....
"
Cellulose 11: 403 411, 2004.
2004 KlulVer Academic Publishers. Printed in /he Ne/her/ands.
I[)
403
Structural investigations of microbial cellulose produced
in stationary and agitated culture
Wojciech Czaja 1,2, Dwight Romanovicz' and R. Malcolm Brown, 1r. 1,*
ISection of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78713, [/SI1;
is currently affiliated with the Institute of Technical Biochemistry, Technical University of Lodz,
Stefanowskiego 4, 10, Lodz 90-924, Poland; * AUlhor for correspondence (e-mail: rmbrown(c~mail.utexas.edu)
2 Author
Received 24 November 2003; accepted in revised form 6 April 2004
Key words: Acetobac/er, Agitated culture, ATCC 53582, Bacterial cellulose, Fermentation, FT-IR, Micro­
bial cellulose
Abstract
Structural characteristics of microbial cellulose synthesized by two different methods have been compared
using FT-IR and X-ray diffraction techniques. Cellulose synthesized by Acetobacter xylinum NQ-5 strain
from agitated culture conditions is characterized by a lower I a mass fraction than cellulose that was
produced statically. Such a decrease was in good correlation with smaller crystallite sizes of microfibrils
produced in agitated culture. Formation of characteristic cellulose spheres during agitation has been
investigated by various electron and light microscopic methods. On this basis, a hypothetical mechanism of
sphere formation and cell arrangement in the agitated culture has been proposed. During agitation, cells are
stacked together in organized groups around the outer surface of the cellulose sphere.
Introduction
Cellulose is the most abundant biopolymer on
earth and is produced by a variety of organ­
isms, ranging from vascular plants to algae and
prokaryotic organisms such as cyanobacteria
(Jonas and Farah 1998; Nobles et al. 2001). In
addition, there are some strains of the
prokaryotic, non-photosynthetic organism, Ace­
tobacter, which have the ability to synthesize
high-quality cellulose organized as twisting rib­
bons of microfibril bundles (Brown et al. 1992).
Bacterial cellulose demonstrates unique proper­
ties including high mechanical strength, high
crystallinity, high water holding capacity and
high porosity, which make it a very useful bio­
material in many different industrial processes
(Brown 1998; Iguchi et al. 2000). So far, the
best-known commercial applications of bacterial
cellulose include: an acoustic transducer dia­
phragm made of dried cellulose sheet (Nishi
et al. 1990), wound dressing material (artificial
skin) made of wet and purified cellulosic mem­
brane (Fontana et al. 1990), or Nata de Coco, a
traditional Philippine fermented dessert, which
became very popular in Japan a few years ago
(Yoshinaga et al. 1997). In recent years, an
interest has developed in producing bacterial
cellulose on a large commercial scale. Some at­
tempts have been made in the area of optimi­
zation of culture conditions (Kouda et al. 1997a,
b), medium composition (Matsuoka et al. 1996),
strain improvement (Ishikawa et al. 1995;
Vandamme et al. 1998), or the scaling-up pro­
cess, but few production yield enhancements
have been reported so far.
There are two methods to produce bacterial
cellulose: (a) stationary culture, which results in
404
the accumulation of a gelatinous membrane of
cellulose on the surface of the medium; and (b)
agitated culture, where cellulose is synthesized in
deep media in the form of fibrous suspensions,
pellets, or irregular masses (Watanabe et at. 1998;
Chao et at. 2000). While stationary culture con­
ditions have been quite successfully investigated
and described, agitated culture of Acetobacter
strains causes many problems, among which
strain instability, non-Newtonian behavior during
mixing of bacterial cellulose, or proper oxygen
supply are the most common (Kouda et at. 1996,
1997a, b). Despite those problems, some
researchers have suggested that agitated culture
might be the most suitable technique for eco­
nomical scale production (Ross et at. 1991;
Yoshinaga et at. 1997).
Detailed structural characteristics carried out
using electron diffraction analyses (Sugiyama et at.
1991) and (CPMAS) l3C NMR (VanderHart and
Atalla 1984; Yamamoto and Horii 1993) revealed
that native cellulose is a composite of two different
crystalline phases called la and Ifl. Normally,
Acetobacter xylinum cellulose displays character­
istics of highly crystalline, I",-rich cellulose (Van­
derHart and Atalla 1984).
In our investigations, we have studied the
synthesis and structural characteristics of bacte­
rial cellulose produced in stationary and agitated
culture by A. xylinum NQ5 strain (ATCC
53582). This particular strain is characterized by
a periodic series of reversals in the direction of
cellulose ribbon synthesis and produces an agar
colony which contains cellulose synthesized in
tunnels (Thompson et at. 1988). It is also unique
for an uncharacteristic absence of spontaneous
mutation during the agitation process (Brown
and Lin 1990: Saxena et at. 1990). X-ray dif­
fraction was used to characterize the effects of
agitation on the crystalline arrangement of glu­
can chains within microfibrils. Furthermore, the
effect of different rotational speeds of the agita­
tion on the structure of the cellulose was inves­
tigated. Estimation of la and Ip cellulose
fractions in ceJlulose samples from different cul­
ture conditions was carried out using FT-IR
spectroscopy. Light and electron microscopic
techniques were used to examine the formation
of cellulose spheres that are characteristic for
this particular strain when grown in agitated
culture.
Materials and methods
Microorganisms
Acetobacter xylinum NQ5 (ATeC 53582) strain
from the collection of Section of Molecular
Genetics and Microbiology, Cniversity of Texas at
Austin, was used in this study.
recon
resoll'
400 Cl
2900 I
The}
the f(
I,
caleul
absor
Culture medium
X-ray
Schramm Hestrin medium (Hestrin and Schramm
1954) without pH adjustment was used in all
experiments unless otherwise specified.
Culture conditions
The cells for the inoculum were cultured in flasks
either statically or on a rotary shaker with
addition of O. J % cellulase enzyme (Celluclast
1.5L™ from Trichoderma resei, Novo Nordisk
Bioindustrials, Inc., Denmark) for 3 days at
28°C. In the first case, a thick gelatinous mem­
brane was squeezed aseptically to remove cells
em bedded inside the pellicle, and the cell sus­
pension was transferred as the inoculum for the
main culture. In the second case, cells were har­
vested by centrifugation, then resuspended in the
fresh culture medium. The main cultures were
grown in the flasks either statically or on a rotary
shaker (Lab-Line Instruments, Inc. USA) oper­
ating at different rotational speeds (in the range
90- 250 rpm), for 7 days at 28°C. The synthe­
sized cellulose was separated from the medium by
filtration. The quantity of cellulose produced was
measured as dry mass of polymer after washing
with 2% sodium hydroxide (overnight) followed
by three changes of distilled water in order to
remove cells and medium embedded in the cel­
lulose material.
FT-IR spectroscopy
Each cellulose sample was air-dried on a glass
slide in the form of a thin film, which was then
placed across a hole in a magnetic holder. FT-IR
spectra were obtained using a Perkin-Elmer
spectrometer (Spectrum 2000). All spectra were
Cellul
glass
diffra'
Cu-K
either
eqUIp
Co un
1720
25 m,
diffra
rome
range
The c
progr
ware
by Sl
(FWI
ski al
Secti<
The I
gluta
then
After
dehyl
follo\
resin­
in ep
ences
for 2'
were
eithel
secti(
obsel
Ultra
stainl
405
12) strain
vIolecular
fTexas at
recorded with the accumulation of 32 scans, a
resolution of 2 cm- I in the range from 4000 to
400 cm- I, normalized using the band at
2900 cm I due to the COC stretching vibration.
The!, fraction of the samples was calculated by
the following equation (Yamamoto et aL 1996):
f~ = 2.55f;R - 0.32, where f;R of cellulose can be
calculated as A 1 '(A~ + All) and A~ and Ap are
absorbencies at 750 and 710 cm- l , respectively.
X-ray diffraction
Schramm
~d in all
. in flasks
ker with
=elluclast
Nordisk
days at
us mem­
ave cells
cell sus­
t1 for the
;vere har­
ed in the
Ires were
a rotary
A) oper­
he range
: synthe­
~dium by
llced was
washing
followed
order to
the cel­
I
a glass
;vas then
". FT-IR
in- Elmer
tra were
Cellulose samples in the form of sheets dried on
glass slides were placed in the X-ray holder. X-ray
diffraction spectra were recorded using Ni-filtered
Cu-KIX radiation (Il
0.15418 nm) produced by
either a Rigaku RINT 2200 X-ray generator
equipped with a Position Sensitive Proportional
Counter (PSPC) as the detector or a Philips PW
1720 X-ray generator operating at 35 kV and
25 mA, equipped with a Philips vertical scanning
diffractometer and a diffracted beam monoch­
rometer. Scans were perfolmed over the 5 40 28
range using steps of either 0.05° or 0.01° in width.
The data were analyzed using the WinFit software
program (Krumm 1997) or the Jade 5 XRD soft­
ware program. The crystallite size was estimated
by substituting the full-width at half-maximum
(FWHM) into the Scherrer equation (Nieduszyn­
ski and Preston 1970; Alexander 1979).
Sectioning for light and electron microscopy
The microbial cellulose material was fixed in 4%
glutaraldehyde, washed in cacodylate buffer, and
then fixed again in 2% osmium tetroxide (OS04).
After washing in distilled water, the cellulose was
dehydrated in stepwise concentrations of ethanol
followed by absolute acetone, then infiltrated with
resin -acetone solutions. Cellulose was embedded
in epoxy resin (EPON; Electron Microscopy Sci­
ences, USA) and allowed to polymerize at 60 ~C
for 24 h. Both thick (about 1 ,LIm) and thin sections
were cut on a Reichert OM2 ultramicrotome, using
either a glass or diamond knife, respectively. Thick
sections were stained with 1% bromo toluidine and
observed with a Zeiss Universal Light Microscope.
Ultra-thin sections were gently placed on the grids,
stained with lead citrate, washed in 0.02 N NaOH
and boiled distilled water and finally post-stained
with 2% uranyl acetate. Grids were then examined
with a Philips 420 transmission electron mIcro­
scope (TEM) operating at 100 kV.
Scanning electron microscope (SEM) observations
Cellulose samples were fixed in 4% glutaraldehyde
followed by 2% osmium tetroxide and dehydrated
using the same procedure as for sectioning. Sam­
ples were either freeze-dried or critical point dried
(Samdri-790, Tousimis Research Corp.) and then
coated with gold (30~OO, Ladd Research Indus­
tries, Inc.). A Hitachi S-4500 field emission scan­
ning electron microscope operating at 10 or 15 kV
was used for examination of the samples.
Results and discussion
Most of the Acetobacter xy/inul11 strains used
worldwide in research synthesize cellulose stati­
cally in the form of a gelatinous membrane. When
these cultures are grown in agitated conditions the
results often give a poor yield (Yoshinaga et aL
1997). Considering the properties of our Aceto­
bacter NQ5 strain, especially its great genetic sta­
bility, it might be one of the best available strains
to apply using large-scale, agitated and aerated
fermentation systems. A time course of cellulose
synthesis both in stationary and agitated culture
shown in Figure 1 indicates that after 7 days of
culture almost the same quantity of cellulose was
produced.
However, after 16 days we did not notice any
further significant increase in cellulose synthesis
under agitated culture, whereas the pellicle grown
in stationary culture reached a dry mass value of
10 gil (data not shown). One possible explanation
may focus on the different ways of oxygen distri­
bution and substrate penetration during growth.
While the mechanism of pellicle formation and
its oxygen profile have been recently investigated
(Verschuren et aL 2000), not much attention has
been paid to cellulose accumulation in agitated
culture conditions. The NQ5 strain in agitated
culture produces cellulose in the form of charac­
teristic spheres, as shown in Figure 2.
Thick sections cut across the fixed and
embedded cellulose spheres revealed a specific
406
3.5
3
____ stationary
----.- agitated
25
<::
~
2
Q)
III
o
:i 1.5
Qj
u
0.5
o._==--.::r---,----,----,----,----,---------,,----------,
192
24
48
72
96
120
168
144
o
cultivation time [hours]
Figure I. Time course of bacterial cellulose synthesis in stationary and agitated culture.
Figure 2. Cellulose spheres formed in the agitated culture
conditions; scale bar = 5
nlnl.
localization of Acetobacter cells. Sections pre­
sented in Figure 3 show that most of the cells are
distributed at the surface of the sphere and only a
few of them are randomly scattered inside.
Such a particular arrangement might be ex­
plained by the following: cells which are intro­
duced into the fresh medium become attached
around the surface of air bubbles existing in the
agitated liquid; cells start to reproduce and syn­
thesize cellulose ribbons forming eventually a
more compact structure shown in Figures 2 and 3.
In this hypothesis the surface distribution of cells
would suggest that the cellulose is synthesized only
at the surface and that the cells fail, somehow, to
become entrapped in the pellicle as in static cul­
ture. Perhaps shear forces during agitation cause
cells to become separated from the surface of the
sphere.
Alternatively, cells may have a periodic ribbon
synthesis phase whereby the cells actually have a
cycle of synthesis, separation from the sphere,
rejoining the sphere and initiating ribbon synthe­
sis. Such a scenario could explain why the center of
the sphere is solid and has only cellulose ribbons
present. Such a specific surface distribution of cells
has been also proved by SEM observations (Fig­
ure 4b) and TEM observations of thin sections
taken from the same specimen (Figure 3c, d).
Another interesting point is a microscopic
comparison of cellulose microfibril structure, syn­
thesized in stationary and agitated culture condi­
tions. Figure 4c, d demonstrates clearly the
differences between both cellulose samples. Gen­
erally, a fine net built of entangled cellulose rib­
bons represents both of them. A close observation
revealed that mostly uniaxially oriented ribbons
characterize cellulose formed in the stationary
culture, whereas cellulose synthesized under agi­
tated conditions demonstrated a structure of dis­
orderly, curved, overlapping ribbons. Such a
disordered microstructure could be a result of
constant motion forces occurring during agitation.
The thickness of the cellulose microfibrils also
seems to differ between those two samples, with
Fi
d
10
sr
tf
sl
If
Cl
c;
X
d
p
sJ
n
CI
Sl
a
ti
fl
r,
a
407
lesized only
)mehow, to
1 static cul­
ation cause
rface of the
)dic ribbon
ally have a
the sphere,
)on synthe­
he center of
)se ribbons
tion of cells
ltions (Fig­
in sections
3c, d).
nicroscopic
Icture, syn­
ture condi­
:learly the
lples. Gen­
llulose rib­
,bservation
ed ribbons
stationary
under agi­
me of dis­
s. Such a
I result of
~ agitation.
fibrils also
nples, with
Figure 3. Thick (a, b) and thin (c, d) sections of cellulose sphere produced in agitated culture; the characteristic ring of cells is situated
close to the surface of the ball (see a); () small groups of entangled ceJls - initial stage of sphere formation; ( ,) some of the cells
localized inside the sphere formed an ordered layer; we can probably assume that these overlapping layers might be another stage of
sphere formation; scale bars: (a) 25 1,m, (b) 60 11m, (c) lOa nm, (d) 100 nm.
the one from agitated culture distinguished by the
slightly thinner microfibrils.
In order to compare the microstructural changes
in cellulose samples from both differenL culture
conditions and especially to estimate if the shaking
causes any disturbance in the crystallization process,
X-ray diffraction was used. Figure 5 presents X-ray
diffraction patterns taken for both cellulose sam­
ples, which represent a typical profile of cell ulose 1.
Quantitive analysis of the reflections corre­
sponding to all three peaks in those X-ray profiles
revealed that they are shifted to wider angles in the
cellulose sample from agitated culture. Compari­
son of 28 angle values revealed also that in case of
agitation, the (110) and (110) reflections are posi­
tioned closer together than in the cellulose profile
from cellulose grown statically.
These changes in the d-spacings appear to rep­
resent a different proportion of Ie< and If! cellulose
allomorphs as reported previously (Yamamoto
et a1. 1989; Watanabe et a1. 1998). The crystallite
sizes calculated for peaks 1, 2 and 3 using the
Scherrer formula (Nieduszynski and Preston 1970)
are shown in Table I. They clearly demonstrate
existence of smaller crystallite sizes in cellulose
from agitated culture.
The conditions of stress occurring during agi­
tation appear to interfere strongly in the process of
nascent microfibrils crystallization, favoring the
formation of smaller size microfibrils and in­
creased 1/3, the more stable allomorph. Such a
hypothesis is in a good agreement with previous
reports (Yamamoto et a1. 1996; Hirai et a1. 1998).
To determine the exact values of mass fractions
of cellulose Ie< and Iii, FT-IR spectroscopy was
applied. The enlarged regions of the FT-IR spectra
shown in Figure 6 present peaks assigned to Ie,
(750 cm- I ) and If! (710 cm- I ) fractions.
Careful observations of those peaks reveal that
the Ix contribution in cellulose synthesized in
408
Table
togra
Cellu
Static
Agita
also
(eM
alan
199~
Abs
Figure 4. SEM miC!"ographs of the bacterial cellulose produced under different cultme conditions: (a) surface of a sphere formed in the
agitated culture, (b) bacteria seen close to the surface of a cracked cellulose sphere, (c) structure of cellulose produced statically, (d)
structure of cellulose produced during agitation.
1400
d3 (200)
1200
'iii'
- ­ stationary
--agitated
1000
Co
~
~
'iii
c::
Ql
800
d, (110)
600
'E
400
200
0
Abs
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
2 theta [degree]
Figure 5. X-ray diffraction patterns obtained from bacterial cellulose samples synthesized in stationary and agitated culture condi­
tions. Three typical diffraction peaks occurring in the region of 10- 25° are labeled as d), d" and d.1.
agitated culture is lower in comparison with sharp
and well defined peaks in the same spectrum of
stationary produced cellulose. The numerical data
from those spectra are presented in Table 2.
According to the formula proposed previously
(Yamamoto et al. 1996) the estimated Ta mass
fraction is lower in the cellulose sample from agi­
tated culture conditions, confirming our X-ray
results.
Besides the external, environmental stresses on
Acetabaeter during agitation, another possible
explanation of such I'l. mass fraction decrease may
Figw
enlar
409
Table I. d-spacings, crystallite sizes and percent crystallinity of different bacterial cellulose samples determined from X-ray diA'rac­
tograms.
Cellulose sample
Sta tionary
Agitated
d-Spacings (A)
Difference in 28 angle
Crystallite sizes (nm)
Percent crystallinity (%)
CI',
Cl'z
CI',
C
7.9
7.9
8.6
6.6
6.7
6.4
89
84
dl
dz
d)
(Peak I -
602
59
5.23
5.17
3.85
383
223
2.15
also be connected with {J-I,4-endogluconase
(CMCax) synthesis, which occurs in the medium
along with the cellulose production (Tahara et al.
1997; Koo el al. 1998). Generally, it has been
peak 2)
reported that this enzyme plays an essential role in
the cellulose formation process (Tonouchi et al.
1995; Koo et al. 1998). It has also been reported
that the CMCase activity at the end of the agitated
1.1
(a)
f
!
Abs
0.5
400
/
'armed in the
,tatica lIy , (d)
J
~ooo
~
3000
2000
1000
400
Wavenumber[cm-1)
0.9 r - - - - - - - - - - - - - - - - - - - T " ' i l " n - - - , r - - - - - - - - - - - - - - - - - ,
(I))
0.8
0.6
Abs
.Iture condi­
0.4
400
I
~J
rom agi­
ur X-ray
3000
.I·esses on
possible
'ease may
I
2000
1000
\
400
Wavenumber[cm-l)
Figure 6. FT-IR spectra of bacterial cellulose from stationary (a) and agitated (b) culture conditions (part of the graph has been
enlarged to highlight the peaks assigned to cellulose) Ix (750 cm- I ) and II' (710 cm· I ).
410
Table 2. Cellulose I" and I, content (%) and crystallinity index
of bacterial cellulose from different culture conditions deter­
mined by FT-IR measurements.
Cellulose
sample
Stationary
Agitated
I,
76
71
It
24
29
IR crystallinity index
(abs. at 1427(895 cm- I )
4.84
4.48
culture was more than 10 times higher than in the
stationary culture (Watanabe et al. 1998). The
same report (Watanabe et al. 1998) showed that
this higher enzyme activity in the agitated culture
was a reason for lower D?w (weight-average de­
gree of polymerization) fractions in that culture.
Considering this fact, we can suggest that such a
high CMCase activity in the agitated process
might also have an influence on I~ and Iii contri­
bution in the cellulose. This mechanism should be
understood in more detail.
'he crystallinity index calculated for our sam­
ples based on the F'T-IR spectra revealed also a
reduction in crystallinity for samples of agitated
cellulose. "he decrease in crystallinity is in good
agreement with data determined based on the X­
ray profiles analysis. jJ1 that case an estimrrted
percent crystallinity for cellulose grown statically
was also slightly higher than for cellulose synthe­
sized in agitated culture.
Cur studies have focused on the structural
investigation of cellulose formed in agitated cul­
ture conditions and on an interesting Aeetobaete,.
behavior and its product accumulation during
agitation. A. xylinum 1,rQ5 has been found to
dfectively synthesize cellulose in agitated culture
in the form of unique, large spheres. :v.any other
strC1ins of Aeetobaete,. undergo mutation to non­
cellulose producing cells under agitation, and this
often is a problem with maintenance of active
cellulose-producing strains; however, with the
NQ5 strain, mutations to the non-cellulose state
do not occur. :.n fact, no mutations to the non­
cellulose producing state have been observed in
more than 20 years growing this strain. This fact
might hu ve a great impact on its further applica­
tion in a large-scale fermentation process with
such strains as NQ5. ~n several studies up to now,
cellulose production in stationary culture has been
investigated, but a quite low productivity and high
prod uction costs were often the limiting factors
(Matsuoka el al. 1996;~' oshinaga et al. 1997). In
addition, while the static technique does not offer
many optimization alternatives, an efficient bac­
terial cellulose synthesis in agitated and aerated
conditions might be a cost-effective technological
system (Ross et al. 1991). It has been reported that
microbial cellulose production and aerobic Aee­
tobaete,. cell growth are strictly related processes
(1\.1arx-Figini and Pion 1974). For improvement of
cellulose productivity, a high oxygen supply in
agitated ami aerated culture is required to increase
the total cell density and consequently to achieve
high production rates (Kouda et al. I997b, 1998;
Yoshinaga et al. 1997). Besides a good production
yield, the novel properties of such a NQ5 cellulose
synthesized under agitated conditions might have
many different advantages useful in industrial
applications.
Acknowledgements
pel
tee
Hestt
Ac,
car
34~
Hirai
ery
pol
vea
21:
Tgud
cel
261
Ishik
19~
gUi
nUl
Bi<
lanai
m"
Koo
tha
nUl
f,mong many colleagues from the lab, authors are
fibl
Kou(
especially grateful to Dr Krystyna Kudlicka for
her advice and fruitful discussion during this re­
search and to Mr Richard Santos for his technical
assistance. We thank Dr Tetsuo Kondo for use of
FT-IR and X-ray diffraction instruments. This
work was supported in part by a Grant to R.M.B.
from the Welch Foundation (F-1217) and the
Johnson & Johnson Centennial Chair Fund.Spe­
cial thanks go to the Polish-American Fulbright
Commission for a grant awarded to Dr Wojciech
Czaja.
Kou(
Ka
bel
References
Krun
htr
Eff
cel
cui
Kou(
Inl
pr<
Bi(
KO\l(
COl
ani
J.
1
MaD
Alexander L.E. 1979. X-ray Diffraction Methods in Polymer
Science. Robert E. Kreiger Publishing Co., Humington, NY,
pp. 423-424.
Brown R.M. Jr. 1998. Microbial cellulose: a new resource for
wood, paper, textiles, food and specialty products (http:!(
www.botany.utexas.edu(facstaff(facpages(mbrown), Posi ti 0 n
Paper.
Brown R.M. Jr., Kudlicka K., Cousins S.K. and Nagy R. 1992.
Gravity effects on cellulose assembly. Am. J. Bot. 79: 1247­
1258
Brown R.M. lr. and Lin F.e. 1990. Multiribbon microbial
cellulose. US Patent 4,954,439.
Chao Y., Ishida T., Sugano Y. and Shoda M. 2000. Bacterial
cellulose production by Ace/obacter xytinum in a 50-1 inter­
nal-loop airlift reactor. Biotechnol. Bioeng. 68(3): 345-352.
Fontana J.D., de Sousa A.M., Fontana e.K., Torriani I.L.,
Moreschi 1.C.. Gallotti BJ., de Sousa SJ., Narcisco G.P.,
Bichara 1.A. and Farah L.F.x. 1990. Acetobacter cellulose
bic
Bi(
Mats
Yo
los
tali
Nied
nal
ishl
Igl
me
lui
411
is not offer
icient bac­
~d aerated
:hnological
)orted that
robic Ace­
I processes
wement of
supply in
to increase
to achieve
97b, 1998;
?roduction
15 cellulose
night have
industrial
uthors are
Idlicka for
~g this re­
s technical
for use of
ents. This
to R.M.B.
) and the
~und. SpeFulbright
. Wojciech
; in Polymer
rington, NY,
resource for
lucts (http://
vn), Position
agy R. 1992.
)t. 79: 1247­
)11
microbial
00. Bacterial
a 50-1 inter­
:): 345-352.
lorriani I. L.,
[rcisco G.P.,
Iler cellulose
pellicle as a temporary skin substitute. Appl. Biochem. Bio­
technol. 24i25: 253-264.
Hestrin S. and Schramm M. 1954. Synthesis of cellulose by
AcelObacler xy/inum: II. Preparation of freeze-dried cells
capable of polymerizing glucose to cellulose. Biochem. J. 58:
345-352
Hirai A., Tsuji M, Yamamoto H. and Horii F. 1998. /n silll
crystallization of bacterial cellulose. III. Influence of different
polymeric additives on the formation of microfibrils as re­
vealed by transmission electron microscopy. Cellulose 5: 201­
213
Iguchi M., Yamanaka S. and Budhiono A 2000. Bacterial
cellulose - a masterpiece of nature's arls. J. Mater. Sci 35:
261-270
Ishikawa A., Matsuoka Tvf .• Tsuchida T. and Yoshinaga F.
1995. Increasing of bacterial cellulose production by sulfo­
guanidine-resistant mutants derived from Acelobauer xy/i­
mlln subsp. sUCI'o(imnen/ans BPR200 I. Biosci. Biotechnol.
Biochem. 59: 2259-2263
Jonas R. and Farab L.F. 1998. Production and application of
microbial cellulose Polym. Degrad. Stabil. 59: 101-106.
Koo H.M., Song S.H., Pyun Y.R. and Kim Y.S. 1998. Evidence
tha t a beW-I,4-endoglucanase secreted by A celobacler xy/i­
num plays an essential role for the formation of cellulose
fiber. Biosci. Biotechnol. Biochem. 62( II): 2257 -2259.
Kouda T.. Naritomi T.. Yano H. and Yoshinaga F [997a.
Effects of oxygen and carbon dioxide pressures on bacterial
cellulose production by Acelobaeter in aerated and agitated
culture. J. Ferment. Bioeng. 84(2): 124- 127.
Kouda T., Naritomi T.. Yano H. and Yoshinaga F. 1998.
Inhibitory effect of carbon dioxide on bacterial cellulose
prodllction by Acelobaeler in agitated culture. J. Ferment.
Bioeng. 85(3): 318-321.
Kouda T., Yano H. and Yoshinaga F. 1997b. Effect of agitator
configuration on bacterial cellulose productivity in aerated
and agitated culture. J. Ferment. Bioeng. 83(4): 371-376.
Kouda T .. Yano H .. Yoshinaga 1-'., Kaminoyama :vl. and
Kamiwano M. 1996. CharacteriLation of non-l'iewtonian
behavior during mixing of bacterial cellulose in a bioreactor.
J. Ferment. Bioeng. 82(4): 382-386
Krumm S, 1997. Web site: http.www.geol.uni-erlangen.de
html,'.
Marx-Figini \iI. and Pion B.G. 1974 Kinetic investigations on
biosynthesis of cellulose by Acelobacle,. xl'/inium Biochim.
Biophys. ,\Cla 338: 382-393.
Matsuoka M., Tsuchida T., Matsushita K., Adachi O. and
Yoshinaga F. [996. A synthetic medium for bacterial cellu­
lose production by Acelobacler xy/in!l111 subsp. sucro(ermen­
/(Jns. Biosci. Biotechnol. Biochem. 60(4): 575-579.
Nieduszynski 1. and Preston R.D. 1970. Crystallite size in
natural cellulose. Nature 225: 273 274.
Nishi Y., Uryu M., Yamanaka S., Watanabe K., Kitamura K,
Iguchi M. and Mitsuhashi S. 1990. The structure and
mecha nica I properties of sheets prepared from bacteria I cel­
lulose. Part 2: improvement of the mechanical properties of
sheets and their applicability to diaphragms of electro­
acoustic transducers. J. Mater. Sci. 25: 2997-300 I.
l'iobles D.R., Romanovicz D.K. and Brown R.M. Jr 2001.
Cellulose in cyanobacteria. Origin of vascular plant cellulose
synthase? Plant Physio\. 127(2): 529 542.
Ross P .. \ilayer R. and Benziman \iI. 1991. Cellulose biosyn­
thesis and function in bacteria. Microbiol. Rev. 55(1): 35-58.
Saxena l.M., Robens E.M. and Brown R.M. Jr 1990. Modi­
Ikation of cellulose normally synthesized by cellulo,c:-pro­
ducing microorganisms. US Patent 4,950,597
Sugiyama J., Persson J. and Chanzy H. 1991. Combined
infrared and electron diffraction study of the polymorphism
of na tive celluloses. Macromolecules 24: 2461-2466.
Tahara J\., Tabuchi M., Watanabe K, Yano H., Morinaga Y.
and Yosl1inaga F. 1997. Degree of polymerization of cellu­
lOSe from Ace/obacler x.r/inum BPR2001 decreased by cellu­
lase produced by the strain. Biosci. Biotechnol. Biochem.
61(11): 1862-1865
Thompson l\.S., Kaustinen II.M .. Carlson J.A. and Uhlin K.I.
1988. Tunnel structures in Acelobacler xy/inum. Int. J. BioI.
Macromol 10: 126-127.
Tonouchi N., Tahara X, Tsuchida T., Yoshinaga F., Beppu T.
and Horinouchi S. [995. Addition of small amount of an
endoglucanase enhances cellulose production by Ace/obac/er
xy/inum, Biosci. Biotechnol. Biochem. 59(5): 805-808.
Vandamme E.J., De Baets S., Vanbaelen A., Joris K. and De
Wulf P. 1998. Improved production of bacterial cellulose and
its applicatiun potential. Polym. Degrad. Stab. 59: 93-99.
VanderHart D.l.. and Atalla R.H. 1984. Studies of micro­
structure in native celluloses using solid-state I'C NMR
Macromolecules 17: 1465-1472.
Verschuren P.G., Cardona T.D., Robert Nout MJ., De Goo­
ijer K.D and Van den Heuvel J.e. 2000. Location and lim­
itation of cellulose production by Ace/obauer :()'/in!l111
established from oxygen profiles. J. Biosci. Bioeng. 89(5):
414419.
Watanabe K., Tabuchi M., Morinaga Y and Yoshinaga F.
1998. Structural features and properties of bacterial cellulose
produced in agitated culture. Cellulose 5: 187 -200.
Yamamoto H, and Horii F. 1993. CP/MAS I'C NMR analysis
of the crystal transformation induced for Valonia cellulose by
annealing at hi!!h temperatures. :vlacromolecules 26: 13[3­
1317
Yamamoto H" Horii r. and Hirai A. 1996. In silu crystaJIiza­
tion of bacterial cellulose. II. Influences of different polymeric
additives on the fonTIation of celluloses I, and I, at the early
stage of incubation. Cellulose 3: 229242.
Yamamoto H., Hurii F. and Odani H. 1989. Structural changes
of native cellulose crystals induced by annealing in aqueous
alkaline and acidic solutions at high temperatures. Macro­
molecules 22: 4130-4132.
Yoshinaga F., Tonouchi :--J. and Watanabe K. 1997. Research
progress in production of bacterial cellulose by aeration and
agitation culture and its application as a new industrial
material. Biosci. Biotechnol Biochem. 61 (2): 219-224.