Cellulose (2009) 16:1033–1045

DOI 10.1007/s10570-009-9346-5

Effect of different additives on bacterial cellulose production

by Acetobacter xylinum and analysis of material property
Kuan-Chen Cheng Æ Jeffrey M. Catchmark Æ
Ali Demirci

Received: 19 December 2008 / Accepted: 15 July 2009 / Published online: 9 August 2009
Ó Springer Science+Business Media B.V. 2009

Abstract Bacterial cellulose (BC) demonstrates 1%. The variation between replicates for all analysis
unique properties including high mechanical strength, was \5%. From XRD analysis, however, the crys-
high crystallinity, and high water retention ability, tallinity and crystal size decreased as CMC addition
which make it an useful material in many industries, increased. FESEM results showed CMC-altered BC
such as food, paper manufacturing, and pharmaceu- produced from agitated culture retained its inter-
tical application. In this study, different additives weaving property. TGA results demonstrated that
including agar, carboxymethylcellulose (CMC), CMC-altered BC had about 98% water retention
microcrystalline cellulose, and sodium alginate were ability, which is higher than BC pellicle produced
added into fermentation medium in agitated culture to with static culture. CMC-altered BC also exhibited
enhance BC production by Acetobacter xylinum. The higher Tmax compared to control. Finally, DMA
optimal additive was chosen based on the amount of results showed that BC from agitated culture loses its
BC produced. The produced BC was analyzed by mechanical strength in both stress at break and
using X-ray diffraction (XRD), field emission scan- Young’s modulus when compared to BC pellicle.
ning electron microscopy (FESEM), thermogravi- This study clearly demonstrated that addition of CMC
metric analysis (TGA), and dynamic mechanical enhanced BC production and slightly changed its
analysis (DMA). Among the evaluated additives, structure.
CMC yielded highest BC production (8.2 g/L) com-
pared to the control (1.3 g/L). The results also Keywords Bacterial cellulose

indicated that CMC-altered BC production increased Acetobacter xylinum

with CMC addition and reached saturation around Cellulose crystallinity, cellulose yield

K.-C. Cheng
J. M. Catchmark (&)
A. Demirci

Introduction
Department of Agricultural and Biological Engineering,
The Pennsylvania State University, University Park,
PA 16802, USA Cellulose is the most abundant macromolecule on
e-mail: [email protected] earth (Brown 2004) and most cellulose is produced
by vascular plants. However, the ever-increasing
A. Demirci
The Huck Institutes of Life Sciences, The Pennsylvania demand of industrialization has imposed extreme
State University, University Park, PA 16802, USA negative pressure on the delicate ecological balance

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1034 Cellulose (2009) 16:1033–1045

of the plant world. Cellulose obtained from wood Calcofluor White ST, a stilbene derivative used as
stock requires the removal of impurities such as an optical brightener for cellulose, increased the rate
lignin, hemicellulose, and pectin. The industrial of glucose polymerization into cellulose, but dis-
process of removal of lignin, a major component of rupted the assembly crystalline cellulose I when the
the plant cell wall, to obtain cellulose accounts for concentration is above 0.1 mM (Benziman et al.
*50% of the total energy consumed in the liberation 1980). The negatively charged water-soluble cellu-
of cellulose from wood, and is often accomplished lose derivative, carboxymethylcellulose (CMC), was
via environmentally hazardous chemicals. widely used to enhance BC production in static
One approach to reduce the demand from plants is culture (Haigler et al. 1982; Hirai et al. 1998; Seifert
the production of cellulose using a microbial system et al. 2003). The improvement of BC production with
(Lynd et al. 2002; Brown 2004). The Gram-negative the presence CMC is varied from its DP and/or (DS).
Acetobacter xylinum has been the subject of the most Water content of CMC-altered BC pellicle also
intensive inquiry, which permits a detailed biochem- increased from 73 to 96% (w/w). Addition of other
ical description of the bacterial cellulose (BC) water soluble polysaccharides such as agar, sodium
biogenesis in this organism (Cook and Colvin 1980; alginate was also reported to enhance BC production
Delmer 1987). A. xylinum produces BC at the air/ in agitated cultivation (Ishida et al. 2003; Bae et al.
liquid interface. Traditional static culture has been 2004; Zhou et al. 2007).
used for BC production, which produces pellicles on Moreover, the mechanical properties of BC pelli-
the surface of fermentation broth. Several cultivation cle from static cultivation were investigated (Yama-
improvements had been presented later; Yoshino naka et al. 1989; Backdahl et al. 2006). Yamanaka
et al. (1996) developed a silicone membrane vessel found that sheets of bacterial cellulose gave Young’s
which provided oxygen from the bottom; the rate of modulus around 15 GPa and future improved to
BC production was doubled using the cylindrical 30 GPa by alkaline and oxidation solution treatment
vessel. Serafica et al. (2002) made bacterial cellulose (Nishi et al. 1990). Backdahl reported that bacterial
in a rotating disk bioreactor that consists of a cellulose exhibited similar stress-strain response of
cylindrical trough with inoculated medium into the carotid artery as a potential scaffold for tissue
which are dipped flat, circular disks mounted on a engineered blood vessels (TEBV). Hsieh et al. (2008)
rotating central shaft. Rotating disk bioreactor is studied on a single filament of bacterial cellulose and
more efficient and reduces the time of a run to about Young’s modulus reached 114 GPa. Mechanical
3.5 days instead of the usual 12–20 days. Hornung properties of cellulose composite such as cellulose/
et al. (2007) developed a novel reactor which both pectin or xyloglucan, and cellulose/CAB (cellulose
glucose and oxygen were fed directly to the BC- acetate butyrate) have also been studied (Astley et al.
producing cells. However, the production is still a 2003; Gindl and Keckes 2004). This collective
function of surface area to volume ratio. research on enhanced production of BC using agita-
An alternative method for BC production is using tion cultivation and different additives has provided a
submerged fermentation, for which several strains of wealth of information, but comprehensive analyses
BC producing bacteria had been screened for aerated on the material properties of these BC products are
and agitated systems (Ishikawa et al. 1995; Toyosaki sparse.
et al. 1995). Instead of cellulose pellicle, small pellets Therefore, the goal of this study was to evaluate
of BC were produced in these submerged/agitated effects of different additives on bacterial cellulose
fermentations. The BC produced in these agitated production by Acetobacter xylinum and analyze
systems exhibits a lower degree of polymerization material properties. This work consisted of the
(DP), crystallinity, and Young’s modulus than that following objectives: (1) to evaluate the yield of
produced under static cultivation. The less-organized BC production in agitation culture with various
form of BC may result from shear stress during additives; (2) to analyze the degree of crystallinity
agitation (Watanabe et al. 1994). and crystal size by x-ray diffraction (XRD); (3) TGA
Addition of different chemicals in BC production to determine its water content and thermal decom-
medium was found to enhance BC production both position behavior; (4) scanning electron microscopy
static and submerged cultivation. Addition of analysis (SEM) for determining the structure of BC;

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Cellulose (2009) 16:1033–1045 1035

and (5) dynamic mechanical analysis (DMA) for cellulose (Cat # 9004346, Lot # P206879308, FMC
determining tensile strength of BC. Co., Newark, DE), and sodium alginate (Cat # A-2033,
Lot # 39H0154, Sigma, St. Louis, MO) were used as
additives at various concentrations.
Materials and methods
Flask fermentation
Bacterial strains
Ten milliliter of the prepared inoculum was added
The bacterial strain used in this study was Acetobac- to 90 mL of fresh CSL-Fru medium supplemented
ter xylinum (ATCC 700178) obtained from the with various additives including agar, carboxymethyl-
American Type Culture Collection (Rockville, MD), cellulose (CMC), microcrystalline cellulose, and
which was grown in an agitated culture as a BC sodium alginate at different concentrations (0.2 and
producer. The cell suspension of A. xylinum was 0.5%) in a 250 mL flask and incubated at 30 °C
stored at -80 °C in a 20% glycerol solution. One and 200 rpm for 5 days. A control experiment with-
milliliter of cell suspension stored at -80 °C was out addition of additives was also performed
added to 100 mL of CSL-Fru medium in a 500 mL simultaneously.
flask and statically cultivated at 30 °C for 3 days. The
cellulose pellicle formed on the surface of the broth Measurement of biomass and bacterial cellulose
was homogenized using a homogenizer (model
7011S, Waring Co., Torrington, CT) at 10,000 rpm At the end of 5-day incubation, the samples of culture
for 1 min and filtered through a sterile gauze to broth were centrifuged at 3,300g for 20 min (Super
remove BC. Ten milliliter of the filtrate containing T-21, Sorvall Co., Norwich, CT). For biomass, the
the cell suspension was added to 90 mL of CSL-Fru BC pellets were added to 90 mL 0.1 M potassium
medium and cultivated 30 °C and 200 rpm for 24 h acetate-acetate buffer (pH 5.0) and 10 mL of 20%
and used as an inoculum. cellulase solution (Cat # C2730, Lot # 074K1156,
Sigma, St. Louis, MO) and incubated at 50 °C with
Medium shaking at 100 rpm for 1 h to hydrolyze BC (Kouda
et al. 1997). Then, the solution was centrifuged at
All the chemicals used were of analytical grade and 3,300g for 20 min. The precipitate was washed with
commercially available unless specified description. deionized water twice and centrifuged. Finally, the
For BC production, corn steep liquor with fructose precipitate was dried in an oven at 80 °C overnight
(CSL-Fru) medium was slightly modified as described and then weighed to determine biomass. For BC
previously (Kouda et al. 1997), and contains the determination, the precipitated BC pellets were
following constituents per liter: 50 g of fructose, treated with 0.1 N NaOH solution at 80 °C for
20 mL of CSL (corn steep liquor, Nihon Starch 30 min to remove the bacterial cells and medium
Industry, Kagoshima, Japan), 1.0 g of KH2PO4, components (Hwang et al. 1999). This NaOH treat-
0.25 g of MgSO4
7H2O, 3.3 g of (NH)2SO4, 3.6 mg ment was repeated three times and then, the solution
of FeSO4
7H2O, 1.5 mg of CaC12
2H2O, 2.4 mg of was centrifuged at 3,300g for 20 min. The purified
Na2MoO2
2H2O, 1.7 mg of ZnSO2
7H2O, 1.4 mg cellulose was dried in an oven at 80 °C overnight and
of MnSO4
5H2O, 0.05 mg of CuSO4
5H2O, 2.0 mg then weighed.
of inositol, 0.4 mg of nicotinic acid, 0.4 mg of
pyridoxinėHCl, 0.4 mg of thiaminėHCl, 0.2 mg of Fructose concentration
pantothenic acid Ca salt, 0.2 mg of riboflavin, 0.2 mg
of pamino-benzoic acid, 0.002 mg of folic acid, and Fructose concentration was determined using a
0.002 mg of biotin. The final pH was adjusted to 5.0. Waters high-performance liquid chromatography
Agar (Cat # 214010, Lot # 6080253, Becton, Dickin- instrument equipped with column heater, autosam-
son Co., Sparks, MD), carboxymethylcellulose (CMC, pler, computer controller, and a refractive index
0.60–0.95 substitution; Cat # 21900, Lot # 434516, detector (Waters, Franklin, MA). Components were
Fluka Co., Buchs SG, Switzerland), microcrystalline separated on a Bio-Rad Aminex HPX-87H column

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1036 Cellulose (2009) 16:1033–1045

(300 mm 9 7.8 mm; Bio-Rad, Richmond, CA) with Thermogravimetric analysis

0.012 N sulfuric acid as a mobile phase at a flow rate
of 0.8 mL/min with an injection volume of 20 lL and The dynamic weight loss tests were conducted on a
a column temperature of 65 °C. Before injection, the thermogravimetric analyzer (TGA) machine (model
samples were centrifuged in a microcentrifuge at Q500, TA instruments-Water LLC, New Castle, DE).
2,000g for 5 min (Model C-1200, National Labnet For water content determination of cellulose, all
Co., Woodbridge, NJ) and filtered through 0.22 lm sample tests were conducted in a N2 purge (40 mL/
nylon filters (13 mm diameter disk filters, Millipore, min) over a temperature range 30–650 °C at an
Bedford, MA) to remove suspended solid particles. increase rate of 10 °C/min. BC samples were placed
on the absorbent wipers to eliminate excess water.
For thermal decomposition behavior test, cellulose
X-ray diffraction
samples were dried at 80 °C and were then tested in a
N2 purge (40 mL/min) over a temperature range of
To determine the crystallinity of the BC, the X-ray
80–650 °C at an increase rate of 10 °C/min. Samples
diffraction (XRD) patterns of the samples were
were initially dehydrated by fixing the temperature at
collected on a Scintag PADV theta-2-theta diffrac-
80 °C and holding until no decrease in sample weight
tometer (Scintag, Cupertino, CA) using a copper
was observed. The weight became the initial 100%
x-ray source. Scans were collected at 2 deg per
weight value.
minute from 5 to 70 degree 2h. Samples of BC were
lyophilized first by using a lab-scale freeze dryer
Mechanical properties of bacterial cellulose
(Model FreeZone 2.5L, Labconco co., Kansas, MO)
and pressed into a thin and flat layer (*1.0 mm) for
The mechanical properties of BC were measured
analysis. MDI Jade 8 software (Materials Data, Inc.,
using a dynamic mechanic analyzer (DMA; model
Livermore, CA) was used to process diffraction
Q800, TA instrument-Water LLC, New Castle, DE).
pattern and to calculate the crystallinity of BC. The
BC from agitated culture after removal of cells was
degree of crystallinity was taken as Cr I = (I200-
freeze dried first and pressed into flat piece at 2,500
Iam)/I200, where I200 is the overall intensity of the
psi using a press. The BC samples were then cut to
peak at 2h (about 22.9°) and Iam is the intensity of the
make 20 9 5 mm pieces for evaluation of tensile
baseline at 2h (about 18°; Mihranyan et al. 2004).
modulus. Samples were mounted between upper (fix)
The crystal size was calculated by the Scherrer
and lower (movable) clamps, and two ends were fixed
equation (Zhang et al. 2003):
to avoid slip. Force was applied to lower clamp to
Kk pull sample in tension. Experiments were run at
bhkl ¼
Lhkl cos hhkl 0.1 N/min. Tests were done at an environmental
temperature of 35 °C. Stress (r) was calculated by
where b is the breadth of the peak of a specific phase
F/A where A is the area measured as width X
(hkl), K is a constant that varies with the method of
thickness of sample and F is force in Newton. Strain
taking the breadth (0.89 \ K \ 1), k is the wave-
(e) was calculated by DL/Lo where DL is exerted
length of incident x-rays, h is the center angle of the
extension from starting point Lo. Young’s modulus
peak, and L is the crystallite length (size).
was calculated by stress/strain.

Field emission scanning electron microscopy Statistical analysis

BC samples after removal of cells were lyophilized All treatments were replicated at least three times.
and then coated with a thin Platinum film around The significant difference of the results was evaluated
5 nm. A LEO 1530 field emission scanning electron using the Generalized Linear Model (GLM; with
microscope (Leo Co., Oberkochen, Germany) oper- P \ 0.05) and Tukey’s honestly significant differ-
ating at 2 kV and imaging magnification about ences (HSD) multiple comparison module of the
50,000 times was used for examination of BC MINITAB Statistical Software package (Release
samples. 13.30; State College, PA, USA).

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Cellulose (2009) 16:1033–1045 1037

Results production reached 8.2 g/L at an optimal CMC

concentrations of 0.8%, which is 6.3-fold greater
Effects of addition of different additives on BC than the control (without CMC). There were no
production in agitated cultures significant differences in the enhanced amount of BC
produced at CMC concentration of 0.8 and 1.0%.
A. xylinum was grown in CSL-Fru medium with
different types and concentration of additives for BC BC structure and physical properties
production in agitated cultures. A control run without
additive was also performed (Fig. 1). BC production As other polymeric additives such as agar, acetan,
by A. xylinum in the control medium without and xanthan, CMC exhibited its ability to hinder
additives was 1.34 g/L, and produced with irregular formation of large clumps of BC and enhanced BC
clumps of different size and shapes. production during submerged fermentation (Ishida
In this study, different additives including sodium et al. 2003; Bae et al. 2004; Zhou et al. 2007). This
alginate, agar, CMC and microcrystalline cellulose can possibly be attributed to an increase in the
were compared for BC production by A. xylinum. BC solubility of BC in the presence of the highly soluble
production in the control case was 1.34 g/L (Fig. 1). CMC, which may bind to the surface of the BC
Among different cultivation conditions, BC produc- fibrils. Moreover, the morphological structures of BC
tion with 0.5% (w/v) CMC was the highest (7.2 g/L) fibers from lyophilized BC samples with and without
and formed small pellets. Addition of microcrystal- different concentration CMC addition were analyzed
line cellulose (0.5%) and agar (0.2%) also demon- by FESEM and shown in Fig. 3. The results showed
strated an improvement of BC production, but were that these samples showed interweaving BC fibers.
only 4.37 and 4.49 g/L, respectively. Addition of The width of BC fibers is around 15–30 nm. As CMC
sodium alginate seemed to have a negative effect on concentration increased, the width of cellulose fiber
this A. xylinum strain and resulted in a decrease of BC decreased slightly and the photographs revealed some
production. As a result, CMC was selected as optimal differences in surface structures. The CMC-altered
additive for the rest of the experiments. BC kept its interweaving property and had some
BC production by A. xylinum in CSL-Fru medium debris on its surface.
containing CMC concentration in the range of 0–1% Haigler et al. (1982) suggested that the addition of
(w/v) is shown in Fig. 2. The results indicated BC CMC can change morphology of BC into separate,

Fig. 1 Bacterial cellulose 10

production by A. xylinum in
9
CSL-Fru medium
containing different 8
Weight of BC (g/L)

concentration of Avicel, 7
CMC, sodium alginate or
6
agar in 250 mL flasks
(n = 3) 5
4
3
2
1
0
C

r
l

el

el

te

te
ro

ga
M

M
ic

ic

na

na
nt

A
av

av

gi

gi
Co

2%

5%

2%
al

al
2%

5%

0.

0.

0.
um

um
0.

0.

di

di
so

so
2%

5%
0.

0.

Types and concentration of additives

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1038 Cellulose (2009) 16:1033–1045

Fig. 2 Bacterial cellulose 10

production by A. xylinum in
CSL-Fru medium
containing different 8

Weight of BC (g/L)
concentration of CMC in
250 mL flasks (n = 3)
6

0
0.0 0.. 2 0.. 5 0.8 1.0
CMC concentration (%)

intertwining bundles of microfibrils but do not sodium alginate reduced from 78 to 59%, the crystal
prevent microfibril crystallization. Although it has size also reduced from 5.8 to 5.1 nm (Zhou et al.
been found that crystallization is indeed not pre- 2007), which probably due to the shear force stress in
vented, we found that the addition of CMC does an agitated bioreactor.
impact both crystallinity and crystal size. The crys-
tallinity index of CMC-altered BC in this study Thermogravimetric analysis
adhered to this conclusion.
X-ray diffraction patterns obtained from our BC To determine the water retention property and infor-
samples demonstrated three main characteristic peaks mation on thermal decomposition behavior of CMC-
standing for crystal plane \-110[, \110[, and altered BC, TGA was performed on CMC-altered BC
\200[, which showed that both CMC-free and samples at the end of cultivation with different
CMC-altered cellulose was mainly in cellulose I-b concentration of CMC addition. The TGA thermo-
form after compared the database of MDI Jade 8 grams of CMC-altered BC samples are shown in
software, which may due to smaller crystallite sizes Fig. 5. The control BC exhibited around 98% water
of microfibrils produced in agitated culture (Fig. 4; retention ability (Fig. 5a), which is higher than both
Czaja et al. 2004). The crystalline indices of CMC- BC (73%) and CMC-altered BC (96%) pellicles
altered BCs were lower than CMC-free BC (Table 1), reported earlier (Seifert et al. 2003). When CMC
and the crystallinity of BC significantly decreased was included in the medium, CMC-altered BC
from 85 to 80% when 0.8% CMC was added in the exhibited similar water retention ability to the control.
medium, which may be due to the incorporation of BC with 0.2–1.0% CMC addition exhibited *98%
CMC into the BC microfibril. The crystal size of water content in the samples. The high water retention
\200[ crystal plane also decreased from 5.2 to ability of BC produced in our studies could possibly
3.8 nm after 1.0% CMC addition. It has been shown arise from differences in the material microstructure
previously that crystallization of cellulose is a rate due to the agitated culture conditions. Increased
limiting step in its microbial production (Haigler microporosity, for example, could result in larger
et al. 1982). The disruption of cellulose crystalliza- volumes for water to collect in the hydrophilic matrix.
tion by the incorporation of CMC may be the main For thermal decomposition behavior of BC samples,
factor in the observed increase in cellulose yield. dried BC samples were used. Two distinct steps for
Although CMC-altered BC exhibited a reduction in weight loss of CMC-altered BC were found, indicat-
its crystallinity and crystal size when CMC was ing the possibility of two types of decomposition
presented in the medium, the impact of CMC is much (Fig. 5b). Yang and Chen (2005) suggested that the
lower when compared to addition of sodium alginate, initial weight loss at lower temperature ranging from
and the crystallinity of BC product with 0.04% 200 to 380 °C is attributed to the removal of small

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Cellulose (2009) 16:1033–1045 1039

Fig. 3 Visualization of BC
and CMC-altered BC,
FESEM pictures of freeze
dried BC samples. a BC
without CMC addition, b
BC with 0.2% CMC, c BC
with 0.5% CMC, d BC with
0.8% CMC and e BC with
1.0% CMC in CSL-Fru
medium in 250 mL flasks

molecular fragments such as hydroxyl and hydroxy- chains and the six-member cyclic structure, pyran.
methyl groups. The second weight loss ranging from CMC altered BC exhibited a bi-stage degradation
360 to 600 °C showed the degradation of polymeric pattern in the second step (see arrow in Fig. 5b),

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1040 Cellulose (2009) 16:1033–1045

Fig. 4 X-ray diffraction

patterns of bacterial
cellulose produced by A.
xylinum in the medium
containing CMC in the
250 mL flasks. a Control, b
0.2%, c 0.5%, d 0.8%, and e
1.0% CMC. I200 peak was
labeled as an arrow and
used for calculation of
crystallinity

Table 1 Summary of
CMC concentration in the medium (%) Crystallinity (%) Crystallite size of \002[ (nm)
crystallinity and crystal size
with different CMC 0.0 85.0±1.2a 5.2±0.3a
addition a
0.2 84.1±0.6 4.5±0.2b
ab
0.5 83.3±0.4 4.1±0.2b
Values (in the same
column) not marked by the 0.8 80.0±0.7b 4.0±0.1b
same letter are significantly 1.0 80.0±1.0b 3.8±0.2b
different (P 2 0.05; n = 3)

which we hypothesize is a combination of decompo- indicated CMC-altered BC samples possess higher

sition of cellulose and CMC. Since the thermal thermal stability and began to degrade at higher
degradation behavior is affected by some structural temperature.
parameters such as molecular weight, crystallinity,
and orientation (Um et al. 2004), the sharp decrease of
CMC-altered BC at the second stage with the presence Mechanical properties
of CMC could be due to its decreased crystallinity and
possibly lower molecular weight. The maximum BC produced in either agitated or air-lifted culture is
decomposition temperature (Tmax), known as a crite- believed to lose its high polymerization property of
rion of thermal decomposition position, was calcu- glucose unit and result in low mechanical strength
lated from different TGA curves. The BC samples (Benziman et al. 1980). As a result, DMA analysis
displayed two peaks (Fig. 6), one at 260 °C and was done to illustrate BC mechanical strength.
another at 530 °C. In the cases of CMC-altered BC, Tensile testing was done at 35 °C with a cross head
the Tmax of first peak increased by about 20 °C, which speed of 0.1 N/min. Three types of BC were

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Cellulose (2009) 16:1033–1045 1041

Fig. 5 The TGA curves of A 100

control BC and CMC-
altered BC. Both control Control
and CMC altered BC CMC 0.2%
exhibited high water 80 CMC 0.5%
retention ability around
CMC 0.8%
98% (a), and a bi-stage
CMC1.0%

We i g h t ( % )
thermal decomposition
behavior (b). (The arrow 60
here indicates a bi-stage
degradation pattern of
CMC-altered BC in second 40
step)

20

0
0 100 200 300 400 500 600
Temperature (°C)

B 100
Control
90
CMC 0.2%
80 CMC 0.5%
70 CMC 0.8%
CMC 1.0%
60
We i g h t ( % )

CMC
50

40

30

20

10

0
0 100 200 300 400 500 600 700
Temperature (°C)

investigated: pellicle from static culture, CMC- experiment results, the stress at break decreased from
altered BC, and control from agitated culture. 34.1 (pellicle) to *2.4 MPa. Young’s modulus
Figure 7 shows the mean value (l) and standard decreased from 2,671 (pellicle) to *320 MPa. The
deviation (r) of the tested samples’ tensile properties strain at break also decreased from 1.3 to 0.8%. The
(n = 5); tensile strength (rmax, MPa), strain at break Young’s modulus showed that mechanical strength of
[emax (%)], and Young’s modulus [E (MPa)] were CMC-altered BC decreased dramatically when com-
measured. BC testers from agitated culture, CMC- pared to BC pellicle, which is directly correlated to
altered BC and control, showed dramatic decrease in the decrease of glucose polymerization. However,
both stress at break and Young’s modulus when there were no significant differences among different
compared to BC pellicle from static culture. From our concentration CMC-altered BC samples.

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1042 Cellulose (2009) 16:1033–1045

Fig. 6 Derivative TGA 1.0

patterns of control BC and Control
CMC-altered BC displayed 0.9 CMC 0.2%
two thermal decomposition CMC 0.5%
peaks 0.8 CMc 0.8%
CMC 1.0%

Deriv. Weight (%/°C)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0
0 100 200 300 400 500 600 700
Temperature (°C)

Figure 8 shows a typical stress-strain response of property but with looser structure. The introduction of
the BC pellicle (A) and CMC-altered BC (B) CMC resulted in a significantly decrease in crystallin-
samples. The extension of our testers showed a ity and crystal size when 0.8% CMC was present in the
linear-elastic behavior. Among different concentra- cultivation medium. The result of increased water
tion CMC-altered BC samples, the elastic deforma- retention ability is also consistent with as earlier study
tion only gave around 1% elongation, where a yield by Seifert et al. (2003). They reported that CMC-
point was seen, which was followed by irreversible altered BC from static culture exhibited higher water
deformation. retention ability (96%) when compared to BC pellicle
(87%). Thermo-gravimetric analysis indicated CMC-
Conclusion altered BC had similar water retention and higher
thermal stability compared to the control. This study
Based on the results of this study, BC produced by showed that the addition of water soluble CMC
A. xylinum can be enhanced and modified by intro- enhanced the thermal stability of BC. However, this
ducing CMC into BC production medium using behavior is not demonstrated by all additions. For
agitated culture. CMC-altered BC was produced in example, the addition of SiO2 was reported to decrease
small pellets and mainly in cellulose I-b form. CMC CMC thermal stability due to the incompatibility of the
addition demonstrated the highest BC production materials (Tsioptsias et al. 2008). Different concen-
among different additives and was correlated with its tration of CMC addition did not affect the mechanical
concentration. BC produced by A. xylinum reached strength of BC pellets produced from agitated culture.
8.2 g/L with optimal 0.8% CMC addition in the However, the mechanical strength of BC pellets
medium after 5 day cultivation, which is 6.3-fold produced by agitated culture is much lower than BC
compared to control. The decrease in crystallinity pellicle produced from static culture.
resulting from the addition of CMC is believed to be a Further study on determining the properties of
major factor in the observed increase in cellulose yield, CMC-altered BC such as gas-penetration ability,
since the rate of crystallization is known to be a metal-ion chelating capacity, and thermal stability are
limiting step in cellulose synthesis. The structure, needed. Moreover, how to increase its mechanical
cellulose type, crystallinity, thermal, and mechanical strength by combination with other materials is an
properties were also investigated. Based on FESEM important issue when producing BC in agitated
images, CMC-altered BC retained its interweaving culture.

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Cellulose (2009) 16:1033–1045 1043

Fig. 7 Results of tensile

test of BC. a Stress at break;
b strain at break; c Young’s
modulus (n = 5)

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1044 Cellulose (2009) 16:1033–1045

Fig. 8 Stress/strain
diagrams of tensile test
results, both BC pellicle (a)
and CMC-altered BC (1%;
b) exhibited a linear-elastic
behavior. a, Elastic
deformation, where stress is
proportional to strain; b,
yield point; c, plastic
deformation, where
polymer chains orient
themselves with the applied
stress; d, break

Acknowledgments This work was supported in part by a Bae S, Sugano Y, Shoda M (2004) Improvement of bacterial
seed grant from the College of Agricultural Sciences at the cellulose production by addition of agar in a jar fermentor.
Pennsylvania State University and the Pennsylvania J Biosci Bioeng 97(1):33–38
Experiment Station. The authors are very grateful to Nichole Benziman M, Haigler C, Brown RM et al (1980) Cellulose
Wonderling from the Materials Research Institute of the biogenesis: polymerization and crystallization are coupled
Pennsylvania State University for her assistance with X-ray process in Acetobacter xylinum. Proc Natl Acad Sci USA
diffraction measurements. Thanks also to Yang Hu and Yufan 77(11):6678–6682
Zeng in the Agricultural and Biological Engineering and Forest Brown RM (2004) Cellulose structure and biosynthesis: what is
Resources department for useful discussion of DMA and TGA in store for the 21th Century? J Poly Sci: Part A: Polymer
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