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Optimization Production of Biocellulose by

Acetobacter Xylinum 0416 Using Response
Surface Methodology

Conference Paper September 2015

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Norliza Abd.Rahman
National University of Malaysia
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 Optimization Production of Biocellulose by Acetobacter Xylinum
0416 Using Response Surface Methodology
S. M. Mohammad, N. Abd. Rahman*a, M. S. Khalil, S. R. S. Abdullah
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia
43600 UKM Bangi, Selangor, Malaysia
a liza@eng.ukm.my

ABSTRACT
Bacterial cellulose (BC) is a biopolymer produced by Acetobacter xylinum has high purity, high water holding capacity, good
mechanical strength, elasticity, high crystallinity and high porosity compared to plant cellulose. However, the production costs
of BC are very high due to the use of quite expensive culture media such as HS medium. In this research, an investigation was
conducted to evaluate the possibility of using varied types of coconut water as nutrient and carbon source to replace pure carbon
sources as the substrate for the synthesis of bacterial cellulose by Acetobacter xylinum 0416. The objective of this study was to
optimize production of bacterial cellulose in coconut water medium using Response Surface Methodology (RSM) based on the
central composite design (CCD). The research utilized samples at difference temperatures (28C, 30C and 32C), pH (4, 5 and
6) and three types of medium (old, young and mixed coconut water). Each sample contained 100 mL of medium in 250mL
conical flask and incubated for 7 days. The bacterial cellulose film produced by Acetobacter xylinum was boiled with 0.5M
Sodium Hydroxide (NaOH) for 30 min, washed with 10% Hydrogen peroxide (H 2O2) and finally washed with deionized water
to neutralize the BC. The results showed that the types of medium, pH and temperature of cultivation had affected the yield of
bacterial cellulose. Through response surface methodology (RSM), the optimum condition for bacterial cellulose was old coconut
water, pH 4.16 and cultivation temperature at 30.06C. Thus, the obtained optimum conditions for bacterial cellulose production
are promising results to overcome high BC production costs.

Keywords: Temperature, pH and medium, Fermentation, Acetobacter xylinum 0416, Coconut water, Bacterial cellulose, RSM

ABSTRAK
Selulosa bakteria (BC) adalah biopolimer yang dihasilkan oleh Acetobacter xylinum di mana mempunyai ketulenan tinggi,
keupayaan pegangan air yang tinggi, kekuatan mekanikal yang baik, keanjalan, penghabluran tinggi dan porositi yang tinggi
berbanding dengan selulosa tumbuhan. Walau bagaimanapun, kos pengeluaran BC adalah sangat tinggi kerana menggunakan
kultur media yang agak mahal seperti medium HS. Dalam penyelidikan ini, satu kajian dijalankan untuk mengkaji penggunaan
air kelapa sebagai nutrien dan sumber karbon bagi menggantikan sumber karbon tulen sebagai substrat untuk sintesis selulosa
bakteria oleh Acetobacter xylinum 0416. Objektif kajian ini adalah untuk mengoptimumkan pengeluaran selulosa bakteria dalam
medium air kelapa dengan menggunakan Kaedah Respon Permukaan (RSM) berdasarkan reka bentuk komposit pusat (CCD).
Kajian ini telah dijalankan dengan menggunakan sampel berdasarkan perbezaan suhu (28C, 30C dan 32C), pH (4, 5 dan 6)
dan tiga jenis medium (air kelapa muda, tua dan campuran). Setiap sampel mengandungi 100ml medium fermentasi dalam setiap
250ml kelalang kon dan dieram dalam inkubator selama 7 hari. Filem selulosa bakteria yang dihasilkan oleh Acetobacter xylinum
dirawat dengan dididihkan didalam 0.5m Natrium Hidroksida (NaOH) selama 30 minit, dilunturkan dengan 10% Hidrogen
peroksida (H2O2) dan kemudian dibasuh dengan air suling untuk meneutralkan BC. Hasil kajian menunjukkan bahawa jenis
medium, pH dan suhu.mempengaruhi penghasilan selulosa bakteria. Dengan menggunakan kaedah permukaan respons (RSM),
keadaan optimum untuk selulosa bakteria adalah dengan menggunakan air kelapa tua sebagai jenis medium fermentasi, pH 4.16
dan suhu pada 30.06C. Oleh itu, keadaan optimum yang diperoleh untuk pengeluaran merupakan kejayaan awal untuk mengatasi
kos pengeluaran BC yang tinggi.

Kata kunci: Suhu, pH dan medium, Fermentasi, Acetobacter xylinum 0416, Air kelapa, Selulosa bakteria, RSM

biotechnology areas. However, to obtain cellulose from plant,

INTRODUCTION
Cellulose, the most abundant biopolymer in the biosphere,
composes glucose monomers with -1, 4 glucosidic bonds.
Cellulose is commonly used in food, biomedical, textile and
separation and purification processes require harsh chemicals,
such as alkali and acidic compounds to remove hemicellulose
and lignin structures. The wastes from these processes cause
environmental pollution and damage the structure of native
cellulose. (Cakar 2014). In the era of declining forests, global

1
climate changes, continuing expansion of industrialization, it
is reasonable to consider the consequences of an alternative
source of cellulose. Thus, use of bacterial cellulose can reduce
the dependency on the plant cellulose.
Bacterial cellulose (BC) is one of pure product in the
form of cellulose produced by bacteria with a formula of
(C6H10O5) n (Petersen & Gatenholm 2011). It is also known as
microbial cellulose. The bacteria which can produce cellulose
are from Acetobacter, Rhizobium, Agrobacterium, Aerobacter,
Achromobacter, Azotobacter, Salmonella, and Sarcina genera
(Chawla et al. 2008). However, Acetobacter is the only species
which can synthesize enough cellulose for commercial
purposes and produce the most efficient bacterial cellulose
(Bielecki et al. 2004).
Nowadays, bacterial cellulose has been used in various
fields including textile, papermaking, food, pharmaceutical,
waste treatment, broadcasting, mining and refining (Chawla et
al. 2009). However, the use of bacterial cellulose is not suitable
to meet the market due to high costs, particularly for the
preparation of the medium (Chawla et al. 2009). Besides that,
the cultivation medium for bacterial cellulose production
mainly consists of glucose and sucrose. Common medium used
for bacterial cellulose production was corn steep liquor-
fructose (CSL-Fru) and, Hestrin and Shramm medium which
contains mixed chemicals and carbohydrate. These types of
medium are cost effective since it uses many types of
chemicals in order to prepare it. Therefore, to develop the
production of bacteria cellulose, the media should be cheap and
easily available.
As we know, in Malaysia, there are a lot of organic
wastes from different stages of agro industrial productions that,
in many cases, cannot be marketed due to their poor quality.
However, they are rich in sugars such as glucose, fructose and
sucrose, as well as nitrogen and vitamins that are useful for
cellulose biosynthesis (Castro et al. 2010). The coconut water
wastescontain high concentration of biodegradable organic
material such as carbohydrate that can be utilized for the
production of organic acid. These waste discharges to the
environment are left untreated causing a serious environmental
pollution. Thus, this study used coconut water by converting it
into useful and higher value added products (BC), in addition
to overcome the problem of disposal of the waste.
The cultivation method and the growth media influence
the ability of A. xylinum to produce BC (Coban and Biyik,
2011). Before coconut water can be selected as fermentation
media for producing BC, their function as the best substrate
should be defined. This work aimed to optimize the bacterial
cellulose production using coconut water as a substrate that
consists of carbon sources and nitrogen sources. In this
research, three variables, which are pH, temperature and types
of medium, were evaluated with intention to search for the
optimum condition to enhance production of bacterial cellulose
using coconut water as medium through Response Surface
Methodology (RSM).
2
MATERIALS AND METHODS
MICROORGANISM
Acetobacter xylinum 0416, an aerobic bacterium that requires
oxygen and nutrient for growth, was used in this study. The
stock culture of A. xylinum was obtained from Malaysian
Agricultural Research and Development Institute (MARDI),
Serdang Selangor.
PREPARATION OF INOCULUM
Media for starter and fermentation of A. xylinum were
prepared. A starter medium was prepared by adding 100mL of
coconut water, 8g glucose and 0.5g ammonium sulphate in a
250 mL of conical flask. Then, it was sterilized in an autoclave
(Hirayama, HV100, Hirayama Manufacturing Corporation and
Japan) at 121oC for 15 min. After that, 10 mL stock culture of
A. xylinum is added into the conical flask. The culture broth
was incubated statically at 30C for 3 days. After 3 days, the
inoculums is ready for the next fermentation process.
PREPARATION OF COCONUT WATER WASTE
The coconut water was collected at Pasar Seksyen 1, Bandar
Baru Bangi. The collected coconut water was then filtered with
a filter paper to separate the impurities and then it was sterilized
using an autoclave at 121oC for 15 min.
OPTIMIZATION BY RESPONSE SURFACE
METHODOLOGY
250 mL conical flask was filled with 100 mL of fermentation
medium consisting of three types of coconut water medium
(old, young and mixed coconut water). Then, the fermentation
medium was sterilized using autoclave.at 121oC for 15 min.
After that, 10mL of the inoculums was then transferred to the
medium. The medium was incubated statically at 30C for 7
days. The lids of the flasks containing A. xylinum culture were
kept loose to ensure transfer of oxygen. After 7 days of
incubation, the pellicles formed at the surface layer are treated
with 0.5 M sodium hydroxide for 20 min at a temperature of
100C, then it was rinsed with distilled water. Lastly, the
weight of the pellicles was measured. All the steps were
repeated at different incubation temperature (28C, 30C and
32C), pH medium (4, 5 and 6) and fermentation medium
(immature , mature and mixed coconut water) according to the
Central Composite Design (CCD) with 39 experimental runs.
The data were analysed and suitable conditions for production
were selected using the Response Surface Methodology.
RESULTS AND DISCUSSION
OPTIMIZATION OF FERMENTATION MEDIUM
In general, the medium composition and environmental factors
affect bacterial cell growth and product formation (Chawla,
2009). Three main fermentation factors, namely temperature
(A), initial pH (B), and type of fermentation medium (C), were
chosen for the optimization of BC production by Acetobacter
xylinum 0416. CCD was used to identify the optimal condition The maximum BC production of 29.75 g/mL is obtained at an
for the medium. A set of 39 experimental runs with six initial pH of 4.16 and temperature of 30.06oC. Figure 2c shows
replicates at central point was conducted. All the experiments the effect of temperature and initial pH on BC production at
were performed in 250 mL Erlenmeyer flask containing 100 mixed fermentation medium. The BC production increases
mL of media. The CCD matrix of the independent variables in with temperature and initial pH up to 32oC and 6.00
experimental design along with predicted and experimental respectively. The maximum BC production of 28.53 g/mL was
values of response are summarized in Table 1. BC production obtained at temperature 28oC and pH 5.93 and 28.4oC.
were determined using multiple regression analysis with .
p<0.05 and used to generate second-order regression model
capable of predicting the amounts of BC DESIGN-EXPERT
production (Y) as aPlot Predicted vs. Actual
BC yieldThe response
function of the three independent variables.
41.23
equation from the above set of experiments can be written as

Y= -5650.06532 +26.56345(A)+1230.04658(B) - 4.4475(A2)

-90.55068(B2)+56.69756(AB)+0.47231(A2B) + 3.1655(AB2)
(1) 33.72

The response obtained under different combinations of

variables and defined experimental design (Table 1) were Predicted
26.22
analysed using the Analysis of Variance (ANOVA)
appropriate to the experimental design. Table 2 indicates that
the sum of squares due to regression (first and second-order
terms) was found to be significant (p <0.05) and no significant 18.72
lack of fit (p>0.05). The quality of fit for the quadratic
2
polynomial equation was examined using R and probability
(p) values. The R2 value provides a measure of how much
variability in the observed response values can be explained by
11.21
the experimental factors and their interactions. The R2 value
2
always lied between 0 and 1. The closer R value to 1.00, the
stronger the model was and the better it predicted the response. 11.21 18.72 26.22 33.72 41.23

In this study, R2 value was found by calculation to be 0.7035.

These values indicates that 70.35% of the variation in BC
Actual
production could be explained by the model (Table 2). As
shown in Table 2, statistical analysis indicates significant
effects of combination independent variables on BC
production. The generated second-order regression model is Figure 1: Parity plot showing distribution of experimental and
adequate in predicting the BC production. From the linear predicted values of BC production (g/ml)
DESIGN-EXPERT Plot
coefficients of second-order equation in Table 3, the
combination of variables show a positive effect BC yield on BC
X = A: Temperature
production, whereas an one variable shows a negative
Y = B: result.
Initial pH
Parity plot (Figure 1) illustrate the distribution
Actual Factor
of
experimental and model predicted values where data points
C: Type are =40.0377
of medium Immature
35.6155
scattered around the diagonal line suggesting the model is
adequate enough to explain BC production. 31.1933

The second-order regression model was used to develop 26.7711

BCyield

response surface plots (Figure 2). Figure 2a shows the response 22.3489
surface curve of BC production as a function of temperature
and initial pH of the medium fermentation with young coconut
water. The BC production increases with declined of
temperature and initial pH. Further increase in both variables 6.00
leads to opposite effects. Maximum BC yield of 35.84 g/mL is 32.00
5.50
obtained at temperature 30oC and pH 6 of medium 31.00
fermentation. Figure 2b depicts the response surface curve of 5.00
30.00
BC production as a function of temperature and initial pH of B: Initial pH 4.50 29.00
A: Temperature
the medium fermentation with matured fermentation medium. 4.00 28.00
o
The BC production increases with temperature up to 30 C
(central level) and then decreases. Whereas, elevated initial pH (a)
of medium fermentation leads to decrease of BC production.

3
N-EXPERT Plot

d ACKNOWLEDGEMENTS
Temperature
nitial pH
Financial support from Universiti Kebangsaan Malaysia under
Factor 32.439 FRGS/2/2013/TK05/UKM/02/1 grant is highly appreciated.
e of medium = Mature
26.8623

21.2856
REFERENCES
15.7088
BCyield

10.1321 Bielecki, S., A. Krystynowicz, M. Turkiewicz, H. Kalinowska.

2004. Bacterial Cellulose. In: Polysaccharides I:
Polysaccharides from prokaryotes. Biopolymers. 15, 37-
45.
6.00 akar, F., I. Ozer, A.O. Aytekin, F. ahin. 2014. Newly
5.50
32.00 Developed Medium and Strategy for Bacterial Cellulose
31.00 Production. Biochemical Engineering Journal.
5.00
30.00 Castro, C., R. Zuluaga,. J.L. Putaux, G. Caro, I. Mondragon
B: Initial pH 4.50 29.00
A: Temperature
and P. Ganan. 2011. Structural characterization of
4.00 28.00 bacterial cellulose produced by Gluconacetobacter
swingsii sp. From Colombian agroindustrial wastes;
(b) Carbohydrate Polymers. 84,96-102.
N-EXPERT Plot Chawla, P. R., I. B. Bajaj, S. A, Survase and R. S Singhal.
d 2008. Microbial Cellulose: Fermentative Production and
Temperature
Initial pH Application. Biotechnology. 47 (2), 107-124
Chawla, P.R, I.B. Bajaj, S.A. Survase and R.S. Singhal. 2009.
Factor 34.8835
e of medium = Mixed Microbial cellulose: fermentative production and
29.4736 applications; Food Technology Biotechnology. 47, 107-
24.0637 124.
18.6538
Coban, E.P & H Biyik. 2011. Effect of various carbon and
nitrogen sources on cellulose synthesis by Acetobacter
BCyield

13.2439
lovaniesis HBB5. African Journal of Biotechnology.
10(27), 5346-5354
Petersen, N. & P. Gatenholm. 2011. Bacterial cellulose-based
materials and medical devices: current state and
6.00
32.00
perspectives. Applied Microbiology Biotechnology, 91,
5.50
31.00
12771286
5.00
30.00
.
B: Initial pH 4.50 29.00
A: Temperature
4.00 28.00

(c)

Figure 2: Three-dimensional response surface plot of BC

production by Acetobacter xylinum 0416: effect of temperature
and initial pH in (a) young, (b) old (c) mixed coconut water.

CONCLUSIONS

As a conclusion, the results showed that the types of

medium, pH and temperature of cultivation were major
parameters that gave main effect on the yield of bacterial
cellulose. Besides that, this result also proved that matured
coconut water (coconut water waste) that contains high glucose
has great potential to be used for the synthesis of bacterial
cellulose. Through response surface methodology (RSM), the
optimum condition for bacterial cellulose was with old coconut
water at pH 4.16 and cultivation temperature at 30.06C. Thus,
the optimum conditions for bacterial cellulose production can
be promising results to overcome high BC production costs.

4
 Parameters Response (BC production g/mL)
Treatment Temperature Initial pH Types Of Experimented Predicted
Medium
1 28 4 Young 40.56 40.04
2 32 4 Young 39.47 35.45
3 28 6 Young 31.31 29.42
4 32 6 Young 41.23 35.85
5 28 5 Young 31.68 34.09
6 32 5 Young 12.94 22.35
7 30 4 Young 37.60 37.37
8 30 6 Young 27.80 28.48
9 30 5 Young 21.90 28.48
10 30 5 Young 32.50 25.96
11 30 4 Young 23.50 25.96
12 30 5 Young 32.60 37.37
13 30 5 Young 33.70 25.96
14 28 6 Mature 27.85 27.43
15 32 6 Old 14.34 15.13
16 28 4 Old 14.16 14.71
17 32 4 Old 27.02 28.78
18 28 5 Old 23.11 22.99
19 32 5 Old 13.76 11.21
20 30 4 Old 32.10 31.52
21 30 6 Old 30.50 28.20
22 30 5 Old 30.90 25.44
23 30 5 Old 21.10 25.44
24 30 4 Old 31.30 31.52
25 30 5 Old 14.70 25.44
26 30 5 Old 32.40 25.44
27 28 4 Mixed 19.55 17.05
28 32 4 Mixed 30.03 29.81
29 28 6 Mixed 30.94 28.83
30 32 6 Mixed 20.82 21.00
31 28 5 Mixed 19.23 23.85
32 32 5 Mixed 13.61 13.66
33 30 4 Mixed 32.60 33.91
34 30 6 Mixed 35.30 31.62
35 30 5 Mixed 35.00 27.35
36 30 5 Mixed 22.30 27.35
37 30 4 Mixed 32.50 33.91
38 30 6 Mixed 26.00 31.62
39 30 5 Mixed 29.40 27.35

Table 1: Experimental design and results of wet weight using central composite design based on the effect of temperature, initial
pH and type of medium fermentation

Source Sum of Squares DF Mean Square F Value Prob > F

Model 1700.47 19 89.5 2.37 0.0335 significant
A 35.18 1 35.18 0.93 0.3463
B 56.57 1 56.57 1.5 0.2357
C 325.13 2 162.57 4.31 0.0286
2
A 224.45 1 224.45 5.95 0.0247
B2 285.5 1 285.5 7.57 0.0127
AB 23.48 1 23.48 0.62 0.4398

5
 AC 2.45 2 1.22 0.032 0.9681
BC 50.82 2 25.41 0.67 0.5216
2
AB 20.09 1 20.09 0.53 0.4744
A2C 242.95 2 121.48 3.22 0.0624
2
AB 160.33 1 160.33 4.25 0.0532
2
BC 9.99 2 5 0.13 0.8767
ABC 286.73 2 143.36 3.8 0.0409
Residual 716.64 19 37.72
Lack of Fit 289.32 7 41.33 1.16 0.391 not significant
Pure Error 427.32 12 35.61
Correlation Total 2417.11 38

DF = Degree of freedom, R2 = 0.7035

Table 2 : Analysis of variance (ANOVA) of BC production