Materials Letters 333 (2023) 133622

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Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Highly crystalline bacterial cellulose production by Novacetimonas hansenii

strain isolated from rotten fruit
S. Neelima a, S. Sreejith b, Shamna Shajahan b, Aparna Raj a, L. Vidya a, V.M. Aparna a, E.
K. Radhakrishnan b, C. Sudarsanakumar a, *
a
School of Pure and Applied Physics, Mahatma Gandhi University, Kottayam, Kerala 686560, India
b
School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala 686560, India

A R T I C L E I N F O A B S T R A C T

Keywords: We have isolated the cellulose-producing Novacetimonas hansenii strain from rotten pomegranate for the first
Bacterial cellulose time. The further characterization of cellulose film using X-ray Diffraction revealed the highest relative crys­
Crystallinity tallinity value of 96.6% observed till date.
Novacetimonas hansenii
Biomaterials
Polymers

1. Introduction 2. Materials and methods

Bacterial cellulose (BC) was used by humans long ago in the form of a
food product called nata de coco. The BC is known for its purity, higher
crystallinity, and enhanced water-holding capacity [1]. As microbial
cellulose has been studied for diverse applications ranging from
biomedical to food packaging, the exploration of novel bacterial sources
for BC production is highly important.
The present study used rotten fruits such as apples, oranges, grapes,
and pomegranates, to isolate cellulose-producing bacteria. Among nine
isolates screened, the P3 isolate from rotten pomegranate showed the
highest cellulose production. Upon 16S rDNA sequencing-based mo­
lecular identification, the P3 isolate was identified as a strain of Nova­
cetimonas hansenii (GenBank Acc. No. OM883845). The BC synthesized
has been characterized by Fourier Transform Infrared Spectroscopy
(FTIR), and X-ray diffraction (XRD), and showed the highest relative
crystallinity with cellulose І structure.
2.1. Selection of fruit samples and primary screening for bacterial
cellulose production
Rotten fruits were collected from the Athirampuzha market near
Kottayam, Kerala, India. Approximately 40 g of each fruit was made into
small dice and incubated separately in 200 mL of sterile medium con­
taining 20 g of brown sugar and 20 g of granulated sugar for five days at
room temperature [2].
2.2. Isolation of cellulose-producing bacteria
From the primary screening, those samples with the formation of
thick pellicles were selected to isolate cellulose-producing bacteria.
Here, a loop full of the sample was further inoculated into 15 mL HS
medium [3] containing (g/L): glucose 20, yeast extract 5, peptone 5,
disodium hydrogen phosphate 2.7, and citric acid 1.15 with supple­
mentation of the antifungal agent cycloheximide (0.02 wt%) [4]

* Corresponding author.
E-mail address: sudarsanakumar@mgu.ac.in (C. Sudarsanakumar).

https://doi.org/10.1016/j.matlet.2022.133622
Received 13 June 2022; Received in revised form 12 September 2022; Accepted 27 November 2022
Available online 2 December 2022
0167-577X/© 2022 Elsevier B.V. All rights reserved.
S. Neelima et al. Materials Letters 333 (2023) 133622

Table 1
The CrI % (peak height method) of various BC samples isolated from different organisms. The highlighted value refers to the current study.
Microorganism Media composition Crystallinity index (%) Reference

Komagataeibacter saccharivorans MD1 HS 87 [11]

Gluconaceter xylinus BNKC19 HS-glucose 82.92 [12]
HS-pure glycerol 84.02
HS-crude glycerol 92.43
Komagataeibacter rhaeticus PG2 HS-glycerol 80.80 [10]
Gluconacetobacter intermedius Cis26 HS-Citrus Waste Solution 75 [13]
Komagataeibacter sucrofermentans DSM 15,973 Solid citrus waste 86.9 ± 2.23 [14]
Komagataeibacter saccharivorans BC1 Optimized medium 82.2 [15]
Acetobacter xylinum ATCC 23,767 Tobacco waste extract medium 85.58 [16]
Komagataeibacter hansenii JR-02 Optimized medium 91.76 [17]
Novacetimonas hansenii P3 HS 96.60 Current study

followed by the incubation for ten days at room temperature. For where I(002) and Iam were the intensities of the peak at 2θ corresponding
bacterial isolation, the test tube with strong pellicle production was
to 22.5◦ and background intensity between 16◦ and 22.5◦ , respectively
further inoculated on HS agar plates and kept for incubation for five
[10]. The FTIR spectra of the dried BC were recorded using PERKIN
days.
ELMER SPECTRUM TWO FT-IR SPECTROMETER.

2.3. Identification of cellulose-producing bacteria 3. Results and discussion

The cellulose-producing bacterial isolate P3 was cultured for ten
days in HS medium under room temperature and further subjected to
genomic DNA isolation using the Macherey Nagel NucleoSpin® Micro­
bial DNA isolation kit (Cat No. 740235.50) by following the manufac­
turer’s instructions. The isolated genomic DNA was analyzed by agarose
gel electrophoresis, and the 16S rDNA amplification was done using the
universal primers 16SF (5′ -AGAGTTTGATCMTGGCTC-3′ ) and 16SR (5′ -
AAGGAGGTGWTCCARCC-3′ ) [5]. The PCR product obtained was
sequenced by Sanger sequencing (AgriGenome Labs Pvt Ltd., Kochi),
and the sequence data were analyzed by using the NCBI Basic Local
Alignment Search Tool (BLAST) [5]. For the phylogenetic analysis, the
maximum likelihood method with 1000 bootstraps of MEGA-X [6] was
used.
2.4. Characterisation of bacterial cellulose
A loop full of P3 isolate was inoculated into 15 mL of sterilized HS
medium and used as a starter culture. After ten days of inoculation, 5 %
(v/v) [7] of the starter culture was further added to 150 mL of sterilized
HS medium and kept at room temperature for 20 days. The thick pellicle
formed at the interface was then harvested and treated with 0.5 M
NaOH, followed by a wash with distilled water several times until the pH
became neutral. The film was dried in the oven at 45 ◦ C until the weight
(dry weight 4.46 g/L) became constant [8] and further used for
characterization.
The XRD patterns of oven-dried BC samples were recorded using
Bruker D8 advance with Cu Kα wavelength (λ = 1.5406 Å), operating
current 35 mA, and voltage 40 kV. The crystallinity index (CrI) % was
calculated by the Peak intensity method (Segal equation) [9] as given
below
I(002) − Iam
CrI(%) = × 100 (1)
I(002)
While screening, the apple and orange samples showed poor film
formation; thus, grape and pomegranate samples were selected for
bacterial isolation. The nine isolates obtained from HS agar plates were
purified, including 6 from pomegranate (P1-P6) and 3 from grape (G1-
G3). Each isolate was inoculated separately into HS medium, and the P3
isolate was found to have maximum film production; hence, it was
selected for detailed identification. By 16S rDNA gene sequencing and
the following BLAST analysis, P3 was identified as Novacetimonas han­
senii strain due to its 99.78 % identity with the Novacetimonas hansenii
strain NBRC 14820. The P3 isolate from pomegranate was inoculated
into HS medium and the film formed was washed, oven dried and used
for characterization.
The XRD pattern of the dried pellicle is given in Fig. (1. a). Here,
three significant peaks (1, 2, and 3) at 14.4◦ , 16.7◦ , and 22.5◦ are ob­
tained corresponding to planes, (1 0 0) plane of Iα or (1–10) of Iβ, (0 1 0)
of Iα or (1 1 0) of Iβ and (1 1 0) of Iα or (2 0 0) of Iβ respectively [11].
Thus, it shows a crystalline form of cellulose І.
The crystallinity index % of alkaline treated BC was calculated by
equation (1), showing 96.6 %, which is remarkable compared to existing
results. The CrI % of various BC samples produced by different micro­
organisms isolated from diverse sources is summarized in Table 1. The
table shows that cellulose produced by Novacetimonas hansenii P3 shows
the highest relative crystallinity.
The FTIR spectrum shows characteristic peaks at 1205 cm− 1 (sym­
metrical stretching vibration of C–O–C bond) [14], 1160 cm− 1
(C–O–C asymmetric stretching vibration), 1107 cm− 1 (C–O–C (1–4)
glycosidic linkages or skeletal vibration) [14,18], 1031 cm− 1 (CO
deformation) [19], 1054 cm− 1 (C–O–C pyranose ring skeletal vibra­
tion) (Fig. 1.b). The bands around 3345 cm− 1, 1425 cm− 1, and 1160
cm− 1 suggest the presence of cellulose I structure [20]. The FTIR results
provide clear evidence of cellulose structure and confirm the presence of
cellulose І.

2
S. Neelima et al. Materials Letters 333 (2023) 133622

Fig. 1. (a) The XRD spectrum of BC showing three significant peaks 1, 2 and 3, corresponding to 14.4◦ , 16.7◦ and 22.5◦ respectively. (b) FTIR spectra of dried BC
film. The absorption bands at 1205 cm− 1, 1160 cm− 1, 1107 cm− 1 and 894 cm− 1 are characteristic of cellulose structure.

3
S. Neelima et al.
4. Conclusion
A cellulose-producing bacteria, Novacetimonas hansenii strain, was
isolated from a rotten pomegranate sample for the first time. The BC
produced shows the highest relative crystallinity with cellulose І
structure.
CRediT authorship contribution statement
S. Neelima: Conceptualization, Methodology, Software, Data cura­
tion, Writing – original draft, Visualization, Investigation. S. Sreejith:
Methodology, Investigation, Resources, Visualization. Shamna Shaja­
han: Methodology, Investigation. Aparna Raj: Investigation, Re­
sources. L. Vidya: Resources, Investigation. V.M. Aparna: Software,
Resources. E.K. Radhakrishnan: Methodology, Validation, Investiga­
tion, Writing – review & editing, Visualization. C. Sudarsanakumar:
Conceptualization, Methodology, Validation, Investigation, Writing –
review & editing, Visualization, Supervision.
Declaration of Competing Interest
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.
Data availability
No data was used for the research described in the article.
Acknowledgment
This work was supported by the Department of Science & Technol­
ogy, Government of India (DST-Inspire) by providing the Junior
research fellowship (JRF) award for Ms. Neelima S. The author Ms.
Aparna VM acknowledges the University Grants Commission, Govern­
ment of India, for the award of JRF. The author also acknowledges DST-
SAIF Cochin for providing XRD facility.
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