Accepted Manuscript

Efficient and economic process for the production of bacterial cellulose from
isolated strain of Acetobacter pasteurianus of RSV-4 bacterium

Vinod Kumar, Devendra Kumar Sharma, Vasudha Bansal, Deepak Mehta,

PII: S0960-8524(18)31703-6
DOI: https://doi.org/10.1016/j.biortech.2018.12.042
Reference: BITE 20799

To appear in: Bioresource Technology

Received Date: 21 October 2018

Please cite this article as: Kumar, V., Sharma, D.K., Bansal, V., Mehta, D., Sangwan, R.S., Yadav, S.K., Efficient
and economic process for the production of bacterial cellulose from isolated strain of Acetobacter pasteurianus of
RSV-4 bacterium, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.042

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Efficient and economic process for the production of bacterial cellulose from isolated strain

of Acetobacter pasteurianus of RSV-4 bacterium

Vinod Kumar1, Devendra Kumar Sharma1, Vasudha Bansal1, Deepak Mehta, Rajender S

Sangwan1,2 and Sudesh Kumar Yadav1*

India
2
Academy of Scientific and Innovative Research (AcSIR), Sector 19, Kamla Nehru Nagar, Ghaziabad, Uttar

Pradesh- 201 002

* Corresponding Author: Email:- sudesh@ciab.res.in

Abstract:

In the present investigation, several residues from agro-forestry industries such as rice straw acid

hydrolysate, corn cob acid hydrolysate, tomato juice, cane molasses and orange pulp were

evaluated as the economical source for the production of bacterial cellulose. The bacterial

cellulose attained the significant yield of 7.8 g/L using tomato juice, followed by 3.6 g/L using

cane molasses and 2.8 g/L using orange pulp after 7 days of incubation. Furthermore, the

optimum pH and temperature of fermentation for maximum production of bacterial cellulose was

4.5 and 30±1oC. The identified bacterium Acetobacter pasteurianus RSV-4 has been deposited at

repository under the accession number MTCC 25117. The produced bacterial cellulose was

characterized through FTIR, SEM, TGA and DSC and found to be of very good quality. The

bacterial cellulose produced by identified strain on these various agro-waste residues could be a

cost effective technology for commercial its production.

Key words: Tomato juice, Bacterial cellulose, Acetobacter pasteurianus, Agro-residues, Scale

up

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1. Introduction:

Cellulose is the most abundant biopolymer on earth and has been recognized as the major

component of plant biomass as well as a representative of bacterial extracellular polymer.

Bacterial cellulose (BC) is structurally similar to that of the plant cellulose (PC) and made up of

β-1, 4 glucan, but has different physical properties. Bacterial synthesis of cellulose offers a more

cost-effective means of supply as microorganisms secretes cellulose in pure form which is free

from lignin, pectin, hemicelluloses, and phytate. While the presence of such components in plant

cellulose (PC) make its recovery difficult and relatively expensive (Numata et al., 2015; Jahan et

al., 2018). Besides high chemical purity, BC possesses many other unique features which make it

superior over PC. BC has numerous advantageous properties in the form of water retention,

compressibility, elongation, mechanical strength, large surface area, high porosity, and bio-

compatibility that can be widely used for biomedical applications (Campano et al., 2016; Costa

et al., 2017). All these properties have made BC a biomaterial of considerable commercial and

scientific interest. Generally, a nutritionally rich medium containing yeast extract and peptone

have been reported to support the production of BC in high quantity by Acetobacter strains.

Several studies have reported the requirement of complex medium such as mixture of glucose,

yeast extract, polypeptone, Na2HPO4.12H2O and citric acid monohydrate for the efficient

production of BC using Acetobacter sp. (Hestrin and Schramn, 1954; Gomes et al., 2013). In

order to find a new, low cost, and nutritionally rich culture medium for the industrial level

production of bacterial cellulose, several studies have been conducted by employing the use of

agriculture wastes such as orange, apple, pineapple, Japanese pear, grapes, fruit juice, maple

syrup, dry olive mill residue, etc and industrial by-products such as corn steep liquor (CSL)

(Kurosumi et al., 2009; Gomes et al., 2013; Lin et al., 2014). Till now, BC has been produced

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using synthetic media alone or in combination with additional components. However, intensive

academic and industrial research has recently been increasingly directed to study the novel and

sustainable ways to re-utilize biomass residues or recover valuables from them (Carreira et al.,

2011). Furthermore, strategies for utilization of agri-food related waste can significantly reduce

the volume of waste and allow the extraction of their ingredients to produce value added

products of edible as well as non-edible nature (Reddy and Yang, 2005). Such smart

technologies will not only reduce the major pollution problems but also provide economic gains.

One of the most widely studied and used cellulose-producing bacterial species is Acetobacter

xylinum including the strains ATCC 23769, 10145, 53582, AX5 and some others (Mohite et al.,

2013).

In this study, a process has been developed for the efficient production of bacterial cellulose

(BC) by using a newly identified strain (RSV-4) of Acetobacter pasteurianus using tomato juice

without employing any peculiar medium components. The developed bacterial cellulose has been

characterizations using TGA, DSC, FTIR, and SEM.

2. Materials and Methods

2.1 Procurement of reagents

Microcrystalline cellulose used for comparison studies was purchased from Sigma-Aldrich

Chemical Co., USA. All other chemicals and reagents used in the present study were of

analytical grade.

2.2 Evaluation of low cost substrates for BC production

Cellulose production was carried out using different low-cost substrates with a view to produce

at a low cost. Here, glucose was replaced with different low cost carbon sources including

industrial and agricultural wastes. The total carbon content of these sources used was equivalent

3
to 2.0 % sucrose. HS medium was used as control for BC production. In a preliminary set of

experiment, 10 g of different substrates mixed with 20 ml of distilled water in a 250 ml

Erlenmeyer flask, was mixed homogenously and sterilized at 121 °C for 30 min. Thereafter, the

flask was cooled at room temperature and inoculated with the cellulose mat slurry (~4%) and

flasks were incubated at 30 °C for 7 days under static conditions.

2.3 Purification of BC

The pellicle formed at the air–liquid interface of the production medium, was collected and

rinsed with water for thrice. It was then treated with 0.5 N NaOH at 80 °C for 30 min. In order to

neutralize NaOH, the pellicle was treated with 0.5% acetic acid solution. It was again washed

with water for thrice. The purified pellicle obtained was dried at room temperature under vacuum

until a constant weight was obtained and then weighed for determining the production of BC

(g/L). The presence of cellulose fibrils in the pellicle was confirmed by SEM, FTIR, TGA, and

DSC. Microcrystalline cellulose (Sigma, USA) was used as reference compound.

2.4 Characterization of BC

2.4.1. Scanning electron microscopy

The morphology of bacterial cellulose was studied by Scanning Electron Microscopy (SEM).

Thin layers of BC samples were coated with gold using an ion sputter coater and observed with a

Jeol JSM-JCM 7000F, Japan microscope operated at 20 kV.

2.4.2 Fourier transform infrared (FT-IR) spectroscopy

FT-IR spectra were recorded on an Agilent Technologies Cary 660 FTIR in the 4000–400 cm−1

range under ATR mode. The spectra were recorded with a resolution of 4 cm−1 along with an

accumulation of 8 scans.

2.4.3 Thermogravimetric analysis (TGA)

4
Thermogravimetric analysis was performed using a thermogravimetric analyzer (Q50 V20.13

Build 39), in an alumina crucible with approximately 5 mg of each sample. During analysis,

temperature was varied between 25 and 600 °C with a heating rate of 10 °C/min in an oxidizing

atmosphere (air) ASTM E-1868.

2.5. Process improvement for maximum production of bacterial cellulose

To obtain the maximum production of BC by the Acetobacter pasteurianus (RSV-4), improved

physiological conditions were optimized following temperature, and inoculum preparation. All

the optimization experiments were carried out in replicates, and the data is presented as the

means of the replicates.

2.6 Scale-up production of bacterial cellulose

The production of BC was carried out in 10 L production medium in trays which were

particularly designed for tray type bioreactor. It was a horizontal prototype equipped with trays

of holding capacity of 10 L production medium. These trays were connected with silicon tube for

circulation of medium and maintenance of medium level during the production of bacterial

cellulose by the isolate. This tray type bioreactor had an in-situ sterilization facility using steam.

The incubation was maintained at optimum temperature for cellulose production.

3 Results and discussion

3.1 Low cost substrates for BC production

Different agro-residues such as orange pulp, cane molasses, corn cob, rice straw and tomato juice

were evaluated for maximum production of bacterial cellulose using Acetobacter pasteurianus

RSV-4. The experiments were planned and executed in 1.0 L of selected different residues as

production media for BC. The HS medium was used as a control for comparison studies. The BC

production was 4.8 g/L of tomato juice in comparison to 3.7 g/L of HS medium (Table 1). While

5
cane molasses was found to be the second most producer of BC to the extent of 3.6 g/L by the

identified bacterial strain. On the other hand, rice straw and corn cob did not support the BC

production. Orange pulp, a low-cost substrate could produce 2.8 g BC/L of the medium. Similar

to the present investigation, few researchers have also utilized cane molasses for BC production

by other Acetobacter strains like Acetobacter xylinum and Acetobacter sp. V6 (Keshk and

Sameshima, 2006; Numata et al., 2015). In addition, use of crude distillery effluent has been

reported for 85 g/100 ml of BC production (Jahan et al., 2018). Similarly BC productivity has

been reported to be increased from 2.2 to 5.9 g/ L after 15 days of incubation by

Gluconacetobacter xylinus in fructose media supplemented with 2% HNTs (Tian et al., 2018).

Also. 4.34 g/L of BC production has been reported using sugarcane molasses (10.77 %) with

corn steep liquor (12.47 %) at 31oC and pH 6.5 (Singh et al., 2017).

3.2 Inoculum preparation

The use of various waste agro-residues has suggested that tomato juice was the best cellulose

production medium for the strain of Acetobacter pasteurianus RSV-4. Therefore, the process

optimization was initiated straight from the inoculum preparation on tomato juice media. The

inoculum was raised under static culture conditions in tomato juice and HS. HS medium was

used as control medium and subsequently, cellulose production in the tomato juice medium was

evaluated. Notably, higher cellulose yield (7.6±0.3 g/L) was found in the tomato juice media

compared to HS medium (4.7 ±0.26 g/L).

3.3. Effect of temperature on BC production

BC production by Acetobacter pasteurianus RSV-4 was examined in the temperature range of 20

to 40 °C. All other experimental conditions were same as described previously. Acetobacter

pasteurianus RSV-4 was observed to produce the maximum amount of BC at 30 oC (7.7 g/L). A

6
decline in BC production was recorded above this temperature. The yield of BC was declined

drastically above 40 oC (1.2 g/L). Earlier studies have reported maximum BC production

between 28-30 oC temperature (Sun et al., 2005; Keshk and Sameshima, 2006; Numata et al.,

2015; Singh et al., 2017; Buldum et al., 2018).

3.4 Scale up production of bacterial cellulose

Cellulose production was scaled upto 10.0 L of the tomato juice production medium in the trays

employing adapted strain of Acetobacter pasteurianus RSV-4. In a tray containing 10.0 L of the

production medium, 2.4 cm thick bacterial cellulose mat with 67.5 g of dry weight (6.8 g/L) was

produced. This yield was slightly decreased to the amount of cellulose produced in 1.0 L of the

production medium. Suwannapinunt et al. (2007) has reported the scale-up in the cellulose

production by Acetobacter xylinum TISTR976 in 1.5 L of the production medium and the yield

was 4.73 g/L. Song et al., (2009) has documented the use of 50 L of bioreactor of spherical type

bubble column and reported 5.6 g/L of BC production in 0.4 % agar, 1vvm air at 3 days.

Hungund et al., (2013) reported the use of bacterial strain Gluconacetobacter persimmonis GH-2

for cellulose production from various natural carbon sources provided as 2% in HS medium.

Some organisms have been reported for the production of 5.75, 5.98, 6.18 and 8.08 g/L of

bacterial cellulose with the usage of molasses, watermelon, orange juice and muskmelon that

were provided as carbon and nutrient sources. In addition, Yoshino et al., (1996) reported the

production of bacterial cellulose using Acetobacter pasteurianus AP-1SK in static culture. They

developed a system in which cellulose could be formed on an oxygen-permeable synthetic

membrane on a liquid surface. Comparative efficacy of pineapple waste medium (PIWAM) and

pawpaw waste medium (PAWAM) in the production of biocellulose by Acetobacter

pasteurianus strain PW1 has also been reported (Adebayo-Tayo et al., 2017). Their yield of

7
bacterial cellulose was reported to be 0.1 to 3.9 g/L and 0.2 to 1.0 g/L for PIWAM and PAWAM

medium, respectively.

3.5 Characterization of bacterial cellulose

The bacterial cellulose (BC) produced by Acetobacter pasteurianus RSV-4 on tomato juice was

characterized by scanning electron microscopy (SEM), fourier transform infrared spectroscopy

(FT-IR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) in order

to evaluate its structural features and elucidate the molecular validity of special type of cellulose.

3.5.1 SEM analysis of BC

SEM analysis of bacterial cellulose was performed on cellulose dried sheets under 5000X and

8500X magnification. The cellulose sheet was found to be fibrous with irregular size and shape.

However, the scanning image of dried sheet of bacterial cellulose under 7000X magnification

showed fine cellulose ribbon like fibrils. The size of the ribbons was in the range of 50-60 nm.

Similar observations have been made on the bacterial cellulose produced by other bacterial

species (Jahan et al., 2012; Jahan et al., 2018).

3.5.2 FT-IR analysis of BC

The analysis of bacterial cellulose produced from the tomato juice by Acetobacter pasteurianus

RSV-4 showed the peaks at 3440 cm-1, 2926 cm-1, 1300 cm-1, 1440 cm-1, 1163 cm-1 and 1040

cm-1. For the pure cellulose spectrum, distinguish peaks of 3350 cm-1 and shouldering around

3400 cm-1 to 3500 cm-1 signified the presence of O-H stretching. Furthermore, peaks originated

from 2800 cm-1 to 2900 cm-1 indicated the stretching of C-H. Similarly, peaks at 1160 cm-1 and

1035 cm-1 to 1060 cm-1 exhibited the stretching of C-O-C as well as C-O. In addition, other

fingerprint regions for cellulose were found in the peaks around 1300 cm-1 and 1400 cm-1 that

revealed the bending of C-H and CH2, respectively (Marchessault and Sundararajan, 1985).

8
Results confirmed that the BC produced by Acetobacter pasteurianus RSV-4 on tomato juice

was cellulose with high purity.

3.5.3 TGA analysis of BC

The thermogravimetric analysis of BC produced by Acetobacter pasteurianus RSV-4 on tomato

juice showed the maximum rate of weight loss around 343 °C. Previous studies has reported the

maximum rate of weight loss around 370 °C (Surma-S´lusarska et al., 2008b). Similarly,

maximum weight loss of BC produced on barley straw has been reported as 333°C (Sun et al.,

2005). In addition, earlier studies have also reported the similar thermal stability and temperature

degradation behavior of bacterial cellulose (Yoshino et al., 1996).

4. Conclusion

In the present study, tomato juice has been found to be useful medium for bacterial cellulose

production by the identified bacterial strain Acetobacter pasteurianus RSV-4. Scale up process

has been optimized for the 10 L production media and the highest production of BC was

achieved to the level of 7.8 g/L. Furthermore, the produced bacterial cellulose has been

characterized and found to be of high purity. Hence, the developed sustainable process of BC

production on agro-residues could be of high commercial significance.

Acknowledgements

Authors are thankful to CEO, CIAB for continuous support and encouragement. The authors

express their sincere thanks to the Department of Biotechnology (DBT), Government of India,

New Delhi and BIRAC, GOI for the financial support in the laboratory.

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 Table and Figure Legends

Table 1: Evaluations of low cost medium for bacterial cellulose production after 7 days
incubations

Table 2: Scale up of cellulose production by Acetobacter pasteurianus RSV-4 upto 10.0 L

Figure 1 TGA analysis of bacterial cellulose produced by Acetobacter pasteurianus RSV-4

on tomato juice.

13
14
Table 1: Evaluations of low-cost medium for bacterial cellulose production after 7 days
incubations

S.No. Production medium Dry wet. Productivity

1. HS Medium control 3.7±0.12 0.52±0.02

3. Orange pulp medium 2.80±0.14 0.40±0.04

3. Cane molasses medium 3.60 ±0.14 0.52±0.19

4. Corn cob medium 1.80±0.04 0.25±0.01

5. Rice straw medium 0.80±0.002 0.11±0.01

6. Tomato juice medium 4.8 ±0.21 0.68±019

15
Table 2: Scale up of cellulose production by Acetobacter pasteurianus RSV-4 upto 10.0 L of the
product

Production size Volume of Final dry weight Bacterial cellulose (g/l) ion
(g) medium
(cm3) the medium (Lit)

30 x 20 x 4.5 1 7.8±2.76 7.8±2.76

85 x 35 x 15 5 37.5 ±2.76 7.5±2.76

85 x 35 x 15 10 67.5±2.76 6.8±2.76

16
Highlights:

Efficient and economic process for production of bacterial cellulose using Acetobacter pasteurianus RSV-

4.

17