Int.J.Curr.Microbiol.App.

Sci (2020) 9(11): 3890-3898

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 9 Number 11 (2020)
Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2020.911.465

Edible Bacterial Cellulose Production by Acetobacter xylinum 2526 with

Response Surface Methodology (RSM) Optimized Processing Parameters in
Medium Prepared Using Tofu Whey, Soymilk Okara and Defatted Soy Flour

Samlesh Kumari*

Centre of Excellence on Soybean Processing and Utilization, ICAR-Central Institute of

Agricultural Engineering, Bhopal-462038, Madhya Pradesh, India

*Corresponding author

ABSTRACT

A biopolymer bacterial cellulose is obtained by Acetobacter species and this

biopolymer has unique properties because of that it is considered better than plant-
Keywords
based polymers. In the present investigation attempt has made for low-cost media
Acetobacter preparation for Acetobacter xylinum NCIM 2526 growth and bacterial cellulose
xylinum, production. Carbohydrate rich fraction was extracted from the soybean industry waste
Bacterial Cellulose, products (Tofu whey, soymilk okara and deffated soy flour) and this fraction was used
Tofu whey, soymilk for the replacement of sugar in the standard medium (Hestrin schramn) composition.
okara and deffated
soy flour
Acetobacter xylinum (NCIM 2526) was cultured in the developed medium and a
process is was developed for the maximum yield of bacterial cellulose. The design of
Article Info the study optimised three conditions: inoculum level 8.47%, incubation temperature
28.85°C and soluble carbohydrate rich fraction 90% (conc.) of soybean by products in
Accepted:
06 October 2020
dilute form. Yield of bacterial cellulose was 4.66gm/ 100ml of medium which was
Available Online: more than the predicted value 4.55gm/ 100ml of RSM. Finally, it is concluded that the
10 November 2020 process developed for the production of edible bacterial cellulose increases with use of
particular carbon sources in some media and selection of medium and growth
conditions greatly affect the yield of edible bacterial cellulose.

Introduction al., 1991). Bacterial cellulose produced at the

air-liquid interface of medium used for the
Bacterial cellulose (BC) is a nano-structured production and it is popularly known as
material synthesized by some species of natade-coco. At the air-liquid interface of
bacteria belonging to the genera Aerobacter, sugary rich medium is popularly known as
Agrobacterium, Rhizobium, nata de-coco. It is an organic high dietary
GluconAcetobacter (formerly called as fiber food product, high in cellulose, low in
Acetobacter xylinum), Acetobacter (Ross et fat and calories and contains no cholesterol.

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The cellulose is recognized by the FDA as (COD) values. Traditionally, fermentation

edible and the GluconAcetobacter is a non- wastewater is treated by activated sludge. In
pathogenic cellulose-producing food grade microbial fermentations, the cost of the
bacterium (Kersters et al., 2006). As bacterial fermentation medium can account for almost
cellulose fibrils are highly amorphous and 30% of the total cost. Medium costs limit
generated as a never-dried membrane in a commercial use of bacterial cellulose or nata.
nearly pure form without lignin and So use of alternative substrates is one way to
hemicelluloses as that of plant cellulose. reduce the cost of production of bacterial
Further, more the purity and quality of cellulose. Development of a cost-effective
bacterial cellulose far better as compared to culture medium to obtain maximum product
plant based cellulose. BC is characterized by yield will solve this problem. Most of the
higher purity, higher degree of studies on BC production by Acetobacter
polymerization, higher crystallinity, higher strains have been carried out in media
water-absorbing and holding capacity, higher containing pure sugar as carbon source, such
tensile strength and good biocapatibility, as glucose, sucrose, fructose, mannitol, and
compared to plant cellulose (Klemm et al., arabitol (Jung et al., 2010). An alternative
2001; Ul-Islam et al., 2012). These enhanced sugary nutrient rich substrate that can be used
properties have made BC become considered for fermentation is soybean industry waste or
a kind of highly functional biopolymer which low value products. Soybean waste is by
has application potential in bio-medicine, product in production of tofu and soymilk.
cosmetics, high-end acoustic diaphragms, Soybean waste commonly dumps directly to
papermaking, food industry and other areas the water sewer and made environmental
(Aramwit and Bang 2014; Shah et al., 2013). problems like eutrotification. Soybean waste
contains 23% hemicellulose, 16% cellulose
In economic terms market potential of thin dan 28% protein.
film bacterial cellulose is in demand including
cbdnfb acoustic diaphragms, artificial skin, pulp and This research aimed to formulate the media
paper industry BC is characterized by higher for production of bacterial cellulose and
purity, higher degree of polymerization, optimize the processing parameters for
higher crystallinity, higher water-absorbing production of good quality bacterial cellulose.
and holding capacity, higher tensile strength Furthermore, analysis of BC produced in the
and good bio-scapatibility, compared to plant formulated media under optimized conditions.
cellulose (Klemm et al., 2001; Ul-Islam et al., Thus, the new media proposed in the present
2012). Although BC has excellent potential as study provides a potentially economical and
material in many novel applications, the low environmentally-friendly process for the
yield and high production cost hinder its production of BC.
industrial-scale production and broad range of
application. Therefore, looking into Materials and Methods
inexpensive feedstock as the culture medium
is helpful to make BC production more cost- Materials collection
effective (Cavka et al., 2013)
Acetobacter xylinum NCIM 2526 was
Low-cost waste of soybean industry is rich in purchased from National Collection of
sugar, low-molecular weight sugars, glycerol, Industrial Microorganisms (NCIM), Pune,
proteins, vitamins and other nutrients, India. Hestrin Schramn (HS) medium and
resulting in high chemical oxygen demand other chemicals were purchased from Hi

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media. Tofu whey, soymilk okara and Acetobacter xylinum (NCIM 2526) grown on
deffated soy flour were collected from Centre HS agar slants was inoculated into sterilized
of Excellence on Soybean Processing & media containing glucose (2g), yeast extract
Utilization, ICAR-Central Institute of (0.5g), peptone (0.5g), citric acid (0.115g),
Agricultural Engineering, Bhopal(India). The disodium hydrogen phosphate (0.11g). The
laboratory work for the present investigation reagents were dissolved in distilled water, the
on the production of cellulose from soybean volume was brought up to 100 mL and pH
industry using Acetobacter xylinum NCIM was adjusted to 6.0 with addition of
2526 was carried out at Centre of Excellence hydrochloric acid or sodium hydroxide and
on Soybean Processing & Utilization. sterilized at 1210C for 15min. The inoculated
media was incubated statically at 30C for 4
Formulation of media days. Sub culturing was done to maintain the
purity and viability of microorganism as
The composition analysis of defatted soy shown in Figure.2.
slour, soymilk by product (okara) and tofu by
product (whey) was done using using Production of bacterial cellulose
standard AACC procedures AACC (2000).
Soybean by products: okara, tofy whey and Processing conditions for production of
defatted soy flour were analysed for their bacterial cellulose processing conditions were
proximate composition. Soluble optimised using response surface
carbohydrates or sugars were extracted from methodology. Dependent variables are
the by products with some modification in the inoculum level, soluble carbohydrates fraction
method given by Rupérez, 2003. The protocol and incubation temperature. As shown in the
used is mentioned in the Figure.1. table.1.

Culture maintenance and preparation of Harvesting of bacterial cellulose

inoculum
The Bacterial Cellulose layer formed after
Acetobacter xylinum (NCIM 2526) culture 15–20 days was harvested when it was about
obtained from National Collection of 0.8–1.0 cm thick, washed repeatedly with
Industrial Microorganisms (NCIM), Pune, water to remove glacial acetic acid. The layer
India. The culture was maintained on HS of Bacterial cellulose was immersed in water
medium with composition as mentioned by for 24 h with repeated changing of water to
Hestrin and Schramm (Sherif et al., 2006). remove the sour odour.
The composition of medium was (g/100mL):
glucose (2g), yeast extract (0.5g), peptone BC purification and quantification
(0.5g), citric acid (0.115g), disodium
hydrogen phosphate (0.11g). After cultivation, the BC membranes were
rinsed three times with deionized water and
The reagents were dissolved in distilled water, then soaked in boiling 2% (w/v) NaOH
the volume was brought up to 100 mL and pH solution for 1 h to remove and dissolve the
was adjusted to 6.0 with addition of bacteria cells entrapped in the microfibers.
hydrochloric acid or sodium hydroxide and
sterilized at 1210C for 15min. A. xylinum was After turning transparent, the BC membranes
streaked on these slants and incubated at 30C were washed with deionized water several
for 4 days. times to make the membranes neutralized.

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The purified cellulose was dried at 60°C Optimization study data were analyzed by
overnight and weighted. For each membrane, Completely Randomized Design as per the
triplicate experiments were performed, and methods described by Steel and Torrie (1980).
the mean values were calculated. Storage study data were examined using
Factorial CRD. The values for microbial
Analysis of bacterial cellulose counts were log transformed before analysis.

Determination of Dry Weight Results and Discussion

Cellulose Harvested microbial cellulose Okara, tofy whey and defatted soy flour used
washed with NaOH solution 2% (w/v) for 30 for the extraction of soluble carbohydrates
min and thoroughly washed with distilled fraction were analysed for their composition
water thoroughly and record the wet cellulose as results shown in the table.1
weight and dried at 75°C in an oven for 6
hours, cooled to ambient temperature and Soluble carbohydrate rich fraction obtained
record the dry cellulose weight. The dry from the soy by products (Okara, tofy whey
weight of the cellulose obtained was and defatted soy flour) was used for the
calculated. formulation of media for Acetobacter xylinum
(NCIM 2526). In this formulation no
Moisture content additional sucrose was added and this portion
of media was replaced with soy by-products
The moisture content (%w/w) of bacterial soluble carbohydrate rich fraction as shown in
cellulose was determined based on the weight the Table.3.
loss of bacterial cellulose when dried at 75°C.
After harvesting, the produced BC in in the
Moisture content % = (wet weight ─ Dry standard and developed media was measured
weight) × 100 Dry weight for its yield in terms of growth Acetobacter
xylinum (NCIM 2526) of which refers to the
Percent yield optical density and pH of the per liter of
medium as shown in the Table.3.
Percent yield of BC was calculated by
following Equation Percent yield = Dry Optimization of processing conditions for
weight of BC × 100 Weight of carbon source production of high yield of bacterial
used in production medium. cellulose using response surface
methodology (RSM)
Statistical analysis
Adequacy of model
Excel was used for feeding and analysis raw
data obtained from different experiments and The adequacy of model was tested using F-
further Graph pad prism was used to represent value and coefficient of determination (R2).
data. Data were analyzed by using completely The model was generally considered adequate
randomized factorial design. Analysis of when (a) the calculated F-ratio was more than
variance was conducted; when significant that of table value; (b) the R2 value of more
effect was detected, the means were separated than 80 percent (Filmore et al., 1976), and (c)
by Fisher Least Square Analysis. an adequate precision value greater than 4.0.

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Table.1 Proximate analysis of soya by-products (Defatted Soy Flour, Soymilk Okara and Tofu
whey

Parameters Defatted Soy Soymilk by product Tofu by product

Flour (Okara) (whey)
Moisture 4.37 ± 0.03 67 ± 0.61 88.33 ± 0.45
Protein 50.12 ± 0.31 18 ± 0.52 0.81 ±0.29
Fat - 08 ±0.44 0.78 ±0.32
Ash 6.31 ± 0.19 4.1 ±0.08 3.51 ±0.11
Carbohydrates 43.12 ±0.22 12 ±0.36 0.95 ±0.21

Table.2 Formulation of media with soy by-products extracts and comparison with commercial
media

S.No. Components Tofu whey –based (SCPS) based media

medium
1. Sucrose 10% -
2. Yeast extract 0.25% 0.25%
3. K2HPO4 0.5% 0.5%
4. (NH4)2SO4 0.6% 0.6%
5. MgSO4 0.2% 0.2%
6. Ammonium 0.25% 0.25%
sulfate
7. Calcium sulfate 0.25% 0.25%
8. Tofu whey 1lit SCRF with distilled
water
pH adjusted to 6.0

Table.3 Formulation of media with soy by-products extracts and comparison with commercial
media

Medium 2526 (OD) 2526 (pH) 2529 (OD) 2529(pH)

HS media 3.666 ± 0.02 4.2±0.04 3.666±0.02 4.2±0.03
Tofu whey –based 1.981 ± 0.03 4.7± 0.04 1.882±0.07 5.1±0.04
medium
(SSP) based media 2.771± 0.05 4.6 ±0.03 1.999±0.04 4.9 ±0.08

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Table.4 Optimized parameters for bacterial cellulose production and summery of model statics

Optimized parameters
Cond. Value pH Dry wt. of BC (g)
Inoculum level 8.47% Predict Exp. Predict Exp.
(%) 3.46793 3.3368 4.55263 4.66
Temperature 28.85°C
(°C)
Carbohydrate 90%
rich fraction
(%)
Model Summary Statistics
Quadratic pH BBC
R-Squared 0.9562 0.9887
Adj R- Squared 0.9168 0.9785
Pred R-Squared 0.8583 0.9103

Fig.1 Extraction of soluble carbohydrates and formulation of media for Acetobacter xylinum
(NCIM 2526)

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Fig.2

Fig.3 Desirability values and plots obtained in RSM

Response Surface optimization of 7.0.0) of Response surface methodology

Variables for production of bacterial technique was applied for the optimizating the
cellulose levels of various unknown variables e.g.
percent of inoculums level, temperature of
As there was scanty information on the incubation and percent carbohydrate rich
production of bacterial cellulose by utilising fraction obtained from soy by-products. To
soybean by products, the Central Composite optimize the yield of bacterial cellulose in the
Rotatory Design package (Design Expert formulated media and growth Acetobacter

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xylinum (NCIM 2526). Central composite suitable for safe environments such as food
design of response surface methodology with and medical applications. Cellulose
three levels was employed and results are production increases with use of particular
mentioned in Table.4. A CCRD was used to carbon sources in some media, but not in
determine the effects and interactions of three others and yield is greatly affected by
factors. selection of media. It could be concluded that
optimized conditions are suitable for bacterial
Highest yield of bacterial cellulose 4.66 cellulose production.
gm/ltr of media was obtained at 8.47%
inoculum level, 28.85°C, and 90 % References
Carbohydrate rich fraction (%) with addition
of distilled water. Moisture content of the Approved laboratory methods. St. Paul:
bacterial cellulose obtained with best American Association of Cereal
optimised conditions was 83.27%. Chemists; 2000.
Aramwit P, Bang N. The characteristics of
Desirability value is 0.877 the range for all bacterial nanocellulose gel releasing
the three parameters in respect of bacterial silk sericin for facial treatment. BMC
cellulose yield and plots obtained in Biotechnol. 2014 [cited 2017 May
application of RSM are shown in the Figure 3. 04];14:1. DOI:10.1186/s12896-014-
The results indicate the optimized process for 0104-x.
the production bacterial cellulose by Cavka A, Guo X, Tang S-J, et al., Production
utilization of soy industry by products is a of bacterial cellulose and enzyme from
potential and economic approach. waste fiber sludge. Biotechnol
Biofuels. 2013 [cited 2017 May
Future Prospects 04];6:1. DOI:10.1186/1754- 6834-6-
25
Due to ever increasing population in urbans, Filmore, E. B., Kramer, A. and Gerald, K.,
the management of food industry waste is a (1976). Response surface techniques.
major challenge. Effective recycling of this In Filmore FE, Kramer A, Gerald K
waste at source is one of the most efficient (eds.), Systems Analysis for Food
waste managements. Thus, using this Industry AVI, Westport, Connecticut,
technology, soybean industry by products or 127.
waste can be recycled at source for Jung, H. I., Lee, O. M., Jeong, J. H., Jeon, Y.
minimizing environmental pollution with D., Park, K., Kim, H. S., An, W. G.
additional economic benefit and employment and Son, H.J., Production and
generation. characterization of cellulose by
Acetobacter sp. V6 using a cost
Bacterial cellulose was produced by effective molasses-corn steep liquor
Acetobacter xylinum (NCIM 2526) using medium. Appl. Biochem. Biotechnol.,
Okara, tofy whey and defatted soy flour as 2010; 162: 486-97.
substrates proved to be an efficient substrate Kersters, K., Lisdiyanti, P., Komagata, K. and
for production of bacterial cellulose when Swings, J., The family
compared with standard medium or substrate. Acetobacteraceae: the genera
In addition, it does not involve toxic and any Acetobacter, Acidomonas, Asaia,
kind of hazardous materials in producing GluconAcetobacter, Gluconobacter
Bacterial cellulose, which is excellent and and Kozakia. (in) The Prokaryotes.

3897
 Int.J.Curr.Microbiol.App.Sci (2020) 9(11): 3890-3898

Springer, New York. 2006; 163-200. 98:1585–1598.

Klemm D, Schumann D, Udhardt U, al., Sherif K., Taha R. and Sameshima K. (2006),
Bacterial synthesized cellulose- “Bacterial Cellulose Production from
artificial blood vessels for Beet Molasses”, African Journal of
microsurgery. Prog Polym Sci. 2001; Biotechnology, 5 (17): 1519-1523.
26:1561–1603. Steel, R. G. D., and Torrie, J. H., (1980).”
Ross P, Mayer R, Benziman M. Cellulose Principles and procedure of statistics -
biosynthesis and function in bacteria. a biometrical approach”. 2nd Ed.
Microbiol Rev. 1991; 55:35–58. McGraw-Hill Kogakusha Ltd.,
Rupérez P, Toledano G: Celery by-products Japan.pp:137.
as a source of mannitol. Eur Food Res Ul-Islam M, Khan T, Park J K. Water holding
Technol 2003, 216:224-226. and release properties of bacterial
Shah N, Ul-Islam M, Khattak WA, al., cellulose obtained by in situ and ex
Overview of bacterial cellulose situ modification. Carbohydr.Polym.
composites: a multipurpose advanced 2012;88:596–603.
material. CarbohydrPolym. 2013;
How to cite this article:

Samlesh Kumari. 2020. Edible Bacterial Cellulose Production by Acetobacter xylinum 2526
with Response Surface Methodology (RSM) Optimized Processing Parameters in Medium
Prepared Using Tofu Whey, Soymilk Okara and Defatted Soy Flour.
Int.J.Curr.Microbiol.App.Sci. 9(11): 3890-3898. doi: https://doi.org/10.20546/ijcmas.2020.911.465

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