultrasonication, and the ultrasonication and homogenization combination,

successfully produced nanoscale fibrils ranging from 13 to 21 nm, better than commercial

NFC. Other NFC plant sources derived from birch kraft pulp, bamboo pulp, and recycled pulp

fiber have a nano-size diameter of 20-300, 58 and 40 nm, respectively (Filipova et al., 2018;

Silos et al., 2019). The fibril size of BNFC is smaller than NFC derived from plant sources,

demonstrating the BC’s potential as a substitute source of BNFC.

Table 2

Mean values of cellulose fibre diameters produced using different treatments

Type of treatments Mean value (nm)

Ultrasonication 15.73±5.58

Homogenization → Ultrasonication 20.76±6.07

Ultrasonication → Homogenization 13.16±4.93

Commercial NFC 26.96

3.3.2 Water retention value (WRV)

WRV is an empirical measure of cellulose's ability to retain water, which has been widely

used to characterize the extent of fiber fibrillation. The fibrillation treatment increased the

cellulose's surface area, increasing the fiber's water retention (Gu et al., 2018; Nechyporchuk

et al., 2016).

Fig. 5(A) shows the WRV values of fibrillated BC that underwent different mechanical

treatments. The BNFC obtained from ultrasonication followed by homogenization treatment

gave WRV of 6105 %, similar to commercial NFC at 6456 %. The ultrasonication process

alone could produce BNFC with a WRV of 5863 %. Meanwhile, BC treated with

homogenization followed by ultrasonication give a WRV of 5358 %. The homogenization

24
process alone has the lowest WRV of 4830 %. The combined effect of shearing, cavitation

and turbulence created homogenizing action. A high-pressure homogenizer or microfluidizer

is commonly used to produce CNF from plant sources and to defibrillate the cellulose.

However, in this study, a conventional homogenizer was applied to test the efficiency of

breaking down the fibril of BC pellicles. Ultrasonication produced smaller fibrils with a larger

surface area than microfibrils cellulose from homogenization. Combination treatments started

with ultrasonication and later homogenization, which have been proven more effective at

disintegrating BC pellicles into single fibers. Thus, increasing the surface area, and its ability

to hold a huge amount of water.

On the other hand, BNFC produced from homogenization followed by ultrasonication

exhibited lower WRV. Principally, the homogenization process disrupted the BC into a

smaller size. As a result, the ultrasonic cavitation was not high enough to produce high

pressure to split the fine particle agglomerates and disperse them more uniformly

(Shojaeiarani et al., 2020). During the sonication process, acoustic cavitation broke the

relatively weak interfaces between the nanofibers, such as van der Waals forces (Zhao et al.,

2007). The ultrasonic energy gradually disintegrates the micron-sized natural fibers into

nanofibers, leading to a higher WRV because there was more water to hold between the fibril

surface.

3.3.3 X-ray diffraction (XRD)

Crystallinity is a major factor influencing the materials' mechanical properties, commonly

measured using XRD. Fig. 5(B) shows the BNC's XRD spectra obtained from different

treatments. These diffraction peaks commonly corresponded to crystalline peaks at 14°, 17°

and 23°. The highest intensity peak at 23° represents cellulose type I. The homogenized BC

and BNFC generated by combining homogenization and ultrasonication exhibit strong peaks

25
in the diffraction bands, indicating high crystallinity of the cellulose. Native BC-OPF+CSL

has a crystallinity index (CI) value of 84 %, which decreased after exposure to mechanical

treatments in the following sequence: homogenized BC, BNFC (homogenization →

ultrasonication), BNFC (ultrasonication) and BNFC (ultrasonication → homogenization) at

the values of 72 %, 69 %, 64 % and 60 %, respectively. The decreasing trend of CI values

indicates that the native BC has fibrillated into BNFC (Daicho et al., 2018). The CI of NFC

obtained from kraft pulp by combining ammonium persulphate treatment with ultrasound and

mechanical processing was higher (74.3 %) (Filipova et al., 2018). In addition, the NFC

crystallinity from bleached bamboo pulp using a supermass colloider was 71 % (Silos et al.,

2019). The shear forces applied by the homogenizer destroyed the cellulose inter-hydrogen

bonds, causing the crystal structure breakdown (Lin et al., 2015). Otherwise, strong

hydrodynamic shear forces resulted in lower crystallinity for smaller-sized BNFC

(ultrasonication) and BNFC (ultrasonication → homogenization). The high kinetic energy of a

liquid jet from the acoustic cavitation scission the micro-length BC fiber (Tischer et al., 2010;

Wong et al., 2012). The reduced CI agrees with Abral et al. (2018), who obtained 65 % CI

after the ultrasonication process of nata de coco. The XRD results obtained here agrees with

the morphological observations. The BNFC's average nanofiber diameter (ultrasonication →

homogenization) was the smallest, resulting in the lowest crystallinity value. The lower CI

indicates that the treatment entirely disintegrated BC into nanometer-scale cellulose fibrils.

The results clearly showed that the average nanofiber size affected the CI. Moreover, CI also

affected BNFC's thermal behaviour in thermogravimetric analysis (TGA). The BNFC’s

(ultrasonication → homogenization) CI value is comparable to commercial NFC (61 %),

indicating that the mechanical treatment protocols applied in this study is a promising

technique to convert BC pellicle into nanofibrillated cellulose.

26
3.3.4. Thermal analysis

Thermogravimetric analysis (TGA) is used to characterize materials that exhibit a weight

change upon heating and detect phase changes caused by decomposition. Fig. 5(C) shows the

thermal properties of BC samples that were treated physically. All samples show two

significant changes in the degradation profile. The first weight loss occurred between 60–

150°C due to the water evaporation (Asrofi et al., 2017). The second event occurred in the

temperature range of 250–383°C. Like Fittipaldi et al. (2017), it is characterized by a series of

cellulose reaction degradation, including dehydration, decomposition, and depolymerization

of the glycoside units. The weight loss in the second event was 47% and 54 % for the BNFC

(ultrasonication→homogenization) and BNFC (homogenization → ultrasonication),

respectively, reflecting the nanofibers' lower thermal stability. This is because the smallest

diameter led to a higher surface area, while the number of free ends in the chain was more

vulnerable to thermal degradation. The TEM images discussed previously proved the smaller

BNFC nanofiber diameter.

Lam et al. (2017) also discovered nanocellulose thermal degradation at a lower temperature

range, starting at 250°C. A higher degradation temperature was observed for the homogenized

BC, BNFC (ultrasonication) and commercial NFC at 76 %, 60 % and 71 %, respectively.

Higher crystallinity may result in greater thermal stability. Homogenized BC had the highest

crystallinity and thermal resistance while BNFC (ultrasonication→homogenization) had the

lowest thermal resistance related to its lowest crystallinity.

27
 A
8000

7000

6000

5000
WRV (%)

4000

3000

2000

1000

0
Homogenization Ultrasonication
Homogenization→Ultrasonication Ultrasonication→Homogenization
Commercial NFC
Data is presented as mean ± SD (n =3)

Commercial NFC B
BNFC (Ultrasonication)

BNFC (Ultrasonication→Homogenization)
Intensity (a.u.)

Homogenized BC

BNFC (Homogenization→Ultrasonication)

10 20 30
2θ (o)

28
 C

Fig. 5. Properties of of nanofibrillated bacterial cellulose (BNFC) converted from BC OPF+CSL.

Water retention value (WRV) (A), X-ray diffraction (XRD) patterns (B) and thermogravimetric

analaysis (C).

The cellulose's thermal resistance is affected by crystallinity and intermolecular hydrogen-

bonded regions (Shebani et al., 2008). A slow weight loss occurred in the third thermal event,

as the temperature increased to 600°C. It is related to the oxidation and breakdown of

carbonaceous residues, yielding low molecular weight gaseous products. About 12 % to 20 %

of BC was left undecomposed up to 600°C. The degradation curve of commercial NFC

derived from plant cellulose sample was also found to have remaining residues of 11 %,

similar to homogenized BC.

4. Conclusion

29
OPF juice is a feasible option because it is inexpensive, abundant and can be utilized as a

substitute feedstock to manufacture BC. Supplementing OPF juice with CSL during static

fermentation resulted in a six-fold increase in BC yield compared to the HS medium.

Moreover, BC-OPF+CSL possessed excellent crystallinity, water-holding capacity, and

thermal stability compared to BC-HS. Its remarkable properties make it an excellent candidate

for a biopolymer and a biocomposite material. This work has developed a simple mechanical

treatment to produce BNFC from BC-OPF+CSL pellicles using a combination of

homogenization and ultrasonication. Without using any chemicals, BNFC was successfully

synthesized from BC and has qualities superior to plant NFC in terms of thermal stability,

crystallinity, and water retention. In conclusion, the BNFC obtained in this study required

less complex preparation and treatment, making it ideal for food or biomedical applications.

E-supplementary data of this work is available in the online version of this paper.

Acknowledgement

The authors would like to acknowledge and thank the Ministry of Higher Education through

the Fundamental Research Grant Scheme (FRGS) FRGS/1/2017/TK10/UNIKL/02/6 for their

financial support.

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Declaration of interests

☐The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Sharifah Soplah Syed Abdullah reports financial support was provided by Malaysia Ministry of Higher
Education.

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Graphical Abstract

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