Springer 2005

Biotechnology Letters (2005) 27: 14351438

DOI 10.1007/s10529-005-1465-y

Determining the water holding capacity of microbial cellulose

S.T. Schrecker & P.A. Gostomski*
Department of Chemical and Process Engineering, University of Canterbury, Christchurch, New Zealand
*Author for correspondence (Fax: +64-3-3642063; E-mail: peter.gostomski@canterbury.ac.nz)
Received 27 June 2005; Accepted 25 July 2005

Key words: Acetobacter xylinus, microbial cellulose, water holding capacity

Abstract
Comparing reported water holding capacities for microbial cellulose is dicult because dierent methods
are used. The dierent methods aect both the average value and the precision. In this new method, a
vacuum of 10 mm H2O (98 Pa) is applied to the wet cellulose to stabilize the sample prior to determining
the wet weight. This simple method lowers the standard deviation by 50% or more over other methods.

Introduction
In recent years, microbial cellulose has received
considerable attention due to its unique properties especially for biomedical applications. Acetobacter xylinus extrudes cellulose microbrils from
pores on its cell envelope. These microbrils
agglomerate along the side of the cell to form a
cellulose ribbon. The bulk membrane or pellicle
is an entangled web of these ribbons. Microbial
cellulose is very hydrophilic holding over 100
times it weight in water and is far stronger
than cellulose derived from plants (Ross et al.
1991, Brown Jr. 1993).
The water holding capacity of microbial cellulose is an important property for medical applications such as wound dressing for chronic wounds
and thermal burns or articial blood vessels for
microsurgery (Klemm et al. 2001, Dalton 2004).
To measure this property, the sample needs to be
stabilized prior to weighing the wet sample. Published techniques include draining the cellulose
pellicle on a mesh for 1 h (Seraca et al. 2002),
wiping o the excess liquid (Budhiono et al. 1999)
and a quick transfer from the broth to a balance
(Watanabe & Yamanaka 1995). The other common method is to suspend the sample and then
allow the water to drain (Schramm & Hestrin

1954, Williams & Cannon 1989). Since the methods vary substantially, the average wet weight can
vary signicantly, making comparisons between
groups dicult. In addition, some of these methods have poor reproducibility because they
depend on the researchers sample handling technique. To overcome these limitations, we have
developed a new sample stabilization method and
compared it to other published methods.

Materials and methods

Acetobacter xylinus, ICMP 15569 was grown for
192 h in a rotating biological contactor, using a
120 mm diameter cylinder that was 16% submerged in medium. The rotational speed of the
cylinder was 7 rpm. The growth medium was
modied from Seraca et al. (2002): it contained
per litre: glucose 50 g, (NH4)2SO4 5 g, NaH2PO4
2.7 g, MgSO47H2O 1 g, yeast extract (Sigma)
0.5 g, citric acid 1.5 g, ethanol 14 ml, trace element solution 2 ml.
Sample preparation
At harvest, the cellulose pellicle was approximately 10 mm thick. The cellulose was removed

1436
from the cylinder and boiled in 1 M NaOH for
15 min to remove cell debris and remaining medium. It was rinsed twice and soaked in deionized
water (DIW) for 48 h. Four 25 mm diameter
round samples were cut for each method using a
wad punch. For the Vertical drain method, additional samples of approximately 90  30 mm
were prepared. Parts of the pellicle with obvious
defects such as holes were not used as samples.
All samples were soaked in DIW until needed.
Wet sample stabilization
The wet weight of the microbial cellulose was
stabilized by six different methods. Some of the
reported methods did not provide enough detail
to reproduce exactly, therefore some assumptions
were required, e.g. sample dimensions. Four samples were assayed for each method.

Shake method: The sample was removed from

the storage container using tweezers. The
sample was shaken twice quickly and then
weighed.
Rapid transfer method: The sample was transferred to the balance swiftly, minimizing the
amount of surface water dripping o
(Watanabe & Yamanaka 1995).
Vertical drain method: Samples were suspended vertically, held by a 32 mm wide foldback clip for 15 min. The rectangular samples
were gripped from the short edge. This
method was slightly modied from Schramm
& Hestrin (1954), where the samples were
hung over a peg.
Horizontal drain method: The sample was laid
horizontally on a at stainless steel mesh with
3 mm diameter openings separated by 1 mm
and allowed to drain for 60 min before weighing. To avoid evaporation, the sample was
covered (Seraca et al. 2002).
Wipe method: The cellulose was removed
from the DIW and the surface water was
wiped o the sample by hand (Budhiono
et al. 1999).
Vacuum method: Tubing connected a 47 mm
diameter lter holder (Millipore) to an open
reservoir (Figure 1). The lter holder and reservoir were lled with DIW and a 0.22 lm
mixed cellulose esters hydrophilic membrane
(Millipore) was placed on the membrane

Fig. 1. Set-up for the Vacuum method: a glass lter holder

connected to a water lled reservoir.

support mesh. Initially, the water level in the

reservoir was set at the same height as the
membrane support to eliminate air bubbles
trapped below the membrane. The reservoir
water level was then lowered 10 mm below
the level of the lter membrane. This height
dierence maintained a 98 Pa vacuum on the
membrane, while maintaining continuous
water connectivity between the lter membrane and the reservoir. Since this pressure
dierence was below the bubble point of the
hydrophilic membrane (P3.52  105 Pa) air
did not ow through the membrane. The
cellulose sample was placed on the membrane
and the lter reservoir was covered. After 4 h,
the sample was weighed.
Dry weight
The samples were dried at room temperature for
48 h, followed by 12 h at 85 C. They were
quickly transferred to the balance for weighing
to minimize possible rehydration (Figure 1).
Sample weights
All wet and dry weights were determined with a
balance having 0.1 mg reproducibility.
Calculation of the water holding capacity
The water holding capacity (WHC) was the mass
of water removed during drying divided by the
dry weight of cellulose (gwater/gdrycellulose). The
standard deviation of the WHC was calculated
based on four samples.

1437
Results
Each method was repeated four times on cellulose samples from the same fermentation run.
The wet weight was measured after applying the
different methods and combined with the dry
weight, to calculate the water holding capacity
(WHC). Since each method produced a different
average WHC, the relative standard deviation
was evaluated for comparison (Table 1).
The Rapid transfer and Shake methods gave
the highest WHC due to the limited drain time
before the measurement. The Vertical and Horizon drain methods gave a lower WHC since
draining was permitted. The Vacuum method
gave the lowest average WHC. Most of the
methods gave similar variability, however the
Vacuum method had approximately 50% less
variability than the other methods.
Discussion
Different methods for stabilizing wet cellulose
prior to weighing were investigated to assess
average value and variability. We found the published methods inadequate especially in the effect
of technique on reported values within our own
group and therefore comparing our results to
other reports was extremely difcult.
All the methods are related by recognizing
that capillary forces hold the water in the cellulose pore structure and when the sample is
removed from the water, some water drains out.
Table 1. A comparison of water holding capacity (WHC)
using dierent methods for stabilizing the wet cellulose prior
to weighing.
Method

Relative standard
Average WHC
(gwater/gdry cellulose) deviation
(r/WHC) (%)

Vacuum
Vertical drain
(rectangular sample)
Rapid transfer
Horizontal drain
Wipe
Vertical drain
(round sample)
Shake

148
285

5.5
9.88

309
204
249
209

11.2
11.4
13.1
13.5

291

15.3

Values are for four repeats of each method.

The different methods stabilized the sample differently including: (1) simple gravity draining of
the sample (Vertical, Horizontal drain), (2)
impressing an additional force to accelerate
draining (Wipe, Shake, Vacuum) or (3) minimizing the loss (Rapid transfer). The different methods gave quite different results, thereby indicating
the need for a clearly dened technique.
The investigated methods left varying
amounts of surface water on the pellicle. The
Rapid transfer technique had the most surface
water. Water dropped off uncontrollably during
the transfer from the beaker to the balance,
which signicantly inuenced the wet weight. The
amount of water lost depended on the speed of
the transfer, the distance and the tweezer pressure. The Shake and Wipe methods suffered
from similar problems, the number and intensity
of shakes/wipes affected the wet weight, and consistent technique was difcult.
The Vertical drain method allowed a large
amount of the water drain off. However, due to
the vertical drying position, the dimensions of
the sample inuenced the wet weight. The two
sample sizes yielded statistically different water
holding capacities (>99% probability at 95%
condence). The amount of water retained by the
cellulose matrix varied along the length, with the
bottom portion remaining essentially saturated
where water dripped off, a common feature when
porous material drains (Selker et al. 1999). Gravity draining should have caused the longer rectangular samples to have equal or lower bulk
water content than the shorter round samples.
However, the clip caused additional localized
drainage. Since the round cellulose samples were
smaller, the clip compression aected a greater
percent of the round samples. Another contributing eect was the more focused point of draining
in the round samples, meaning a smaller portion
of the sample remained saturated. Therefore, the
sample size, shape and support technique possibly inuence the Vertical drain technique.
The Horizontal drain method eliminated
many of the inuencing factors of the Vertical
drain method, since the vertical dimension was
the sample thickness and the whole sample was
supported evenly. The drain time was longer
than the Vertical drain, which is the most likely
cause for the lower average water content.
However, this method does not rapidly reach

1438
equilibrium (Seraca et al. 2002). Additionally,
the draining process depended heavily on the
aperture size and width of the mesh. Smaller
holes would not let the water pass though easily,
mostly due to surface tension. The majority of
the water drained from the side of the pellicle,
not from the bottom.
Jamye & Rothamel (1948), developed a centrifugation method that was used by Klemm
et al. (2001), and Krystynowicz et al. (2002) for
microbial cellulose. This method was not reproduced in our work. The recommended 800  g
centrifugal force would drain signicantly more
than just the surface water from the pellicle.
Accelerations between 2 and 5 g would generate
similar forces to the Vacuum method, but most
commercial centrifuges do not spin this slowly,
thus requiring a custom centrifuge apparatus.
The centrifugation method at appropriate speeds
should give similar results to the Vacuum method, but would not reach equilibrium signicantly faster.
The Vacuum method was designed to counter
the problems identied with other methods. During its development, we decided to use readily
available laboratory components. The key variables for the method included the magnitude and
duration of the vacuum applied to the sample.
Previous studies have shown that the water
release rate from samples can be quite variable
(Seraca et al. 2002). Steady state was normally
achieved in 12 h (data not shown), so 4 h was
chosen to account for sample dierences.
The 10 mm hanging water column or 98 Pa
vacuum removed free water from the surface of
the cellulose thereby minimizing dripping during
transfer to the balance. The applied vacuum
eliminated edge/surface saturation in the sample
compared to the Vertical or Horizontal drain
methods. However, even at this low vacuum,
the sample thickness decreased slightly as water
drained from the pores and the cellulose structure partially collapsed. The sample thickness
probably decreased in the other methods but
was not observed because of less water loss.
The Vacuum method yielded the lowest relative
standard deviation of all the methods, because
the technique was more repeatable and longer
equilibration times. Four hours of equilibration
was longer than applied to the Horizontal or

Vertical drain methods and the variability of

those methods may have improved if the samples were equilibrated longer. However, we concluded that the Vacuum method was much less
susceptible to technique, sample size or apparatus inuence, thereby allowing better comparison of WHC between research groups.

Acknowledgements
The authors gratefully acknowledge the support
provided by technical staff in the Department of
Chemical and Process Engineering and School of
Biological Sciences at the University of Canterbury.

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