World J Microbiol Biotechnol (2010) 26:125–131
DOI 10.1007/s11274-009-0151-y
ORIGINAL PAPER
The influence of fermentation conditions and post-treatment
methods on porosity of bacterial cellulose membrane
Weihua Tang Æ Shiru Jia Æ Yuanyuan Jia Æ
Hongjiang Yang
Received: 18 January 2009 / Accepted: 4 August 2009 / Published online: 20 August 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Bacterial cellulose has been found to be
attractive as a novel scaffold material due to its unique
material properties. Porosity is the most important morpho-
logical parameter in the design of scaffolds for tissue engi-
neering. The effects of fermentation conditions (cultivation
time and inoculation volume) and post-treatment methods
(alkali treatment and drying methods) on the porosities of
bacterial cellulose membranes were investigated. With
extended cultivation time and increased inoculation volume,
more micro-fibrils were secreted by bacteria, which resulted
in a more compact structure and diminished porosity. The
porosities of alkali-treated bacterial cellulose membranes
was in the order of K2CO3 [ Na2CO3 [ KOH [ NaOH.
Freeze-dried membranes had much higher porosity (92%)
than the hot air-dried ones (65%). The experimental results
suggested that bacterial cellulose with controlled porosities
could be prepared by varying fermentation conditions and
post-treatment methods. The resulting bacterial cellulose is
regarded as a scaffold material of great potentialities.
Keywords Gluconacetobacter xylinus

Bacterial cellulose
Porosity
Fermentation conditions

Post-treatment methods
Introduction
Bacterial cellulose (BC) is receiving great attention and
presently being widely investigated as a new type of scaf-
fold material due to its fine fiber network, biocompatibility,
W. Tang
S. Jia (&)
Y. Jia
H. Yang
Key Laboratory of Industrial Microbiology, Ministry of
Education, Tianjin University of Science and Technology,
300457 Tianjin, People’s Republic of China
e-mail: jiashiru@tust.edu.cn; tangweihua79@hotmail.com
high water holding capacity, and high tensile strength
(Hong et al. 2006; Putra et al. 2008).
Helenius and coworkers implanted bacterial cellulose
subcutaneously in rats for 1, 4, and 12 weeks, respectively,
and found that the biocompatibility of bacterial cellulose
was good and the material had potential to be used as a
scaffold in tissue engineering (Helenius et al. 2006).
Svensson declared that bacterial cellulose was a potential
scaffold for tissue engineering of cartilage (Svensson et al.
2005). Several groups (Charpentier et al. 2006; Klemm
et al. 2001) have recently studied bacterial cellulose for use
as a blood vessel. Wet cellulose could be used as a tem-
porary artificial skin to treat severe skin burn (Farah 1990).
Fabricating a scaffold with the desired pore size and
porosity is of great importance in tissue engineering (Li
et al. 2009). For bacterial cellulose scaffold, the definition
of a specific pore size in a bacterial cellulose fibrous
hydrogel is not relevant because the nanofibrils can be
pushed aside by migrating cells (Bäckdahl et al. 2006).
Bacterial cellulose has potentialities to be an appropriate
scaffold for different types of tissue and organ. So how to
control the porosity of bacterial cellulose according to
individual requirements is a task of great significance. Few
researchers have tried to alter the porosity of bacterial cel-
lulose networks. In this paper, the effect of fermentation
conditions and post-treatment methods on porosities of
bacterial cellulose membranes has been investigated.
Materials and methods
Bacterial strain
Gluconacetobacter xylinus (TCCC No. 12003) used in this
study was obtained from Tianjin University of Science &
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Technology Culture Collection Center (TCCC), Tianjin,
China.
Medium and cultivation
The medium for inocula preparation and membrane forma-
tion used consisted of the following (v/v): carbon source
(glucose, fructose, sucrose, or glycerol) 2.5%, peptone
0.75%, yeast extract 1%, disodium phosphate 1%, acetate
acid 1%, initial pH value was adjusted to 6.0. For seed culture,
colonies of G. xylinus were inoculated into 100 ml of medium
in a 500 ml flask shaken at 160 rev/min and cultured at 30°C
for 24–30 h. Fermentation cultures performed in 90 mm
(i.d.) petridishes containing 25 ml medium with a 6%(v/v)
cell suspension were incubated statically at 30°C for 5 days.
Bacterial cellulose purification
After incubation, bacterial cellulose membranes produced
on the surface of each medium were harvested and washed
with water, and then boiled in each of 0.1 M alkali solu-
tions (K2CO3, Na2CO3, KOH, or NaOH) for 30 min,
neutralized with 1% acetic acid and washed with distilled
water, successively. It was then dried in an oven at 80°C or
freeze-dried with a vacuum freeze dryer (1-4LP-2,
Gefriertrocknungsang/agen, Germany).
Analytical methods
Cell mass
The cell mass was estimated by measuring the optical
density at 600 nm after treating the culture broth with 5%
(v/v) cellulase at 50°C for 2 h to release the cells from
bacterial cellulose membranes (Hwang et al. 1999).
Porosity
Porosity was calculated using the equation (Kitaoka et al.
1997):
Porosity (%) = (wet weight-dry weight)/(wet weight-
weight in water) 9 100.
Dried bacterial cellulose samples were soaked in
deionized water for more than 12 h at room temperature,
and the weight in water was measured by harnessing the
sample in a device, which suspended the sample in water
(Mancini et al. 2001).
Thickness
Thickness of the bacterial cellulose membranes was
measured at ten different positions by a thickness gauge
(CH-1-ST. Shanghai, China), and the values were averaged.
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World J Microbiol Biotechnol (2010) 26:125–131
Concentration of carbon sources
The glucose and fructose concentrations in the supernatant
of broths were analysed using the modified dinitrosalicylic
acid (DNS) method (Ghose 1987). The sucrose and glycerol
concentrations were determined by HPLC (Yang et al.
1998).
Scanning electron microscopy
The purified bacterial cellulose samples as described above
were sputter coated with gold. Scanning electron micro-
scope observation was performed using FEI Quanta 200.
Atomic force microscopy
The bacterial cellulose membrane was directly observed by
the ATM (JSPM-5200, Japan) at room temperature.
Results and discussion
Characterization of bacterial cellulose membrane
The SEM images of two sides of bacterial cellulose mem-
brane are shown in Fig. 1a, b. The denser surface side
formed at the interface between air and culture medium,
showed a smooth surface with no visible pores (at magni-
fication 10,0009). The porous opposite side was relatively
loose and possessed larger pores. It was also found that
bacterial cellulose has a three-dimensional network, which
retains a lot of water and favors the transportation of
nutrient. According to Bodin, smooth surfaces have been
shown to improve adhesion and proliferation of cells. The
porous structure and open fibril network are advantageous
for ingrowths of cells (Bodin et al. 2007).
Cross section image of the bacterial cellulose membrane
shows a layered structure (Fig. 1c). In the zone near to air-
medium side, it is much denser. This is consistent with the
difference between two sides of bacterial cellulose mem-
brane. The AFM-image reveals that microfibrils arrange in
ribbons with a diameter of approximate 60–100 nm, as
seen in Fig. 1d. The ribbons highly entangle to be finally
assembled as bacterial cellulose membrane.
Effect of carbon sources on porosities of bacterial
cellulose membranes
The effect of various carbon sources on the porosities and
thicknesses of the bacterial cellulose membranes was
examined (Fig. 2). The result showed that when sucrose as
the sole carbon source, the porosity of bacterial cellulose
membrane was up to 77%, followed by glucose, fructose
World J Microbiol Biotechnol (2010) 26:125–131
Fig. 1 Images of bacterial
cellulose membranes, a Denser
surface side by SEM b Porous
opposite side by SEM c Cross
section of bacterial cellulose
membrane by SEM d AFM
image of bacterial cellulose
ribbons. Bacterial cellulose
membrane obtained after 5 days
cultivation with 6% (v/v)
inoculation volume
and glycerol. Since G. xylinus has no sucrose synthase,
there are at least four enzyme steps in the pathway from
sucrose to UDP-glucose (Nakai et al. 1999). If sucrose was
used as the sole carbon source, it led to the limited growth
of bacteria, which was verified by cell mass measurement;
the cell mass was only 50% of the case when glucose was
used as the sole carbon source (Table 1). As a conse-
quence, fewer amounts of microfibrils were produced,
which explained the least cell mass and the lowest thick-
ness together with highest porosity. When the sole carbon
source was glycerol, bacterial cellulose membrane showed
the lowest porosity. There might be no gluconic acid pro-
duction during glycerol metabolism (Jonas and Farah
1998), so the pH remained stable (Table 1), which pro-
vided a favorable condition for bacteria growth. Massive
microfibrils were secreted by bacteria, which led to a
compact structure. From Table 1, it was found that when
glucose was used as the sole carbon source, the bacterial
cellulose yield against unit cell mass (g bacterial cellulose
produced/OD600) from glucose was higher than the other
carbon sources. When glycerol was used as the sole carbon
source, the bacterial cellulose yield, cell mass and bacterial
127
cellulose yield against sugar consumed from glycerol were
higher than that from other carbon sources. Considering
both production and porosity of bacterial cellulose mem-
brane, in following experiments glucose was applied as the
sole carbon source.
Effect of culture conditions on porosities of bacterial
cellulose membranes
The effect of culture time and inoculation volume on
porosities and thicknesses of bacterial cellulose membranes
is shown in Figs. 3 and 4. In the initial stage of cultivation,
e.g., the first day, the bacterial cellulose membranes showed
high porosities. During cultivation, the porosities of bacte-
rial cellulose decreased successively from the first day to the
third day. Then the porosities decreased rarely in the fourth
day and the fifth day. Small inoculation volume led to high
porosities of bacterial cellulose membranes. The porosities
of bacterial cellulose membranes dropped from 71.4 to 60%
with increasing inoculation volume (v/v) from 2 to 8%.
During cultivation, the yield of bacterial cellulose and
cell mass increased with time, but porosities decreased due
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90 90 6

80
80 porosity
5
70

porosity (%) thickness (µm)

thickness

BC yield (g/L) OD600

70 60 4
porosity (%) thickness (µm)

60 50
3
40
50
30 2

40
30
20
10
0
glucose fructose
Fig. 2 Effect of carbon sources on porosities and thicknesses of
bacterial cellulose membranes produced after 5 days cultivation with
6% (v/v) inoculation volume. Error bars represent standard devia-
tions (n = 3)
to accumulation of more fibrils (Fig. 3). Inoculation vol-
ume had a similar trend as discussed above. Generally,
production of a large number of microfibrils often led to a
compact structure and lower porosity for bacterial cellulose
membrane.
Although we found that culture conditions, such as
inoculation volume and culture time could significantly
influence porosity of the bacterial cellulose membrane, we
have not yet managed to fully understand the controlling
mechanism of bacterial cellulose porosity.
According to the recent structural model, several tens of
cellulose -synthesizing sites are located in the cytoplasmic
membrane, being aligned parallel with the longitudinal axis
of the bacterial cell. Cellulose synthetases in the respective
sites produce 12–15 cellulose chains and extrude them into
the culture medium as fine fibrils with a lateral width of
about 1.5 nm (such fine fibrils are often called subele-
mentary fibrils) through small pores in the outer membrane.
These subelementary fibrils aggregate to form microfibrils
20
1
10
0 0
1 2 3 4 5
culture time (d)
porosity thickness BC yield OD600
Fig. 3 Effect of culture time on porosities, thicknesses, yields of
bacterial cellulose membranes and cell growth of G. xylinus. Bacterial
sucrose glycerol cellulose membranes produced with inoculation volume of 6% (v/v).
Error bars represent standard deviations (n = 3)
3–6 nm in width, and the resulting microfibrils further
aggregate to produce a typical ribbon assembly with a
lateral width of 40–60 nm (Hirai et al. 1998).
The secreted cellulose is randomly deposited behind the
moving micro-organism to produce a certain porosity of
bacterial cellulose membrane with three-dimensional net-
work. The movement of single cells is caused by the
inverse force of the secretion of cellulose nanofibers (Hesse
and Kondo 2005). Watanabe and Yamanaka (1995) found
that oxygen tension in the gaseous phase under static cul-
ture conditions affected network formation of BC, and
density of network in the gelationous membrane could be
controlled. Then by changing oxygen tension we might
produce bacterial cellulose membranes with desired
porosities for various applications. This might be a topic
for further investigation.
Effect of alkali treatment on porosities of bacterial
cellulose membranes
There were protein, medium ingredients, and bacterial cells
in native bacterial cellulose membranes. In general, alkali

Table 1 Effect of carbon sources on bacterial cellulose production and cell growth of G. xylinus
Carbon BC Cell mass Final pH BC yield against unit cell BC yield against sugar consumed
source yield (g/l) (OD600) mass (g BC produced/OD600) (g BC produced/g- sugar consumed)

Glucose 5.54 2.74 3.56 2.02 0.258

Fructose 5.26 2.65 3.32 1.98 0.247
Sucrose 2.72 1.37 3.00 1.98 0.131
Glycerol 6.20 3.32 5.12 1.87 0.521

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80
70
porosity (%) thickness (µm)
60
50
40
30
20
10
0 0
2 4 6 8
inoculation volume (%)
porosity thickness BC yield
Fig. 4 Effect of inoculation volume on porosities, thicknesses, yields
of bacterial cellulose membranes and cell growth of G. xylinus.
Bacterial cellulose membranes produced with culture time of 5 days.
Error bars represent standard deviations (n = 3)
treatment was used to process the biochemically complex
pellicles into clean cellulose membranes. Having been
treated with different alkali solutions, porosities of bacte-
rial cellulose membranes were found to be in the order of
K2CO3 [ Na2CO3 [ KOH [ NaOH (Fig. 5).
George et al. (2005) found that oxygen transmission
rates of bacterial cellulose membranes were in the order of
K2CO3 [ Na2CO3 [ KOH [ NaOH. They suggested that,
probably due to higher swelling, the mean diameter of the
fibrils in NaOH-treated membranes had increased, thus
reducing the effective pore size available in the mem-
branes, causing a reduction in the transmission of oxygen
through the membrane.
The diameters of ribbons in NaOH-treated bacterial
cellulose were in the range of 40–120 nm with an average
size of 76.10 ± 37.8 nm, while that of K2CO3-treated
bacterial cellulose were in the range of 20–100 nm with an
average size of 53.10 ± 34.8 nm (Fig. 6). This may
explain the fact that the porosity and oxygen transmission
rate of the NaOH-treated bacterial cellulose were the
lowest, while that of K2CO3-treated cellulose was the
highest.
Effect of drying methods on porosities of bacterial
cellulose membranes
Dry reticulated bacterial cellulose can be prepared using
freeze-drying and hot air-drying techniques. Freeze-dried
membranes had much higher porosity (92%) than hot
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6 80
70
5
60
porosity (%)
BC yield (g/L) OD600
4
50
3 40
30
2
20
NaOH KOH Na2CO3 K2CO3
1
Fig. 5 Porosities of chemically treated cellulose membranes. Error
bars represent standard deviations (n = 3)
air-dried ones (65%). Freeze-drying was effective in pre-
OD600
venting the shrinkage of pores during drying (Marabi and
Saguy 2004; Svensson et al. 2005). The collapse was very
severe in air drying for high water content materials, while
the collapse was usually less severe or negligible in freeze
drying (Karathanos et al. 1996).
Observed by SEM, freeze-dried bacterial cellulose had a
more uniform pore size distribution and a higher porosity
(Fig. 7b), in comparison with hot air-dried bacterial cel-
lulose (Fig. 1b). It was obvious that there was remarkable
difference between the two sides of freeze-dried mem-
brane, as seen in Fig. 7a, b.
Conclusions
The porosity of bacterial cellulose membrane is feasible to
be controlled by changing the culture conditions and post-
treatment methods. The growth of bacteria is affected
under varying culture time and inoculation volume, then
the porosities and thicknesses of bacterial celluloses
secreted by G. xylinus are altered.
The mild alkali treatment applied to obtain a pure cel-
lulose membrane also influences the porosity of bacterial
cellulose membrane as the average diameters of fibrils are
changed. The diameter of NaOH-treated BC, which has the
lowest porosity, was in the range of 40–120 nm with an
average size of 76.10 ± 37.8 nm; while for K2CO3-treated
BC, which has the highest porosity, was in range of 20–
100 nm with an average size of 53.10 ± 34.8 nm. Freeze-
dried bacterial cellulose membranes had a more uniform
pore size distribution and a higher porosity than the hot air-
dried ones.
Three-dimensional (3D) network of bacterial cellulose
scaffolds with controlled porosities could be prepared with
the fermentation and post-treatment technique discussed
above. It is concluded that the porous bacterial cellulose
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Fig. 6 AFM images, diameter (b)

(a)
distribution and average
diameter of chemically treated
cellulose microfibrils, a AFM
image of NaOH-BC membrane
b AFM image of Na2CO3-BC
membrane c Diameter
distribution of NaOH-BC
membrane d Diameter
distribution of Na2CO3-BC
membrane

(c) 35
d=76.10± 37.8
30
25
Count(%) 20
15
10
5
0
20 40 60 80 100 120
diameter (nm)

(d) 50
45 d=53.10± 34.8
40
35
Count(%)

30
25
20
15
10
5
0
20 40 60 80 100 120
diameter (nm)

Fig. 7 SEM images of freeze-

dried bacterial cellulose
membrane, a Denser surface
side b Porous opposite side

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would be a potential scaffold material suitable for cells
originating from different tissues and organs.
Acknowledgments This work was funded by the National Basic
Research Program of China (973. Program) under No. 2007CB714305.
It is also supported by Tianjin University of Science and Technology
under Contract No. 20070443.
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