Cellulose 11: 403411, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands. 403

-1

Structural investigations of microbial cellulose produced

in stationary and agitated culture

Wojciech Czaja1,2, Dwight Romanovicz1 and R. Malcolm Brown, Jr.1,*

1
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78713, USA;
2
Author is currently aliated with the Institute of Technical Biochemistry, Technical University of Lodz,
Stefanowskiego 4/10, Lodz 90-924, Poland; *Author for correspondence (e-mail: rmbrown@mail.utexas.edu)

Received 24 November 2003; accepted in revised form 6 April 2004

Key words: Acetobacter, Agitated culture, ATCC 53582, Bacterial cellulose, Fermentation, FT-IR, Micro-
bial cellulose

Abstract

Structural characteristics of microbial cellulose synthesized by two dierent methods have been compared
using FT-IR and X-ray diraction techniques. Cellulose synthesized by Acetobacter xylinum NQ-5 strain
from agitated culture conditions is characterized by a lower Ia mass fraction than cellulose that was
produced statically. Such a decrease was in good correlation with smaller crystallite sizes of microbrils
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 include: an acoustic transducer dia-

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 microbril 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 dierent industrial processes
(Brown 1998; Iguchi et al. 2000). So far, the
best-known commercial applications of bacterial
phragm made of dried cellulose sheet (Nishi
et al. 1990), wound dressing material (articial
skin) made of wet and puried 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 brous suspensions,
pellets, or irregular masses (Watanabe et al. 1998;
Chao et al. 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 al. 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 al. 1991;
Yoshinaga et al. 1997).
Detailed structural characteristics carried out
using electron diraction analyses (Sugiyama et al.
1991) and (CPMAS) 13 C NMR (VanderHart and
Atalla 1984; Yamamoto and Horii 1993) revealed
that native cellulose is a composite of two dierent
crystalline phases called Ia and Ib . Normally,
Acetobacter xylinum cellulose displays character-
istics of highly crystalline, Ia -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 al. 1988). It is also unique
for an uncharacteristic absence of spontaneous
mutation during the agitation process (Brown
and Lin 1990; Saxena et al. 1990). X-ray dif-
fraction was used to characterize the eects of
agitation on the crystalline arrangement of glu-
can chains within microbrils. Furthermore, the
eect of dierent rotational speeds of the agita-
tion on the structure of the cellulose was inves-
tigated. Estimation of Ia and Ib cellulose
fractions in cellulose samples from dierent 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 (ATCC 53582) strain
from the collection of Section of Molecular
Genetics and Microbiology, University of Texas at
Austin, was used in this study.
Culture medium
SchrammHestrin medium (Hestrin and Schramm
1954) without pH adjustment was used in all
experiments unless otherwise specied.
Culture conditions
The cells for the inoculum were cultured in asks
either statically or on a rotary shaker with
addition of 0.1% cellulase enzyme (Celluclast
1.5LTM from Trichoderma resei, Novo Nordisk
Bioindustrials, Inc., Denmark) for 3 days at
28
C. In the rst case, a thick gelatinous mem-
brane was squeezed aseptically to remove cells
embedded 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 asks either statically or on a rotary
shaker (Lab-Line Instruments, Inc. USA) oper-
ating at dierent rotational speeds (in the range
90250 rpm), for 7 days at 28
C. The synthe-
sized cellulose was separated from the medium by
ltration. 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 lm, 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

recorded with the accumulation of 32 scans, a
resolution of 2 cm1 in the range from 4000 to
400 cm1 , normalized using the band at
2900 cm1 due to the COC stretching vibration.
The fa fraction of the samples was calculated by
the following equation (Yamamoto et al. 1996):
fa = 2.55fIR IR
a  0:32, where fa of cellulose can be
calculated as Aa /(Aa + Ab ) and Aa and Ab are
absorbencies at 750 and 710 cm1 , respectively.
X-ray diraction
Cellulose samples in the form of sheets dried on
glass slides were placed in the X-ray holder. X-ray
diraction spectra were recorded using Ni-ltered
Cu-Ka radiation (k = 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
diractometer and a diracted beam monoch-
rometer. Scans were performed over the 540
2h
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 xed in 4%
glutaraldehyde, washed in cacodylate buer, and
then xed again in 2% osmium tetroxide (OsO4 ).
After washing in distilled water, the cellulose was
dehydrated in stepwise concentrations of ethanol
followed by absolute acetone, then inltrated with
resinacetone 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 lm) and thin sections
were cut on a Reichert OM2 ultramicrotome, using
either a glass or diamond knife, respectively. Thick
sections were stained with 1% bromotoluidine 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
405
and boiled distilled water and nally 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 xed 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 (30800, Ladd Research Indus-
tries, Inc.). A Hitachi S-4500 eld 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 xylinum 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 signicant increase in cellulose synthesis
under agitated culture, whereas the pellicle grown
in stationary culture reached a dry mass value of
10 g/l (data not shown). One possible explanation
may focus on the dierent ways of oxygen distri-
bution and substrate penetration during growth.
While the mechanism of pellicle formation and
its oxygen prole 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 xed and
embedded cellulose spheres revealed a specic
406
3.5
stationary
3 agitated
cellulose [g/l] 2.5
1.5
0.5
0
0 24
Figure 1. Time course of bacterial cellulose synthesis in stationary and agitated culture.
Figure 2. Cellulose spheres formed in the agitated culture
conditions; scale bar = 5 mm.
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
48 72 96 120 144 168 192
cultivation time [hours]
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 specic 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 microbril structure, syn-
thesized in stationary and agitated culture condi-
tions. Figure 4c, d demonstrates clearly the
dierences between both cellulose samples. Gen-
erally, a ne 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 microbrils also
seems to dier between those two samples, with
 407

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 cells 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 lm, (b) 60 lm, (c) 100 nm, (d) 100 nm.

the one from agitated culture distinguished by the
slightly thinner microbrils.
In order to compare the microstructural changes
in cellulose samples from both dierent culture
conditions and especially to estimate if the shaking
causes any disturbance in the crystallization process,
X-ray diraction was used. Figure 5 presents X-ray
diraction patterns taken for both cellulose sam-
ples, which represent a typical prole of cellulose I.
Quantitive analysis of the reections corre-
sponding to all three peaks in those X-ray proles
revealed that they are shifted to wider angles in the
cellulose sample from agitated culture. Compari-
son of 2h angle values revealed also that in case of
agitation, the (1
10) and (110) reections are posi-
tioned closer together than in the cellulose prole
from cellulose grown statically.
These changes in the d-spacings appear to rep-
resent a dierent proportion of Ia and Ib cellulose
allomorphs as reported previously (Yamamoto
et al. 1989; Watanabe et al. 1998). The crystallite
sizes calculated for peaks 1, 2 and 3 using the
Scherrer formula (Nieduszynski and Preston 1970)
are shown in Table 1. 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 microbrils crystallization, favoring the
formation of smaller size microbrils and in-
creased Ib , the more stable allomorph. Such a
hypothesis is in a good agreement with previous
reports (Yamamoto et al. 1996; Hirai et al. 1998).
To determine the exact values of mass fractions
of cellulose Ia and Ib , FT-IR spectroscopy was
applied. The enlarged regions of the FT-IR spectra
shown in Figure 6 present peaks assigned to Ia
(750 cm1 ) and Ib (710 cm1 ) fractions.
Careful observations of those peaks reveal that
the Ia contribution in cellulose synthesized in
408

Figure 4. SEM micrographs of the bacterial cellulose produced under dierent culture 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 stationary
agitated
1000
intensity [cps]

800
d1 (110)
600 d3 (110)

400

200

0
5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
2 theta [degree]

Figure 5. X-ray diraction patterns obtained from bacterial cellulose samples synthesized in stationary and agitated culture condi-
tions. Three typical diraction peaks occurring in the region of 1025
are labeled as d1 , d2 , and d3 .

agitated culture is lower in comparison with sharp
and well dened 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 Ia mass
fraction is lower in the cellulose sample from agi-
tated culture conditions, conrming our X-ray
results.
Besides the external, environmental stresses on
Acetobacter during agitation, another possible
explanation of such Ia mass fraction decrease may
 409

Table 1. d-spacings, crystallite sizes and percent crystallinity of dierent bacterial cellulose samples determined from X-ray dirac-
tograms.

Cellulose sample d-Spacings (A) Dierence in 2h angle Crystallite sizes (nm) Percent crystallinity (%)

d1 d2 d3 (Peak 1  peak 2) cr1 cr2 cr3 c

Stationary 6.02 5.23 3.85 2.23 7.9 8.6 6.7 89

Agitated 5.9 5.17 3.83 2.15 7.9 6.6 6.4 84

also be connected with b-1,4-endogluconase reported that this enzyme plays an essential role in
(CMCax) synthesis, which occurs in the medium the cellulose formation process (Tonouchi et al.
along with the cellulose production (Tahara et al. 1995; Koo et al. 1998). It has also been reported
1997; Koo et al. 1998). Generally, it has been that the CMCase activity at the end of the agitated

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) Ia (750 cm1 ) and Ib (710 cm1 ).
410
Table 2. Cellulose Ia and Ib content (%) and crystallinity index
of bacterial cellulose from dierent culture conditions deter-
mined by FT-IR measurements.
Cellulose Ia Ib IR crystallinity index
sample (abs. at 1427/895 cm1 )
Stationary 76 24 4.84
Agitated 71 29 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 DPw (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 inuence on Ia and Ib contri-
bution in the cellulose. This mechanism should be
understood in more detail.
The crystallinity index calculated for our sam-
ples based on the FT-IR spectra revealed also a
reduction in crystallinity for samples of agitated
cellulose. The decrease in crystallinity is in good
agreement with data determined based on the X-
ray proles analysis. In that case an estimated
percent crystallinity for cellulose grown statically
was also slightly higher than for cellulose synthe-
sized in agitated culture.
Our studies have focused on the structural
investigation of cellulose formed in agitated cul-
ture conditions and on an interesting Acetobacter
behavior and its product accumulation during
agitation. A. xylinum NQ5 has been found to
eectively synthesize cellulose in agitated culture
in the form of unique, large spheres. Many other
strains of Acetobacter 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. In fact, no mutations to the non-
cellulose producing state have been observed in
more than 20 years growing this strain. This fact
might have a great impact on its further applica-
tion in a large-scale fermentation process with
such strains as NQ5. In several studies up to now,
cellulose production in stationary culture has been
investigated, but a quite low productivity and high
production costs were often the limiting factors
(Matsuoka et al. 1996; Yoshinaga et al. 1997). In
addition, while the static technique does not oer
many optimization alternatives, an ecient bac-
terial cellulose synthesis in agitated and aerated
conditions might be a cost-eective technological
system (Ross et al. 1991). It has been reported that
microbial cellulose production and aerobic Ace-
tobacter cell growth are strictly related processes
(Marx-Figini and Pion 1974). For improvement of
cellulose productivity, a high oxygen supply in
agitated and aerated culture is required to increase
the total cell density and consequently to achieve
high production rates (Kouda et al. 1997b, 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 dierent advantages useful in industrial
applications.
Acknowledgements
Among many colleagues from the lab, authors are
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 diraction 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.
References
Alexander L.E. 1979. X-ray Diraction Methods in Polymer
Science. Robert E. Kreiger Publishing Co., Humington, NY,
pp. 423424.
Brown R.M. Jr. 1998. Microbial cellulose: a new resource for
wood, paper, textiles, food and specialty products (http://
www.botany.utexas.edu/facsta/facpages/mbrown), Position
Paper.
Brown R.M. Jr., Kudlicka K., Cousins S.K. and Nagy R. 1992.
Gravity eects on cellulose assembly. Am. J. Bot. 79: 1247
1258.
Brown R.M. Jr. and Lin F.C. 1990. Multiribbon microbial
cellulose. US Patent 4,954,439.
Chao Y., Ishida T., Sugano Y. and Shoda M. 2000. Bacterial
cellulose production by Acetobacter xylinum in a 50-l inter-
nal-loop airlift reactor. Biotechnol. Bioeng. 68(3): 345352.
Fontana J.D., de Sousa A.M., Fontana C.K., Torriani I.L.,
Moreschi J.C., Gallotti B.J., de Sousa S.J., Narcisco G.P.,
Bichara J.A. and Farah L.F.X. 1990. Acetobacter cellulose

pellicle as a temporary skin substitute. Appl. Biochem. Bio-
technol. 24/25: 253264.
Hestrin S. and Schramm M. 1954. Synthesis of cellulose by
Acetobacter xylinum: II. Preparation of freeze-dried cells
capable of polymerizing glucose to cellulose. Biochem. J. 58:
345352.
Hirai A., Tsuji M., Yamamoto H. and Horii F. 1998. In situ
crystallization of bacterial cellulose. III. Inuence of dierent
polymeric additives on the formation of microbrils as re-
vealed by transmission electron microscopy. Cellulose 5: 201
213.
Iguchi M., Yamanaka S. and Budhiono A. 2000. Bacterial
cellulose a masterpiece of natures arts. J. Mater. Sci 35:
261270.
Ishikawa A., Matsuoka M., Tsuchida T. and Yoshinaga F.
1995. Increasing of bacterial cellulose production by sulfo-
guanidine-resistant mutants derived from Acetobacter xyli-
num subsp. sucrofermentans BPR2001. Biosci. Biotechnol.
Biochem. 59: 22592263.
Jonas R. and Farah L.F. 1998. Production and application of
microbial cellulose. Polym. Degrad. Stabil. 59: 101106.
Koo H.M., Song S.H., Pyun Y.R. and Kim Y.S. 1998. Evidence
that a beta-1,4-endoglucanase secreted by Acetobacter xyli-
num plays an essential role for the formation of cellulose
ber. Biosci. Biotechnol. Biochem. 62(11): 22572259.
Kouda T., Naritomi T., Yano H. and Yoshinaga F. 1997a.
Eects of oxygen and carbon dioxide pressures on bacterial
cellulose production by Acetobacter in aerated and agitated
culture. J. Ferment. Bioeng. 84(2): 124127.
Kouda T., Naritomi T., Yano H. and Yoshinaga F. 1998.
Inhibitory eect of carbon dioxide on bacterial cellulose
production by Acetobacter in agitated culture. J. Ferment.
Bioeng. 85(3): 318321.
Kouda T., Yano H. and Yoshinaga F. 1997b. Eect of agitator
conguration on bacterial cellulose productivity in aerated
and agitated culture. J. Ferment. Bioeng. 83(4): 371376.
Kouda T., Yano H., Yoshinaga F., Kaminoyama M. and
Kamiwano M. 1996. Characterization of non-Newtonian
behavior during mixing of bacterial cellulose in a bioreactor.
J. Ferment. Bioeng. 82(4): 382386.
Krumm S. 1997. Web site: http/www.geol.uni-erlangen.de/
html/.
Marx-Figini M. and Pion B.G. 1974. Kinetic investigations on
biosynthesis of cellulose by Acetobacter xylinium. Biochim.
Biophys. Acta 338: 382393.
Matsuoka M., Tsuchida T., Matsushita K., Adachi O. and
Yoshinaga F. 1996. A synthetic medium for bacterial cellu-
lose production by Acetobacter xylinum subsp. sucrofermen-
tans. Biosci. Biotechnol. Biochem. 60(4): 575579.
Nieduszynski I. and Preston R.D. 1970. Crystallite size in
natural cellulose. Nature 225: 273274.
Nishi Y., Uryu M., Yamanaka S., Watanabe K., Kitamura N.,
Iguchi M. and Mitsuhashi S. 1990. The structure and
mechanical properties of sheets prepared from bacterial cel-
lulose. Part 2: improvement of the mechanical properties of
411
sheets and their applicability to diaphragms of electro-
acoustic transducers. J. Mater. Sci. 25: 29973001.
Nobles D.R., Romanovicz D.K. and Brown R.M. Jr. 2001.
Cellulose in cyanobacteria. Origin of vascular plant cellulose
synthase? Plant Physiol. 127(2): 529542.
Ross P., Mayer R. and Benziman M. 1991. Cellulose biosyn-
thesis and function in bacteria. Microbiol. Rev. 55(1): 3558.
Saxena I.M., Roberts E.M. and Brown R.M. Jr. 1990. Modi-
cation of cellulose normally synthesized by cellulose-pro-
ducing microorganisms. US Patent 4,950,597.
Sugiyama J., Persson J. and Chanzy H. 1991. Combined
infrared and electron diraction study of the polymorphism
of native celluloses. Macromolecules 24: 24612466.
Tahara N., Tabuchi M., Watanabe K., Yano H., Morinaga Y.
and Yoshinaga F. 1997. Degree of polymerization of cellu-
lose from Acetobacter xylinum BPR2001 decreased by cellu-
lase produced by the strain. Biosci. Biotechnol. Biochem.
61(11): 18621865.
Thompson N.S., Kaustinen H.M., Carlson J.A. and Uhlin K.I.
1988. Tunnel structures in Acetobacter xylinum. Int. J. Biol.
Macromol. 10: 126127.
Tonouchi N., Tahara N., Tsuchida T., Yoshinaga F., Beppu T.
and Horinouchi S. 1995. Addition of small amount of an
endoglucanase enhances cellulose production by Acetobacter
xylinum. Biosci. Biotechnol. Biochem. 59(5): 805808.
Vandamme E.J., De Baets S., Vanbaelen A., Joris K. and De
Wulf P. 1998. Improved production of bacterial cellulose and
its application potential. Polym. Degrad. Stab. 59: 9399.
VanderHart D.L. and Atalla R.H. 1984. Studies of micro-
structure in native celluloses using solid-state 13 C NMR.
Macromolecules 17: 14651472.
Verschuren P.G., Cardona T.D., Robert Nout M.J., De Goo-
ijer K.D. and Van den Heuvel J.C. 2000. Location and lim-
itation of cellulose production by Acetobacter xylinum
established from oxygen proles. 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: 187200.
Yamamoto H. and Horii F. 1993. CP/MAS 13C NMR analysis
of the crystal transformation induced for Valonia cellulose by
annealing at high temperatures. Macromolecules 26: 1313
1317.
Yamamoto H., Horii F. and Hirai A. 1996. In situ crystalliza-
tion of bacterial cellulose. II. Inuences of dierent polymeric
additives on the formation of celluloses Ia and Ib at the early
stage of incubation. Cellulose 3: 229242.
Yamamoto H., Horii 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: 41304132.
Yoshinaga F., Tonouchi N. 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): 219224.